Synthesis and Structure–Activity Relationships for Glutamate Transporter Allosteric Modulators

Abstract

Excitatory amino acid transporters (EAATs) are essential CNS proteins that regulate glutamate levels. Excess glutamate release and alteration in EAAT expression are associated with several CNS disorders. Previously, we identified positive allosteric modulators (PAM) of EAAT2, the main CNS transporter, and have demonstrated their neuroprotective properties in vitro. Herein, we report on the structure–activity relationships (SAR) for the analogs identified from virtual screening and from our medicinal chemistry campaign. This work identified several selective EAAT2 positive allosteric modulators (PAMs) such as compounds 4 (DA-023) and 40 (NA-014) from a library of analogs inspired by GT949, an early generation compound. This series also provides nonselective EAAT PAMs, EAAT inhibitors, and inactive compounds that may be useful for elucidating the mechanism of EAAT allosteric modulation.

Introduction

Glutamate is the major excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS) and is critical for normal brain function.¹ Glutamatergic neurotransmission is terminated by reuptake of l-glutamate (l-Glu) from the synaptic cleft by the action of Na⁺-dependent excitatory amino transporters (EAATs).^2,3 There are five distinct subtypes of EAATs, including (rat/human homologue): GLAST/EAAT1/, GLT-1/EAAT2, EAAC1/EAAT3, EAAT4, and EAAT5, which allow for precise spatial and temporal regulation of l-Glu neurotransmission.⁴ The function and expression of these transporters may be dysregulated at the genetic, epigenetic, transcriptional, or translational levels, leading to high levels of extracellular glutamate and excitotoxicity,^2,5−9 and dysfunction of the EAATs plays a role in development of neurological disorders.^5,10,11

EAAT1 is highly expressed in the glial cells of the cerebellar Purkinje cell layer^12,13 and generally is expressed at higher levels in astrocytes and oligodendrocytes compared to microglia.¹⁴ The most abundant subtype, EAAT2,¹⁵ is primarily expressed in astrocytes, select neurons, and oligodendrocytes of the brain and spinal cord.^16,17 EAAT2 accounts for approximately 95% of the total l-Glu transport activity and 1% of total brain protein in the CNS^15,18−21 demonstrating that EAAT2 plays a key role in the maintenance of extracellular glutamate homeostasis and prevention of excitotoxicity.^22,23 The neuronal EAAT3 is ubiquitously expressed in the brain and is important in maintaining low local concentrations of glutamate, where its predominant postsynaptic localization can buffer nearby glutamate receptors and modulate excitatory neurotransmission and synaptic plasticity.²⁴ The glial EAAT1 and neuronal EAAT4 are the two predominant EAATs responsible for maintaining low extracellular glutamate levels and preventing neurotoxicity by limiting mGluR1 signaling in the cerebellum, the brain region essential for motor control.²⁵ EAAT5 mediates glutamate release and light responses in depolarizing bipolar cells in the retina.²⁶

Under healthy conditions, the activation of the l-glutamate receptors is involved in important neurophysiological processes underlying memory and learning, motor functions, and neural plasticity and development.^27,28 However, failure of the proper removal of glutamate by astrocytes from the synapses, results in glutamate excitotoxicity, characterized by sustained elevation of l-Glu levels. This results in excessive activation of postsynaptic Glu receptors that can lead to apoptotic or necrotic cell death.²⁹l-Glu-mediated excitotoxicity has been observed in several disorders such as epilepsy,¹¹ traumatic brain injury,^2,30−32 and stroke,³³⁻³⁵ among others. Additionally, dysregulation of EAAT2 expression has been observed during exposure to drugs of abuse such as methamphetamine or cocaine, which contributes to the enhanced l-Glu transmission in brain reward circuits that facilitate dependence and relapse.^21,36l-Glu is also involved in the transmission of pain, as suggested by studies linking dysregulation of EAAT2 levels in animal models of neuropathic pain.³⁷⁻⁴⁰

We have focused on the mitigation of glutamate excitotoxicity through the removal of excess l-Glu and the restoration of glutamate homeostasis by stimulating EAAT2 with the help of a positive allosteric modulator (PAM).⁴¹⁻⁴³ Our initial interest started from working with a venom from the South American social spider Parawixia bistriata that produces a venom containing complex organic compounds that induce paralysis in termites⁴⁴ and stimulates l-Glu uptake in rat synaptosomes.⁴⁵l-Glu is the major neurotransmitter at the insect neuromuscular junction⁴⁶ and at the mammalian CNS,⁴⁷ suggesting a modulation of the glutamatergic system by the venom. In previous work, we studied the main high-performance liquid chromatography (HPLC) Parawixin (Pwx) fractions that demonstrate this bioactivity, components Pwx1, 2, and 10. Pwx1 stimulates l-Glu uptake through the main transporter in the brain, EAAT2, and is neuroprotective in in vivo glaucoma models.⁴⁵ Pxw2 inhibits GABA and glycine uptake in synaptosomes and inhibits seizures and neurodegeneration,^48,49 and Pwx10 increases l-Glu uptake in synaptosomes and is neuroprotective and anticonvulsant, shown in in vivo epilepsy models.⁵⁰ We elucidated and confirmed the structure of the active acylpolyamine in Pwx10 through synthesis and comparison of bioactivity, and demonstrated that this compound acted as a PAM of astrocytic transporters EAAT1 and EAAT2.⁴¹ Thus, the Parawixia bistriata spider venom provided proof-of-principle to identify small molecule modulators of neurotransmitter systems as potential therapeutic agents, which justified studying more drug-like compounds as modulators of glutamate transporters.

Previous studies revealed that each subunit of the trimeric glutamate transporter is composed of two domains: the scaffold domain composed of transmembrane helices TM1, TM2, TM4, and TM5, and the transport domain composed of TM3, TM6, TM7, and TM8 and the helical hairpins HP1 and HP2.^51,52 The scaffold domains of the three subunits, also called the trimerization domain, are strongly associated in the trimeric transporter, serving as a support for the elevator-like movements of the transport domains between the outward-facing (OF) and inward-facing (IF) conformations of each subunit, either symmetrically or asymmetrically.^53,54 Structure–function studies,⁵⁵ which utilized Pwx1, identified the putative allosteric region of the transporter most susceptible to synergizing with a small molecule to modulate transporter activity. This region is at the interface between the scaffold and transport domains of the subunits, suggesting that compounds binding at that location might be accelerating the “elevator”, thus increasing the rate of glutamate transport.⁵⁴

Subsequently, a hybrid structure-based (HSB) approach was used for virtual screening of small molecules that would interact with the transporter based on the crystal structure of the homologue archaeal transporter, Glt_Ph. This virtual screening approach identified the hit compound, GT949, which selectively stimulated EAAT2-mediated l-Glu uptake with an EC₅₀ = 0.26 ± 0.03 nM, with no effect on EAAT1 and EAAT3 in COS-7 expressing cell lines.⁴³ GT949 increased the rate of l-Glu removal by ∼70%, which was comparable to that of Pwx1. Separation and evaluation of racemic GT949 into its individual enantioenriched isomers demonstrated a ∼20-fold improvement in activity of GT949A (EC₅₀ = 0.041 ± 0.01 nM) compared to GT949B (EC₅₀ = 0.89 ± 0.42 nM).⁴³ Kinetic assays revealed that the V_max of glutamate transport was increased and no changes were observed in the K_m in the presence of GT949, i.e., the affinity for substrate remained unchanged, which is indicative of allosteric modulation.⁴³ Further, mutagenesis studies suggested critical residues in transmembrane domains (TM) 2, 5, and 8 to comprise the allosteric site, which supported the premise that PAMs would act at this interface to facilitate movement of the transport domain.⁵⁵ Compound GT949 was also evaluated in in vitro primary culture models of excitotoxicity to investigate potential neuroprotective effects by increasing the activity of EAAT2 through allosteric modulation and it displayed neuroprotective properties after acute and prolonged glutamate-mediated excitotoxicity.⁴² We propose that GT949 prevents excess glutamate signaling by increasing the rate of glutamate clearance by EAAT2, thereby preventing excitotoxic damage and cell death. Thus, a drug-like CNS penetrant EAAT2 PAM holds potential for translation in in vivo animal models of glutamate-mediated excitotoxicity, drugs of abuse disorders, and neuropathic pain.

In this manuscript, we describe the results from our lead optimization efforts to identify and characterize a series of allosteric modulators of EAATs, characterizing EAAT1–3 selectivity and identifying PAMs and negative allosteric modulators (NAMs) as tool compounds to study the molecular mechanisms of regulation of the glutamate transporters. EAAT2 PAMs are expected to be efficacious in diseases related to excess glutamate since activation of astrocytic EAAT2 provides neuroprotection by restoring glutamate homeostasis.⁴²

Results

We report here lead optimization efforts of the selective EAAT2 PAM, GT949^42,43 focused on optimizing its molecular properties to identify a CNS-permeable lead series suitable for in vivo validation. In Table 1, we show structure–activity relationships for GT949-like commercially available quinoline-2-one containing analogs for EAAT2 potency and mechanism of action (PAM, NAM, or inactive). The lipophilic N-cyclohexyl ring attached to the piperazine can be replaced by an aromatic ring to provide an analog, GT951, with similar stimulating potency to racemic GT949. Substitution with an N-benzyl dioxolyl piperazine moiety and introduction of a methyl group at the 7-position of the quinolinone ring, GT835, resulted in a less potent EAAT2 NAM, while methyl substitution at positions 5, 6, and 8 of the quinolinone ring, i.e., GT867, resulted in a compound inactive on l-Glu uptake. N-Capping the piperazine with a furanamide and methyl groups at positions 6 and 8 of the quinolinone ring provided a modest NAM of l-Glu uptake, i.e., GT729, while methyl group substitution at positions 5 and 7 resulted in a compound, GT996, inactive on l-Glu uptake. GT949 and GT951 are selective EAAT2 PAMs, not active on EAAT1 or 3. GT876 and GT996 are inactive on EAAT2, and GT835 and GT729 are NAMs of EAAT2. GT949 was evaluated for metabolic stability in mouse and human liver microsomes (MLM t_1/2 <1.4 min; HLM t_1/2 = 5.5 min) and for water solubility (Aq solubility = 301 μg/mL). Its water solubility is in a suitable range; however, its short half-life in liver microsomes suggested that it would have modest in vivo exposure due to rapid metabolic clearance. Therefore, a set of tetrazole analogs were synthesized to develop additional SAR with a focus on physiochemical properties to improve CNS activity, such as reducing mol wt (<400) and focusing on balancing lipophilicity and polarity (LogP ∼2.5–3.5; polar surface area (tPSA <70).

Table 1. Structure–Activity Relationships (SAR) for the Effect of GT949 Analogs on EAAT2-Mediated Glutamate Uptake.

^aEnantiomer enriched isomer A.

^bEnantiomer enriched isomer B.

^cMechanism of the compound upon EAAT2-mediated l-Glu uptake performed in transfected COS-7 cells, which is either PAM (Positive Allosteric Modulator), which is associated with an EC₅₀ value; NAM (negative allosteric modulator), with an associated IC₅₀ value, or inactive. Selectivity studies on EAAT1 and EAAT3-mediated l-Glu uptake on these compounds are described in the text.

The Ugi-Azide reaction (Scheme 1),⁵⁶⁻⁵⁸ which was used to synthesize GT949,⁴³ was also used for the synthesis of analogs in Table 2. These analogs were designed to provide SAR for EAAT1–3 selectivity and mechanism of action by modifying the side chain appended from the tetrazole and piperazine motifs, and changes to the size and polarity of the quinolinone ring (Table 2). Mechanism of action was classified as positive versus negative allosteric modulation (PAM or NAM), or no effect (inactive) on the activity of the glutamate transporters EAAT1–3. While an ideal compound for neuroprotection would be selective for EAAT2, some EAAT1 PAM activity may be tolerated since they are both astrocytic transporters that contribute to uptake of l-Glu to glial cells, where l-Glu is converted to glutamine, exported to neurons and reconverted to glutamate.^59,60 On the other hand, activity on EAAT3 may be a concern since they are localized to the postsynaptic neurons and their modulation could lead to glutamate receptor activation.

Scheme 1. Ugi-Azide Reaction Used for GT949 Analog Synthesis.

Table 2. Structure–Activity Relationships (SAR) for the Effect of Tetrazole-Pyridinyl Analogs on EAAT2-Mediated Glutamate Uptake.

^aNAM: Negative Allosteric Modulator, with an IC₅₀ value associated.

^bEAAT2 expressing COS-7 cells.

Tetrazole Series Structure–Activity Data (Table 2)

The analog, 1, replaces the larger and more lipophilic N-cyclohexane ring appended from the piperazine with an N-methyl group resulting in a modest loss of EAAT2 PAM potency and inactive at EAAT1 and EAAT3. We synthesized additional analogs to explore reducing the size of the 6-methoxy-quinolin-2-one ring with a 3-pyridinyl. Compound 2 replaces the 6-methoxy-quinolin-2-one ring with a 3-pyridinyl while maintaining the piperazine N-cyclohexyl ring, resulting in good EAAT2 PAM potency, with EAAT1 PAM activity (EC₅₀ = 0.6 ± 0.8 nM), and inactive at EAAT3. Shortening the tetrazole side chain (N-benzyl versus N-phenethyl), i.e., 3, maintained or slightly improved EAAT2 PAM activity, and inactive at EAAT1 and EAAT3.

Replacement of the piperazine N-cyclohexyl ring with an N-methyl group and maintaining the tetrazole N-phenethyl side chain provided 4 (DA-023) showing single digit nanomolar EAAT2 PAM potency, and inactive against EAAT1 and EAAT3. Shortening the tetrazole side chain to N-benzyl provided 5, which maintained nanomolar EAAT2 PAM potency and EAAT1 PAM activity (EC₅₀ = 0.02 ± 0.008 nM) but is inactive at EAAT3. Several additional analogs were synthesized to further probe the SAR for changes to the piperazine N-side chain and for 2- and 4-pyridinyl changes. Removal of the piperazine N-substituent and tetrazole N-phenethyl side chain provided 6, which is inactive. The piperazine N-Boc and tetrazole N-phenethyl side chain analog, 7, was inactive on l-Glu uptake. The piperazine N–H and tetrazole N-benzyl side chain analog, i.e., 8, was a modest nonselective NAM of EAAT1–3 (EAAT1 IC₅₀ = 716 ± 140 nM, EAAT2 IC₅₀ = 924 ± 17 nM and EAAT3 IC₅₀ = 1487 ± 70 nM). Moving the pyridinyl N to the 2-position, the piperazine N-methyl, tetrazole N-phenethyl, and benzyl side chain analogs, 9 and 10, resulted in inactive compounds at EAAT2 and EAAT3, and 10 is a NAM at EAAT1 (IC₅₀ = 1.8 ± 1 μM). Moving the pyridinyl N to the 4-position in the piperazine N-methyl and tetrazole N-benzyl side chain analog, 11, maintained potent EAAT2 and EAAT1 PAM activity (EC₅₀ = 2.24 ± 1 nM). Four 4-substituted-3-pyridinyl analogs were evaluated. Compounds 12 and 13, which contains the piperazine N-methyl and tetrazole N-phenethyl side chain but differ in 3-pyridinyl 4-substitution with a −F or −OH, respectively, are both similar in EAAT2 PAM potency and also have PAM EAAT1 activity (EC₅₀ = 2.75 ± 2.25 nM and EC₅₀ = 2 ± 1 nM, respectively) and EAAT3 activity (EC₅₀ = 2.5 ± 2.4 nM and EC₅₀ = 1.55 ± 2.05 nM, respectively). However, the compounds, 14 and 15, which contain the piperazine N-methyl and tetrazole N-benzyl side chain, and 3-pyridinyl 4-substitution with a −OH or −Br, respectively, are inactive on l-Glu uptake.

