TITLE Serotonin 5-HT2A, 5-HT2B, and 5-HT2C Receptor Inverse Agonists CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63/221,920, filed 14 July 2021, which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 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. BACKGROUND 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-HT _2A , 5-HT _2B , and 5-HT _2C receptors (Rs), which represent promising neurotherapeutic targets. However, extensive structural homology complicates the design of selective therapeutic agents. For example, 5- HT ₂ -type receptors share 60-70% amino acid identity within structurally conserved regions, and 27-31% identity with histamine receptors (H ₁ Rs), (Pandy-Szekeres, et al., 2018). Antagonism of 5-HT _2A Rs 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-HT _2C Rs, 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-HT _2A /5-HT _2C R 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-HT _2C Rs to PIMA's efficacy is unclear (Stahl, 2016). Meanwhile, activation of 5-HT _2B Rs is linked to valvular heart disease (Rothman, et al.,2000; Ayme-Dietrich, et al.,2017), and deficiency or antagonism of 5-HT _2B Rs 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-HT _2B Rs may be undesirable for antipsychotic medications. Likewise, H ₁ Rs represent a common 'off-target' for CNS-penetrating drugs (Weiner, et al., 2001) and H ₁ R 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-HT _2A , 5-HT _2B and 5-HT _2C 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. SUMMARY The present technology provides novel 4-phenyl-2-dimethylaminotetralin (4-PAT) compounds that are shown to provide inverse agonism at one or more 5-HT _2A-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: (Formula I); wherein Y is selected from the group consisting of and wherein covalent bond z is attached at any carbon atom of Y; wherein Y is unsubstituted or is substituted with one or more moieties V, each of the one or more moieties V independently selected from the group consisting -F, - Cl, -Br, -I, -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -NH(CH ₂ CH ₃ ), -N(CH ₂ CH ₃ ) ₂ , -C=NH, -C=NNH ₂ , -C=ONH ₂ , -NO ₂ , -NO, -CN, -N ₃ , -N=C=O, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C=OOH, - CH ₂ C=OOH, -S=OCH ₃ , -S(=O) ₂ CH ₃ , -S(=O) ₂ OH, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(CH ₃ ) ₂ , -OH, -OCN, -OCH ₃ , -OCH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CHOHCH ₂ OH, -CHOHCH ₃ , -SH, - SCN, -SCH ₃ , -SCH ₂ CH ₃ , -CH ₂ SH, -CH ₂ CH ₂ SH, -CHSHCH ₂ SH, -CHSHCH ₃ , and substituted or unsubstituted thiophene, furanyl, phenyl and pyridyl; and wherein the compound comprises at least 50% of a single stereoisomer selected from the group of stereoisomers consisting of 2R4R, 2S4S, 2R4S, and 2S4R; or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 2. The compound of feature 1, wherein one or more moieties V are independently selected from the group consisting of and wherein V is attached to Y via a covalent bond to any one of carbons 5-7 of V; and wherein V is substituted with one or more substituents W, each of the one or more substituents W independently selected from the group consisting of -F, -Cl, - Br, -I, -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -NH(CH ₂ CH ₃ ), -N(CH ₂ CH ₃ ) ₂ , -C=NH, -C=NNH ₂ , - C=ONH ₂ , -NO ₂ , -NO, -CN, -N ₃ , -N=C=O, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C=OOH, - CH ₂ C=OOH, -S=OCH ₃ , -S(=O) ₂ CH ₃ , -S(=O) ₂ OH, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(CH ₃ ) ₂ , -OH, -OCN, -OCH ₃ , -OCH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CHOHCH ₂ OH, -CHOHCH ₃ , -SH, - SCN, -SCH ₃ , -SCH ₂ CH ₃ , -CH ₂ SH, -CH ₂ CH ₂ SH, -CHSHCH ₂ SH and -CHSHCH ₃ . 3. The compound of any of the preceding features, wherein the compound comprises at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of said single stereoisomer. 4. The compound of any of the preceding features, wherein Y is bound to C through the bond z attached at carbon atom x of Y. 5. The compound of any of the preceding features, wherein the compound is selected from the group consisting of the following compounds:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 6. The compound of any of the preceding features, wherein the compound is a neutral antagonist or an inverse agonist at one or more of the 5-HT2A and 5-HT2C receptors. 7. The compound of any of the preceding features, wherein the compound does not cause sedation when administered to a subject at physiologically relevant levels. 8. The compound of any of the preceding features, wherein the compound comprises a greater binding affinity at 5-HT2A receptor and/or 5-HT2C receptor than at 5-HT2B receptor. 9. The compound of any of the preceding features, wherein the compound has a greater binding affinity for 5-HT2A receptor and 5-HT2C receptor than for 5-HT1A, 5- HT2B, 5-HT7, D2, D3, alpha1A, and/or alpha1B receptors. 10. The compound of any of the preceding features, wherein the compound is a neutral antagonist or an inverse agonist at a histamine (H1) receptor at physiologically relevant levels. 11. The compound of any of the preceding features, wherein the compound comprises a greater binding affinity at 5-HT2A receptors and/or 5-HT2C receptors than at the H1 receptor. 12. The compound of any of the preceding features, wherein the one or more moieties V and/or W comprise a positive and/or a negative charge at a physiological pH. 13. The compound of feature 12, comprising a pharmaceutically acceptable anion comprising acetate, adipate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, caprate, caproate, caprylate, carbonate, chloride, citrate, decanoate, dodecylsulfate, edetate, esylate, formate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, octanoate, oleate, oxalate, palmitate, pamoate, pantothenate, phosphate, dihydrogen phosphate dodecahydrate, dihydrogen phosphate dihydrate, polygalacturonate, propionate, sabacate, salicylate, stearate, acetate, succinate, sulfate, tartrate, teoclate, thiocyanate, tosylate, or undecylenate. 14. The compound of feature 12, comprising a pharmaceutically acceptable cation comprising aluminum, arginine, benzathine, calcium, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, lysine, magnesium, histidine, lithium, meglumine, potassium, procaine, sodium, triethylamine, or zinc. 15. The compound of any of the preceding features, wherein the compound comprises a hydrate or a solvate comprising one or more water molecules and/or one or more solvent molecules associated via hydrogen bonding and/or ionic bonding to the compound and/or to an anion or cation associated with the compound. 16. The compound of any of the preceding features, wherein the compound comprises one or more of ¹⁸ F, ¹⁹ F, ⁷⁵ Br, ⁷⁶ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹¹ C, ¹³ C, ¹³ N, ¹⁵ O, or ³ H. 17. The compound of any of the preceding features, wherein the compound selectively modulates a physiological activity of 5-HT2A and/or 5-HT2C receptors over a physiological activity of one or more of 5-HT1A, 5-HT2B, 5HT7, D2, D3, α1A, and α1B receptiors. 18. The compound of feature 17, wherein said selective modulation is associated with a difference in binding affinity, inverse agonism, agonism, partial agonism, 20. The pharmaceutical composition of feature 19 comprising an amount of said compound that aids in treating psychosis, fragile X syndrome, autism, substance use disorder, or an impulsive behavior. 21. The pharmaceutical composition of feature 19 comprising an amount of said compound that aids in treating hypertension, migraine, obesity, irritable bowel syndrome, Parkinson's disease, attention deficit hyperactivity disorder, anxiety or generalized anxiety, depression, schizophrenia, binge eating, opioid use disorder, amphetamine use disorder, panic disorder, social anxiety disorder, obsessive- compulsive disorder, pain, Alzheimer's disease, or Huntington's disease. 22. The pharmaceutical composition of feature 19, wherein the compound comprises (2R,4S)-(trans)-4-(3-(thiophen-2-yl)phenyl)-N,N-dimethyl-1,2 ,3,4- tetrahydronaphthalen-2-amine, (2R,4S)-(trans)-4-(3-(furan-2-yl)phenyl)-N,N- dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, or (2S,4S)-(cis)-4-([1,1'-biphenyl]-3- yl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine. 23. A method to aid in treating a disease or disorder, the method comprising administering an effective amount of a compound of any of features 1-18 to a mammalian subject in need thereof. 24. The method of feature 23, wherein the compound is administered as the pharmaceutical composition of any of features 19-22. 25. The method of feature 23 or feature 24, wherein said administering does not cause sedation, dizziness, and/or orthostatic hypotension. 26. The method of feature 23, wherein the disease or disorder is a neuropsychiatric disorder is selected from the group consisting of psychosis, fragile X syndrome, autism, substance use disorder, and impulsive behaviors. 27. The method of feature 23, wherein the disease or disorder is selected from the group consisting of hypertension, migraine, obesity, irritable bowel syndrome, Parkinson's disease, attention deficit hyperactivity disorder, anxiety or generalized anxiety, depression, schizophrenia, binge eating, opioid use disorder, amphetamine use disorder, panic disorder, social anxiety disorder, obsessive-compulsive disorder, pain, Alzheimer's disease, or Huntington's disease. 28. The method of any of features 23-27, wherein said administering results in selective modulation of a serotonin 5-HT2A or 5-HT2C receptor in the subject. 29. The method of feature 18, wherein the selective modulation comprises inverse agonism, agonism, partial agonism, allosteric agonism, antagonism, partial antagonism, allosteric antagonism, or a difference in binding affinity compared to a different receptor type. 30. Use of the compound of any of features 1-18 or the composition of any of features 19-22 to treat or prevent psychosis, fragile X syndrome, autism, substance use disorder, impulsive behaviors, hypertension, migraine, obesity, irritable bowel syndrome, Parkinson's disease, attention deficit hyperactivity disorder, anxiety or generalized anxiety, depression, schizophrenia, binge eating, opioid use disorder, amphetamine use disorder, panic disorder, social anxiety disorder, obsessive- compulsive disorder, pain, Alzheimer's disease, and/or Huntington's disease in a mammalian subject. 31. The use of feature 30, wherein said use does not cause sedation in the subject. 32. The use of feature 30, wherein said use does not agonize 5-HT2B receptors and/or does not antagonize H1 receptors in the subject. 33. A compound for selective modulation of one or more of peripheral serotonin 5- HT2A, 5-HT2B, and 5-HT2C receptors, the compound having a structure according to Formula I:

(Formula I); wherein E is a quaternary amine selected from the group consisting of - N ⁺ (CH ₃ ) ₃ , -N ⁺ (CH ₃ ) ₂ (CH ₂ CH ₃ ), -N ⁺ (CH ₃ )(CH ₂ CH ₃ ) ₂ , and -N ⁺ (CH ₂ CH ₃ ) ₃ ; wherein Y is selected from the group consisting of: and ; wherein covalent bond z is attached at any carbon atom of Y; wherein Y is unsubstituted or is substituted with one or more moieties V, each of the one or more moieties V independently selected from the group consisting -F, - Cl, -Br, -I, -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -NH(CH ₂ CH ₃ ), -N(CH ₂ CH ₃ ) ₂ , -C=NH, -C=NNH ₂ , -C=ONH ₂ , -NO ₂ , -NO, -CN, -N ₃ , -N=C=O, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C=OOH, - CH ₂ C=OOH, -S=OCH ₃ , -S(=O) ₂ CH ₃ , -S(=O) ₂ OH, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(CH ₃ ) ₂ , -OH, -OCN, -OCH ₃ , -OCH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CHOHCH ₂ OH, -CHOHCH ₃ , -SH, - SCN, -SCH ₃ , -SCH ₂ CH ₃ , -CH ₂ SH, -CH ₂ CH ₂ SH, -CHSHCH ₂ SH, -CHSHCH ₃ , and substituted or unsubstituted thiophene, furanyl, phenyl and pyridyl; and wherein the compound comprises at least 50% of a single stereoisomer selected from the group of stereoisomers consisting of 2R4R, 2S4S, 2R4S, and 2S4R; or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 34. The compound of feature 33, wherein one or more moieties V are independently selected from the group consisting of: and wherein V is attached to Y via a covalent bond to any one of carbons 5-7 of V; and wherein V is substituted with one or more substituents W, each of the one or more substituents W independently selected from the group consisting of -F, -Cl, - Br, -I, -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -NH(CH ₂ CH ₃ ), -N(CH ₂ CH ₃ ) ₂ , -C=NH, -C=NNH ₂ , - C=ONH ² , -NO ² , -NO, -CN, -N ³ , -N=C=O, -CH ³ , -CH ² CH ³ , -CH(CH ³ ) ² , -C=OOH, - CH ₂ C=OOH, -S=OCH ₃ , -S(=O) ₂ CH ₃ , -S(=O) ₂ OH, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(CH ₃ ) ₂ , -OH, -OCN, -OCH ₃ , -OCH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CHOHCH ₂ OH, -CHOHCH ₃ , -SH, - SCN, -SCH ₃ , -SCH ₂ CH ₃ , -CH ₂ SH, -CH ₂ CH ₂ SH, -CHSHCH ₂ SH and -CHSHCH ₃ . 35. The compound of feature 33 or 34, wherein the compound comprises at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of said single stereoisomer. 36. The compound of any of features 33-35, wherein Y is bound to C through the bond z attached at the carbon atom x of Y. 37. The compound of any of features 33-36, wherein the compound is selected from the group consisting of:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof. 38. The compound of any of features 33-37, wherein the compound is an antagonist, a neutral antagonist or an inverse agonist at one or more of the 5-HT2A, 5-HT2B, and 5-HT2C receptors. 39. The compound of any of features 33-38, wherein the compound has a greater binding affinity for 5-HT2A, 5-HT2B, and/or 5-HT2C receptors than for 5-HT1A, 5- HT7, D2, D3, alpha1A, and/or alpha1B receptors. 40. The compound of any of features 33-39, comprising a pharmaceutically acceptable anion selected from the group consisting of acetate, adipate, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate, bitartrate, bromide, camsylate, caprate, caproate, caprylate, carbonate, chloride, citrate, decanoate, dodecylsulfate, edetate, esylate, formate, fumarate, gluceptate, gluconate, glutamate, glycolate, hexanoate, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, octanoate, oleate, oxalate, palmitate, pamoate, pantothenate, phosphate, dihydrogen phosphate dodecahydrate, dihydrogen phosphate dihydrate, polygalacturonate, propionate, sabacate, salicylate, stearate, acetate, succinate, sulfate, tartrate, teoclate, thiocyanate, tosylate, and undecylenate. 41. The compound of any of features 33-40, wherein the compound comprises one or more of ¹⁸ F, ¹⁹ F, ⁷⁵ Br, ⁷⁶ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ¹¹ C, ¹³ C, ¹³ N, ¹⁵ O, or ³ H. 42. The compound of any of features 33-41, wherein the compound selectively modulates a physiological activity of 5-HT2A, 5-HT2B, and/or 5-HT2C receptors over a physiological activity of one or more of 5-HT1A, 5-HT7, 5HT7, D2, D3, α1A, and α1B receptors. 43. The compound of feature 42, wherein said selective modulation is associated with a difference in binding affinity, inverse agonism, agonism, partial agonism, allosteric agonism, antagonism, partial antagonism, or allosteric antagonism. 44. A pharmaceutical composition comprising a compound of any of features 33- 43 and an excipient. 45. The pharmaceutical composition of feature 44, wherein the compound comprises (2R,4S)-(trans)-4-(3-(thiophen-2-yl)phenyl)-N,N,N-trimethyl- 1,2,3,4- tetrahydronaphthalen-2-amine, (2R,4S)-(trans)-4-(3-(furan-2-yl)phenyl)-N,N,N- trimethyl-1,2,3,4-tetrahydronaphthalen-2-amine, or (2S,4S)-(cis)-4-([1,1'-biphenyl]-3- yl)-N,N,N-trimethyl-1,2,3,4-tetrahydronaphthalen-2-amine. 46. A method to aid in treating a disease or disorder, the method comprising administering an effective amount of a compound of any of features 33-43 or the pharmaceutical composition of any of features 44-45, to a mammalian subject in need thereof. 47. The method of feature 46, wherein the disease or disorder is selected from the group consisting of hypertension, thrombosis, deep vein thrombosis, pulmonary embolus, atrial fibrillation, atherosclerosis, valvular atherosclerosis, cardiac fibrosis, obesity, irritable bowel syndrome, and lack of bladder control. 48. The method of feature 47, wherein the subject further suffers from a neuropsychiatric disease or disorder, such as depression. 49. The method of any of features 46-48, wherein the method results in inverse agonism, antagonism, partial antagonism, or allosteric antagonism at a peripheral 5- HT-2A, 5-HT2B, and/or 5-HT2C receptor. As used herein, the term room temperature refers to a temperature within the range of about 15-30°C. 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". BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1A shows structures of the 4-phenyl-2-dimethylaminotetralin (4-PAT, 1) chemotype and derivatives with halogen (2a-2b', 3a-3b') or aryl substituents (2c-k and 3c-k) at the C(4)-phenyl meta position (Y). Fig. 1B shows structures of the 4-phenyl-2-dimethylaminotetralin (4-PAT, 1) chemotype and derivatives with halogen (2a-2b', 3a-3b') or aryl substituents (2c-k and 3c-k) at the C(4)-phenyl meta position (Y). Examples of charged substituent E or quaternary amine substituent E (top right) are -N ⁺ (CH ₃ ) ₃ , -N ⁺ (CH ₃ ) ₂ (CH ₂ CH ₃ ), -N ⁺ (CH ₃ )(CH ₂ CH ₃ ) ₂ , and - N ⁺ (CH ₂ CH ₃ ) ₃ ; Fig.2A shows exploratory functional screening results for reference ligands and 4-PAT analogs at 5-HT ₂ -type receptors (Rs) 5-HT _2A , 5-HT _2B , 5-HT _2C and H ₁ Rs. The percent change from basal accumulation of inositol monophosphate (IP1) in clonal cells transiently expressing human wild-type 5-HT _2A , 5-HT _2B , 5-HT _2C , or H ₁ Rs following incubation with 10 µM of the indicated ligand is displayed in Fig. 2A. Reference ligands (top) include 5-HT (5- hydroxytryptamine), HIS (histamine), DOX (doxepine), RIT (ritanserin), and pimavanserin (PIMA). The mean percent change in basal signaling for each compound is normalized to the change for the reference agonist (i.e., 5-HT or histamine), and is shown numerically in each cell as the mean of at least two independent experiments performed using three technical replicates. Crossed spaces indicate no data. Figs. 2B-2E show comparative functional assessments of PIMA and aryl substituted 4-PATs at 5-HT _2A , 5-HT _2C , 5-HT _2B and H ₁ Rs expressed in clonal cells. In Figs. 2B-2C, PIMA, (2S,4R)-2k, and (2R,4R)-3h demonstrate inverse agonist activity at (Fig.2B) constitutively activated C322K ^6.34 5-HT _2A Rs and (Fig.2C) WT 5-HT _2C Rs by attenuating basal (dotted line) IP1 accumulation. At Fig.2D, 5-HT _2B Rs, PIMA, (2S,4R)-2k, and (2R,4R)-3h competitively antagonize 5-HT-stimulated IP1 accumulation. At Fig.2E H1Rs, (2S,4R)-2k and (2R,4R)-3h competitively antagonize histamine-stimulated IP1 accumulation, with minimal competition by PIMA. Concentration-response curves represent the mean ± SD of n = 5 independent experiments performed using three (Fig.2B, Fig.2C) or two (Fig.2D, Fig.2E) technical replicates. Figs.3A-3C show comparative assessments of PIMA, (2S,4R)-2k and (2R,4R)-3h in male C57BL/6J mice. Fig.3A shows head-twitch response elicited by 1 mg*kg ^-1 (±)-DOI (s.c.) following pretreatment with vehicle, PIMA, (2S,4R)-2k, or (2R,4R)-3h. Fig. 3B shows locomotor activity of mice administered 1 mg*kg ^-1 (±)-DOI (s.c.) following pretreatment with vehicle, PIMA, (2S,4R)-2k, or (2R,4R)-3h. Fig.3C shows, after a 6-week washout period, the locomotor activity of the same mice from (3A) and (3B) is reassessed following administration (s.c.) of vehicle or 3 mg*kg ^-1 PIMA, (2S,4R)-2k, or (2R,4R)-3h. Data represent the mean ± SD of n = 6 to 7 treatments, individual values are shown for each condition. Significance is determined using one-way ANOVA, with Tukey's correction for multiple comparisons, *p< 0.05. Figs.4A-4C show proposed binding modes of PIMA, (2S,4R)-2k, and (2R,4R)-3h at a model of the 5-HT _2A R. Fig.4A shows the structure of PIMA at top and the proposed binding mode at the 5-HT _2A R at bottom. Fig. 4B shows the structure of (2S,4R)-2k at top and the proposed binding at the 5-HT _2A R mode at bottom. Fig.4C shows the structure of (2R,4R)-3h at top and the proposed binding mode at the 5-HT _2A R at bottom. Side chains within 4.0 Å of each ligand are shown, as well as F213 ^4.63 and D231 ^5.35 which are experimentally point mutated herein. For clarity, only the anchoring side chain of D155 ^3.32 is shown for residues in TM3. Figs.5A-5B show molecular dynamics studies demonstrating the structural basis of 4- PAT stereoselectivity at histamine H ₁ Rs. Fig. 5A shows the proposed binding mode of (2S,4R)-2k at the H ₁ R resembles that observed at the 5-HT _2A R (see Fig. 4B), where the aminotetralin moiety could form aromatic T-stacking interactions with the side chain of W428 ^6.48 , while the aryl substituent extends in a cavity between TM4 and TM5 where it could participate in aromatic T-stacking interactions with the side chain of W158 ^4.56 . Fig.5B shows the proposed binding mode of (2R,4R)-3h at the H1R indicates that a stereochemical restriction at the C(2)-position causes the aryl substituent to position between TM5 and TM6, thus disfavoring productive aromatic interactions between the ligand and the side chains of W158 ^4.56 and W428 ^6.48 . Figs.6A and 6B show X-ray crystal structures of (2R,4R)-3b (Fig.6A) and (2S,4S)-0U{ (Fig.6B). Coordinates are provided in the Examples section. Figs.7A-7B show follow up in vitro assessment of potential (2S,4R)-2k and (2R,4S)- 2c agonist activity at 5-HT _2B and 5-HT _2C Rs, respectively. Fig.7A shows analog (2S,4R)-2k compared to 5-HT at 5-HT _2B Rs. Fig.7B shows analogs (2S,4R)-2c and (2R,4S)-2c compared to 5-HT at 5-HT _2C Rs. Data are represented as the individual mean values from 5 - 8 independent experiments performed in triplicate, and the mean ± SD of all experiments for a given concentration of ligand. An asterisk * indicates statistically significant effects (p < 0.05) of ligand concentration on the accumulation of IP1, as determined by either an ordinary one- way ANOVA or Kruskal-Wallis test; ns, not significant. Fig.8 shows superimposed 5-HT _2A Rs stabilized in an inactive state by PIMA, (2S,4R)- 2k, or (2R,4R)-3h (light gray, dark gray, and medium gray receptor, respectively). Insets highlight features of an inactive GPCR, including the orientation of the W336 ^6.48 toggle switch being perpendicular to the lipid bilayer, as well as the state of the PIF motif, and the distance between R173 ^3.50 and E318 ^6.30 of the E/DRY domain being close enough such that an ionic bond could form. Fig. 9 shows activity of (2S,4R)-2a at constitutively activated C322K ^6.34 5-HT _2A Rs, showing analog (2S,4R)-2a behaves as an inverse agonist at C322K ^6.34 5-HT _2A Rs, eliciting a reduction in basal IP accumulation by ~50%. Data are shown as the mean ± SD of n = 5 independent experiments performed using three technical replicates. Fig.10A shows a top view (from extracellular to cytosol view) of (2S,4R)-2k after a 100 ns molecular dynamics simulation at the 5-HT _2A R shows the steric tolerance afforded by the side chain of G238 ^5.42 between TM4 and TM5. Fig.10B shows a plot of the minimum distance between (2S,4R)-2k and G238 ^5.42 showing the interaction is stable over 100 ns (X-axis). Figs. 11A-11G show visualizations of the change in potency of 5-HT and various antagonists at point mutated 5-HT _2A Rs. Fig.11A shows change in functional potency of 5-HT at point-mutated 5-HT _2A Rs. Fig.11B shows concentration response for 5-HT at WT and point mutated 5-HT _2A Rs, normalized to the percent change from the basal concentration inositol monophosphates (IP1). Figs. 11C-11G show antagonism of 1 µM 5-HT-mediated IP1 accumulation by five 5-HT _2A R antagonists (spanning three medicinal chemical chemotypes) at WT and point-mutated 5-HT _2A Rs. Data in (11A) are presented as the mean ΔpEC ₅₀ ± SD, data in (11C-11F) are presented as the mean ApKb _ SD, where ΔpEC ₅₀ - ΔpEC _{50(mutant) --} For clarity, the concentration response curves of 5-HT in Fig.11B are shown as the mean ± SEM. The number of independent experiments (n) performed for each condition is 5 19, with exact n shown in Table 2. The asterisk * indicates statistical significance (p < 0.05) between the wild-type and mutant receptor parameters, determined by an unpaired t-test or Mann-Whitney U-test where appropriate. Figs.12A-12B show superimpositions of a zotepine bound 5-HT _2A R (PDB: 6A94), LSD bound 5-HT _2B R (PDB: 5TVN), and ritanserin bound 5-HT _2C R (PDB: 6BQH). The structures indicate that a unique rotamer of F ^5.38 exists in the 5-HT _2A R (black ellipse, Fig.12A), where it is raised toward the extracellular end of TM4 through hydrophobic interactions with the non- conserved residue F ^4.63 (K ^4.63 and I ^4.63 in 5-HT _2B and 5-HT _2C Rs, respectively). Fig.13 shows comparative dynamics of F ^5.38 in 5-HT _2A and 5-HT _2B Rs. The side chain of F ^5.38 is more dynamic (measured by RMSD) in WT 5-HT _2A Rs (black trace) than it is in WT 5-HT _2B Rs (gray trace). When residue D5.35 in the 5-HT _2A R is mutated in silico to the equivalent residue (F5.35) in 5-HT _2B RS (gray trace), the dynamics of F ^5.38 in the D5.35F 5- HT _2A R bear greater resemblance to that of F ^5.38 in WT 5-HT _2B RS.

Figs. 14A-14D show exploratory saturation binding results indicating that specific binding of [ ³ H]mesulergine (Fig. 14A, Fig. 14B), [ ³ H]ketanserin (Fig. 14C), or [ ³ H]spiperone (Fig. 14D) cannot be detected for HEK293 cells transfected with cDNA encoding human D231 F ^5.35 5-HT _2A RS. Data are presented as single experiments with three technical replicates for total (black circles) and nonspecific binding (gray squares, determined using 30 μM mianserin and 30 μM risperidone).

DETAILED DESCRIPTION

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 5HT _2C agonists with 5HT _2A/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 , Fig. 1 B), thereby increasing the targeting efficacy of the technology. Mechanisms of single enantiomer specificity are elucidated. Synthesis and purification are described in detail. The technology can provide stereochemically-pure compounds having Cis-2R4R, Cis-2S,4S, trans- 2R4S, or trans-2S4R stereochemistry. In the examples, there are compounds for use disclosed herein that are (2R,4S)-trans-4-(3-(thiophen-2-yl)phenyl)-N,N-dimethyl-1 ,2,3,4- tetrahydronaphthalen-2-amine (2c) and (2R,4S)-trans-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl- 1 ,2,3,4-tetrahydronaphthalen-2-amine (2d), and (2S,4S)-Cis-4-([1 ,1'-biphenyl]-3-yl)-N,N- dimethyl-1 ,2,3,4-tetrahydronaphthalen-2-amine, (3f) (Fig. 1A, Table 1 ).

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 Fig. 1 B is charged and can be a quaternary amine or other charged moiety; for example, E can be -N ⁺ (CH ₃ ) ₃ , -N ⁺ (CH ₃ ) ₂ (CH ₂ CH ₃ ), - N ⁺ (CH ₃ )(CH ₂ CH ₃ ) ₂ , or -N ⁺ (CH ₂ CH ₃ ) ₃ . US 10548856B2, which is hereby incorporated by reference in its entirety, describes charged 5-PAT compounds and methods for modulating serotonin receptors in the periphery. Using compounds of the present technology, the peripherally restricted, charged 4-PAT compounds can preventing side effects caused by binding to 5-HT receptor sites in the CNS. In another example, 5HT _2B is not expressed in the brain for a desired treatment, and the technology may treat a peripheral disease.

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.

5HT _2C agonists with 5HT _2A/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 patents US 8586634B2, US 9024071 B2, US 9862674B2 and US 10017458B2, 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-HT _2A and 5-HT _2c are important neurotherapeutic targets, though, obtaining selectivity over 5-HT _2B and closely related histamine H ₁ Rs is challenging. Herein are delineated molecular determinants of selective binding to 5-HT _2A and 5-HT _2C Rs using novel 4-PATs.

Example compounds are compared to 5-HT, HIS, DOX, RIT and PIMA in exploratory functional screening at 5-HT ₂ -type receptors (Rs) 5-HT _2A , 5-HT _2B , 5-HT _2C and H ₁ Rs in Fig. 2A. The percent change from basal accumulation of inositol monophosphate (IP1) in clonal cells transiently expressing human wild-type 5-HT _2A , 5-HT _2B , 5-HT _2c , or H ₁ Rs following incubation with 10 μM is shown in the form of a heat map (Fig. 2A). Further comparisons are made in Figs. 2B-2C, wherein PIMA, (2S,4R)-2k, and (2R,4R)-3h (Table 1) show inverse agonist activity at (Fig. 2B) constitutively activated C322K ^6.34 5-HT _2A Rs and (Fig. 2C) WT 5-HT _2c Rs by attenuating basal (dotted line) IP1 accumulation. In Fig. 2D, 5-HT _2B Rs, PIMA, (2S,4R)-2k, and (2R,4R)-3h competitively antagonize 5-HT -stimulated IP1 accumulation. In Fig. 2E H ₁ Rs, (2S,4R)-2k and (2R,4R)-3h competitively antagonize histamine-stimulated IP1 accumulation, with minimal competition by PIMA. Affinity, function, molecular modeling, and 5-HT _2A R mutagenesis studies are undertaken to understand structure-activity relationships at 5-HT ₂ - type and H ₁ Rs. Lead 4-PAT selective 5-HT _2A /5-HT _2C R inverse agonists are compared to PIMA, a selective 5-HT _2A /5-HT _2C R inverse agonist approved to treat psychoses, in the mouse head twitch response (Fig. 3A) and locomotor activity assays (Fig. 3B, Fig. 3C), as models relevant to antipsychotic drug development.

Most 4-PAT diastereomers in the (2S,4R)-configuration bind non-selectively to 5-HT _2A , 5-HT _2C , and H ₁ Rs, with >100-fold selectivity over 5-HT _2B RS, whereas diastereomers in the (2R,4R)-configuration bind preferentially to 5-HT _2A over 5-HT _2c Rs and have > 100-fold selectivity over 5-HT _2B and H ₁ Rs. Results suggest that G238 ^5.42 and V235 ^5.39 (Figs. 4A-4C) in 5-HT _2A RS (conserved in 5-HT _2C RS) are important for high affinity binding, whereas interactions with T194 ^5.42 and W158 ^4.56 (Figs. 5A-5B) are important for H ₁ Rs. The 4-PAT (2S,4R)-2k, a potent and selective 5-HT _2A /5-HT _2C R inverse agonist, has activity like PIMA in the mouse head-twitch response assay but is distinct in not suppressing locomotor activity. The 4'-NMe ₂ - C ₆ H ₄ substituent on ring C (2k, 3k; Table 1 ), can be used as an example substituent to explore electronic and steric effects.

The novel 4-PAT chemotype can yield selective 5-HT _2A /5-HT _2C R 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 H ₁ Rs to help circumvent sedative effects. High homology between 5-HT ₂ -type and histamine H ₁ Rs could prevent development of antipsychotics without sedative effects.

