Patent Application
20240327353
G protein-coupled receptors (GPCRs) are targeted by about 34% of FDA approved drugs, many of which mediate aminergic neurotransmission (Hauser, et al., 2017). Among at least thirteen serotonin (5-hydroxytryptamine, 5-HT) GPCRs are 5-HT2A, 5-HT2B, and 5-HT2C receptors (Rs), which represent promising neurotherapeutic targets. However, extensive structural homology complicates the design of selective therapeutic agents. For example, 5-HT2-type receptors share 60-70% amino acid identity within structurally conserved regions, and 27-31% identity with histamine receptors (H1Rs), (Pandy-Szekeres, et al., 2018).
Antagonism of 5-HT2ARs is associated with the improved efficacy of so-called atypical antipsychotics toward schizophrenia, and hallucinations and delusions in psychosis (Meltzer, 1999; Weiner, et al., 2001; Hacksell, et al., 2014). Additionally, inverse agonism of 5-HT2CRs, a mainstay of atypical antipsychotic polypharmacology, might be pharmacotherapeutic for generalized anxiety, major depression, and schizophrenia (Chagraoui, et al., 2016; Demireva, et al., 2018).
The selective 5-HT2A/5-HT2CR inverse agonist pimavanserin (PIMA) is approved to treat hallucinations and delusions associated with Parkinson's disease psychosis (Meltzer, 1999; Cummings, et al., 2014), though the contribution of 5-HT2CRs to PIMA's efficacy is unclear (Stahl, 2016). Meanwhile, activation of 5-HT2BRs is linked to valvular heart disease (Rothman, et al., 2000; Ayme-Dietrich, et al., 2017), and deficiency or antagonism of 5-HT2BRs is linked to psychotic-like and impulsive behaviors in laboratory animals and humans (Bevilacqua, et al., 2010; Pitychoutis, et al., 2015). Thus, engagement of 5-HT2BRs may be undesirable for antipsychotic medications. Likewise, H1Rs represent a common ‘off-target’ for CNS-penetrating drugs (Weiner, et al., 2001) and H1R antagonism is associated with sedative-hypnotic effects (Nicholson, et al., 1991; Stahl, 2008). Notably, PIMA has nil affinity for H1Rs and does not appear to cause daytime sleepiness in humans (Cummings, et al., 2014; Meltzer, et al., 2010; Ancoli-Israel, et al., 2011; Fava, et al., 2019). Structural homology of receptors, broad distribution of 5-HT receptors and various side effects such as QT interval prolongation, locomotion problems, and nonspecific receptor binding prevent accurate targeting of indications in subjects. Modulators of 5-HT2A, 5-HT2B and 5-HT2C receptors with improved selectivity are needed.
Through receptors in the central nervous system (CNS) and the periphery, serotonin can modulate many organ systems in the body, including cardiac functions, the cardiovascular system, the gastrointestinal (GI) system, genitourinary systems, the endocrine system, metabolism, reproductive function and pregnancy as well as the CNS. Targeting 5-HT receptors in the periphery can affect multiple systems in the body.
The present technology provides novel 4-phenyl-2-dimethylaminotetralin (4-PAT) compounds that are shown to provide inverse agonism at one or more 5-HT2A-C receptors. The compounds do not cause sedation at doses that are antipsychotic. The technology demonstrates mechanisms that can be predictive of the selective efficacy of substituents and stereochemistry of 4-PAT compounds. The technology also provides novel serotonin receptor-modulating compounds that do not substantially accumulate in the brain (or CNS) and therefore are useful in treating diseases or disorders of the periphery.
The technology can be further summarized by the following list of features.
1. A compound for selective modulation of one or more of serotonin 5-HT2A and 5-HT2C receptors, the compound having a structure according to Formula I:
As used herein, the term room temperature refers to a temperature within the range of about 15-30° C.
As used herein, the term “about” refers to a range of within plus or minus 10%, 5%, 1%, or 0.5% of the stated value.
As used herein, “consisting essentially of” allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with the alternative expression “consisting of” or “consisting essentially of”.
The present technology provides novel 4-phenyl-2-dimethylaminotetralin (4-PAT) compounds that can be utilized in treating or preventing neuropsychiatric disorders. Similar to other 5HT2C agonists with 5HT2A/2B antagonist/inverse agonist compounds of the 2-aminotetralin chemotype, which are active in rodent and monkey models of psychosis, the present 4-PAT compounds can be used to treat, for example, fragile X syndrome, autism, impulsive behaviors such as occur with attention deficit hyperactivity disorder and binge eating, and substance use disorder (particularly, opioid use disorder and amphetamines use disorder). There are no drugs currently approved to treat psychosis associated with the neurodevelopmental disorder fragile X syndrome (orphan therapeutic indication) or autism. Current approved antipsychotic medications cause sedation (in non-fragile X patients) or other neurological side effects. The present 4-PAT compounds do not cause sedation.
The present technology provides examples of at least 42 new chemical entities that are single enantiomer drug compounds. The examples of at least 42 new chemical entities that are single enantiomer drug compounds can optionally be configured so that the compound or composition does not substantially accumulate in the human brain (e.g., Scheme 11,
The present technology provides novel 4-phenyl-2-dimethylaminotetralin (4-PAT) compounds and compositions that can be utilized in treating or preventing a variety of disorders of the periphery, in particular because their distribution within the body of the subject is restricted to the periphery, i.e., the compound does not substantially accumulate in the human brain. The ‘E’ group shown in
The compound or composition is not rapidly metabolized in the periphery. In various examples, the compounds and compositions, or formulations thereof, deliver a physiological amount of the present compound or composition to the periphery for at least about 6 hours, or at least about 9 hours, or at least about 12 hours, or at least about 15 hours, or at least about 18 hours, or at least about 21 hours, or at least about 24 hours. In various examples, the compounds and compositions, or formulations thereof, deliver a physiological amount of the present compound or composition to the periphery for about 6 hours, or about 9 hours, or about 12 hours, or about 15 hours, or about 18 hours, or about 21 hours, or about 24 hours.
The centrally-acting 4-PAT compounds of the present technology can be used to aid in treating or preventing, for example, migraine, Parkinson's disease, attention deficit hyperactivity disorder, anxiety or generalized anxiety, depression, schizophrenia, binge eating, opioid use disorder, fragile X syndrome, amphetamine use disorder, panic disorder, social anxiety disorder, obsessive-compulsive disorder, pain, Alzheimer's disease, or Huntington's disease.
The peripherally acting 4-PAT compounds of the present technology can be used to aid in treating or preventing, for example, hypertension, thrombosis, deep vein thrombosis, pulmonary embolus, atrial fibrillation, atherosclerosis, valvular atherosclerosis, cardiac fibrosis, obesity, irritable bowel syndrome, and lack of bladder control.
5HT2C agonists with 5HT2A/2B antagonist/inverse agonist activity of the 2-aminotetralin chemotype demonstrate high efficacy and safety in rodent and monkey animal models of psychoses and substance use disorders (amphetamines and opioids), as described in U.S. Pat. No. 8,586,634B2, U.S. Pat. No. 9,024,071B2, U.S. Pat. No. 9,862,674B2 and U.S. Ser. No. 10/017,458B2, each of which is hereby incorporated by reference in its entirety. There are no drugs approved to treat psychosis associated with the neurodevelopmental disorder fragile X syndrome (an orphan therapeutic indication) or autism, and the 4-PAT chemotypes disclosed herein demonstrate outstanding potential for these therapeutic indications.
Synthetic methods described herein include Friedel-Crafts cycli-acyl/alkylation of the commercially available 3-bromostyrene and phenylacetyl chloride to give the intermediate tetralone. The tetralone is subjected to reductive amination to afford a separable mixture of 3′-Br-4-phenyl-2-aminotetralin diastereomers. Reductive amination produces racemic cis or trans-4-phenyl-2-aminotetralins that are separated via silica gel column chromatography. Substituents are introduced at the 3′-position via Suzuki-Miyaura coupling of the 3′-Br-4-PAT diastereomers with the corresponding boronic acids. The ‘bench-top stable’ MIDA ester is used successfully to introduce thiophen-2′-yl and furan-2′-yl fragments into the 3′-Br-4-PATs. The racemic mixtures of trans-analogs are separated by a semi-preparative chiral HPLC column using conditions and solvents specific to each analog to elute the trans-(2R,4S) and trans-(2S,4R) enantiomers at representative retention times t1 and t2, respectively, with absolute stereochemistry assigned according to retention time of the previously published trans-3′CI-4-PAT analog. Chiral HPLC separation yields the (2R,4S)-trans-4-(3-(thiophen-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine and (2R,4S)-trans-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine.
The compounds presented herein are water soluble and can be administered as oral formulations. The technology includes pharmaceutical compositions and formulations including the compounds. Examples of formulations can include drug in capsule with or without excipients additives or buffers, subcutaneous and IV formulations, mixtures with citric acid, lactic acid, solvents, propylene glycol, osmolality adjusting salts or sugars, purified water, or other excipients.
The centrally acting (e.g., uncharged) compounds presented herein cross into the brain when administered to rodents and engage serotonin 5HT2 receptors to produce antipsychotic effects. The compounds do not cause neurological side effects at doses that are antipsychotic. The 5-HT receptor (5-HTR) subtypes 5-HT2A and 5-HT2C are important neurotherapeutic targets, though, obtaining selectivity over 5-HT2B and closely related histamine H1Rs is challenging. Herein are delineated molecular determinants of selective binding to 5-HT2A and 5-HT2CRs using novel 4-PATs.
Example compounds are compared to 5-HT, HIS, DOX, RIT and PIMA in exploratory functional screening at 5-HT2-type receptors (Rs) 5-HT2A, 5-HT2B, 5-HT2C and H1Rs in
Most 4-PAT diastereomers in the (2S,4R)-configuration bind non-selectively to 5-HT2A, 5-HT2C, and H1Rs, with >100-fold selectivity over 5-HT2BRs, whereas diastereomers in the (2R,4R)-configuration bind preferentially to 5-HT2A over 5-HT2CRs and have >100-fold selectivity over 5-HT2B and H1Rs. Results suggest that G2385.42 and V2355.39 (
The novel 4-PAT chemotype can yield selective 5-HT2A/5-HT2CR inverse agonists for antipsychotic drug development by optimizing ligand-receptor interactions in transmembrane domain 5. It is shown that chirality can be exploited to attain selectivity over H1Rs to help circumvent sedative effects. High homology between 5-HT2-type and histamine H1Rs could prevent development of antipsychotics without sedative effects.
The 4-phenyl-2-dimethylaminotetralin (4-PAT,
aData are presented as the mean pKi and range for n independent experiments, shown in superscript. Where n = 5 independent experiments, the standard deviation is provided.
bND = not determined
Structure-activity relationships (SAR) for 4-PATs acting at 5-HT2-type and H1Rs are developed using competitive radioligand displacement and functional assays. The data reveal that aryl substituted 4-PATs are potent 5-HT2A-preferring 5-HT2A/5-HT2CR inverse agonists with selectivity over 5-HT2B and H1Rs. To understand the molecular determinants for selectivity and inverse agonism at 5-HT2ARs, in silico molecular modeling is performed and used to guide site-directed mutagenesis of residues in receptor transmembrane (TM) domains 4 and 5. The potency and selectivity of certain aryl substituted 4-PATs at 5-HT2A and 5-HT2CRs (e.g., [2S,4R]-2k, and [2R,4R]-3h) resemble PIMA, thus, a comparison is made of 4-PATs to PIMA in vitro and in silico. Comparative in vivo assessments in mice are also performed, utilizing the (±)-2,5-dimethoxy-4-iodoamphetamine (DOI)-elicited head twitch response as a model to screen for central 5-HT2AR engagement and antipsychotic-like activity, and locomotor activity assays to assess behavioral disruption and untoward motor effects.
The optimized synthetic methods demonstrated herein generate novel cis- and trans-4-PAT enantiomers separable by chiral-HPLC. Competitive radioligand binding studies at 5-HT2-type and H1Rs indicate that small meta-halo substituents (e.g., —F, —Cl, —Br) on ring C (
The affinity (pKi) of the FDA-approved antipsychotic drugs PIMA and risperidone under that same assay conditions used for 4-PATs are assessed in Table 1. Notably, while the 4-PAT leads (2S,4R)-2k and (2R,4R)-3h, as well as PIMA and risperidone, have moderate to high selectivity over 5-HT2B (˜50-3,000-fold) and H1Rs (˜40-15,000-fold), only risperidone displays high affinity at all receptors tested. The 4-PAT leads (2S,4R)-2k and (2R,4R)-3h have high affinity and inverse agonist activity at 5-HT2A and 5-HT2CRs comparable to PIMA and risperidone. While (2S,4R)-2k trends toward agonist activity at 5-HT2BRs, this ligand may not pose cardiovascular concerns given its low potency and efficacy (Unett, et al., 2013).
At 5-HT2ARs, no rank order in the radioligand-derived affinity (pKi) is observed. However, when comparing the functionally derived affinity (pKb), a clear rank order in potency appears (i.e., PIMA >[2S,4R]-2k>[2R,4R]-3h). The observed discontinuities are not investigated further but may involve differences in the biological milieu between experimental formats using a membrane preparation (radioligand binding assays) and whole live cells (functional assays). Biological milieu, including the membrane environment and effector expression, may vary between cell lines (Symons, et al., 2021; Zhang, et al., 2017) and transfections (Lee, et al., 2019) to impact functional signaling (Gutierrez, et al., 2016; Lefkowitz, et al., 2002). The results highlight the necessity of implementing orthogonal assays and reference ligands (e.g., PIMA and risperidone) to characterize ligand affinity and function (Tran, et al., 2019).
Comparing the affinity of 4-PATs at 5-HT2-type and H1Rs in Table 1, previous lab work shows that meta-halo substituted trans-4-PATs, including (2S,4R)-2a, display higher affinity at 5-HT2-type receptors than the parent unsubstituted analog (2S,4R)-1 (Sakhuja, et al., 2015). However, the impact of meta-halo substitution on the affinity of cis-4-PATs at 5-HT2-type receptors has not been reported. Here, it is shown that cis-diastereomers of the previously reported meta-Br substituted 4-PAT, 2a, and its meta-CI and meta-F congeners (3a, 3b and 3b′, respectively) demonstrate moderate (pKi=6.5-7.5) to low (pKi<6.5) affinity at 5-HT2-type receptors in the (2R,4R)-configuration. These values resemble those for the unsubstituted cis-4-PATs previously reported (Booth, Fang et al., 2009), though larger, more polarizable substituents have higher affinity (pKi 3a>3b>3b′). The corresponding (2S,4S)-enantiomers, on the other hand, exhibit high affinity (pKi>7.5) at H1Rs, with robust selectivity (40-280-fold) over 5-HT2-type receptors. Due to their unimpressive affinity at, and selectivity for, 5-HT2-type receptors, analogs 3a, 3b or 3b′ are not explored further (Table 1).
It is hypothesized that the chiral nature of the 4-PAT chemotype combined with steric bulk at the meta-position of ring C (
Heteroaromatic substitution on ring C (i.e., 2c-e and 3c-e) yields five-membered heterocycles (2c-d, 3c-d) which bind to 5-HT2A, 5-HT2C, and H1Rs in the (2S,4R)-configuration with high affinity and moderate selectivity for 5-HT2A and 5-HT2C over 5-HT2BRs (˜20-40-fold). In contrast to analogs (2S,4S)-3a-b′, the (2S,4S)-3c meta-thiophen-2′-yl analog has low affinity at, and no selectivity for, H1Rs. Meanwhile, (2R,4R)-3c preferentially binds to 5-HT2A and 5-HT2CRs, with highest affinity at 5-HT2ARs and moderate selectivity (˜30-60-fold) over 5-HT2B and H1Rs. The meta-pyridin-2′-yl (2e, 3e) analogs have moderate to low affinity at 5-HT2-type receptors, however, (2S,4R)-2e and (2R,4R)-3e have high affinity at H1Rs, thus, 2e, 3e are not pursued in further investigation.