In Table 3, the SAR for tetrazole-substituted aryl analogs is presented evaluating the effect of halogen, electron-donating, and electron-withdrawing substituents on the aryl ring, which replaces the 6-methoxy-quinolin-2-one ring in GT949. Compounds 16, 17, and 18 contain a 4-chlorophenyl and probe the effect of the piperazine N-cyclohexyl versus N-methyl, and tetrazole N-phenethyl versus benzyl side chains. These three compounds are inactive at EAAT2; however, compound 18 shows EAAT1 NAM activity (IC₅₀ = 4.2 ± 3.7 μM). Compounds 19 and 20 contain 4-fluorophenyl, tetrazole N-phenethyl, and piperazine N-cyclohexyl and N-methyl side chains. Compound 19 with the more lipophilic piperazine N-cyclohexyl ring is inactive. The less lipophilic piperazine N-methyl analog, 20, shows selective EAAT2 PAM activity. Compounds with electron-donating substituents on the phenyl ring, i.e., 21–27 showed mixed results. Compound 21, which contains a 4-methoxyphenyl, tetrazole N-phenethyl, and piperazine N-methyl side chain, is inactive. However, the 4-hydroxyphenyl compound 22 is a selective EAAT2 PAM (low efficacy of 127 ± 9%). It is worth noting that, except for 22, all EAAT2 PAMs (Tables 1–5) have comparable efficacies of 159 ± 4% (average ± SEM, not statistically different among all compounds, when comparing at least three independent experiments for the effect of each compound). 22 has a lower efficacy of 127 ± 9%, suggesting it is not ideal. Compound 23 containing a 4-methoxyphenyl, tetrazole N-benzyl, and piperazine N-cyclohexyl side chain is inactive. Compound 24 contains a 3,4-dimethoxyphenyl, tetrazole N-benzyl, and piperazine N-cyclohexyl side chain and is inactive. The piperazine N-methyl analog, 25, activity is a selective EAAT3 PAM EC₅₀ = 132 ± 127 nM. Compounds 26 and 27 contain a 3,5-dimethoxyphenyl ring and are inactive. Compounds 28, 29, and 30 contain an electron-withdrawing 4-cyanophenyl ring. Compound 28, containing a tetrazole N-phenethyl and piperazine N-cyclohexyl side chain, is inactive. Piperazine N-methyl analogs, 29, is a PAM at EAAT1 (EC₅₀ = 4 ± 0.02 nM) and EAAT2; 30 is an EAAT1–3 PAM (EAAT1 EC₅₀ = 1.6 ± 0.9 nM; EAAT2 EC₅₀ = 1.2 ± 0.7 nM and EAAT3 EC₅₀ = 0.2 ± 0.02 nM, respectively). Compounds 31 and 32 containing an electron-withdrawing 4-trifluoromethylphenyl and a piperazine N-cyclohexyl ring are inactive. Overall, the piperazine N-methyl analogs with reduced lipophilicity compared to the piperazine N-cyclohexyl analogs are expected to have better drug-like properties. For this limited set of analogs, it appears that steric hindrance affected activity greater than electronic effects in the aromatic ring.

Table 3. Structure–Activity Relationships (SAR) for the Effects of Tetrazole-Substituted Aryl Analogs on EAAT2-Mediated Glutamate Uptake.

^aEAAT2 expressing COS-7 cells.

Table 5. Structure–Activity Relationships (SAR) for Amides-4-Cyanophenyl Analogs on EAAT2-Mediated Glutamate Uptake.

	EAAT2a				LogP; tPSA; cLogP	selectivity
ID	EC50 (nM)	n	R1	Mol Wt	ChemDraw	EAAT1	EAAT3
39	6.1 ± 4.7	1	–H	348.44	2.5; 59.4; 2.8	PAM	inactive
40 (NA-014)	3.0 ± 2.0	2	–F	380.46	2.9; 59.4; 3.2	inactive	inactive
41	inactive	1	–F	366.43	2.7; 59.4; 3.0	inactive	inactive
42	inactive	1	–OMe	378.47	2.4; 68.6; 2.7	inactive	inactive

^aEAAT2 expressing COS-7 cells.

A set of amide analogs were synthesized to replace the tetrazole with the bioisosteric amide bond (Tables 4 and 5). Synthesis of the amide series is shown in Scheme 2, exemplified for compound 40 (NA-014). Commercially available ethyl 2-(4-cyanophenyl)acetate was converted to benzyl bromide, VY-11-097, by reaction with NBS in refluxing CCl₄ with catalytic AIBN. The bromide was then alkylated with 1-methylpiperazine to provide VY-11-098. Hydrolysis with lithium hydroxide provided the acid VY-12-012, the low yield was due to competing nitrile hydrolysis. Coupling of the carboxylic acid, VY-12-012, with 2-(4-fluorophenyl) ethan-1-amine with HATU provided racemic compound 40 (NA-014).

Table 4. Structure–Activity Relationships (SAR) for the Effect of Amides-Pyridinyl Analogs on EAAT2-Mediated Glutamate Uptake.

	EAAT2b				LogP; tPSA; cLogP	selectivity
ID	EC50 or EC50 (nM)	n	R1	Mol Wt	ChemDraw	EAAT1	EAAT3
33	inactive	2	–H	338.45	1.4; 47.9; 2.2	inactive	inactive
34a	1039 ± 36	1	–H	324.42	1.1; 47.9; 1.9	inactive	inactive
35a	344 ± 25	2	–Cl	372.90	1.9; 47.9; 2.9	NAM	NAM
36	15 ± 4	2	–F	356.44	1.5; 47.9; 2.3	PAM	PAM
37	inactive	1	–F	342.10	1.3; 47.0; 2.1	inactive	inactive
38	inactive	1	–OMe	354.45	1.0; 57.2; 1.7	inactive	inactive

^aNAM: Negative Allosteric Modulator, with an IC₅₀ value associated.

^bEAAT2 expressing COS-7 cells.

Scheme 2. Synthesis of the Amide Series Exemplified by 40 (NA-014).

Reagents and conditions: (i) NBS, AIBN (cat), CCl₄, reflux, 2h, 89%; (ii) 1-methylpiperazine, TEA, CH₃CN, 0 °C to RT, 4h, 92%; (iii) LiOH, EtOH, H₂O, 0 °C to RT, 18%; (iv) 2-(4-fluorophenyl)ethan-1-amine, HATU, DIPEA, DMF, 0 °C to RT, 37.9%.

The 3-pyridinyl, piperazine N-methyl, and amide N-phenethyl side chain surprisingly resulted in an inactive compound i.e., 33 (Table 4). The analog, 34, with a 3-pyridinyl and amide N-benzyl side chain resulted in EAAT2 NAM activity (IC₅₀ = 1039 ± 36 nM) and was inactive at EAAT1 and EAAT3. 4-Chlorophenyl substitution on the amide N-phenethyl resulted in EAAT1–3 NAM activity, i.e., 35 (EAAT1 IC₅₀ = 344 ± 25 nM and EAAT3 IC₅₀ = 125 ± 85 nM, respectively). 4-Fluorophenyl substitution on the amide N-phenethyl provided good EAAT1–3 PAM activity, i.e., 36 (EAAT1 EC₅₀ = 58 ± 11 nM and EAAT3 EC₅₀ = 4.3 ± 4.3 nM). Shortening the side chain length to the amide N-benzyl provided an inactive compound, 37, and substitution with a 4-methoxyphenyl amide N-benzyl also resulted in an inactive compound, 38. An additional set of analogs was synthesized in the amide a series with a 4-cyanophenyl moiety (Table 5). The amide N-benzyl analog, 39, is an EAAT1 PAM EC₅₀ = 5.3 ± 4.7 nM and EAAT2 PAM. The 4-fluorophenyl analog 40 (NA-014) resulted in a selective EAAT2 PAM. Shortening the side chain to an N-benzyl lost activity, i.e., 41 is inactive and the 4-methoxyphenyl N-benzyl analog, 42, is inactive.

A selected subset of analogs were evaluated for metabolic stability in mouse liver microsomes (MLM) and for hERG binding (Table 6) to see if there was an improvement over the poorly stable GT949. Replacement of the 6-methoxy-quinolin-2-one ring with a 3-pyridinyl, i.e., 3, provided modest-to-good stability. The two analogs 4 (DA-023) and 5 replace the lipophilic N-cyclohexyl group with an N-methyl and have either an N-phenethyl or N-benzyl side chain off the tetrazole. Compound 4 (DA-023) shows poor stability in the MLM assay, but the N-benzyl analog provides a compound with good stability, which is improved over GT949 and 3. Thus, reducing lipophilicity (N-cyclohexyl to N-methyl) and modification to the side chain (tetrazole N-phenethyl versus N-benzyl) provided a compound, i.e., 5, stable to metabolic degradation in MLM, which may be predictive of reduced clearance in a mouse due to metabolism. Compound 30 MLM stability is consistent with the tetrazole N-benzyl analogs 3 and 5. The nitrile of 30 may be a modest metabolic liability, since we found it has some chemical stability concerns. Compound 40 (NA-014) is an example in the amide series which is selective for EAAT2 and shows good MLM stability, which may be improved by replacement of the nitrile due to the modest chemical stability issues with this moiety.

Table 6. Mouse Liver Microsome Stability, Aqueous Solubility, and hERG Channel Activity for Select Analogs.

The hERG binding assay, Predictor hERG Tracer Red binding to Predictor hERG Membrane (Invitrogen;⁶¹ data obtained from Reaction Biology, Inc.; Malvern PA), showed only modest hERG inhibition for GT949 and weak hERG inhibition for compound 4 (DA-023). The modest hERG binding for 29, 30, 36, and 40 (NA-014) suggests that hERG inhibition is not a concern with this series.

Dose–Response Curves and Kinetic Properties of EAAT2 PAMs and NAMs on l-Glu Uptake

Dose–response curves for the activity of compounds 4 (DA-023) and 40 (NA-014) are shown in Figure 1A,B, respectively, in EAAT1–3 transfected COS-7 cells. EC₅₀ and efficacy values for DA-023 are 1.0 ± 0.8 nM and 157.3 ± 10.3% and for compound 40 (NA-014) are 3.5 ± 2.0 nM and 167.3 ± 8.3%, respectively. No effects on EAAT1- or EAAT3-mediated uptakes were observed, illustrating their selective EAAT2 PAM activity. Compound 4 (DA-023) and compound 40 (NA-014) were further screened in kinetic assays to evaluate their effects on V_max and K_m of glutamate transport in EAAT2-transfected COS-7 cells (Figure 1C,D). Compound 4 (DA-023) was evaluated in EAAT2-transfected cells, and V_max values were increased from 303 ± 43 pmol/well/min for control (vehicle) conditions to 407 ± 18, 709 ± 95, and 975 ± 95 pmol/well/min in the presence of increasing concentrations of 4 (10, 100, and 500 nM) (Figure 1C). K_m values were not significantly different among conditions. For compound 40 (NA-014), V_max values were significantly increased from 279 ± 76 pmol/well/min for control (vehicle) conditions to 359 ± 17, 675 ± 52, and 1413 ± 46 pmol/well/min in the presence of increasing concentrations of compound 40 (NA-014) (10, 100, and 500 nM) (Figure 1D). These results validate the mechanism of stimulation to be through positive allosteric modulation of EAAT2 since the affinity for the substrate (K_m values) was not significantly different among conditions (One-way ANOVA followed by Dunnett’s posthoc test comparing to the vehicle), with an average ± SD of 63 ± 8.5 μM for EAAT2. Next, we evaluated the effect of these compounds in glia cultures, an environment that expresses endogenous l-Glu transporters. Dose–response curves of 4 (DA-023) and 40 (NA-014) show an EC50 of 0.50 ± 0.04 nM and an efficacy of 261 ± 27% and EC50 = 13.4 ± 10.2 nM and efficacy = 189.4 ± 53%, respectively (Figure 2A,B).

Figure 1.

Dose–response curves of 4 (DA-023, A) and 40 (NA-014, B) on l-Glu transport mediated by EAAT1, EAAT2, or EAAT3, showing that compounds do not modulate EAAT1- or EAAT3-mediated transport but increase EAAT2 selectively. EC₅₀s and efficacies are indicated. Cells were incubated with varied concentrations of compounds for 10 min at 37 °C and 10 min with 50 nM ³H-l-glutamate. Results are normalized to percentage of control (vehicle) and expressed as mean ± SEM of three independent experiments. Kinetic analyses of 4 (DA-023, C) and 40 (NA-014, D) on l-Glu uptake in EAAT2-transfected COS-7 cells preincubated with 10, 100, and 500 nM compounds. V_max and K_m values are indicated in the table; K_m was not statistically different between the varying concentrations of compounds. V_max is increased in the presence of 10 nM 4 (*p <0.05), 10 nM 40 (**p <0.01), 100 nM 4 and 40 (***p <0.001), and 500 nM 4 and 40 (****p <0.0001, One-way ANOVA compared to the vehicle). Results are expressed in pmol/well/min (V_max) and μM (K_m) and expressed as mean ± SEM of three independent experiments.

Figure 2.

Dose–response curves of 4 (DA-023, A) and 40 (NA-014, B) on l-Glu transport in glia cultures. EC₅₀s and efficacies are indicated. Cells were incubated with varied concentrations of compound for 10 min at 37 °C and 10 min with 50 nM ³H-l-glutamate. Results are normalized to percentage of the control (vehicle) and expressed as mean ± SEM of three independent experiments. Kinetic analyses of 4 (C) and 40 (D) on l-Glu uptake in glia cultures. Cells preincubated with 10, 100, and 500 nM compounds. V_max and K_m values are indicated in the table; K_m was not statistically different between the varying concentrations of compounds. V_max is increased in the presence of 10 nM 4 (*p <0.05), 10 nM 40 (**p <0.01), 100 nM 4 and 40 (***p <0.001), and 500 nM 4 and 40 (****p <0.0001, One-way ANOVA compared to the vehicle). Results are expressed in pmol/well/min (V_max) and μM (K_m) and expressed as mean ± SEM of three independent experiments.

Kinetic experiments performed in cultured glia in the presence of different concentrations of compound 4 (DA-023) show that the V_max values were significantly increased from 13.27 ± 2.5 pmol/well/min for control (vehicle) conditions to 17.4 ± 2.5, 22.7 ± 3.7, and 29.9 ± 2.7 pmol/well/min in the presence of 10, 100, and 500 nM compound 4 (Figure 2C). For compound 40 (NA-014), V_max values were significantly increased from 14.9 ± 1.75 pmol/well/min for control (vehicle) conditions to 18.9 ± 1.1, 21 ± 2.7, and 28 ± 2.4 pmol/well/min in the presence of 10, 100, and 500 nM compound 40 (NA-014), respectively (Figure 2D). Similarly, to the results in transfected cells, the K_m values were not significantly different among conditions (One-way ANOVA followed by Dunnett’s posthoc test comparing to the vehicle), with an average ± SD of 16.9 ± 5 μM, suggesting once more that these compounds act through positive allosteric modulation of l-Glu transporters.

We next evaluated one of the inhibitor compounds, 34 (Table 4). The dose–response curves in EAAT2-transfected COS-7 cells (Figure 3A) and in glia (Figure 3C) show IC₅₀ values of 10.4 ± 0.3 μM in COS-7 cells and 752 ± 34 nM in glia, and efficacy values for inhibition of 23 ± 7% in COS-7 cells and 46 ± 11% in glia. Compound 34 is inactive in EAAT1- and EAAT3-transfected COS-7 cells (Table 4).

Figure 3.

Dose–response and kinetic analysis for an EAAT2 NAM. Dose–response curves for 34 (NA-10) on l-glu transport in COS-7 cells (A) and glia (C) show that the compound decreases l-Glu uptake, IC_50s and efficacies are indicated. Cells were incubated with varied concentrations of compound 34 for 10 min at 37 °C and 10 min with 50 nM ³H-l-Glu. Results are normalized to percentage of the control (vehicle) and expressed as Mean ± SEM of at least three independent experiments. Kinetic analysis of l-Glu uptake in the presence of 34 (NA-10) in COS-7 transfected with EAAT2 (B) and glia (D). Cells were preincubated with 10, 100 nM, and 1 μM compound. V_max and K_m values are indicated in the table; K_m was not statistically different between the varying concentrations of compounds. V_max is decreased in the presence of 10 nM (*p <0.05), 100 nM (**p <0.01), and 1 μM (***p <0.001) in COS-7 cells, and in the presence of 100 nM (*p <0.05) and 1 μM (**p <0.01) of 34 (NA-10), One-way ANOVA compared to the vehicle.

Kinetic experiments of l-Glu transport are shown (Figure 3B,D). In EAAT2-transfected COS-7 cells (Figure 3B), compound 34 decreased the V_max from 249 ± 8 pmol/well/min (control conditions) to 214 ± 19, 188 ± 18, and 164 ± 18 pmol/well/min in the presence of 10, 100 nM, and 1 μM compound 34, respectively. The K_m was not significantly different among conditions. In glia cultures (Figure 3D), the V_max of l-Glu uptake was decreased from 13 ± 4 (control conditions) to 7 ± 2 and 3.4 ± 1.7 pmol/well/min in the presence of 100 nM and 1 μM compound 34, respectively. Likewise, the K_m was not significantly different among conditions. These experiments demonstrate that compound 34 decreases V_max without altering K_m, validating its mechanism as an EAAT2-selective NAM.

EAAT2 Inhibitors Abolish the Effects of EAAT2 PAMs and NAMs

The effects of the compounds 4, 34, and 40 are abolished in the presence of WAY 213613, a nonsubstrate inhibitor of EAAT2 (Figure S1). Glutamate transport assays in COS-7 cells transiently transfected with either empty vector CMV or EAAT2, incubated in the presence of 10 μM 4 or 40 or 100 μM 34 with or without 1 μM WAY 213613, a concentration that only blocks EAAT2 but not EAAT1 or EAAT3, suggesting that the mechanism of the compounds involve direct modulation of EAAT2.