The 4-phenyl-2-dimethylaminotetralin (4-PAT, Fig. 1A) configured in a (2S,4R)-1 chemotype can yield 5-HT _2C R agonists with antagonist/inverse agonist activity at 5-HT _2A , 5- HT _2B , and H ₁ Rs (Moniri, et al., 2004; Booth, et al., 2009). However, (2S,4R)-1 configurations can bind to H ₁ Rs with high affinity, and to 5-HT ₂ -type receptors with only moderate affinity. Bromine substitution at the meta position of 4-PAT ring C yields (2S,4R)-2a (Fig. 1A, Table 1) a ligand with lower affinity at H ₁ Rs, higher affinity at 5-HT ₂ -type receptors (Canal, et al., 2014; Sakhuja, et al., 2015), yet nil subtype-selectivity. Notably, (2S,4R)-2a demonstrates antipsychotic-like activity in several mouse models (Canal, et al., 2014). The current work can attain 4-PATs with selectivity for 5-HT _2A and/or 5-HT _2C RS over 5-HT _2B and H ₁ Rs (Canal, et al., 2014; Sakhuja, et al., 2015). Herein, 42 novel 4-PAT analogs are synthesized, including diastereomers of meta-halo substituted 4-PATs (3a-b', Fig. 1A), as well as aryl substituted analogs (2c-k, 3c-k, Fig. 1A).

Table 1. Affinity (p/Ki) of reference ligands and novel 2 -aminotetralins at 5-HT _2A , 5-HT _2B , 5- HT _2C , and H ₁ Rs ^a .

Structure-activity relationships (SAR) for 4-PATs acting at 5-HT ₂ -type and H ₁ Rs are developed using competitive radioligand displacement and functional assays. The data reveal that aryl substituted 4-PATs are potent 5-HT _2A -preferring 5-HT _2A /5-HT _2C R inverse agonists with selectivity over 5-HT _2B and H ₁ Rs. To understand the molecular determinants for selectivity and inverse agonism at 5-HT _2A RS, 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-HT _2A and 5-HT _2c Rs (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-HT _2A R 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- HT ₂ -type and H-iRs indicate that small meta-halo substituents (e.g., -F, -Cl, -Br) on ring C (Fig. 1 A) impart selectivity to bind H ₁ Rs over 5-HT ₂ -type receptors in the c/s-(2S,4S)-configuration (~40-280-fold). In contrast, aryl substituted 4-PATs in the Cis-(2R,4R)-configuration selectively bind 5-HT _2A RS over 5-HT _2B RS (-6-415-fold), 5-HT _2c Rs (~2-40-fold), and H ₁ Rs (-2-1, 300-fold), with the greatest selectivity over all receptors observed for the 3'-F-C ₆ H ₄ substituted analog (2R,4R)-3h (Table 1). Interestingly, aryl substituted 4-PATs in the trans- (2S,4R)-configuration selectively bind to 5-HT _2A and 5-HT _2c Rs over 5-HT _2B RS (-15-180-fold), although only minimal selectivity is achieved over H ₁ Rs (≤5-fold). An exception is the attainment of 38-fold selectivity for 5-HT _2A RS over H ₁ Rs with the 4'-NMe ₂ -C ₆ H ₄ substituted analog (2S,4R)-2k, potentially due to the 4'-NMe ₂ moiety forming unique van der Waals interactions within the binding pocket of 5-HT _2A and 5-HT _2c Rs. Notably, the -C ₆ H ₄ substituted analog (2S,4R)-2f, a nonselective 5-HT _2A / H ₁ R antagonist with ~70-fold selectivity over 5- HT _2B RS and ~5-fold selectivity over 5-HT _2c Rs, yields results here distinct from a prior report where it displayed high affinity and selectivity for 5-HT _2C Rs (Sakhuja, et al. , 2015). The reason for this discontinuity is unclear, although variability in the assay conditions (radioligand K _d , buffer, incubation time, and temperature) may be contributing factors. Whereas a previous report explored the trans-enantiomers of 2f as the sole aryl substituted 4-PAT, here are explored all four stereoisomers of 9 distinct aryl substituted 4-PATs, which yield stereochemically consistent SAR across all aryl substituted 4-PATs in the (2 S,4R)- configuration.

The affinity (p/Ki) 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-HT _2B (-50-3, 000-fold) and H ₁ Rs (-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-HT _2A and 5-HT _2c Rs comparable to PIMA and risperidone. While (2S,4R)-2k trends toward agonist activity at 5-HT _2B RS, this ligand may not pose cardiovascular concerns given its low potency and efficacy (Unett, et al., 2013). At 5-HT _2A RS, no rank order in the radioligand-derived affinity (pKii) is observed. However, when comparing the functionally derived affinity (pK _b ), 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-HT ₂ -type and H ₁ Rs in Table 1 , previous lab work shows that meta-halo substituted trans-4-PATs, including (2S,4R)-2a, display higher affinity at 5-HT ₂ -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 c/s-4-PATs at 5-HT ₂ -type receptors has not been reported. Here, it is shown that c/s-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 (pKii = 6.5-7.5) to low (pKii < 6.5) affinity at 5-HT ₂ -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 (pKii 3a > 3b > 3b'). The corresponding (2S,4S)-enantiomers, on the other hand, exhibit high affinity (pKii > 7.5) at H ₁ Rs, with robust selectivity (40-280-fold) over 5-HT ₂ -type receptors. Due to their unimpressive affinity at, and selectivity for, 5-HT ₂ -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 (Fig. 1A), reveals important SAR information about ligand binding at 5-HT ₂ -type and H ₁ Rs. Therefore, a variety of aromatic moieties are substituted at the meta-position of ring C, yielding 36 aryl substituted 4-PAT analogs (2c-k, 3c-k), and their affinity at 5-HT ₂ -type and H-iRs is determined (Table 1 ).

Heteroaromatic substitution on ring C (i.e., 2c-e and 3c-e) yields five-membered heterocycles (2c-d, 3c-d) which bind to 5-HT _2A , 5-HT _2C , and H ₁ Rs in the (2S,4R)- configuration with high affinity and moderate selectivity for 5-HT _2A and 5-HT _2c over 5-HT _2B RS (~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, H ₁ Rs. Meanwhile, (2R,4R)-3c preferentially binds to 5-HT _2A and 5-HT _2c Rs, with highest affinity at 5-HT _2A RS and moderate selectivity (~30- 60-fold) over 5-HT _2B and H ₁ Rs. The meta-pyridin-2'-yl (2e, 3e) analogs have moderate to low affinity at 5-HT ₂ -type receptors, however, (2S,4R)-2e and (2R,4R)-3e have high affinity at H ₁ Rs, 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-HT _2c Rs (Sakhuja, at al, 2015), it binds non- selectively to 5-HT _2A , 5-HT _2C , and H ₁ Rs, with moderate selectivity over 5-HT _2B RS (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-C ₆ H ₄ substituted diastereomer, (2R,4R)-3h, shows highest affinity at 5-HT _2A RS, with 33-fold selectivity over 5-HT _2c Rs, 415-fold selectivity over 5-HT _2B RS, and ~1 ,300-fold selectivity over H ₁ Rs, making it the most 5-HT _2A R-selective 4-PAT-type compound reported herein. Its diastereomer, (2S,4R)-2h, exhibits high affinity at 5-HT _2A and 5-HT _2c Rs, with ~30-fold selectivity over 5-HT _2B RS, and no selectivity over H ₁ Rs. 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-HT _2A RS.

The 4'-F-C ₆ H ₄ substituted analog (2S,4R)-2i has high affinity at 5-HT _2A and 5-HT _2C RS, 140-fold selectivity for 5-HT _2A RS over 5-HT _2B RS, though, selectivity over H ₁ Rs is modest (5- fold). Similarly, the 4'-CI-C ₆ H ₄ substituted analog (2S,4R)-2j has 143-fold selectivity for 5- HT _2A Rs over 5-HT _2B RS, and no selectivity over H ₁ Rs. The diastereomer, (2R,4R)-3j, retains high affinity for 5-HT _2A and 5-HT _2c Rs, and shows ~100- and ~225-fold selectivity for 5-HT _2A RS over 5-HT _2B and H ₁ Rs, respectively.

To explore electronic and steric effects, a 4'-NMe ₂ -C ₆ H ₄ substituent is introduced on ring C (2k, 3k), yielding stereoisomers with a computationally determined logP~5.69 (logD~3.77). Notably, these are slightly lower than that of 2i, 3i (logP~5.85, logD~3.86), 2j, 3j (logP~6.36, logD~4.41), and the other lead, (2R,4R)-3h (logP~5.85, logD~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-HT _2A RS over 5-HT _2c Rs (~7-fold) and high selectivity over 5-HT _2B and H ₁ Rs (>350-fold). In contrast, (2S,4R)-2k has similarly high affinity at both 5-HT _2A and 5-HT _2C RS, with high selectivity over 5-HT _2B Rs (~180-fold) and moderate selectivity over H ₁ Rs (~38-fold). Since (2S,4R)-2k portrays dual 5-HT _2A /5-HT _2C R activity with selectivity over 5-HT _2B and H ₁ Rs, (2S,4R)-2k is selected, along with the 5-HT _2A R 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-HT ₂ -type and H ₁ Rs. In this work, PIMA exhibited high affinity for 5-HT _2A RS, with modest selectivity (12-fold) over 5- HT _2C RS, and high selectivity over 5-HT _2B and H ₁ Rs (>3, 000-fold). Risperidone is equipotent to PIMA at 5-HT _2A RS and has high affinity at the other receptors. The results are consistent with those in the literature (Vanover, etaL, 2006; Chopko & Lindsley, 2018). Investigations of the functional activity of 4-PATs and PIMA at 5-HT ₂ RS and H ₁ Rs are conducted with the exploratory functional screening presented in Fig. 2A and continued to develop mechanistic specificity as is now described. To understand the impact of 4-PAT substitution and stereochemistry on the efficacy vector of functional signaling at 5-HT ₂ -type and H ₁ Rs, analogs 3a-k and 2c-k are subjected to exploratory screening at 10 μM in inositol monophosphate (IP1 ) accumulation assays in Fig. 2A. Fig. 2A displays the percent change from basal accumulation of IP1 in clonal cells transiently expressing human wild-type 5-HT _2A , 5-HT _2B , 5-HT _2C , or H ₁ Rs following incubation with 10 μM of the ligand indicated at the left. Reference ligands (top) include 5-HT (5-hydroxytryptamine), HIS (histamine), DOX (doxepine), RIT (ritanserin) and PIMA. The mean percent change in basal signaling for each compound is normalized to the change for the reference agonist (i.e., 5-HT or histamine), and is shown numerically in each cell as the mean of at least two independent experiments performed using three technical replicates. Crossed spaces indicate no data. In Fig.2A, no 4- PAT-type compound tested activates 5-HT _2A RS or H ₁ Rs. At 5-HT _2B RS, however, analogs (2S,4R)-2c, (2S,4S)-3f, and (2S,4R)-2k appear to display partial agonist efficacy (12-14% 5- HT), with lead, (2S,4R)-2k, displaying -13% efficacy. Therefore, full concentration-response assays are undertaken for (2S,4R)-2k at 5-HT _2B RS (Fig. 7 A), and while a trend toward receptor activation is observed in Fig. 7A, the response did not reach statistical significance due to unequal variance between concentrations. In Fig. 2A at 5-HT _2C RS, only (2R,4S)-2c, (2R,4S)- 2d, and (2S,4S)-3f display apparent partial agonist efficacy (19-31% 5-HT). In concentrationresponse assays, however, one of the most efficacious analogs, (2R,4S)-2c provides no consistent effect on IP1 -accumulation (Fig. 7B). In contrast, the (2S,4R)-2c enantiomer demonstrates inverse agonism at 5-HT _2c Rs (pIC ₅₀ = 6.60 ± 0.27, l _max = 58% below basal; Fig. 7B). Thus, the novel 4-PAT-type ligands reported here have neutral antagonist or inverse agonist activity at 5-HT ₂ -type and H ₁ Rs.

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- HT _2A and 5-HT _2C RS. Therefore, these compounds are comparatively assessed in concentration-response assays using clonal cells (Fig. 2B) expressing constitutively activated C322K ^6. - ³⁴ 5-HT _2A RS (Egan, et a/., 1998) or WT 5-HT _2C Rs (Figs. 2C-2E). In Figs. 2B-2C, PIMA, (2S,4R)-2k, and (2R,4R)-3h demonstrate inverse agonist activity at constitutively activated C322K ^6.34 5-HT _2A RS (Fig. 2B) and WT 5-HT _2C Rs (Fig. 2C) by attenuating basal (dotted line) IP1 accumulation. In Fig. 2D, 5-HT _2B RS, PIMA, (2S,4R)-2k, and (2R,4R)-3h competitively antagonize 5-HT-stimulated IP1 accumulation. In Fig. 2E, H ₁ Rs, (2S,4R)-2k and (2R,4R)-3h competitively antagonize histamine-stimulated IP1 accumulation, with minimal competition by PIMA. Concentration-response curves represent the mean ± SD of n = 5 independent experiments performed using three (Fig. 2B, Fig. 2C) or two (Fig. 2D, Fig. 2E) technical replicates. It is found that PIMA has 5- and 20-fold higher potency at C322K ^6.34 5-HT _2A RS than

(2S,4R)-2k or (2R,4R)-3h (plC ₅₀ = 8.12 ± 0.18, 7.43 ± 0.17, and 6.81 ± 0.39, respectively, Fig.

2B), with each compound showing comparable inverse agonist efficacy (-60% below basal).

The pIC ₅₀ values are in excellent agreement with the corresponding pK _b values at WT 5-

HT _2A RS (Table 2). At 5-HT _2c Rs, PIMA and (2S,4R)-2k are equipotent (pIC ₅₀ = 6.55 ± 0.42 and

6.64 ± 0.21 , respectively) and similarly efficacious (-75% below basal), whereas an IC ₅₀ value of (2R,4R)-Cis-3h at 5-HT _2c Rs cannot be detected, although it exhibits inverse agonism (Fig.

2C). At off-target 5-HT _2B RS, PIMA, (2S,4R)-2k, and (2R,4R)-3h display low-potency competitive antagonism of 5-HT-mediated IP1 accumulation (pK _b = 5.86 ± 0.64, 5.59 ± 0.50, and 5.34 ± 0.24, respectively), although only PIMA reduces IP accumulation to basal levels at the concentrations used (Fig. 2D). Low-potency competitive antagonism is also observed with

(2S,4R)-2k and (2R,4R)-3h at H ₁ Rs (pK _b = 6.33 ± 0.24 and 5.82 ± 0.22, respectively), although the concentration of IP1 is not reduced to basal, and little, if any, antagonism is observed with

PIMA (Fig. 2E).

Table 2. Functional potencies of reference and 2-aminotetralin antagonists (pK _b ), and 5-HT (pEC ₅₀ ), at 5-HT _2A R variants ^a,b . aThe equilibrium dissociation constant (p/K _b ) of 5-HT _2A R antagonists is determined in the presence of 1 μM 5-HT. ^b Data are presented as the pK _b ± SD or pEC ₅₀ ± SD for the number of independent experiments indicated in parenthesis, see Figs. 11A-11G for data visualization. ^c Not calculable. Asterisk * indicates statistical significance (p < 0.05) between the wild-type and mutant receptor parameters using an unpaired t-test.

In Figs. 3A-3C comparative assessments of PIMA, (2S,4R)-2k and (2R,4R)-3h in male

C57BL/6J mice are shown. In Fig. 3A, in vivo studies comparing PIMA to (2S,4R)-2k, and

(2R,4R)-3h indicate that each ligand attenuates the (±)-DOI-elicited head twitch response, a model sensitive to antipsychotic-like activity, apparently through action at 5-HT _2A /5-HT _2C RS and not 5-HT _1A , α _1A -, D ₂ , or D ₃ Rs (Canal & Morgan, 2012). When administered alone, PIMA also suppresses locomotor activity in mice, whereas (2S,4R)-2k and (2R,4R)-3h are behaviorally selective to attenuate the head twitch response while preserving general locomotor ability (Fig. 3B, Fig. 30). Fig. 3A shows head-twitch response elicited by 1 mg*kg ^-1 (±)-DOI (s.c.) following pretreatment with vehicle, PIMA, (2S,4R)-2k, or (2R,4R)-3h. Fig. 3B shows locomotor activity of mice administered 1 mg*kg ^-1 (±)-DOI (s.c.) following pretreatment with vehicle, PIMA, (2S,4R)-2k, or (2R,4R)-3h. Fig. 3C shows, after a 6-week washout period, the locomotor activity of the same mice from (3A) and (3B) is reassessed following administration (s.c.) of vehicle or 3 mg*kg ^-1 PIMA, (2S,4R)-2k, or (2R,4R)-3h. Data represent the mean ± SD of n = 6 to 7 treatments, individual values are shown for each condition. Significance is determined using one-way ANOVA, with Tukey’s correction for multiple comparisons, *p < 0.05. Compared to mice pretreated with saline before injection of 1 mg*kg" ¹ DOI, locomotor suppression in mice pretreated with 0.3 mg*kg ^-1 PIMA is observed, but not 0.3 mg*kg ^-1 (2S,4R)-2k (Fig. 3B). In fact, the distance traveled by mice pretreated with 0.3 mg*kg ^-1 PIMA is significantly less than that of mice pretreated with 0.3 mg*kg ^-1 (2S,4R)-2k.