The reassessment of (2S,4R)-2f indicates that, contrary to a 2015 report where the ligand displayed high affinity and selectivity for 5-HT2CRs (Sakhuja, et al., 2015), it binds non-selectively to 5-HT2A, 5-HT2C, and H1Rs, with moderate selectivity over 5-HT2BRs (70-fold). Substitution effects on phenyl ring D are then investigated using a ‘fluorine-walk’ approach, wherein, fluorine is mono-substituted at each position (2g-i and 3g-i). The 3′-F—C6H4 substituted diastereomer, (2R,4R)-3h, shows highest affinity at 5-HT2ARs, with 33-fold selectivity over 5-HT2CRs, 415-fold selectivity over 5-HT2BRs, and ˜1,300-fold selectivity over H1Rs, making it the most 5-HT2AR-selective 4-PAT-type compound reported herein. Its diastereomer, (2S,4R)-2h, exhibits high affinity at 5-HT2A and 5-HT2CRs, with ˜30-fold selectivity over 5-HT2BRs, and no selectivity over H1Rs. Thus, (2R,4R)-3h is chosen as a lead for further characterization in vivo, as well as in vitro and in silico to identify molecular determinants for selective binding to 5-HT2ARs.
The 4′-F—C6H4 substituted analog (2S,4R)-2i has high affinity at 5-HT2A and 5-HT2CRs, 140-fold selectivity for 5-HT2ARs over 5-HT2BRs, though, selectivity over H1Rs is modest (5-fold). Similarly, the 4′-CI—C6H4 substituted analog (2S,4R)-2j has 143-fold selectivity for 5-HT2ARs over 5-HT2BRs, and no selectivity over H1Rs. The diastereomer, (2R,4R)-3j, retains high affinity for 5-HT2A and 5-HT2CRs, and shows ˜100- and ˜225-fold selectivity for 5-HT2ARs over 5-HT2B and H1Rs, respectively.
To explore electronic and steric effects, a 4′-NMe2—C6H4 substituent is introduced on ring C (2k, 3k), yielding stereoisomers with a computationally determined log P˜5.69 (log D˜3.77). Notably, these are slightly lower than that of 2i, 3i (log P˜5.85, log D˜3.86), 2j, 3j (log P˜6.36, log D˜4.41), and the other lead, (2R,4R)-3h (log P˜5.85, log D˜3.79). Like other aryl substituted 4-PATs in the (2R,4R)-configuration (i.e., 3c-3k), compound (2R,4R)-3k shows modest selectivity to bind 5-HT2ARs over 5-HT2CRs (˜7-fold) and high selectivity over 5-HT2B and H1Rs (>350-fold). In contrast, (2S,4R)-2k has similarly high affinity at both 5-HT2A and 5-HT2CRs, with high selectivity over 5-HT2BRs (˜180-fold) and moderate selectivity over H1Rs (˜38-fold). Since (2S,4R)-2k portrays dual 5-HT2A/5-HT2CR activity with selectivity over 5-HT2B and H1Rs, (2S,4R)-2k is selected, along with the 5-HT2AR selective analog (2R,4R)-3h (above), for further investigation in vitro, in vivo, and in silico.
To compare the affinity of 4-PATs with FDA-approved antipsychotic drugs, an assessment is made of the affinity of PIMA and risperidone at 5-HT2-type and H1Rs. In this work, PIMA exhibited high affinity for 5-HT2ARs, with modest selectivity (12-fold) over 5-HT2CRs, and high selectivity over 5-HT2B and H1Rs (>3,000-fold). Risperidone is equipotent to PIMA at 5-HT2ARs and has high affinity at the other receptors. The results are consistent with those in the literature (Vanover, et al., 2006; Chopko & Lindsley, 2018).
Investigations of the functional activity of 4-PATs and PIMA at 5-HT2Rs and H1Rs are conducted with the exploratory functional screening presented in
The binding and functional screening results indicate that PIMA, (2S,4R)-2k, and (2R,4R)-3h exhibit similar affinity, and inverse agonist efficacy, at therapeutically favorable 5-HT2A and 5-HT2CRs. Therefore, these compounds are comparatively assessed in concentration-response assays using clonal cells (
aThe equilibrium dissociation constant (pKb) of 5-HT2AR antagonists is determined in the presence of 1 μM 5-HT.
bData are presented as the pKb ± SD or pEC50 ± SD for the number of independent experiments indicated in parenthesis, see FIGS. 11A-11G for data visualization.
cNot calculable.
In
This leads to the question if the apparent greater effect of 0.3 mg*kg−1 PIMA in the DOI-assay might result from behaviorally disruptive effects on locomotor activity. Thus, each compound is administered alone at 3 mg*kg−1, and it is found that PIMA, but not (2S,4R)-2k or (2R,4R)-3h, elicits locomotor suppression (
Some 2-aminotetralins substituted at the C(5)- or C(8)-position have high affinity at 5-HT1A and 5-HT7Rs (Perry, et al., 2020), while others target D2-like receptors (Seiler & Markstein, 1984). Furthermore, affinity at 5-HT2B and α1B-adrenergic receptors may predict ligand promiscuity (Peters, et al., 2012), and high affinity antagonism of central α1A/1B-adrenergic receptors is associated with adverse events such as orthostatic hypotension, dizziness, and sedation (Andersson & Gratzke, 2007). Similarly, antagonism of central H1Rs is linked to sedation in humans (Nicholson, et al., 1991; Stahl, 2008; Valk & Simons, 2009). The lead 4-PATs from this study, (2S,4R)-2k and (2R,4R)-3h, display high selectivity to bind 5-HT2A/5-HT2CRs over 5-HT1A, 5HT2B, 5HT7, D2, D3, α1A- and α1B-adrenergic receptors (>100-fold), whereas selectivity over H1Rs is moderate for (2S,4R)-2k (˜38-fold).
To understand how aryl substituted 4-PATs and PIMA bind to 5-HT2ARs, molecular modeling studies are performed using a model of the 5-HT2AR (
Site-directed mutagenesis is used to validate the proposed ligand-receptor interactions. Indeed, the mutagenesis studies reveal that (2S,4R)-2k and (2R,4R)-3h have nil affinity (pKb) at G238S5.42 5-HT2ARs, whereas the affinity of (2S,4R)-2a, which lacks aryl ring D, is less affected. Confounding is that 5-HT2BRs also present G5.42, yet aryl substituted 4-PATs do not bind with high affinity to 5-HT2BRs. The molecular modeling results with the 5-HT2AR bound to inverse agonists PIMA, (2S,4R)-2k and (2R,4R)-3h reveal similar binding modes for each ligand (
Interestingly, aryl substituted 4-PATs in the (2S,4R)-configuration have high affinity for H1Rs (Table 1), despite the presence of T1945.42, which possesses a bulkier side chain than serine. The molecular modeling results suggest that W1584.56, a residue unique to H1Rs, might form stereospecific aromatic interactions with 4-PATs to impart high affinity (
Overall, PIMA, (2S,4R)-2k, and (2R,4R)-3h stabilize an inactive-like conformation of the 5-HT2AR, typified by an ionic lock between R1733.50 and E3186.30 within the E/DRY domain (
The models also indicate that the isobutoxybenzyl moiety of PIMA and aryl ring D of (2S,4R)-2k and (2R,4R)-3h occupy a side cavity between TM4 and TM5, unimpeded by the small side chain of G2385.42, a residue unique to 5-HT2-type receptors among aminergic GPCRs. Furthermore, in all models, F2345.38 assumes a rotamer conformation oriented away from G2385.42, which is suggested to extend the side cavity (Kimura, et al., 2019). Several amphipathic and hydrophobic side chains in this region of the binding pocket (I2104.60, V2355.39, G2385.42, and S2425.46) are close enough to the isobutoxybenzyl of PIMA and aryl ring D of (2S,4R)-2k and (2R,4R)-3h to facilitate interactions (Table 3), thus providing a potential structural basis for the observed selectivity of these ligands to bind 5-HT2ARs. In Table 3, conserved residues are highlighted in parenthesis; results for point-mutated 5-HT2AR residues in bold (with quotation marks) are reported herein.
aWithin 4 Å of only (2S,4R)-2k
bOnly within 4 Å of PIMA
cOnly within 4 Å of (2R,4R)-3h
To validate the molecular modeling results, residues are point-mutated in and around the 5-HT2AR side-extended cavity (Kimura, et al., 2019) and quantified the antagonist affinity (pKb) of (2S,4R)-2k and (2R,4R)-3h, as well as (2S,4R)-2a (which lacks 5-HT2R subtype selectivity) at 5-HT2AR variants to understand how stereochemistry and aryl ring D impact ligand-receptor interactions. Notably, like PIMA, (2S,4R)-2k, and (2R,4R)-3h, key analog (2S,4R)-2a demonstrate inverse agonist activity at C322K6.34 5-HT2ARs (
A G238S5.42 5-HT2AR is generated to test the hypothesis that the large side chain of serine precludes ligand access to the side extended cavity, as suggested by the molecular modeling results (
The experiments are extended by asking if the attenuated affinity of (2S,4R)-2a, (2S,4R)-2k, and (2R,4R)-3h at G238S5.42 5-HT2ARs translates to aminergic GPCRs natively presenting S5.42. Table 4 shows that (2S,4R)-2k and (2R,4R)-3h have >1,000-fold selectivity for 5-HT2ARs over 5-HT1A, 5-HT7, D2L, α1A- and α1B-adrenergic GPCRs. It is noted that (2S,4R)-2k has 270-fold selectivity over D3Rs, whereas (2R,4R)-3h has >1,000-fold selectivity. In contrast, (2S,4R)-2a exhibits moderate-to-high affinity for 5-HT7, D2L, D3, and α1A-adrenergic receptors.
aData are presented as the mean pKi and range for n independent experiments, shown in superscript.
Alignment of 5-HT2A, 5-HT2B and 5-HT2CR crystal structures (
Further inspection of the 5-HT2-type receptor crystal structures reveals that one helical turn above F5.38 in 5-HT2A and 5-HT2CRs exists a non-conserved residue in 5-HT2BRs (D2315.35 D2115.35, and F214535, respectively). The root-mean-square deviation (RMSD) of the F5.38 side chain in WT 5-HT2A and 5-HT2BRs is tracked in silico and found that F5.38 exhibited large transient variations in RMSD in WT 5-HT2ARs. Interestingly, the RMSD of F5.38 in D231F5.35 5-HT2ARs recapitulates the restricted pattern observed in silico for WT 5-HT2BRs, indicating that D2315.35 may facilitate flexibility in the side chain of F5.38 (
It is therefore hypothesized that D2315.35 may modulate the side chain rotamer of F2345.38 in 5-HT2ARs to mediate subtype selective binding. To test this hypothesis, a D231F5.35 5-HT2AR is generated, however, D231F5.35 5-HT2ARs are insufficiently responsive to 5-HT for competitive antagonism studies (
Residues in TM4 and TM5 lining the side-extended cavity of 5-HT2ARs and in proximity to PIMA, (2S,4R)-2k, and (2R,4R)-3h (Table 3) are then investigated. Among these are the side chains of I2104.60, V2355.39, and S2425.46. Importantly, the side chains of I2104.60 and V2355.39 are conserved in 5-HT2CRs, while S2425.46 is unique to 5-HT2ARs. It is hypothesized that selectivity to bind 5-HT2A and 5-HT2CRs over 5-HT2BRs may involve interactions with the side chains of I4.60, V5.39, or the 5-HT2AR-specific residue S2425.46. In fact, a significant increase in the affinity of PIMA and (2S,4R)-2k at V235M5.39 5-HT2ARs is observed, with no change in affinity for any antagonist at I210V4.60 or S242A5.46 5-HT2ARs (Table 2,
To explain the observed selectivity of PIMA, (2S,4R)-2k, and (2R,4R)-3h to bind 5-HT2ARs over 5-HT2BRs, the focus is made on non-conserved residues which line the 5-HT2AR side extended cavity. The results indicate that the affinity and selectivity of inverse agonists to bind 5-HT2ARs does not individually involve the side chains of I2104.60, F2134.63, V2355.39, or S2425.46, since point mutation to the equivalent residues in 5-HT2BRs does not attenuate their affinity. Importantly, the finding that selective inverse agonists have similar affinity at F213K4.63 and WT 5-HT2ARs suggests a refinement to the current understanding of subtype selective binding at 5-HT2ARs (Kimura, et al., 2019). An attempt is made to identify a 5-HT2AR residue other than F2134.63 which may impact the side chain rotamer of F5.38, and the computational results directed this work to D231.35. However, D231F5.35 5-HT2ARs lack sufficient Gaq functionality and cannot bind various radioligands, which, to the best of knowledge of the literature, is reported here for the first time. Each ligand is 9-10 Å from D2315.35 in the molecular models herein, thus, direct interactions are unlikely. Instead, it is speculated that D231F5.35 may hinder proper protein folding and trafficking of the receptor to the membrane.
In an embodiment, the 4′-NMe2—C6H4 substituent introduced on ring C (2k, 3k) in the stereoisomers is utilized to determine molecular determinants of selective binding to 5-HT2A and 5-HT2CRs including substituents of a length about that of NMe2 on ring D (
Like the observations with 5-HT2BRs, the selectivity of some aryl substituted 4-PATs to bind 5-HT2A over H1Rs cannot be explained by the results with G238S5.42 5-HT2ARs. For example, H1Rs present T1945.42, yet aryl substituted 4-PATs in the (2S,4R)-configuration, but not the (2R,4R)-configuration, bind to H1Rs with high affinity. Molecular dynamics studies revealing the structural basis of 4-PAT stereoselectivity at histamine H1Rs are shown in
The research disclosed here details the discovery of a novel series of selective 5-HT2A/5-HT2CR inverse agonists (i.e., aryl substituted 4-PATs), which are behaviorally active at comparable doses to PIMA, an FDA-approved drug. Efforts to detail the mechanism of their selectivity over 5-HT2BRs are challenging, though evidence is provided herein that F2134.63 is not a molecular determinant of subtype-selective inverse agonist binding at 5-HT2ARs. Also G228S5.42 5-HT2ARs are identified as a point mutated 5-HT2AR which may predict ligand selectivity over several aminergic GPCRs. In most cases, the mutagenesis studies reveal only minor changes in antagonist affinity, i.e., Δ(pKb)≤0.5. Therefore, it is surmised that ensembles of non-conserved residues, rather than individual residues, mediate subtype-selective antagonist or inverse agonist binding at 5-HT2ARs. In line with this, herein is reported a non-conserved, stereochemically sensitive molecular ensemble for H1R recognition. Taken together, the results warrant further investigation into the ability of aryl substituted 4-PAT diastereomers to unlock the molecular determinants of subtype-selective binding at 5-HT2ARs, and preclinical characterization of analogs like (2S,4R)-2k in animal models of psychosis.
These data show that antagonist activity at G238S5.42 5-HT2ARs is predictive of ligand selectivity over several aminergic GPCRs, and non-conserved residues in transmembrane domains 4 and 5 of the H1R mediate stereoselective ligand binding. The clinical significance is relevant because understanding the molecular determinants of selective binding over H1Rs may yield non-sedating antipsychotic medications, and aryl substituted 4-PATs demonstrate pharmacology like PIMA yet do not alter locomotor activity in mice.
Examples of psychiatric therapeutic indications
An example of psychiatric therapeutic indications for 5-HT2A and 5-HT2C inverse agonists or antagonists is schizophrenia. As documented in Casey, et al., 2022 and references therein, reduction of serotonin 5-HT2A receptor signaling via receptor antagonism and/or inverse agonism reported for the compounds in Casey, et al., 2022 is associated with clinical drug efficacy to treat schizophrenia and other conditions involving psychosis characterized by hallucinations and delusions (references in Casey, et al., 2022: Hacksell, et al., 2014; Meltzer, 1999 and Weiner, et al., 2001).
In addition, reduction of serotonin 5-HT2C receptor signaling via receptor antagonism and/or inverse agonism reported for the novel compounds in Casey et al., 2022 also is associated clinically with drug efficacy to treat schizophrenia (references in Casey, et al., 2022: Chagraoui, et al., 2016).