Pharmacokinetic Evaluation of Compounds 4 (DA-023) and 40 (NA-014)

We evaluated the EAAT2-selective PAMs, compound 4 (DA-023) and compound 40 (NA-014), in mouse pharmacokinetic studies (Chempartners) to determine plasma and brain distribution concentrations after a single IP administration to male CD1 mice. We have also obtained plasma protein binding and brain tissue binding to calculate free unbound compound in plasma, fu plasma, and brain tissue, fu brain, and provide the concentration of unbound compound in the brain compared to plasma represented by the unbound partition coefficient Kp, uu.^62,63 The unbound brain-to-plasma concentration ratio provides a more meaningful value for the extent of blood-brain barrier (BBB) transport, where brain exposure is normalized to systemic exposure.^64,65 After a single IP dose at 10 mg/kg in male CD1 mice, compound 4 (DA-023) showed a C_max at 15 min (10 mg/kg; IP; mouse plasma C_max = 824 ng/mL; AUC _0-∞ (ng_*h/mL) = 413). Compound 4 (DA-023) was poorly brain penetrant (mouse brain C_max = 31.9 ng/mL; AUC _0-∞ (ng_*h/mL) = 38.7), with a brain-to-plasma ratio = 0.54, estimated from drug concentrations at the 1 h time point. If we used the AUC to estimate the brain-to-plasma ratio, then the brain-to-plasma ratio = 0.09. Compound 4 (DA-023) was cleared rapidly resulting in a short half-life (t_1/2 = 0.9 h, plasma and 0.4 h, brain). The concentration of unbound 4 in the brain compared to plasma, Kp, uu = 0.045. The short half-life is consistent with the poor metabolic stability observed in the mouse liver microsome assay (Figure S2). In comparison, after a single IP dose at 10 mg/kg in male CD1 mice, compound 40 (NA-014) showed a C_max at 30 min (10 mg/kg; IP; mouse plasma C_max = 330 ng/mL; AUC _0-∞ (ng_*h/mL) = 624). Compound 40 (NA-014) was about 10-fold more brain penetrant (mouse brain C_max = 279 ng/mL; AUC _0-∞ (ng_*h/mL) = 515), with a brain-to-plasma ratio = 1.32, estimated from drug concentrations at the 1 h time point. If we used the AUC, then the brain-to-plasma ratio = 0.82. Compound 40 (NA-014) was cleared with a half-life (t_1/2 = 1.19 h, plasma and 1.23 h, brain). The concentration of unbound 40 in the brain compared to plasma, Kp, uu = 0.207 (Figure 4). Thus, compound 40 (NA-014), although not optimal, is a brain penetrant compound with exposure better than DA-023.

Figure 4.

Mouse pharmacokinetic study with Compound 40 (NA-014). (A) Mean plasma and brain concentration–time profiles of Compound 40 after a single IP dose at 10 mg/kg in male CD1 mice (N = 3/time point). (B) In-life parameters for plasma and brain concentrations for Compound 40 (NA-014). Plasma protein binding and brain tissue binding provided free drug concentration in plasma fu,plasma = 0.427 and free drug concentration in brain fu,brain = 0.107. The brain-to-plasma ratio at 1 h = 1.32.

Off-Target Screening for Compound 40 (NA-014)

Compound 40(NA-014) was screened against GABA, glycine, monoamine transporters, and alanine transporters and does not have any activity toward these neurotransmitter transporters (Figure S3). Compound 40 (NA-014) did not have any activity against glutamate receptors NMDA, AMPA, mGluR1, and mGluR5 (Eurofins) (Table S1). Compound 40 (NA-014) was also evaluated in the NIMH-Psychoactive Drug Screening Program (PDSP)⁶⁶ for screening on binding assays for serotonin, adrenergic, BZP; dopamine, opioid, histamine, muscarinic, benzodiazepine, and sigma receptors, which revealed a lack of effect on any of the receptors evaluated (Tables S2 and S3).

Chiral Separation and Evaluation of Single-Enriched Enantiomers of Compound 40 (NA-014)

Since we had previously observed that GT949⁴³ (Table 1) was enantioselective at stimulating EAAT2, we wanted to evaluate the single-enriched enantiomers of compound 40 for enantioselective EAAT2 stimulation in our functional assays. Therefore, we contracted for the chiral separation of compound 40 (NA-014) (Chempartners, Inc.) to provide each single-enriched enantiomer of compound 40 (NA-014). Evaluation of racemic 40 (NA-014) on a chiral column shows two peaks with retention times at 0.78 min and another at 1.36 min with a 1:1 ratio confirming good separation (Figure S4). Each enantiomer was purified showing a single peak by a chiral column analysis and by analytical HPLC after solvent evaporation to confirm that the single-enriched enantiomers were pure and did not racemize. Then, each single enantiomer of 40 (NA-014) was evaluated for functional activity for effects on EAAT2-mediated glutamate transport. After analysis for EAAT2 PAM activity, each single enantiomer showed relatively the same activity (no statistical differences between EC₅₀s or efficacies). This is in contrast to the results with single enantiomers of GT949.⁴³ Therefore, in an attempt to understand the result, we evaluated docking of each single enantiomer of GT949 and 40 (NA-014) to human EAAT2 structures.

Molecular Docking of the R- and S-Enantiomers of GT949 and 40 (NA-014) onto EAAT2

We utilized our computational expertise with our EAAT2 model, which predicted binding poses for (R)-AS-1 and (R)-GT949 docking into the allosteric site.⁶⁷ This putative EAAT2 allosteric site is located in the transport domain, which undergoes a concerted elevator-like movement along with gating events when the transporter reconfigures between its outward-facing (OF) and inward-facing (IF) states. We hypothesize that small molecules binding in this allosteric site can either enhance (PAM) or disrupt (NAM) this movement. Our original virtual docking study at this allosteric site provided the GT949 series of compounds.⁴³ The orthosteric site residues D475, R478, and N482 define where glutamate binds in the transporter, and the allosteric site important residues are K299, W472, M86, D83, K90, and P443. The ligand-binding sites and binding poses were determined using AutoDock Vina,⁶⁸ based on the EAAT2 OF conformer, following the previous approach.⁶⁷ Simulations indicate that GT949-S (Figure 5A) and GT949-R (Figure 5B) bind at the interface between the scaffold domain and transport domain, i.e., the allosteric site of EAAT2 previously identified.⁴³ The view of the allosteric site in comparison to the orthosteric site is also shown for clarity (Figure 5C), where the orthosteric site residues are colored black, GT949-R is colored by the atom type, and the key residues in the allosteric site are in gray.

Figure 5.

Comparison of the most favorable molecular docking pose of single enantiomers of GT949 in the allosteric site of EAAT2. The binding poses of GT949-S (A) and GT949-R (B) are predicted to bind at the allosteric site at the interface between the scaffold domain and transport domain of human EAAT2, with binding free energies of −7.5 and −8.0 kcal/mol, respectively. Residues D83, K90, and P443 are labeled to orient the binding view. The pi–cation interaction in GT949-R, the unfavorable steric interaction in GT949-S, and lower free energy predict that GT949-R is the more potent enantiomer. The EAAT2 protein was modeled after the EAAT1 OF conformer (PDB: 5LLU). (C) Structure of the EAAT2 homology model pointing out the binding site for GT949-R and colored in black are orthosteric site residues D475, R478, and N482 define where glutamate binds. The structure was visualized using Molsoft ICM Browser (Molsoft, L.L.C., San Diego, CA, USA).

The corresponding calculated binding energies are −7.5 kcal/mol (GT949-S) and −8.0 kcal/mol (GT949-R). The view for the GT949 enantiomers is oriented so that the phenethyl tetrazole is in the same relative position for docking of both the R and S poses. The residues D83, K90, and P443 are shown to aid in orienting the view. GT949-S prefers a conformation where the 6-methoxyquinolin-2(1H)-one aromatic group can bind in a pocket in the upper left quadrant (Figure 5A). While in the GT949-R-enantiomer, the 6-methoxyquinolin-2(1H)-one aromatic group binds in the lower left quadrant where it is engaged in a favorable pi–cation interaction with the lysine residue, K90. This favorable interaction is not available for the GT949-S enantiomer. For GT949-S binding pose, the K90 epsilon nitrogen atom is forced to be close to the piperazine ring N atom so it can be involved in a H-bond with the quinolin-2(1H)-one carbonyl group, potentially causing the modest electronic repulsion. Thus, the favorable pi–cation interaction seen in GT949-R may provide the extra binding energy evident from the calculated lower binding free energy. Also, the potential steric interaction of the two aromatic rings in GT949-S makes this a less favorable ligand for EAAT2. These observations may explain why we observe a ∼20-fold difference in functional potency for the single-enriched enantiomers of GT949, although we have not unequivocally determined their configuration since they were resolved by chiral chromatography.

We also evaluated the binding poses of the 40 (NA-014) enantiomers. NA-014-S (Figure 6A) and NA-014-R (Figure 6B) are predicted to bind in the allosteric site of human EAAT2, in generally the same region. The calculated binding free energies are −6.5 kcal/mol (NA-014-S) and −7.4 kcal/mol (NA-014-R). Residues D83, K90, and P443 are labeled to orient the binding view. The NA-014 enantiomers are predicted to bind in a site slightly lower than for the GT949 enantiomers. The view of the docked binding poses of the NA-014 enantiomers are oriented so the 4-fluoro-phenethyl moiety is in the same relative orientation, in the back of the binding pocket. In the NA-014-S docking pose, the 4-cyano-phenyl moiety is in the upper left-hand quadrant (Figure 6A), while in the NA-014-R-enantiomer docking pose, the 4-cyano-phenyl moiety is in the lower left quadrant (Figure 6B). Compound 40 (NA-014) has a smaller aromatic group compared to GT949 and a smaller less lipophilic N-methyl piperazine instead of the bulky N-cyclohexyl piperazine. As noted above, experimentally, we find that both enantiomers of 40 (NA-014) appear equally active in our functional assay to monitor EAAT2 stimulation.

Figure 6.

Comparison of the most favorable molecular docking pose of single enantiomers of NA-014 in the allosteric site of EAAT2. The binding poses of NA-014-S (A) and NA-014-R (B) are predicted to bind at the interface between the scaffold domain and transport domain of human EAAT2, with binding free energies of −6.5 and −7.4 kcal/mol, respectively. Residues D83, K90, and P443 are labeled to orient the binding view. The NA-014 enantiomers are predicted to bind in a site slightly lower than for GT949 enantiomers. The EAAT2 protein was modeled after the EAAT1 OF conformer (PDB: 5LLU). The structure was visualized using Molsoft ICM Browser (Molsoft, L.L.C., San Diego, CA, USA).

Thus, both enantiomers appear to bind equally well without any obvious advantage for one enantiomer over the other and both can engage in an H-bond with the amide NH of M240. The docking model predicts that the NA-014-R-enantiomer would bind with higher affinity due to the lower binding free energy. However, our functional assay cannot distinguish the difference between these two similar enantiomers. The docking simulation helps us to explain why both enantiomers of compound 40 (NA-014) appear equally potent in the functional assays, in contrast to GT949, and suggests that additional modified analogs that clearly differentiate the enantiomers may be enantiospecific, like GT949.⁴³

Discussion

One of the key objectives of this study was to optimize the compound, GT949, identified as a PAM of EAAT2. GT949 is a highly lipophilic molecule that displays poor water solubility, and poor metabolic stability as measured in mouse liver microsomes. The compound 4 (DA-023) series was readily synthesized using similar chemistry to GT949, and the focus was to reduce the lipophilicity to improve the physiochemical properties of the series. Compound 4 (DA-023) was identified as a selective EAAT2 PAM with a reduced LogP of 2.5 compared to 4.2 for GT949. Notably, the structure–activity relationships (SAR) for EAAT modulation are complex for this series. The length of the side chain is very important for selectivity of EAAT2 modulation. A shorter side chain, benzyl versus phenethyl, resulted in loss of EAAT2 selectivity. The regiochemistry of the pyridine side chain was also sensitive to EAAT modulation. The change of the pyridine to a substituted aromatic ring increased the lipophilicity compared to 4 (DA-023), but also provided 20 and 22, which emerged as selective EAAT2 PAMs. In general, 4 (DA-023) emerged as the best compound from this series based on EAAT2 selectivity and calculated properties. GT949 had poor metabolic stability in the mouse liver microsome (MLM) assay, and a key objective was to improve metabolic stability. However, 4 (DA-023) and 5, two analogs from this series with LogP values between 2.0 and 2.5 showed mixed results in the MLM assay. Compound 4 (DA-023), containing a phenethyl side chain off the tetrazole nitrogen, showed poor MLM stability compared to 5, which has a benzyl moiety off the tetrazole nitrogen, which has very good stability of over 1 h in the MLM assay. It may be that the phenethyl moiety is more readily available for an attack by metabolic enzymes leading to N-dealkylation compared to the more compact benzyl side chain. We evaluated a set of amide isosteric analogs of the compound 4 (DA-023) series where the amide was used to replace the tetrazole. Synthesis of the direct analog to compound 4 (DA-023), 38, resulted in an inactive compound, perhaps due to its inability to bind in the allosteric site in an analogous manner as 4 (DA-023). However, 36, which contains a 4-fluorophenethyl side chain, and is almost analogous to 4 (DA-023), was found to be a nonselective EAAT PAM. Interestingly, the amide analogs, with a shorter side chain, 34 and 35 were NAMs. Replacement of the pyridine side chain in the amide series produced two PAMs. The benzyl side chain analog, 39, was a nonselective PAM, while 40 (NA-014) containing the 4-fluorophenethyl side chain was a selective EAAT2 PAM. We evaluated 40 (NA-014) for physiochemical properties and found it has good stability in the MLM assay, moderate water solubility, and has better pharmacokinetic properties including better brain penetration than 4 (DA-023). Thus, compound 40 (NA-014) may serve as a tool compound to study EAAT2 modulation in vivo; until EAAT2 PAMs with better pharmacokinetic properties are identified. In vitro neuroprotection studies suggest that the EAAT1 and EAAT2 PAM, as well as the selective EAAT2 PAM, display neuroprotection against glutamate and oxygen-glucose deprivation insults (unpublished data from ACKF).

We attempted to determine enantioselectivity of 40 (NA-014) by separating the racemic mixture into each enantioenriched isomer using chiral chromatography. However, evaluation of each single-enriched enantiomer did not clearly demonstrate enantioselectivity using our l-Glu uptake assays, although GT949 activity was shown to be enantioselective using the same analysis. Thus, we evaluated the binding poses of the NA-014 single enantiomers and compared these to the single enantiomers of GT949 using sophisticated computational methods. Interestingly, the computational analysis predicts that the R-enantiomer of both GT949 and 40 (NA-014) is the more potent enantiomer. However, in contrast to GT949, which has a larger aromatic group and clearly suggests a favorable pi–cation interaction for the R-enantiomer, the single enantiomers of 40 (NA-014) appear to bind equally well and there are no distinguishing favorable interaction suggesting one enantiomer is preferred over the other. Potentially, the functional assays that we employ to study these allosteric modulators may not be sensitive enough to distinguish the potency of each enantiomer if they bind in the allosteric site in a very similar fashion. In addition, 40 (NA-014) appears to bind in a position slightly lower than GT949 in the allosteric site. Thus, it is compelling to ask if binding affinity against a radio-labeled 40 (NA-014), in a competitive binding assay, would be sensitive enough to observe the potential differences in affinity between each single enantiomer of 40 (NA-014), which we are currently investigating.

The putative EAAT2 allosteric site is located in the transport domain, which undergoes a concerted elevator-like movement along with gating events when the transporter reconfigures between its outward-facing (OF) and inward-facing (IF) states. Thus, we hypothesize that small molecules binding in this allosteric site can potentially enhance (PAM) or disrupt (NAM) this movement. Future studies are aimed at studying rates of conformational transitions underlying transport using fluorescence-based approaches, with the hypothesis that the transport rate of EAATs is also controlled by the rate of transport domain transitions and that PAMs and NAMs modulate these rates. Previous studies on an archaeal homologue of EAATs, GltPh,⁵¹ have suggested that the rate of substrate transport is limited by the frequency of the elevator-like movements of the transport domain between the extracellular and cytoplasmic positions.⁵⁴ These motions, first inferred from crystal structures, were visualized using single-molecule FRET (smFRET) and other spectroscopic techniques.⁶⁹

When considering potential future translational studies, it is worth noting that the PAM activity of compounds 2, 5, 11, 29, and 39 on EAAT1 and EAAT2 is desirable, as targeting astrocytic glutamate transporters over neuronal transporters may be beneficial over activation of neuronal transporters, such as EAAT3 and EAAT4. An EAAT2-selective PAM is most desirable since EAAT2 is responsible for more than 90% of glutamate uptake and it is expected that any abnormality in the expression or function of this transporter could lead to increased extracellular glutamate concentration leading to neuronal death. Therefore, selectively targeting EAAT2 may provide an optimal potential therapeutic approach toward maintaining glutamate homeostasis and preventing disorders due to increased glutamate concentration. Compounds GT949 (early generation) and 4 (DA-023), 1, 20, and 40 (NA-014; later generation) are examples of selective EAAT2 PAMs. Nonselective PAMs include compounds such as 30, which is a broad EAAT1–3 PAM, and can be helpful as a pharmacological tool to further understand how these different molecules affect EAAT activity. It is noteworthy that NAMs in this study show the modest μM range potency, compared to the PAMs that are in the nM range. Nonetheless, EAAT2 NAMs, such as 34, could be used to study disorders such as Rett’s syndrome, a severe and progressive neurodevelopmental disorder characterized by reduced glutamatergic activity during early maturation.⁷⁰ This suggests that enhancement of glutamatergic neuronal activity with an EAAT2 NAM could prevent the onset of molecular and cellular phenotypes of Rett’s syndrome, and future studies exploring the utility of EAAT2 NAMs for this disorder are warranted.

