Discovery of Dihydropyrrolo[1,2-a]pyrazin-3(4H)-one-Based Second-Generation GluN2C- and GluN2D-Selective Positive Allosteric Modulators (PAMs) of the N-Methyl-d-Aspartate (NMDA) Receptor

Abstract

The N-methyl-d-aspartate receptor (NMDAR) is an ion channel that mediates the slow, Ca²⁺-permeable component of glutamatergic synaptic transmission in the central nervous system (CNS). NMDARs are known to play a significant role in basic neurological functions, and their dysfunction has been implicated in several CNS disorders. Herein, we report the discovery of second-generation GluN2C/D-selective NMDAR-positive allosteric modulators (PAMs) with a dihydropyrrolo[1,2-a]pyrazin-3(4H)-one core. The prototype, R-(+)-EU-1180–453, exhibits log unit improvements in the concentration needed to double receptor response, lipophilic efficiency, and aqueous solubility, and lowers cLogP by one log unit compared to the first-generation prototype CIQ. Additionally, R-(+)-EU-1180–453 was found to increase glutamate potency 2-fold, increase the response to maximally effective concentration of agonist 4-fold, and the racemate is brain-penetrant. These compounds are useful second-generation in vitro tools and a promising step toward in vivo tools for the study of positive modulation of GluN2C- and GluN2D-containing NMDA receptors.

Graphical Abstract

INTRODUCTION

The N-methyl-d-aspartate receptor (NMDAR) belongs to the family of mammalian ligand-gated excitatory ionotropic glutamate receptors (iGluRs) that includes 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptors (AMPARs) and (2S,3S,4S)-3-(carboxymethyl)-4-prop-1-en-2-ylpyrrolidine-2-carboxylic acid (kainate) receptors.¹ NMDARs are known to play a role in learning,^2,3 memory,⁴ brain development,⁵ and synaptic plasticity^6,7 and have been implicated in numerous neurological conditions including schizophrenia,^8,9 Alzheimer’s disease,¹⁰ Parkinson’s disease,¹¹ Huntington’s chorea,¹² epilepsy,¹³ neuropathic pain,¹⁴ ischemic brain injury,^15–17 and depression.^18,19 This has driven interest in the development of both positive and negative modulators of the NMDAR for potential therapeutic gain.^20,21

Structurally, NMDARs are heterotetrameric assemblies of two GluN1 and two GluN2 protein subunits, which bind co-agonists glycine and glutamate, respectively. Each subunit contains four semiautonomous domains: an amino terminal domain (ATD), an agonist-binding domain (ABD), a transmembrane domain (TMD), and a carboxyl terminal domain (CTD).¹ Upon co-agonist binding at the ABD, membrane depolarization occurs, which removes Mg²⁺ block^22,23 from the pore. This allows Ca²⁺ and Na⁺ ions to flow into the cell, which contribute to postsynaptic signal transmission.²⁴ The four isoforms of the GluN2 subunit (GluN2A-D) are expressed with different spatiotemporal patterns in the brain^25–27 and endow the receptor with unique pharmacological properties including open probability,^28–31 agonist potency,³² and deactivation time.³³ For example, GluN2C- and GluN2D-containing NMDA receptors are expressed more prominently in the cerebellum, basal ganglia, thalamus, and cortical and hippocampal interneurons,^25,26,34 compared to GluN2A- and GluN2B-containing receptors. Functionally, GluN2C/GluN2D-containing NMDA receptors show increased glutamate and glycine sensitivity^32,35 and reduced open probability,^29,30,36 Mg²⁺ sensitivity,²⁵ Ca²⁺ permeability, and single-channel conductance compared to GluN2A/GluN2B-containing receptors.³⁷

Because these subunit-specific expression patterns and GluN2-specific functional properties allow NMDARs to serve unique roles at different synapses, subunit-selective modulators provide an opportunity to target the aforementioned diseases through selective modification of specific circuits.³⁸ However, FDA-approved drugs that target NMDARs nonselectively block the highly conserved NMDAR pore, irrespective of subunit composition. This results in the inhibition of all NMDAR subtypes, potentially leading to the neurological side effects that include altered cardiovascular activity, hallucinations, delusions, and impaired motor function.³⁹