Regarding psychoses, as documented in Casey, et al., 2022, inverse agonist activity at the serotonin 5-HT2C receptor reported for the novel compounds in Casey, et al., 2022 is associated clinically with treatment of psychoses (reference in Casey, et al., 2022: Chagraoui, et al., 2016). For example, the selective 5-HT2A/5-HT2C receptor inverse agonist PIMA (Zuplazid®) is FDA approved to treat hallucinations and delusions associated with Parkinson's disease psychosis (reference in Casey, et al., 2022: Cummings, et al., 2014).
In addition, the novel compounds reported in Casey, et al., 2022 likely will be effective to treat psychosis and dementia associated with Alzheimer's disease and other disorders characterized dementia. For example, the selective 5-HT2A/5-HT2C receptor inverse agonist PIMA is undergoing clinical assessment for efficacy to treat psychosis in Alzheimer's disease (Caraci, et al., 2020; Ballard, et al., 2020; Ballard, et al., 2018; Tariot, et al., 2021).
As documented in Casey, et al., 2022, the novel compounds reported demonstrate inverse agonism of 5-HT2C receptors which is thought to be pharmacotherapeutic for major depression (reference in Casey, et al., 2022; Demireva et al., 2018).
Regarding anxiety, as documented in Casey, et al., 2022, the novel compounds reported demonstrate inverse agonism of 5-HT2C receptors which is thought to be pharmacotherapeutic for generalized anxiety (reference in Casey, et al., 2022: Demireva, et al., 2018). Note that anxiety behavior is associated with a wide variety of neuropsychiatric (including substance use disorder), neurodegenerative (AD, PD), and neurodevelopmental (autism) disorders, all of which may be therapeutic indications for the novel compounds reported in Casey, et al., 2022 and analogs thereof (Fluyau, et al., 2022; Simonoff, et al., 2008; El Haj, et al., 2020; Wen, et al., 2016).
Examples of non-psychiatric therapeutic indications:
Thrombosis or the prevention thereof (which can be referred to as ‘blood thinners’) is most often treated with drugs that affect the blood clotting cascade such as heparin (a natural product used intravenously) and warfarin (an orally-active natural product anticoagulant) and the newer synthetic and much more expensive ‘blood thinning’ drugs apixaban (Eliquis), dabigatran (Pradaxa), edoxaban (Savaysa), and rivaroxaban (Xarelto). They are used to prevent blood clots associated with deep vein thrombosis, pulmonary emboli, atrial fibrillation and other heart and cardiovascular system disorders where pathophysiology involves blood clots. Activation of 5HT2A receptors on platelets activates membrane bound phospholipase C to produce the second messengers inositol phosphates and diacylgylcerol which cause the platelets to become ‘sticky’—the platelets adhere to each other and form a clot.
Although thrombosis pathophysiology and potential pharmacotherapy involving 5HT2A receptor antagonism is known (e.g., Lin, et al., 2014), most 5HT2A antagonists also enter the brain to produce psychopharmacological effects that are not necessary and may be counterproductive for cardiovascular disorders. In Lin, the authors note that the 5HT2A antagonists they mention are antidepressants (not commonly used as such because there are much better ones such as the SSRIs that work differently) thus they suggest the therapeutic indication should be depressed patients who have thrombosis disorders. A 4PAT-type 5HT2A antagonist or inverse agonist is claimed to prevent this thrombosis or reverse it in patients where psychotropic actions are not needed.
5-HT2B inverse agonists/antagonists can be utilized for treatment of atherosclerosis. Activation of 5HT2B causes atherosclerosis of cardiac valves. In Janssen, et al., 2015, for example, 5-HT2B receptor antagonists have been investigated to inhibit fibrosis and protect from RV (right ventricular) heart failure. The present technology can provide selectivity between the CNS and the periphery with ligand specificity.
D. Hypertension treatment by 5-HT2A antagonists
It is reported in Casey, et al., 2022 that the new chemical entities are potent 5-HT2A antagonists. It is known for several decades that antagonism of 5-HT2A receptor leads to beneficial cardiovascular effects including reduced platelet aggregation and vasodilation. For example, the 5-HT2A antagonist ketanserin is approved to treat hypertension (Hedner & Persson, 1988; Bellos, et al., 2020; Nagatomo, et al., 2004).
E. Migraine treatment by 5-HT2B antagonists.
It is reported in Casey, et al., 2022 that the new chemical entities are potent 5-HT2B antagonists and inverse agonists. Exploiting their expression in the CNS vascular system, 5-HT2B antagonists are proposed to treat migraine headache (Padhariya, et al., 2017).
F. Obesity treatment by 5-HT2B antagonists
It is reported in Casey, et al., 2022 that the new chemical entities are potent 5-HT2B antagonists and inverse agonists. Exploiting their expression in the gastrointestinal system, 5-HT2B antagonists are proposed to obesity (Padhariya, et al., 2017).
G. Irritable bowel syndrome (IBS) treatment by 5-HT2B antagonists
It is reported in Casey, et al., 2022 that the new chemical entities are potent 5-HT2B antagonists and inverse agonists. Exploiting their expression in the gastrointestinal system, 5-HT2B antagonists are proposed to treat irritable bowel syndrome (IBS) (Padhariya, et al., 2017).
H. Safety of the novel chemical entities reported in Casey et al., 2022.
The novel chemical entities reported in Casey, et al., 2022 and analogs thereof are unlikely to demonstrate untoward cardiovascular effects because it is documented in the paper that the compounds are inverse agonists at the serotonin 5-HT2B receptor. Agonists at 5-HT2B receptors have potential to cause valvular heart disease and other untoward cardiovascular effects (references in Casey, et al., 2022: Ayme-Dietrich, et al., 2017; Rothman, et al., 2000).
Also as reported in Casey et al., 2022, the novel compounds did not demonstrate inhibition of locomotor activity (bradykinesia) indicating it is unlikely the compounds and analogs thereof will cause sedation or neurological disorders (Berardelli, et al., 2001).
All commercially available regents and solvents were purchased and used without purification, unless otherwise specified. Flash column chromatography was performed with the use of Agela Technologies 230-400 mesh silica gel. Analytical thin-layer chromatography (TLC) was carried out on Agela Technologies silica gel 60F254 plates. Final compounds were converted from free base to HCl salt either before or after NMR analysis, as noted. All spectra were recorded by a Varian 500 MHz, 400 MHz NMR in CDCl3 or CD3OD as noted and are expressed as chemical shift (6) values in parts per million (ppm). Coupling constants (J) are presented in Hertz. Abbreviations used in the reporting of NMR spectra include s=singlet, bs=broad singlet, d=doublet, t=triplet, q=quartet, dd=doublet of doublets, qd=quartet of doublets, dt=doublet of triplets, m=multiplets. High resolution mass spectrometry (HRMS) was performed with an LTQ Orbitrap XL (Thermo Fisher Scientific) instrument using electron spray ionization (ESI). Sample data were acquired with MS1 scan (m/z 50-500) at 30,000 resolution using the Orbitrap as the MS detector. HPLC separation of both enantiomers was determined by UV Trace 220/254 nm on a Shimadzu™ instrument equipped with a semi-preparative (s-prep)-RegisCell™ (5 μm, 25 cm×10 mm i.d.) chiral (polysaccharide-based) column.
To efficiently produce novel 4-PAT derivatives, designing and implementing an improved synthetic route than reported earlier was accomplished (Sakhuja, et al., 2015). Previously, key compounds like 2a were synthesized using a four-step procedure from commercially available 3-bromostyrene and trifluoroacetyl phenylacetyl anhydride (Canal, et al., 2014; Sakhuja, et al., 2015). The overall yield for this pathway, however, was low, and reproducibility in large scale was unreliable. The modified synthetic pathway utilized here involved Friedel-Crafts cycli-acylalkylation of the commercially available 3-bromostyrene and phenylacetyl chloride to give the intermediate tetralone 6a (Scheme 1). The tetralone was subjected to reductive amination to afford a separable mixture of 3′-Br-4-PAT diastereomers with improved overall yields (2a 32% yield, 3a 25% yield). The reductive amination step was further optimized to obtain racemic cis-4-PATs (e.g., [2S,4S], [2R,4R]) as the major isomer. The modified protocol involved reduction of a preformed enamine with sodium borohydride (Scheme 2).
Diastereomeric 3′-Br-4-PATs 2a and 3a (Scheme 1,
The absolute stereochemistry of optically pure cis-4-PAT isomers (separated by chiral HPLC) was determined using X-ray crystallography on crystals of (2R,4R)-3b and (2S,4S)-3b′ (
4-(3-bromophenyl)-3,4-dihydronaphthalen-2(1H)-one 6a: To an oven dried 100 mL round bottom flask with stir bar was added anhydrous AlCl3 (800 mg, 6.0 mmol) and CH2Cl2 (20 ml) under nitrogen atmosphere. The resulting suspension was transferred to an ice bath and cooled for 10 minutes. To the reaction flask was slowly added phenylacetyl chloride 5 (661 μL, 5.0 mmol). The resulting mixture was stirred for 10 minutes under nitrogen atmosphere in an ice bath. To the reaction mixture was then added 3-bromostyrene 4 (664 μL, 5.1 mmol) and the resulting solution was stirred for 30 minutes on ice bath. Water (50 mL) was added to the flask and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (20 mL×3). The combined organic layer was washed with saturated aq NaHCO3 (30 mL×2) followed by brine (30 mL) and dried over Na2SO4. After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography (97:3 hexanes: ethyl acetate) to afford 4-(3-bromophenyl)-3,4-dihydronaphthalen-2(1H)-one 6a as colorless solid with 60% yield.
Trans-4-(3-bromophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 2a and cis-4-(3-bromophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 3a: To an oven dried 100 mL round bottom flask with stir bar was added ketone 6 (1.12 g, 3.72 mmol), dimethylamine hydrochloride (3.0 g, 37.2 mmol), Tetrahydrofuran (28 mL) and methanol (37 mL). The resulting mixture was stirred at room temperature under nitrogen atmosphere until all solid dissolved. To the reaction mixture was added sodium cyanoborohydride (1.18 g, 18.8 mmol) and the flask was transferred to an oil bath. The resulting reaction mixture was stirred for 16 h under nitrogen atmosphere at 50° C. The solvent was evaporated and to the residue was added saturated aq NaHCO3 (50 mL) and ethyl acetate (25 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (25 mL×4). The combined organic layer was washed with brine (30 mL) and dried over Na2SO4. After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) to afford cis-3a as colorless oil with 42% yield and trans-2a as colorless oil with 54% isolated yield.
General procedure to synthesize amine 3a, 3b and 3b′: To an oven dried 10 mL round bottom flask with stir bar was added ketone 6 (1.0 mmol), 10M solution of dimethylamine in ethanol (130 μL, 1.3 mmol), 4 Å molecular sieves and toluene (1.0 mL). To the resulting mixture was added glacial acetic acid (11 μL, 0.2 mmol). The reaction mixture was stirred at room temperature for 24 h under nitrogen atmosphere (Scheme 4). In an another over dried flask with stir bar was added sodium borohydride (95 mg, 2.5 mmol) and methanol (3 mL). The flask was cooled to −78° C. under nitrogen atmosphere. To the cooled mixture was added previously formed imine reaction mixture through a filter syringe. Resulting reaction mixture was stirred at −78° C. for 3 h, then the reaction flask was slowly warmed to rt. The reaction mixture was stirred overnight at rt under nitrogen atmosphere. The solvent was evaporated and to the residue was added saturated aq NaHCO3 (15 mL) and ethyl acetate (10 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (10 mL×4). The combined organic layer was washed with brine (15 mL) and dried over Na2SO4. After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography to afford cis-amines as colorless oil.
The racemic mixtures of cis-analogs were separated by semi-preparative chiral HPLC Regiscell™ column using conditions and solvents specific to each analog to elute the cis-(2S,4S) and -(2R,4R) enantiomers at retention time t1 at t2, respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-cis-3′-CI-4-PAT, 3b and (2S,4S)-cis-3′-F-4-PAT, 3b′ analogs (
Cis-4-(3-bromophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3a: amine 3a was synthesized from ketone 6a following the general procedure as described above. The crude reaction mixture (cis:trans 10:1) was purified by silica gel column chromatography (4:2:0.1 hexanes: ethyl acetate: triethylamine) to afford racemic cis-4-(3-bromophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 3a as colorless oil with 45% isolated yield. 1H-NMR (500 MHz; CDCl3): δ 7.38 (d, J=7.9 Hz, 1H), 7.33 (s, 1H), 7.19 (t, J=7.8 Hz, 1H), 7.15-7.10 (m, 3H), 7.03 (t, J=7.3 Hz, 1H), 6.73 (d, J=7.8 Hz, 1H), 4.07 (dd, J=12.2, 5.2 Hz, 1H), 3.04-3.00 (m, 1H), 2.95-2.90 (m, 1H), 2.80 (tdd, J=11.5, 4.7, 2.3 Hz, 1H), 2.37 (s, 6H), 2.33 (ddd, J=9.9, 5.2, 2.5 Hz, 1H), 1.68 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 149.14, 138.64, 136.49, 131.86, 130.29, 129.68, 129.63, 129.28, 127.57, 126.46, 126.17, 122.74, 60.56, 47.10, 41.60, 36.93, 33.08.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 13.01 (s, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.31 (s, 1H), 7.23-7.17 (m, 3H), 7.13-7.10 (m, 2H), 6.77 (d, J=7.8 Hz, 1H), 4.19 (dd, J=12.1, 5.2 Hz, 1H), 3.65-3.59 (m, 1H), 3.43-3.39 (m, 1H), 3.31-3.26 (m, J=13.6 Hz, 1H), 2.85-2.84 (m, 6H), 2.69 (dt, J=12.2, 2.6 Hz, 1H), 2.00 (q, J=12.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 146.64, 137.05, 132.01, 131.67, 130.58, 130.47, 129.52, 129.38, 127.57, 127.42, 127.33, 122.95, 61.76, 45.82, 39.76, 39.44, 34.24, 30.19. Calculated C18H21BrN for [M+H]+: 330.0858. Found: 330.0859. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=1.5 mL/min; t1=14.41 min, t2=19.71 min.
4-(3-chlorophenyl)-3,4-dihydronaphthalen-2(1H)-one 6b: Ketone 6b was synthesized from 3-chlorostyrene 4b and phenylacetyl chloride 5 in presence of AlCl3 following the procedure as described above for 6a. The crude reaction mixture was purified by silica gel column chromatography (95:5 hexanes: ethyl acetate) to afford 4-(3-chlorophenyl)-3,4-dihydronaphthalen-2(1)-one 6b as colorless oil with 60% isolated yield. 1H and 13C NMRs are in agreement with previously published data (Vincek & Booth, 2009).
Cis-4-(3-chlorophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3b: amine 3b was synthesized from ketone 6b following the general procedure as described above. The crude reaction mixture (cis:trans 7:1) was purified by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) to afford racemic cis-4-(3-chlorophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 6b as colorless oil with 40% isolated yield. 1H-NMR (500 MHz; CDCl3): δ 7.25-7.21 (m, 2H), 7.17-7.12 (m, 3H), 7.07 (d, J=7.1 Hz, 1H), 7.03 (t, J=7.2 Hz, 1H), 6.73 (d, J=7.8 Hz, 1H), 4.08 (dd, J=12.2, 5.1 Hz, 1H), 3.03 (ddd, J=15.6, 4.5, 1.9 Hz, 1H), 2.93 (dd, J=12.4, 11.4 Hz, 1H), 2.81 (tdd, J=11.5, 4.6, 2.3 Hz, 1H), 2.38 (s, 6H), 2.35-2.31 (m, J=2.6 Hz, 1H), 1.69 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 148.80, 138.62, 136.43, 134.42, 129.93, 129.60, 129.24, 128.92, 127.07, 126.72, 126.43, 126.13, 60.54, 47.08, 41.55, 36.87, 33.02.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.88 (s, 1H), 7.29-7.27 (m, 2H), 7.21-7.15 (m, 3H), 7.12-7.07 (m, 2H), 6.77 (d, J=7.7 Hz, 1H), 4.21 (dd, J=12.0, 5.1 Hz, 1H), 3.64 (td, J=11.0, 1.9 Hz, 1H), 3.42-3.39 (m, 1H), 3.31-3.26 (m, 1H), 2.85 (s, 6H), 2.70-2.67 (m, 1H), 2.00 (q, J=12.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 146.36, 137.06, 134.67, 132.03, 130.24, 129.48, 129.33, 128.76, 127.48, 127.34, 127.26, 127.08, 61.72, 45.79, 39.75, 39.43, 34.16, 30.19. Calculated C18H21ClN for [M+H]+: 286.1363. Found: 286.1359. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH:nPrOH (80:10:5:5) 0.1% TEA (modifier) 0.1% TFA (modifier); flow rate=1.5 mL/min; t1=11.0 min, t2=13.0 min.