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 (Fig. 3C). These results indicate that (2S,4R)-2k is behaviorally selective in modulating the head twitch response.

Some 2-aminotetralins substituted at the C(5)- or C(8)-position have high affinity at 5- HTIA and 5-HT/Rs (Perry, et al., 2020), while others target D ₂ -like receptors (Seiler & Markstein, 1984). Furthermore, affinity at 5-HT _2B and α _1B -adrenergic receptors may predict ligand promiscuity (Peters, et al., 2012), and high affinity antagonism of central α _1A/1B - adrenergic receptors is associated with adverse events such as orthostatic hypotension, dizziness, and sedation (Andersson & Gratzke, 2007). Similarly, antagonism of central H ₁ Rs 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-HT _2A /5-HT _2C RS over 5-HTIA, 5HT _2B , 5HT ₇ , D ₂ , D3, α _1A - and α _1B -adrenergic receptors (>100- fold), whereas selectivity over H ₁ Rs is moderate for (2S,4R)-2k (~38-fold).

To understand how aryl substituted 4-PATs and PIMA bind to 5-HT _2A RS, molecular modeling studies are performed using a model of the 5-HT _2A R (Figs. 4A-4C). Figs. 4A-4C show proposed binding modes of PIMA, (2S,4R)-2k, and (2R,4R)-3h in the model of the 5- HT _2A R. The mechanism by which (2S,4R)-2k and (2R,4R)-3h selectively bind 5-HT _2A /5- HT _2C RS involves aryl ring D occupying a cavity in 5-HT _2A and 5-HT _2c Rs afforded by the small side chain of G ^5.42 , a residue unique to 5-HT ₂ -type receptors and key structural determinant of polypharmacology (Peng, et al., 2018). Residue G ^5.42 is depicted in Figs. 4A-4C in the model of the 5-HT _2A R. The SAR results indicate that larger aryl meta-substituents on ring C (e.g., 3a, 3b, 3b', Table 1 ) can yield moderate to high selectivity to bind 5-HT _2A RS over 5-HT _2B RS in the (2S,4R)-configuration, and over 5-HT _2B , 5-HT _2C and H ₁ Rs in the (2R,4R)-configuration. PIMA selectively binds 5-HT _2A RS over 5-HT _2B and H ₁ Rs, with moderate selectivity over 5-HT _2c Rs. Fig. 4A shows the structure of PIMA at top and the proposed binding mode at the 5-HT _2A R at bottom. Fig. 4B shows the structure of (2S,4R)-2k at top and the proposed binding at the 5- HT _2A R mode at bottom. Fig. 40 shows the structure of (2R,4R)-3h at top and the proposed binding mode at the 5-HT _2A R at bottom. Side chains within 4.0 A of each ligand are shown, as well as F213 ^4.63 and D231 ^5.35 which are experimentally point mutated herein. For clarity, only the anchoring side chain of D155 ^3.32 is shown for residues in TM3.

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 (pK _b ) at G238S ^5.42 5-HT _2A RS, whereas the affinity of (2S,4R)-2a, which lacks aryl ring D, is less affected. Confounding is that 5-HT _2B RS also present G ^5.42 , yet aryl substituted 4- PATs do not bind with high affinity to 5-HT _2B RS. The molecular modeling results with the 5- HT _2A R bound to inverse agonists PIMA, (2S,4R)-2k and (2R,4R)-3h reveal similar binding modes for each ligand (Fig. 4A, Fig. 4B, Fig. 4C). All compounds dock close enough to the side chain of D155 ^3.32 (Ballesteros-Weinstein numbering system, Ballesteros, 1995) such that an ionic bond can form with their respective basic amine moieties, a highly conserved interaction critical to the binding of most ligands across aminergic GPCRs (Kristiansen, et al., 2000; Vass, et al., 2019). Each ligand can also interact with conserved residues in TM3, including V156 ^3.33 , S169 ^3.36 , and T160 ^3.37 (Table 3).

Interestingly, aryl substituted 4-PATs in the (2S,4R)-configuration have high affinity for H ₁ Rs (Table 1), despite the presence of T194 ^5.42 , which possesses a bulkier side chain than serine. The molecular modeling results suggest that W158 ^4.56 , a residue unique to H ₁ Rs, might form stereospecific aromatic interactions with 4-PATs to impart high affinity (Fig. 5A, Fig. 5B). For example, ring D of (2S,4R)-2k positions close to TM4, where it could form optimal T- shaped interactions with W158 ^4.56 , while ring B of the aminotetralin core could form edge-to- face aromatic interactions with W428 ^6.48 . In contrast, ring D of (2R,4R)-3h orients toward TM5, potentially, due to a stereochemical restriction at the C(2)-position. Interactions with residues in TM5 might be disfavored by a negative steric interaction with T194 ^5.42 , and cause ring D to position between TM5 and TM6, precluding optimal aromatic interactions between ring B and the side chain of W428 ^6.48 . To validate the proposed model, a W158I ^4.56 H ₁ R is generated. However, W158I ^4.56 H ₁ Rs are unable to stimulate IP1 accumulation in response to histamine, and specific binding of [ ³ H]mepyramine or [ ³ H]ketanserin cannot be detected.

Overall, PIMA, (2S,4R)-2k, and (2R,4R)-3h stabilize an inactive-like conformation of the 5-HT _2A R, typified by an ionic lock between R173 ^{3 50} and E318 ^6.30 within the E/DRY domain (Fig. 8). The ionic lock may restrict the intracellular end of TM6 from outward displacement, and thus inhibit productive Gα _q -coupling and accumulation of inositol phosphates (Shapiro, et al., 2002). Clues to how the inactive state is stabilized are found at the ligand -receptor interface. For example, the simulations indicate that the fluorobenzyl ring of PIMA, as well as the aminotetralin core of (2S,4R)-2k and (2R,4R)-3h, may situate deep in the hydrophobic cleft of the orthosteric binding pocket. In this way, the fluorobenzyl and aminotetralin moieties can interact directly with I163 ^3.40 and F332 ^6.44 of the conserved P246 ^5.50 -I163 ^3.40 -F322 ^6.44 motif, thought to be involved in the activation mechanism of 5-HT ₂ -type GPCRs (Kim, et al., 2020; Kimura, et al., 2019; Peng, et al., 2018). Similar interactions are observed between these moieties and the side chain of W336 ^6.48 , a ‘toggle switch’ potentially mediating on/off states of class A GPCRs (Kim, et al., 2020; Peng, et al., 2018; Rasmussen, et al., 2011; Visiers, et al., 2002) (Fig. 4A, Fig. 4B, Fig. 4C, Fig. 8). Moreover, PIMA, (2S,4R)-2k, and (2R,4R)-3h can form edge-to-face aromatic interactions with the side chains of F243 ^5.47 and F340 ^6.52 , while π -cation interactions can form between the side chain of F339 ^6.51 and the basic nitrogen of the PIMA piperidine fragment or the tertiary amine of (2S,4R)-2k and (2R,4R)-3h.

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 G238 ^5.42 , a residue unique to 5-HT ₂ -type receptors among aminergic GPCRs. Furthermore, in all models, F234 ^5.38 assumes a rotamer conformation oriented away from G238 ^5.42 , 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 (1210 ^4.60 , V235 ^5.39 , G238 ^5.42 , and S242 ^5.46 ) 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-HT _2A RS. In Table 3, conserved residues are highlighted in parenthesis; results for point-mutated 5-HT _2A R residues in bold (with quotation marks) are reported herein.

Table 3. Residues within 4.0 A of docked PIMA, (2S,4R)-2k, or (2R,4R)-3h at 5-HT _2A RS following molecular dynamics simulations, and equivalent residues at 5-HT _2B , 5-HT _2C , and Hi GPCRs.

To validate the molecular modeling results, residues are point-mutated in and around the 5-HT _2A R side-extended cavity (Kimura, et al., 2019) and quantified the antagonist affinity (pK _b ) of (2S,4R)-2k and (2R,4R)-3h, as well as (2S,4R)-2a (which lacks 5-HT ₂ R subtype selectivity) at 5-HT _2A R 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 C322K ^6.34 5-HT _2A Rs (Fig. 9). An assessment is also made of the antagonist affinity of PIMA and risperidone at point-mutated 5-HT _2A RS, which represent selective and promiscuous 5-HT _2A R ligands, respectively.

A G238S ^5.42 5-HT _2A R 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 (Fig. 10A, Fig. 10B) and reported elsewhere for PIMA (Kimura, et al., 2019). Compared to WT 5-HT _2A RS, a modest, but significant, decrease in the pK _b of risperidone and (2S,4R)-2a at G238S ^5.42 5-HT _2A RS is observed. Moreover, the affinity of PIMA, (2S,4R)-2k, and (2R,4R)-3h is nearly abolished at G238S ^5.42 5-HT _2A Rs (Table 2, Fig. 11F). Notably, the A(pK _b ) for (2S,4R)-2a is less than that of (2S,4R)-2k and (2R,4R)-3h suggesting a size- dependent negative steric interaction between the 4-PAT ring C substituent and S ^5.42 . Also, a significant reduction in the potency is observed, but not the efficacy, of 5-HT at G238S ^5.42 compared to WT 5-HT _2A RS (Table 2, Fig. 11 A, Fig. 11 B), consistent with previous reports (Kimura, et al., 2019).

The experiments are extended by asking if the attenuated affinity of (2S,4R)-2a, (2S,4R)-2k, and (2R,4R)-3h at G238S ^5.42 5-HT _2A Rs translates to aminergic GPCRs natively presenting S ^5.42 . Table 4 shows that (2S,4R)-2k and (2R,4R)-3h have >1 ,000-fold selectivity for 5-HT _2A RS over 5-HTIA, 5-HT ₇ , D2L, CHA- and aiB-adrenergic GPCRs. It is noted that (2S,4R)- 2k has 270-fold selectivity over DsRs, whereas (2R,4R)-3h has >1 ,000-fold selectivity. In contrast, (2S,4R)-2a exhibits moderate-to-high affinity for 5-HT?, D2L, DS, and a-iA-adrenergic receptors.

Table 4. Affinities (pKi) of (2S,4R)-2a, (2S,4R)-2k, and (2R,4R)-3h at aminergic GPCRs natively presenting s ^5.42a .

Alignment of 5-HT _2A , 5-HT _2B and 5-HT _2c R crystal structures (Fig.12A, Fig. 12B) indicates that a side chain rotamer of F234 ^5.38 , unique to 5-HT _2A RS (Kimura, et al., 2019), orients toward the extracellular end of TM4 to form a side-extended cavity potentially due to hydrophobic interactions with F213 ^4.63 . In contrast, K193 ^4.63 and I192 ^4.63 in 5-HT _2B and 5- HT _2C RS, respectively, do not form productive interactions with F ^5.38 and may restrict the side cavity (Kimura, et al., 2019). However, no experimental studies have been reported to test this hypothesis. Therefore, a F213K ^4.63 5-HT _2A R is generated to test the hypothesis that the selectivity of PIMA, (2S,4R)-2k, and (2R,4R)-3h to bind 5-HT _2A RS relies on an interaction between F213 ^4.63 and F234 ^5.38 . Unexpectedly, a change in the pK _b of any antagonist tested at F213K ^4.63 5-HT _2A RS is not observed (Table 2, Fig. 11 D). However, a decrease in the potency of 5-HT at F213K ^4.63 5-HT _2A RS is observed, but not its efficacy (Table 2, Fig. 11 A, Fig. 11 B). These results do not support the hypothesis that F213 ^4.63 mediates subtype selective binding of inverse agonists at 5-HT _2A RS, however, F213 ^4.63 may be involved in 5-HT binding.

Further inspection of the 5-HT ₂ -type receptor crystal structures reveals that one helical turn above F ^5.38 in 5-HT _2A and 5-HT _2c Rs exists a non-conserved residue in 5-HT _2B RS (D231 ^5.35 , D211 ^5.35 , and F214 ^5.35 , respectively). The root-mean-square deviation (RMSD) of the F ^5.38 side chain in WT 5-HT _2A and 5-HT _2B RS is tracked in silico and found that F ^5.38 exhibited large transient variations in RMSD in WT 5-HT _2A RS. Interestingly, the RMSD of F ^5.38 in D231 F ^5.35 5- HT _2A RS recapitulates the restricted pattern observed in silico for WT 5-HT _2B RS, indicating that D231 ^5.35 may facilitate flexibility in the side chain of F ^5.38 (Fig. 13).

It is therefore hypothesized that D231 ^5.35 may modulate the side chain rotamer of F234 ^5.38 in 5-HT _2A RS to mediate subtype selective binding. To test this hypothesis, a D231 F ^5.35 5-HT _2A R is generated, however, D231 F ^5.35 5-HT _2A RS are insufficiently responsive to 5-HT for competitive antagonism studies (Fig. 11 B), thus, antagonist activity cannot be experimentally determined. Furthermore, no specific binding is detected for [ ³ H]ketanserin, [ ³ H]mesulergine, or [ ³ H]spiperone in exploratory studies using membranes from cells transfected with cDNA encoding D231 F ^5.35 5-HT _2A RS (Figs. 14A-14D).

Residues in TM4 and TM5 lining the side-extended cavity of 5-HT _2A RS and in proximity to PIMA, (2S,4R)-2k, and (2R,4R)-3h (Table 3) are then investigated. Among these are the side chains of I21O ^4.60 , V235 ^5.39 , and S242 ^5.46 . Importantly, the side chains of I210 ^4.60 and V235 ^5.39 are conserved in 5-HT _2c Rs, while S242 ^5.46 is unique to 5-HT _2A RS. It is hypothesized that selectivity to bind 5-HT _2A and 5-HT _2C RS over 5-HT _2B RS may involve interactions with the side chains of I ^4.60 , V ^5.39 , or the 5-HT _2A R-specific residue S242 ^5.46 . In fact, a significant increase in the affinity of PIMA and (2S,4R)-2k at V235M ^5.39 5-HT _2A RS is observed, with no change in affinity for any antagonist at 1210V ^4.60 or S242A ^5.46 5-HT _2A Rs (Table 2, Fig. 11C, Fig. 11 E, Fig. 11 G). Interestingly, the potency of 5-HT (but not the efficacy) is attenuated at V235M ^5.39 and S242A ^5.46 but not 1210V ^4.60 5-HT _2A RS, consistent with other reports investigating 1210V ^4.60 and S242A ^5.46 5-HT _2A RS (Table 2, Fig. 11 A, Fig. 11 B) (Kimura, et a/., 2019).

To explain the observed selectivity of PIMA, (2S,4R)-2k, and (2R,4R)-3h to bind 5- HT _2A RS over 5-HT _2B RS, the focus is made on non-conserved residues which line the 5-HT _2A R side extended cavity. The results indicate that the affinity and selectivity of inverse agonists to bind 5-HT _2A RS does not individually involve the side chains of 1210 ^4.60 , F213 ^4.63 , V235 ^5.39 , or S242 ^5.46 , since point mutation to the equivalent residues in 5-HT _2B RS does not attenuate their affinity. Importantly, the finding that selective inverse agonists have similar affinity at F213K ^4.63 and WT 5-HT _2A RS suggests a refinement to the current understanding of subtype selective binding at 5-HT _2A Rs (Kimura, et al., 2019). An attempt is made to identify a 5-HT _2A R residue other than F213 ^4.63 which may impact the side chain rotamer of F ^5.38 , and the computational results directed this work to D231 ^5.35 . However, D231 F ^5.35 5-HT _2A Rs lack sufficient Ga _q 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 A from D231 ^5.35 in the molecular models herein, thus, direct interactions are unlikely. Instead, it is speculated that D231 F ^5.35 may hinder proper protein folding and trafficking of the receptor to the membrane.

