This mouse study explored the effects of a number of synthetic psychoactive drugs, such as the 2C-family, on the serotonin receptor 5-HT7R. The ability of a particular 2C compound to bind 5-HT7R over other subtypes and reduce self-grooming time in mice suggests that 5-HT7R could be a potential target for treating autism.
5-HT7R belongs to a family of G protein-coupled receptors and is associated with a variety of physiological processes in the central nervous system via the activation of the neurotransmitter serotonin (5-HT). To develop selective and biased 5-HT7R ligands, we designed and synthesized a series of pyrazolyl-diazepanes 2 and pyrazolyl-piperazines 3, which were evaluated for binding affinities to 5-HTR subtypes and functional selectivity for G protein and β-arrestin signaling pathways of 5-HT7R. Among them, 1-(3-(3-chlorophenyl)-1H-pyrazol-4-yl)-1,4-diazepane 2c showed the best binding affinity for 5-HT7R and selectivity over other 5-HTR subtypes. It was also revealed as a G protein-biased antagonist. The self-grooming behavior test was performed with 2c in vivo with Shank3-/- transgenic (TG) mice, wherein 2c significantly reduced self-grooming duration time to the level of wild-type mice. The results suggest that 5-HT7R could be a potential therapeutic target for treating autism spectrum disorder stereotypy.
G protein-coupled receptors (GPCRs) mediate many physiological signals through G proteins and through β-arrestin–dependent pathways; ligands that preferentially activate one pathway over the other (biased ligands) can yield therapeutic benefits and fewer side effects. Among serotonin receptors, the 5-HT7 receptor (5-HT7R) is a Gs-coupled GPCR that elevates intracellular cAMP, is highly expressed in the central nervous system, and has been implicated in thermoregulation, circadian rhythm, cognition and neurodevelopmental disorders including autism spectrum disorders (ASD). Previous work identified β-arrestin–biased and conventional agonists/antagonists for 5-HT7R, but, according to the extracted text, a ligand with preferential activation of the Gs protein pathway had not been reported. Lee and colleagues set out to design, synthesise and pharmacologically evaluate a series of 6-chloro-2′-methoxy biphenyl derivatives bearing different amine moieties to identify Gs protein–biased ligands at 5-HT7R. The study combined medicinal chemistry, in vitro binding and functional assays (Gs-mediated cAMP production and β-arrestin recruitment), selectivity profiling across serotonin receptor subtypes, in vitro ADME (CYP inhibition and microsomal stability), in vivo pharmacokinetics in mice, and a behavioural test (self-grooming) in Shank3 transgenic mice to probe a potential link between 5-HT7R signalling bias and ASD-relevant stereotyped behaviour.
This work combined synthetic chemistry with in vitro pharmacology, ADME assays, in vivo pharmacokinetics and behavioural testing. Chemically, three scaffolds were prepared: 6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl-N-alkylmethanamine derivatives (series 1), the one-carbon-homologated ethyleneamines (series 2), and pyrrolidine derivatives (series 3). Representative synthetic routes included iodination, Suzuki cross-coupling to install the biphenyl core, reductive amination and SN2 reactions; full synthetic and characterisation procedures (NMR, LC/MS, HRMS, HPLC purity ≥95%) are reported in the Experimental Section. Binding and functional assays were performed in cell systems. Binding affinities (K i ) at 5-HT7R were measured by [3H]LSD radioligand competition using crude membrane fractions from transfected HEK293 cells; IC50 values were converted to K i. Gs protein–mediated activity was assessed by a luminescence cAMP assay (GloSensor) in HEK293 cells transiently transfected with human 5-HT7R and the GloSensor plasmid; compounds were tested over multiple concentrations and at least in triplicate. β-Arrestin recruitment was measured in HTLA cells (HEK293 derivative stably expressing a tTA-dependent luciferase reporter and a β-arrestin2–TEV fusion) transiently transfected with a 5-HT7R construct (Tango assay); again serial dilutions and triplicate determinations were used. Reported readouts included EC50 (concentration producing half-maximal response) and Emax (maximal observed efficacy), and relative functional bias was quantified using ΔΔlog(τ/KA) and bias factors compared with reference ligands (5-HT and E-55888). Selectivity across other serotonin receptor subtypes (5-HT1A, 1B, 1D, 1E, 2A, 2B, 2C, 3, 5A, 6) was assessed by radioligand binding (K i values). Drug–drug interaction potential and metabolic stability were probed with in vitro CYP450 inhibition assays (CYP1A2, 2C9, 2C19, 2D6, 3A4) and human liver microsomal stability assays, respectively. Pharmacokinetic (PK) parameters for lead compound 2b were obtained after intravenous (1 mg/kg) and intraperitoneal (10 mg/kg) dosing in male ICR mice; plasma was collected at multiple timepoints and analysed by LC–MS/MS, with non-compartmental PK analysis to estimate clearance, half-life, AUC and bioavailability. The self-grooming behavioural assay was performed in wild-type and Shank3 transgenic male mice (7–9 weeks old); drugs were administered intraperitoneally (2b at 5 mg/kg; SB269970 at 30 mg/kg) and total grooming duration and counts were recorded over 30 minutes. The extracted text does not clearly report animal group sizes or the number of replicates for behavioural cohorts.
Synthesis yielded 16 target compounds across series 1–3 with high purities. In binding assays, 15 of the 16 compounds bound 5-HT7R with K i values ranging from 1.6 to 110 nM. Compound 2a had the best binding affinity (K i = 1.6 nM), comparable to reference agonist E-55888 (K i = 1.3 nM); 2b showed K i = 2.8 nM. Functional profiling separated balanced agonists from Gs-biased ligands. In the cAMP (Gs) assay, many compounds showed potency and efficacy: examples include 1a (EC50 = 460 nM, Emax = 72%), 2a (EC50 = 73 nM, Emax = 77%), and 2b (EC50 = 180 nM, Emax = 91%). In the β-arrestin (Tango) assay, fewer compounds were active; three balanced agonists were identified (1a, 2a, 2e), while six compounds (1b, 1c, 2b, 2c, 2f, 2g) showed activity in cAMP production but no detectable β-arrestin recruitment and were therefore classified as Gs protein–biased in this series. Among these, 2b was highlighted as the best Gs-biased ligand on the basis of potency and efficacy in the cAMP assay (EC50 = 180 nM; Emax = 91%), with 1b as a secondary Gs-biased example (EC50 = 770 nM; Emax = 71%). Quantitative bias assessment using ΔΔlog(τ/KA) yielded a bias factor for 2b of 461, indicating strong Gs bias relative to the reference ligand E-55888, which had a bias factor <1 and thus was relatively biased toward β-arrestin in these analyses. Selectivity testing against other 5-HT receptor subtypes showed variable profiles. Compounds 1a–1e generally lacked selectivity over 5-HT2B (K i values near low tens of nM), raising potential safety considerations, whereas many compounds in series 2 exhibited improved selectivity over 5-HT2B; notably, 2a and 2b displayed more than 40-fold selectivity over 5-HT2B. Some compounds had poor selectivity over 5-HT1D. In in vitro ADME assays, 2b exhibited minimal inhibition of five major CYP isoforms (remaining enzyme activities >88.7%), suggesting low potential for CYP-mediated drug–drug interactions in these assays. Microsomal stability testing showed 71.5% remaining concentration for 2b after incubation with human liver microsomes, indicating relative metabolic stability in vitro. PK studies in male ICR mice indicated rapid systemic clearance after intravenous administration of 2b (CL = 477.7 mL/min/kg; T1/2 = 0.5 h; AUC_last = 32.9 h·ng/mL). After intraperitoneal dosing, a longer half-life (T1/2 = 1.0 h) and a larger AUC_last (442.3 h·ng/mL) were reported, yielding an apparent bioavailability of about 134% (authors present this value; no further discussion of causes is given in the extracted text). In the self-grooming behavioural assay, administration of 2b (5 mg/kg, i.p.) markedly increased grooming duration in Shank3 transgenic mice (t = 553 s) relative to saline; co-administration of the selective 5-HT7R antagonist SB269970 (30 mg/kg, given 10 min before 2b) reduced the grooming duration toward saline levels (reported t = 70.8 s in WT and 166 s in TG mice), indicating that the 2b-induced increase in grooming was blocked by a 5-HT7R antagonist. SB269970 alone did not produce significant changes compared with saline in the reported measures (WT t = 29.3 s; TG t = 181 s). The extracted text does not report statistical tests, p-values or the number of animals per group in the behavioural results.