4-(3-fluorophenyl)-3,4-dihydronaphthalen-2(1H)-one 6b′: Ketone 6b′ was synthesized from 3-fluorostyrene 4b′ and phenylacetyl chloride 5 in presence of AlCl3 following the procedure as described above for 6a. The crude reaction mixture was purified by silica gel column chromatography (95:5 hexanes: ethyl acetate) to afford 4-(3-fluorophenyl)-3,4-dihydronaphthalen-2(1)-one 6b′ as colorless oil with 50% isolated yield. 1H and 13C NMRs are in agreement with previously published data (Vincek & Booth, 2009).
Cis-4-(3-fluorophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3b′: amine 3b′ was synthesized from ketone 6b′ following the general procedure as described above. The crude reaction mixture (cis:trans 10:1) was purified by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) to afford racemic cis-4-(3-fluorophenyl)-N, N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 6b′ as colorless oil with 35% isolated yield. 1H-NMR (500 MHz; CDCl3): 7.26-7.23 (m, 1H), 7.15-7.12 (m, 2H), 7.08-7.04 (m, 1H), 6.95-6.91 (m, 2H), 6.80 (d, J=9.3 Hz, 1H), 6.71 (d, J=7.7 Hz, 1H), 4.09 (dd, J=12.2, 5.2 Hz, 1H), 3.06-3.02 (m, 1H), 2.94 (dd, J=12.4, 11.2 Hz, 1H), 2.79-2.74 (m, 1H), 2.39 (s, 6H), 2.34-2.31 (m, 1H), 1.69 (q, J=12.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 164.18, 166.66, 148.95, 148.88, 138.71, 133.94, 130.47, 130.39, 129.35, 129.04, 126.71, 126.95, 124.07, 114.75, 114.53, 113.42, 113.21, 60.75, 47.92, 41.53, 36.53, 33.19.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.70 (brs, 1H), 7.31-7.26 (m, 1H), 7.18-7.15 (m, 2H), 7.10-7.06 (m, 1H), 6.98-6.94 (m, 2H), 6.84 (d, J=9.4 Hz, 1H), 6.75 (d, J=7.7 Hz, 1H), 4.22 (br d, J=9.1 Hz, 1H), 3.68-3.60 (m, 1H), 3.38 (d, J=14.0 Hz, 1H), 3.29-3.24 (m, 1H), 2.84 (s, 6H), 2.67 (d, J=9.5 Hz, 1H), 2.03-1.96 (m, 1H). 13C-NMR (100 MHz, CDCl3): δ 164.39, 161.93, 146.82, 146.75, 137.16, 131.95, 130.58, 130.50, 129.57, 129.42, 127.41, 127.35, 124.67, 115.77, 115.55, 114.44, 114.23, 61.98, 45.99, 39.81, 39.47, 34.33, 30.37. Calculated C18H21FN for [M+H]+: 270.1659. Found: 270.1655. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH:nPrOH (80:10:5:5) 0.1% TEA (modifier) flow rate=1.5 mL/min; t1=11.77 min, t2=13.14 min.
Racemic 2f-k were prepared. To an oven dried 25 mL round bottom flask with stir bar was added racemic trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), Aryl boronic acid (0.3 mmol) and toluene (1.5 mL). The resulting mixture was degassed by sparging with N2 gas for 45 minutes. To the reaction mixture was added potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol). The flask was fitted with a reflux condenser and the reaction mixture was heated to 110° C. for 4 h under nitrogen atmosphere (Scheme 5). The reaction was quenched with 1N NaOH aq (3 mL) and ethyl acetate (4 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (5 mL×4). The combined organic layer was washed with brine (10 mL) and dried over Na2SO4. After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) to afford racemic 2f-k.
The racemic mixtures of trans-analogs were separated by semi-preparative chiral HPLC Regiscell column using conditions and solvents specific to each analog to elute the trans-(2R,4S) and -(2S,4R) enantiomers at retention time t1 at t2, respectively, with absolute stereochemistry assigned according to retention time of the previously published trans-3′-CI-4-PAT analog (Sakhuja, et al., 2015). Both enantiomers were converted to HCl salts for use in pharmacological assays by adding 2M HCl solution in ether to the solution of free amine in ether.
Trans-4-([1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 2f: trans-amine 2f was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), phenylboronic acid (37 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2f as colorless oil with 82% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.52 (d, J=7.3 Hz, 2H), 7.41 (dd, J=7.5, 7.5 Hz, 3H), 7.32 (dd, J=7.7, 7.7 Hz, 2H), 7.27 (s, 1H), 7.20-7.16 (m, 2H), 7.11 (t, J=6.9 Hz, 1H), 6.98 (dd, J=14.5, 7.6 Hz, 2H), 4.44 (t, J=5.1 Hz, 1H), 3.07 (dd, J=16.2, 4.7 Hz, 1H), 2.90 (dd, J=16.1, 9.5 Hz, 1H), 2.75 (tt, J=9.1, 4.5 Hz, 1H), 2.32 (s, 6H), 2.23-2.15 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 147.27, 141.37, 141.25, 137.79, 136.14, 130.15, 129.49, 128.83, 128.72, 127.81, 127.62, 127.34, 127.32, 126.57, 126.29, 125.12, 56.64, 44.20, 41.83, 34.83, 32.25. 1H and 13C NMRs are in agreement with previously published data (Sakhuja, et al., 2015).
Trans-4-(2′-fluoro-[1,1′-biphenyl]-3-yl)-N, N-dimethyl-1,2,3,4-tetrahydronaphtha-len-2-amine, 2g: trans-amine 2g was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 2-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2g as colorless oil with 98% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.39-7.36 (m, 2H), 7.32 (t, J=7.7 Hz, 1H), 7.29-7.24 (m, 2H), 7.18-7.15 (m, 3H), 7.13-7.09 (m, 2H), 6.99 (d, J=7.5 Hz, 2H), 4.43 (t, J=5.2 Hz, 1H), 3.05 (dd, J=16.2, 4.8 Hz, 1H), 2.88 (dd, J=16.2, 9.5 Hz, 1H), 2.71 (tt, J=9.1, 4.5 Hz, 1H), 2.28 (s, 6H), 2.21-2.13 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 161.10, 158.63, 147.08, 137.91, 136.45, 135.75, 130.91, 130.87, 130.15, 129.54, 129.51, 129.48, 129.35, 129.21, 129.01, 128.93, 128.35, 128.19, 126.88, 126.85, 126.48, 126.18, 124.40, 124.37, 116.29, 116.06, 56.44, 44.22, 41.97, 35.04, 32.40.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.65 (br s, 1H), 7.39-7.30 (m, 4H), 7.26-7.25 (m, 2H), 7.22-7.18 (m, 3H), 7.15-7.10 (m, 1H), 7.07 (d, J=7.5 Hz, 1H), 6.88 (d, J=6.9 Hz, 1H), 4.59 (br s, 1H), 3.53 (br d, J=13.7 Hz, 1H), 3.42 (br s, 1H), 3.29-3.24 (m, 1H), 2.73 (s, 6H), 2.48 (br s, 2H). 13C-NMR (100 MHz, CDCl3): δ 160.90, 158.44, 144.62, 136.15, 135.39, 132.76, 130.73, 130.70, 130.14, 129.47, 129.30, 129.22, 128.97, 128.94, 128.88, 128.66, 128.53, 127.69, 127.57, 127.55, 127.45, 127.25, 124.55, 124.51, 116.23, 116.00, 58.75, 43.61, 40.99, 38.93, 31.57, 30.73. Calculated C24H25FN for [M+H]+: 346.1971. Found: 346.1969. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier) flow rate=3.0 mL/min; t, =8.0 min, t2=17.65 min.
Trans-4-(3′-fluoro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphtha-len-2-amine, 2h: trans-amine 2h was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 3-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2h as colorless oil with 98% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.39-7.37 (m, 1H), 7.35-7.29 (m, 3H), 7.27 (s, 1H), 7.23-7.16 (m, 3H), 7.12-7.09 (m, 1H), 7.03-6.96 (m, 3H), 4.43 (t, J=5.3 Hz, 1H), 3.04 (dd, J=16.3, 4.8 Hz, 1H), 2.88 (dd, J=16.2, 9.3 Hz, 1H), 2.70-2.64 (m, 1H), 2.28 (s, 6H), 2.17 (t, J=6.1 Hz, 2H). 13C-NMR (100 MHz, CDCl3): δ 164.46, 162.01, 147.75, 143.75, 143.67, 139.95, 139.93, 137.92, 136.54, 130.28, 130.19, 130.06, 129.52, 128.79, 128.45, 127.55, 126.51, 126.18, 124.97, 122.91, 122.89, 114.25, 114.16, 114.03, 113.95, 56.45, 44.23, 42.06, 35.28, 32.39. HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.75 (s, 1H), 7.43 (d, J=7.6 Hz, 1H), 7.43-7.34 (m, 2H), 7.29-7.26 (m, 3H), 7.23-7.17 (m, 3H), 7.06-7.02 (m, 2H), 6.89 (d, J=7.6 Hz, 1H), 4.60 (br s, 1H), 3.53 (dd, J=15.5, 4.2 Hz, 1H), 3.41-3.40 (m, 1H), 3.24 (dd, J=14.9, 11.7 Hz, 1H), 2.72 (br s, 6H), 2.49-2.44 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 164.32, 161.88, 145.18, 143.00, 142.92, 140.35, 140.33, 135.40, 132.70, 130.37, 130.29, 130.06, 129.45, 129.24, 127.87, 127.48, 127.27, 126.97, 125.73, 122.86, 122.83, 114.36, 114.15, 114.11, 113.89, 58.63, 43.60, 40.67, 38.98, 31.53, 30.45. Calculated C24H25FN for [M+H]+: 346.1971. Found: 346.1969. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier) flow rate=3.0 mL/min; t1=8.23 min, t2=13.42 min.
Trans-4-(4′-fluoro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphtha-len-2-amine, 2i: trans-amine 2i was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 4-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2i as colorless oil with 92% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.47 (td, J=6.0, 2.7 Hz, 2H), 7.35 (d, J=7.8 Hz, 1H), 7.31 (dd, J=7.6, 7.6 Hz, 1H), 7.23 (br s, 1H), 7.20-7.16 (m, 2H), 7.12-7.07 (m, 3H), 6.97 (t, J=7.6 Hz, 2H), 4.43 (t, J=5.3 Hz, 1H), 3.06 (dd, J=16.2, 4.8 Hz, 1H), 2.89 (dd, J=16.2, 9.4 Hz, 1H), 2.74-2.69 (m, 1H), 2.31 (s, 6H), 2.20-2.17 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.76, 161.30, 147.66, 140.24, 138.02, 137.55, 137.52, 136.55, 130.09, 129.51, 128.86, 128.79, 128.73, 127.88, 127.50, 126.49, 126.17, 124.89, 115.76, 115.55, 56.47, 44.26, 42.05, 35.27, 32.38.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.76 (s, 1H), 7.46 (dd, J=8.6, 5.3 Hz, 2H), 7.40 (d, J=7.7 Hz, 1H), 7.34 (t, J=7.6 Hz, 1H), 7.29-7.20 (m, 3H), 7.14-7.09 (m, 3H), 7.06 (d, J=7.6 Hz, 1H), 6.86 (d, J=7.5 Hz, 1H), 4.60 (br s, 1H), 3.52 (dd, J=15.5, 3.1 Hz, 1H), 3.45-3.38 (m, 1H), 3.24 (dd, J=14.4, 12.0, 1H), 2.72 (br s, 6H), 2.52-2.44 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 163.84, 161.38, 145.12, 140.75, 136.89, 136.85, 135.50, 132.72, 130.17, 129.52, 129.23, 128.87, 128.79, 127.54, 127.33, 126.98, 125.72, 115.85, 115.64, 58.70, 43.70, 40.75, 39.11, 31.63, 30.49. Calculated C24H25FN for [M+H]+: 346.1971. Found: 346.1968. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier) flow rate=3.0 mL/min; t, =9.96 min, t2=16.64 min.
Trans-4-(4′-chloro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphtha-len-2-amine, 2j: trans-amine 2j was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 4-chlorophenylboronic acid (47 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2j as colorless oil with 97% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.45 (d, J=8.5 Hz, 2H), 7.37 (d, J=8.6 Hz, 3H), 7.31 (t, J=7.6 Hz, 1H), 7.24 (s, 1H), 7.26-7.20 (m, 2H), 7.10 (t, J=7.1 Hz, 1H), 6.97 (t, J=7.9 Hz, 2H), 4.42 (t, J=5.3 Hz, 1H), 3.04 (dd, J=16.3, 4.8 Hz, 1H), 2.87 (dd, J=16.2, 9.3 Hz, 1H), 2.68-2.65 (m, 1H), 2.28 (s, 6H), 2.17 (t, J=6.0 Hz, 2H).
13C-NMR (100 MHz, CDCl3): δ 147.74, 139.98, 139.86, 137.95, 136.54, 133.40, 130.08, 129.52, 128.96, 128.80, 128.54, 128.20, 127.46, 126.51, 126.18, 124.86, 56.44, 44.24, 42.06, 35.29, 32.34.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.77 (br s, 1H), 7.44-7.38 (m, 5H), 7.34 (t, J=7.6 Hz, 1H), 7.29-7.26 (m, 2H), 7.25-7.20 (m, 1H), 7.15 (s, 1H), 7.05 (d, J=7.5 Hz, 1H), 6.87 (d, J=7.5 Hz, 1H), 4.60 (br s, 1H), 3.51 (dd, J=15.4, 4.5 Hz, 1H), 3.47-3.43 (m, 1H), 3.23 (dd, J=15.0, 11.7 Hz, 1H), 2.71 (d, J=4.6 Hz, 6H), 2.52-2.43 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 145.19, 140.48, 139.19, 135.45, 133.67, 132.70, 130.15, 129.51, 129.28, 129.00, 128.49, 127.63, 127.54, 127.33, 126.93, 125.67, 58.65, 43.66, 40.57, 39.04, 31.59, 30.40. Calculated C24H25ClN for [M+H]+: 362.1676. Found: 362.1673. HPLC (s-prep): Solvent System: hexanes: iPrOH (98:2) 0.1% TEA (modifier), flow rate=2.0 mL/min; t, =12.42 min, t2=14.52 min.
