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Amino acid hot spots of halogen bonding–a combined theoretical and experimental case study of the 5-HT7 receptor Rafa# Kurczab, Vittorio Canale, Grzegorz Sata#a, Pawe# Zajdel, and Andrzej J. Bojarski J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00828 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 6, 2018

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Journal of Medicinal Chemistry

Amino acid hot spots of halogen bonding–a combined theoretical and experimental case study of the 5-HT7 receptor Rafał Kurczaba,*, Vittorio Canaleb, Grzegorz Satałaa, Paweł Zajdelb, Andrzej J. Bojarskia

a

Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of

Sciences, 12 Smętna Street, 31-343 Krakow, Poland b

Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna

Street, 30-688 Krakow, Poland

KEYWORDS: Halogen bonding, drug design, 5-HT7R, molecular interactions, medicinal chemistry

ABSTRACT

A computational approach combining a structure-activity relationship library of halogenated and the corresponding unsubstituted ligands (called XSAR) with QM-based molecular docking and binding free energy calculations was used to search for amino acids frequently targeted by halogen bonding (hot spots) in a 5-HT7R as a case study. The procedure identified two sets of hot spots, extracellular (D2.65, T2.64, and E7.35) and transmembrane (C3.36,

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T5.39 and S5.42), which were further verified by a synthesized library of halogenated arylsulfonamide derivatives of (aryloxy)ethyl piperidines. It was found that a halogen bond formed between T5.39 and a bromine atom at 3-position of the aryloxy fragment, caused the most remarkable, 35-fold increase in binding affinity for 5-HT7R when compared to the nonhalogenated analog. The proposed paradigm of halogen bonding hot spots was additionally verified on D4 dopamine receptor showing that it can be used in rational drug design/optimization for any protein target.


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Introduction In the range of intermolecular interactions, halogen bonding (XB) is one of the most intensively investigated in recent years.1–8 Halogen bonds are ruled by the same mechanism as hydrogen bonds, i.e., they show mostly electrostatic character and are highly directional, and their length is shorter than the sum of the van der Waals radii of their constituent atoms.9 A halogen bond can be defined as a directional bond between a covalently bound halogen atom (acting as a donor) and a Lewis base, the bond acceptor. It originates from the anisotropy of the electron density distribution around the halogen atom,10–14 in particular from the fundamental properties of the covalent σ-bond between atoms in the C–X group. Halogen atoms have five electrons occupying the p atomic orbitals of their valence shell (according to molecular orbital theory) and the single valence electron of the pz orbital is involved in the creation of a covalent σ-bond with a carbon atom. As a result, the depopulation of this orbital opposite the C–X σ-bond leaves a hole that partially exposes the positive nuclear charge. This so-called σ-hole accounts for the electropositive crown and polar flattening associated with polarization effects (anisotropy in charge distribution), whereas the four electrons remaining in the px and py orbitals account for the electronegative ring lying perpendicular to the σ-bond. This arrangement leads to attractive interactions between C–X moieties and classical hydrogen bond acceptors (Figure 1). Halogen bonds are strong enough to control the aggregation of organic molecules in solid,15 liquid,16 and gas phases17 and have been intensively investigated in recognition processes.18–21 They are also considered a new tool in materials science22 or a novel interaction for rational drug design.2,7,8,23–27 Among fourteen 5-hydroxytryptamine receptor (5-HTR) subtypes, 5-HT7R is the mostly recently identified.28 It is canonically coupled to Gαs or Gα12 proteins, promoting signal transduction through cAMP/PKA- and ERK-dependent pathways.29 Distribution studies



revealed a correlation between the localization of 5-HT7R in the central nervous system (CNS), especially in the thalamus, hypothalamus (suprachiasmatic nucleus), hippocampus and cerebral cortex, and its functions, suggesting an involvement in physiological (specifically, regulation of sleep and circadian rhythm and learning and memory processes) as well as pathophysiological phenomena.30,31 Several studies have confirmed that 5-HT7R agonists may produce beneficial effects in the treatment of dysfunctional memory in neurodegenerative disease (e.g., Alzheimer’s disease),32 X fragile syndrome, ADHD, and pain.33,34 On the other hand, preclinical findings revealed that the genetic inactivation or pharmacological blockade of 5-HT7R produced an antidepressant-like effect.35–37 A large body of evidence underlines the involvement of 5-HT7R antagonists in improving memory processes, suggesting a potential therapeutic benefit in the treatment of cognitive impairment in depression38 and the negative symptoms of schizophrenia.39 This work presents a universal computational algorithm developed to identify the amino acids frequently targeted by halogen bonding (called hot spots) in the 5-HT7R binding site used as a case study. The theoretical predictions were evaluated experimentally by using newly synthetized library of halogenated arylsulfonamide derivatives of (aryloxy)ethyl piperidines. The results confirmed the existence of amino acids preferred for halogen bonding and provide evidence for the rational design of new bioactive molecules using halogen atoms.

Results and Discussion Study design A computational workflow (Figure 2) was developed to identify amino acids that frequently form halogen bonds with ligands. In the first stage, a library containing unsubstituted and halogenated analog(s) (called XSAR) whose affinities for 5-HT7R are


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available in the ChEMBL database40 was built. Next, a positive subset of the XSAR library (i.e., sets showing improved activity after halogenation) was used to probe the binding site using a combination of QM-based molecular docking (i.e., quantum-polarized ligand docking, QPLD) to multiple 5-HT7R homology models and binding free energy (∆G) calculations (i.e., generalized-Born/surface area, GBSA). In the final stage, all amino acids involved in the formation of halogen bonds that yielded higher ∆G values for halogenated analogs than unsubstituted structures (∆∆G 1.00). The docking to 5HT7R homology models was performed by the use of the QPLD/GBSA procedure, which showed very good performance in reproducing halogenated ligand–receptor (L–R) complexes



stored in the protein database (PDB).42 An analysis of the best docking poses for each XSAR set showed a common interaction pattern with 5-HT7R, i.e., a salt bridge with D3.32 and hydrophobic/aromatic interactions with an aromatic cluster consisting of F6.51, F6.52, and W6.48. Additional contacts were specific and determined by the chemotype and substituents. The selected XSAR sets highlight different roles of halogen atoms that result in the halogenated analogs having higher activity than the nonhalogenated analogs (Figure 3). Set8 is represented by the 3- (magenta) and 4-Cl (cyan) derivatives of the N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamide43 chemotype and showed an Xeffect value slightly greater than one. The binding mode revealed that no halogen bond was formed; however, the position change in the chlorine atom decreased (3-Cl, approximately 11-fold and ∆∆G = +3.04 kcal/mol) and increased (4-Cl, approximately 1.5-fold and ∆∆G = –0.28 kcal/mol) the activity. This change was caused by a change on the chlorobenzene ring orientation triggered by a steric interaction of the chlorine atom. Compounds belonging to set1444 (with a chlorinated fragment exposed toward the extracellular part of the receptor) had only slightly higher activity for halogenated ligand (1.1-fold and ∆∆G = –1.81 kcal/mol) than the nonhalogenated analog. Set1145 displayed the highest Xeffect value (97.1-fold higher activity for the halogenated derivative and ∆∆G = –9.15 kcal/mol), which was related to the formation of two halogen bonds, one with the backbone carbonyl oxygen of T5.39 (distance 3.15 Å, σhole angle = 168.5°) and one with the backbone carbonyl oxygen of S5.42 (distance 2.95 Å, σ-hole angle = 155.4°). The halogenated derivatives of long-chain 4-substituted piperazines linked to a quinazoline system (set1546) confirmed that the T5.39 is a preferred amino acid for the creation of halogen bonds and illustrates the high geometric preferences for their formation. The 3-Cl derivative showed an approximately 7-fold increase in affinity (∆∆G = – 4.78 kcal/mol) as a result of the formation of halogen bonding (distance 3.22 Å and σ-hole angle = 157.2°); however, when the chlorine atom was switched to the 4-position, the activity


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decreased approximately 8-fold from that of the nonhalogenated form (∆∆G = +13.05 kcal/mol). The reason for this lower activity by the 4-Cl derivative is steric hindrance that leads to the destabilization of the complex. Based on the analysis of the interactions of all XSAR sets with the 5-HT7R (Figure S2, Figure S3 and Table S1), it can generally be concluded that the increases in the Xeffect value resulting from steric (the median fold is 2.1, Table S1) or hydrophobic (the median fold change is 1.75, Table S1) interactions of the ligand with the protein were smaller than those originating from halogen bonding interactions (the median fold change is 5.5, Table S1). On the other hand, the decrease in the activity of halogenated derivatives (Xeffect < 1.0) was caused by unfavorable steric interactions (the median fold change is –5.0, Table S1) or a docking failure (the median fold change is –5.0, Table S1) of a given derivative to the receptor model(s).