We have dedicated some extra effort to characterizing 40 (NA-014), our lead selective EAAT2 PAM. Based on its pharmacokinetic properties, 40 (NA-014) is shown to be brain penetrant, and although not optimal, it serves as a tool compound to study EAAT2 pharmacology since it has no obvious off-target activities and is selective against other neurotransmitter transporters such as GABA, glycine, monoamines, and glutamate receptors such as NMDA, AMPA, mGluR1 and mGluR5 that could confound interpreting results from in vivo studies. It is noteworthy to mention that selectivity for NMDA is critical since previous studies revealed that NMDA receptor blockers, although neuroprotective in vitro, caused severe side effects in vivo, such as dissociative effects and hallucinations.⁷¹⁻⁷³ Finally, 40 (NA-014) showed no off-target activity in the PSDP program from NIMH against a panel of receptor binding assays including serotonin, adrenergic, benzylpiperazine (BZP), dopamine, opioid, histamine, muscarinic, benzodiazepine, and sigma receptors.

Overall, 40 (NA-014) appears to be highly selective at EAAT2, and our experiments strongly suggest that it acts as a positive allosteric modulator (PAM). Allosteric modulation holds potential to provide a rapid approach to maintaining the highly precise temporal and spatial aspects of glutamatergic synaptic transmission,⁷⁴ in contrast to EAAT expression enhancers,^75,76 and a range of neurological and neuropsychiatric disorders could benefit from this approach. Collectively, these results encourage us to progress to in vivo studies. Hence ongoing and future directions will include in vivo studies in models of drugs of abuse disorders, epilepsy, neuropathic pain, and stroke, signifying promising translational avenues for this class of compounds.

Experimental Section

General Methods for Chemistry

All solvents and chemicals were used as purchased without further purification. The progress of all reactions was monitored on Merck precoated silica gel plates (with fluorescence indicator UV254) using the solvent system indicated. Column chromatography was performed with silica gel 60 (230–400 mesh ASTM) or performed using an automated Biotage Isolera one automated flash purification system with the solvent mixtures specified in the corresponding experiment. TLC plates were visualized by irradiation with ultraviolet light (254 nm). Proton (¹H) and carbon (¹³C) NMR spectra were recorded on a Bruker AVANCE III 400 High-Performance Digital NMR Spectrometer and 300 and 500 MHz Varian Unity Inova NMR systems. Chemical shifts are reported in parts per million (ppm, δ) using the residual solvent line as a reference. Splitting patterns are designated using the following abbreviations: s, singlet; d, doublet; t, triplet; dd, doublet of doublet; m, multiplet; br, broad. Coupling constants (J) are reported in hertz (Hz). Compound purity was determined by LCMS and NMR. LCMS was obtained on a Waters Acquity QDa UPLC/MS mass spectrometer with an electrospray ionization (ESI) source and a PDA detector (210–400 nm). The purity of all final compounds was 95% or higher. High-resolution accurate mass LCMS/MS data were acquired on a Thermo Q Exactive Plus mass spectrometer coupled with a Waters Nano-Acquity UPLC system.

3-((4-Cyclohexylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-6-methoxyquinolin-2(1H)-one (DA-015; GT949)

To a stirred solution of 2-hydroxy-6-methoxyquinoline-3-carbaldehyde (100 mg, 0.46 mmol) in 7 mL of isopropanol were added 1-cyclohexylpiperazine (85 mg, 0.50 mmol) and a catalytic amount of trifluoroacetic acid, and the reaction mixture was stirred at reflux; after 3 h, trimethylsilylazide (60 mg, 0.46 mmol) and (2-isocyanoethyl)benzene (53 mg, 0.46 mmol) were added to the reaction mixture and reflux continued for an additional 24 h. After completion of the reaction, as indicated by TLC monitoring, the mixture was cooled to room temperature, the solvent was evaporated under vacuum, and the crude residue was purified by flash chromatography using (0–15% methanol/dichloromethane) to obtain the pure compound (157 mg, 65.01% yield) as a pale-yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ 11.95 (s, 1H), 8.02 (s, 1H), 7.33 (d, J = 2.7 Hz, 1H), 7.30–7.08 (m, 7H), 5.56 (s, 1H), 4.69 (t, J = 7.4 Hz, 2H), 4.13 (s, 6H), 3.78 (s, 3H), 3.26–3.07 (m, 2H), 2.92 (s, 4H), 2.70 (d, J = 7.8 Hz, 3H), 2.57 (s, 2H), 1.96–1.66 (m, 4H), 1.56 (d, J = 12.0 Hz, 1H), 1.33–1.12 (m, 4H), 1.12–0.95 (m, 1H). ¹H NMR (300 MHz, acetone-d₆) δ 8.21 (s, 1H), 7.39–7.30 (m, 2H), 7.29–7.21 (m, 4H), 7.21–7.11 (m, 2H), 5.52 (s, 1H), 4.87–4.73 (m, 2H), 3.88 (s, 3H), 3.31 (t, J = 7.5 Hz, 2H), 2.53 (d, J = 15.4 Hz, 8H), 2.20 (s, 1H), 1.73 (d, J = 9.2 Hz, 4H), 1.57 (d, J = 11.7 Hz, 1H), 1.21–1.07 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆) δ 161.21, 154.84, 153.89, 139.34, 137.50, 133.20, 129.26, 128.90, 127.31, 127.14, 120.86, 119.73, 116.79, 109.98, 63.74, 55.96, 54.77, 48.72, 35.35, 27.55, 25.61, 25.20.

MS (ESI): m/z 528.4 [M+1]⁺.

HRMS (ESI m/z) for C₃₀H₃₈O₂N₇, calcd 528.30087, found 528.30810 [M+1]⁺. LCMS (ESI): m/z 528.33 [M+1]⁺.

6-Methoxy-3-((4-methylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)quinolin-2(1H)-one (1 (DA-025))

The title compound was prepared according to the procedure for GT949 from 2-hydroxy-6-methoxyquinoline-3-carbaldehyde (100 mg, 0.456 mmol), 1-methylpiperazine (50 mg, 0.50 mmol), trimethylsilylazide (60 mg, 0.456 mmol), and (2-isocyanoethyl)benzene (53 mg, 0.46 mmol to obtain the pure compound 1 (DA-025) (145 mg, 69.38% yield) as a pale-yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.23 (s, 1H), 7.29–7.21 (m, 1H), 7.22–7.08 (m, 6H), 7.01 (d, J = 2.6 Hz, 1H), 5.44 (s, 1H), 4.81–4.60 (m, 2H), 3.85 (s, 3H), 3.26 (t, J = 7.4 Hz, 2H), 2.70–2.48 (m, 7H), 2.34 (s, 3H), 2.03 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 176.08, 162.78, 155.48, 154.27, 141.28, 136.35, 132.44, 128.85, 127.23, 126.03, 121.52, 120.23, 116.89, 109.02, 55.69, 54.79, 54.55, 49.85, 48.96, 45.26, 36.10, 21.82.

MS (ESI): m/z 460.3 [M+1]⁺.

HRMS (ESI m/z) for C₂₅H₃₀O₂N₇, calcd 460.24555, found 460.24530 [M+1]⁺. LCMS (ESI): m/z 460.34 [M+1]⁺.

1-Cyclohexyl-4-((1-phenethyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)piperazine (2 (DA-019))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.933 mmol), 1-cyclohexylpiperazine (173 mg, 1.02 mmol), trimethylsilylazide (0.12 mL, 0.933 mmol), and (2-isocyanoethyl)benzene (0.12 mL, 0.933 mmol) to obtain the pure compound 2 (DA-019) (290 mg, 72.45% yield) as a pale-yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.57–8.53 (m, 1H), 8.34 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.31–7.25 (m, 4H), 7.06–6.99 (m, 2H), 4.86–4.75 (m, 1H), 4.74–4.64 (m, 1H), 4.27 (s, 1H), 3.26–3.14 (m, 2H), 2.54 (brs, 4H), 2.30 (d, J = 16.5 Hz, 4H), 2.18 (s, 1H), 1.78 (t, J = 15.0 Hz, 4H), 1.60 (d, J = 13.1 Hz, 1H), 1.34–1.00 (m, 6H). ¹³C NMR (100 MHz, CDCl₃) δ 153.51, 150.34, 149.92, 137.36, 136.75, 129.13, 128.80, 128.70, 127.51, 123.30, 63.34, 61.65, 50.08, 49.44, 48.73, 36.20, 28.86, 28.82, 26.17, 25.73.

MS (ESI): m/z 432.3 [M+1]⁺.

HRMS (ESI m/z) for C₂₅H₃₄N₇, calcd 432.28702, found 432.28664 [M+1]⁺. LCMS (ESI): m/z 432.44 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)-4-cyclohexylpiperazine (3 (DA-036))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.933 mmol), 1-cyclohexylpiperazine (173 mg, 1.02 mmol), trimethylsilylazide (0.12 mL, 0.933 mmol), and (2-isocyanomethyl)benzene(0.11 mL, 0.933 mmol) to obtain the pure compound 3 (DA-036) (320 mg, 82.26% yield) as a pale-yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 8.53 (dd, J = 4.8, 1.5 Hz, 1H), 8.36 (d, J = 2.0 Hz, 1H), 7.83 (d, J = 8.0 Hz, 1H), 7.39–7.31 (m, 3H), 7.29–7.23 (m, 1H), 7.17–7.06 (m, 2H), 5.77–5.64 (m, 2H), 4.71 (s, 1H), 2.65–2.45 (m, 4H), 2.45–2.25 (m, 4H), 2.17 (t, J = 11.0 Hz, 1H), 1.78 (t, J = 12.4 Hz, 4H), 1.61 (d, J = 12.6 Hz, 2H), 1.30–1.00 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 153.59, 150.35, 149.99, 137.31, 133.26, 129.22, 128.95, 128.80, 127.40, 123.34, 63.27, 62.15, 51.52, 50.55, 48.62, 28.92, 26.21, 25.77.

MS (ESI): m/z 418.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₄H₃₂N₇, calcd 418.27137, found 418.27157 [M+1]⁺. LCMS (ESI): m/z 418.33 [M+1]⁺.

1-Methyl-4-((1-phenethyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)piperazine (4 (DA-023))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.933 mmol), 1-methylpiperazine (85 mg, 1.03 mmol), trimethylsilylazide (107 mg, 0.933 mmol), and (2-isocyanoethyl)benzene (122 mg, 0.933 mmol) to obtain the pure compound 4 (DA-023) (285 mg, 84.07% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.56 (d, J = 4.7 Hz, 1H), 8.35 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.33–7.23 (m, 4H), 7.03 (s, 2H), 4.85–4.63 (m, 2H), 4.28 (s, 1H), 3.27–3.13 (m, 2H), 2.70–2.04 (m, 11H). ¹³C NMR (100 MHz, CDCl₃) δ 153.43, 150.33, 149.97, 137.27, 136.75, 129.14, 128.80, 128.61, 127.52, 123.25, 61.57, 54.84, 49.59, 49.44, 45.80, 36.18.

MS (ESI): m/z 364.3 [M+1]⁺.

HRMS (ESI m/z) for C₂₀H₂₆N₇, calcd 364.22442, found 364.22399 [M+1]⁺. LCMS (ESI): m/z 364.32 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)-4-methylpiperazine (5 (DA-037))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.933 mmol), 1-methylpiperazine (103 mg, 1.03 mmol), trimethylsilylazide (107 mg, 0.933 mmol), and (2-isocyanomethyl)benzene (107 mg, 0.933 mmol) to obtain the pure compound 5 (DA-037) (267 mg, 82.15% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.53 (dd, J = 4.8, 1.6 Hz, 1H), 8.38 (d, J = 2.1 Hz, 1H), 7.86–7.80 (m, 1H), 7.36 (dd, J = 4.9, 1.6 Hz, 3H), 7.29–7.23 (m, 1H), 7.13 (dd, J = 6.5, 2.8 Hz, 2H), 5.78–5.63 (m, 2H), 4.73 (s, 1H), 2.54–2.28 (m, 7H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 153.52, 150.27, 150.05, 137.21, 133.20, 129.24, 129.01, 128.93, 127.41, 123.36, 62.07, 54.73, 51.53, 49.99, 45.78.

MS (ESI): m/z 350.4 [M+1]⁺.

HRMS (ESI m/z) for C₁₉H₂₄N₇, calcd 350.20877, found 350.20849 [M+1]⁺. LCMS (ESI): m/z 350.32 [M+1]⁺.

1-((1-Phenethyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)piperazine (6 (DA-055))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.93 mmol), piperazine (103 mg, 1.03 mmol), trimethylsilylazide (0.12 mL, 0.93 mmol), and (isocyanoethyl)benzene (0.11 mL, 0.93 mmol) to obtain the pure compound 6 (DA-055) (267 mg, 82.15% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 3.5 Hz, 1H), 8.38–8.31 (m, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.34–7.22 (m, 4H), 7.05–6.95 (m, 2H), 4.82–4.71 (m, 1H), 4.69–4.59 (m, 1H), 4.32 (s, 1H), 3.97 (brs, 1H), 3.27–3.11 (m, 2H), 2.95–2.83 (m, 3H), 2.46–2.14 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 153.23, 150.20, 150.12, 137.19, 136.64, 129.18, 128.79, 127.57, 123.42, 61.76, 49.87, 49.45, 45.27, 36.16.

HRMS (ESI m/z) for C₁₉H₂₄N₇, calcd 350.20877, found 350.20789 [M+1]⁺. LCMS (ESI): m/z 350 0.15 [M+1]⁺.

tert-Butyl 4-((1-benzyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)piperazine-1-carboxylate (7 (VY-3-133))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (160 mg, 1.49 mmol), tert-butyl piperazine-1-carboxylate (279 mg, 1.49 mmol), trimethylsilylazide (0.20 mL, 1.49 mmol), and (2-isocyanomethyl)benzene (0.181 mL, 1.49 mmol) to obtain the pure compound 7 (VY-3-133) (400 mg, 61.63% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.55 (dd, J = 4.8, 1.5 Hz, 1H), 8.38 (d, J = 2.0 Hz, 1H), 7.79 (dt, J = 7.9, 1.8 Hz, 1H), 7.40–7.32 (m, 3H), 7.27 (t, J = 6.4 Hz, 1H), 7.12 (dd, J = 6.4, 2.7 Hz, 2H), 5.72–7.58 (m, 2H), 4.79 (s, 1H), 3.44–3.26 (m, 4H), 2.40–2.18 (m, 4H), 1.41 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 154.37, 153.26, 150.21, 150.15, 137.14, 133.07, 129.29, 129.10, 128.93, 127.30, 123.49, 79.93, 61.90, 51.51, 49.85, 43.41, 28.32.

HRMS (ESI m/z) for C₂₃H₃₀N₇O₂, calcd 436.24555, found 436.24502 [M+1]⁺. LCMS (ESI): m/z 436 0.24 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(pyridin-3-yl)methyl)piperazine (8 (VY-3-136))

The title compound was prepared according to the procedure for GT949 from pyridine-3-carbaldehyde (100 mg, 0.93 mmol), piperazine (103 mg, 1.03 mmol), trimethylsilylazide (0.12 mL, 0.93 mmol), and (isocyanomethyl)benzene (0.11 mL, 0.93 mmol) to obtain the pure compound 8 (VY-3-136) (267 mg, 82.15% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.54 (d, J = 4.7 Hz, 1H), 8.38 (s, 1H), 7.81 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 4.3 Hz, 3H), 7.31–7.21 (m, 1H), 7.13 (d, J = 4.0 Hz, 2H), 5.79–5.57 (m, 2H), 4.75 (s, 1H), 2.94–2.73 (m, 4H), 2.50–2.33 (m, 4H), 2.12 (brs, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 153.37, 150.27, 150.11, 137.20, 133.14, 129.28, 129.07, 128.97, 127.37, 123.44, 62.33, 51.53, 50.69, 45.49.

HRMS (ESI m/z) for C₁₈H₂₁N₇, calcd 336.19312, found 336.19232 [M+1]⁺. LCMS (ESI): m/z 336.26 [M+1]⁺.

1-Methyl-4-((1-phenethyl-1H-tetrazol-5-yl)(pyridin-2-yl)methyl)piperazine (9 (DA-056))

The title compound was prepared according to the procedure for GT949 from pyridine-2-carbaldehyde (100 mg, 0.933 mmol), 1-methylpiperazine (103 mg, 1.03 mmol), trimethylsilylazide (0.12 mL, 0.933 mmol), and (2-isocyanoethyl)benzene (0.13 mL, 0.933 mmol) to obtain the pure compound 9 (DA-056) (197 mg, 65.44% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 8.50–8.42 (m, 1H), 7.66–7.58 (m, 1H), 7.49 (dd, J = 7.8, 0.9 Hz, 1H), 7.28–7.09 (m, 6H), 4.83–4.72 (m, 3H), 3.28–3.08 (m, 2H), 2.32 (brs, 8H), 2.17 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 155.40, 153.83, 149.48, 137.03, 128.89, 127.14, 124.36, 123.49, 67.32, 54.89, 51.05, 49.48, 45.83, 35.97.