Trans-4-(4′-(dimethylamino)-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetra-hydronaph-thalen-2-amine, 2k: trans-amine 2k was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 4-(dimethylamino)phenylboronic acid (50 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic trans-amine 2k as colorless oil with 90% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.43 (d, J=8.7 Hz, 2H), 7.37 (d, J=7.7 Hz, 1H), 7.28-7.23 (m, 2H), 7.18-7.14 (m, 2H), 7.09 (t, J=7.1 Hz, 1H), 6.99 (d, J=7.6 Hz, 1H), 6.88 (d, J=7.6 Hz, 1H), 6.77 (d, J=8.7 Hz, 2H), 4.40 (t, J=5.1 Hz, 1H), 3.04 (dd, J=16.2, 4.7 Hz, 1H), 2.97 (s, 6H), 2.86 (dd, J=16.2, 9.5 Hz, 1H), 2.71-2.66 (m, 1H), 2.28 (s, 6H), 2.20-2.11 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 150.07, 147.28, 141.14, 138.26, 136.51, 130.16, 129.48, 129.39, 128.53, 127.85, 126.89, 126.61, 126.36, 126.11, 124.14, 112.87, 56.51, 44.30, 42.07, 40.72, 35.06, 32.60.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.67 (s, 1H), 7.84 (d, J=8.2 Hz, 2H), 7.66 (d, J=8.1 Hz, 2H), 7.39 (dt, J=15.2, 7.6 Hz, 2H), 7.31-7.22 (m, 3H), 7.15 (s, 1H), 7.07 (d, J=7.5 Hz, 1H), 6.94 (d, J=7.3 Hz, 1H), 4.62 (br s, 1H), 3.50-3.43 (m, 2H), 3.21-3.11 (m, 7H), 2.73 (dd, J=8.0, 4.8 Hz, 6H), 2.62 (br d, J=12.0 Hz, 1H), 2.44 (td, J=11.9, 5.3 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 164.93, 145.28, 139.70, 135.38, 132.64, 130.14, 129.54, 129.36, 129.36, 129.11, 128.11, 127.57, 127.31, 127.17, 125.73, 120.66, 58.51, 46.19, 45.97, 43.54, 40.14, 39.29, 31.60, 30.00. Calculated C26H31N2 for [M+H]*: 371.2487. Found: 371.2483. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH:nPrOH (80:10:5:5) 0.1% TEA (modifier), flow rate=1.7 mL/min; t, =11.55 min, t2=12.93 min.
The cis analogues 3f-3k (Scheme 6) were synthesized from racemic cis-3′Br-4-PAT 3a and corresponding Aryl boronic acid following the general procedure as described above for trans 2f-k. The racemic mixtures of cis-analogs were separated by semi-preparative chiral HPLC Regiscell column using conditions and solvents specific to each analog to elute the cis-(2S,4S) and -(2R,4R) enantiomers at retention time t1 at t2, respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-cis-3′CI-4-PAT, 3b and (2S,4S)-cis-3′F-4-PAT, 3b′ analogs. Both enantiomers were converted to HCl salts for use in pharmacological assays by adding 2M HCl solution in ether to the solution of free amine in ether.
Cis-4-([1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3f: cis-amine 3f was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), phenylboronic acid (37 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3f as colorless oil with 85% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.58-7.56 (m, 2H), 7.48 (d, J=7.7 Hz, 1H), 7.47-7.37 (m, 4H), 7.37-7.30 (m, 1H), 7.16 (d, J=8.2 Hz, 2H), 7.12 (t, J=7.3 Hz, 1H), 7.02 (t, J=7.4 Hz, 1H), 6.82 (d, J=7.8 Hz, 1H), 4.16 (dd, J=12.2, 5.1 Hz, 1H), 3.05 (dd, J=15.6, 2.9 Hz, 1H), 2.96 (dd, J=15.6, 11.3 Hz, 1H), 2.84 (tdd, J=11.4, 4.8, 2.3 Hz, 1H), 2.42-2.38 (m, 7H), 1.79 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 147.21, 141.63, 141.29, 139.35, 136.39, 129.53, 129.40, 129.12, 128.83, 127.84, 127.77, 127.37, 127.33, 126.26, 126.07, 125.41, 60.72, 47.45, 41.60, 36.99, 33.18.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.93 (s, 1H), 7.55 (d, J=7.4 Hz, 2H), 7.52 (d, J=7.6 Hz, 1H), 7.44-7.35 (m, 4H), 7.36-7.33 (m, 1H), 7.20-7.19 (m, 2H), 7.16-7.10 (m, 2H), 6.86 (d, J=7.8 Hz, 1H), 4.28 (d, J=9.2 Hz, 1H), 3.70-3.63 (m, 1H), 3.47-3.44 (m, 1H), 3.34-3.29 (m, 1H), 2.86 (s, 6H), 2.73-2.71 (m, 1H), 2.08 (q, J=11.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 144.83, 142.00, 140.90, 137.77, 132.03, 129.52, 129.44, 128.89, 127.71, 127.60, 127.58, 127.30, 127.14, 126.15, 62.02, 46.31, 39.86, 39.67, 34.29, 30.45. Calculated C24H26N for [M+H]*: 328.2066. Found: 328.2059. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (90:5:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t, =10.17 min, t2=24.59 min.
Cis-4-(2′-fluoro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3g: cis-amine 3g was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), 2-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3g as colorless oil with 96% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.45-7.38 (m, 4H), 7.31-7.27 (m, 1H), 7.19-7.11 (m, 5H), 7.03 (t, J=7.3 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H), 4.16 (dd, J=12.2, 5.0 Hz, 1H), 3.04 (dd, J=15.5, 2.6 Hz, 1H), 2.95 (dd, J=15.5, 11.4 Hz, 1H), 2.83 (tdd, J=11.5, 4.7, 2.2 Hz, 1H), 2.42-2.38 (m, 7H), 1.78 (q, J=12.1 Hz, 1H). 13C NMR (100 MHz; CDCl3): δ 161.10, 158.60, 146.9, 139.30, 136.40, 136.10, 130.95, 130.91, 129.65, 129.62, 129.52, 129.42, 129.09, 129.00, 128.80, 128.20, 127.28, 127.25, 126.30, 126.10, 124.43, 124.39, 116.30, 116.10, 60.70, 47.40, 41.60, 37.00, 33.20.
HCl salt: 1H-NMR (500 MHz; CDCl3): δ 12.69 (s, 1H), 7.46 (d, J=7.5 Hz, 1H), 7.41 (t, J=7.1 Hz, 2H), 7.35 (s, 1H), 7.30 (dd, J=14.9, 9.0 Hz, 1H), 7.21-7.08 (m, 6H), 6.85 (d, J=7.7 Hz, 1H), 4.27 (br d, J=8.3 Hz, 1H), 3.70-3.65 (m, 1H), 3.43 (br d, J=14.3 Hz, 1H), 3.32-3.27 (m, 1H), 2.85 (d, J=11.5 Hz, 7H), 2.70 (d, J=9.8 Hz, 1H), 2.07 (q, J=11.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 160.92, 158.45, 144.49, 137.65, 136.42, 132.03, 130.84, 130.81, 129.46, 129.42, 129.39, 129.27, 129.19, 129.04, 128.04, 127.95, 127.92, 127.21, 127.08, 124.49, 124.46, 116.24, 116.02, 61.89, 46.12, 39.78, 39.48, 34.15, 30.40. Calculated C24H25FN for [M+H]*: 346.1971. Found: 346.1968. HPLC (s-prep): Solvent System: hexanes:iPrOH (94:6) 0.2% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t, =13.11 min, t2=29.15 min.
Cis-4-(3′-fluoro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3h: cis-amine 3h was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), 3-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3h as colorless oil with 97% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.46 (d, J=7.7 Hz, 1H), 7.41-7.34 (m, 4H), 7.28-7.26 (m, 1H), 7.20-7.12 (m, 3H), 7.04-7.01 (m, 2H), 6.80 (d, J=7.8 Hz, 1H), 4.17 (dd, J=12.2, 5.1 Hz, 1H), 3.06 (dd, J=15.6, 2.6 Hz, 1H), 2.97 (dd, J=15.6, 11.4 Hz, 1H), 2.88-2.83 (m, 1H), 2.41-2.39 (m, 7H), 1.78 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 164.49, 162.04, 147.45, 143.62, 143.55, 140.37, 140.35, 139.22, 136.46, 130.31, 130.22, 129.58, 129.34, 129.25, 128.45, 127.65, 126.32, 126.08, 125.35, 122.95, 122.92, 114.29, 114.24, 114.07, 114.03, 60.71, 47.45, 41.63, 37.08, 33.20.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 13.00 (s, 1H), 7.49 (d, J=7.7 Hz, 1H), 7.44-7.32 (m, J=8.0 Hz, 4H), 7.23 (s, 1H), 7.20-7.19 (m, 3H), 7.13-7.09 (m, 1H), 7.04 (td, J=8.2, 1.1 Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 4.28 (dd, J=11.8, 4.5 Hz, 1H), 3.69-3.65 (m, 1H), 3.44 (d, J=14.0 Hz, 1H), 3.34-3.29 (m, 1H), 2.85 (dd, J=4.9, 4.9 Hz, 6H), 2.74-2.72 (m, 1H), 2.08 (q, J=12.2 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 164.33, 161.88, 145.02, 143.10, 143.02, 140.52, 140.50, 137.60, 132.05, 130.34, 130.26, 129.46, 129.39, 129.32, 128.26, 127.43, 127.15, 127.04, 125.94, 122.87, 122.84, 114.32, 114.12, 114.11, 113.91, 61.80, 46.11, 39.58, 34.20, 30.23. Calculated C24H25FN for [M+H]+: 346.1971. Found: 346.1967. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (90:5:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t1=10.07 min, t2=16.80 min.
Cis-4-(4′-fluoro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3i: cis-amine 3i was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), 4-fluorophenylboronic acid (42 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3i as colorless oil with 98% isolated yield. 1H-NMR (500 MHz; CDCl3): δ 7.52 (dd, J=8.6, 5.4 Hz, 2H), 7.43-7.36 (m, 3H), 7.17-7.07 (m, 5H), 7.02 (t, J=7.4 Hz, 1H), 6.81 (d, J=7.8 Hz, 1H), 4.16 (dd, J=12.2, 5.1 Hz, 1H), 3.05 (dd, J=15.7, 3.0 Hz, 1H), 2.98-2.93 (m, 1H), 2.83 (tdd, J=11.4, 4.6, 2.2 Hz, 1H), 2.40-2.38 (m, 7H), 1.78 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 163.78, 161.32, 147.35, 140.64, 139.29, 137.40, 136.46, 129.56, 129.35, 129.18, 128.89, 128.81, 127.87, 127.57, 126.28, 126.06, 125.26, 115.78, 115.56, 60.70, 47.45, 41.65, 37.08, 33.21.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.77 (s, 1H), 7.51 (dd, J=7.6, 5.8 Hz, 2H), 7.45 (d, J=7.6 Hz, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.34 (s, 1H), 7.18-7.08 (m, 6H), 6.83 (d, J=7.7 Hz, 1H), 4.27 (dd, J=11.3, 3.6 Hz, 1H), 3.69-3.65 (m, 1H), 3.42 (br d, J=13.9 Hz, 1H), 3.34-3.28 (m, 1H), 2.85 (dd, J=6.8, 4.8 Hz, 6H), 2.72 (br d, J=9.9 Hz, 1H), 2.08 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 163.81, 161.35, 144.91, 140.94, 137.67, 136.98, 132.00, 129.47, 129.45, 129.44, 128.88, 128.80, 127.68, 127.41, 127.27, 127.14, 125.98, 115.83, 115.62, 61.91, 46.23, 39.68, 39.53, 34.31, 30.29. Calculated C24H25FN for [M+H]*: 346.1971. Found: 346.1967. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (90:5:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=2.0 mL/min; t1=16.53 min, t2=23.22 min.
Cis-4-(4′-chloro-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3j: cis-amine 3j was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), 4-chlorophenylboronic acid (47 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3j as colorless oil with 98% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.49 (d, J=8.5 Hz, 2H), 7.45-7.37 (m, 5H), 7.18-7.12 (m, 3H), 7.03 (t, J=7.3 Hz, 1H), 6.80 (d, J=7.8 Hz, 1H), 4.17 (dd, J=12.1, 4.9 Hz, 1H), 3.06 (dd, J=15.5, 2.5 Hz, 1H), 3.00-2.94 (m, 1H), 2.90-2.86 (m, 1H), 2.41-2.38 (m, 7H), 1.79 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 147.32, 140.43, 139.71, 139.16, 136.22, 133.49, 129.59, 129.36, 129.29, 128.99, 128.59, 128.20, 127.54, 126.38, 126.16, 125.30, 60.73, 47.39, 41.52, 36.93, 33.02.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.88 (s, 1H), 7.49-7.46 (m, 3H), 7.42-7.38 (m, 3H), 7.35 (s, 1H), 7.19-7.15 (m, 3H), 7.12-7.10 (m, 1H), 6.84 (d, J=7.8 Hz, 1H), 4.28 (br d, J=8.2 Hz, 1H), 3.69-3.64 (m, 1H), 3.43 (br d, J=14.2 Hz, 1H), 3.34-3.29 (m, 1H), 2.86 (s, 6H), 2.73 (br d, J=10.4 Hz, 1H), 2.08 (q, J=11.7 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 145.01, 140.70, 139.30, 137.64, 133.64, 132.00, 129.56, 129.46, 129.01, 128.53, 128.02, 127.39, 127.30, 127.17, 125.96, 61.92, 46.24, 39.72, 39.52, 34.34, 30.28. Calculated C24H25CIN for [M+H]*: 362.1676. Found: 362.1673. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=1.5 mL/min; t, =23.00 min, t2=30.59 min.
Cis-4-(4′-(dimethylamino)-[1,1′-biphenyl]-3-yl)-N,N-dimethyl-1,2,3,4-tetrahydro-naphthalen-2-amine, 3k: cis-amine 3k was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol), 4-(dimethylamino)phenylboronic acid (50 mg, 0.3 mmol) in presence of potassium phosphate (85 mg, 0.4 mmol), Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (12 mg, 0.03 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:2:0.1 hexanes:ethyl acetate:triethylamine) afforded racemic cis-amine 3k as colorless oil with 92% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.48 (d, J=8.7 Hz, 2H), 7.44 (d, J=7.7 Hz, 1H), 7.37 (s, 1H), 7.34 (t, J=7.6 Hz, 1H), 7.13 (dt, J=14.7, 7.4 Hz, 2H), 7.06 (d, J=7.5 Hz, 1H), 7.01 (t, J=7.4 Hz, 1H), 6.83 (d, J=7.8 Hz, 1H), 6.79 (d, J=8.7 Hz, 2H), 4.14 (dd, J=12.3, 4.9 Hz, 1H), 3.07-3.03 (m, 1H), 2.98-2.94 (m, 7H), 2.88-2.85 (m, 1H), 2.42-2.38 (m, 7H), 1.79 (q, J=12.1 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 150.07, 146.95, 141.57, 139.50, 136.26, 129.46, 129.28, 128.99, 127.85, 126.96, 126.55, 126.17, 126.05, 124.52, 112.83, 60.70, 47.45, 41.51, 40.71, 36.83, 33.13.
HCl salt 1H-NMR (500 MHz; CD30D): δ 7.85-7.80 (m, 2H), 7.75-7.70 (m, 2H), 7.59-7.55 (m, 2H), 7.47 (t, J=6.8 Hz, 1H), 7.30-7.24 (m, 2H), 7.17 (t, J=7.1 Hz, 1H), 7.07 (t, J=7.3 Hz, 1H), 6.77 (d, J=7.6 Hz, 1H), 4.41-4.40 (m, 1H), 3.87-3.86 (m, 1H), 3.21 (q, J=7.0 Hz, 2H), 2.98 (s, 6H), 2.60-2.59 (m, 1H), 2.17-2.15 (m, 1H). 13C-NMR (100 MHz, CDCl3): 164.94, 144.93, 140.15, 136.62, 132.41, 129.43, 129.34, 128.96, 128.45, 128.11, 127.50, 127.23, 127.02, 125.72, 120.63, 62.69, 48.27, 44.74, 43.91, 40.25, 32.12, 30.42. Calculated C26H31N2 for [M+H]+: 371.2487. Found: 371.2486. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5), 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t1=10.76 min, t2=21.76 min.
Racemic compounds 2c-d were synthesized. To an oven dried 25 mL round bottom flask with stir bar was added trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), Aryl MIDA boronate (0.3 mmol) and dioxane (2.4 mL). The resulting mixture was sparged with N2 for 30 min. To the flask was added palladium(II) acetate (2 mg, 0.01 mmol), SPhos (8 mg, 0.02 mmol) and aq K3PO4 (3.0 M, 0.5 mL, degassed by sparging with N2 for 30 min). The resulting reaction mixture was stirred for 20 h under nitrogen atmosphere at 60° C. (Scheme 7). The reaction was quenched with 1 N NaOH aq (3 mL) and ethyl acetate (4 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (5 mL×4). The combined organic layer was washed with brine (10 mL) and dried over Na2SO4. After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography (4:1:0.1 hexanes: dichloromethane:triethylamine) to afford racemic 2c-d.