The 5-HT7R halogen bonding hot spots The QPLD/GBSA docking procedure and XSAR data were used to probe the 5-HT7R binding site to determine the amino acids most frequently targeted by halogen bonding. Since the 5-HT7R has not been crystallized, a set of homology models built on eight templates close to the 5-HT7R was used. For each template, amino acids were classified into two categories, namely, the primary (i.e., those commonly forming halogen bonding) and the secondary hot spots (less frequently engaged). For each category, halogen bonding contacts with the carbonyl oxygens of the protein backbone or with Lewis bases in side chains were distinguished (Figure S4). All amino acids classified as halogen bond acceptors were averaged over all templates, and a final hypothesis was generated (Figure 4) independently for side chains and backbone carbonyl oxygens.



We found 2 primary and 2 secondary halogen bonding hot spots targeting the backbone carbonyl oxygen of amino acids, and 4 primary and 2 secondary halogen bonding contacts targeting side chains. The most frequently engaged carbonyl oxygens were those of S5.42 and T5.39. Interestingly, S5.42 was indicated as a primary hot spot by 7 out of 8 sets of 5-HT7R homology models (for details see Table S4) and as a secondary hot spot by the remaining set. Additionally, T2.64 and C3.36 were indicated to form halogen bonds with carbonyl oxygens less frequently but at a significant level. For amino acid side chains as halogen bonding acceptors, D2.65, C3.36, S5.42 and E7.35 were indicated as primary hot spots, and T2.64 and S6.55 were indicated as secondary XB acceptors. In this category, the role of S5.42 was additionally highlighted as a key anchoring point for halogen bonding in the 5-HT7R binding site (indicated by almost all homology models generated, Figure S4). We previously suggested the potential role of the C3.36 side chain in interactions with halogenated 5-HT7R ligands.47 However, this interaction is limited by competitive contact with the carbonyl oxygen of S5.42 or T5.39 (the detailed analysis is in the Supporting Information, Figure S5). Published analyses of PDB LR complexes with halogen bonds showed that the most frequently observed halogen bonds are halogen−carbonyl oxygen,1,26,27,48 followed by halogen−π1,49,50 interactions, while side chains are only rarely targeted.26 Additionally, current experimentally identified halogen bonds in L–R complexes are mostly targeted to peptide oxygens.51–53 Moreover, due to the anisotropy of the electron density on halogens, they may act as hydrogen bond acceptors. Thus, halogenated ligands compete with hydrogen bond donors (e.g., the hydroxyl groups in serine, threonine, and tyrosine), and additionally, entropic disadvantages stemming from side chain flexibility54 significantly limit the number of effective targetable side chains. Thus, in light of these findings, we can conclude that the most significant halogen bond acceptor in 5-HT7R is the carbonyl oxygens of T5.39 and S5.42.


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Synthesis of a library of halogenated derivatives The library used to evaluate the predicted hot spots was designed on the basis of set24, which had been identified in the XSAR library. The synthesis of designed compounds (58– 88) was performed according to a multistep procedure reported in Scheme 1. First, the alkylation of the commercially available phenols 1–19 under biphasic conditions in refluxing acetone yielded (aryloxy)ethyl bromide derivatives 20–38. Next, these alkylating agents reacted with Boc-protected 4-aminopiperidine, yielding intermediates 39–57. After the removal of the Boc group, the final arylsulfonamides 58–88 were obtained upon treatment of primary amines with the selected arylsulfonyl chlorides.

Evaluation of 5-HT7R halogen bonding hot spots using synthesized library An analysis of the binding mode for an unsubstituted compound (58) in multiple 5-HT7R homology models showed that the phenylsulfonyl fragment was generally exposed toward the extracellular part of the receptor, whereas the aryloxy fragment was pointing into the binding crevice formed by helices 3, 5 and 6 (Figure 5A). Thus, the introduction of a halogen or hydrophobic substituents into different positions in both terminal aromatic fragments allows a systematic validation of the halogen bonding hot spots located in different parts of the binding site (Figure 4). At first, the ability of the extracellular fragment (i.e., D2.65, T2.64, E7.35, and C45.x50) of the 5-HT7R to target the halogen bonding hot spots was evaluated by docking halogenated derivatives bearing Cl, Br, and I atoms in all positions at the phenylsulfonyl fragment. Each compound was docked to a set of 5-HT7R homology models using a combination of QPLD and GBSA methods. The results indicated that only 2-halogenated analogs form halogen bonds with either the D2.65 and/or T2.64 side chains from one side or C45.x50, which is located in the ECL2 on another side (Figure 5B). For both 3- and 4-halogenated derivatives,



only steric/hydrophobic interactions were found (Figure 5C, D). An energetic analysis showed that the formation of halogen bonds increased the binding free energy (depending on the binding pose, the ∆∆G ranged from –4.9 to –2.1 kcal/mol) more significantly than the steric/hydrophobic interactions found for the 3- and 4-analogs (depending on the binding pose, the ∆∆G ranged from –2.8 to –0.7 kcal/mol) relative to the unsubstituted analog (58). These findings were confirmed by binding affinity results obtained for the synthesized derivatives (Table 1). In general, all compounds with a halogen atom in the 2-position displayed higher affinity for 5-HT7R than their 4- and 3-halogenated analogs (59 vs 62 and 64, 60 vs 63 and 65). However, for 2-halogenated derivatives, the predicted increase in affinity with increasing size of the halogen atom was not observed, i.e., the bromine derivative is slightly preferred over derivatives with the larger iodine and smaller chlorine. The experimental data highlight a plateau of affinity in this series and that the type of halogen (Cl, Br, I) has an important influence on the affinity (Xeffect).

Next, a theoretical investigation of the derivatives with 2-halogenated aryloxy fragments targeting the remaining XB hot spots (i.e., C3.36, T5.39 and S5.42) indicated that no halogen bonds were formed (Figure 6A) and that the increase in the binding free energy is a result of fitting to the volume and shape of the binding crevice formed by helices 3, 5 and 6 (Figure 6B). This hypothesis led to the calculation of the molecular volume (Vm, Table 2) for halogens (i.e., Cl, Br, and I) as well as several hydrophobic substituents (i.e., CH3, CF3, i-Pr, t-butyl, and phenyl). The results of these theoretical investigations showed that for halogens, the increase in the atom size causes an increase in the binding free energy; however, for hydrophobic substituents, this correlation was not so clear and probably depends on the shape of the substituent (e.g., substitution with the larger t-butyl gained less ∆∆G than that with the smaller i-Pr substituent). Interestingly, the binding results for synthesized 2-substituted