Subunit-selective NMDAR modulators have therefore become a focus of the research community, given their potential to reduce side effects by targeting one subunit in specific areas of the brain for therapeutic gain. Specifically, NMDAR-positive allosteric modulators (PAMs) could be useful in conditions associated with reduced NMDAR activity such as schizophrenia,^40–42 autism spectrum disorders,^43,44 anti-NMDAR encephalitis,^45–47 and age-related memory loss.^2,38,48,49 Further, there is evidence suggesting that positive therapeutic outcomes could be achieved with the development of GluN2C- and/or GluN2D-subtype-selective PAMs. One hypothesis is that the hypofunction of GluN2C-containing receptors plays a role in schizophrenic symptoms⁵⁰ and, thus, GluN2C-selective PAMs could have therapeutically relevant actions. Additionally, postmortem examinations of schizophrenic patients with a specific NRG1 gene polymorphism revealed reduced GluN2C expression in the right cerebellum.⁵¹ GluN2D-containing receptors may also play a role in schizophrenia.^51–54 For example, there is evidence to suggest that decreased inhibitory drive can lead to hyperactivity of principal cells and enhanced dopamine release.⁵⁵ Expression of GluN2D in interneurons^25,56,57 suggests that selective enhancement of GluN2D-containing receptor function could potentially rectify this hypothesized circuit imbalance. Finally, (3-chlorophenyl)(6,7-dimethoxy-1-((4-methoxyphenoxy)-methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (CIQ, 1), a GluN2C/D-selective PAM previously developed in our lab (Figure 1),^58,59 has shown promising results in behavioral mouse models. This includes the ability to recover striatal synaptic plasticity deficit in a Parkinson’s model,⁶⁰ facilitate the retention of fear and extinction learning,⁶¹ and prevent MK-801 (a noncompetitive antagonist of NMDARs)-induced prepulse inhibition deficit,⁶² suggesting the clinical potential of potent positive modulators of these subtypes.

Figure 1.

PAMs of the NMDAR.

There have been numerous other nonselective and subunit-selective NMDAR PAMs identified in addition to CIQ (Figure 1). For example, we have previously identified a GluN2B-preferring class of isopropoxy-substituted tetrahydroisoquinolines (TIQs) based on CIQ (prototype compound 2).⁶³ Wang et al. published GNE-9278 (3), a nonselective benzenesulfonamide-based PAM that acts on each of the GluN1/GluN2 subtypes in the low micromolar (3–16 μM) range.⁶⁴ Structural determinants suggest a possible binding site near the GluN1 pre-M1 helix, and it was shown to potentiate the receptor by increasing co-agonist affinity, increasing receptor response at saturating co-agonist concentrations, and slowing deactivation after glutamate removal. Other nonselective PAMs include the neurosteroid pregnenolone sulfate (PS, 4)⁶⁵ and 24(S)-hydroxycholesterol (5).⁶⁶ Genentech also published a GluN2A-selective series of thiazole derivatives that bind in a similar pocket to the N-(4-(2-benzoylhydrazine-1-carbonyl)-benzyl)-3-chloro-4-fluorobenzenesulfonamide (TCN) class of negative allosteric modulators (NAMs). These compounds potentiate via stabilization of the agonist-bound state.^67,68 Significant optimization resulted in GNE-5729 (6), a PAM of GluN2A-containing receptors (EC₅₀ = 37 nM) showing improved AMPAR selectivity and unbound clearance and an excellent secondary pharmacology profile.⁶⁹ The only known series of compounds able to distinguish between GluN2C- and GluN2D-containing receptors are the pyrrolidinone-based (prototype PYD-106 (7)) GluN2C-selective PAMs.^70,71 These compounds appear to increase mean open time and open probability via actions on the ATD and S1 domains.