The racemic mixtures of trans-analogs were separated by semi-preparative chiral HPLC Regiscell column using conditions and solvents specific to each analog to elute the trans-(2R,4S) and -(2S,4R) enantiomers at retention time t1 at t2, respectively, with absolute stereochemistry assigned according to retention time of the previously published trans-3′CI-4-PAT analog (Sakhuja, et al., 2015). Both enantiomers were converted to HCl salts for use in pharmacological assays by adding 2M HCl solution in ether to the solution of free amine in ether.
Trans-4-(3-(thiophen-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 2c: trans-amine 2c was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol) and 2-thiopheneboronic acid MIDA ester (72 mg, 0.3 mmol) in presence of aq. potassium phosphate Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (8 mg, 0.02 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:1:0.1 hexanes:dichloromethane:triethylamine) afforded racemic trans-amine 2c as colorless oil with 60% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.43 (d, J=7.6 Hz, 1H), 7.32 (s, 1H), 7.26-7.23 (m, 3H), 7.20-7.16 (m, 2H), 7.10 (t, J=6.9 Hz, 1H), 7.05 (dd, J=4.7, 3.9 Hz, 1H), 6.96 (d, J=7.6 Hz, 1H), 6.89 (d, J=7.7 Hz, 1H), 4.39 (t, J=5.2 Hz, 1H), 3.04 (dd, J=16.2, 4.7 Hz, 1H), 2.87 (dd, J=16.2, 9.4 Hz, 1H), 2.66 (tt, J=9.0, 4.5 Hz, 1H), 2.28 (s, 6H), 2.17-2.14 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 147.66, 144.67, 137.85, 136.49, 134.40, 130.10, 129.49, 128.83, 128.08, 128.06, 126.53, 126.44, 126.21, 124.84, 123.92, 123.20, 56.50, 44.15, 42.04, 34.98, 32.48. HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.71 (s, 1H), 7.47 (d, J=7.7 Hz, 1H), 7.29-7.21 (m, 7H), 7.07-7.03 (m, 2H), 6.77 (d, J=7.7 Hz, 1H), 4.57-4.55 (m, 1H), 3.53 (dd, J=15.5, 4.8 Hz, 1H), 3.40-3.36 (m, 1H), 3.25 (dd, J=15.3, 11.5 Hz, 1H), 2.71 (t, J=5.1 Hz, 6H), 2.48-2.45 (m, 2H). 13C-NMR (100 MHz, CDCl3): δ 145.33, 143.88, 135.30, 134.95, 132.78, 130.21, 129.54, 129.39, 128.22, 127.61, 127.52, 127.39, 125.78, 125.25, 124.69, 123.57, 58.77, 43.64, 41.08, 38.82, 31.51, 30.83. Calculated C22H24NS for [M+H]+: 334.1630. Found: 334.1628. HPLC (s-prep): Solvent System: hexanes:iPrOH (98:2) 0.1% TEA (modifier), flow rate=2.0 mL/min; t, =10.55 min, t2=12.20 min.
Trans-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 2d: trans-amine 2d was synthesized from trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol) and 2-furanboronic acid MIDA ester (67 mg, 0.3 mmol) in presence of aq. potassium phosphate Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (8 mg, 0.02 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:1:0.1 hexanes:dichloromethane:triethylamine) afforded racemic trans-amine 2d as colorless oil with 60% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.49 (d, J=7.7 Hz, 1H), 7.44 (s, 1H), 7.39 (s, 1H), 7.27-7.24 (m, 2H), 7.21-7.17 (m, 2H), 7.11 (t, J=6.9 Hz, 1H), 6.96 (d, J=7.6 Hz, 1H), 6.86 (d, J=7.5 Hz, 1H), 6.59 (d, J=3.2 Hz, 1H), 6.45-6.44 (m, 1H), 4.41 (t, J=5.0 Hz, 1H), 3.09 (d, J=15.1 Hz, 1H), 2.91 (dd, J=15.9, 9.6 Hz, 1H), 2.72 (br s, 1H), 2.32 (s, 6H), 2.22-2.17 (m, 2H). 13C-NMR (126 MHz, CDCl3): δ 154.17, 147.46, 142.07, 137.97, 136.52, 130.87, 130.07, 129.45, 128.62, 127.98, 126.45, 126.16, 124.18, 121.73, 111.70, 105.07, 56.49, 44.13, 42.12, 35.02, 32.64.
HCl-salt 1H-NMR (500 MHz; CDCl3): δ 12.71 (br s, 1H), 7.52 (d, J=7.7 Hz, 1H), 7.45 (s, 1H), 7.31-7.28 (m, 4H), 7.22-7.19 (m, 1H), 7.03 (d, J=7.7 Hz, 1H), 6.75 (d, J=7.7 Hz, 1H), 6.61 (d, J=3.2 Hz, 1H), 6.47-6.46 (m, 1H), 4.58-4.56 (br m, 1H), 3.56 (dd, J=15.6, 4.6 Hz, 1H), 3.43-3.36 (m, 1H), 3.25 (dd, J=15.2, 11.7 Hz, 1H), 2.71 (s, 6H), 2.47-2.44 (m, 2H). 13C-NMR (126 MHz, CDCl3): δ 153.39, 145.08, 142.30, 135.32, 132.76, 131.29, 130.10, 129.45, 129.10, 127.45, 127.34, 127.28, 123.45, 122.44, 111.78, 105.60, 58.68, 43.62, 41.05, 38.67, 31.37, 30.78. Calculated C22H24NO for [M+H]+: 318.1858. Found: 318.1858. HPLC (s-prep): Solvent System: hexanes:iPrOH (98:2) 0.1% TEA (modifier), flow rate=2.0 mL/min; t1=12.96 min, t2=15.57 min.
The cis analogues 3c and 3d (Scheme 8) were synthesized from cis-3′Br-4-PAT 3a and corresponding Aryl MIDA boronates following the general procedure as described above for trans 2c and 2d. The racemic mixtures of cis-analogs were separated by semi-preparative chiral HPLC Regiscell column using conditions and solvents specific to each analog to elute the cis-(2S,4S) and -(2R,4R) enantiomers at retention time t1 at t2, respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-cis-3′CI-4-PAT, 3b and (2S,4S)-cis-3′F-4-PAT, 3b′ analogs. Both enantiomers were converted to HCl salts for use in pharmacological assays by adding 2M HCl solution in ether to the solution of free amine in ether.
Cis-4-(3-(thiophen-2-yl) phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3c: cis-amine 3c was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol) and 2-thiopheneboronic acid MIDA ester (72 mg, 0.3 mmol) in presence of aq. potassium phosphate Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (8 mg, 0.02 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:1:0.1 hexanes:dichloromethane:triethylamine) afforded racemic cis-amine 3c as colorless oil with 60% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.51 (d, J=7.8 Hz, 1H), 7.45 (s, 1H), 7.33 (t, J=7.7 Hz, 1H), 7.30-7.27 (m, 2H), 7.17-7.12 (m, 2H), 7.09-7.05 (m, 2H), 7.05-7.02 (m, 1H), 6.81 (d, J=7.8 Hz, 1H), 4.15 (dd, J=12.6, 5.4 Hz, 1H), 3.10-2.98 (m, 3H), 2.46-2.39 (m, 7H), 1.80 (q, J=11.8 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 147.39, 144.52, 139.17, 136.43, 134.76, 129.56, 129.36, 129.31, 128.08, 128.03, 126.55, 126.31, 126.10, 124.91, 124.29, 123.31, 60.72, 47.35, 41.68, 36.89, 33.27.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 13.00 (br s, 1H), 7.54 (d, J=7.8 Hz, 1H), 7.42 (s, 1H), 7.35 (t, J=7.7 Hz, 1H), 7.30 (m, J=4.7 Hz, 2H), 7.19 (d, J=4.0 Hz, 2H), 7.12-7.05 (m, 3H), 6.84 (d, J=7.8 Hz, 1H), 4.24 (dd, J=12.1, 5.4 Hz, 1H), 3.68-3.64 (m, 1H), 3.48-3.44 (m, 1H), 3.34-3.28 (m, 1H), 2.87-2.84 (m, 6H), 2.72-2.69 (m, 1H), 2.06 (q, J=12.5 Hz, 1H). 13C-NMR (100 MHz, CDCl3): δ 145.02, 143.97, 137.55, 135.08, 132.03, 129.63, 129.46, 128.16, 127.81, 127.31, 127.18, 126.36, 125.16, 124.97, 123.56, 61.92, 46.17, 39.68, 39.49, 34.08, 30.38. Calculated C22H24NS for [M+H]+: 334.1630. Found: 334.1629. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t1=8.58 min, t2=25.12 min.
Cis-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, 3d: cis-amine 3d was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol) and 2-furanboronic acid MIDA ester (67 mg, 0.3 mmol) in presence of aq. potassium phosphate Palladium(II) acetate (2 mg, 0.01 mmol) and SPhos (8 mg, 0.02 mmol) following general procedure described above. Purification of crude reaction mixture by silica gel column chromatography (4:1:0.1 hexanes:dichloromethane:triethylamine) afforded racemic cis-amine 3d as colorless oil with 60% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 7.57 (d, J=7.8 Hz, 1H), 7.54 (s, 1H), 7.46 (s, 1H), 7.35 (t, J=7.7 Hz, 1H), 7.15 (dt, J=15.4, 7.6 Hz, 2H), 7.08 (d, J=7.6 Hz, 1H), 7.03 (t, J=7.4 Hz, 1H), 6.81 (d, J=7.8 Hz, 1H), 6.65 (d, J=3.2 Hz, 1H), 6.48-6.45 (m, 1H), 4.15 (dd, J=12.1, 4.8 Hz, 1H), 3.07 (dd, J=15.5, 2.8 Hz, 1H), 2.98 (dd, J=15.8, 11.4 Hz, 1H), 2.88-2.83 (m, 1H), 2.40-2.37 (m, 7H), 1.79 (q, J=12.2 Hz, 1H). 13C-NMR (126 MHz, CDCl3): δ 154.05, 142.10, 139.22, 136.37, 131.22, 129.52, 129.34, 129.09, 127.93, 126.26, 126.08, 124.26, 122.12, 111.74, 105.20, 60.69, 47.38, 41.62, 36.73, 33.21.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 12.66 (s, 1H), 7.55 (d, J=7.7 Hz, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 7.31 (t, J=7.7 Hz, 1H), 7.17-7.12 (m, 2H), 7.05-7.01 (m, 2H), 6.77 (d, J=7.8 Hz, 1H), 6.62 (d, J=3.0 Hz, 1H), 6.44-6.42 (m, 1H), 4.21 (d, J=8.1 Hz, 1H), 3.67-3.60 (br m, 1H), 3.39 (d, J=14.4 Hz, 1H), 3.30-3.25 (m, 1H), 2.81 (d, J=9.8 Hz, 6H), 2.65-2.63 (m, 1H), 2.07-2.01 (m, 1H). 13C-NMR (126 MHz, CDCl3): δ 153.50, 144.75, 142.26, 132.01, 131.49, 129.42, 129.38, 129.37, 127.75, 127.12, 127.01, 124.02, 122.77, 112.57, 111.82, 105.55, 61.91, 46.20, 39.75, 39.50, 33.99, 30.39. C22H24NO for [M+H]+: 318.1858. Found: 318.1859. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t1=9.27 min, t2=27.19 min.
Trans-4-(3-(pyridin-4-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 2e (Knapp, et al., 2009): To an oven dried 25 mL round bottom flask with stir bar was added trans-3′Br-4-PAT 2a (66 mg, 0.2 mmol), 4-pyridinylboronic acid (30 mg, 0.24 mmol) and dioxane (2.4 mL). The resulting mixture was sparged with N2 for 30 min. To the flask was added Tris(dibenzylideneacetone)dipalladium(0) (9 mg, 0.01 mmol), PCy3 (7 mg, 0.024 mmol) and aq K3PO4 (3.0 M, 0.5 mL, degassed by sparging with N2 for 30 min). The resulting reaction mixture was stirred for 12 h under nitrogen atmosphere at 95° C. (Scheme 9). The reaction was quenched with 1N NaOH aq (3 mL) and ethyl acetate (4 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (5 mL×4). The combined organic layer was washed with brine (10 mL) and dried over Na2SO4.
After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography (1:1:0.2 hexanes: ethyl acetate:triethylamine) to afford racemic 2e as colorless oil with 60% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 8.63 (dd, J=4.6, 1.4 Hz, 2H), 7.47-7.43 (m, 3H), 7.37 (t, J=7.7 Hz, 1H), 7.32 (br s, 1H), 7.21-7.17 (m, 2H), 7.13-7.10 (m, 1H), 7.07 (d, J=7.7 Hz, 1H), 6.96 (d, J=7.6 Hz, 1H), 4.46 (t, J=5.2 Hz, 1H), 3.05 (dd, J=16.3, 4.8 Hz, 1H), 2.89 (dd, J=16.2, 9.3 Hz, 1H), 2.67 (tt, J=8.8, 4.4 Hz, 1H), 2.29 (s, 7H), 2.20-2.16 (m, 1H). 13C-NMR (100 MHz, CDCl3): δ 150.32, 148.55, 148.06, 138.21, 136.52, 130.04, 129.65, 129.61, 129.08, 127.42, 126.65, 126.27, 124.91, 121.81, 56.42, 44.24, 41.97, 35.23, 32.23.
HCl salt 1H-NMR (500 MHz; CD3OD): δ 8.87 (d, J=6.7 Hz, 2H), 8.39 (d, J=6.7 Hz, 2H), 7.87 (d, J=7.8 Hz, 1H), 7.75 (s, 1H), 7.57 (t, J=7.8 Hz, 1H), 7.36 (d, J=7.7 Hz, 1H), 7.28 (dd, J=16.5, 7.9 Hz, 2H), 7.22 (t, J=7.4 Hz, 1H), 7.04 (d, J=7.6 Hz, 1H), 4.75 (t, J=4.1 Hz, 1H), 3.64-3.60 (m, 1H), 3.43 (dd, J=15.8, 4.8 Hz, 1H), 3.35 (s, 1H), 3.19 (dd, J=15.8, 10.9 Hz, 1H), 2.89 (d, J=11.2 Hz, 6H), 2.59-2.48 (m, 2H). 13C-NMR (100 MHz, CDCl3): 148.83, 148.02, 147.86, 136.95, 134.92, 129.60, 129.51, 129.26, 128.02, 126.85, 126.93, 126.00, 124.28, 121.58, 57.62, 45.35, 39.75, 39.54, 32.87, 30.01. C23H25N2 for [M+H]+: 329.2018. Found: 329.2018. HPLC (s-prep): Solvent System: hexanes:MeOH:iPrOH:EtOH (70:10:10:10) 0.1% TEA (modifier), flow rate=3.0 mL/min; t1=9.27 min, t2=14.68 min.
Cis-4-(3-(pyridin-4-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 3e: cis-amine 3e was synthesized from cis-3′Br-4-PAT 3a (66 mg, 0.2 mmol) and 4-pyridinylboronic acid (30 mg, 0.24 mmol) in presence of aq. potassium phosphate Tris(dibenzylideneacetone)dipalladium(0) (9 mg, 0.01 mmol) and PCy3 (7 mg, 0,024 mmol) following procedure described above for trans-2e (Scheme 10). Purification of crude reaction mixture by silica gel column chromatography (1:1:0.2 hexanes: ethyl acetate:triethylamine) afforded racemic cis-amine 3e as colorless oil with 55% isolated yield.