Lee and colleagues interpret their data as the identification of a novel chemical series of 6-chloro-2′-methoxy biphenyl derivatives that yield both balanced and Gs protein–biased ligands at 5-HT7R. They emphasise that, prior to this work, a Gs protein–biased ligand for 5-HT7R had not been reported, and that compound 2b represents a potent Gs-biased agonist as judged by cAMP efficacy and the calculated bias factor (bias factor = 461 versus E-55888). Structure–activity observations are reported: mono-ethyl substitution on the amine appears to favour Gs bias in this scaffold, whereas mono-methyl substitution (as in 2a) produced a balanced agonist. In terms of translational relevance, the authors link the pharmacology to behaviour by showing that systemic administration of the Gs-biased agonist 2b increased self-grooming in Shank3 transgenic mice, an ASD-relevant stereotypy model, and that the effect was reversed by the selective 5-HT7R antagonist SB269970. From these findings the investigators suggest that 5-HT7R signalling, and specifically Gs-mediated activation, may modulate stereotyped behaviours and could be considered in future work targeting ASD symptoms. Safety and developability issues are addressed to a degree: many compounds showed acceptable selectivity profiles, with series 2 showing improved selectivity over the 5-HT2B receptor that is associated with valvulopathy; 2b showed little CYP inhibition in vitro and reasonable microsomal stability. The authors also report PK parameters indicating relatively rapid systemic elimination, and they present bioavailability data for 2b after intraperitoneal dosing. The extracted text notes that more experiments are required to clarify the association between 5-HT7R and autism, which the authors acknowledge as a limitation of the current behavioural findings. No additional limitations or statistical detail are presented in the extracted Results/Discussion text.
Lee and colleagues conclude that they synthesised a set of 6-chloro-2′-methoxy biphenyl derivatives and identified multiple 5-HT7R ligands with distinct functional profiles: three balanced agonists (1a, 2a, 2e) and six Gs protein–biased ligands (1b, 1c, 2b, 2c, 2f, 2g). Compound 2b emerged as the best Gs protein–biased ligand in the series (EC50 = 180 nM; Emax = 91%). In vivo, administration of 2b increased self-grooming in Shank3 transgenic mice, an effect that was reversed by the selective 5-HT7R antagonist SB269970. On the basis of these results the authors suggest that 5-HT7R is associated with ASD-relevant stereotypy and could be a therapeutic target for treating such behaviours, while noting that further studies are needed.
G protein-coupled receptors (GPCRs) are a family of cell surface receptors with seven highly conserved, transmembrane helical proteins, and there are over 800 individual genes encoded in the human genome.These receptors are expressed extensively in various tissues, and they participate in multiple physiological signal transductions through interactions with the heterotrimeric G proteins that transfer the signals to canonical transducer proteins, arrestins, kinases, ion channels, and scaffolding proteins. Thus, they have been the pharmaceutical targets of various drugs.Canonical GPCR signaling is mediated via coupling to intracellular transducers, that is, G proteins, which are formed by G α , G β , and G γ subunits.A ligand binds to a GPCR and activates the G protein, which is followed by the activation of the downstream signaling known as the G protein signaling pathway. Then, GPCR recruits β-arrestin to block or desensitize the activated signal. In addition to the classical G protein signaling pathway mediated by G proteins, it has been suggested recently that β-arrestin is able to initiate G protein-independent cellular signals, including the activation of various MAPKs, such as ERK,which is referred to as the βarrestin signaling pathway. Thus, there have been active efforts to produce biased ligands in order to elucidate the complicated mechanism of GPCRs and to develop novel GPCR drugs with fewer side effects. These biased ligands can activate either the G protein or the β-arrestin signaling pathway selectively, thereby yielding the desired effects of drugs and blocking unwanted side effects due to the stimulation of other signaling pathways.There are 14 distinct serotonin receptors (5-HTRs) encoded in the human genome. Among the 5-HTR subtypes, the 5-HT 7 receptor (5-HT 7 R) was the last to be identified, and it has been shown to be highly expressed in the central nervous system (CNS), for example, in the hypothalamus, hippocampus, and cortex. 5-HT 7 R belongs to a family of GPCRs and binds positively to adenylate cyclase (AC) through the activation of Gs protein, resulting in an extracellular increase of cyclic adenosine monophosphate (cAMP), and it also displays a high constitutive AC activity.On the basis of the distribution of 5-HT 7 R in the CNS, it has been proposed that it is involved in various important functional roles, such as thermoregulation, circadian rhythm, sleep, learning and memory, autism, cognition, and schizophrenia.In addition, numerous studies have suggested that the altered 5-HT system that includes abnormal levels of 5-HT, morphological changes in the serotonergic fibers, and decreased expression of 5-HTR might be a major marker of abnormalities in autism spectrum disorders (ASDs).Recent reports have provided evidence that 5-HT 7 R has an essential role in regulating severe behavioral symptoms, which are represented as autistic-like behaviors in animal models, such as the Fmr1 KO mouse model of the fragile X syndrome and the Mecp2 KO mouse model of the Rett syndrome. The 5-HT 7 R agonist restored the long-term depression level in an Fmr1 KO mouse to the level of wild type (WT), which is applicable to ASDs; also, the treatment of 5-HT 7 R agonist improved the behavioral impairments and cognition in Mecp2 KO mice.However, no studies have been conducted in which the 5-HT 7 Rbiased ligand was used in testing autism behavior. Although many potent agonists (E-55888,AS-19,and 1a) and antagonists (SB-269970) against 5-HT 7 R have been reported, they are considered to be agonists/antagonists that activate/inhibit the G protein signaling pathway, while no activation/inhibition of the β-arrestin signaling pathway other than SB-269970 has been reported(Figure). Recently, Kim et al.have discovered an azepine derivative, that is, 3-(4chlorophenyl)-1,4,5,6,7,8-hexahydropyrazolo [3,4-d]azepine, as a β-arrestin-biased ligand against 5-HT 7 R (K i 30 nM), IC 50 (G protein) 7800 nM, and EC 50 (β-arrestin) 162 nM, whereas, to date, no Gs protein-biased ligand of 5-HT 7 R has been discovered. In this study, we designed and synthesized biphenyl derivatives 1, 2, and 3 (Figure), and 2b was found to be a potent Gs protein-biased agonist of 5-HT 7 R, and it was used in the self-grooming behavior test that was conducted with Shank3 transgenic (TG) mice to investigate the association between 5-HT 7 R and ASD.