In an embodiment, the 4'-NMe ₂ -C ₆ H ₄ substituent introduced on ring C (2k, 3k) in the stereoisomers is utilized to determine molecular determinants of selective binding to 5-HT _2A and 5-HT _2c Rs including substituents of a length about that of NMe ₂ on ring D (Fig. 1A, Fig. 1 B, lower right), for example, -F, -Cl, -Br, -I, -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -N ⁺ (CH ₃ ) ₃ , , - N ⁺ (CH ₃ ) ₂ (CH ₂ CH ₃ ), -N ⁺ (CH ₃ )(CH ₂ CH ₃ ) ₂ , -N ⁺ (CH ₂ CH ₃ ) ₃ , -NH(CH ₂ CH ₃ ), -N(CH ₂ CH ₃ ) ₂ , -C=NH, - C=NNH ₂ , -C=ONH ₂ , -NO ₂ , -NO, -CN, -N ₃ , -N=C=O, -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C=OOH, - CH ₂ C=OOH, -S=OCH ₃ , -S(=O) ₂ CH ₃ , -S(=O) ₂ OH, -S(=O) ₂ NH ₂ , -S(=O) ₂ N(CH ₃ ) ₂ , -OH, -OCN, - OCH ₃ , -OCH ₂ CH ₃ , -CH ₂ OH, -CH ₂ CH ₂ OH, -CHOHCH ₂ OH, -CHOHCH ₃ , -SH, -SON, -SCH ₃ , - SCH ₂ CH ₃ , -CH ₂ SH, -CH ₂ CH ₂ SH, -CHSHCH ₂ SH, and -CHSHCH ₃ . In an embodiment, carbon- hydrogen (C-H) bonds have a length of about 1.09 A, while C-N single bonds have a length of about 1.48 A. The length of a fluorine atom is about 1.47 A, and hydrogen is about 1.2 A. In an embodiment, the substituent introduced on ring D can have a length measured extending from ring D, not including the bond from substituent to ring D, in the range from about 1 .0 A to about 25 A, or in the range from about 1.0 A to about 10 A.

Like the observations with 5-HT _2B RS, the selectivity of some aryl substituted 4-PATs to bind 5-HT _2A over H ₁ Rs cannot be explained by the results with G238S ^5.42 5-HT _2A RS. For example, H-iRs present T194 ^5.42 , yet aryl substituted 4-PATs in the (2S,4R)-configuration, but not the (2R,4R)-configuration, bind to H ₁ Rs with high affinity. Molecular dynamics studies revealing the structural basis of 4-PAT stereoselectivity at histamine H ₁ Rs are shown in Figs. 5A-5B. Fig. 5A shows the proposed binding mode of (2S,4R)-2k at the H1R resembles that observed at the 5-HT _2A R (Fig. 4B), where the aminotetralin moiety could form aromatic T- stacking interactions with the side chain of W428 ^6.48 , while the aryl substituent extends in a cavity between TM4 and TM5 where it could participate in aromatic T-stacking interactions with the side chain of W158 ^4.56 . Fig. 5B shows the proposed binding mode of (2R,4R)-3h at the H1R indicates that a stereochemical restriction at the C(2)-position causes the aryl substituent to position between TM5 and TM6, thus disfavoring productive aromatic interactions between the ligand and the side chains of W158 ^4.56 and W428 ^6.48 . The modeling studies indicate that high affinity binding of aryl substituted 4-PATs in the (2 S,4R)- configuration may be afforded by stereospecific aromatic interactions with W158 ^4.56 , a residue unique to H ₁ Rs among aminergic GPCRs and critical to histamine, mepyramine and (2S,4R)- 1 binding (Cordova-Sintjago, et al., 2012). Such interactions may position ring D of aryl substituted 4-PATs in the (2S,4R)-configuration in a cleft between TM4 and TM5, toward TM4 and away from T194 ^5.42 . In contrast, T194 ^5.42 may preclude high affinity binding of aryl substituted 4-PATs in the (2R,4R)-configuration due to a stereochemical restraint at the C(2)- position, causing ring D to disfavor aromatic interactions with W158 ^4.56 . It is speculated that these findings can also explain the selectivity of (2S,4S)-3a, 3b, and 3b' to bind H ₁ Rs, as the meta-halo substituent on ring C may form van der Waals interactions with W158 ^4.56 , while interactions with l/V ^4.56 in 5-HT ₂ -type receptors might be too weak to support high affinity binding. Together, the results echo work from other labs showing T194 ^5.42 mediates stereoselective binding of H1R antagonists, such as: (R)- and (S)-hydroxyzine, (R)- and (S)- cetirizine, as well as, (R)-ubc-29992 and (S)-ubc-29993 (Gillard, et al., 2002; Moguilevsky, et al., 1995). These authors speculated that residues such as K191 ^5.39 may influence stereospecific interactions with T194 ^5.42 (Gillard, et al., 2002), while others suggested that W158 ^4.56 may impact selective binding to H ₁ Rs (Shimamura, et al., 2011). Based on knowledge of the literature, this is the first report to suggest W158 ^4.56 influences the way stereoisomers interact with T194 ^5.42 .

The research disclosed here details the discovery of a novel series of selective 5- HT _2A /5-HT _2C R 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-HT _2B RS are challenging, though evidence is provided herein that F213 ^4.63 is not a molecular determinant of subtype-selective inverse agonist binding at 5-HT _2A RS. Also G228S ^5.42 5-HT _2A RS are identified as a point mutated 5-HT _2A R which may predict ligand selectivity over several aminergic GPCRs. In most cases, the mutagenesis studies reveal only minor changes in antagonist affinity, i.e., A(pK _b >) ≤ 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-HT _2A RS. In line with this, herein is reported a nonconserved, stereochemically sensitive molecular ensemble for H ₁ R 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-HT _2A RS, and preclinical characterization of analogs like (2S,4R)-2k in animal models of psychosis.

These data show that antagonist activity at G238S ^5.42 5-HT _2A RS is predictive of ligand selectivity over several aminergic GPCRs, and non-conserved residues in transmembrane domains 4 and 5 of the H-iR mediate stereoselective ligand binding. The clinical significance is relevant because understanding the molecular determinants of selective binding over H ₁ Rs 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

1. Schizophrenia.

An example of psychiatric therapeutic indications for 5-HT _2A and 5-HT _2C inverse agonists or antagonists is schizophrenia. As documented in Casey, et al., 2022 and references therein, reduction of serotonin 5-HT _2A 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-HT _2c 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). 2. Psychoses.

Regarding psychoses, as documented in Casey, et al., 2022, inverse agonist activity at the serotonin 5-HT _2c 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-HT _2A /5-HT _2C 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-HT _2A /5-HT ₂ C receptor inverse agonist PIMA is undergoing clinical assessment for efficacy to treat psychosis in Alzheimer’s disease (Caraci, et al., 2020; Ballard, et a/., 2020; Ballard, et al., 2018; Tariot, et al., 2021).

3. Depression.

As documented in Casey, et al., 2022, the novel compounds reported demonstrate inverse agonism of 5-HT _2c receptors which is thought to be pharmacotherapeutic for major depression (reference in Casey, et al., 2022; Demireva et al., 2018).

4. Anxiety.

Regarding anxiety, as documented in Casey, et al., 2022, the novel compounds reported demonstrate inverse agonism of 5-HT _2C 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, eta/., 2022 and analogs thereof (Fluyau, et al., 2022; Simonoff, eta/., 2008; El Haj, et a/., 2020; Wen, et a/., 2016).

Examples of non-psychiatric therapeutic indications:

B. Thrombosis

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 5HT _2A 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 5HT _2A receptor antagonism is known (e.g., Lin, et al., 2014), most 5HT _2A 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 5HT _2A antagonist or inverse agonist is claimed to prevent this thrombosis or reverse it in patients where psychotropic actions are not needed.

C. 5HT2B Inverse Agonists/Antagonists to Treat Cardiac Fibrosis-Related Conditions.

5-HT _2B inverse agonists/antagonists can be utilized for treatment of atherosclerosis. Activation of 5HT _2B causes atherosclerosis of cardiac valves. In Janssen, et al., 2015, for example, 5-HT _2B 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-HT _2A antagonists. It is known for several decades that antagonism of 5-HT _2A receptor leads to beneficial cardiovascular effects including reduced platelet aggregation and vasodilation. For example, the 5-HT _2A 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-HT _2B 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-HT _2B antagonists and inverse agonists. Exploiting their expression in the gastrointestinal system, 5- HT _2B 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-HT _2B antagonists and inverse agonists. Exploiting their expression in the gastrointestinal system, 5- HT _2B 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-HT _2B receptor. Agonists at 5-HT _2B 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).

EXAMPLES

Example 1. Chemical Syntheses.

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 60 F254 plates. Final compounds were converted from free base to HCI salt either before or after NMR analysis, as noted. All spectra were recorded by a Varian 500MHz, 400 MHz NMR in CDCI ₃ or CD ₃ OD as noted and are expressed as chemical shift (5) 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 semipreparative (s-prep)-RegisCell™ (5 qm, 25 cm x 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).

Scheme 1 . Synthesis of 3'-Br-4-PAT 2a and 3a diastereomers.

Scheme 2. Optimized synthesis of 4-PAT analogs (3a, 3b, 3b‘).

Diastereomeric 3'-Br-4-PATs 2a and 3a (Scheme 1 , Fig. 1A) served as key intermediates to generate 4-PAT analogs substituted at the meta-position of ring C with a variety of aryl substituents (2c-k, 3c-k, Table 1 ) via coupling with commercially-available boronic acids (7e-k) or esters (8c, d). Substituted phenyl rings were introduced (rac-2f-k and rac-3f-k, 82-98%, Scheme 3) with excellent yields via Suzuki-Miyaura coupling of respective

3'-Br-4-PAT diastereomers with the corresponding boronic acids (7e-k), in the presence of

Pd(ll)acetate and SPhos (Scheme 3) (Altman & Buchwald, 2007). Coupling performed with the slow reacting pyridine boronic acid in the presence of Pd ₂ (dba) ₃ and PCy ₃ gave analogs

2e and 3e, with moderate yields (55-60%, Scheme 3) (Kudo, et al., 2006). Conventional

Suzuki-Miyaura coupling failed to give thiophen-2'-yl or furan-2-yl analogs (2c, d and 3c, d) due to in situ decomposition of the unstable heterocyclic boronic acids. Pioneering cross- coupling by Burke, with the less nucleophilic ‘bench-top stable’ MIDA ester was used successfully to introduce thiophen-2'-yl and furan-2'-yl fragments into the 3'-Br-4-PATs with

-60% yield (Scheme 3) (Knapp, et al., 2009).

Scheme 3. Synthesis of aryl substituted analogs 2cqk and 3cqk.

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‘

(Fig. 6A, Fig. 6B). The absolute stereochemistry of trans-4-PAT stereoisomers (i.e., [2S,4R],

[2R,4S]) has been reported (Booth, Fang, et al., 2009; Sakhuja, et al., 2015).

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 AICI ₃ (800 mg, 6.0 mmol) and CH ₂ CI ₂ (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 qb, 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 qb, 5.1 mmol) and the resulting solution was stirred for 30 minutes on ice bath. Water (50 ) was admdeLd to the flask and the organic layer was separated. The aqueous layer was extracted with CH ₂ CI ₂ (20 mL 3). The combined organic layer was washed with saturated aq NaHCO ₃ (30 mL x 2) followed by brine (30 mL) and dried over Na ₂ SO ₄ . 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, /V-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine 2a and

Cis-4-(3-bromophenyl)-/V, /V-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 ) and m mLethanol (37 mb). 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 NaHCO ₃ (50 ) m anLd ethyl acetate (25 ). Them oLrganic layer was separated, and the aqueous layer was extracted with ethyl acetate (25 4). TmheL combined organic layer was washed with brine (30 )m aLnd dried over Na ₂ SO ₄ . 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 c/s-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 m roLund bottom flask with stir bar was added ketone 6 (1.0 mmol), 10M solution of dimethylamine in ethanol (130 μL, 1.3 mmol), 4 A 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 NaHCO ₃ (15 ) and ethyl acetate (10 mL ). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (10 mL x4). The combined organic layer was washed with brine (15 ) and m drLied over Na ₂ SO ₄ . After evaporation of solvent the crude reaction mixture was purified by silica gel column chromatography to afford cis- amines as colorless oil.

Scheme 4. Optimized synthesis of Cis-analogues (3a, 3b, 3b').

The racemic mixtures of c/s-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 ti at t ₂ , respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-c/s-3'-CI-4-PAT, 3b and (2S,4S)-c/s-3'-F-4-PAT, 3b' analogs (Fig. 6A, Fig. 6B). Both enantiomers were converted to HCI salts for use in pharmacological assays by adding 2M HCI solution in ether to the solution of free amine in ether.

Cis-4-(3-bromophenyl)- /V, /V-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 c/s-4-(3-bromophenyl)-/V, N- dimethyl-1 ,2,3,4-tetrahydronaphthalen-2-amine 3a as colorless oil with 45% isolated yield. 1H-NMR (500 MHz; CDCI ₃ ): δ 7.38 (d, J = 7.9 Hz, 1 H), 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, 1 H), 4.07 (dd, J = 12.2, 5.2 Hz, 1 H), 3.04-3.00 (m, 1 H), 2.95-2.90 (m, 1 H), 2.80 (tdd, J = 11.5, 4.7, 2.3 Hz, 1 H), 2.37 (s, 6H), 2.33 (ddd, J = 9.9, 5.2, 2.5 Hz, 1 H), 1.68 (q, J = 12.1 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 13.01 (s, 1 H), 7.42 (d, J = 8.0 Hz, 1H), 7.31 (s, 1 H), 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, 1 H), 3.65-3.59 (m, 1H), 3.43-3.39 (m, 1 H), 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, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 Ci ₈ H ₂ iBrN for [M+H] ⁺ : 330.0858. Found: 330.0859. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate = 1 .5 mL/min; t ₁ = 14.41 min, t ₂ = 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 AlCh 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(1/7)-one 6b as colorless oil with 60% isolated yield. ¹ H and ¹³ C NMRs are in agreement with previously published data (Vincek & Booth, 2009).

C/s-4-(3-chlorophenyl)-/V, /V-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)-/V, N- dimethyl-1 ,2,3,4-tetrahydronaphthalen-2-amine 6b as colorless oil with 40% isolated yield. ¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 3.03 (ddd, J = 15.6, 4.5, 1.9 Hz, 1 H), 2.93 (dd, J = 12.4, 11.4 Hz, 1H), 2.81 (tdd, J = 11.5, 4.6, 2.3 Hz, 1 H), 2.38 (s, 6H), 2.35-2.31 (m, J = 2.6 Hz, 1H), 1.69 (q, J = 12.1 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.88 (s, 1 H), 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, 1 H), 4.21 (dd, J = 12.0, 5.1 Hz, 1H), 3.64 (td, J = 11.0, 1.9 Hz, 1 H), 3.42-3.39 (m, 1 H), 3.31-3.26 (m, 1 H), 2.85 (s, 6H), 2.70-2.67 (m, 1H), 2.00 (q, J = 12.2 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 CI ₈ H ₂ ICIN for [M+H] ⁺ : 286.1363. Found: 286.1359. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH: ⁿ PrOH (80:10:5:5) 0.1% TEA (modifier) 0.1% TFA (modifier); flow rate = 1.5 mL/min; t ₁ = 11.0 min, t ₂ = 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 AlCh 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. ¹ H and ¹³ C NMRs are in agreement with previously published data (Vincek & Booth, 2009).