MS (ESI): m/z 364.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₀H₂₆N₇, calcd 364.22442, found 364.22497 [M+1]⁺. LCMS (ESI): m/z 364.25 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(pyridin-2-yl)methyl)-4-methylpiperazine (10 (DA-057))

The title compound was prepared according to the procedure for GT949 from pyridine-2-carbaldehyde (100 mg, 0.933 mmol), 1-methylpiperazine (103 mg, 1.03 mmol), trimethylsilylazide (0.12 mL, 0.933 mmol), and (isocyanomethyl)benzene (0.13 mL, 0.933 mmol) to obtain the pure compound 10 (DA-057) (240 mg, 73.61% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.50 (dd, J = 4.0, 0.8 Hz, 1H), 7.68 (td, J = 7.7, 1.7 Hz, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.36–7.29 (m, 3H), 7.27–7.18 (m, 3H), 6.02 (d, J = 15.3 Hz, 1H), 5.80 (d, J = 15.3 Hz, 1H), 5.05 (s, 1H), 2.51–2.20 (m, 8H), 2.21 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 155.09, 154.00, 149.38, 136.90, 134.12, 128.86, 128.47, 127.78, 124.32, 123.45, 67.15, 54.65, 51.63, 50.93, 45.80.

MS (ESI): m/z 350.5 [M+1]⁺.

HRMS (ESI m/z) for C₁₉H₂₄N₇, calcd 350.20877, found 350.20953 [M+1]⁺. LCMS (ESI): m/z 350.25 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(pyridin-4-yl)methyl)-4-methylpiperazine (11 (DA-058))

The title compound was prepared according to the procedure for GT949 from pyridine-4-carbaldehyde (100 mg, 0.93 mmol), 1-methylpiperazine (103 mg, 1.03 mmol), trimethylsilylazide (0.12 mL, 0.93 mmol), and (isocyanomethyl)benzene (0.11 mL, 0.93 mmol) to obtain the pure compound 11 (DA-058) (280 mg, 85.88% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.52 (dd, J = 4.5, 1.5 Hz, 2H), 7.38–7.29 (m, 3H), 7.21 (dd, J = 4.6, 1.5 Hz, 2H), 7.09 (dd, J = 7.1, 2.2 Hz, 2H), 5.68 (s, 2H), 4.75 (s, 1H), 2.50–2.28 (m, 8H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 153.04, 150.03, 142.18, 133.19, 129.20, 128.97, 127.34, 124.02, 63.53, 54.72, 51.46, 50.20, 45.77, 25.39.

MS (ESI): m/z 350.5 [M+1]⁺.

HRMS (ESI m/z) for C₁₉H₂₄N₇, calcd 350.20877, found 350.20892 [M+1]⁺.

1-((6-Fluoropyridin-3-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (12 (VY-3-285))

The title compound was prepared according to the procedure for GT949 from 6-fluoronicotinaldehyde (63 mg, 0.50 mmol), 1-methylpiperazine (51 mg, 0.50 mmol), trimethylsilylazide (0.066 mL, 0.50 mmol), and (isocyanoethyl)benzene (0.069 mL, 0.50 mmol) to obtain the pure compound 12 (VY-3-285) (280 mg, 85.88% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.98–7.90 (m, 1H), 7.86 (d, J = 2.1 Hz, 1H), 7.30–7.22 (m, 3H), 6.99 (dd, J = 6.4, 2.9 Hz, 2H), 6.92 (dd, J = 8.5, 2.9 Hz, 1H), 4.87–4.69 (m, 2H), 4.18 (s, 1H), 3.30–3.15 (m, 2H), 2.49–2.16 (m, 11H). ¹³C NMR (100 MHz, CDCl₃) δ 163.84 (d, J_C–F = 242.0 Hz), 153.11, 148.00 (d, J_C–F = 15.0 Hz), 142.08 (d, J_C–F = 8.4 Hz), 136.27, 129.23, 128.80, 127.67, 127.37 (d, J_C–F = 4.6 Hz), 110.39 (d, J_C–F = 38.0 Hz), 59.45, 53.60, 49.37, 46.91, 46.37, 43.16, 35.89.

HRMS (ESI m/z) for C₂₀H₂₅FN₇, calcd 382.21499, found 382.21415 [M+1]⁺. LCMS (ESI): m/z 382.32 [M+1]⁺.

5-((4-Methylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)pyridin-2-ol (13 (VY-3-286))

The title compound was prepared according to the procedure for GT949 from 6-hydroxynicotinaldehyde (62 mg, 0.50 mmol), 1-methylpiperazine (51 mg, 0.50 mmol), trimethylsilylazide (0.066 mL, 0.50 mmol), and (isocyanoethyl)benzene (0.069 mL, 0.50 mmol) to obtain the pure compound 13 (VY-3-286) (139 mg, 78% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.52 (dd, J = 9.5, 2.3 Hz, 1H), 7.32–7.21 (m, 3H), 7.12 (d, J = 1.8 Hz, 1H), 7.01–6.88 (m, 2H), 6.53 (d, J = 9.5 Hz, 1H), 4.81–4.66 (m, 2H), 3.94 (s, 2H), 3.33–3.15 (m, 2H), 2.50–2.16 (m, 11H). ¹³C NMR (100 MHz, CDCl₃) δ 164.79, 153.25, 142.83, 136.73, 135.12, 129.16, 128.82, 127.51, 119.84, 112.52, 60.18, 54.77, 49.43, 49.27, 45.71, 36.22.

HRMS (ESI m/z) for C₂₀H₂₆N₇O, calcd 380.21934, found 380.21840 [M+1]⁺. LCMS (ESI): m/z 380.32 [M+1]⁺.

5-((1-Benzyl-1H-tetrazol-5-yl)(4-methylpiperazin-1-yl)methyl)pyridin-2-ol (14 (VY-3-218))

The title compound was prepared according to the procedure for GT949 from 6-hydroxynicotinaldehyde (100 mg, 0.812 mmol), 1-methylpiperazine (82 mg, 0.812 mmol), trimethylsilylazide (0.107 mL, 0.812 mmol), and (isocyanomethyl)benzene (0.098 mL, 0.812 mmol) to obtain the pure compound 14 (VY-3-218) (201 mg, 68% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.58 (dd, J = 9.5, 2.5 Hz, 1H), 7.39–7.31 (m, 3H), 7.11 (dd, J = 5.7, 3.5 Hz, 3H), 6.49 (d, J = 9.5 Hz, 1H), 5.78–5.63 (m, 2H), 4.43 (s, 1H), 2.46–2.26 (m, 8H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.68, 153.39, 142.64, 135.07, 133.19, 129.30, 129.06, 127.35, 120.10, 112.71, 60.86, 54.69, 51.52, 49.79, 45.74.

HRMS (ESI m/z) for C₁₉H₂₄N₇O, calcd 366.20368, found 366.20282 [M+1]⁺. LCMS (ESI): m/z 366.15 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(6-bromopyridin-3-yl)methyl)-4-methylpiperazine (15 (VY-3-246))

The title compound was prepared according to the procedure for GT949 from 6-bromonicotinaldehyde (160 mg, 0.860 mmol), 1-methylpiperazine (86 mg, 0.860 mmol), trimethylsilylazide (0.114 mL, 0.860 mmol), and (isocyanomethyl)benzene (0.104 mL, 0.860 mmol) to obtain the pure compound 15 (VY-3-246) (290 mg, 79% yield) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (d, J = 2.4 Hz, 1H), 7.68 (dd, J = 8.3, 2.5 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.39–7.32 (m, 3H), 7.15–7.07 (m, 2H), 5.79–5.64 (m, 2H), 4.70 (s, 1H), 2.59–2.35 (m, 8H), 2.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 153.11, 150.50, 142.56, 139.66, 133.13, 129.30, 129.06, 128.39, 127.95, 127.36, 60.93, 54.43, 51.59, 49.31, 45.35.

HRMS (ESI m/z) for C₁₉H₂₃BrN₇, calcd 428.11983, found 428.11889 [M+1]⁺. LCMS (ESI): m/z 430.14 [M+2]⁺.

1-((4-Chlorophenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-cyclohexylpiperazine (16 (DA-033))

The title compound was prepared according to the procedure for GT949, using 4-chlorobenzaldehyde (100 mg, 0.711 mmol), 1-cyclohexylpiperazine (119 mg, 0.78 mmol), trimethylsilylazide (0.1 mL, 0.711 mmol), and (2-isocyanoethyl)benzene (0.09 mL, 0.711 mmol) to obtain the pure compound 16 (DA-033) (260 mg, 78.78% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.33–7.25 (m, 5H), 7.22 (d, J = 8.5 Hz, 2H), 7.04 (dd, J = 7.1, 2.2 Hz, 2H), 4.77–4.51 (m, 2H), 4.40 (s, 1H), 3.15 (t, J = 7.2 Hz, 2H), 2.54 (brs, 4H), 2.38 (brs, 2H), 2.33–2.12 (m, 3H), 1.87–1.71 (m, 4H), 1.61 (d, J = 12.4 Hz, 1H), 1.30–1.00 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 154.02, 136.71, 134.52, 132.25, 130.55, 129.05, 128.77, 128.74, 127.36, 63.95, 63.28, 50.88, 49.16, 48.78, 35.93, 28.98, 28.91, 26.24, 25.78.

MS (ESI): m/z 465.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₆H₃₄N₆Cl, calcd 465.25280, found 465.25381 [M+1]⁺. LCMS (ESI): m/z 465.23 [M+1]⁺.

1-((4-Chlorophenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (17 (DA-040))

The title compound was prepared according to the procedure for GT949 from 4-chlorobenzaldehyde (100 mg, 0.711 mmol), 1-methylpiperazine (78 mg, 0.783 mmol), trimethylsilylazide (0.09 mL, 0.711 mmol), and (isocyanoethyl)benzene (0.10 mL, 0.711 mmol) to obtain the pure compound 17 (DA-040) (245 mg, 86.87% yield) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.36–7.19 (m, 6H), 7.11–7.03 (m, 2H), 4.76–4.65 (m, 1H), 4.62–4.53 (m, 1H), 4.47–4.39 (m, 1H), 3.24–3.12 (m, 2H), 2.65–2.34 (m, 6H), 2.33–2.18 (m, 5H). ¹³C NMR (100 MHz, CDCl3) δ 153.99, 136.66, 134.60, 132.36, 130.45, 129.07, 128.81, 128.77, 127.40, 63.87, 54.88, 50.32, 49.16, 45.81, 35.90.

MS (ESI): m/z 397.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₁H₂₆N₆Cl, calcd 397.19020, found 397.18958 [M+1]⁺. LCMS (ESI): m/z 397.34 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(4-chlorophenyl)methyl)-4-methylpiperazine (18 (DA-066))

The title compound was prepared according to the procedure for GT949 from 4-chlorobenzaldehyde (50 mg, 0.356 mmol), 1-methylpiperazine (39 mg, 0.39 mmol), trimethylsilylazide (0.05 mL, 0.356 mmol), and (isocyanomethyl)benzene (0.04 mL, 0.356 mmol) to obtain the pure compound 18 (DA-066) (120 mg, 88.23% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.29 (m, 3H), 7.26–7.19 (m, 4H), 7.10–7.04 (m, 2H), 5.76–5.47 (m, 2H), 4.69 (s, 1H), 2.58–2.26 (m, 8H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 154.13, 134.64, 133.29, 132.28, 130.51, 129.15, 128.85, 128.77, 127.35, 64.12, 54.76, 51.30, 50.47, 45.77.

MS (ESI): m/z 383.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₀H₂₄ClN₆, calcd 383.17455, found 383.17506 [M+1]⁺. LCMS (ESI): m/z 383.15 [M+1]⁺.

1-Cyclohexyl-4-((4-fluorophenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)piperazine (19 (DA-046))

The title compound was prepared according to the procedure for GT949, using 4-fluorobenzaldehyde (100 mg, 0.806 mmol), 1-cyclohexylpiperazine (149 mg, 0.887 mmol), trimethylsilylazide (0.1 mL, 0.806 mmol), and (2-isocyanoethyl)benzene (0.1 mL, 0.806 mmol) to obtain the pure compound 19 (DA-046) (267 mg, 73.9% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.33–7.22 (m, 5H), 7.08–6.96 (m, 4H), 4.66–4.55 (m, 1H), 4.51–4.42 (m, 1H), 4.40 (s, 1H), 3.20–3.05 (m, 2H), 2.82 (brs, 4H), 2.62–2.36 (m, 5H), 1.95 (brs, 2H), 1.84 (brs, 2H), 1.64 (d, J = 12.0 Hz, 1H), 1.34–1.18 (m, 4H), 1.17–1.04 (m, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 162.85 (d, J_C–F = 247.0 Hz), 154.36, 136.50, 130.88 (d, J_C–F = 8.2 Hz), 129.77 (d, J_C–F = 3.3 Hz), 129.10, 128.76, 127.50, 115.87 (d, J_C–F = 21.0 Hz), 64.15, 63.57, 49.38, 49.06, 48.43, 35.84, 27.92, 25.67, 25.42.

MS (ESI): m/z 449.4 [M+1]+.

HRMS (ESI m/z) for C₂₆H₃₄N₆F, calcd 449.28235, found 449.28277 [M+1]⁺. LCMS (ESI): m/z 449.34 [M+1]⁺.

1-((4-Fluorophenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (20 (DA-044))

The title compound was prepared according to the procedure for GT949 from 4-Fluorobenzaldehyde (100 mg, 0.806 mmol), 1-methylpiperazine (89 mg, 0.887 mmol), trimethylsilylazide (0.10 mL, 0.806 mmol), and (isocyanoethyl)benzene (0.10 mL, 0.806 mmol) to obtain the pure compound 20 (DA-044) (276 mg, 90.19% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.32–7.23 (m, 5H), 7.07–6.97 (m, 4H), 4.74–4.50 (m, 2H), 4.44 (s, 1H), 3.15 (t, J = 7.2 Hz, 2H), 2.60–2.19 (m, 11H). ¹³C NMR (100 MHz, CDCl₃) δ 162.66 (d, J_C–F = 247.0 Hz), 154.22, 136.69, 130.89 (d, J_C–F = 8.2 Hz), 129.65 (d, J_C–F = 3.3 Hz), 129.05, 128.77, 127.38, 115.55 (d, J_C–F = 21.0 Hz), 63.80, 54.89, 53.46, 50.28, 49.12, 45.80, 35.89.

MS (ESI): m/z 381.4[M+1]⁺.

HRMS (ESI m/z) for C₂₁H₂₆FN₆, calcd 381.21975, found 381.21904 [M+1]⁺. LCMS (ESI): m/z 381.32 [M+1]⁺.

1-((4-Methoxyphenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (21 (DA-043))

The title compound was prepared according to the procedure for GT949, using 4-methoxybenzaldehyde (100 mg, 0.734 mmol), 1-methylpiperazine (81 mg, 0.807 mmol), trimethylsilylazide (0.10 mL, 0.734 mmol), and (2-isocyanoethyl)benzene (0.10 mL, 0.734 mmol) to obtain the pure compound 21 (DA-043) (245 mg, 85.36% yield) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.34–7.24 (m, 3H), 7.28–7.19 (m, 2H), 7.10–7.04 (m, 2H), 6.89–6.82 (m, 2H), 4.67–4.57 (m, 1H), 4.54–4.47 (m, 1H), 4.45 (s, 1H), 3.78 (s, 3H), 3.09 (d, J = 8.0 Hz, 2H), 2.54–2.32 (m, 6H), 2.31–2.20 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 159.77, 154.74, 136.72, 130.22, 129.01, 128.78, 127.33, 126.17, 114.11, 64.29, 55.32, 54.90, 50.59, 48.95, 45.77, 35.82.

MS (ESI): m/z 393.4[M+1]⁺.

HRMS (ESI m/z) for C₂₂H₂₉N₆O, calcd 393.23974, found 393.23914 [M+1]⁺. LCMS (ESI): m/z 393.25 [M+1]⁺.

4-((4-Methylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)phenol (22 (DA-045))

The title compound was prepared according to the procedure for GT949, using 4-hydroxybenzaldehyde (100 mg, 0.819 mmol), 1-methylpiperazine (90 mg, 0.9 mmol), trimethylsilylazide (0.11 mL, 0.819 mmol), and (2-isocyanoethyl)benzene (0.11 mL, 0.819 mmol) to obtain the pure compound 22 (DA-045) (253 mg, 81.87% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.33–7.24 (m, 4H), 7.04–6.93 (m, 3H), 6.60 (d, J = 8.4 Hz, 2H), 4.64–4.34 (m, 2H), 4.28 (s, 1H), 3.18–2.98 (m, 2H), 2.70–2.35 (m, 6H), 2.34–2.17 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 157.11, 154.86, 136.74, 130.57, 129.05, 128.81, 127.36, 123.14, 115.53, 63.86, 54.78, 49.09, 45.57, 35.95, 25.36.