Although CIQ has made a significant impact as a first-in-class tool compound, it exhibits several liabilities limiting its use both in vitro and in vivo. These include modest potency, modest potentiation, low aqueous solubility, and high lipophilicity.^58,59 Herein, we report the discovery of second-generation dihydropyrrolo[1,2-a]pyrazin-3(4H)-one-based GluN2C/D-selective PAMs that are the result of successive core changes from CIQ. First, we modified the TIQ scaffold to a 1,4-dihydroisoquinolin-3(2H)-one core (Figure 2, left), which resulted in improved concentrations needed to double receptor response (doubling concentration, 0.5 log unit improvement on average) and lipophilic ligand efficiency (LLE, defined here as LLE = −log₁₀(doubling conc.) −cLogP, 0.4 log unit improvement on average). We have chosen to report the doubling concentration alongside the EC₅₀ throughout this manuscript as it captures both the potency and potentiation contributions of a PAM’s biological activity in one directly comparable number. Since it considers both contributions, we believe it to be more useful than EC₅₀ in describing the biological activity of these PAMs, as the EC₅₀ describes only the potency without information on maximal achievable response. We have also chosen to define LLE using the doubling concentration (compared to the standard definition using EC₅₀) due to its increased relevance. Finally, we have chosen 2-fold as the cutoff for several reasons: (1) it is currently unknown what percent potentiation of GluN2C/D-containing receptors will lead to pharmacologically relevant actions, (2) this percent potentiation was achieved by most compounds studied, (3) it is a convenient and memorable cutoff, and (4) to be consistent with previous reports.⁶³ Building on this core modification resulted in a dihydropyrrolo[1,2-a]pyrazin-3(4H)-one series (Figure 2, right), with derivatives showing roughly log unit improvements in doubling concentration, aqueous solubility, and LLE, and a log unit decrease in cLogP compared to CIQ. The selectivity for GluN2C and GluN2D is maintained at subsaturating concentrations of co-agonists for both new cores. The enantiomers of promising derivatives were separated via chiral high-performance liquid chromatography (HPLC) and show stereo-dependent activity, with one enantiomer inactive against all examined NMDAR subtypes. The active enantiomers exhibit improved potency over the racemic mixture in each case. Finally, a crystal structure was solved for prototypical compound R-(+)-EU-1180–453 that allowed for assignment of the absolute configuration of the active enantiomers. Therefore, the improvements in activity and physicochemical properties observed in these two new structural modifications compared to the parent CIQ have created improved in vitro tool compounds for GluN2C- and GluN2D-containing NMDA receptors.

Figure 2.

Labeled 1,4-dihydroisoquinolin-3(2H)-one (left) and dihydropyrrolo[1,2-a]pyrazin-3(4H)-one (right) to distinguish between the A-, B-, and C-rings.

RESULTS AND DISCUSSION

Two-Electrode Voltage Clamp (TEVC) Recordings.

All compounds synthesized were evaluated via TEVC recordings of Xenopus laevis oocytes expressing four recombinant NMDAR subtypes: GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, and GluN1/GluN2D. Each compound was co-applied at 30 μM with saturating concentrations of glutamate (100 μM) and glycine (30 μM). When the mean potentiated response exceeded 130% of control, concentration–effect curves were generated by co-applying increasing concentrations of compound with saturating co-agonists. We determined the pEC₅₀ value and maximal degree of potentiation by fitting the concentration–effect curves with eqs 1 and 2 (see Experimental Procedures). Each concentration–effect curve was generated with data from 6–19 oocytes from 2–3 frogs; all inactive compounds were tested in 4–10 oocytes from 1 to 2 frogs. Tables 1–7 summarize the mean pEC₅₀ value with 95% confidence interval and average maximum potentiation (max) for each active compound at GluN1/GluN2C and GluN1/GluN2D receptors. The negative log of the doubling concentration (eqs 3 and 4; see Experimental Procedures), aqueous solubility (see the Supporting Information for Methods), and LLE (eq 5; see Experimental Procedures) are also reported when appropriate. Table 1 compares the doubling concentration of select compounds reported here to previously reported compounds. For compounds in which a pEC₅₀ value was not determined (ND), the maximum potentiation is reported as the average potentiation at 30 μM drug as a percent of control. The average percent potentiation is defined as the mean ratio of current upon application of drug at the stated concentration to the current response in its absence. The doubling concentration is defined as the concentration of test compound at which the response to maximally effective concentration of co-applied glutamate and glycine is increased 2-fold over the response to glutamate and glycine alone. All compounds were also tested at GluN1/GluN2A and GluN1/GluN2B receptors at 10 or 30 μM and were typically found to have no effect (4–17 oocytes from 1 to 3 frogs). Only two compounds (12c–d) exhibited potentiation above 130% at GluN2A- or GluN2B-containing receptors and neither exceeded 2-fold potentiation (Supporting Information Table S1).