To discover Gs protein-biased ligands of 5-HT 7 R, we referred three structurally similar agonists: well-known agonists AS-19 and E-55888, with a biaryl core and an ethyleneamine moiety, and 1a with a biphenyl core and a methyleneamine moiety, which was identified in our previous work as a potent and selective agonist against 5-HT 7 R(Figure). According to our study of the structure-activity relationship (SAR), compounds with a 6-chloro-2′-methoxy biphenyl core showed good binding affinities, so this core structure was retained in this study. We planned to synthesize and biologically evaluate 6-chloro-2′methoxy-[1,1′-biphenyl]-3-yl-N-alkylmethanamine derivatives 1, 2-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-1-amine derivatives 2 and 3-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidine derivatives 3 as shown in Figure, and the introduction of secondary/tertiary amines with alkyl chains and cyclic amines to the amine moiety followed by SAR study was our main concern. Synthesis. 6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl-N-alkylmethanamine derivatives 1 were synthesized in three steps starting from 3-chloro-4-iodobenzaldehyde 4 (Scheme 1), which is iodinated from 4-chlorobenzaldehyde 3 through electrophilic aromatic substitution of aldehyde by using I 2 , NaIO 3 , and sulfuric acid.Suzuki cross-coupling reaction between 4 and 2-methoxyphenylboronic acid 5 provided biphenyl core 6 in 64-98% yields. This biphenyl benzaldehyde 6 was then converted to N-alkylamines 1a-1g via reductive amination by the treatment of NaBH(OAc) 3 and various alkylamines in 16-93% yields. With iodinated starting material 4, the syntheses of 2-(6chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-1-amine derivatives 2 were accomplished efficiently, as shown in Scheme 2. One carbon homologation of the iodoaldehyde 4 was obtained by the introduction of cyanide and reduction. The iodobenzaldehyde 4 was reduced by NaBH 4 , which is brominated, and this was followed by the S N 2 reaction with NaCN to provide an intermediate 9 with the cyanide group. Next, the Suzuki cross-coupling of 9 produced biphenyl cyanide 10, which subsequently was reduced by LiAlH 4 and AlCl 3 to produce one-carbon-homologated ethyleneamine 11. Preparation of dialkyl compounds was established by reductive amination using formaldehyde/formic acid or the S N 2 reaction using alkyliodide/K 2 CO 3 . Reductive amination was conducted to synthesize the dimethyl derivative 2e, and the S N 2 reaction was performed to obtain diethyl compound 2f and dipropyl compound 2g. In order to obtain monoalkylated compounds, ethyleneamine 11 was protected by the Boc group to afford an intermediate 12, which was treated with NaH and alkyliodide to provide Boc-protected monoalkyl compounds 13. The use of 1 N HCl in diethyl ether to eliminate the protection of the Boc group allowed us to obtain the final monoalkylated compounds 2a-2d. We also synthesized 3-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidine derivatives 3a and 3b, as shown in Scheme 3. Biphenylaldehyde 6 underwent the nitroaldol reaction by treatment with nitromethane and ammonium acetate at 100 °C to afford a nitrovinyl derivative 14. To incorporate the malonate ester into the nitrovinyl position by Michael addition, NaH and diethyl malonate were used to provide a malonated Binding Affinity and Functional Activity against 5-HT 7 R. Binding affinities of 1, 2, and 3 for 5-HT 7 R were determined by the [ 3 H] D-lysergic acid diethylamide ([ 3 H]LSD) radioligand binding assay in transfected HEK293 cells.Tableshows the binding affinities (K i ) and functional activities in the Gs protein/β-arrestin signaling pathways against 5-HT 7 R. Among the biphenyl derivatives 1 with the methyleneamine moiety, compounds 1a-c (R 1 = Me, Et, and Pr, R 2 = H) showed binding affinities against 5-HT 7 R with K i values of 5.2, 9.3, and 6.8 nM, respectively. However, the longer carbon chain of the Nalkyl moiety, like a butyl chain, reduces the affinity for 5-HT 7 R. Compound 1d with the monobutyl amine moiety (R 1 = Bu, R 2 = H) had a K i value of 52 nM. Dimethyl derivative 1e (R 1 = Me, R 2 = Me) showed binding affinity with a K i value of 18 nM, while the diethyl and dipropyl compounds 1f and 1g had decreased affinity (K i = 110 nM) or no activity up to 10 μM, respectively. Interestingly, compared with the binding affinities of compounds 1, one-carbon-elongated compounds 2 mostly main-tained potency of binding affinities to 5-HT 7 R. Compounds 2a and 2b (R 1 = Me and Et) showed binding affinity with K i values of 1.6 and 2.8 nM for 5-HT 7 R, respectively, comparable to those of 1a and 1b. Compounds 2c and 2d (R 1 = Pr and Bu) had almost the same binding affinities with K i values of 8.7 and 64 nM in comparison to the corresponding compounds 1c and 1d, while dialkyl compounds 2e-g (R 1 , R 2 = Me, Et, and Pr) showed at least 4 times better binding affinities with K i values of 2.5, 25, and 20 nM, respectively, than the corresponding compounds 1e-g. Compounds 3a and 3b (R 1 = H and Me) with a pyrrolidine moiety showed binding affinities with K i values of 2.8 and 30 nM, respectively. Among the synthesized compounds, compound 2a had the best binding affinity value (K i = 1.6 nM), which was comparable to that of E-55888 (K i = 1.3 nM). To investigate the functional activities of compounds on the Gs protein and β-arrestin signaling pathways, we conducted two types of cell-based functional assays, that is, bioluminescencebased assays to measure Gs-mediated cAMP production (cAMP assay) and β-arrestin recruitment (Tango assay) with synthesized compounds 1, 2, and 3, and an endogenous agonist 5-HT and a 5-HT 7 R agonist E-55888 were used as reference compounds. All compounds were evaluated by both cAMP assay and Tango assay. The cAMP assay was done in transiently transfected HEK293 cells with 5-HT 7 R plasmid/GloSensor plasmid, while, in the Tango assay, the HEK293-derived cell line was used, and it contained stable interactions of a tTAdependent luciferase reporter and a β-arrestin 2-TEV fusion gene (HTLA cells). Compounds 1a-c showed potency and efficacy in the cAMP assay (EC 50 = 460, 770, and 1200 nM, respectively, and E max = 72, 71, and 68%, respectively), while the E max value of compound 1e is relatively low with 11%, even though the EC 50 value of 1e was 1100 nM. Compounds 1d, 1f, and 1g show no functional activities in the cAMP assay. In βarrestin recruitment Tango assay, only compounds 1a and 1e showed functional activities with EC 50 values of 150 and 480 nM, respectively. Compounds 1b and 1c had activities in cAMP production but no activities in β-arrestin recruitment, which can be called Gs protein-biased ligands. In the series of compounds 2, compounds 2a and 2b had EC 50 values of 73 and 180 nM, which showed better activities than compounds 1a and 1b in the cAMP assay. Efficacy of 2b in cAMP production is the best with an E max value of 91% among the synthesized compounds. Compound 2c showed marginal activity in cAMP production with an EC 50 value of 1000 nM and an E max value of 53% and compound 2d showed no activity. Compared with compounds 1e-g, compounds 2e-g showed improved activities in the cAMP assay (EC 50 = 84, 1900, and 1400 nM, E max = 67, 76, and 72%). In β-arrestin recruitment Tango assay, only compounds 2a and 2e showed functional activities with EC 50 values of 71 and 56 nM. Therefore, compounds 2b, 2c, 2f, and 2g had activities in cAMP production but no activities in β-arrestin recruitment, which can also be called Gs protein-biased ligands. Compounds 3a and 3b displayed better activities in the Tango assay (EC 50 = 75 and 650 nM, E max = 54 and 51%) than they did in the cAMP assay (EC 50 = 92 and 560 nM, E max = 29 and 9.8%). There are three balanced agonists 1a, 2a, and 2e like E-55888, among which 2a is the best in the view of potency and efficacy in both assays, while there are six G protein-biased ligands 1b, 1c, 2b, 2c, 2f, and 2g, among which 2b is the best in the cAMP assay (EC 50 = 180 nM, E max = 91%) and 1b is the next (EC 50 = 770 