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derivatives showed good correlation with theoretical indications – an increase in the volume of the halogen atom improved the affinity for 5-HT7R (66 vs 67 vs 68). However, for the hydrophobic substituents, the increase in the affinity was not directly correlated with the volume, and the shape of the substituent can also have important contributions. This result is in line with our previously reported data,55 in which a compound with a methyl group (Vm = 1070.5 bohr3/mol) displayed lower affinity than compounds with the sterically hindered isopropyl and phenyl analogs (69 vs I and II, Ki = 534, and 36 and 45 nM, respectively). Molecular modeling showed that the 3-halogenated derivatives can form halogen bonds and adopt three orientations in the binding site (the same result was identified in an analysis of the XSAR sets, see Figure S4), each being stabilized via halogen bonding (Figure 6C). The binding free energy calculations indicated that the most preferred orientation is with these derivatives halogen bonding with T5.39 (∆∆G = –4.84 kcal/mol), followed by S5.42 (∆∆G = –3.20 kcal/mol) and C3.36 (∆∆G = –1.98 kcal/mol). In contrast, the presence of hydrophobic substituents at the 3-position significantly decreased the binding free energy from the value in the corresponding 2-analogs, which resulted from steric hindrances obstructing the ligand from adopting the optimal position in the binding site. The binding of synthesized 3-analogs revealed, however, that only the 3-Br derivative (73) exhibited high affinity (Ki = 41 nM, Xeffect = 34.6). Interestingly, in contrast to the theoretical predictions, experimental data of this series of halogenated derivatives also indicated a plateau of affinity. Theoretical predictions for hydrophobic substituents in the 3-position were in line with experimental results, as these compounds showed substantially lower affinity than 2-analogs. Finally, as docking results for 4-substituted derivatives indicated unfavorable steric interactions (positive ∆∆G), only two 4-halogenated derivatives (Cl and Br) were synthesized. Indeed, the 5-HT7R affinity for 4-Cl and 4-Br derivatives of 58 was significantly lower than that of the parent structure (Xeffect = 0.6 and 0.4, respectively).



Additionally, the interaction sphere plotted onto the backbone carbonyl oxygen of T5.39 was used to explain the decrease in activity upon changing the location of the bromine atom in the aryloxy fragment (Figure 6D). The generated interaction sphere confirmed that the Br atom in the 3-position lies within the energetically favorable region of the sphere and the Br atoms in the 2- and 4-position were outside the sphere, ruling out the formation of halogen bonds. These experimental data are consistent with the calculated binding free energy values: the 3Br derivative (73) showed the highest gain in the binding free energy (∆∆G = −4.84 kcal/mol), followed by the 2-Br and 4-Br analogs (∆∆G = –2.55 and +6.14 kcal/mol, respectively).

Combined effect of two halogens Based on the theoretical and experimental results obtained for monosubstituted aryloxy and phenylsulfonyl fragments, compound 73 (with the preferred substitution pattern at the aryloxy fragment, i.e., 3-Br) was further modified to study additive effects resulting from the presence of another halogen atom. For this purpose, selected derivatives containing an additional halogen atom at either the aryloxy moiety or the terminal phenylsulfonyl fragment were investigated theoretically and then experimentally (Table 3). The theoretical investigation showed that the introduction of an additional halogen atom generally reduced the binding free energy of the L–R interactions (Table 3) from its value for compound 73. Halogen bonding between the carbonyl oxygen of T5.39 and the bromine atom of the aryloxy fragment was detected for all studied derivatives (Figure 7A–C). For the phenylsulfonyl fragment, only the 3-Br analog (86) formed a halogen bond with the D2.64 side chain or the carbonyl oxygen of E7.35 (Figure 7B), which may be associated with the 3-Br analog having a higher binding free energy than the 2- and 4-Br derivatives (84 and 88, respectively). The binding results showed that compounds containing two halogens in the aryloxy fragment also


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had slightly lower affinity for 5-HT7R than their monosubstituted derivatives (82 and 83 vs 68 and 73, Table 3). Moreover, the addition of a halogen atom at the arylsulfonyl fragment decreased the affinity for 5-HT7R. Interestingly, in the case of compounds with halogen atoms placed at two terminal positions (84–88), a halogen at the 3-position at the arylsulfonyl fragment was more favorable for binding than a halogen at either the 2- or 4-positions (85 vs 87 and 86 vs 84 and 88), which is in line with theoretical predictions.

Insight from molecular dynamics simulations into the plateau effect The theoretical study indicated that a halogen at only the 3-position in aryloxy fragment had suitable geometric conditions for halogen bonding; however, experimental tests showed that increasing the size of the halogen atom did not improve the affinity (a plateau effect). To obtain further insight into the potential molecular mechanism of this effect, molecular dynamics (MD) simulations for complexes of 2-Br/I and 3-Br/I aryloxy analogs were performed. Because the carbonyl oxygen of T5.39 was found as a target for halogen bonding, the distance and the σ-hole angle between the halogen atom and carbonyl oxygen of T5.39 were extracted from the MD trajectories. The results showed (Figure 8) that in the 2-position, both bromine and iodine have unfavorable geometric parameters for halogen bonding with T5.39, the closest identified hot spot. For the 3-position, a majority of XB geometric parameters extracted from MD simulations of the Br analog are clustered in a favorable region for halogen bonding; however, in the case of iodine, a weaker halogen bond appears to be created with T5.39 (favorable angles but larger XB distances), probably due to its large size. Another plausible explanation of the plateau effect was provided by Lange et al.,56 who suggested that dispersive CH-X interactions can affect the negative electrostatic potential of the halogen and thus compensate for the loss of the halogen bond. For the smaller atom



chlorine, these effects can even overcompensate for the halogen bond, while bromine is indifferent, and the potential repulsion of the larger iodine leads to a reduced affinity.

Evaluation of the impact of halogen bonding on 5-HT7/1AR selectivity The impact of halogen substitution pattern on selectivity over the 5-HT1AR subtype (which shows a high degree of amino acid homology to 5-HT7R57) was also investigated. The binding data showed that structural modifications at the arylsulfonamide fragment were not crucial for binding to 5-HT7R and 5-HT1A/7R selectively. In contrast, an increase in the volume of halogen at the 2-position in the aryloxy fragment improved the selectivity ratio (66 vs 67 vs 68), and these parameters are even more strongly correlated for the hydrophobic substituents (the best selectivity was obtained for the t-butyl group, S1A/7 = 50.5). Among all 3-substituted analogs, only compound 73, with a bromine atom, exhibited a significantly high selectivity ratio. Since molecular modeling revealed that no compound from the whole series docked in a proper way to 5-HT1AR homology models, 73 having the highest selectivity (as well as Xeffect) can be attributed to the creation of the halogen bond with T5.39 at the 5HT7R binding pocket. The introduction of a second halogen atom into the compound generally led to moderate 5-HT7R affinity and selectivity, and only compound 83 (2-I, 5-Br) showed a further improvement in selectivity for 5-HT1AR (S1A/7 = 96.4).

XB hot spot strategy verification on the D4 dopamine receptor To validate the developed algorithm and the XB hot spot paradigm, an experimentally solved target structure was used. The dopamine D4 receptor was selected because (i) it belongs to the same aminergic subfamily of class A GPCRs as the 5-HT7R, (ii) it has a high resolution crystal structure (1.962 Å), (iii) it has an appropriate number of XSAR sets to