C/s-4-(3-fluorophenyl)-/V, /V-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 c/s-4-(3-fluorophenyl)-/V, N- dimethyl-1 ,2,3,4-tetrahydronaphthalen-2-amine 6b' as colorless oil with 35% isolated yield. ¹ H-NMR (500 MHz; CDCI ₃ ): 7.26-7.23 (m, 1 H), 7.15-7.12 (m, 2H), 7.08-7.04 (m, 1 H), 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, 1 H), 3.06-3.02 (m, 1H), 2.94 (dd, J = 12.4, 11.2 Hz, 1 H), 2.79-2.74 (m, 1 H), 2.39 (s, 6H), 2.34-2.31 (m, 1 H), 1.69 (q, J = 12.2 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.70 (brs, 1 H), 7.31-7.26 (m, 1 H), 7.18-7.15 (m, 2H), 7.10-7.06 (m, 1 H), 6.98-6.94 (m, 2H), 6.84 (d, J = 9.4 Hz, 1H), 6.75 (d, J = 7.7 Hz, 1 H), 4.22 (br d, J = 9.1 Hz, 1 H), 3.68-3.60 (m, 1 H), 3.38 (d, J = 14.0 Hz, 1 H), 3.29-3.24 (m, 1H), 2.84 (s, 6H), 2.67 (d, J = 9.5 Hz, 1 H), 2.03-1.96 (m, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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:'PrOH: ⁿ PrOH (80:10:5:5) 0.1% TEA (modifier) flow rate = 1.5 mL/min; t ₁ = 11.77 min, t ₂ = 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 N ₂ gas for 45 minutes. To the reaction mixture was added potassium phosphate (85 mg, 0.4 mmol), Palladium(ll) 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 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 (4:2:0.1 hexanes:ethyl acetate:triethylamine) to afford racemic 2f-k.

Scheme 5. General Suzuki-coupling procedure for analogs trans-2f-k (Altman & Buchwald, 2007).

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 ti at t2, respectively, with absolute stereochemistry assigned according to retention time of the previously published trans-3'-C\- 4-PAT analog (Sakhuja, et al., 2015). Both enantiomers were converted to HCI salts for use in pharmacological assays by adding 2M HCI solution in ether to the solution of free amine in ether.

Trans-4-([1,1'-biphenyl]-3-yl)-/V,/V-dimethyl-1,2,3,4-tet rahydronaphthalen-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 7.20-7.16 (m, 2H), 7.11 (t, J = 6.9 Hz, 1 H), 6.98 (dd, J = 14.5, 7.6 Hz, 2H), 4.44 (t, J = 5.1 Hz, 1 H), 3.07 (dd, J = 16.2, 4.7 Hz, 1 H), 2.90 (dd, J = 16.1 , 9.5 Hz, 1 H), 2.75 (tt, J = 9.1 , 4.5 Hz, 1 H), 2.32 (s, 6H), 2.23-2.15 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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. ¹ H and 1 ³ C NMRs are in agreement with previously published data (Sakhuja, et al., 2015). Trans-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-N, /V-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.39-7.36 (m, 2H), 7.32 (t, J = 7.7 Hz, 1 H), 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, 1 H), 3.05 (dd, J = 16.2, 4.8 Hz, 1 H), 2.88 (dd, J = 16.2, 9.5 Hz, 1 H), 2.71 (tt, J = 9.1, 4.5 Hz, 1 H), 2.28 (s, 6H), 2.21-2.13 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.65 (br s, 1 H), 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, 1 H), 4.59 (br s, 1 H), 3.53 (br d, J = 13.7 Hz, 1 H), 3.42 (br s, 1 H), 3.29-3.24 (m, 1 H), 2.73 (s, 6H), 2.48 (br s, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1969. HPLC (s- prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1 % TFA (modifier) flow rate = 3.0 mL/min; t ₁ = 8.0 min, t ₂ = 17.65 min.

Trans-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)- /V /V -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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.39-7.37 (m, 1 H), 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, 1 H), 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). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.75 (s, 1 H), 7.43 (d, J = 7.6 Hz, 1 H), 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, 1 H), 3.53 (dd, J = 15.5, 4.2 Hz, 1 H), 3.41-3.40 (m, 1 H), 3.24 (dd, J = 14.9, 11.7 Hz, 1H),

2.72 (br s, 6H), 2.49-2.44 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1969. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier) flow rate = 3.0 mL/min; t ₁ = 8.23 min, (2 = 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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 7.23 (br s, 1 H), 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, 1 H), 3.06 (dd, J = 16.2, 4.8 Hz, 1 H), 2.89 (dd, J = 16.2, 9.4 Hz, 1H), 2.74-2.69 (m, 1 H), 2.31 (s, 6H), 2.20-2.17 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.76 (s, 1 H), 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, 1 H), 7.29-7.20 (m, 3H), 7.14-7.09 (m, 3H), 7.06 (d, J = 7.6 Hz, 1 H), 6.86 (d, J = 7.5 Hz, 1H), 4.60 (br s, 1 H), 3.52 (dd, J = 15.5, 3.1 Hz, 1H), 3.45-3.38 (m, 1 H), 3.24 (dd, J = 14.4, 12.0, 1H), 2.72 (br s, 6H), 2.52-2.44 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1968. HPLC (s- prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1 % TFA (modifier) flow rate = 3.0 mL/min; t ₁ = 9..96 min, t ₂ = 16.64 min. Trans-4-(4'-chloro-[1 ,1 '-biphenyl]-3-yl)-/V,/\/-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.45 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.6 Hz, 3H), 7.31 (t, J = 7.6 Hz, 1 H), 7.24 (s, 1 H), 7.26-7.20 (m, 2H), 7.10 (t, J = 7.1 Hz, 1 H), 6.97 (t, J = 7.9 Hz, 2H), 4.42 (t, J = 5.3 Hz, 1 H), 3.04 (dd, J = 16.3, 4.8 Hz, 1 H), 2.87 (dd, J = 16.2, 9.3 Hz, 1 H), 2.68- 2.65 (m, 1 H), 2.28 (s, 6H), 2.17 (t, J = 6.0 Hz, 2H).

¹³ C-NMR (100 MHz, CDCI ₃ ): 5 147.74, 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.77 (br s, 1 H), 7.44-7.38 (m, 5H), 7.34 (t, J = 7.6 Hz, 1 H), 7.29-7.26 (m, 2H), 7.25-7.20 (m, 1 H), 7.15 (s, 1 H), 7.05 (d, J = 7.5 Hz, 1 H), 6.87 (d, J = 7.5 Hz, 1 H), 4.60 (br s, 1 H), 3.51 (dd, J = 15.4, 4.5 Hz, 1 H), 3.47-3.43 (m, 1 H), 3.23 (dd, J = 15.0, 11.7 Hz, 1 H), 2.71 (d, J = 4.6 Hz, 6H), 2.52-2.43 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 5 145.19, 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 C ₂₄ H ₂₅ CIN for [M+H] ⁺ : 362.1676. Found: 362.1673. HPLC (s-prep): Solvent System: hexanes: 'PrOH (98:2) 0.1% TEA (modifier), flow rate = 2.0 mL/min; t ₁ = 12.42 min, t ₂ = 14.52 min.

Trans- 4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,Af-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.43 (d, J = 8.7 Hz, 2H), 7.37 (d, J = 7.7 Hz, 1 H), 7.28-7.23 (m, 2H), 7.18-7.14 (m, 2H), 7.09 (t, J = 7.1 Hz, 1 H), 6.99 (d, J = 7.6 Hz, 1 H), 6.88 (d, J = 7.6 Hz, 1 H), 6.77 (d, J = 8.7 Hz, 2H), 4.40 (t, J = 5.1 Hz, 1 H), 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). ¹³ C- NMR (100 MHz, CDCI ₃ ): 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. HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.67 (s, 1 H), 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, 1 H), 7.07 (d, J = 7.5 Hz, 1 H), 6.94 (d, J = 7.3 Hz, 1 H), 4.62 (br s, 1 H), 3.50-3.43 (m, 2H), 3.21-3.1 1 (m, 7H), 2.73 (dd, J = 8.0, 4.8 Hz, 6H), 2.62 (br d, J = 12.0 Hz, 1 H), 2.44 (td, J = 11 .9, 5.3 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₆ H ₃ IN ₂ for [M+H] ⁺ : 371.2487. Found: 371.2483. HPLC (s- prep): Solvent System: hexanes:MeOH:'PrOH: ⁿ PrOH (80:10:5:5) 0.1 % TEA (modifier), flow rate = 1 .7 mL/min; t ₁ = 11 .55 min, f ₂ = 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 ti at t2, respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-c/s-3'CI-4-PAT, 3b and (2S,4S)-c/s-3’F-4-PAT, 3b' analogs. Both enantiomers were converted to HCI salts for use in pharmacological assays by adding 2M HCI solution in ether to the solution of free amine in ether.

Scheme 6. General Suzuki-coupling procedure for analogs c/s-3f-k.

C/s-4-([1,1'-biphenyl]-3-yl)-Af,/V-dimethyl-1 ,2,3,4-tetrahydronaphthalen-2-amine, 3f: cisamine 3f was synthesized from c/s-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(ll) 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. ¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.58-7.56 (m, 2H), 7.48 (d, J = 7.7 Hz, 1 H), 7.47-7.37 (m, 4H), 7.37-7.30 (m, 1 H), 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, 1 H), 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, 1 H), 2.84 (tdd, J = 11.4, 4.8, 2.3 Hz, 1 H), 2.42-2.38 (m, 7H), 1.79 (q, J = 12.1 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.93 (s, 1 H), 7.55 (d, J = 7.4 Hz, 2H), 7.52 (d, J = 7.6 Hz, 1 H), 7.44-7.35 (m, 4H), 7.36-7.33 (m, 1 H), 7.20-7.19 (m, 2H), 7.16-7.10 (m, 2H), 6.86 (d, J = 7.8 Hz, 1 H), 4.28 (d, J = 9.2 Hz, 1 H), 3.70-3.63 (m, 1 H), 3.47-3.44 (m, 1 H), 3.34-3.29 (m, 1 H), 2.86 (s, 6H), 2.73-2.71 (m, 1H), 2.08 (q, J = 11.8 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₆ N for [M+H] ⁺ : 328.2066. Found: 328.2059. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (90:5:5) 0.1% TEA (modifier), 0.1 % TFA (modifier), flow rate = 3.0 mL/min; t ₁ = 10.17 min, t ₂ = 24.59 min.

Cis-4-(2'-fluoro-[1,1'-biphenyl]-3-yl)-/V,/V-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.45-7.38 (m, 4H), 7.31-7.27 (m, 1 H), 7.19-7.11 (m, 5H), 7.03 (t, J = 7.3 Hz, 1H), 6.83 (d, J = 7.8 Hz, 1 H), 4.16 (dd, J = 12.2, 5.0 Hz, 1 H), 3.04 (dd, J = 15.5, 2.6 Hz, 1 H), 2.95 (dd, J = 15.5, 11.4 Hz, 1 H), 2.83 (tdd, J = 11.5, 4.7, 2.2 Hz, 1 H), 2.42-2.38 (m, 7H), 1.78 (q, J = 12.1 Hz, 1H). ¹³ C NMR (100 MHz; CDCI ₃ ): 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.

HCI salt: ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.69 (s, 1H), 7.46 (d, J = 7.5 Hz, 1 H), 7.41 (t, J = 7.1 Hz, 2H), 7.35 (s, 1 H), 7.30 (dd, J = 14.9, 9.0 Hz, 1H), 7.21-7.08 (m, 6H), 6.85 (d, J = 7.7 Hz, 1 H), 4.27 (br d, J = 8.3 Hz, 1H), 3.70-3.65 (m, 1 H), 3.43 (br d, J = 14.3 Hz, 1H), 3.32-3.27 (m, 1 H), 2.85 (d, J = 11 .5 Hz, 7H), 2.70 (d, J = 9.8 Hz, 1 H), 2.07 (q, J = 11.8 Hz, 1 H).

¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1968. HPLC (s-prep): Solvent System: hexanes:'PrOH (94:6) 0.2% TEA (modifier), 0.1% TFA (modifier), flow rate = 3.0 ml/min; t ₁ = 13.11 min, t ₂ = 29.15 min.

Cis-4-(3'-fluoro-[1,1'-biphenyl]-3-yl)-/V, /V-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.46 (d, J = 7.7 Hz, 1 H), 7.41-7.34 (m, 4H), 7.28-7.26 (m, 1 H), 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, 1 H), 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). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 13.00 (s, 1 H), 7.49 (d, J = 7.7 Hz, 1 H), 7.44-7.32 (m, J = 8.0 Hz, 4H), 7.23 (s, 1 H), 7.20-7.19 (m, 3H), 7.13-7.09 (m, 1H), 7.04 (td, J = 8.2, 1.1 Hz, 1 H), 6.84 (d, J = 7.8 Hz, 1 H), 4.28 (dd, J = 11 .8, 4.5 Hz, 1 H), 3.69-3.65 (m, 1 H), 3.44 (d, J = 14.0 Hz, 1 H), 3.34-3.29 (m, 1H), 2.85 (dd, J = 4.9, 4.9 Hz, 6H), 2.74-2.72 (m, 1 H), 2.08 (q, J = 12.2 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 5 164.33, 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1967. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (90:5:5) 0.1 % TEA (modifier), 0.1 % TFA (modifier), flow rate = 3.0 mL/min; t ₁ = 10.07 min, t ₂ = 16.80 min.

Cis-4-(4 ^, -fluoro-[1,1'-biphenyl]-3-yl)-/V,/V-dimethyl-1,2,3,4-t etrahydronaphthalen-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(ll) 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. ¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 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, 1 H), 2.98-2.93 (m, 1 H), 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). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.77 (s, 1 H), 7.51 (dd, J = 7.6, 5.8 Hz, 2H), 7.45 (d, J = 7.6 Hz, 1 H), 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, 1 H), 4.27 (dd, J = 11.3, 3.6 Hz, 1 H), 3.69-3.65 (m, 1 H), 3.42 (br d, J = 13.9 Hz, 1 H), 3.34-3.28 (m, 1 H), 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). ¹³ C-NMR (100 MHz, CDCI ₃ ): 5 163.81 , 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 C ₂₄ H ₂₅ FN for [M+H] ⁺ : 346.1971. Found: 346.1967. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (90:5:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate = 2.0 mL/min; t ₁ = 16.53 min, = 23.22 min.

C/s-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 4.17 (dd, J = 12.1, 4.9 Hz, 1 H), 3.06 (dd, J = 15.5, 2.5 Hz, 1H), 3.00-2.94 (m, 1 H), 2.90-2.86 (m, 1 H), 2.41-2.38 (m, 7H), 1.79 (q, J = 12.1 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.88 (s, 1 H), 7.49-7.46 (m, 3H), 7.42-7.38 (m, 3H), 7.35 (s, 1 H), 7.19-7.15 (m, 3H), 7.12-7.10 (m, 1 H), 6.84 (d, J = 7.8 Hz, 1H), 4.28 (br d, J = 8.2 Hz, 1 H), 3.69-3.64 (m, 1H), 3.43 (br d, J = 14.2 Hz, 1 H), 3.34-3.29 (m, 1H), 2.86 (s, 6H), 2.73 (brd, J= 10.4 Hz, 1H), 2.08 (q, J= 11.7 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₄ H ₂₅ CIN for [M+H] ⁺ : 362.1676. Found: 362.1673. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1% TEA (modifier), flow rate = 1 .5 /mminL; t ₁ = 23.00 min, t2 = 30.59 min.

Cis-4-(4'-(dimethylamino)-[1,1'-biphenyl]-3-yl)-N,N-dimet hyl-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 7.13 (dt, J = 14.7, 7.4 Hz, 2H), 7.06 (d, J = 7.5 Hz, 1 H), 7.01 (t, J = 7.4 Hz, 1 H), 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, 1 H), 3.07-3.03 (m, 1 H), 2.98-2.94 (m, 7H), 2.88-2.85 (m, 1 H), 2.42-2.38 (m, 7H), 1.79 (q, J = 12.1 Hz, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CD3OD): 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, 1 H), 7.30-7.24 (m, 2H), 7.17 (t, J = 7.1 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1 H), 6.77 (d, J = 7.6 Hz, 1 H), 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, 1 H), 2.17-2.15 (m, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₆ H ₃ iN ₂ for [M+H] ⁺ : 371.2487. Found: 371.2486. HPbC ( s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5), 0.1 % TEA (modifier), 0.1% TFA (modifier), flow rate = 3.0mL /min; t ₁ = 10.76 min, t ₂ = 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 N ₂ for 30 min. To the flask was added palladium(ll) acetate (2 mg, 0.01 mmol), SPhos (8 mg, 0,02 mmol) and aq K3PO4 (3.0 M, 0.5 m, dLegassed by sparging with N ₂ 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 )m aLnd ethyl acetate (4 ).m TLhe 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 Na ₂ SO ₄ . 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.

Scheme 7. General procedure for cross-coupling with heteroaromatic Ml DA boronates (Kudo, et al., 2006): analogues 2c and 2d.