nM, E max = 71%) (Figure). Among 16 synthesized compounds, 15 compounds bound to 5-HT 7 R with K i values between 1.6 and 110 nM, and 12 compounds showed potency and efficacy in the Gs protein signaling pathway, while only six compounds were active in βarrestin signaling pathway. Among those, 2a is the best balanced agonist and 2b is the best G protein-biased ligand. Selectivity over Other 5-HTR Subtypes. We investigated the binding affinities of compounds 1, 2, and 3 for other serotonin subtype receptors, such as 5-HT 1A R, 5-HT 1B R, 5-HT 1D R, 5-HT 1E R, 5-HT 2A R, 5-HT 2B R, 5-HT 2C R, 5-HT 3 R, 5-HT 5A R, and 5-HT 6 R to determine their selectivity for 5-HT 7 R. Compounds 1a-e showed selectivity over 5-HT 1A R, 5-HT 1B R, 5-HT 1D R, 5-HT 1E R, 5-HT 2A R, 5-HT 3 R, 5-HT 5A R, and 5-HT 6 R, except 5-HT 2B R and some cases of 5-HT 2C R. Compounds 1a-e had binding affinities for 5-HT 2B R with K i values of 13, 19, 12, 15, and 11 nM, respectively, resulting in little selectivity of compounds 1a-e over 5-HT 2B R. Compounds 2a and 2b had at least 19-fold selectivity over other 5-HTR subtypes except 5-HT 1D R. Compounds 2d, 2f, and 2g had almost no selectivity over 5-HT 1A R and 5-HT 1D R. It is very interesting that compounds 1 had little selectivity over 5-HT 2B R, while compounds 2 except 2d had at least 12-fold selectivity over 5-HT 2B R, but little selectivity over 5-HT 1D R. Specially, compounds 2a and 2b showed more than 40-fold selectivity over 5- HT 2B R. Compound 3a indicated at least 9-fold selectivity over other 5-HTR subtypes, except for 5-HT 1D R, while compound 3b showed more than 30-fold selectivity over 5-HT 1E R, 5-HT 2A R, 5-HT 2C R, 5-HT 3 R, and 5-HT 5A R. It was reported that 5-HT 2B R was associated with cardiac valvulopathy, resulting in potential cardotoxicity,while the 5-HT 1D R agonist, triptan, shows a pharmacological benefit in migraine pain.Functional Selectivity. Based on their pharmacological profiles (Table) and selectivity profiles (Table), we selected potent and functionally distinct ligands 1b, 2a, and 2b to determine ligand bias toward the Gs protein signaling pathway or the β-arrestin signaling pathway compared with their functional selectivity profile of 5-HT and E-55888. The results indicated that E-55888 had almost full efficacy in the Gs protein pathway (EC 50 = 83 nM, E max = 99%) compared to 5-HT, while showed a partial agonistic effect in the β-arrestin pathway (EC 50 = 21 nM, E max = 54%). Since compound 2a activated both the Gs protein pathway and the β-arrestin pathway, 2a was shown to be a balanced agonist with good potency and efficacy (cAMP assay: EC 50 = 73 nM, E max = 77%, and Tango assay: EC 50 = 71 nM, E max = 67%). By contrast, compounds 1b and 2b, both of which have a mono-ethyl group as the alkyl substitution at the methyleneamine and ethyleneamine moieties, exhibited no activity in the β-arrestin pathway compared to 5-HT (100%) and E-55888(54%). Compound 2b acted as almost full agonist in the Gs protein pathway with an E max of 91%, while compound 1b had a lower E max value with 71% than compound 2b. Using 5-HT as a positive control and E-55888 as a reference ligand to evaluate our compounds, we found that compound 2a with mono-methyl moiety acted as a balanced agonist, whereas compounds 1b and 2b, with mono-ethyl moiety, showed functional selectivity with preference to the production of Gs protein-mediated cAMP, which means the two compounds, that is, 1b and 2b, are Gs protein-biased ligands. Based on the potency and efficacy, 2b exhibited a better Gs protein-biased ligand, that is, 5-HT 7 R, than compound 1b. It could be suggested that alkyl substitution is important to selectively activate the Gs protein signaling pathway, and in particular, ethyl group at the amine in this series of compounds leads to the Gs protein bias. To determine ligand biasness of a better G protein-biased ligand 2b, we evaluated the differences ΔΔlog(τ/K A ) between the Δlog(τ/K A ) values of 2b and E-55888 in both G protein signaling pathway and β-arrestin signaling pathway.As shown in Table, E-55888 had ΔΔlog(τ/K A ) = -0.47, which was converted to a bias factor. The bias factor of E-55888 is less than 1, which means that E-55888 is more biased to the β-arrestin signaling pathway, not the Gs protein signaling pathway. 2b had ΔΔlog(τ/K A ) = 2.66, of which the bias factor is 461. By the calculation of the bias factors of those two compounds E-55888 and 2b, it became more obvious that 2b is a Gs protein-biased ligand of 5-HT 7 R. CYP Activity and Microsomal Stability. The in vitro cytochrome P450 (CYP450) inhibition assay, including five major isoforms, that is, CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, was performed to assess the potential drug-drug interaction of 2b (Table). As shown in Table, the percentage of remaining activities of 2b in the five CYP isozymes is greater than 88.7%, and compound 2b has little effect on the CYP isozymes, indicating that it might not have a drug-drug interaction. In addition, the metabolic stability of compound 2b was determined through the human liver microsomal stability test. Since the metabolic stability of drug is a key factor that affects both the efficacy and toxicity of the drug, as well as its pharmacokinetic (PK) parameters, it can be tested in an in vitro assay prior to conducting an in vivo study. Compound 2b showed 71.5% as the percent-remaining concentration, which means that 2b is barely decomposed and is stable after incubation with human liver microsomes. Thus, the PK Bias factor was calculated with Prism 4.0 program (GraphPad software, San Diego). b 5-HT was used as the reference agonist. parameters of 2b were evaluated after intravenous injection (dose 1 mg/kg) and intraperitoneal (i.p.) administration (dose 10 mg/kg) in male ICR mice (Table). After the intravenous injection, the mean clearance rate (CL) was measured as 477.7 mL/min/kg, the half-life (T 1/2 ) of 2b was 0.5 h, and the AUC last was 32.9 h ng/mL. According to the half-life and CL, 2b seems to be rapidly eliminated from the body. In i.p. administration, the half-life (T 1/2 ) was 1.0 h, and AUC last was 442.3 h ng/mL. The bioavailability of 2b was about 134%. Self-Grooming Behavior. One of the symptoms in patients with ASD is stereotypy, which is observed as restricted and repetitive patterns of behavior.Currently, studies of the correlation between ASD and 5-HT 7 R are emerging, and there are several reported results indicating that 5-HT 7 R ligands correct phenotypic deficits in various heterogeneous mouse models, such as Fmr1 KO mice,Mecp2 TG mice,and C58/J mice,which exhibit spontaneous self-grooming. Thus, we conducted a self-grooming behavior test using Shank3 -/- (Shank3 TG) male micethat are a well-known ASD animal model. We tested SB269970, also known as 5-HT 7 R selective antagonist, at a dose of 30 mg/kg in i.p. administration to WT and Shank3 TG mice. The administration of SB269970 showed duration times in both WT (t = 29.3 s) and TG mice (t = 181 s) without any significant changes compared with those of saline (Figure). After i.p. administration, the Gs proteinbiased agonist 2b with a dose of 5 mg/kg showed a highly increased duration of the self-grooming time in Shank3 TG mice (t = 553 s) compared to that of saline (Figure). To determine the direct effect of 5-HT 7 on self-grooming behavior, SB269970 was administered 10 min prior to the injection of 2b. Figureshows that the co-treatment of SB269970 (30 mg/ kg) and 2b (5 mg/kg) reduced the duration of self-grooming (t = 70.8 s in WT mice and 166 s in TG mice) to the normal duration with saline administration, which demonstrates that SB269970 inhibits the effect of 2b. Overall, the Gs proteinbiased agonist 2b prominently increased the duration time of self-grooming, which was suppressed by the effect of the 5-HT 7 R selective antagonist, SB269970. Although more experiments are needed regarding the association of 5-HT 7 R and autism, these results support the conclusion that 5-HT 7 R is involved in a modulatory role to stereotypy in ASD (Table).