MS (ESI): m/z 379.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₁H₂₇N₆O, calcd 379.22409, found 379.22550 [M+1]⁺. LCMS (ESI): m/z 379.32 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(4-methoxyphenyl)methyl)-4-cyclohexylpiperazine (23 (DA-006))

The title compound was prepared according to the procedure for GT949 from 4-methoxybenzaldehyde (100 mg, 0.734 mmol), 1-cyclohexylpiperazine (123 mg, 0.734 mmol), trimethylsilylazide (0.1 mL, 0.734 mmol), and (2-isocyanoethyl)benzene (0.088 mL, 0.734 mmol) to obtain the pure compound 23 (DA-006) (243 mg, 67.5% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.36–7.29 (m, 3H), 7.20 (d, J = 8.6 Hz, 2H), 7.12–7.04 (m, 2H), 6.80 (d, J = 8.6 Hz, 2H), 5.67 (d, J = 15.4 Hz, 1H), 5.45 (d, J = 15.4 Hz, 1H), 4.63 (s, 1H), 3.77 (s, 3H), 2.53 (brs, 4H), 2.44 (brs, 2H), 2.31 (brs, 2H), 2.16 (t, J = 10.8 Hz, 1H), 1.85–1.71 (m, 4H), 1.60 (d, J = 12.2 Hz, 1H), 1.28–1.00 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 159.75, 154.92, 133.50, 130.47, 129.04, 128.69, 127.43, 125.82, 113.97, 64.50, 63.31, 55.31, 51.23, 51.13, 48.70, 28.97, 26.26, 25.82.

MS (ESI): m/z 447.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₆H₃₅N₆O, calcd 447.28669, found 447.28611 [M+1]⁺. LCMS (ESI): m/z 447.44 [M+1]⁺.

1-Cyclohexyl-4-((3,4-dimethoxyphenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)piperazine (24 (DA-050))

The title compound was prepared according to the procedure for GT949, using 3,4-dimethoxybenzaldehyde (100 mg, 0.602 mmol), 1-cyclohexylpiperazine (111 mg, 0.662 mmol), trimethylsilylazide (0.08 mL, 0.602 mmol), and (2-isocyanoethyl)benzene (0.08 mL, 0.602 mmol) to obtain the pure compound 24 (DA-050) (155 mg, 52.54% yield) as a white solid. ¹H NMR (500 MHz, CDCl₃) δ 7.33–7.27 (m, 2H), 7.27–7.25 (m, 2H), 7.09–7.04 (m, 2H), 6.87–6.76 (m, 3H), 4.66–4.54 (m, 1H), 4.52–4.45 (m, 1H), 4.43 (s, 1H), 3.87–3.84 (m, 3H), 3.83–3.80 (m, 3H), 3.14–3.04 (m, 2H), 2.66–2.54 (m, 3H), 2.53–2.39 (m, 2H), 2.38–2.27 (m, 2H), 1.91–1.82 (m, 2H), 1.82–1.74 (m, 2H), 1.65–1.53 (m, 4H), 1.28–1.14 (m, 4H). ¹³C NMR (100 MHz, CDCl₃) δ 154.74, 149.30, 149.29, 136.67, 128.97, 128.74, 127.29, 126.85, 121.39, 111.92, 110.85, 64.85, 63.35, 56.03, 55.94, 51.42, 48.87, 48.76, 35.80, 28.97, 28.87, 26.23, 25.79.

MS (ESI): m/z 491.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₈H₃₉N₆O₂, calcd 491.31290, found 491.31345 [M+1]⁺. LCMS (ESI): m/z 491.35 [M+1]⁺.

1-((3,4-Dimethoxyphenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (25 (DA-049))

The title compound was prepared according to the procedure for GT949, using 3,4-dimethoxybenzaldehyde (100 mg, 0.602 mmol), 1-methylpiperazine (66 mg, 0.662 mmol), trimethylsilylazide (0.08 mL, 0.602 mmol), and (2-isocyanoethyl)benzene (0.08 mL, 0.602 mmol) to obtain the pure compound 25 (DA-049) (210 mg, 82.67% yield) as a brown solid. ¹H NMR (400 MHz, CDCl₃) δ 7.35–7.21 (m, 3H), 7.06 (d, J = 6.3 Hz, 2H), 6.91–6.77 (m, 3H), 4.67–4.57 (m, 1H), 4.56–4.45 (m, 1H), 4.44 (s, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.10 (t, J = 7.0 Hz, 2H), 2.56–2.34 (m, 6H), 2.33–2.20 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 154.69, 149.26, 136.64, 128.95, 128.72, 127.27, 126.83, 121.36, 111.80, 110.85, 64.61, 55.99, 55.91, 54.86, 50.75, 48.85, 45.77, 35.73.

MS (ESI): m/z 423.5 [M+1]⁺.

HRMS (ESI m/z) for C₂₃H₃₁N₆O₂, calcd 423.25030, found 423.25025 [M+1]⁺. LCMS (ESI): m/z 423.34 [M+1]⁺.

1-Cyclohexyl-4-((3,5-dimethoxyphenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)piperazine (26 (DA-052))

The title compound was prepared according to the procedure for GT949 from 3,5-dimethoxybenzaldehyde (100 mg, 0.602 mmol), 1-cyclohexylpiperazine (111 mg, 0.662 mmol), trimethylsilylazide (0.08 mL, 0.602 mmol), and (2-isocyanoethyl)benzene (0.08 mL, 0.602 mmol) to obtain the pure compound 26 (DA-052) (268 mg, 90.84% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.33–7.27 (m, 3H), 7.03–6.94 (m, 2H), 6.39 (t, J = 1.9 Hz, 1H), 6.33 (d, J = 2.0 Hz, 2H), 4.50–4.40 (m, 1H), 4.28–4.19 (m, 1H), 4.02 (s, 1H), 3.73 (s, 6H), 3.34 (d, J = 11.5 Hz, 2H), 3.11–3.04 (m, 2H), 3.01–2.91 (m, 3H), 2.76 (t, J = 11.3 Hz, 2H), 2.45 (t, J = 11.9 Hz, 1H), 2.31 (t, J = 12.0 Hz, 1H), 2.07 (d, J = 10.1 Hz, 2H), 1.92 (d, J = 12.8 Hz, 2H), 1.71 (d, J = 13.0 Hz, 1H), 1.45–1.21 (m, 5H). ¹³C NMR (100 MHz, CDCl3) δ 161.07, 154.39, 136.88, 136.61, 128.96, 128.79, 127.30, 106.95, 100.23, 65.22, 63.49, 55.42, 53.45, 51.33, 48.96, 48.69, 35.78, 28.79, 28.69, 26.14, 25.73.

HRMS (ESI m/z) for C₂₈H₃₉N₆O₂, calcd 491.31290, found 491.31198 [M+1]⁺. LCMS (ESI): m/z 491.33 [M+1]⁺.

1-((3,5-Dimethoxyphenyl)(1-phenethyl-1H-tetrazol-5-yl)methyl)-4-methylpiperazine (27 (DA-051))

The title compound was prepared according to the procedure for GT949, using 3,5-dimethoxybenzaldehyde (100 mg, 0.602 mmol), 1-methylpiperazine (66 mg, 0.662 mmol), trimethylsilylazide (0.08 mL, 0.602 mmol), and (2-isocyanoethyl)benzene (0.08 mL, 0.602 mmol) to obtain the pure compound 27 (DA-051) (182 mg, 71.65% yield) as a brown solid. ¹H NMR (400 MHz MeOD-d₄) δ 7.14–7.05 (m, 3H), 6.92–6.85 (m, 2H), 6.38 (t, J = 2.0 Hz, 1H), 6.34 (d, J = 2.1 Hz, 2H), 4.82 (s, 2H), 4.64 (s, 1H), 4.55–4.36 (m, 2H), 3.64 (s, 6H), 3.33–3.19 (m, 2H), 3.02–2.93 (m, 2H), 2.91–2.82 (m, 1H), 2.74 (s, 3H), 2.65 (d, J = 10.0 Hz, 1H), 2.44–2.33 (m, 1H), 2.31–2.20 (m, 1H). ¹³C NMR (100 MHz, MeOD-d₄) δ 161.53, 154.40, 136.77, 135.90, 128.62, 128.52, 126.88, 106.90, 100.28, 62.76, 54.55, 53.35, 48.59, 46.86, 42.00, 35.04.

HRMS (ESI m/z) for C₂₃H₃₁N₆O₂, calcd 423.25030, found 423.24970 [M+1]⁺. LCMS (ESI): m/z 423.34 [M+1]⁺.

4-((4-Cyclohexylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)benzonitrile (28 (DA-039))

The title compound was prepared according to the procedure for GT949, using 4-cyanobenzaldehyde (100 mg, 0.762 mmol), 1-cyclohexylpiperazine (141 mg, 0.838 mmol), trimethylsilylazide (0.1 mL, 0.762 mmol), and (2-isocyanoethyl)benzene (0.09 mL, 0.762 mmol) to obtain the pure compound 28 (DA-039) (238 mg, 68.58% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.30–7.22 (m, 3H), 7.05–6.98 (m, 2H), 4.85–4.59 (m, 2H), 4.35 (s, 1H), 3.21 (t, J = 6.9 Hz, 2H), 2.53 (brs, 3H), 2.41–2.11 (m, 5H), 1.78 (t, J = 11.9 Hz, 4H), 1.61 (d, J = 12.3 Hz, 2H), 1.30–0.99 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 153.21, 138.63, 136.72, 132.09, 130.13, 129.12, 128.80, 127.44, 118.30, 112.42, 63.73, 63.27, 50.51, 49.39, 48.76, 36.06, 28.94, 28.90, 26.21, 25.75.

HRMS (ESI m/z) for C₂₇H₃₄N₇, calcd 456.28702, found 456.28602 [M+1]⁺. LCMS (ESI): m/z 456.34 [M+1]⁺.

4-((4-Methylpiperazin-1-yl)(1-phenethyl-1H-tetrazol-5-yl)methyl)benzonitrile (29 (DA-035))

The title compound was prepared according to the procedure for GT949, using 4-cyanobenzaldehyde (100 mg, 0.762 mmol), 1-methylpiperazine (84 mg, 0.838 mmol), trimethylsilylazide (0.1 mL, 0.762 mmol), and (2-isocyanoethyl)benzene (0.09 mL, 0.762 mmol) to obtain the pure compound 29 (DA-035) (248 mg, 84.06% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.60 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 7.27 (dd, J = 5.9, 2.5 Hz, 3H), 7.01 (dd, J = 6.3, 2.9 Hz, 2H), 4.80–4.59 (m, 2H), 4.39 (s, 1H), 3.21 (t, J = 6.9 Hz, 2H), 2.48–2.19 (m, 11H). ¹³C NMR (100 MHz, CDCl₃) δ 153.18, 138.69, 136.67, 132.16, 130.03, 129.14, 128.79, 127.48, 118.25, 112.51, 63.66, 54.83, 49.93, 49.38, 45.77, 36.02.

MS (ESI): m/z 388.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₂H₂₆N₇, calcd 388.22442, found 388.22472 [M+1]⁺. LCMS (ESI): m/z 388.43 [M+1]⁺.

4-((1-Benzyl-1H-tetrazol-5-yl)(4-methylpiperazin-1-yl)methyl)benzonitrile (30 (DA-038))

The title compound was prepared according to the procedure for GT949, using 4-cyanobenzaldehyde (100 mg, 0.762 mmol), 1-methylpiperazine (84 mg, 0.838 mmol), trimethylsilylazide (0.1 mL, 0.762 mmol), and (isocyanomethyl)benzene (0.09 mL, 0.762 mmol) to obtain the pure compound 30 (DA-038) (216 mg, 76.05% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.54 (d, J = 8.3 Hz, 2H), 7.40 (d, J = 8.3 Hz, 2H), 7.37–7.29 (m, 3H), 7.10–7.03 (m, 2H), 5.74–5.62 (m, 2H), 4.78 (s, 1H), 2.54–2.27 (m, 8H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 153.34, 138.87, 133.23, 132.14, 130.02, 129.21, 128.94, 127.29, 118.21, 112.47, 64.06, 54.73, 51.44, 50.29, 45.79.

HRMS (ESI m/z) for C₂₁H₂₄N₇, calcd 374.20877, found 374.20829 [M+1]⁺. LCMS (ESI): m/z 374.22 [M+1]⁺.

1-Cyclohexyl-4-((1-phenethyl-1H-tetrazol-5 yl)(4(trifluoromethyl)phenyl)methyl)piperazine (31 (DA-029))

The title compound was prepared according to the procedure for GT949, using 4-trifluoromethylbenzaldehyde (100 mg, 0.574 mmol), 1-cyclohexylpiperazine (106 mg, 0.632 mmol), trimethylsilylazide (0.08 mL, 0.574 mmol), and (2-isocyanoethyl)benzene (0.08 mL, 0.574 mmol) to obtain the pure compound 31 (DA-029) (223 mg, 78.24% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.57 (d, J = 8.1 Hz, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.31–7.22 (m, 3H), 7.07–6.99 (m, 2H), 4.84–4.73 (m, 1H), 4.71–4.61 (m, 1H), 4.49 (s, 1H), 3.19 (t, J = 7.0 Hz, 2H), 2.55 (brs, 4H), 2.37 (brs, 2H), 2.29 (brs, 2H), 2.23–2.14 (m, 1H), 1.85–1.71 (m, 4H), 1.60 (d, J = 12.2 Hz, 1H), 1.27–1.03 (m, 5H). ¹³C NMR (100 MHz, CDCl₃) δ 153.72, 142.75, 137.43, 136.77, 130.59 (q, J_C–F = 32.0 Hz), 129.82, 129.05, 128.80, 128.63, 127.38, 125.35 (q, J_C–F = 4.0 Hz), 63.83, 63.29, 50.57, 49.33, 48.79, 35.98, 28.89, 26.22, 25.77.

MS (ESI): m/z 499.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₇H₃₄N₆F₃, calcd 499.27916, found 499.279872 [M+1]⁺. LCMS (ESI): m/z 499.23 [M+1]⁺.

1-((1-Benzyl-1H-tetrazol-5-yl)(4-(trifluoromethyl)phenyl)methyl)-4-cyclohexylpiperazine (32 (DA-010))

The title compound was prepared according to the procedure for GT949, using 4-trifluoromethylbenzaldehyde (100 mg, 0.574 mmol), 1-cyclohexylpiperazine (97 mg, 0.632 mmol), trimethylsilylazide (0.076 mL, 0.574 mmol), and (2-isocyanoethyl)benzene (0.069 mL, 0.574 mmol) to obtain the pure compound 32 (DA-010) (201 mg, 72.30% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 6.5 Hz, 3H), 7.05 (d, J = 5.9 Hz, 2H), 5.74–5.59 (m, 2H), 4.77 (s, 1H), 2.56 (brs, 4H), 2.44 (brs, 2H), 2.34 (brs, 2H), 2.20 (t, J = 10.5 Hz, 1H), 1.79 (t, J = 14.8 Hz, 4H), 1.61 (d, J = 12.4 Hz, 1H), 1.33–0.99 (m, 5H). ¹³C NMR (100 MHz, MeOD-d₄) δ 153.59, 137.41, 133.95, 130.65 (q, J_C–F = 32.0 Hz), 129.87, 128.66, 128.20, 127.03, 125.25 (q, J_C–F = 3.8 Hz), 122.56, 65.41, 61.76, 50.61, 43.63, 36.13, 26.58, 25.28, 24.59, 24.54.

MS (ESI): m/z 485.4 [M+1]⁺.

HRMS (ESI m/z) for C₂₆H₃₂F₃N₆, calcd 485.26351, found 485.26307 [M+1]+. LCMS (ESI): m/z 485.33 [M+1]⁺.

Ethyl 2-(Pyridin-3-yl)acetate (VY-3-129)

To a solution of 2-(pyridin-3-yl) acetic acid (1737 mg, 10 mmol) in 20 mL of ethanol was added a catalytic amount of sulfuric acid. The reaction mixture refluxed for 4 h and then cooled to room temperature. The solvent was evaporated, crude residue was diluted with aqueous sodium bicarbonate solution and extracted into ethyl acetate (3 × 75 mL), and the combined organic layer was concentrated and purified by flash chromatograph using ethyl acetate and hexane as an eluent to obtain the pure compound. ¹H NMR (400 MHz, CDCl₃) δ 8.56–8.49 (m, 2H), 7.65 (dd, J = 7.8, 1.7 Hz, 1H), 7.27 (dd, J = 8.1, 4.5 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.62 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H).

Ethyl 2-Bromo-2-(pyridin-3-yl)acetate (NA-002)

To a solution of ethyl-4-pyridyl acetate (1651 mg, 10 mmol) in 150 mL of anhydrous THF at 0 °C under a nitrogen atmosphere was added DBU (1674 mg, 1.10 mmol). The reaction mixture was allowed to warm to room temperature over 30 min, and then it was cooled to −78 °C before addition of carbon tetrabromide (3648 mg, 1.10 mmol) and stirring continued at −78 °C for 2 h. After completion of the reaction as indicated by TLC, aqueous ammonium chloride was added to quench the reaction, then ethyl acetate was added, and layers were separated; the organic layer was dried over sodium sulfate and concentrated under vacuum to obtain orange color liquid, which was directly used for the next step without purification.