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 ti at t2, respectively, with absolute stereochemistry assigned according to retention time of the previously published trans-3’C\-4- PAT analog (Sakhuja, et al., 2015). Both enantiomers were converted to HCI salts for use in pharmacological assays by adding 2M HCI solution in ether to the solution of free amine in ether.

Trans-4-(3-(thiophen-2-yl)phenyl)-N,/V-dimethyl-1,2,3,4-t etrahydronaphthalen-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(ll) 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 transamine 2c as colorless oil with 60% isolated yield.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 4.39 (t, J = 5.2 Hz, 1 H), 3.04 (dd, J = 16.2, 4.7 Hz, 1 H), 2.87 (dd, J = 16.2, 9.4 Hz, 1 H), 2.66 (tt, J= 9.0, 4.5 Hz, 1 H), 2.28 (s, 6H), 2.17-2.14 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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. HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.71 (s, 1 H), 7.47 (d, J = 7.7 Hz, 1 H), 7.29-7.21 (m, 7H), 7.07-7.03 (m, 2H), 6.77 (d, J = 7.7 Hz, 1 H), 4.57-4.55 (m, 1 H), 3.53 (dd, J = 15.5, 4.8 Hz, 1 H), 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). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₂ H ₂₄ NS for [M+H] ⁺ : 334.1630. Found: 334.1628. HPLC (s-prep): Solvent System: hexanes: 'PrOH (98:2) 0.1% TEA (modifier), flow rate = 2.0 mL/min; t ₁ = 10.55 min, t ₂ = 12.20 min.

7rans-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetra hydronaphthalen-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 6.96 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1 H), 6.59 (d, J = 3.2 Hz, 1 H), 6.45-6.44 (m, 1 H), 4.41 (t, J = 5.0 Hz, 1 H), 3.09 (d, J = 15.1 Hz, 1 H), 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). ¹³ C- NMR (126 MHz, CDCI ₃ ): 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.

HCI-salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.71 (br s, 1 H), 7.52 (d, J = 7.7 Hz, 1 H), 7.45 (s, 1 H), 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, 1 H), 6.61 (d, J = 3.2 Hz, 1H), 6.47-6.46 (m, 1 H), 4.58-4.56 (br m, 1 H), 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). ¹³ C-NMR (126 MHz, CDCI ₃ ): 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 C ₂₂ H ₂₄ NO for [M+H] ⁺ : 318.1858. Found: 318.1858. HPLC (s-prep): Solvent System: hexanes: 'PrOH (98:2) 0.1% TEA (modifier), flow rate = 2.0 mL/min; t ₁ = 12.96 min, f ₂ = 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 c/s-analogs were separated by semi- preparative chiral HPLC Regiscell column using conditions and solvents specific to each analog to elute the c/s-(2S,4S) and -(2R,4R) enantiomers at retention time ti at t ₂ , respectively. The absolute stereochemistry was assigned by correlating retention time to the x-ray crystal structure of (2R,4R)-c/s-3'CI-4-PAT, 3b and (2S,4S)-c/s-3’F-4-PAT, 3b' analogs. Both enantiomers were converted to HCI salts for use in pharmacological assays by adding 2M HCI solution in ether to the solution of free amine in ether.

Scheme 8. Synthesis of cis analogues 3c and 3d.

C/s-4-(3-(thiophen-2-yl) phenyl)-/V,/V-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(ll) 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 cisamine 3c as colorless oil with 60% isolated yield.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.51 (d, J = 7.8 Hz, 1 H), 7.45 (s, 1 H), 7.33 (t, J = 7.7 Hz, 1 H), 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, 1 H), 4.15 (dd, J = 12.6, 5.4 Hz, 1 H), 3.10-2.98 (m, 3H), 2.46-2.39 (m, 7H), 1.80 (q, J = 11.8 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 13.00 (br s, 1 H), 7.54 (d, J = 7.8 Hz, 1 H), 7.42 (s, 1 H), 7.35 (t, J = 7.7 Hz, 1 H), 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, 1 H), 4.24 (dd, J = 12.1 , 5.4 Hz, 1H), 3.68-3.64 (m, 1 H), 3.48-3.44 (m, 1H), 3.34-3.28 (m, 1H), 2.87-2.84 (m, 6H), 2.72-2.69 (m, 1 H), 2.06 (q, J = 12.5 Hz, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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 C ₂₂ H ₂₄ NS for [M+H] ⁺ : 334.1630. Found: 334.1629. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate = 3.0 ml/min; t ₁ = 8.58 min, t ₂ = 25.12 min. Cfs-4-(3-(furan-2-yl)phenyl)-N,N-dimethyl-1,2,3,4-tetrahydro naphthalen-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(ll) 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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 7.57 (d, J = 7.8 Hz, 1 H), 7.54 (s, 1 H), 7.46 (s, 1 H), 7.35 (t, J = 7.7 Hz, 1 H), 7.15 (dt, J = 15.4, 7.6 Hz, 2H), 7.08 (d, J = 7.6 Hz, 1 H), 7.03 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 7.8 Hz, 1 H), 6.65 (d, J = 3.2 Hz, 1 H), 6.48-6.45 (m, 1H), 4.15 (dd, J = 12.1, 4.8 Hz, 1 H), 3.07 (dd, J = 15.5, 2.8 Hz, 1 H), 2.98 (dd, J = 15.8, 11 .4 Hz, 1 H), 2.88-2.83 (m, 1 H), 2.40-2.37 (m, 7H), 1.79 (q, J = 12.2 Hz, 1 H). ¹³ C-NMR (126 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 12.66 (s, 1 H), 7.55 (d, J = 7.7 Hz, 1H), 7.47 (s, 1 H), 7.41 (s, 1H), 7.31 (t, J = 7.7 Hz, 1 H), 7.17-7.12 (m, 2H), 7.05-7.01 (m, 2H), 6.77 (d, J = 7.8 Hz, 1 H), 6.62 (d, J = 3.0 Hz, 1 H), 6.44-6.42 (m, 1 H), 4.21 (d, J = 8.1 Hz, 1 H), 3.67-3.60 (br m, 1 H), 3.39 (d, J = 14.4 Hz, 1 H), 3.30-3.25 (m, 1H), 2.81 (d, J = 9.8 Hz, 6H), 2.65-2.63 (m, 1 H), 2.07-2.01 (m, 1 H). ¹³ C-NMR (126 MHz, CDCI ₃ ): 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. C ₂₂ H ₂₄ NO for [M+H] ⁺ : 318.1858. Found: 318.1859. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH (85:10:5) 0.1% TEA (modifier), 0.1% TFA (modifier), flow rate = 3.0 m/mLin; t ₁ = 9.27 min, t ₂ = 27.19 min.

Trans- 4-(3-(pyridin-4-yl)phenyl)- N,/V-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 N ₂ for 30 min. To the flask was added Tris(dlbenzylideneacetone)dipalladium(0) (9 mg, 0.01 mmol), PCy ₃ (7 mg, 0.024 mmol) and aq K ₃ PO ₄ (3.0 M, 0.5 m, dLegassed by sparging with N ₂ 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 04). The combined organic layer was washed with brine (10 mL) and dried over Na ₂ SO ₄ .

Scheme 9. Example Synthesis of trans-2a → trans-2e.

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.

¹ H-NMR (500 MHz; CDCI ₃ ): δ 8.63 (dd, J = 4.6, 1.4 Hz, 2H), 7.47-7.43 (m, 3H), 7.37 (t, J = 7.7 Hz, 1 H), 7.32 (br s, 1 H), 7.21-7.17 (m, 2H), 7.13-7.10 (m, 1 H), 7.07 (d, J = 7.7 Hz, 1H), 6.96 (d, J = 7.6 Hz, 1 H), 4.46 (t, J = 5.2 Hz, 1 H), 3.05 (dd, J = 16.3, 4.8 Hz, 1 H), 2.89 (dd, J = 16.2, 9.3 Hz, 1 H), 2.67 (tt, J= 8.8, 4.4 Hz, 1 H), 2.29 (s, 7H), 2.20-2.16 (m, 1H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-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, 1 H), 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, 1 H), 7.04 (d, J = 7.6 Hz, 1 H), 4.75 (t, J = 4.1 Hz, 1 H), 3.64-3.60 (m, 1H), 3.43 (dd, J = 15.8, 4.8 Hz, 1 H), 3.35 (s, 1 H), 3.19 (dd, J = 15.8, 10.9 Hz, 1 H), 2.89 (d, J = 11.2 Hz, 6H), 2.59-2.48 (m, 2H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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. C23H ₂₅ N2 for [M+H] ⁺ : 329.2018. Found: 329.2018. HPLC (s-prep): Solvent System: hexanes:MeOH:'PrOH:EtOH (70:10:10:10) 0.1% TEA (modifier), flow rate = 3.0 ml/min; t ₁ = 9.27 min, t ₂ = 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 c/s-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(dibenzyiideneacetone)dipaliadium(0) (9 mg, 0.01 mmol) and PCy ₃ (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. ¹ H-NMR (500 MHz; CDCI ₃ ): 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, 1 H), 7.15 (dt, J = 13.7, 7.0 Hz, 2H), 7.03 (t, J =

7.4 Hz, 1 H), 6.77 (d, J = 7.8 Hz, 1H), 4.19 (dd, J = 12.2, 4.8 Hz, 1 H), 3.08 (d, J = 14.6 Hz, 1 H), 3.06-2.87 (m, 2H), 2.38-2.37 (m, 7H), 1.87-1.75 (m, 1 H). ¹³ C-NMR (126 MHz, CDCI ₃ ): 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.

HCI salt ¹ H-NMR (500 MHz; CDCI ₃ ): δ 8.64-8.62 (m, 2H), 7.52 (d, J = 8.1 Hz, 1 H), 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, 1 H), 6.77 (d, J = 7.8 Hz, 1 H), 4.19 (dd, J = 12.2, 4.8 Hz, 1H), 3.08 (d, J = 14.6 Hz, 1 H), 3.06-2.87 (m, 2H), 2.38-2.37 (m, 7H), 1.87-1.75 (m, 1 H). ¹³ C-NMR (100 MHz, CDCI ₃ ): 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. C23H ₂₅ N2 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; t ₁ = 18.60 min, t ₂ = 23.79 min.

Scheme 10. Example Synthesis of Cis-3a → Cis-3e.

Examples of synthesized and purified chemical structures are shown below:

Example 2. Functional assays.

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-HT _2A , 5-HT _2C , α _1A - or α _1B -adrenergic receptors, whereas 5-HT?(a)Rs were stably expressed in HEK293 cells. H ₁ Rs were transiently expressed, and D2RS were stably expressed in CHO cells. 5-HT _2B and 5-HTIARS in HEK293T cells were transiently expressed because HEK293 cells failed to provide sufficient expression. D ₃ Rs 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 pg of cDNA and 40 pg 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 1X 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, #1 1668027 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 pg 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-HT _2A , 5-HT _2B , 5-HT _2C , or H ₁ Rs 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 I Q- 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 2.5x 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-HCI (pH=7.4, 4 °C) before being oven dried and placed into scintillation vials containing 1 ml SX18-4 ScintiVerse™ BD Cocktail (Fisher Chemical,

Fair Lawn, NJ). Scintillation was detected using a Tri-Carb 2910 TR Liquid Scintillation Analyzer (Perkin Elmer, Boston, MA).

Table 5. Radioligand binding assay conditions for membranes used in this study, including the binding parameters used to derive K, values and the results of saturation binding assays.

Original data are represented as the mean ± SEM from the indicated number of individual experiments performed in triplicate. Buffer: ^a 50 mM Tris-HCI, 10 mM MgCI ₂ , 0.1 mM EDTA, pH = 7.4, ^b 50 mM HEPES, 50 mM NaCI, 5 mM MgCI ₂ , 0.5 mM EDTA, 0.1% BSA, pH = 7.4, ^c 20 mM Tris-HCI, 145 mM NaCI, pH = 7.4 ^d Values obtained from (Armstrong, Casey et al., 2020). ^e Values obtained from (Roth, 2013).

Table 6. Number of independent radioligand binding experiments performed for each ligand at 5-HT _2A , 5-HT _2B , 5-HT2c, and H ₁ Rs listed in Table 1.

The pharmacological parameters (e.g., pEC ₅₀ , pK _b , pIC ₅₀ , I _max ) of agonist and antagonist-mediated signal transduction through Ga _q -coupled 5-HT _2A , 5-HT _2B , 5-HT _2C , and H ₁ Rs 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 1x PBS, dissociated in 10 ml of 1x 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 2x compound diluted in stimulation buffer, was added to each well. For competitive antagonism ( pK _b ) experiments, 2x antagonist was diluted in stimulation buffer containing 2x reference agonist (e.g., 2 μM 5- HT for WT and point mutated 5-HT _2A Rs, 20 nM 5-HT for 5-HT _2B Rs, and 20 μM histamine for H ₁ Rs) 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-l P1 antibody covalently bound to a terbi um-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-l P1-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 Wl). Purified DNA was sequence validated by Psomagen Inc. (Cambridge, MA).

7A. Experimental conditions used to perform site-directed mutagenesis.

81 Table 7B. Primer Sequences from Table 7A.

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.

Equation 1 :

The antagonist equilibrium dissociation constant (K _b ), determined in parallel with the EC ₅₀ of reference agonists, was calculated using the following Equation 2 (Cheng, 2001 ): Equation 2: where IC ₅₀ 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-HT _2A , 5-HT _2B , and H ₁ Rs, respectively), EC ₅₀ is the concentration of the reference agonist which elicited a half maximal response, and K is the Hill slope of the reference agonist. The IC ₅₀ 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 IC ₅₀ of inverse agonists in the absence of A. In contrast, the EC ₅₀ was derived from a log([agonist]) vs. response-variable slope (four parameters)’ model to simultaneously obtain K. Each K _b , IC ₅₀ , and EC ₅₀ value was logarithmically transformed into the pK _b , pIC ₅₀ , and pEC ₅₀ , respectively for ease of presentation and statistical analyses. Since standard deviations tend to be symmetrically distributed around log normal pharmacological parameters (e.g., pK _b , pIC ₅₀ , and pEC ₅₀ ), but not equilibrium constants (e.g., K _b , IC ₅₀ , and EC ₅₀ ), 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 pKi values at the 5-HT _2A R (pKi = 6.99 for [2S,4S]-3h, pKi = 9.84 and 9.65 for [2S,4R]-2i), one of 125 pKi values at the 5-HT _2B R (pKi, = 7.32 for (2S,4R)-2k), and two of 119 pKi values at the 5-HT _2C R ( 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 (K _b ) 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-HT _2A RS, WT 5-HT _2B RS, WT 5-HT _2C RS, and WT H ₁ Rs. The number of independent experiments performed at WT 5-HT _2A Rs to determine the EC ₅₀ of 5-HT, and the K _b 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- HT _2A Rs (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-HT _2B or 5-HT _2c Rs, respectively, indicated by an exploratory screening effort (Fig. 2A), was assessed using a one-way ANOVA comparison of the mean percent change in basal response for n ≥ 5 independent experiments performed in triplicate. One-way ANOVA was also used to compare the impact of compound treatment on the DOI- elicited head twitch response and locomotor activity, in mice relative to 1 mg*kg ^-1 (±)-DOI or vehicle control, respectively. Tukey’s or Dunnett’s multiple comparison tests were used to complement these comparisons of in vivo data. The impact of individual point mutations within the 5-HT _2A R on antagonist affinity (pK _b ) was assessed using unpaired parametric t-tests between the pK _b(WT) and pK _b(mutant) values for each antagonist, as well as between the pEC _50(WT) and pEC _(mutant) of 5-HT.

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).

Example 3. Comparative in vivo assessment of PIMA, (2S,4R)-2k and (2R,4R)-3h.

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 lightdark 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 pm 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 x 43 cm, Med Associates, St. Albans, VT). Trials were videotaped 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 (Fig. 3A). Similar to previous reports (Canal, et al., 2014; Canal, et al., 2015), treatment naive subjects at 9 weeks of age were administered either vehicle or compound and returned to their home cage for 15 minutes. Subjects were then injected once more, this time with 1 mg kg ^-1 (±)-DOI and returned to their home cage for 10 minutes before being placed in the open field arena for the 10-minute test session. Two trained observers (A.B.C and R.P.M) counted the head twitch response, defined as rapid, discrete back-and-forth twisting of the head. Among 43 trials, 34.9% of the scores were entirely consistent, whereas scores differed by 1 , 2, 3, 4, 5, 6 or 7 head twitches in 23.3%, 18.6%, 14%, 4.7%, 2.3%, 0%, and 2.3% of trials. All scores by the two observers were averaged for each subject.