We synthesized a series of 6-chloro-2'-methoxy biphenyl derivatives 1, 2, and 3 bearing various amine moieties and evaluated binding affinities of these synthesized compounds for 5-HT 7 R along with other serotonin receptor subtypes. Most of the compounds had binding affinities to 5-HT 7 R. By conducting cAMP production assay and β-arrestin recruitment Tango assay, three balanced agonists 1a, 2a, and 2e like E-55888 were found, among which 2a is the best in the view of potency and efficacy in both assays, while there are six G protein-biased ligands 1b, 1c, 2b, 2c, 2f, and 2g, among which 2b is the best in the cAMP assay (EC 50 = 180 nM, E max = 91%). In the animal study with Shank3 TG mice, the self-grooming behavior test of 2b was performed, and 2b showed an increased duration of self-grooming, which was reversed by the selective 5-HT 7 R antagonist, SB269970. Based on the results of the self-grooming behavior, we further suggested that 5-HT 7 R is associated with ASD and could be a therapeutic target for the treatment of stereotypy in ASD.
Chemistry. All reactions were carried out under dry nitrogen unless otherwise indicated. Commercially available reagents were used without further purification. Solvents and gases were dried according to standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator. Analytical thin layer chromatography (TLC) was performed using glass plates pre-coated with silica gel (0.25 mm). TLC plates were visualized by exposure to UV light and then were visualized with a KMnO 4 , ninhydrin, and p-anisaldehyde staining solution followed by brief heating on a hot plate. Flash column chromatography was performed using silica gel 60 (230-400 mesh, Merck) with the indicated solvents.H andC NMR spectra were recorded on Bruker 300 or 400 NMR spectrometers.H NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, br s = broad singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constant (J) in hertz. Liquid chromatography-mass spectrometry (LC/MS) and high-resolution mass spectrometry (HRMS) analyses were performed on an Agilent 6410 Triple Quad system and the Bruker Compact ESI+ positive mode, respectively. The purity of all the tested compounds was checked on a Waters HPLC e2695 instrument equipped with a UV/vis 2489 detector and a Capcell Pak 3 μm C18 MG-II (4.6 × 75 mm) column and was at least 95% for all tested compounds. Standard conditions were as follows: eluents system A (CH 3 CN), system B (H 2 O/0.1 M AcOH); a flow rate of 1 mL/min; a gradient of (10-100%) A over 20 min; and detection at 254 and 280 nm. 4-Chloro-3-iodobenzaldehyde (4). Iodine (1.59 g, 6.26 mmol) and NaIO 3 (620 mg, 3.13 mmol) were suspended in 95% concentrated H 2 SO 4 . The solution was stirred for 30 min at room temperature (rt). 4-Chlorobenzaldehyde 3 (2 g, 14.23 mmol) was added to the dark brown iodinating solution and the mixture was stirred for an additional 1 h at rt. The reaction mixture was poured slowly into ice-water. The crude solid product was collected by filtration with EtOH to afford the desired product 4 (2.43 g, 9.12 mmol) in 64% yield.H NMR (400 MHz, CDCl 3 ): δ 9.92 (s, 1H), 8.34 (d, J = 1.9 Hz, 1H), 7.80 (dd, J = 8.2, 1.9 Hz, 1H), 7.61 (d, J = 8.2 Hz, 1H);C NMR (100 MHz, CDCl 3 ): δ 189.41, 145.07, 141.37, 135.68, 130.00, 129.95, 98.83. 6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-carbaldehyde (6). 2-Methoxyphenylboronic acid 5 (2.25 mmol), Pd(PPh 3 ) 4 (0.02 mmol), and Na 2 CO 3 (2.82 mmol) were added to a solution of benzaldehyde 4 (1.88 mmol) in DMF (15 mL). The mixture was stirred overnight at 80 °C. After cooling down to rt, the reaction mixture was quenched by the addition of the saturated solution of NaHCO 3 and then partitioned with EtOAc. The combined organic layers were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (hexanes/EtOAc = 10:1) to obtain desired product 6 in 82% yield.6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl-methanamine Derivatives (1). The mixture of 6-chloro-2′-methoxy-[1,1′-biphenyl]-3carbaldehyde 6 (1 equiv) and appropriate amine (2 equiv) in MeOH (20 mL) was stirred at rt for 2 h. NaBH(OAc) 3 (3 equiv) was added and the solution was stirred overnight at rt. The reaction mixture was quenched by the addition of a saturated solution of NaHCO 3 and extracted with dichloromethane (DCM). The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 10:1) to afford the desired final product 1 in 16-93% yields. 1-(2′-Methoxy-[1,1′-biphenyl]-3-yl)-N-methylmethanamine (1a). Compound 1a was synthesized according to the general procedure of 1. Yield: 37%; HPLC: purity 100%, t R = 5.5 min;N-((6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)methyl)-N-ethylethanamine (1f). Compound 1f was synthesized according to the general procedure of 1. Yield: 20%; HPLC: purity 100%, t R = 6.1 min; 1 H NMR (400 MHz, CDCl 3 ): δ 7.40-7.35 (m, 2H), 7.28-7.26 (m, 2H), 7.20 (dd, J = 7.4, 1.8 Hz, 1H), 7.04-6.97 (m, 2H), 3.78 (d, 3H), 7.58 (s, 2H), 2.55 (q, J = 7.4 Hz, 4H), 1.04 (t, J = 7.0 Hz, 3H);(4-Chloro-3-iodophenyl)methanol (7). 4-Chloro-3-iodophenylbenzaldehyde 6 (7.62 mmol) was added in portions to a suspension of sodium borohydride (7.62 mmol) in methanol (50 mL). The mixture was stirred for 2 h at rt. The solution was quenched by the addition of water and then extracted with DCM. The combined organic layers were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc = 5:1) to obtain the desired product 7 (6.70 mmol, 88%).H NMR (400 MHz, CDCl 3 ): δ 7.85 (d, J = 1.9 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.26 (dd, J = 8.2, 2.0 Hz, 1H), 4.62 (d, J = 4.0 Hz, 2H), 1.98 (t, J = 6.0 Hz, 1H);4-(Bromomethyl)-1-chloro-2-iodobenzene (8). (4-Chloro-3iodophenyl)methanol 7 (7.45 mmol) was dissolved in DCM (50 mL) and triphenylphosphine (8.94 mmol) was added to the solution at rt and then carbon tetrabromide (8.94 mmol) was slowly added to the mixture. The reaction mixture was stirred for 2 h at rt, at which time, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc = 50:1) to obtain the desired product 8 (6.94 mmol, 93%).2-(4-Chloro-3-iodophenyl)acetonitrile (). 4-(Bromomethyl)-1chloro-2-iodobenzene 8 (4.71 mmol) was dissolved in toluene (30 mL) and sodium cyanide (7.06 mmol) was added to the solution. Tetrabutylammonium bromide (0.047 mmol) dissolved in water (1 mL) was added to the mixture and then the reaction mixture was stirred overnight at 50 °C. Based on TLC analysis, sodium cyanide (2.36 mmol) and water were additionally added to the mixture. After stirring for 5 h, the solution was quenched by the addition of water and extracted with EtOAc. The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc = 7:1) to afford the desired final product 9 (3.60 mmol, 77%).2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)acetonitrile (10). 2-Methoxyphenylboronic acid 5 (7.57 mmol), Pd(PPh 3 ) 4 (0.06 mmol), and Na 2 CO 3 (9.46 mmol) were added to a solution of 2-(4-chloro-3iodophenyl)acetonitrile 9 (6.31 mmol) in DMF (30 mL). The mixture was stirred overnight at 110 °C. After cooling down to rt, the reaction mixture was quenched with a saturated solution of NaHCO 3 and then extracted with EtOAc. The combined organic layers were dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (hexanes/EtOAc = 10:1) to afford the desired product 10 (5.04 mmol, 80%).2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethan-1-amine (11). 