Table 1.

1,4-Dihydroisoquinolin-3(2H)-one Compounds and TIQ Counterparts

#	R1	R2	R3	X	Y	pEC50 [95% CI] (max) (%)a		p(doubling conc.)b		Δp(doubling conc.)		cLogP	LLE		ΔLLE
						GluN2C	GluN2D	GluN2C	GluN2D	GluN2C	GluN2D		GluN2C	GluN2D	GluN2C	GluN2D
CIQc	OMe	OMe	CI	O	-	5.3 (230)	5.3 (220)	4.9	4.5	0.2	0.6	5.6	−0.7	−1.1	0.1	0.5
12m				-	O	5.3 [5.1–5.4] (270)	5.0 [4.9–5.1] (340)	5.1	5.1			5.7	−0.6	−0.6
117c	OMe	H	CI	O	-	5.9 (240)	6.0 (220)	5.5	5.3	0.1	0.4	5.9	−0.4	−0.6	0.1	0.4
12e				-	O	5.7 [5.6–5.8] (270)	5.6 [5.5–5.8] (330)	5.6	5.7			5.9	−0.3	−0.2
2*c,d	OiPr	H	CI	O	-	5.7 (240)	5.5 (250)	5.3	5.2	1.3	1.4	6.7	−1.4	−1.5	1.2	1.3
12a				-	O	6.4 [6.3–6.5] (350)	6.3 [6.1–6.6] (370)	6.6	6.6			6.8	−0.2	−0.2
88c	OiBu	H	CI	O	-	6.0 (450)	5.9 (520)	6.4	6.3	−0.2	0.1	7.3	−0.9	−1.0	−0.3	0
12d				-	O	6.4 [6.3–6.4] (280)	6.2 [6.1–6.3] (370)	6.2	6.4			7.4	−1.2	−1.0
127c	OiPr	H	CF3	O	-	5.8 (380)	5.6 (360)	6.0	5.8	0.3	0.5	6.9	−0.9	−1.1	0.3	0.5
12b				-	O	6.4 [6.2–6.6] (280)	6.2 [6.2–6.3] (310)	6.3	6.3			6.9	−0.6	−0.6
114c	OiPr	H	F	O	-	5.2 (370)	5.1 (430)	5.4	5.5	0.6	0.3	6.1	−0.7	−0.6	0.5	0.2
12c				-	O	6.0 [5.9–6.0] (320)	5.7 [5.6–5.8] (330)	6.0	5.8			6.2	−0.2	−0.4

^aFitted pEC₅₀ values are shown to two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding fitted pEC₅₀ mean value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. Data are between 8 and 19 oocytes from 2 to 3 frogs for each compound and receptor.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cPreviously published^59,63 data for compounds CIQ, 117, 2*, 88, 127, and 114 were included for comparison. The numbering corresponds to the compound’s numbering in its respective publication.

^dCompound 2* is compound 2 from Santangelo et al.⁵⁹

Table 7.

Stereodependence of Select Pyrrolopyrazine Cores^e

				pEC50 [95% CI] (max) (%)a
#	X	Y	R	GluN2C	GluN2D	p(doubling conc.) (GluN2C/2D)b	sol. (μM)c	cLogP	LLE (GluN2C/2D)
CIQd (TIQ core)			Cl	5.3 (230)	5.3 (220)	4.9/4.5	8	5.6	−0.7/−1.1
S-(−)-31a		O	CF3	ND (84)	ND (88)		57	5.2
R-(+)-31a		O	CF3	6.1 [6.0–6.3] (280)	6.1 [6.0–6.2] (330)	6.0/6.2	57	5.2	0.8/1.0
S-(−)-25i	O		Cl	ND (98)	ND (99)		36	4.9
R-(+)-25i	O		Cl	5.7 [5.7–5.8] (410)	5.7 [5.7–5.8] (450)	6.1/6.2	36	4.9	1.2/1.3
S-(−)-25j	O		CF3	ND (99)	ND (98)		58	5.1
R-(+)-25j	O		CF3	5.9 [5.8–6.0] (460)	5.9 [5.8–5.9] (540)	6.3/6.4	58	5.1	1.2/1.3
S-(−)-EU-1180-453		O	F	ND (100)	ND (88)		74	4.5
R-(+)-EU-1180-453		O	F	5.5 [5.4–5.6] (410)	5.5 [5.4–5.6] (390)	5.9/5.8	74	4.5	1.4/1.3