Ethyl 2-(4-Methylpiperazin-1-yl)-2-(pyridin-3-yl)acetate (NA-003)

The crude ethyl 2-bromo-2-(pyridin-3-yl)acetate (2440 mg, 10 mmol) was dissolved in 20 mL of dichloromethane. To this, N-methyl piperazine (4006 mg, 40 mmol) and triethylamine (4050 mg, 40 mmol) were added, and reaction mixture was refluxed for overnight. After disappearance of the starting material, water was added and organic layers were separated and concentrated under vacuum to obtain crude residue, which was purified to obtain a colorless liquid. ¹H NMR (300 MHz, CDCl₃) δ 8.71–8.47 (m, 2H), 7.97–7.73 (m, 1H), 7.40–7.14 (m, 2H), 4.33–4.07 (m, 2H), 2.49 (s, 8H), 2.28 (d, J = 1.2 Hz, 3H), 1.22 (td, J = 7.1, 1.2 Hz, 2H).

2-(4-Methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetic Acid (NA-004)

To a solution of ethyl 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl)acetate dissolved 10 mL of methanol:tetrahydrofuran:water (3:3:1) was added 3 equiv of lithium hydroxide, and reaction mixture was stirred at room temperature. After completion of reaction, solvent was evaporated, and crude residue was dissolved in 3 mL of water, acidified with 4N hydrochloric acid, and extracted into dichloromethane (3 × 100 mL); the combined organic layer was evaporated under vacuum to obtain the NA-004 as a white solid.

2-(4-Methylpiperazin-1-yl)-N-phenethyl-2-(pyridin-3-yl) Acetamide (33 (NA-019))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and finally phenylethylamine (121 mg, 1.1 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 33 (NA-019) (189 mg, 56% yield) as a brown solid. ¹H NMR (400 MHz, MeOD-d₄) δ 8.39 (dd, J = 6.7, 1.6 Hz, 2H), 7.77–7.71 (m, 1H), 7.32 (dd, J = 7.9, 4.9 Hz, 1H), 7.16–7.10 (m, 2H), 7.09–7.00 (m, 3H), 3.75 (s, 1H), 3.46–3.32 (m, 2H), 2.73–2.66 (m, 2H), 2.52–2.05 (m, 11H). ¹³C NMR (100 MHz, MeOD-d₄) δ 170.99, 149.15, 148.51, 138.83, 137.25, 132.94, 128.52, 128.10, 126.02, 123.86, 72.37, 54.17, 50.18, 44.26, 39.97, 34.69.

HRMS (ESI m/z) for C₂₀H₂₇N₄, calcd 339.21794, found 339.21744 [M+1]⁺. LCMS (ESI): m/z 339.16 [M+1]⁺.

N-Benzyl-2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetamide (34 (NA-010))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol and benzylamine (107.5 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water, extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer, washed with saturated sodium bicarbonate solution, and dried over anhydrous Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent eluent to obtain the pure compound 34 (NA-010) (197 mg, 61% yield) as a white solid. ¹H NMR (400 MHz, MeOD-d₄) δ 8.62 (s, 1H), 8.54 (d, J = 3.7 Hz, 1H), 7.99–7.95 (m, 1H), 7.47 (dd, J = 7.9, 4.9 Hz, 1H), 7.32–7.23 (m, 3H), 7.22–7.18 (m, 2H), 4.44–4.33 (m, 2H), 4.08 (s, 1H), 2.94–2.83 (m, 4H), 2.70–2.50 (m, 7H). ¹³C NMR (100 MHz, MeOD-d₄) δ 170.69, 149.12, 148.75, 138.32, 137.20, 132.77, 128.17, 127.16, 126.96, 123.95, 71.76, 53.80, 49.14, 43.34, 42.69.

HRMS (ESI m/z) for C₁₉H₂₄N₄O, calcd 325.20229, found 325.20162 [M+1]⁺. LCMS (ESI): m/z 325.06 [M+1]⁺.

N-(4-Chlorophenethyl)-2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetamide (35 (VY-3-171))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) waere added at 0 °C. After 10 min DIPEA (0.398 mL, 2 mmol) and 4-chlorophenylethylamine (155 mg, 1.0 mmol) were added. Then the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 35 (VY-3-171) (193 mg, 52% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.53 (dd, J = 4.7, 1.4 Hz, 1H), 8.47 (d, J = 1.6 Hz, 1H), 7.54–7.46 (m, 1H), 7.29 (d, J = 1.8 Hz, 1H), 7.26–7.21 (m, 1H), 7.19 (t, J = 5.4 Hz, 1H), 7.12 (d, J = 8.3 Hz, 2H), 3.88 (s, 1H), 3.66–3.52 (m, 2H), 2.84 (t, J = 6.8 Hz, 2H), 2.58–2.43 (m, 1H), 2.41–2.24 (m, 7H), 2.23 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.34, 150.24, 149.42, 137.06, 136.55, 132.46, 130.92, 130.08, 128.83, 123.36, 72.97, 55.00, 51.07, 45.79, 39.78, 34.72.

HRMS (ESI m/z) for C₂₀H₂₆ClN₄O, calcd 373.17897, found 373.17840 [M+1]⁺. LCMS (ESI): m/z 373.15 [M+1]⁺.

N-(4-Fluorophenethyl)-2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetamide (36 (NA-005))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and 4-fluorophenylethylamine (139 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 36 (NA-005) (207 mg, 58% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.54 (dd, J = 4.8, 1.5 Hz, 1H), 8.47 (d, J = 1.9 Hz, 1H), 7.53–7.47 (m, 1H), 7.26–7.22 (m, 1H), 7.19–7.09 (m, 3H), 7.01 (t, J = 8.7 Hz, 2H), 3.89 (s, 1H), 3.67–3.51 (m, 2H), 2.84 (t, J = 6.8 Hz, 2H), 2.45–2.25 (m, 8H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 170.32, 161.71 (d, J_C–F = 243.0 Hz), 150.26, 149.45, 136.57, 134.25 (d, J_C–F = 3.2 Hz), 130.93, 130.14 (d, J_C–F = 7.8 Hz), 123.36, 115.53 (d, J_C–F = 21.0 Hz), 73.00, 55.01, 51.08, 45.77, 40.02, 34.61.

HRMS (ESI m/z) for C₂₀H₂₆FN₄O, calcd 357.20852, found 357.20789 [M+1]⁺. LCMS (ESI): m/z 357.25 [M+1]⁺.

N-(4-Fluorobenzyl)-2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetamide (37 (NA-008))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) was added and finally and 4-fluorobenzylamine (125 mg, 1.0 mmol) was added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 37 (NA-008) (161 mg, 47% yield) as a white solid. ¹H NMR (400 MHz, MeOD-d₄) δ 8.94 (s, 1H), 8.86 (d, J = 4.9 Hz, 1H), 8.63 (d, J = 8.1 Hz, 1H), 8.07 (dd, J = 8.0, 5.7 Hz, 1H), 7.37–7.26 (m, 2H), 7.13–6.96 (m, 2H), 4.51 (s, 1H), 4.47–4.34 (m, 2H), 3.57–3.40 (m, 2H), 3.31–3.17 (m, 2H), 3.17–3.06 (m, 1H), 2.98–2.92 (m, 1H), 2.91 (s, 3H), 2.77–2.62 (m, 1H), 2.58–2.46 (m, 1H). ¹³C NMR (100 MHz, MeOD-d₄) δ 168.29, 162.18 (d, J_C–F = 243.0 Hz), 145.46, 142.62, 142.38, 136.18 (d, J_C–F = 3.0 Hz), 134.15, 129.38 (d, J_C–F = 8.2 Hz), 126.77, 114.90 (d, J_C–F = 22.0 Hz), 69.36, 53.23, 42.29, 42.05.

HRMS (ESI m/z) for C₁₉H₂₄FN₄O, calcd 343.19287, found 343.19238 [M+1]⁺. LCMS (ESI): m/z 343.26 [M+1]⁺.

N-(4-Methoxybenzyl)-2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) Acetamide (38 (NA-009))

To a stirred solution of 2-(4-methylpiperazin-1-yl)-2-(pyridin-3-yl) acetic acid (NA-004, 235 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and 4-methoxybenzylamine (137 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 38 (NA-009) (149 mg, 42% yield) as a brown solid. ¹H NMR (400 MHz, MeOD-d₄) δ 8.60 (s, 1H), 8.52 (d, J = 4.1 Hz, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.46 (dd, J = 7.8, 4.9 Hz, 1H), 7.14 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 4.40–4.20 (m, 2H), 3.98 (s, 1H), 3.77 (s, 3H), 2.73–2.37 (m, 8H), 2.33 (s, 3H). ¹³C NMR (100 MHz, MeOD-d₄) δ 170.88, 159.08, 149.11, 148.58, 137.24, 133.10, 130.34, 128.54, 123.89, 113.52, 72.41, 54.28, 54.14, 50.18, 44.24, 42.15.

HRMS (ESI m/z) for C₂₀H₂₇N₄O₂, calcd 355.21285, found 355.21225 [M+1]⁺. LCMS (ESI): m/z 355.15 [M+1]⁺.

Ethyl 2-(4-Cyanophenyl)acetate (NA-001)

To a solution of 4-cyanophenylacetic acid (1616 mg, 10 mmol) in 20 mL of ethanol was added a catalytic amount of sulfuric acid. The reaction mixture refluxed for 4 h and then cooled to room temperature. The solvent was evaporated, and crude residue was diluted with aqueous sodium bicarbonate solution and extracted into ethyl acetate (3 × 75 mL); the combined organic layer concentrated and purified by flash chromatograph using ethyl acetate and hexane as an eluent to obtain pure compound. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8.3 Hz, 2H), 7.41 (d, J = 8.3 Hz, 2H), 4.17 (q, J = 7.1 Hz, 2H), 3.68 (s, 2H), 1.26 (t, J = 7.1 Hz, 3H).

Ethyl 2-Bromo-2-(4-cyanophenyl)acetate (NA-011)

To a solution of ethyl 2-(4-cyanophenyl)acetate (1000 mg, 10 mmol) in 150 mL of anhydrous THF at 0 °C under a nitrogen atmosphere was added DBU (824 mg, 1.10 mmol). The reaction mixture was allowed to warm to room temperature over 30 min, and then it was cooled to −78 °C before addition of carbon tetrabromide (1926 mg, 1.10 mmol) and stirring continued at −78 °C for 2 h. After completion of the reaction as indicated by TLC, aqueous ammonium chloride was added to quench the reaction, ethyl acetate was added, layers were separated, and then the organic layer was dried over sodium sulfate and concentrated under vacuum to obtain orange color liquid, which was directly used for the next step without purification.

Ethyl 2-(4-Cyanophenyl)-2-(4-methylpiperazin-1-yl)acetate (NA-012)

Compound ethyl 2-bromo-2-(4-cyanophenyl)acetate (2810 mg, 10 mmol) was dissolved in 20 mL of dichloromethane. To this, N-methyl piperazine (4230 mg, 40 mmol) and triethylamine (4276 mg, 40 mmol) were added, and reaction mixture was refluxed for overnight. After disappearance of the starting material at TLC, water was added and organic layers were separated and concentrated under vacuum to obtain crude residue, which was purified to obtain a colorless liquid. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (dd, J = 21.1, 8.3 Hz, 4H), 4.26–4.08 (m, 2H), 4.04 (s, 1H), 2.47 (brs, 7H), 2.28 (s, 3H), 1.21 (t, J = 7.1 Hz, 3H).

2-(4-Cyanophenyl)-2-(4-methylpiperazin-1-yl) Acetic Acid (NA-013)

To a solution of ethyl 2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl)acetate in 10 mL of methanol:tetrahydrofuran:water (3:3:1), 3 equiv of lithium hydroxide was added and reaction mixture was stirred at room temperature. After completion of reaction, the solvent was evaporated, and crude residue was dissolved in 3 mL of water, acidified with 4N hydrochloric acid, and extracted into dichloromethane (3 × 100 mL), and combined organic layer was evaporated under vacuum to obtain the NA-013 as a white solid.

N-Benzyl-2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl) Acetamide (39 (NA-018))

To a stirred solution of 2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl) acetic acid (NA-013, 259 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and 4-phenylmethanamine (107 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 39 (NA-018) (167 mg, 48% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.38–7.28 (m, 3H), 7.21 (d, J = 6.5 Hz, 2H), 4.55–4.39 (m, 2H), 4.00 (s, 1H), 2.50–2.31 (m, 8H), 2.25 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.89, 140.75, 138.05, 132.25, 129.79, 128.81, 127.69, 127.66, 118.50, 112.09, 75.29, 54.99, 51.32, 45.80, 43.35.

HRMS (ESI m/z) for C₂₁H₂₅N₄O, calcd 349.20229, found 349.20180 [M+1]⁺. LCMS (ESI): m/z 349.26 [M+1]⁺.

2-(4-Cyanophenyl)-N-(4-fluorophenethyl)-2-(4-methylpiperazin-1-yl) Acetamide (40 (NA-014))

To a stirred solution of 2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl) acetic acid (NA-013, 259 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and 4-fluorophenylethylamine (139 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 40 (NA-014) (144 mg, 37.9% yield) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.21 (t, J = 5.5 Hz, 1H), 7.79 (d, J = 8.0 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.15 (t, J = 6.8 Hz, 2H), 7.04 (t, J = 8.7 Hz, 2H), 3.84 (s, 1H), 3.40–3.20 (m, 2H), 2.69 (t, J = 6.8 Hz, 2H), 2.38–2.24 (m, 4H), 2.24–2.17 (m, 4H), 2.15 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.44, 161.28 (d, J_C–F = 240.0 Hz), 143.65, 135.75 (d, J_C–F = 3.0 Hz), 132.50, 130.91 (d, J_C–F = 7.9 Hz), 129.94, 119.22, 115.28 (d, J_C–F = 21.0 Hz), 110.86, 74.52, 54.88, 50.85, 45.92, 34.32.

HRMS (ESI m/z) for C₂₂H₂₆FN₄O, calcd 381.20852, found 381.20765 [M+1]⁺. LCMS (ESI): m/z 381.25 [M+1]⁺.

2-(4-Cyanophenyl)-N-(4-fluorobenzyl)-2-(4-methylpiperazin-1-yl) Acetamide (41 (NA-016))

To a stirred solution of 2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl) acetic acid (NA-013, 259 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and (4-fluorophenyl)methanamine (125 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 41 (NA-016) (153 mg, 42% yield) as a white solid. ¹H NMR (400 MHz, MeOD-d₄) δ 8.81 (t, J = 5.8 Hz, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.54 (d, J = 8.3 Hz, 2H), 7.09 (dd, J = 8.5, 5.4 Hz, 2H), 6.88 (t, J = 8.8 Hz, 2H), 4.31–4.19 (m, 2H), 4.05 (s, 1H), 3.48–3.27 (m, 2H), 3.17–2.93 (m, 3H), 2.77 (s, 3H), 2.73–2.61 (m, 1H), 2.57–2.45 (m, 1H), 2.32–2.16 (m, 1H). ¹³C NMR (100 MHz, MeOD-d₄) δ 170.13, 162.08 (d, J_C–F = 243.0 Hz), 141.20, 134.33 (d, J_C–F = 3.2 Hz), 132.24, 129.42, 129.13 (d, J_C–F = 8.2 Hz), 117.96, 114.78 (d, J_C–F = 22.0 Hz), 112.11, 73.07, 53.25, 42.05, 42.01.

HRMS (ESI m/z) for C₂₁H₂₄FN₄O, calcd 367.19287, found 367.19184 [M+1]⁺. LCMS (ESI): m/z 367.25 [M+1]⁺.

2-(4-Cyanophenyl)-N-(4-methoxybenzyl)-2-(4-methylpiperazin-1-yl) Acetamide (42 (NA-017))

To a stirred solution of 2-(4-cyanophenyl)-2-(4-methylpiperazin-1-yl) acetic acid (NA-013s, 259 mg, 1 mmol) in dry DMF (5 mL), HATU (758.98 mg, 2.0 mmol) was added at 0 °C. After 10 min, DIPEA (0.398 mL, 2 mmol) and (4-methoxyphenyl)methanamine (137 mg, 1.0 mmol) were added. Then, the resulting mixture was stirred at room temperature for 8–10 h and then the reaction mixture was quenched with ice-cold water and extracted in 15% IPA/dichloromethane (4 × 50 mL) from the ice-cold aqueous layer. The organic layer was washed with saturated sodium bicarbonate solution and dried over Na₂SO₄. The organic layer was evaporated under vacuum, and the resulting product was purified by column chromatography employing MeOH/DCM as an eluent to obtain the pure compound 42 (NA-017) (154 mg, 41% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.62 (d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H), 7.31–7.25 (m, 1H), 7.13 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 4.46–4.32 (m, 2H), 3.97 (s, 1H), 3.81 (s, 3H), 2.47–2.37 (m, 6H), 2.25 (s, 3H), 2.11–1.99 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.82, 159.13, 140.85, 132.27, 130.07, 129.75, 129.03, 118.51, 114.16, 112.08, 75.28, 55.32, 54.94, 51.19, 45.74, 42.84.