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determine the role of halogen atoms in L–R interactions, and (iv) a halogen bond is present in the crystallized L–R complex. Application of our algorithm to D4 receptor ligands stored in the ChEMBL database yielded 53 XSAR sets (all sets are provided in the Supporting Information), where halogenation improved the activity relative to the unsubstituted structure (i.e., Xeffect > 1.00). Structural clustering of the XSAR library indicated 14 different clusters (molecular scaffolds), whose centroids were next used to tune the crystal structure of the D4 receptor conformations using an induced-fit docking procedure, (described in the Experimental Section, all data are provided in the Supporting Information). Based on the analysis of the interactions of all XSAR sets with a set of D4 receptor conformations, a different role of halogen atoms in L–R complexes was identified (Figure S6, Figure 9). First, the most frequently targeted amino acids by halogen bonding were identified, i.e., the primary – V5.40 (c) and the secondary – S5.43 (c), S5.461 (c, s) and H6.55 (s). The prediction of the primary XB hot spot (V5.40) was confirmed by a majority of XSAR sets; however, the most significant evidence came from the experimental structure of the D4 receptor complexed with nemonapride, where aromatic chlorine forms a halogen bond to the backbone carbonyl of V5.40 (Figure 9A, distance 3.68 Å, σ-hole angle = 163.2°). Worthy of note were L–R complexes of XSAR sets having a particularly high Xeffect value (Figure S6; i.e., set44 and set51) because they all contained diCl derivatives which formed halogen bonds with the carbonyl oxygen of V5.40 (Figure 9B, distance 3.37 Å, σ-hole angle = 167.4°), and with the side chain of H6.55 (Figure 9B, distance 3.19 Å, σ-hole angle = 148.5°). Moreover, the analysis also indicated that H6.55 was generally targeted by 2-halogenated aryl-piperazine/piperidine derivatives. The last two secondary XB hot spots were less accessible for a halogenated fragment of ligands and were detected only for several chemotypes. For instance, in the case of set53 (Figure 9C), an approximately 39-fold increase of affinity for halogenated analog can result from the



formation of two medium halogen bonds, i.e. with the backbone of S5.43 (distance 3.56 Å, σhole angle = 142.6°), and the side chain of S5.461 (distance 3.41 Å, σ-hole angle = 141.3°). In XSAR sets, which had only a slightly higher activity for halogenated analogs and no possibility to form halogen bonds, a visual inspection revealed that the halogenated fragment of a ligand was usually exposed toward the extracellular part of the receptor (Figure 9D).

Conclusions The hypothesis of certain amino acids being targeted for halogen bonding (i.e., hot spots) was examined first in silico and then verified experimentally. For this purpose, a new computational approach was developed to search for hot spots of halogen bonding by probing the binding site using a structure-activity relationship library containing pairs of halogenated and unsubstituted ligands (so-called XSAR) with known affinity for the 5-HT7R. The algorithm identified a set of primary and secondary XB hot spots located in two distinct parts of the receptor, namely, the extracellular (including D2.65, T2.64, and E7.35) and transmembrane parts (i.e., C3.36, T5.39 and S5.42). To verify these hypotheses, N-[1-(2phenoxyethyl)piperidin-4-yl]benzenesulfonamide was used as a molecular framework for the generation of a library of halogenated derivatives. The rationale behind the choice of this structure resulted from its low affinity for 5-HT7R, its having a known synthetic route enabling the facile generation of halogenated derivatives, and its binding mode exposing the two terminal aromatic rings toward the part of the receptor where sets of XB hot spots were found. Notably, a complementary strategy that uses halogen-enriched fragment libraries (HEFLibs) has recently been developed and used for lead discovery.58,59 HEFLibs contain small halogenated fragments, which as molecular probes can explore binding sites for


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favorable halogen bond interactions to identify unique binding modes (halogen bonding hot spots). In general, the theoretical predictions made by the developed computational approach were in good agreement with the experimental validation. However, among all identified XB hot spots for 5-HT7R, only several were found to make an important contribution to halogen bonding (i.e., D2.65, T2.64, and T5.39). Although S5.42 was indicated by the XSAR library to be the most frequently targeted XB hot spot (an amino acid neighboring T5.39 and buried deeper than it in the binding site), T5.39 had the most significant contribution to the affinity increase for the synthesized library. A plausible explanation for this difference may result from the molecular scaffold influencing the spatial orientations and conformational restrictions of a ligand in the binding site; moreover, it should be emphasized that the majority of the XSAR subsets contained a halogenated arylpiperazine fragment that was more rigid than the aryloxyethyl fragment. The lower impact of extracellular XB hot spots than transmembrane regions on the affinity change can be explained by the higher flexibility of the extracellular fragment and its greater exposure to the solvent, which can lead to the creation of weaker and less frequent XB contacts. The detailed analysis of molecular interactions involved in the stabilization of the L–R complex of both XSAR and synthesized libraries revealed that halogen atoms can play different roles in the L–R interaction, i.e., they can halogen bond and provide hydrophobic interactions and

steric

hindrance.

The

interplay between

halogen

bonding and

hydrophobic/steric interactions is difficult to quantify; thus, hydrophobic substituents with a diverse range of volumes and shapes were used to verify the differences in XB vs. hydrophobic effects. Interestingly, this approach showed that in the case where XB was detected in L–R complexes, the affinity increase was significantly higher for derivatives containing halogen than those containing hydrophobic substituents, and in the case where



only steric/hydrophobic effects dominated the L–R complexes, the gain in affinity was well correlated with the volume and shape of the substituent. Besides the evaluation of L–R interactions with halogenated derivatives, other important effects, i.e., the synergy of halogen bonding, the plateau of an affinity, and the selectivity for the similar subtype receptor 5-HT1AR, were considered in this study. The initial hypothesis of halogen bonding being additive was investigated using di-substituted derivatives targeting hot spots belonging to the different regions (i.e., extracellular and transmembrane). Interestingly, the theoretical method indicated that two halogen bonds were formed (86); however, probably due to induced conformational changes, the L–R complex with 86 had a lower binding free energy than the complex with monosubstituted compound 73, which was in line with experimental data. A purely theoretical assumption states that halogen bonding strength increases from chlorine to bromine to iodine; however, among derivatives with a halogen atom localized in a favorable position to form a halogen bond (2- and 3-positions at the phenylsulfonyl and aryloxy fragments, respectively), a plateau of affinity was reached with bromine. Unfortunately, in both series (for aryloxy and phenylsulfonyl fragments), iodine provided no significant increase in affinity, which means that in this system, other factors counteracted. The results of MD simulations supported the experimental data for these findings and showed that steric restrictions have a key impact on the stabilization of the L–R complex by halogen bonding. Moreover, to the best of our knowledge, this report was the first use of MD simulations to study the plateau effect in halogen bonding. Finally, among all synthesized monohalogenated derivatives, the most selective also showed the highest affinity gain from halogenation. Unfortunately, due to its very low affinity to the second receptor, no coherent binding mode was obtained, and thus it cannot be concluded that


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halogen bonding in one receptor is crucial for selectivity over the second one. However, it should be noted that halogen bonding was previously verified as a selectivity trigger.2 Analysis of the DrugBank database for the presence of halogen atoms in drug molecules shows that they are a frequently used modification by medicinal chemists (the percentages of all drug molecules containing –Cl, –Br, –I, –CH3, –OCH3, –i-Pr, and –t-butyl groups are 11.1, 2.6, 1.3, 4.7, 6.3, 1.2 and 0.5%, respectively). The computational algorithm developed and tested in this study showed a high potential to indicate the most favorable halogen substitution position in the aromatic ring to efficiently maximize biological activity. Thus, this algorithm might be used to rationalize synthetic protocols and to improve virtual screening for any target.



Experimental Section Workflow for searching for halogen-bonding hot spots A calculation procedure (Figure 2) was developed to identify key amino acids that improve the activity of a ligand by participating in the formation of halogen bonding with it. The first step was to create a library (called XSAR) containing sets, each containing an unsubstituted molecule and all of its halogenated analogs, whose biological activity data for a given receptor are available. Next, the XSAR sets that showed greater activity for the halogenated derivative(s) were used to probe the binding site using QM-based molecular docking and the binding free energy (∆G) calculations. In the final stage, all amino acids (XB hot spots) involved in the formation of halogen bonding contacts that resulted in greater ∆G values for halogenated analogs than for their corresponding unsubstituted structure and fulfilled the geometric criteria for halogen bonding (both in terms of distance and σ-hole angle) were identified. The JChem Suite v17.23.060 and MayaChemTools v961 libraries for computational drug discovery were used.