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding log-fitted pEC₅₀ value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. For compounds in which a pEC₅₀ value was not determined, maximum potentiation is reported as percent potentiation at 30 μM drug. Data are between 6 and 13 oocytes from 2 frogs for each compound and receptor.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cAqueous solubilities reported from racemic mixtures.

^dPreviously published data for CIQ were included for comparison.

^eND = not determined.

Design of NMDAR Potentiators.

Previous computational studies on a CIQ analogue suggested that potency could be improved if the A-ring could be directed to adopt an s-trans conformation within the binding site, with s-cis/s-trans defined by the orientation of the amide carbonyl with respect to the stereocenter.⁶³ To investigate this hypothesis, a novel 1,4-dihydroisoquinolin-3(2H)-one scaffold was designed and synthesized (Scheme 1). We predicted that translocation of the amide carbonyl of CIQ into the TIQ ring would provide enough steric influence to favor the s-trans conformation and therefore increase the fraction of drug in the predicted more active pose (Figure 3). This phenomenon can be visualized via a crystal structure of prototype R-(+)-EU-1180–453. The crystallographic data and methods can be viewed in Table S2 and the Crystal Structure Methods portion of the Supporting Information, respectively.

Scheme 1.

Synthesis of Dihydroisoquinolinones^a

^aReagents and conditions: (a) chloroacetyl chloride, SnCl₄, 1,2-dichloroethane (DCE), reflux, 90 min; (b) 4-methoxyphenol, KI, K₂CO₃, reflux, 4 h; (c) substituted primary amines, Ti(OiPr)₄, NaBH(OAc)₃, DCE, 120 °C, mw, 20 min; (d) (i) PtO₂, H₂ (balloon), EtOH, rt, 10 h, (ii) Pd/C, H₂ (50 psi), EtOH, rt, 24 h; (e) NaN₃, CH₂O (37% in H₂O), AcOH, cat. sodium ascorbate, cat. CuSO₄, tetrahydrofuran (THF), rt, 24 h.

Figure 3.

Model of the steric encumbrance upon translocation of the carbonyl in the 1,4-dihydroisoquinolin-3(2H)-one series. The s-cis conformation was hypothesized to create steric clash between the carbonyl and A-ring, creating preference for the more active s-trans conformation (left). This is visualized on this scaffold through a crystal structure of R-(+)-EU-1180–453 (right).

Chemistry.

The synthesis of 1,4-dihydroisoquinolin-3(2H)-one analogues is shown in Scheme 1. Methyl esters 8a and 8b were prepared according to modified literature procedures,⁶³ while all other similar intermediates were commercially available. α-Chloro ketones 9a–d were prepared via Friedel–Crafts acylation of the appropriate methyl esters with chloroacetyl chloride and tin(IV) chloride. The resulting α-chloroamide was then reacted with 4-methoxyphenol and potassium iodide to give ketones 10a–d, which were then cyclized in a microwave reactor using the appropriate amine, titanium(IV) isopropoxide, and sodium triacetoxyborohydride to afford the penultimate eneamide intermediates 11a–n as inconsequential mixtures of their E and Z isomers. Each eneamide could then either be reduced via hydrogenation at 50 psi using palladium on carbon or in cases with aryl chlorides, hydrogenated with platinum oxide at 1 atm to give the final 1,4-dihydroisoquinolin-3(2H)-one compounds (12a–m).