HRMS (ESI m/z) for C₂₂H₂₇N₄O₂, calcd 379.21285, found 379.21221 [M+1]⁺. LCMS (ESI): m/z 379.25 [M+1]⁺.

General Methods for Biology

Methods have been adapted from the scientific literature to maximize reliability and reproducibility. Reference standards were run as an integral part of each assay to ensure the validity of the results obtained. Assays were performed under conditions described in the accompanying “Methods” section of this report. Where presented, IC₅₀ values were determined by a nonlinear, least-squares regression analysis using MathIQTM (ID Business Solutions Ltd., UK). Where inhibition constants (K_i) are presented, the K_i values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W.H., Biochem. Pharmacol. 22:3099–3108, 1973) using the observed IC50 of the tested compound, the concentration of radioligand employed in the assay, and the historical values for the KD of the ligand (i.e., obtained experimentally at Eurofins Panlabs, Inc.). Where presented, the Hill coefficient (nH), defining the slope of the competitive binding curve, was calculated using MathIQTM. Hill coefficients significantly different than 1.0 may suggest that the binding displacement does not follow the laws of mass action with a single binding site. Where IC50, K_i, and/or nH data are presented without Standard Error of the Mean (SEM), data are insufficient to be quantitative, and the values presented (K_i, IC50, nH) should be interpreted with caution.

All research studies involving animals were performed in accordance with institutional guidelines as defined by the Institutional Animal Care and Use Committee for U.S. institutions or equivalent regulatory committee in other countries.

Effect of Compounds on Glutamate Transporters in Cell Lines

The effects of compounds were examined on glutamate transporter studies in cell lines, according to previous studies.^41,43 Briefly, COS-7 cells (ATCC, Manassas, VA, USA) were maintained in DMEM containing 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified incubator with 5% CO₂ at 37 °C. Subconfluent cells were transiently transfected with 0.5 μg of plasmid DNA (EAAT1, EAAT2, EAAT3, or empty vector pCMV-5 for the background) per well using TransIT-LT1 transfection reagent (Mirus Bio LLC, Madison, WI, USA) and plated at a density of 50,000 cells/well. Uptake experiments were performed 2 days after plating. For dose–response assays, cells were washed with room-temperature phosphate buffer PBS-CM (2.7 mM KCl; 1.2 mM KH₂PO₄, 138 mM NaCl; 8.1 mM Na₂HPO₄, added 0.1 mM CaCl₂ and 1 mM MgCl₂, pH 7.4) and incubated for 10 min at 37 °C with 0.1 nM–10 μM final concentrations of compounds. Uptake reactions were initiated by the addition of ³H-l-glutamate (PerkinElmer, Boston, MA, USA) at a final concentration of 50 nM, and 10 min later, uptake was terminated by removal of solution, followed by two washes with PBS-CM and lysis with 1% SDS/0.1 M NaOH for 20 min. Radioactivity in the lysate was quantified in a scintillation counter LS 6500 (Beckman Coulter, Brea, CA). For kinetic assays, cells were transfected as described above; 2 days later, plates were preincubated with compounds for 10 min at the indicated concentrations. Uptake reactions were initiated by the addition of unlabeled l-glutamate and ³H-l-glutamate (1–1000 μM, final concentration, 99% unlabeled and 1% labeled). After 10 min, uptake was terminated, and radioactivity was counted as above.

Effect of Compounds on Glutamate Transporters in Cultured Glia

Glia Preparation

Glia was prepared and cultured according to a previous study.⁴¹ Briefly, cerebral cortices from 2–4 day old Sprague–Dawley rat pups were dissected under sterile conditions, minced in buffer, digested in 0.25% trypsin, triturated with 60 μg/mL DNase, centrifuged, and cultured in a medium (90% DMEM, 10% FBS and 50 μg/mL gentamicin) in a 37 °C incubator (5–10% CO2). After growth for 10 days in vitro (DIV), cells were plated at the density of 10,000 cells/well in polylysine-coated 96-well plates and grown for 14 DIV before uptake assays.

Uptake Assays in Glia Cultures

Assays were performed as described,⁷⁷ using an Elx50 Biotek plate washer (Winooski, VT, USA). The vehicle and several concentrations of selected compounds were added and incubated for 10 min at 37 °C, uptake assays are initiated by adding 50 nM ³H-l-glutamate, and 10 min later, reactions were stopped by two washes and the addition of scintillation fluid. Nonspecific uptake was obtained in the presence of 10 μM DL-TBOA. Radioactivity was counted in a Microplate Scintillation and Luminescence Counter (Wallac, Shelton, CT, USA).

Off-Target Screening

For evaluation of the effect of compounds on other neurotransmitter transporters in cell lines, COS-7 cells were transfected with pcDNA3.1 (as the empty vector) and GAT-1, GAT-3, GLYT1, or GLYT2 in 24-well plates, and uptakes were performed as described above for glutamate transport assays but using ³H-GABA and ³H-glycine (PerkinElmer, Boston, MA, USA) as substrates. Additionally, stable hDAT, hNET, hSERT transfected Madin-Darby Canine Kidney (MDCK) cells were used, as well naïve MDCK cells for obtaining the background, and ³H-dopamine, ³H-noradrenaline, and ³H-5HT (PerkinElmer, Boston, MA, USA) as substrates, respectively, as previously described.⁷⁸ Furthermore, HEK293 cells (ATCC, Manassas, VA, USA) were transiently transfected with ASCT1 and ASCT2 [(SLC1A5(NM_005628.3) and SLC1A4(NM_003038.5), respectively, from Genecopeia, Rockville, MD, USA)], or CMV for obtaining the background, and uptakes were performed as described above for COS-7 cells, but with 50 nM ³H-alanine (American Radiolabeled Chemicals, St. Louis, MO, USA).

Further, compound 40 (NA-014) was evaluated for the effect on binding on a battery of receptors (serotonin, adrenergic, benzylpiperazine (BZP); dopamine, opioid, histamine, muscarinic, benzodiazepine, and sigma), by the National Institute of Mental Health’s Psychoactive Drug Screening Program (PDSP), using methods previously described.⁶⁶ Briefly, radioligand binding assays were performed using the Integrated Sample Storage, Retrieval & Liquid Handling Robotic System in 96-well plates on stably or transiently transfected cell lines expressing mainly human recombinant receptors, monoamine transporters, or ion channels. Primary assays were done using 10 μM compound, and secondary assays were done using a range of the compound concentration. Every radioligand binding assay had a positive control and negative control. Full-detailed protocols can be found at PDSP webpage.⁷⁹

HERG Binding Assay (Reaction Biology)⁶¹

Membrane Preparations

The hERG-T-REx 293 cell line (Invitrogen, Carlsbad, CA) was used to generate membrane preparations for testing the affinity of fluorescent tracer molecules. Cells were maintained following the manufacturer’s recommended protocol. FP assays; Tracer evaluation was conducted by incubating diluted membrane preparations and the fluorescent tracer in the presence or absence of 10 μM dofetilide (Sequoia Research Products, Pangbourne, UK) to assess the degree of hERG-specific (and displaceable) tracer binding. Optimal FP assay buffer composition consisted of 25 mM HEPES (pH 7.5), 15 mM KCl, 1 mM MgCl2, and 0.05% Pluronic F-127. Compound displacement assays were performed by first dispensing 10 μL of assay buffer with or without test compounds to wells of a 384-well untreated polystyrene assay plate and then adding 10 μL of a mixture of membrane preparation and tracer at twice the final assay concentration. Reactions were incubated for 2–6 h and then read on a microplate reader using polarized excitation and emission filters or monochromator settings that were appropriate to the tracer being evaluated. Optimal conditions for FP assays were determined by titrating a matrix of membrane protein against varying concentrations of the fluorescent tracer in the presence or absence of 30 μM E-4031 (Tocris Bioscience) to determine a 50% effective concentration for membrane concentration and a ΔmP value. Assay conditions contained 1 nM Predictor hERG Tracer Red and 85 μg/mL membrane protein (Bmax of membrane preparation ∼450 pmol/mg) in a 20 μL final assay volume. Assay wells were excited at 530 nm, and emission was measured at 585 nm (20 nm bandwidth) using a microplate reader.

Mouse Liver Microsome Stability Assay

Test compounds (0.5 μM) were incubated with liver microsomes (0.5 μg/mL) and an NADPH-regenerating system (cofactor solution), and samples were taken at various time points, quenched with an acetonitrile solution containing an internal standard, and then analyzed by LCMS/MS. These data provide half-life of parent remaining and intrinsic clearance (CLint) determined from the first-order elimination constant by nonlinear regression. Thirty μL of 1.5 μM spiking solution containing 0.75 mg/mL microsomes solution was dispensed to the assay plates designated for different time points (0-, 5-, 15-, 30-, 45 min) on ice. For 0 min, 135 μL of ACN containing internal standard (IS) was added to the wells of 0 min plate and then added 15 μL of NADPH stock solution (6 mM). All other plates were preincubated at 37 °C for 5 min. Fifteen μL of NADPH stock solution (6 mM) was added to the plates to start the reaction and timing. At 5 min, 15 min, 30 min, and 45 min, 135 μL of ACN containing IS was added to the wells of corresponding plates, respectively, to stop the reaction. After quenching, the plates were shaken at the vibrator (IKA, MTS 2/4) for 10 min (600 rpm/min) and then centrifuged at 5594g for 15 min (Thermo Multifuge ×3R). 50 μL of supernatant was transferred from each well into a 96-well sample plate containing 50 μL of ultrapure water (Millipore, ZMQS50F01) for LC/MS analysis.

Mouse Pharmacokinetics

(Chempartners) A pharmacokinetic study was performed to determine plasma and brain distribution concentration of test compounds after single IP administration to male CD1 mice. Compounds were prepared prior to use in 5% DMSO, plus 10% Solutol HS 15, plus 85% PBS in a concentration of 1 mg/mL, i.e., weighing 4.28 mg of NA-014 into a new vial then adding 0.214 mL of DMSO into the vial containing the compound and vortexing the vial for 1 min and sonicating for 5 min. Then, adding 0.428 mL Solutol HS 15 into the vial containing the compound and vortexing the vial for 2 min, then adding 3.638 mL of PBS buffer into the vial containing the compound with vortexing the vial for 1 min and sonication for 0.5 min. CD1 mice, 6–8 weeks, 29–31 g, male, N = 9, were purchased from Shanghai JiHui Laboratory Animal Co. Ltd. Animals were fasted overnight and fed at 4 h post dosing. Compound was administered 10 mg/kg (10 mL/kg) via intraperitoneal injection (N = 6). Sampling was done at 0.25, 0.5, 1, 2, 4, and 6 h post dose, 6 time points in total, stagger bleeding for plasma and brain collection at 1, 3, and 6 h post dose, 3 time points in total. Blood collection: approximately 110 μL blood/time point was collected into K₂EDTA tubes via facial vein. The blood sample was put on wet ice and centrifuged to obtain plasma sample (2000g, 5 min under 4 °C) within 15 min. Brain collection: after blood collection, a midline incision was made in the animal’s scalp and skin retracted. The skull overlying the brain was removed. The whole brain was collected, rinsed with cold saline, dried on filtrate paper, and weighed; snap frozen by placing into dry ice. For brain samples: The sample was homogenized with 3 volumes (v/w) of MeOH:PBS Buffer (1:1). The diluted factor was 4. The following operation was the same as nondiluted ones. For nondiluted samples: (1) an aliquot of 30 μL sample was mixed with 200 μL of IS (Diclofenac, 40 ng/mL) in ACN. (2) The mixture was vortexed for 1 min and centrifuged at 5800 rpm for 10 min. (3) 100 μL supernatant was transferred to a new plate. (4) 0.5 μL of solvent was injected for LCMS/MS analysis.

Structural Modeling and Ligand Docking Simulations

Human EAAT2 protein (L35-R509; UniProt ID P43004) was modeled by the SWISS-MODEL⁸⁰ using the resolved EAAT1 protomer (PDB: 5LLU(81)) as the template. The ligand-binding sites and binding poses were determined using AutoDock Vina.⁶⁸ Simulations were carried out using grids with dimensions 32 × 32 × 32 ų set to encapsulate the region of interest, following our previous protocol.⁶⁷ The total number of runs (exhaustiveness parameter in Autodock Vina) was set to 50 and the algorithm returned 20 binding poses. Binding sites were rank ordered based on binding affinities calculated using Vina. The same docking simulation protocol was adopted to GT949-S, GT949-R, NA-014-R, and NA-014-S.

Data analysis

All data were analyzed using GraphPad Prism version 10.1.2 for Windows (GraphPad Software, La Jolla, CA, USA). Nonspecific transport (background) obtained from transfection with empty vector was subtracted. Dose–response curves were fitted by nonlinear regression analysis using the Hill equation; the Hill slope was allowed to vary to obtain a better fit to the data. EC₅₀ and IC₅₀ values (as the concentration of compound resulting in 50% of the maximum observed stimulation or inhibition, respectively), and efficacies are obtained by bottom and top parameters (best fit values) generated in GraphPad Prism and are given as means ± SEM of three or four independent assays performed in triplicate and normalized to percentage of control (vehicle). Michaelis–Menten kinetics was assumed for calculations of K_m and V_max, and statistical significance was assessed using One-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni multiple comparisons post hoc tests with the vehicle (for analysis of the effect of compounds) as the control (*p <0.05).

Glossary

Abbreviations

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

BZP

benzylpiperazine

CNS

central nervous system

hDAT

human dopamine transporter

DL-TBOA

DL-threo-β-benzyloxy aspartic acid

DMF

dimethylformamide

PBS-CM

Dulbecco’s phosphate-buffered saline with 0.1 mM CaCl₂ and 1 mM MgCl₂ added

EAAT1–5

excitatory amino acid transporters 1–5

EAAC1

excitatory amino acid carrier 1, rodent homologue of EAAT3

GAT-1

GABA transporter subtype 1

GAT-3

GABA transporter subtype 3

GLYT1

glycine transporter subtype 1

GLYT2

glycine transporter subtype 2

GLAST

glutamate aspartate transporter, rodent homologue of EAAT1

GLT-1

glutamate transporter 1, rodent homologue of EAAT2

GltPh

glutamate transporter homologue from Pyrococcus horikoshii

hERG

the human ether-à-go-go-related gene

hNET

human noradrenaline transporter

HSB

hybrid structure based approach

hSERT

human serotonin transporter

HPLC

ultrahigh-performance liquid chromatography

LCMS/MS

ultrahigh-performance liquid chromatography-MS/MS

mGluR

metabotropic glutamate receptor

MLM

mouse liver microsomes

MS/MS

tandem mass spectrometry (MS/MS)

NAM

negative allosteric modulation

MDCK

Madin–Darby Canine Kidney

NaOH

sodium hydroxide

NMDA

N-methyl-d-aspartate

NMR

nuclear magnetic resonance

PAM

positive allosteric modulation

PDSP

Psychoactive Drug Screening Program

SDS

sodium dodecyl sulfate

TM

transmembrane domains.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01909.

• (Figure S1) Evaluation of 4 (DA-23), 40 (NA-014), and 34 (NA-010) on glutamate uptake in the presence of selective EAAT2 inhibitor WAY 213613; (Figure S2) mouse pharmacokinetic study with Cpd 4 (DA-023); (Figure S3) evaluation of 40 (NA-014) for off-target activities on GABA, glycine, monoamine transporters, and alanine transporters; (Table S1) evaluation of 40 (NA-014) effects on glutamate receptors; (Table S2) evaluation of 40 (NA-014) on serotonin, adrenergic, BZP; dopamine, opioid, histamine, muscarinic, benzodiazepine, and sigma receptors; (Table S3) secondary binding assays of 40 (NA-014) on dopamine receptor D5 and histamine receptor H1; (Figure S4) chiral chromatography for compound 40 (NA-014); and compounds characterization spectra, ¹H and ¹³C NMR spectra, LCMS, and high-resolution mass spectra (HRMS) of final compounds (PDF)

• EAAT compound data table including batch ID, manuscript ID, activity on EAAT expressing COS cells, and molecular strings (SMILES String) (CSV)

• Homology (Docking) Model for R enantiomer of GT949, EAAT on 5LLU, S1A (PDB)

• Homology (Docking) Model for S enantiomer of GT949, EAAT on 5LLU, S1A (PDB)

• Homology (Docking) Model for R enantiomer of NA-014, site S1A, EAAT, outward facing (PDB)

• Homology (Docking) Model for S enantiomer of NA-014, site S1A, EAAT, outward facing (PDB)

Author Contributions

Project design and search for funding: A.C.K.F., S.M.R., and J.M.S.; chemical synthesis: A.N.R.P., Y.V.V.S., J.G., N.A., and D.A.; bioassays: A.C.K.F., A.K., and K.L.R.; computational modeling: M.H.C. and I.B.; manuscript writing and revision: S.M.R., I.B., A.C.K.F., and J.M.S.

The authors declare the following competing financial interest(s): JMS owns equity in Alliance Discovery, Inc., Barer Institute, Context Therapeutics, and consults for Syndeavor Therapeutics, Inc. The other authors declare no competing interests.