Generation of structure-activity relationship datasets for halogenated analogs (XSAR) Compounds whose activity for 5-HT7R had been determined were fetched from the ChEMBL v22 database.40 However, only molecules whose activities were quantified by Ki, pKi, IC50 or pIC50 and had been tested in human protein assays were taken into account. Next, an algorithm was developed and used to find all pairs containing halogenated structures and their corresponding unsubstituted structures (a detailed description of the algorithm is in the Supporting Information, Figure S1). To describe the influence of halogenation on the biological activity of the unsubstituted (parent) molecule, the Xeffect parameter was calculated as a ratio of parent compound to its halogenated derivative activity. Xeffect values


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between 0 and 1 denote the negative influence of halogenation (a decrease in the activity upon halogenation), whereas values higher than 1 mean that the activity increased after halogen substitution. To visualize the collected XSAR data, an R script was prepared and used to generate a heat matrix. For individual XSAR subsets, the influence of halogenation (both the type of halogen and the place of substitution) was represented by a field whose spectrum of colors (purple to blue to red) were assigned to denote increases in the Xeffect level. The field was black when the Xeffect had been decreased by halogenation. The columns of the XSAR matrix indicate the Xeffect value of the halogenation type in the aromatic ring. A single row shows the Xeffect value for a given substitution position in the aromatic ring; however, if an XSAR set contains more than one halogenation position, the halogenation at each position was written in a separate row.

Homology modeling of 5-HT7R Building of homology models. The sequence of the human 5-HT7 receptor (ID: P34969) was obtained from the UniProtKB/Swiss-Prot database.62 Homology models of the 5-HT7 receptor were built based on class A GPCR crystal structures retrieved from the Protein Data Bank (Table S2, Similarity/identity scores before sequences are shown in Figure S7). Sequences of the modeled receptor and selected templates were aligned manually (Figure S8) using Accelrys Discovery Studio v3.0, making sure that the most conserved amino acids in each helix and motifs characteristic for class A GPCRs were in equivalent positions. Ranges of helices were determined on the basis of crystal structures; loop regions were modeled, but not refined. For each aligned template, a series of 200 models were generated using Modeller 9v8 software63 employing an approach previously utilized in our laboratory.64,65 Validation. The modeled structures were validated using a flexible ligand docking method (performed using Glide software in XP precision mode) and enrichment calculations. Sets of



ligands with known activities (Ki 5-HT7 < 100 nM) and inactivities (Ki 5-HT7 > 1000 nM) were retrieved from the ChEMBL database (version 22).40 All active compounds were hierarchically clustered using Moldprint2D, the Tanimoto metric and the Complete Cluster Linking Method implemented in Canvas v2.0.66 The centroid was selected from each cluster containing more than two members. Finally, all centroids of active compounds (121 for 5HT7R) and the whole set of inactive compounds (1634) constituted the input for docking. The 3-dimensional structures of the synthesized compounds were prepared using LigPrep v3.067 and the appropriate ionization states at pH = 7.4±1.0 were assigned using Epik v2.8.68 Protein Preparation Wizard was used to assign the bond orders, check for steric clashes and assign appropriate amino acid ionization states for each receptor model. The receptor grids were generated (the OPLS3 force field69) by centering the grid box of the size of 12 Å on the D3.32. Automated docking was performed using Glide v6.370 at the XP level with the flexible docking option turned on. Selection of final models. The homology models were evaluated by calculating the ROC curve based on the Glide Score values of docked compounds (the undocked actives and inactives were assigned as false negatives and true negatives, respectively). The final model quality was determined by the area under the ROC curve (AUROC), based on which ten models per template with the highest AUROC value were selected for further studies.

QM-polarized ligand docking Quantum mechanic/molecular mechanic (QM/MM) docking was performed using the QPLD implemented in Schrödinger Suite.71 QPLD combines the Glide docking algorithm with QM/MM calculations performed by the Q-Site program,72,73 which uses the Jaguar74 and Impact programs for the QM (ligand) and MM (protein) regions, respectively. At the initial


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stage of the QPLD procedure, the ligands were docked into a rigid protein using Glide SP. Next, the resulting binding poses were used to calculate the partial atomic charges of the ligand by a single-point calculation in Q-Site. The B3PW91 functional,75–78 which was applied in several theoretical studies of halogen bonding systems,79–82 was used in conjunction with the cc-pVTZ basis set for Cl and Br and the cc-pVTZ-pp basis set for I-containing ligands.83 Thus, the effect of the polarization of the charges on the ligand by the receptor was considered in the final docking stage, where the partial charges derived from the QM calculations of the ligand were used. During the QPLD calculations, no protein flexibility was present; however, the ligands are treated as flexible in each of the two docking stages. The number of returned poses per ligand in each docking stage was set to 10.

Binding free energy calculations GBSA was used to calculate the binding free energy based on the L–R complexes generated by the QPLD procedure. The ligand pose energies were minimized using the local optimization feature in Prime, the flexible residue distance from a ligand pose was set to 4.0 Å, and the ligand charges obtained in the QPLD stage were used. The energies of complexes were calculated with the OPLS369 force field and GBSA continuum solvent model. To assess the influence of a given substituent on the binding, the ∆∆G was calculated as a difference between the binding free energy (∆G) of a halogenated compound and its unsubstituted (parent) analog.

Identification of halogen bonding hot spots The docking results for the XSAR sets (which showed that the activity of the halogenated derivatives was greater than that of the parent) were used to determine the number of halogen bonding interactions with the side chains and carbonyl oxygen atoms of ACS Paragon Plus Environment


amino acids. Only amino acids forming a halogen bond with a distance lower than 4.00 Å, a σ-hole angle larger than 140° and a ∆∆G lower than 0 were accepted. For individual receptor conformations, the halogen bonding interaction frequency (calculated as a number of halogen bonds formed by a given amino acid divided by all halogen bonds detected overall) was obtained to distinguish the most commonly interacting amino acids, which were depicted as hot spots (a frequency > 40% threshold was used), and those that were involved in the formation of the halogen bonding less frequently (20% < frequency < 40%). All other amino acids were rejected as insignificant (mainly singletons). In the next step, the results of amino acid classification were averaged over all the models generated on a given template using the same thresholds as were used in the first step.

Plotting interaction spheres for halogen bonding To visualize (plotting interaction spheres) the possible contribution of halogen bonding to L–R complexes, the halogen bonding web server was used (accessed 01-02-2018, http://www.halogenbonding.com/).

Molecular dynamics simulations MD simulations (60 ns) were performed using Schrödinger Desmond software. The L–R complexes, selected in molecular docking analysis, were immersed into a phosphatidylcholine (POPC, 300 K) membrane bilayer and positioned using the PPM web server (http://opm.phar.umich.edu/server.php, accessed 05-12-2017).84 Each system was solvated by water molecules described by the TIP4P potential, and the OPLS3 force field parameters were used for all atoms. Additionally, 0.15 M NaCl was added to mimic the ionic strength inside the cell.


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Optimization of the binding site of the D4 receptor using an induced-fit docking procedure The dopamine 4 receptor (PDB ID: 5WIU85) was retrieved from the PDB database and was then optimized using the induced-fit docking (IFD) protocol from Schrödinger. The IFD combines flexible ligand docking (using the Glide algorithm) and side chain refinement in Prime. A set of halogenated analogs representing each XSAR set and exhibiting an Xeffect > 1.0 was hierarchically clustered using Moldprint2D, the Tanimoto metric and the Complete Cluster Linking Method implemented in Canvas v2.0. The centroid was selected from each cluster and used as input for IFD. In each case, the grid box was anchored on D3.32 and allowed on residues refinement within 12 Å from ligand. Then, for each centroid the ten topscored L–R complexes were inspected visually to select those showing the closest compliance with the common binding mode for D4 receptor ligands.85 The final validation of the selected receptor conformations was performed by QPLD docking of the XSAR library, retaining at least one receptor conformation per cluster centroid from the XSAR library.