1H-NMR (500 MHz; CDCl3): δ 8.64-8.62 (m, 2H), 7.52 (d, J=8.1 Hz, 1H), 7.48 (dd, J=4.6, 1.4 Hz, 2H), 7.45-7.42 (m, 2H), 7.26-7.24 (m, 1H), 7.15 (dt, J=13.7, 7.0 Hz, 2H), 7.03 (t, J=7.4 Hz, 1H), 6.77 (d, J=7.8 Hz, 1H), 4.19 (dd, J=12.2, 4.8 Hz, 1H), 3.08 (d, J=14.6 Hz, 1H), 3.06-2.87 (m, 2H), 2.38-2.37 (m, 7H), 1.87-1.75 (m, 1H). 13C-NMR (126 MHz, CDCl3): δ 150.33, 148.28, 147.07, 138.67, 138.54, 129.60, 129.30, 127.43, 126.64, 126.45, 125.48, 121.80, 60.98, 47.05, 41.06, 36.39, 32.29.
HCl salt 1H-NMR (500 MHz; CDCl3): δ 8.64-8.62 (m, 2H), 7.52 (d, J=8.1 Hz, 1H), 7.48 (dd, J=4.6, 1.4 Hz, 2H), 7.45-7.42 (m, 2H), 7.26-7.24 (m, 1H), 7.15 (dt, J=13.7, 7.0 Hz, 2H), 7.03 (t, J=7.4 Hz, 1H), 6.77 (d, J=7.8 Hz, 1H), 4.19 (dd, J=12.2, 4.8 Hz, 1H), 3.08 (d, J=14.6 Hz, 1H), 3.06-2.87 (m, 2H), 2.38-2.37 (m, 7H), 1.87-1.75 (m, 1H). 13C-NMR (100 MHz, CDCl3): 149.05, 148.88, 147.95, 140.69, 134.84, 129.88, 129.15, 127.55, 126.98, 126.32, 125.59, 121.92, 62.12, 45.98, 39.01, 39.26, 34.65, 30.15. C23H25N2 for [M+H]*: 329.2018. Found: 329.2018. HPLC (s-prep): Solvent System: hexanes:EtOH (85:15) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate=3.0 mL/min; t1=18.60 min, t2=23.79 min.
Examples of synthesized and purified chemical structures are shown below:
For cell culture and transfection, HEK293 (ATCC no. CRL-1573) and HEK293T cells (ATCC no. CRL-3216) were maintained in MEM and DMEM (Corning), respectively, supplemented with 10% regular FBS and 1% penicillin/streptomycin; CHO cells (ATCC no. CRL-61) were maintained in Kaighn's modification of Ham's F-12K (Gibco) using the same supplementation. All cells were grown adherently in 10 cm plates in a humidified incubator kept at 37° C. and 5% carbon dioxide. All human wild type aminergic GPCR clones, encoded in a pcDNA3.1(+) vector, were obtained from the cDNA Resource Center (cdna.orq). Transient transfections were used to express 5-HT2A, 5-HT2C, α1A- or α1B-adrenergic receptors, whereas 5-HT7(a)Rs were stably expressed in HEK293 cells. H1Rs were transiently expressed, and D2Rs were stably expressed in CHO cells. 5-HT2B and 5-HT1ARs in HEK293T cells were transiently expressed because HEK293 cells failed to provide sufficient expression. D3Rs stably expressed in HEK293 cells were generously provided by Dr. David Sibley's laboratory.
Transient transfections were performed on cells in the log growth phase (70-90% confluence). First, a transfection cocktail was prepared by adding 10 μg of cDNA and 40 μg of linear polyethylenimine (˜40,000 g/mol; Polysciences Inc.) separately to two 2.5 mL aliquots of Opti-MEM (Gibco, Ref. 31985-070). Each solution was mixed by inversion before combining, mixed by inversion again, and incubated at 37° C. for 30 minutes. Cells were then washed with 1 mL 1×PBS, followed by the gentle addition of 5 mL of transfection cocktail and 5 mL of cell culture medium with 5% (final) dialyzed FBS. Transfections were performed for 48 hours. This represents an economic modification of the previous transfection methods using lipofectamine or Turbofect (Invitrogen, #11668027 and Thermo Scientific, #R0532, respectively), which gave similar levels of expression for the receptors studied here. Cell membranes were homogenized as previously described (Perry, et al., 2020).
The affinity of ligands was determined via radioligand binding techniques using human recombinant receptors expressed in mammalian clonal cells. Details on assay conditions, radioligand, nonspecific binding and receptor expression are shown in Table 5. Number of independent radioligand binding experiments is shown in Table 6. In Table 6, all independent experiments listed were performed using 3 technical replicates.
Ligand affinity was assessed using established methodology and 2-5 μg of protein per well, determined by the Pierce bicinchoninic acid protein assay according to the manufacturers protocol (Thermo Scientific), (Perry, et al., 2020; Roth, 2013). Saturation binding experiments were performed on membranes expressing 5-HT2A, 5-HT2B, 5-HT2C, or H1Rs in triplicate across 8 concentrations. Competition binding assays were performed in at least triplicate with approximate Kd concentrations of radioligand. Total and nonspecific binding were determined in octet. Each compound was assessed in at least two independent experiments across 10-14 concentrations in half-log units (1 μM-100 μM) where the center of the concentration range was the approximate pKi. Serial dilutions of unlabeled compound were made in assay buffer at 2.5× the final concentration using 10 mM DMSO stocks (final [DMSO]≤1%). Assays were terminated using rapid filtration via Whatman GF/B Fired Filters (Brandel Inc., Gaithersburg, MD) soaked in 0.3% (w/v) PEI, using an automated Tomtec Harvester 96 (Hamden, CT).
Filters were washed with 50 mM Tri-HCl (pH=7.4, 4° C.) before being oven dried and placed into scintillation vials containing 1 mL SX18-4 ScintiVerseTM BD Cocktail (Fisher Chemical, Fair Lawn, NJ). Scintillation was detected using a Tri-Carb 2910 TR Liquid Scintillation Analyzer (Perkin Elmer, Boston, MA).
a50 mM Tris-HCl, 10 mM MgCl2, 0.1 mM EDTA, pH = 7.4,
b50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.1% BSA, pH = 7.4,
c20 mM Tris-HCl, 145 mM NaCl, pH = 7.4
dValues obtained from (Armstrong, Casey et al., 2020).
eValues obtained from (Roth, 2013).
The pharmacological parameters (e.g., pEC50, pKb, pIC50, Imax) of agonist and antagonist-mediated signal transduction through Gaq-coupled 5-HT2A, 5-HT2B, 5-HT2C, and H1Rs was quantified using the Cisbio (Bedford, MA) IP-One homogeneous time resolved fluorescence (HTRF) immunoassay. The protocol used was consistent with that recommended by the manufacturer for suspension cells in 384-well plate format, with minor modifications. Immediately following transfection, cells were washed twice with 10 mL of prewarmed 1×PBS, dissociated in 10 mL of 1×PBS, and centrifuged at 270 g for 5 minutes at room temperature. Cells (˜2,000 cells/μL, >90% viability, determined by the Corning® Cell Counter) were resuspended in the manufacturer's stimulation buffer (pH=7.4, 37° C.) modified to include 0.1% bovine serum albumin stabilizer (PerkinElmer, part #: CR84-100) and added to a white 384-well OptiPlate (PerkinElmer). Next, stimulation buffer, or 2× compound diluted in stimulation buffer, was added to each well. For competitive antagonism (pKb) experiments, 2× antagonist was diluted in stimulation buffer containing 2× reference agonist (e.g., 2 μM 5-HT for WT and point mutated 5-HT2ARs, 20 nM 5-HT for 5-HT2BRs, and 20 μM histamine for H1Rs) such that agonist and antagonist were added to the cells simultaneously. Cells were then incubated in the dark for two hours at 37° C. to ensure equilibrium was obtained. Plates were covered with an aluminum foil seal to prevent evaporation.
Following incubation, the manufacturer's lysis solution (detection buffer) containing inositol monophosphate (IP1) covalently bound to a fluorescent acceptor dye (d2) was added to each well. This process generated a homogenous mixture of cellular and d2-labeled IP1, in a ratio dependent on the concentration- and activity-dependent efficacy of test ligands to modulate cellular IP1 levels. Next, detection buffer containing an anti-IP1 antibody covalently bound to a terbium-cryptate, which serves as a fluorescent acceptor, was added to each well. A one-hour incubation at room temperature was used to allow competitive interactions between cellular and d2-labeled IP1 to equilibrate with anti-IP1-cryptate. Time-resolved fluorescent resonance energy transfer (TR-FRET) was then quantified by a Synergy H1 plate reader equipped with a HTRF filter cube (BioTek). Light pulse at 320 or 340 nm was used to excite the fluorescent donor, and emission at 615 and 665 nm was detected. The relative levels of TR-FRET were used to obtain an emission ratio at 665/620 nm, which was then used to interpolate the concentration of IP1 in each well.
Site directed mutagenesis experiments were performed using 5′-phosphorylated, PAGE-purified custom primers (Life Technologies, Carlsbad CA) and the Quikchange Site-Directed Mutagenesis kit (Agilent, Santa Clara CA) according to the manufacturer's protocol. Reactions were performed in thin-walled PCR tubes using a TOne Gradient 96 Thermal Cycler (Biometra). Primer sequences and optimized reaction conditions are provided in Table 7A, and the primer sequences are provided in Table 7B. Following Dpn1 digestion, 2 μL of the PCR product mixture was transformed into 50 μL XL1-Blue competent cells using a 45 second pulse of heat in a 42° C. water bath. The transformed bacteria were then incubated for 1 hour at 37° C. in 0.5 mL of LB broth before being plated on LB-agar plates and incubated overnight at 37° C. The next day, two colonies for each mutated receptor were grown overnight in LB broth at 37° C. The mutated cDNA was extracted the following day using the PureYield™ Plasmid Maxiprep System (Promega Corp., Madison WI). Purified DNA was sequence validated by Psomagen Inc. (Cambridge, MA).
GraphPad Prism (La Jolla, CA) version 9.1.1 was used to analyze all experimental data in this work. To analyze data from saturation binding studies, the radioligand counts per minute (cpm) were normalized to fmol/mg protein bound and then fit the data to a ‘specific binding with hill slope’ model. For competitive radioligand displacement studies, a baseline correction was performed by subtracting the mean nonspecific binding value from the radioligand binding values with and without competitor to obtain specific binding values. Specific binding values were then normalized such that total binding in the absence of competitor represented 100% radioligand binding, and the radioactivity associated with each concentration of competitor was a percentage of total binding. The normalized data was then fit to the ‘one-site Fit Ki’ nonlinear regression model. Ligand selectivity is reported as the fold change between mean affinity (Ki) values.
The functional activity of each compound was determined by incubating cells expressing recombinant receptors with or without a compound of interest in parallel with well containing only buffer and 8 known concentrations of IP1 to generate a standard curve for each experiment. The IP1 concentration in cell-containing wells was then interpolated using nonlinear regression and the ‘log(inhibitor) vs. response (three parameters)’ model in Prism. To control for variation between assays, the resulting concentrations were transformed into molar units and change from basal was calculated using the following equation (Equation 1), where B is the basal concentration of IP, and Y is the concentration of IP generated by cells incubated with compound.
The antagonist equilibrium dissociation constant (Kb), determined in parallel with the EC50 of reference agonists, was calculated using the following Equation 2 (Cheng, 2001):
where IC50 is the concentration of antagonist which inhibited 50% of the IP1 elicited by a constant concentration (A) of reference agonist (i.e., 1 μM 5-HT, 10 nM 5-HT, or 10 μM histamine for 5-HT2A, 5-HT2B, and H1Rs, respectively), EC50 is the concentration of the reference agonist which elicited a half maximal response, and K is the Hill slope of the reference agonist. The IC50 of antagonists was determined using the ‘log([inhibitor]) vs. response (three parameter)’ model, where cells stimulated only by A represented the lowest X value (−12). The same model was used to calculate the IC50 of inverse agonists in the absence of A. In contrast, the EC50 was derived from a log([agonist]) vs. response-variable slope (four parameters)’ model to simultaneously obtain K. Each Kb, IC50, and EC50 value was logarithmically transformed into the pKb, pIC50, and pEC50, respectively for ease of presentation and statistical analyses. Since standard deviations tend to be symmetrically distributed around log normal pharmacological parameters (e.g., pKb, pIC50, and pEC50), but not equilibrium constants (e.g., Kb, IC50, and EC50), statistical comparisons of in vitro functional data were only performed using log normal values (Neubig, et al., 2003).
No blinding or randomization methods were used for in vitro studies since these measurements are insensitive to time of day or experimenter interpretation.
Data exclusion, sample size and statistical analyses were utilized. For purposes of efficient lead identification, each competitive radioligand displacement assay condition was performed using three or four technical replicates in at least two independent experiments. Some results from radioligand binding assays were not included in the reported data, for example, when excessive or incomplete radioligand displacement (>30%) occurred at the lowest or highest concentration of unlabeled ligand, respectively. Among these experiments were three of 130 pK values at the 5-HT2AR (pK=6.99 for [2S,4S]-3h, pKi=9.84 and 9.65 for [2S,4R]-2i), one of 125 pKi values at the 5-HT2BR (pKi=7.32 for (2S,4R)-2k), and two of 119 pKi values at the 5-HT2CR (pKi=9.08 for [2S,4R)-2c, and pKi=9.17 for [2S,4R]-2d). These results were attributed to experimenter error during assay optimization or chemical contamination.
The number of concentrations (7-9) used between receptors varied in functional assays. Three technical replicates were used in all functional assays except for antagonism (Kb) assays, which utilized two technical replicates. Five independent experiments were performed to define the functional concentration-response relationships for ligands acting at point mutated 5-HT2ARs, WT 5-HT2BRs, WT 5-HT2CRs, and WT H1Rs. The number of independent experiments performed at WT 5-HT2ARs to determine the EC50 of 5-HT, and the Kb of antagonists, however, varied (n=14-19) since these experiments were performed in parallel with each other and often in parallel with analogous experiments at point mutated 5-HT2ARs (see Table 2 for exact n). For all in vivo conditions (Example 3), n=6 treatments were administered, except mice treated with vehicle+(±)-DOI (n=7) due to an additional mouse being procured in case of error.
The data and statistical analyses in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis, et al., 2018). Where n ? 5, all pharmacological parameters are presented according to Hopkin's two-digit rule (Hopkins, et al., 2011). Parametric one-way ANOVA and unpaired t-tests were used to analyze the data in this work. The p<0.05 was considered as statistically significant, and only performed statistical analyses on groups with n>5 independent samples. The potential agonist activity of (2S,4R)-2k or (2R,4S)-2c at 5-HT2B or 5-HT2CRs, respectively, indicated by an exploratory screening effort (
To ensure data adhered to assumptions of ordinary one-way ANOVA and unpaired parametric t-tests, diagnostic statistics were performed using Brown-Forsythe and F-tests to measure variance distribution, and Shapiro-Wilk tests for normality. Data abided by these assumptions in the majority of cases with few deviations, and where deviations occurred, nonparametric tests (Mann-Whitney U or Kruskal-Wallis) were performed to assess the robustness of a result. No deviations were endemic to a particular compound, receptor variant, or experimental technique, and no comparison violated both assumptions.
All tritiated radioligands were purchased from PerkinElmer (Boston, MA) and are shown in Table 5. 5-hydroxytryptamine hydrochloride and doxepine hydrochloride were purchased from Alfa Aesar (Ward Hill, MA). (±)-2,5-Dimethoxy-4-iodoamphetamine hydrochloride, chlorpromazine hydrochloride, histamine dihydrochloride, and tripolidine hydrochloride were purchased from Sigma Aldrich (St. Louis, MO). Mianserin hydrochloride, and risperidone (free base), were purchased from Tocris Biosciences (Bristol BS11 OQL, UK). PIMA tartrate was purchased from Selleck Chemical LLC (Houston, TX).
For nomenclature of targets and ligands, key protein targets and ligands are hyperlinked to corresponding entries in (guidetopharmacology.org), the common portal for data from the IUPHAR/BPS Guide to Pharmacology (Harding, et al., 2018), and are permanently archived in the Concise Guide to Pharmacology 2017/18 (Alexander, et al., 2017).