Locomotor activity assays were performed (Fig. 3B, Fig. 3C). In accordance with the principles of reduction, replacement and refinement of animal welfare, the subjects used in the head twitch response assay were also used to assess compound-induced alterations in spontaneous locomotion following a 6-week washout period (15 weeks of age). Subjects were randomized, pretreated with vehicle or 3 mg*kg ^-1 compound, and placed in their home cage for 15 minutes before vehicle administration. Ten minutes later, subjects were placed in an open field for a 10-minute test session while the experimenter stepped out of view. This format was intended to mirror the conditions used in the (±)-DOI-elicited head twitch response assay while allowing for the measurement of ligand-induced locomotor activity in isolation. It was aimed to mirror the conditions in 2.6.1 since the associated locomotor results indicated that

PIMA had a higher propensity to induce locomotor suppression compared to 4-PATs when PIMA was co-administered with (±)-DOI.

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-HT _2A R engagement sensitive to antipsychotic-like activity (Canal & Morgan, 2012). Acute administration of each compound significantly attenuated the head twitch response at every dose tested (Fig. 3A). The lower dose of PIMA (i.e., 0.3 mg*kg ^-1 ) was significantly more effective at attenuating the head twitch response than the same dose of (2S,4R)-2k. In contrast, no differences were observed between 3 mg*kg ^-1 PIMA and 3 mg*kg ^-1 (2S,4R)-2k, 3 mg*kg ¹ PIMA and 5.6 mg*kg" ¹ (2R,4R)-3h, or 3 mg*kg ¹ (2S,4R)-2k and 5.6 mg*kg ¹ (2R,4R)-3h.

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.

Example 4. Molecular modeling and site-directed mutagenesis.

For computational chemistry and molecular modeling, physicochemical parameters (logP and logD) 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 Schrodinger, 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/6-31 G* 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-HT _2A R (PDB: 6A94) and H ₁ R (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 80x80x80 points, 0.375 A 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-HT _2A , 5-HT _2B , 5-HT _2C and Hi 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, eta/., 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, lipidl 7, 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 (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/A ² ) 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/A ² during the heating process, and weakened to 10 kcal/mol/A ² , 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 A cutoff.

The SAR results reported above and elsewhere (Canal, et al., 2014; Sakhuja, et al., 2015) indicated that selectivity to bind 5-HT _2A and/or 5-HT _2c Rs 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-HT _2A RS over 5- HT _2B RS in the (2S,4R)-configuration, and over 5-HT _2B , 5-HT _2C and H ₁ Rs in the (2R,4R)- configuration. Meanwhile, PIMA selectively bound 5-HT _2A RS over 5-HT _2B and H ₁ Rs, with moderate selectivity over 5-HT _2c Rs. To understand how aryl substituted 4-PATs and PIMA bind to 5-HT _2A RS, molecular modeling studies were performed using a model of the 5-HT _2A R (Figs. 4A-4C). Site-directed mutagenesis was used to validate the proposed ligand-receptor interactions. The molecular modeling results with the 5-HT _2A R bound to inverse agonists PIMA, (2S,4R)-2k and (2R,4R)-3h revealed similar binding modes for each ligand (Fig. 4A, Fig. 4B, Fig. 4C). All compounds docked close enough to the side chain of D155 ^3.32 (Ballesteros-Weinstein numbering system, Ballesteros, 1995) such that an ionic bond could form with their respective basic amine moieties, a highly conserved interaction critical to the binding of most ligands across aminergic GPCRs (Kristiansen, et al., 2000; Vass, et al., 2019). Each ligand could also interact with conserved residues in TM3, including V156 ^3.33 , S169 ^3.36 , and T160 ^3.37 (Table 3).

Overall, PIMA, (2S,4R)-2k, and (2R,4R)-3h stabilized an inactive-like conformation of the 5-HT _2A R, typified by an ionic lock between R173 ^3.50 and E318 ^6.30 within the E/DRY domain (Fig. 8). The ionic lock may restrict the intracellular end of TM6 from outward displacement, and thus inhibit productive Gα _q -coupling and accumulation of inositol phosphates (Shapiro, et al., 2002). Clues to how the inactive state was stabilized are found at the ligand-receptor interface. For example, the simulations indicated that the fluorobenzyl ring of PIMA, as well as the aminotetralin core of (2S,4R)-2k and (2R,4R)-3h, may situate deep in the hydrophobic cleft of the orthosteric binding pocket. In this way, the fluorobenzyl and aminotetralin moieties could interact directly with 1163 ³⁴⁰ and F332 ^6.44 of the conserved P246 ^5.50 -l163 ³⁴⁰ -F322 ^6.44 motif, thought to be involved in the activation mechanism of 5-HT ₂ -type GPCRs (Kim, et al., 2020; Kimura, et al., 2019; Peng, et al., 2018). Similar interactions were observed between these moieties and the side chain of W336 ^6.48 , a ‘toggle switch’ potentially mediating on/off states of class A GPCRs (Kim, et al., 2020; Peng, et al., 2018; Rasmussen, et al., 2011 ; Visiers, et al., 2002) (Fig. 4A, Fig. 4B, Fig. 4C, Fig. 8). Moreover, PIMA, (2S,4R)-2k, and (2R,4R)-3h could form edge-to-face aromatic interactions with the side chains of F243 ^5.47 and F340 ^6.52 , while u-cation interactions could form between the side chain of F339 ^6.51 and the basic nitrogen of the PIMA piperidine fragment or the tertiary amine of (2 S,4R)-2k and (2R,4R)-3h.

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 G238 ^5.42 , a residue unique to 5-HT ₂ -type receptors among aminergic GPCRs. Furthermore, in all models, F234 ^5.38 assumed a rotamer conformation oriented away from G238 ^5.42 , 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 (1210 ^4.60 , V235 ^5.39 , G238 ^5.42 , and S242 ^5.46 ) 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-HT _2A RS.

To validate the molecular modeling results, residues were point-mutated in and around the 5-HT _2A R side-extended cavity (Kimura, et al., 2019) and quantified the antagonist affinity (pK _b ) of (2S,4R)-2k and (2R,4R)-3h, as well as (2S,4R)-2a (which lacks 5-HT ₂ R subtype selectivity) at 5-HT _2A R 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 C322K ^6.34 5-HT _2A RS (Fig. 9). An assessment was also made of the antagonist affinity of PIMA and risperidone at point-mutated 5-HT _2A RS, which represent selective and promiscuous 5-HT _2A R ligands, respectively.

A G238S ^5.42 5-HT _2A R 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 (Fig. 10A, Fig. 10B) and reported elsewhere for PIMA (Kimura, et al., 2019). Compared to WT 5-HT _2A Rs, a modest, but significant, decrease in the pK _b of risperidone and (2S,4R)-2a at G238S ^5.42 5-HT _2A Rs was observed. Moreover, the affinity of PIMA, (2S,4R)-2k, and (2R,4R)-3h was nearly abolished at G238S ^5.42 5-HT _2A Rs (Table 2, Fig. 11 F). Notably, the A(pK _b ) for (2S,4R)-2a was less than that of (2S,4R)-2k and (2R,4R)-3h suggesting a sizedependent negative steric interaction between the 4-PAT ring C substituent and S ^5.42 . Also, a significant reduction in the potency was observed, but not the efficacy, of 5-HT at G238S ^5.42 compared to WT 5-HT _2A Rs (Table 2, Fig. 11 A, Fig. 11 B), consistent with previous reports (Kimura, et al., 2019).

The above experiments were extended by asking if the attenuated affinity of (2S,4R)- 2a, (2S,4R)-2k, and (2R,4R)-3h at G238S ^5.42 5-HT _2A Rs translates to aminergic GPCRs natively presenting S ^5.42 . Table 4 shows that (2S,4R)-2k and (2R,4R)-3h had >1 , 000-fold selectivity for 5-HT _2A RS over 5-HT-IA, 5-HT?, D ₂ L, α _1A - and aiB-adrenergic GPCRs. It was noted that (2S,4R)-2k had 270-fold selectivity over D ₃ Rs, whereas (2R,4R)-3h had >1 , 000-fold selectivity. In contrast, (2S,4R)-2a exhibited moderate-to-high affinity for 5-HT?, D ₂ L, D ₃ , and aiA-adrenergic receptors.

Interestingly, aryl substituted 4-PATs in the (2S,4R)-configuration had high affinity for H ₁ Rs (Table 1), despite the presence of T194 ^5.42 , which possesses a bulkier side chain than serine. The molecular modeling results suggested that W158 ^4.56 , a residue unique to H ₁ Rs, might form stereospecific aromatic interactions with 4-PATs to impart high affinity (Fig. 5A, Fig. 5B). For example, ring D of (2S,4R)-2k positioned close to TM4, where it could form optimal T-shaped interactions with W158 ^4.56 , while ring B of the aminotetralin core could form edge-to-face aromatic interactions with W428 ^6.48 . In contrast, ring D of (2R,4R)-3h oriented toward TM5, potentially, due to a stereochemical restriction at the C(2)-position. Interactions with residues in TM5 might be disfavored by a negative steric interaction with T194 ^5.42 , and cause ring D to position between TM5 and TM6, precluding optimal aromatic interactions between ring B and the side chain of W428 ^6.48 . To validate the proposed model, a W158I ^4.56 H ₁ R was generated. However, W158I ^4.56 H ₁ Rs were unable to stimulate IP1 accumulation in response to histamine, and specific binding of [ ³ H]mepyramine or [ ³ H]ketanserin could not be detected. Alignment of 5-HT _2A , 5-HT _2B and 5-HT _2c R crystal structures (Fig.12A, Fig. 12B) indicated that a side chain rotamer of F234 ^5.38 , unique to 5-HT _2A RS (Kimura, et al., 2019), orients toward the extracellular end of TM4 to form a side-extended cavity potentially due to hydrophobic interactions with F213 ^4.63 . In contrast, K193 ^4.63 and I192 ^4.63 in 5-HT _2B and 5- HT _2C RS, respectively, do not form productive interactions with F ^5.38 and may restrict the side cavity (Kimura, et al., 2019). However, no experimental studies have been reported to test this hypothesis. Therefore, a F213K ^4.63 5-HT _2A R was generated to test the hypothesis that the selectivity of PIMA, (2S,4R)-2k, and (2R,4R)-3h to bind 5-HT _2A RS relies on an interaction between F213 ^4.63 and F234 ^5.38 . Unexpectedly, a change in the pK _b of any antagonist tested at F213K ^4.63 5-HT _2A RS was not observed (Table 2, Fig. 1 1 D). However, a decrease in the potency of 5-HT at F213K ^4.63 5-HT _2A RS was observed, but not its efficacy (Table 2, Fig. 11 A, Fig. 11B). These results do not support the hypothesis that F213 ^4.63 mediates subtype selective binding of inverse agonists at 5-HT _2A RS, however, F213 ^4.63 may be involved in 5-HT binding.

Further inspection of the 5-HT ₂ -type receptor crystal structures revealed that one helical turn above F ^{5 38} in 5-HT _2A and 5-HT _2c Rs exists a non-conserved residue in 5-HT _2B RS (D231 ^5.35 , D211 ^5.35 , and F214 ^5.35 , respectively). The root-mean-square deviation (RMSD) of the F ^5.38 side chain in WT 5-HT _2A and 5-HT _2B RS was tracked in silico and found that F ^5.38 exhibited large transient variations in RMSD in WT 5-HT _2A RS. Interestingly, the RMSD of F ^5.38 in D231 F ^5.35 5-HT _2A RS recapitulated the restricted pattern observed in silico for WT 5-HT _2B RS, indicating that D231 ^5.35 may facilitate flexibility in the side chain of F ^5.38 (Fig. 13).

It was therefore hypothesized that D231 ^5.35 may modulate the side chain rotamer of F234 ^5.38 in 5-HT _2A RS to mediate subtype selective binding. To test this hypothesis, a D231 F ^5.35 5-HT _2A R was generated, however, D231 F ^5.35 5-HT _2A RS were insufficiently responsive to 5-HT for competitive antagonism studies (Fig. 11 B), thus, antagonist activity could not be experimentally determined. Furthermore, no specific binding was detected for [ ³ H]ketanserin, [ ³ H]mesulergine, or [ ³ H]spiperone in exploratory studies using membranes from cells transfected with cDNA encoding D231 F ^5.35 5-HT _2A Rs (Figs. 14A-14D).

Residues in TM4 and TM5 lining the side-extended cavity of 5-HT _2A Rs and in proximity to PIMA, (2S,4R)-2k, and (2R,4R)-3h (Table 3) were then investigated. Among these were the side chains of 1210 ^4.60 , V235 ^5.39 , and S242 ^5.46 . Importantly, the side chains of 1210 ^4.60 and V235 ^5.39 are conserved in 5-HT _2c Rs, while S242 ^5.46 is unique to 5-HT _2A RS. It was hypothesized that selectivity to bind 5-HT _2A and 5-HT _2c Rs over 5-HT _2B RS may involve interactions with the side chains of I ^4.60 , V ^5.39 , or the 5-HT _2A R-specific residue S242 ^5.46 . In fact, a significant increase in the affinity of PIMA and (2S,4R)-2k at V235M ^5.39 5-HT _2A RS was observed, with no change in affinity for any antagonist at 1210V ^4.60 or S242A ^5.46 5-HT _2A Rs (Table 2, Fig. 11C, Fig. 11 E, Fig. 11 G). Interestingly, the potency of 5-HT (but not the efficacy) was attenuated at V235M ^5.39 and S242A ^5.46 but not 1210V ^4.60 5-HT _2A RS, consistent with other reports investigating 1210V ^4.60 and S242A ^5.46 5-HT _2A RS (Table 2, Fig. 11A, Fig. 11 B) (Kimura, et a/., 2019).

Example 5. X-ray Crystal Structures.

X-ray crystal data for compound 3b was acquired. The computing details were as follows: data collection: APEX3 (Bruker, 2016); cell refinement: SA//VTV8.40A (Bruker, 2016); data reduction: SAINT V8.40A (Bruker, 2016); program(s) used to solve structure: SheIXT (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_0m.

Table 8. Crystal data for compound 3b.

Table 9. Data collection for compound 3b.

Table 10. Refinement for compound 3b.

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 l.s. planes. Refinement. Refined as a 2-component inversion twin.

Table 11. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (A ² ) for (2R,4R)-3b.

Table 12. Atomic displacement parameters (A ² ) for (mukherjee_neu2_0m).

Table 13. Geometric parameters (A, ⁰ ) for (mukherjee_neu2_0m).

X-ray crystal data for compound 3b' was acquired. The identification code used below is: mukherjee_neu1_0m.

Table 14. Crystal data for compound 3b’.

Table 15. Data collection for compound 3b’.

Table 16. Refinement for compound 3b’. Special details were as follows: geometry. All esds (except the esd in the dihedral angle between two l.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 l.s. planes. Refinement. Refined as a 2-component inversion twin.

Table 17. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (A ² ) for (2S,4S)-3b'.

Table 18. Atomic displacement parameters (A ² ) for (mukherjee_neu1_0m).

Table 19. Geometric parameters (A, °) for (mukherjee_neu1_0m).

Example 6. Synthesis of Charged Substituent on Tetralin Core.

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 R ⁴ can represent ring ‘C’ including substituents described above or shown in Fig. 1B.

Scheme 11. Example Quaternary Amino Derivatization at Tetralin Position 2.

Examples compounds that can be synthesized, with ‘E’ representing a charged group or charged amine, are shown below:

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Example 7. Compounds Bearing a Postively Charged Amino Group at the 2 Position of the Tetralin Core Do Not Readily Cross the Blood-Brain Barrier.

In an example to demonstrate the compound or composition does not substantially accumulate in the human brain, adult, male, C57BI/6J mice, approximately six months old, and treatment-naive 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 (4x starting volume) and internal standard (e.g. (-)-MBP ⁶⁸ ) followed by centrifugation at 14,000 g for 5 minutes at4°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 x 50 mm, 5 μ) 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.

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