The procedure for the synthesis of B-ring derivatives is outlined in Scheme 2. The synthesis began with commercially available 3,4-dimethoxyphenethylamine (13), which was acylated with chloroacetyl chloride to give α-chloroamide 14. This was then reacted with sodium hydride and 4-methoxyphenol to give α-hydroxy amides 15a–d. Intermediates 15a and 15d were then cyclized to their respective imine intermediate using modified Bischler–Napieralski conditions and subsequently reduced via sodium borohydride or hydrogenation to yield their respective TIQ amines (16a in the case of 15a). These TIQ amines were then acylated using the appropriate benzoyl chloride, deprotected to their respective primary alcohols 17a–b, and further functionalized using coupling or S_NAr conditions to produce the final compounds (18a–c). Alternatively, in the case where the final B-ring substitution was installed in step (b), such as with 15b–c, the respective secondary amine (16b–c) obtained via the Bischler–Napieralski cyclization was directly acylated with 3-(trifluoromethyl)benzoyl chloride to give the final compounds (18d–e).

Scheme 2.

Synthesis of B-Ring Derivatives^a

^aReagents and conditions: (a) chloroacetyl chloride, Et₃N, dichloromethane (DCM), 0 °C, 30 min; (b) R₁OH, NaH or Cs₂CO₃; (c) POCl₃, ACN, reflux then NaBH₄, MeOH, 0 °C, 10 min; (d) substituted benzoyl chloride, Et₃N, DCM, rt, 1 h; (e) Pd/C, H₂ (50 psi), MeOH, o/n; (f) methane sulfonic acid, DCM, rt, 6 h; (g) heteroaryl fluoride, NaH, THF, reflux, o/n, (ii) heteroaryl iodide, 1,10-Phen, CuI, Cs₂CO₃, PhMe, 80 °C, o/n.

Heterocyclic derivatives of the C-ring were synthesized utilizing a modified synthesis of CIQ⁵⁹ described in Scheme 3. Ethylamine starting materials 21a–e were synthesized from the corresponding aldehyde via a Henry-type nitroalkenylation and subsequent reduction via in situ generated alane (Scheme 3, step a and step b). 2-Methoxy-5-formylthiophene was synthesized according to the literature.⁷² The remaining synthesis proceeded as described with only slight modifications.⁵⁹

Scheme 3.

Synthesis of Heterocyclic C-Rings^a

^aReagents and conditions: (a) cat. butylamine, cat. AcOH, 4 Å MS, MeNO₂, reflux, 30 min; (b) LiAlH₄/H₂SO₄, THF, 0 °C to reflux, 5 min; (c) chloroacetyl chloride, Et₃N, DCM, 0 °C, 30 min; (d) 4-methoxyphenol, Cs₂CO₃, o/n, 79–93%; (e) POCl₃, ACN, reflux, o/n then NaBH₄, MeOH, 0 °C, 10 min; (f) (i) acid chloride, Et₃N, DCM, rt, 1 h, (ii) carboxylic acid, EDCI, 4-dimethylaminopyridine (DMAP), rt, o/n; (g) Lawesson’s reagent, PhMe, 150 °C, mw, 2 h.

Finally, pyrrolopyrazinone derivatives were synthesized as described in Scheme 4. This synthesis is a modified version of the synthesis described in Scheme 1, where the pyrrole starting material was made through the Clauson–Kaas reaction starting with glycine methyl ester hydrochloride.

Scheme 4.

Synthesis of Pyrrolopyrazinones^a

^aReagents and conditions: (a) 2,5-dimethoxytetrahydrofuran, NaOAc, 2:1 v/v AcOH:water, reflux, 4 h; (b) chloroacetyl chloride, DBN, PhMe, 115 °C, 4 h; (c) 4-methoxyphenol, KI, K₂CO₃, acetone, rt, o/n; (d) substituted primary amines, Ti(OiPr)₄, sodium triacetoxyborohydride (STAB), DCE, 120 °C, mw, 2 h; (e) Pd/C, H₂ (1 atm), EtOAc, rt, o/n; (f) Lawesson’s reagent, PhMe, 150 °C, mw, 2 h.

1,4-Dihydroisoquinolin-3(2H)-one Compounds Show Improved Doubling Concentrations Over TIQ Counterparts.

As predicted, compound 12m showed improved potentiation (270–340%) of GluN2C/D-containing NMDAR responses to saturating co-agonists compared to CIQ (220–230%) (Table 1). Compound 12m also exhibited similar potency (pEC₅₀ = 5.0–5.3 at GluN2C/D) and subunit selectivity (no activity at GluN2A/B) to CIQ, resulting in improved doubling concentrations (p(doubling conc.) = 5.1). Selectivity for GluN2C and GluN2D was also maintained upon application of 12m and subsaturating levels of co-agonists glutamate and glycine (Table S3). Based on this promising result, we designed and synthesized a targeted library of dihydroisoquinolinones to demonstrate that this trend is maintained irrespective of substitution.