1 M LiAlH 4 in anhydrous THF (4.3 mL) was added dropwise under N 2 to the solution of AlCl 3 (3.49 mmol) in THF while stirring. The mixture was cooled down to 0 °C before dropwise addition of the solution of 2-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)acetonitrile 10 (3.88 mmol) in THF (10 mL). The solution was kept stirring for 2 h at 0 °C, at which time, MeOH was slowly added to quench the reaction. The mixture was concentrated under reduced pressure and the residue was dissolved in DCM and washed with water. The organic phase was partitioned with 1 N solution of HCl. The combined aqueous layer was washed with DCM and neutralized by the addition of 10 N solution of NaOH. The mixture was partitioned with DCM and the pooled organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to obtain the desired product 11 (3.88 mmol, 68.9%).H NMR (400 MHz, CDCl 3 ): δ 7.35-7.30 (m, 2H), 7.15 (dd, J = 7.5, 1.7 Hz, 2H), 7.12 (br s, 2H), 7.10 (d, J = 2.2 Hz, 1H), 6.97 (td, J = 7.4, 0.9 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 3.73 (s, 3H), 3.16 (t, J = 7.8 Hz, 2H), 3.01 (t, J = 7.8 Hz, 2H);
-biphenyl]-3-yl)ethan-1-amine 11 (2.67 mmol) and di-tert-butyl dicarbonate (2.67 mmol) were dissolved in anhydrous THF (10 mL). An aqueous solution of NaHCO 3 (2 mL) was added to a mixture and stirred overnight at rt. The mixture was partitioned with water and DCM and the combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc = 5:1) to afford the desired product 12 (2.67 mmol, 77%).H NMR (400 MHz, CDCl 3 ): δ 7.39-7.35 (m, 2H), 7.18 (dd, J = 7.5, 1.7 Hz, 1H), 7.12 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.1, 2.2 Hz, 1H), 7.01 (td, J = 7.4, 1.0 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 4.58 (br s, 1H), 3.78 (s, 3H), 3.38 (q, J = 6.5 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 1.43 (s, 9H);(13). A solution of tert-butyl (2-(6chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)carbamate 12 (1 equiv) in DMF (15 mL) was added dropwise to a sodium hydride 60% in oil (1.25 equiv) in DMF at 0 °C and stirred for an hour. Alkyliodide (2.4 equiv) was then slowly added to a mixture at 0 °C and the stirring was continued for an additional 2 h. The reaction mixture was quenched by the addition of methanol and partitioned with water and EtOAc. The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (hexanes/EtOAc = 5:1) to obtain the desired product 13 in 41-57% yields. tert-Butyl (2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)ethyl)-(methyl)carbamate (13a). Compound 13a was synthesized according to the general procedure of 13. Yield: 50%.2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-alkylethan-1amine (2). The protected amine was dissolved in 1 N HCl solution (1.5 equiv) in diethyl ether and the reaction mixture was stirred at rt overnight. A white precipitate formed after several minutes and additional HCl was added as needed. After completing the reaction, the precipitate was filtered, washed with diethyl ether, and concentrated under reduced pressure. The desired products were obtained in 80-95% yields. 2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-methylethan-1amine (2a). Compound 2a was synthesized according to the general procedure of 2. Yield: 93%; HPLC: purity 100%, t R = 6.0 min;2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-propylethan-1amine (2c). Compound 2c was synthesized according to the general procedure of 2. Yield: 92.2%; HPLC: purity 100%, t R = 6.5 min; 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.70 (br s, 2H), 7.47 (d, J = 8.2 Hz, 1H), 7.41 (ddd, J = 8.0, 7.3, 1.8 Hz, 2H), 7.28 (dd, J = 8.2, 2.2 Hz, 1H), 7.23 (d, J = 2.1 Hz, 1H), 7.15 (dd, J = 7.5, 1.8 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 7.03 (t, J = 7.4 Hz, 1H), 3.73 (s, 3H), 3.17 (t, J = 8.1 Hz, 2H), 2.96 (t, J = 8.1 Hz, 2H), 2.88 (t, J = 7.7 Hz, 2H), 1.62 (sext, J 7.6 Hz, 2H), 0.92 (t, J = 7.4 Hz, 3H);2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N-butyllethan-1amine (2d). Compound 2d was synthesized according to the general procedure of 2. Yield: 95%; HPLC: purity 100%, t R = 6.3 min; 1 H NMR (400 MHz, DMSO-d 6 ): δ 8.66 (br s, 2H), 7.49 (d, J = 8.1 Hz, 1H), 7.44-7.40 (m, 1H), 7.29 (dd, J = 8.3, 1.7 Hz, 1H), 7.24 (d, J = 1.6 Hz, 1H), 7.17-7.12 (m, 2H), 7.04 (t, J = 7.4 Hz, 1H), 3.74 (s, 3H), 3.19 (t, J = 8.1 Hz, 2H), 2.98-2.90 (m, 4H), 1.63-1.55 (m, 2H), 1.34 (t, J = 7.3 Hz, 2H), 0.91 (t, J = 7.3 Hz, 3H);2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N,N-dimethylethan-1-amine (2e). A solution of 2-(6-chloro-2′-methoxy-[1,1′biphenyl]-3-yl)ethan-1-amine 11 (0.44 mmol) in formic acid (2.22 mmol, 88% in water solution) and formaldehyde (2.22 mmol, 37% in water solution) was stirred at 80 °C for 20 h. The reaction mixture was cooled down to rt, and the mixture was diluted with water, adjusted to pH 10 with K 2 CO 3 , and extracted with DCM. The organic layer was washed with brine, dried over MgSO 4 , and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/ MeOH = 10:1) to obtain the desired product 2e (0.19 mmol, 43%); HPLC: purity 100%, t R = 5.4 min; 1 H NMR (400 MHz, CDCl 3 ): δ 7.37 (t, 2H), 7.19-7.17 (m, 3H), 7.03-6.97 (m, 2H), 3.78 (s, 3H), 2.81 (t, J = 8.0 Hz, 2H), 2.61 (t, J = 8.0 Hz, 2H), 2.33 (s, 6H); amine 11 (0.50 mmol) was dissolved in acetonitrile (3 mL). Iodoethane (0.6 mmol) and K 2 CO 3 (1.99 mmol) were added to the solution and the reaction mixture was stirred for 15 h at 65 °C. The mixture was cooled down to rt and quenched with saturated solution of NaHCO 3 and then extracted with EtOAc. The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (DCM/MeOH = 20:1) to obtain the desired product 2f (0.32 mmol, 63%); HPLC: purity 100%, t R = 6.5 min; 1 H NMR (400 MHz, CDCl 3 ): δ 7.40-7.36 (m, 2H), 7.19-7.15 (m, 3H), 7.02 (td, J = 7.5, 1.7 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.78 (s, 3H), 3.10-3.05 (m, 4H), 3.02 (q, J = 7.3 Hz, 4H), 1.32 (t, J = 7.3 Hz, 6H);C NMR (100 MHz, CDCl 3 ): δ 156.6, 138.3, 135.1, 132.8, 130.9, 129.8, 129.6, 128.9, 128.0, 120.4, 111.0, 55.7, 55.6, 46.9, 40.2, 30.1, 9.2; HRMS (ESI+): calcd for C 19 H 25 ClNO + [M + H] + , 318.1625; found, 318.1621. 2-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-N,N-dipropylethan-1-amine (2g). Compound 2g was synthesized according to the general procedure of 2f. Yield: 30%; HPLC: purity 100%, t R = 7.9 min; 1 H NMR (400 MHz, CDCl 3 ): δ 7.43-7.38 (m, 2H), 7.22 (dd, J = 7.4, 1.2 Hz, 1H), 7.16-7.14 (m, 2H), 7.06 (t, J = 7.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 3.82 (s, 3H), 2.82-2.73 (m, 4H), 2.49 (t, J = 7.6 Hz, 4H), 1.52 (sext, J = 7.5 Hz, 4H), 0.91 (t, J = 7.4 Hz, 6H);2-Chloro-2′-methoxy-5-(2-nitrovinyl)-1,1′-biphenyl (14). Ammonium acetate (3.91 mmol) was added to a solution of 6-chloro-2′methoxy-[1,1′-biphenyl]-3-carbaldehyde 6 (3.91 mmol) in nitromethane (31.3 mL). The mixture was stirred for 6 h at 100 °C. After cooling down to rt, the reaction mixture was concentrated under reduced pressure and then the residue was purified by flash column chromatography (hexanes/EtOAc = 20:1) to afford the desired product 14 (2.59 mmol, 66.2%).