Adult male C57BL/6J mice were procured from Jackson Laboratories (Bar Harbor, ME) at 8 weeks of age, and housed 4/cage inside sterile ventilated caging by Innovive (San Diego, CA) on irradiated corn cob. All cages were changed in an animal transfer station using forceps soaked in Clidox-S® solution (Genestil). Animals were maintained in an SPF facility on a 12-hr light:dark cycle with ad libitum access to pre-filled acidified water (Innovive) and irradiated rodent diet (Prolab Isopro). After at least one week at Northeastern University, mice were transported two floors above their vivarium to a testing facility kept at approximately 22° C. with a constant background noise level of 62 dB and fluorescent lighting. All animals were habituated to the novel environment for a minimum of one hour before handling. To eliminate bias during in vivo studies, mice were marked on the tail with a permanent marker and placed in an alphanumeric home cage. A random number generator was used to select the order of mice undergoing treatment, and treatments were blinded to the administrator and observers.
Compounds were prepared in vehicle (5% (v/v) DMSO in MilliQ water) and filtered through a 0.22 μm syringe filter. All injections were performed s.c. on the neck at 0.1 mL/10 g body weight. PIMA and (2S,4R)-2k were administered at 0.3 or 3 mg kg−1, whereas (2R,4R)-3h was administered at elevated doses (3.0 or 5.6 mg kg−1) as pilot studies indicated analogs in the (2R,4R)-configuration were less active in vivo. For all testing procedures, mice were pretreated with compound, placed in their home cage for the time indicated below, and then in an open field arena (43 cm×43 cm, Med Associates, St. Albans, VT). Trials were video-taped using a ceiling-mounted video tracking system connected to Noldus Ethovision XT9 software (Noldus Information Technology, Leesburg, VA) allowing for locomotor activity tracking (distance traveled, cm). Animals were sacrificed by cervical dislocation after being anesthetized with isoflurane.
All behavioral procedures comply with the Guide for the Care and Use of Laboratory Animals (Council, 2011) and were approved by the Northeastern University Institutional Animal Care and Use Committee. The animal care and use program is fully accredited by AAALAC, International and holds an Assurance with OLAW. Moreover, these studies are in accordance with the ARRIVE 2.0 guidelines (Percie du Sert, et al., 2020) and recommendations made by the British Journal of Pharmacology.
DOI-elicited head twitch response assays were performed (
Locomotor activity assays were performed (
To compare the in vivo activity of PIMA, (2S,4R)-2k, and (2R,4R)-3h the DOI-elicited head twitch response assay in mice was used as a model of central 5-HT2AR engagement sensitive to antipsychotic-like activity (Canal & Morgan, 2012). Acute administration of each compound significantly attenuated the head twitch response at every dose tested (
Notably, only male mice were used in this study, and it is unclear if the behavioral findings generalize to female mice, although sex differences in the sensitivity of mice to DOI are nil (Canal & Morgan, 2012). Future studies investigating the efficacy and safety of novel 4-PATs in more comprehensive animal models of psychosis should consider sex as an experimental variable.
For computational chemistry and molecular modeling, physicochemical parameters (log P and log D) were determined computationally using StarDrop™ (Optibrium) version 6.5 and the freebase form of 4-PAT-type compounds (Optibrium). All molecular modeling images were generated using the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC (Schrodinger, 2015).
In molecular docking work, the 3D PAT analogs were built using Maestro (Schrodinger, LLC) and were optimized using an ab initio quantum chemistry method at the HF/6-31G* level, followed by single point energy calculations of molecular electrostatic potential for charge fitting using Gaussian 16 (Gaussian, Inc.) (Bayly, et al., 1993). The atomic charges derived from ab initio calculations were used for molecular docking simulations. The crystal structures for 5-HT2AR (PDB: 6A94) and H1R (PDB: 3RZE) were processed to add missing sidechains and loops with Discovery Studio software (BIOVIA). AutoDock 4.2 (Morris, et al., 1998) was used to dock molecules into the receptors with selected sidechain flexible residues in the binding pocket. A grid map was generated for the receptor using C, H, N, O, S, F, Cl, Br, I (i.e., carbon, hydrogen, nitrogen, oxygen, sulfur, fluorine, chlorine, bromine, and iodine) elements sampled on a uniform grid containing 80×80×80 points, 0.375 Å apart. The Lamarckian genetic algorithm was selected to identify ligand binding conformations. For each ligand, 100 docking simulations were performed. The final docked ligand conformations were selected based on binding energies and cluster analysis.
The molecular dynamics simulations were performed was follows: the protonation states of the titratable residues of 5-HT2A, 5-HT2B, 5-HT2C and H1 structures were calculated at pH=7.4 using the H++ server (biophysics.cs.vt.edu/). The PAT-bound receptor complexes obtained from molecular docking studies were inserted into a simulated lipid bilayer composed of POPC:POPE:cholesterol (2:2:1), (Grossfield, et al., 2008) and a water box using CHARMM-GUI Membrane Builder webserver (charmm-gui.org). Sodium chloride (150 mM) and extra neutralizing counter ions were added into the systems. The PMEMD.CUDA program of AMBER 16 was used to conduct MD simulations. The Amber ff14SB, lipid17, and TIP3P force field was used for the receptors, lipids, and water. The parameters of PAT analogs were generated using general AMBER force field by the Antechamber module of AmberTools 17. The partial charges for the compounds were calculated using a restrained electrostatic potential charge-fitting scheme by ab initio quantum chemistry at the HF/6-31G* level 15 (Gaussian 16) (Bayly, et al., 1993). System topology and coordinate files were generated by using the tleap module of Amber. The systems were energy minimized by 500 steps (with position restraint of 500 kcal/mol/A2) followed by 2000 steps (without position restraint) using the steepest descent algorithm. Subsequently, the systems were heated from 0-303 K using Langevin dynamics with a collision frequency of 1 ps−1. Receptor complexes were position-restrained using an initial constant force of 500 kcal/mol/Å2 during the heating process, and weakened to 10 kcal/mol/Å2, allowing lipid and water molecules free movement. Next, systems went through 5 ns equilibrium MD simulations. Finally, a total of 100-1000 ns MD simulations were conducted, and coordinates were saved every 100 ps for analysis. The MD simulations were conducted under NPT (constant temperature and pressure). Pressure was regulated using an isotropic position scaling algorithm with the pressure relaxation time fixed at 2.0 ps. Long range electrostatics was calculated by a particle mesh Ewald method (Darden, 1993) with a 10 Å cutoff.
The SAR results reported above and elsewhere (Canal, et al., 2014; Sakhuja, et al., 2015) indicated that selectivity to bind 5-HT2A and/or 5-HT2CRs is negligible for 4-PATs with small meta-substituents on ring C (e.g., 3a, 3b, 3b, Table 1). In contrast, larger aryl substituents at this position can yield moderate to high selectivity to bind 5-HT2ARs over 5-HT2BRs in the (2S,4R)-configuration, and over 5-HT2B, 5-HT2C and H1Rs in the (2R,4R)-configuration. Meanwhile, PIMA selectively bound 5-HT2ARs over 5-HT2B and H1Rs, with moderate selectivity over 5-HT2CRs. To understand how aryl substituted 4-PATs and PIMA bind to 5-HT2ARs, molecular modeling studies were performed using a model of the 5-HT2AR (
Overall, PIMA, (2S,4R)-2k, and (2R,4R)-3h stabilized an inactive-like conformation of the 5-HT2AR, typified by an ionic lock between R1733.50 and E3186.30 within the E/DRY domain (
The models also indicated that the isobutoxybenzyl moiety of PIMA and aryl ring D of (2S,4R)-2k and (2R,4R)-3h occupied a side cavity between TM4 and TM5, unimpeded by the small side chain of G2385.42, a residue unique to 5-HT2-type receptors among aminergic GPCRs. Furthermore, in all models, F2345.38 assumed a rotamer conformation oriented away from G2385.42, which has been suggested to extend the side cavity (Kimura, et al., 2019). Several amphipathic and hydrophobic side chains in this region of the binding pocket (I2104.60, V2355.39, G2385.42, and S2425.46) were close enough to the isobutoxybenzyl of PIMA and aryl ring D of (2S,4R)-2k and (2R,4R)-3h to facilitate interactions (Table 3), thus providing a potential structural basis for the observed selectivity of these ligands to bind 5-HT2ARs.
To validate the molecular modeling results, residues were point-mutated in and around the 5-HT2AR side-extended cavity (Kimura, et al., 2019) and quantified the antagonist affinity (pKb) of (2S,4R)-2k and (2R,4R)-3h, as well as (2S,4R)-2a (which lacks 5-HT2R subtype selectivity) at 5-HT2AR variants to understand how stereochemistry and aryl ring D impact ligand-receptor interactions. Notably, like PIMA, (2S,4R)-2k, and (2R,4R)-3h, key analog (2S,4R)-2a demonstrated inverse agonist activity at C322K6.34 5-HT2ARs (
A G238S5.42 5-HT2AR was generated to test the hypothesis that the large side chain of serine precludes ligand access to the side extended cavity, as suggested by the molecular modeling results (
The above experiments were extended by asking if the attenuated affinity of (2S,4R)-2a, (2S,4R)-2k, and (2R,4R)-3h at G238S5.42 5-HT2ARs translates to aminergic GPCRs natively presenting S5.42. Table 4 shows that (2S,4R)-2k and (2R,4R)-3h had >1,000-fold selectivity for 5-HT2ARs over 5-HT1A, 5-HT7, D2L, α1A- and α1B-adrenergic GPCRs. It was noted that (2S,4R)-2k had 270-fold selectivity over D3Rs, whereas (2R,4R)-3h had >1,000-fold selectivity. In contrast, (2S,4R)-2a exhibited moderate-to-high affinity for 5-HT7, D2L, D3, and α1A-adrenergic receptors.
Interestingly, aryl substituted 4-PATs in the (2S,4R)-configuration had high affinity for H1Rs (Table 1), despite the presence of T1945.42, which possesses a bulkier side chain than serine. The molecular modeling results suggested that W1584.56, a residue unique to H1Rs, might form stereospecific aromatic interactions with 4-PATs to impart high affinity (
Alignment of 5-HT2A, 5-HT2B and 5-HT2CR crystal structures (
Further inspection of the 5-HT2-type receptor crystal structures revealed that one helical turn above F5.38 in 5-HT2A and 5-HT2CRs exists a non-conserved residue in 5-HT2BRs (D2315.35, D2115.35, and F2145.35, respectively). The root-mean-square deviation (RMSD) of the F5.38 side chain in WT 5-HT2A and 5-HT2BRs was tracked in silico and found that F5.38 exhibited large transient variations in RMSD in WT 5-HT2ARs. Interestingly, the RMSD of F5.38 in D231F5.35 5-HT2ARs recapitulated the restricted pattern observed in silico for WT 5-HT2BRs, indicating that D2315.35 may facilitate flexibility in the side chain of F5.38 (
It was therefore hypothesized that D2315.35 may modulate the side chain rotamer of F2345.38 in 5-HT2ARs to mediate subtype selective binding. To test this hypothesis, a D231F5.35 5-HT2AR was generated, however, D231F5.35 5-HT2ARs were insufficiently responsive to 5-HT for competitive antagonism studies (
Residues in TM4 and TM5 lining the side-extended cavity of 5-HT2ARs and in proximity to PIMA, (2S,4R)-2k, and (2R,4R)-3h (Table 3) were then investigated. Among these were the side chains of 12104-6, V2355.39, and S2425.46. Importantly, the side chains of 12104.60 and V2355.39 are conserved in 5-HT2CRs, while S2425.46 is unique to 5-HT2ARs. It was hypothesized that selectivity to bind 5-HT2A and 5-HT2CRs over 5-HT2BRs may involve interactions with the side chains of 14.60, V5.39, or the 5-HT2AR-specific residue S2425.46. In fact, a significant increase in the affinity of PIMA and (2S,4R)-2k at V235M5.39 5-HT2ARs was observed, with no change in affinity for any antagonist at 1210V4.60 or S242A5.46 5-HT2ARs (Table 2,
X-ray crystal data for compound 3b was acquired. The computing details were as follows: data collection: APEX3 (Bruker, 2016); cell refinement: SAINT V8.40A (Bruker, 2016); data reduction: SAINT V8.40A (Bruker, 2016); program(s) used to solve structure: ShelXT (Sheldrick, 2015); program(s) used to refine structure: SHELXL (Sheldrick, 2015); molecular graphics: Olex2 (Dolomanov et al., 2009); software used to prepare material for publication: Olex2 (Dolomanov et al., 2009). The identification code was: mukherjee_neu2_Om.
Special details were as follows: Geometry. All esds (except the esd in the dihedral angle between two I.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving I.s. planes. Refinement. Refined as a 2-component inversion twin.
Special details were as follows: geometry. All esds (except the esd in the dihedral angle between two I.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving I.s. planes. Refinement. Refined as a 2-component inversion twin.
Each of the compounds or formulas disclosed herein can be derivatized to a corresponding positively charged quaternary amine, for example, by the addition of a third alkyl group to the amine to render it impermeable to the blood-brain barrier and specific for peripheral 5-HT receptors. For example, any of the compounds or formulas described herein may be derivatized at an amino group at the 2 position of the tetralin core via Scheme 11 shown below, wherein R4 can represent ring ‘C’ including substituents described above or shown in
Examples compounds that can be synthesized, with ‘E’ representing a charged group or charged amine, are shown below:
In an example to demonstrate the compound or composition does not substantially accumulate in the human brain, adult, male, C57Bl/6J mice, approximately six months old, and treatment-naïve for at least six weeks prior to testing, can be injected sc with any of the compounds described herein, bearing a positively charged amino group at the 2 position of the tetralin core (the “test compound”) at a dose of about 3.0 mg/kg and returned to their home cages. At 30, 60, or 90 min later, mice are euthanized by rapid cervical dislocation and decapitation. Trunk blood is collected in pre-chilled, heparin-coated tubes. Brains are quickly excised and frozen in liquid nitrogen. Plasma is collected from blood after centrifugation for 5 min at 13,000 g. Whole brain samples are wrapped in foil, and brain and plasma samples are labeled and stored at −80° C. until liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays are performed. Frozen brain samples are weighed and homogenized in phosphate buffered saline (PBS), pH 7.4. After the first analysis, the extra brain homogenate is stored at −80° C. until they are thawed for a second, more dilute, analysis. Plasma samples are used directly upon arrival. The proteins from each plasma sample and a portion of each brain homogenate are immediately precipitated with 1:1 methanol:acetonitrile (4× starting volume) and internal standard (e.g. (−)-MBP68) followed by centrifugation at 14,000 g for 5 minutes at 4° C. The resulting supernatants from each sample are dried under nitrogen. Each sample is reconstituted in methanol, vortexed, sonicated briefly, and centrifuged prior to LC-MS/MS analysis. Calibration curves are constructed from the ratios of the peak areas of test compound versus internal standard in extracted standards made in mouse plasma or homogenized mouse brain.
LC-MS/MS analysis can be performed using an Agilent 1100 series HPLC and a Thermo Finnigan Quantum Ultra triple quad mass spectrometer. Example mobile phases used are 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) in a 5 minute gradient. Samples of 10 μL each are injected onto a Phenomenex Gemini C18 column (2×50 mm, 5μ) with a C18 guard column. The test compound and its internal standard ((−)-MBP) were ionized in ESI+ and detected in SRM mode. Internal standards are used for quantification of the test compound level per g tissue or per μL plasma.
The test compounds are not expected to accumulate in the brain and, rather, are expected to be more prevalent in the plasma.
Each of the compounds disclosed herein can be derivatized to a corresponding positively charged quaternary amine, for example, by the addition of a third alkyl group, to render it impermeable to the blood-brain barrier and specific for peripheral 5-HT receptors.
This application claims priority to U.S. Provisional Application No. 63/221,920, filed 14 Jul. 2021, which is hereby incorporated by reference in its entirety.
This invention was made with government support under Grant No. RO1 DA030989, RO1 DA047130, and RO1 MH081193 awarded by the NIH National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/037220 | 7/14/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63221920 | Jul 2021 | US |