Chemistry Organic syntheses were carried out at ambient temperature, unless otherwise indicated. The organic solvents used in this study (Sigma-Aldrich and Chempur) were of reagent grade and were used without purification. All other commercially available reagents were of the highest purity (from Sigma-Aldrich, Fluorochem, and TCI). All workup and purification procedures were performed with reagent-grade solvents under ambient atmosphere. Column chromatography was performed using Merck 60 silica gel (70–230 mesh ASTM). Mass spectra were recorded on a UPLC-MS/MS system consisting of a Waters ACQUITY® UPLC® (Waters Corporation, Milford, MA, USA) coupled to a Waters TQD mass spectrometer (electrospray ionization (ESI)-tandem quadrupole). Chromatographic separations were performed using an Acquity UPLC BEH (bridged ethyl hybrid) C18 column



(2.1 × 100 mm, 1.7-µm particle size) equipped with an Acquity UPLC BEH C18 VanGuard pre-column (2.1 × 5 mm, 1.7-µm particle size). The column was maintained at 40°C, and elution was performed under gradient conditions from 95% to 0% of eluent A (water/formic acid (0.1%, v/v); eluent B was acetonitrile/formic acid (0.1%, v/v)) over 10 min at a flow rate of 0.3 mL min-1. Chromatograms were obtained using a Waters eλ PDA detector. The spectra were analyzed in the 200–700 nm range with a 1.2 nm resolution and a sampling rate of 20 points/s. The MS detection settings of the Waters TQD mass spectrometer were as follows: source temperature, 150°C; desolvation temperature, 350°C; desolvation gas flow rate, 600 L h-1; cone gas flow, 100 L h-1; capillary potential, 3.00 kV; and cone potential, 40 V. Nitrogen was used as both the nebulizing gas and the drying gas. The data were obtained in a scan mode ranging from 50 to 2000 m/z at 1.0 s intervals. MassLynx V 4.1 (Waters) was used as the data acquisition software. The UPLC/MS purity of all the final compounds was confirmed to be 95% or higher. 1

H NMR and

13

C NMR spectra were obtained with a Varian BB 200 spectrometer

using TMS (0.00 ppm) as the internal standard in CDCl3 and were recorded at 300 and 75 MHz, respectively. The J values are reported in Hertz (Hz), and the splitting patterns are designated as follows: s (singlet), br.s. (broad singlet), d (doublet), t (triplet), dd (doublet of doublets), dt (doublet of triplets), dq (double of quartets), td (triplet of doublets), ddd (doublet of doublet of doublets), dtd (doublet of triplets of doublets), and m (multiplet). The general procedures used for the synthesis of intermediate and final compounds were in accordance with previously reported methodology.86,87

Characterization data for representative final compounds 2-Iodo-N-[1-(2-phenoxyethyl)piperidin-4-yl]benzenesulfonamide (61)


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Brown oil, 110 mg (yield 70%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 97%, tR = 4.69, C19H23IN2O3S, MW 486.37, Monoisotopic Mass 486.05, [M+H]+ 487.2. 1H NMR (300 MHz, CDCl3) δ 1.47–1.60 (m, 2H), 1.71–1.80 (m, 2H), 2.11–2.21 (m, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.78–2.85 (m, 2H), 3.13–3.23 (m, 1H), 4.03 (t, J = 5.6 Hz, 2H), 5.11 (d, J = 7.6 Hz, 1H), 6.83–6.89 (m, 2H), 6.90–6.97 (m, 1H), 7.22–7.30 (m, 2H), 7.38–7.51 (m, 2H), 7.71–7.75 (m, 1H), 8.16 (dd, J = 7.6, 1.8 Hz, 1H). 3-Chloro-N-[1-(2-phenoxyethyl)piperidin-4-yl]benzenesulfonamide (62) Yellow oil, 120 mg (yield 82%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 96%, tR = 4.61, C19H23ClN2O3S, MW 394.92, Monoisotopic Mass 394.11, [M+H]+ 395.1. 1H NMR (300 MHz, CDCl3) δ 1.43–1.58 (m, 2H), 1.73–1.85 (m, 2H), 2.14–2.27 (m, 2H), 2.76 (t, J = 5.8 Hz, 2H), 2.85 (dt, J = 12.2, 3.3 Hz, 2H), 3.15–3.30 (m, 1H), 4.05 (t, J = 5.8 Hz, 2H), 4.59 (d, J = 7.7 Hz, 1H), 6.85–6.89 (m, 2H), 6.94–6.96 (m, 1H), 7.24–7.29 (m, 2H), 7.29–7.41 (m, 1H), 7.69 (ddd, J = 7.9, 1.9, 1.0 Hz, 1H), 7.78–7.83 (m, 1H), 8.03 (t, J = 1.8 Hz, 1H). N-[1-(3-Bromophenoxyethyl)piperidin-4-yl]benzenesulfonamide (73) Brown oil, 90 mg (yield 78%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC /MS purity 97%, tR = 4.61, C19H23BrN2O3S, MW 439.37, Monoisotopic Mass 438.06, [M+H]+ 439.0. 1H NMR (300 MHz, CDCl3) δ 1.40–1.55 (m, 2H), 1.77 (dd, J = 12.9, 4.1 Hz, 2H), 2.12–2.22 (m, 2H), 2.73 (t, J = 5.9 Hz, 2H), 2.76–2.85 (m, 2H), 3.13–3.26 (m, 1H), 4.01 (t, J = 5.9 Hz, 2H), 4.45 (d, J = 7.6 Hz, 1H), 6.77–6.82 (m, 1H), 7.02–7.08 (m, 2H), 7.08–7.15 (m, 1H), 7.48–7.61 (m, 3H), 7.86–7.91 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 29.7, 32.9, 50.5, 51.7, 56.8, 66.1, 113.0, 117.8, 122.8, 123.9, 126.8, 129.1, 130.5, 132.6, 141.2, 159.4.



N-{1-[2-(3-Tolyloxy)ethyl]piperidin-4-yl}benzenesulfonamide (75) Brown oil, 80 mg (yield 65%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 97%, tR = 4.03, C21H26N2O3S, MW 374.50, Monoisotopic Mass 374.17, [M+H]+ 361.1. 1H NMR (300 MHz, CDCl3) δ 1.45–1.56 (m, 2H), 1.72–1.84 (m, 2H), 2.13–2.23 (m, 2H), 2.31 (s, 3H), 2.74 (t, J = 5.8 Hz, 2H), 2.83 (dt, J = 12.1, 3.3 Hz, 2H), 3.14–3.25 (m, 1H), 4.02 (t, J = 5.8 Hz, 2H), 4.58 (br.s., 1H), 6.64–6.70 (m, 2H), 6.73–6.79 (m, 1H), 7.14 (t, J = 7.9 Hz, 1H), 7.47–7.61 (m, 3H), 7.86–7.92 (m, 2H). N-{1-[2-(3-Isopropylphenoxy)ethyl]piperidin-4-yl}benzenesulfonamide (77) Brown oil, 80 mg (yield 65%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 96%, tR = 4.09, C22H30N2O3S, MW 402.55, Monoisotopic Mass 402.20, [M+H]+ 403.3. 1H NMR (300 MHz, CDCl3) δ 1.22 (d, J = 6.5 Hz, 6H), 1.41–1.55 (m, 2H), 1.69–1.82 (m, 2H), 2.12–2.22 (m, 2H), 2.74 (t, J = 5.9 Hz, 2H), 2.80–2.90 (m, 3H), 3.15–3.25 (m, 1H), 4.03 (t, J = 5.6 Hz, 2H), 4.47 (br.s., 1H), 6.68 (dd, J = 8.1, 2.3 Hz, 1H), 6.74–6.76 (m, 1H), 6.81 (d, J = 7.6, 1H), 7.18 (t, J = 7.9 Hz, 1H), 7.47– 7.61 (m, 3H), 7.85–7.91 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 23.9, 32.9, 34.2, 50.6, 52.3, 57.1, 65.7, 111.3, 113.1, 119.1, 126.9, 129.1, 129.2, 132.6, 141.2, 150.6, 158.7. N-(1-{2-[3-(tertbutyl)phenoxy]ethyl}piperidin-4-yl)benzenesulfonamide (78) Brown oil, 80 mg (yield 65%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 97%, tR = 5.35, C23H32N2O3S, MW 416.58, Monoisotopic Mass 416.21, [M+H]+ 417.3. 1H NMR (300 MHz, CDCl3) δ 1.29 (s, 9H), 1.44– 1.56 (m, 2H), 1.73–1.83 (m, 2H), 2.12–2.22 (m, 2H), 2.75 (t, J = 5.9 Hz, 2H), 2.80–2.89 (m, 2H), 3.16–3.25 (m, 1H), 4.04 (t, J = 5.9 Hz, 2H), 4.44 (br.s., 1H), 6.65–6.70 (m, 1H), 6.90– 6.92 (m, 1H), 6.98 (dd, J = 7.0, 1.8 Hz, 1H), 7.17–7.23 (m, 1H), 7.48–7.58 (m, 3H), 7.86– 7.91 (m, 2H).