(15). Sodium hydride (1.38 mmol) was added to a solution of diethyl malonate (2.76 mmol) in THF (3 mL) and then the mixture was stirred for 15 min at rt. A solution of 2-chloro-2′methoxy-5-(2-nitrovinyl)-1,1′-biphenyl 14 (0.69 mmol) in THF (2 mL) was then added to the mixture and the mixture was stirred for 3 h at rt. The reaction mixture was quenched by the addition of saturated solution of NH 4 Cl and then extracted with EtOAc. The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (hexanes/EtOAc = 10:1) to obtain the desired product 15 (0.33 mmol, 48.3%).H NMR (CDCl 3 , 400 MHz): δ 7.43-7.41 (m, 1H), 7.40-7.38 (m, 1H), 7.20 (d, J = 7.4 Hz, 2H), 7.17 (dd, J = 7.5, 1.8 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 4.95 (dd, J = 13.2, 4.9 Hz, 1H), 4.88 (dd, J = 9.1, 13.3 Hz), 4.30-4.20 (m, 3H), 4.08 (q, J = 7.1 Hz, 2H), 3.83 (d, J = 9.2 Hz, 1H), 3.78 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H), 1.11 (t, J = 7.1 Hz, 3H);C NMR (100 MHz, CDCl 3 ): δ 167. 33, 166.76, 156.67, 138.31, 134.56, 133.97, 130.92, 129.82, 129.64, 128.18, 127.84, 120.34, 111.08, 62.24, 62.04, 55.48, 54.89, 42.33, 13.96, 13.), 1.32 (t, J = 7.1 Hz, 3H);C NMR (100 MHz, CDCl 3 ): δ 175.7, 169.9, 157.7, 147.0, 137. 1, 130.8, 129.8, 129.3, 129.0, 128.6, 125.6, 121.5, 116.6, 60.9, 60.6, 56.1, 39.8, 29.4, 14.1. 4-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidin-2-one (17). To the solution of ethyl 4-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-2-oxopyrrolidine-3-carboxylate 16 (0.90 mmol) in EtOH (3 mL) was added 1 N NaOH (1 mL) at rt. After the mixture was stirred for an hour, the reaction mixture was concentrated under reduced pressure and the residue was added to 5 N HCl and the aqueous phase was partitioned with DCM. The combined organic layer was dried over MgSO 4 , filtered, and concentrated under reduced pressure to obtain corresponding carboxylic acid (0.81 mmol, 90%). The solution of carboxylic acid (0.81 mmol) in toluene (10 mL) was refluxed at 140 °C for 6 h. After cooling down to rt, the mixture was concentrated under reduced pressure and the residue was purified by flash column chromatography (DCM/MeOH = 7:1) to obtain the desired product 17 (0.43 mmol, 67%).H NMR (400 MHz, CDCl 3 ): δ 7.42-7.35 (m, 2H), 7.17-7.14 (m, 3H), 7.01 (td, J = 7.5, 0.8 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 3.77 (s, 3H), 3.67 (t, J = 7.9 Hz, 1H), 3.44-3.40 (m, 1H), 2.73 (q, J = 8.6 Hz, 1H), 2.49 (q, J = 8.6 Hz, 1H), 2.28 (br s, 1H);3-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidine (3a). Aluminum chloride (3.38 mmol) in THF (2 mL) was mixed with lithium aluminum hydride 1.0 M in THF (3.38 mmol) at 0 °C. 4-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)pyrrolidin-2-one 17 (0.85 mmol) was added dropwise to the mixture at 0 °C and then the reaction mixture was stirred at rt for an hour. After an hour, the reaction mixture refluxed at 80 °C overnight. The mixture was carefully quenched by the addition of MeOH at 0 °C and concentrated under reduced pressure. The residue was diluted by DCM and washed with water. The organic phase was partitioned with 1 N HCl aqueous solution. The combined aqueous layer was washed with DCM and neutralized by the addition of 10 N solution of NaOH. The mixture was extracted with DCM and the combined organic layer was dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure to afford the desired product 3a (0.62 mmol, 73%); HPLC: purity 100%, t R = 6.4 min; 1 H NMR (400 MHz, CDCl 3 ): δ 8.34 (br s, 1H), 7.44-7.37 (m, 2H), 7.29 (t, J = 9.0 Hz, 1H), 7.24-7.19 (m, 1H), 7.17-7.14 (m, 1H), 7.02 (dd, J = 7.4, 3.1 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 3.78 (d, J = 11.5 Hz, 3H), 3.64-3.50 (m, 2H), 3.45-3.37 (m, 1H), 3.31-3.20 (m, 1H), 2.48-2.39 (m, 1H), 2.13 (sep, J = 10.5 Hz, 1H);C NMR (100 MHz, CDCl 3 ): δ 156.63, 139. 27, 138.32, 136.69, 133.16, 130.89, 129.86, 128.53, 127.18, 125.58, 120.43, 111.03, 55.61, 53.48, 50.55, 45.19, 43.75, 43.08, 32.43 3-(6-Chloro-2′-methoxy-[1,1′-biphenyl]-3-yl)-1-methylpyrrolidine (3b). A solution of 3-(6-chloro-2′-methoxy-[1,1′-biphenyl]-3yl)pyrrolidine 3a (0.45 mmol) in formic acid (3.16 mmol, 88% in water solution) and formaldehyde (3.16 mmol, 37% in water solution) was stirred at 80 °C for 20 h. After cooling down to rt, the reaction mixture was diluted with water, adjusted to pH 10 with K 2 CO 3 , and extracted with DCM. The organic layer was washed with brine, dried over MgSO 4 , and concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM/MeOH = 10:1) to obtain the desired product 3b (0.17 mmol, 37%); HPLC: purity 100%, t R = 6.2 min;H]LSD radioligand was diluted to 5 times the assay concentration in standard binding buffer. Aliquots (50 mL) of the radioligand were dispensed into the wells of a 96-well plate containing 100 mL of standard binding buffer. Triplicate aliquots (50 mL) of the test and reference compound dilutions were then added. Finally, crude membrane fractions (50 mL) of cells expressing a recombinant target were dispensed into each well. Totally, 250 mL of the reaction mixtures was incubated at rt and shielded from light for 1.5 h and then harvested by rapid filtration onto Whatman GF/B glass fiber filters presoaked with 0.3% polyethyleneimine, by using a 96-well Brandel harvester. Four rapid washes were performed with chilled standard binding buffer (500 mL) to decrease nonspecific binding. Filters were placed in 6 mL scintillation tubes and allowed to dry overnight. The next day, 4 mL of EcoScint scintillation cocktail (National Diagnostics) was added to each tube. The tubes were capped, labeled, and counted by liquid scintillation counting. The filter mats were dried, and the scintillant was melted onto the filters, and then the radioactivity retained on the filters was counted in a MicroBeta scintillation counter. The IC 50 values were obtained by using the Prism 4.0 program (GraphPad software) and converted into K i values. Each compound was tested in triplicate at least. Gs Protein-Mediated cAMP Assay. HEK293 cells were harvested in 150 mm dishes and the medium was changed from Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin to DMEM with 10% dialyzed FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. After 5-6 h, those cells were transfected (via calcium phosphate) with 10 μg of human 5-HT 7 R plasmid and 10 μg of GloSensor-22F plasmid (Promega). Transiently transfected HEK293 cells were seeded (20,000-30,000 cells/20 μL/well) into white, clearbottom 384-well plates (Greiner) in the same medium. After 5-6 h of recovery, the medium was removed from the wells and the cells were treated with 3% GloSensor cAMP reagent, luciferin (Promega) 20 μL in filter-sterilized assay buffer including 1× Hanks' balanced salt solution (HBSS), 20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), and 3rd distilled water, pH 7.4. Then, the reference agonist (5-HT, 400 nM) or the compounds to be tested were prepared by serial dilution (0.04 nM, 0.12 nM, 0.4 nM, 1.2 nM, 4 nM, 12 nM, 40 nM, 120 nM, 400 nM, 1.2 μM, 4 μM, 12 μM, 40 μM, and 120 μM) in the above assay buffer with 0.1% bovine serum albumin. After 30 min, the cells were treated with 10 μL of drugs prepared above (the final ligand concentrations are 100 nM of serotonin