We calculated the doubling concentration using eq 3 for all synthesized 1,4-dihydroisoquinolin-3(2H)-ones with corresponding TIQs^59,63 to demonstrate the trend of improved activity for 1,4-dihydroisoquinolin-3(2H)-one analogues (Table 1). The doubling concentration at GluN2C or GluN2D was improved in the 1,4-dihydroisoquinolin-3(2H)-one scaffold in 92% (11/12) of cases. The only case in which it decreased (compare 12d and 88 at GluN2C) resulted in only a 0.2 log unit drop. The average doubling concentration improvement was 0.5 log units, which led to a 0.4 log unit improvement in LLE due to the similar cLogP values of the isomeric scaffolds. These data demonstrate the ability of the 1,4-dihydroisoquinolin-3(2H)-one core to improve activity over the TIQ core.

The modification to a dihydroisoquinolinone core improved doubling concentrations substantially over the previous TIQ series; however, there were still opportunities to improve the physicochemical properties of the series (e.g., its high lipophilicity). Prototypical compounds (e.g., CIQ, 12m, etc.) exhibited cLogP values near 6 (and >8 for the most potent analogues) and aqueous solubilities in the single-digit micromolar range (<10 μM for CIQ). The highly lipophilic nature of CIQ is also likely the cause of its low free fraction that limits its concentration and receptor occupancy in vivo,⁶³ making optimization of this property vital in medicinal chemistry efforts. Highly lipophilic compounds also typically exhibit liabilities including high clearance,^73,74 low membrane permeability,⁷⁵ and low oral bioavailability.⁷⁶ There have been scattered previous attempts at decreasing the lipophilicity of the series,⁵⁹ but in nearly each case, this led to a substantial or complete loss in potency. With this goal in mind, we developed a series of hydrophilic-focused changes to the three phenyl rings of CIQ, which resulted in the discovery of a brain-penetrant pyrrolopyrazinone-based scaffold with dramatically improved lipophilic efficiency for GluN2C- and GluN2D-containing NMDARs. Prototypical compounds also display an approximate order of magnitude improvement in aqueous solubility, a relatively clean off-target profile, and improved ease of analysis due to elimination of rotamers observed with CIQ. From this point forward, we assume that the structure–activity relationship (SAR) from the TIQ series is transferrable to the dihydroisoquinolinone series. A subset of analogues is provided to demonstrate the ability to extrapolate between scaffolds (Table S4).

Evaluation of A-Ring Replacements.

First, we explored replacements of the A-ring on the dihydroisoquinolinone scaffold aimed at lowering cLogP using the synthesis described in Scheme 1. Of the alternative substituents examined, only alkyl trifluoromethyl derivative 12f (p(doubling conc.) = 5.0–5.1) (Table 2) was tolerated. Despite lowering cLogP values by nearly two log units, 12f still displayed doubling concentrations in the single-digit micromolar range. Although this result is below desired potency ranges, this substitution may prove to be useful for future promising analogues to fine-tune absorption, distribution, metabolism, and excretion (ADME) properties due to the relatively favorable change in LLE (LLE = 0.9–1.0). Other A-ring substitutions such as aliphatic chains (12g, pEC₅₀ < 4.5 or 12h, max = 94–97%), heterocycles (12l, max = 98–110% or 12i, max = 85%), and heterolinear derivatives (12j, max = 98–100% or 12k, max = 96–110%) were not tolerated. These results provide evidence that hydrophobic effects dominate receptor–ligand interactions in this portion of the molecule. Therefore, based on previous optimization,^59,63 meta-Cl- or meta-CF₃-substituted benzyl derivatives were synthesized from this point forward almost exclusively since they provided the most promising combination of activity and lipophilic efficiency.

Table 2.