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N-[1-(4-Chlorophenoxyethyl)piperidin-4-yl]benzenesulfonamide (80) Brown oil, 110 mg (yield 69%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.7 v/v); UPLC/MS purity 97%, tR = 4.53, C19H23ClN2O3S, MW 394.92, Monoisotopic Mass 394.11, [M+H]+ 395.2. 1H NMR (300 MHz, CDCl3) δ 1.45–1.55 (m, 2H), 1.73–1.83 (m, 2H), 2.12–2.22 (m, 2H), 2.73 (t, J = 5.8 Hz, 2H), 2.78–2.86 (m, 2H), 3.14–3.27 (m, 1H), 4.00 (t, J = 5.8 Hz, 2H), 4.55 (br.s., 1H), 6.71–6.80 (m, 2H), 7.32–7.39 (m, 2H), 7.48–7.61 (m, 3H), 7.86–7.91 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 32.8, 50.5, 52.3, 56.9, 65.4, 115.8, 125.7, 126.8, 128.7, 129.3, 132.6, 141.2, 157.23. N-{1-[2-(3-Bromo-2-iodophenoxy)ethyl]piperidin-4-yl}benzenesulfonamide (82) Brown oil, 120 mg (yield 73%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.5 v/v); UPLC/MS purity 97%, tR = 5.08, C19H22BrIN2O3S, MW 565.26, Monoisotopic Mass 563.96, [M+H]+ 565.0. 1H NMR (300 MHz, CDCl3) δ 1.41–1.55 (m, 2H), 1.77 (dd, J = 12.9, 3.5 Hz, 2H), 2.19–2.30 (m, 2H), 2.79–2.85 (m, 2H), 2.85–2.92 (m, 2H), 3.10–3.26 (m, 1H), 4.07 (t, J = 5.3 Hz, 2H), 4.75 (d, J = 7.0 Hz, 1H), 6.65 (dd, J = 8.2, 1.2 Hz, 1H), 7.08–7.15 (m, 1H), 7.22–7.29 (m, 1H), 7.46–7.62 (m, 3H), 7.83–7.94 (m, 2H). 13

C NMR (75 MHz, CDCl3) δ 33.0, 50.5, 52.5, 56.6, 68.4, 94.9, 109.9, 110.0, 125.3, 126.9,

129.1, 130.1, 131.1, 132.6, 141.1, 159.3. 2-Bromo-N-{1-[2-(3-bromophenoxy)ethyl]piperidin-4-yl}benzenesulfonamide (84) Brown oil, 110 mg (yield 74%) following chromatographic purification over silica gel with CH2Cl2/MeOH (9/0.5 v/v); LC/MS purity 98%, tR = 4.94, C19H22Br2N2O3S, MW 518.26, Monoisotopic Mass 519.97, [M+H]+ 519.1. 1H NMR (300 MHz, CDCl3) δ 1.46–1.56 (m, 2H), 1.70–1.80 (m, 2H), 2.10–2.20 (m, 2H), 2.72 (t, J = 5.7 Hz, 2H), 2.78–2.83 (m, 2H), 3.13–3.23 (m, 1H), 4.00 (t, J = 5.7 Hz, 2H), 5.10 (br.s., 1H), 6.77–6.81 (m, 1H), 7.02–7.08 (m, 2H), 7.09–7.15 (m, 1H), 7.39–7.51 (m, 2H), 7.73 (dd, J = 7.7, 1.5 Hz, 1H), 8.16 (dd, J = 7.7, 1.5



Hz, 1H).

13

C NMR (75 MHz, CDCl3) δ 32.5, 50.8, 52.2, 56.8, 66.2, 113.5, 117.9, 119.7,

122.8, 123.9, 127.9, 130.5, 131.2, 133.7, 135.1, 140.0, 159.4.


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AUTHOR INFORMATION Corresponding Author *(R.K.). E-mail: [email protected]

ACKNOWLEDGMENT The

study

was

supported

by

the

National

Science

Center Grant

No.

DEC-

2014/15/D/NZ7/01782.

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TABLES Table 1. Binding affinities of the synthesized compounds 58–65 for 5-HT7 and 5-HT1ARs.

∆∆Gc

Ki [nM] ± SEM Cmpd

R1

Xeffect 5-HT7

5-HT1A

S1A/7a

b

[kcal/mol]

58

H

1417 ± 152

3614 ± 294

2.6

–

–

59

2-Cl

290 ± 31

2058 ± 187

7.1

4.9

–3.1

60

2-Br

204 ± 19

2077 ± 142

8.8

6.0

–3.7

61

2-I

258 ± 27

496 ± 33

1.9

5.5

–3.2

62

3-Cl

1671 ± 193

1428 ± 79

0.9

0.8

–0.7

63

3-Br

1154 ± 72

770 ± 67

0.7

1.2

–1.3

64

4-Cl

1154 ± 104

3198 ± 421

2.9

1.2

–1.8

65

4-Br

902 ± 86

1961 ± 245

2.2

2.6

–2.2

a

Ratio of affinities for 5-HT1A and 5-HT7Rs

b

Ratio of affinities for unsubstituted and X-substituted compounds

c

Averaged after all correct poses were found in the docking results for all homology models



Page 44 of 56

Table 2. Binding affinities of synthesized compounds 66–81 for 5-HT7 and 5-HT1ARs.

Vm

∆∆Gd

(bohr3/mol)

[kcal/mol]

Ki [nM] ± SEM Cmpd

R

Xeffect 5-HT7

5-HT1A

S1A/7a

b

58

H

1417 ± 152

3614 ± 294

2.6

–

839.1

–

66

2-Cl

1029 ± 138

1285 ± 150

1.2

1.4

1176.5

–1.6

67

2-Br

415 ± 36

2349 ± 322

5.7

3.4

1265.1

–2.1

68

2-I

64 ± 9

619 ± 68

9.7

22.1

1333.5

–4.9

69

2-CH3

534 ± 79

2368 ± 177

4.4

2.7

1070.5

–2.5

70

2-CF3

213 ± 24

4358 ± 632

20.5

6.7

1121.3

–3.3

Ic

2-i-Pr

36± 4

249 ± 42

6.9

39.4

1206.4

–5.6

71

2-t-butyl

172 ± 19

8687 ± 652

50.5

8.2

1356.3

–2.7

IIc

2-phenyl

45 ± 4

299 ± 20

6.6

31.5

1497.6

–4.7

72

3-Cl

1342 ± 81

1637 ± 246

1.2

1.1

1176.5

–1.4

73

3-Br

41 ± 6

2211 ± 197

53.9

34.6

1265.1

–4.5

74

3-I

203 ± 26

509 ± 72

2.5

7.0

1333.5

–4.8

75

3-CH3

885 ± 95

2108 ± 181

2.4

1.6

1070.5

–1.1

76

3-CF3

1580 ± 113

1951 ± 329

1.2

0.9

1121.3

0.4

77

3-i-Pr

547 ± 84

388 ± 63

0.7

2.6

1206.4

–1.5

78

3-t-butyl

931 ± 73

1545 ± 93

1.7

1.5

1356.3

–2.1

79

3-phenyl

602 ± 42

1456 ± 222

2.4

2.4

1497.6

–0.9

80

4-Cl

2544 ± 206

38290 ± 3229

15.1

0.6

1176.5

4.7

81

4-Br

3187 ± 413

52610 ± 7688

16.5

0.4

1265.1

5.9

a

Ratio of affinities for 5-HT1A and 5-HT7Rs Ratio of affinities for unsubstituted and X-substituted compounds c Data taken from previous work55 d Averaged after all correct poses were found in the docking results for all homology models b