and 0.01 nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, and 30 μM of test compounds). The luminescence intensity of the accumulated cAMP level was measured by using a microplate reader (FlexStation 3 or SpectraMax i3, Molecular Devices). The sigmoidal dose-response graph of the obtained data was obtained by using the Prism 6.0 program (GraphPad software) to calculate the EC 50 and IC 50 values. Each compound was tested in triplicate at least. β-Arrestin Recruitment Tango Assay. HTLA cells (a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a βarrestin 2-TEV fusion gene) were plated in 150 mm dishes and the medium was changed from DMEM with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 μg/mL puromycin, and 100 μg/ mL hygromycin B to DMEM with 10% dialyzed FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin to transfect the plasmid. After 5-6 h, those HTLA cells were transfected (via calcium phosphate) with 20 μg of a 5-HT 7 R-TCS-tTA construct. The next day, transiently transfected HTLA cells were plated in white, clear-bottom, 384-well plates (Greiner; 30,000-40,000 cells/well, 50 μL/well) in DMEM containing 1% dialyzed FBS, 100 U/mL penicillin, and 100 μg/mL. After 6 h, the cells were challenged with 10 μL/well of reference agonist (serotonin, 6 μM) or the compounds to be tested prepared by serial dilution (0.06 nM, 0.18 nM, 0.6 nM, 1.8 nM, 6 nM, 18 nM, 60 nM, 180 nM, 600 nM, 1.8 μM, 6 μM, 18 μM, 60 μM, and 180 μM) in HBSS, 20 mM HEPES, and pH 7.4 (the final ligand concentrations are 1 μM of serotonin and 0.01 nM, 0.03 nM, 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, 10 μM, and 30 μM of test compounds) and incubated for about 22 h. The medium was removed and replaced with 1× BrightGlo reagent (Promega), and luminescence was read using a SpectraMax i3 (Molecular Devices). The sigmoidal dose-response graph of the obtained data was obtained by using the Prism 6.0 program (GraphPad software) to calculate the EC 50 and IC 50 values. Each compound was tested in triplicate at least. CYP450 Assay. All of CYP450, microsomal stability, and PK data were provided by the New Drug Development Center or Institutional Animal Care and Use Committees (IACUC) in Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) (Daegu, Republic of Korea). To human liver microsomes (0.25 mg/mL), 0.1 M phosphate buffer (pH 7.4), a cocktail of five probe substrates (phenacetin 50 μM, diclofenac 10 μM, S-mephenytoin 100 μM, dextromethorphan 5 μM, and midazolam 2.5 μM), and tested compounds were added at concentrations of 0 μM (as a control) and 10 μM. After incubation at 37 °C for 5 min, reduced nicotinamide adenine dinucleotide phosphate (NADPH) generation system solution was also added and incubated at 37 °C for 15 min again. To terminate the reaction, acetonitrile including the internal standard (terfenadine) was added and the solution was centrifuged for 5 min (14,000 rpm, 4 °C). The supernatant was then injected into the LC/MS system to simultaneously analyze the metabolites of the probe substrates and evaluate the % CYP inhibition of the tested compounds. Microsomal Stability Assay. To human liver microsomes (0.5 mg/mL), 0.1 M phosphate buffer (pH 7.4) and tested compounds (1 μM) were added. After incubation at 37 °C for 5 min, the NADPH generation system solution was also added and incubated at 37 °C for 30 again. To terminate the reaction, including internal standard (chloprapamide) was added and the solution was centrifuged for 5 min (14,000 rpm, 4 °C). The supernatant was then injected into the LC/MS system to analyze the microsomal stability of the tested compounds. PK Studies. All animal experiments were evaluated and approved by the DGMIF IACUC. ICR mice (7-8 weeks of age) weighing 30 ± 5 g were used for the PK and tissue distribution studies and were purchased from Orient Co. (South Korea). The mice were kept at rt controlled at 23 ± 3 °C with relative humidity controlled at about 55 ± 10%, fed with standard solid composite feedstuff, and received tap water. Compound 2b at a dose of 1 or 10 mg/kg was administered intravenously or intraperitoneally, respectively, to male ICR mice. Blood samples were collected via carotid artery at 0 (to serve as a control), 0.08 (IV only), 0.25, 0.5, 1, 2, 4, 6, and 8 h after administration of each compound. After centrifugation at 12,000 rpm for 3 min, plasma samples were stored at -70 °C until analysis. PK parameters were determined by a noncompartmental analysis using WinNonlin v6.4 (Pharsight Corporation, Mountain View, CA) program. The total area under the plasma concentration-time curve from time zero to the last measured time (AUC last ) was calculated by the trapezoidal rule-extrapolation method. Standard methods were used to calculate the following PK parameters:the time-averaged total body clearance (CL), total area under the first moment of plasma concentration and time curve from time zero to time infinity (AUC 0-∞ ), terminal half-life, mean residence time (MRT), and apparent volume of distribution at steady state (V dss ). The concentrations of each compound in the above samples were analyzed using LC-MS/MS. To a 20 μL aliquot of plasma sample, an 80 μL aliquot of acetonitrile containing 2 μM of internal standard (chloropropramide) was added. After vortex mixing and centrifugation at 15,000 rpm for 5 min, a 2 μL of supernatant was injected into the LC-MS/MS system. The LC-MS/MS system consisted of an Agilent 1290 infinity series HPLC system (Agilent, Santa Clara, CA) and API5500 triple-quadrupole mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada). The HPLC mobile phases consisted of 0.1% formic acid in 100% deionized water (A) and 0.1% formic acid in 100% acetonitrile (B). Chromatographic separation was achieved on a reversed-phase Kinetex C18 column (100 × 2.1 mm, 1.7 μm, Phenomenex) using gradient elution at a flow rate of 0.3 mL/ min. The lower limit of quantitation of each compound in rat plasma was 100 ng/mL. The values of coefficients of correlation (R) were more than 0.9971. Self-Grooming Behavior Test. All animal experiments were conducted according to the guidelines of the KIST IACUC. WT and Shank3 TG male mice, 7 to 9 week old, were obtained from KIST (Korea Institute of Science and Technology, Seoul, Korea). They were housed at a temperature of 22 ± 2 °C with a humidity of 55 ± 5% and maintained in a 12/12 h light/dark cycle under 5 dB. All mice had ad libitum access to food and water. Drugs were dissolved in 5% dimethyl sulfoxide in saline solution and injected into the peritoneal cavity before 30 min starting the experiment. Each mouse was placed in a 30 × 20 cm opaque acrylic container with 1-2 cm bedding on the floor under 40 lux lighting. The total duration and number of self-grooming behavior were recorded for 30 min. After the test, the container was cleaned with 70% ethanol, and the floor was covered with new bedding for the next mouse recording. Analyze the video by stopwatch program with the function of snap, being careful not to count sniffing behavior. In case of cotreatment, after 10 min of SB269970 injection, 2b was administrated. The behavior started to be recorded after 30 min of 2b injection.
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