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Table 3. Binding affinities of the synthesized compounds 82–88 for 5-HT7R and 5-HT1ARs.

∆∆Gc

Ki [nM] ± SEM Cmpd

R1

R

Xeffect 5-HT7

5-HT1A

S1A/7a

b

[kcal/mol]

58

H

H

1417 ± 152

3614 ± 294

2.6

–

–

68

H

2-I

64 ± 9

619 ± 68

9.7

22.1

–4.9

73

H

3-Br

41 ± 6

2211 ± 197

53.9

34.6

–4.5

82

H

3-Br,2-I

110 ± 9

2677 ± 447

24.3

12.9

–3.3

83

H

5-Br,2-I

93 ± 14

8966 ± 1131

96.4

15.2

–3.2

84

2-Br

3-Br

228 ± 17

462 ± 63

2.0

6.2

–2.9

85

3-Cl

3-Br

86 ± 13

1411 ± 198

16.4

16.5

–3.2

86

3-Br

3-Br

67 ± 7

1266 ± 184

18.9

21.1

–3.9

87

4-Cl

3-Br

408 ± 56

4067 ± 497

10.0

3.5

–2.4

88

4-Br

3-Br

313 ± 21

2669 ± 325

8.5

4.5

–2.8

a

Ratio of affinities for 5-HT1A and 5-HT7Rs Ratio of affinities for unsubstituted and X-substituted compounds c Averaged after all correct poses were found in the docking results for all homology models b



FIGURES

Figure 1. The illustration of the σ-hole concept. The formation of a covalent carbon−halogen bond (a C–X σ-bond) by pairing of the electrons from the valence orbitals of the two atoms. As a result, the portion of the pz orbital of the halogen opposite the σ-bond becomes depopulated, resulting in an electropositive crown (in blue), whereas the px and py orbitals retain their complement of electrons to account for the overall negative charge of the halogen (in red).


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Figure 2. Workflow of computational algorithm to identify amino acids (hot spots) that are common anchoring points for halogen bonding with halogenated ligands that show greater activity than their unsubstituted analog.



Figure 3. Illustration of an XSAR matrix generated for 25 sets showing the positive changes in activity against 5-HT7R upon halogenation. Several selected examples were depicted to represent the different roles of halogen atoms in ligand–receptor binding. The complexes are shown in the homology model of the 5-HT7 receptor based on the D3 template. In each case, the geometries of unsubstituted (green) and halogenated (cyan, magenta) structures were presented together with the substituted analog activity and corresponding change in the binding free energy (∆∆G). At the bottom, the newly synthesized library designed on the basis of set24 is shown.


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Figure 4. The results of searching for halogen bonding hot spots of 5-HT7R. The algorithm was performed on a set of homology models generated on the eight templates. Considering the frequency that a given amino acid contributes to the formation of a halogen bond, two classes of amino acids were distinguished: the primary (yellow) and secondary (magenta). Additionally, the targeting of halogen bonding contacts to side chains (s) and backbone carbonyl oxygens (c) is indicated.



Scheme 1. Synthesis of compounds 58–88. Reagents and conditions: i) 1,2-dibromoethane, K2CO3, KI, (CH3)2CO, 60°C, 24–48 h; ii) Boc-4-aminopiperidine, K2CO3, KI, (CH3)2CO, 60°C, 24 h; iii) TFA/CH2Cl2 (80/20; v/v), r.t., 2 h; and iv) arylsulfonyl chloride, TEA, CH2Cl2, 0°C, 2–6 h.


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Figure 5. (A) The illustration of the highly scored binding modes of an unsubstituted compound (58, orange) in a homology model (build on D3 template) of 5-HT7R. The green and blue circles denote the locations of modified fragments of 58 in the binding site. (B) The two possible binding modes of the 2-Br analog (60, green): where the bromine atom forms halogen bonds with the side chains of T2.64 and/or D2.65 (XB distances are marked by yellow dotted lines), and where bromine is halogen-bonded to the carbonyl oxygen of C45.x50 from ECL2. (C) An illustration of different conformations of 3-Br (63, cyan) and (D) 4-Br (65, raspberry) analogs in the 5-HT7R binding site.



Figure 6. The interactions of derivatives differently substituted at the aryloxy fragment with 5-HT7R. (A) A comparison of the binding modes of a 2-I derivative (68, dirty violet) and unsubstituted analog 58 (orange). (B) A comparison of the binding modes of analogs with hydrophobic substituents at the 2-position of the aryloxy moiety, i.e., derivatized with phenyl (II, brown), i-Pr (I, blue), t-butyl (71, green) and iodine (68, violet). (C) Illustration of possible orientation of 3-bromo aryloxy fragment of 73 in the binding site. The XB distances and the change in the binding free energy (please note that ∆∆G values are for a given receptor model, whereas in Table 2 the average values are given) for each halogen bonding arrangement are shown. (D) A superposition of the poses of 2-Br (67, amber), 3-Br (73, cyan) and 4-Br (81, dark green) analogs against a putative halogen binding pocket interaction sphere. The bromine–oxygen theoretical interaction sphere illustrates the projected qualities of the formed L–R halogen bonds.


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Figure 7. An illustration of docking poses of 3-Br aryloxy derivatives with an additional bromine atom located at the 2- (A, 84–navy), 3- (B, 86–brown), and 4-positions (C, 88– raspberry) of the phenylsulfonyl fragment in a 5-HT7R homology model (build on D3 template). For each derivative, a highly scored orientations were shown and compared with the pose of the corresponding derivative singly substituted at the 3-position of the aryloxy fragment (73, cyan). The yellow dotted line denotes the halogen bonding contacts.



Figure 8. The halogen bonding distance–angle change extracted from 60 ns long MD simulations for analogs with 2- or 3-Br/I at the aryloxy fragment. Each dot represents data extracted from one MD frame (simulation interval was set to 10 fs), while red rectangles denote the favorable distance–angle regions for halogen bond formation.


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Figure 9. The human D4 dopamine receptor in complex with nemonapride (PDB ID: 5WIU), where a halogen bond with V5.40 backbone is present (A). Examples representing the different roles of halogen atoms in ligand–receptor binding: halogen bonds with primary V5.40 and secondary H6.55 hot spots (B, XSAR set51), halogen bonds with secondary S5.461 and S5.43 hot spots (C, XSAR set53), and the steric/hydrophobic (D, XSAR set1). In each case, the geometries of unsubstituted (green) and halogenated (cyan) structures are presented together with the substituted analog activity, the Xeffect value, and the corresponding change in the binding free energy (∆∆G). The chlorine–oxygen theoretical interaction sphere illustrates the projected qualities of the formed L–R halogen bonds.



Table of Contents Graphic


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Amino Acid Hot Spots of Halogen Bonding: A ... - ACS Publications

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