Hydrophilic A-Ring Substitutions^d

#	R1	R2	X	Y	pEC50 [95% CI] (max) (%)a		p(doubling conc.) (GluN2C/2D)b	cLogP	LLE (GluN2C/2D)
					GluN2C	GluN2D
CIQc	OMe		O	-	5.3 (230)	5.3 (220)	4.9/4.5	5.6	−0.7/−1.1
117c	H		-	O	5.9 (240)	6.0 (220)	5.5/5.3	5.9	−0.4/−0.6
12f	H		-	O	5.1 [4.9–5.3] (290)	4.7 [4.5–4.9] (450)	5.0/5.1	4.1	0.9/1.0
12g	H		-	O	<4.5 (330)	<4.5 (160)	<4.5/ID	4.1	-
12h	H		-	O	ND (97)	ND (94)	-	4.0	-
12l	H		-	O	ND (110)	ND (98)	-	2.9	-
12i	H		-	O	ND (85)	ND (85)	-	5.1	-
12j	H		-	O	ND (98)	ND (100)	-	3.1	-
12k	H		-	O	ND (110)	ND (96)	-	3.6	-

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding fitted pEC₅₀ mean value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. For compounds in which a pEC₅₀ value was not determined, maximum potentiation is reported as percent potentiation as a percent of control at 30 μM drug. Data are between 6 and 11 oocytes from 2 frogs for each compound and receptor.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cPreviously published data for CIQ and 117 were included for comparison. Single methoxy substitution on the C-ring maintains similar or better activity to dimethoxy substitution.⁵⁹

^dND = not determined.

Evaluation of Hydrophilic B-Ring Replacements.

Next, we examined hydrophilic replacements of the B-ring via the divergent synthesis described in Scheme 2. Upon TEVC evaluation, none of the derivatives synthesized were tolerated at any GluN2 subtype. Nitrogen heterocycles such as the 2-pyridinyl (18b), 3-pyridinyl (18e), and 2-pyrimidinyl (18c) derivatives all showed responses when co-applied with agonist that were 88–97% of control for GluN2C- and GluN2D-containing receptors. Incorporation of saturation (18d), bioisosteric replacement with thiophene (18a), and removal of the B-ring completely (17b) were also not tolerated. Para-methoxyphenyl substitution was therefore maintained for subsequent analogues based on previous optimization (Table 3).^59,63

Table 3.

Hydrophilic B-Ring Substitutions^b

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. For compounds in which a pEC₅₀ value was not determined, maximum potentiation is reported as percent potentiation as a percent of control at 30 μM drug. Data are from 4 oocytes from 1 frog.

^bPreviously published data for compound 198 were included for comparison.⁵⁹

Effect of Bioisosteric Replacement of the C-Ring.

Although derivatization of the three rings of CIQ has led to useful tool compounds,^59,63 major changes to the C-ring and TIQ core itself have not been thoroughly explored. We viewed this as an underdeveloped area of the SAR in which opportunities for decreasing lipophilicity and improving affinity for the target protein could emerge, assuming appropriate choice of a hydrophilic core.

Due to thiophene’s known use as a classical phenyl isostere, tetrahydrothienopyridine 25a was synthesized (Scheme 3) and its activity against each of the GluN2 subunits (Table 4) was examined. Compound 25a showed improved maximum potentiation (280–350%) while maintaining similar potency (pEC₅₀ = 5.4) and selectivity (no activity at GluN2A/B) to CIQ. Due to the improved potentiation, 25a showed improved doubling concentrations and LLE (p(doubling conc.) = 5.3–5.6 and LLE = −0.3 to 0.0) in comparison to CIQ (p(doubling conc.) = 4.5–4.9 and LLE = −0.7 to −1.1). A regioisomer of 25a, compound 25h, also showed improved LLE over CIQ (ΔLLE = 0.6–0.9) due to improved potentiation (max = 330–340%). Using these results as a starting point, several tetrahydrothienopyridine-based derivatives were subsequently explored (Table 4 and Scheme 3). First, bioisosteric 2-chlorothiophene replacement of the A-ring provided compound 25b, which was similarly tolerated (LLE = −0.1 to 0.2) to 25a (LLE = −0.3 to 0.0). The trifluorocrotonic acid derivative 25c suffered a loss in potency (pEC₅₀ = 4.8–4.9) compared to CIQ, but resulted in a large improvement in LLE (LLE = 1.2–1.3) due to its notable decrease in cLogP (cLogP = 3.6). Similar to 12f, this may be an attractive substitution for fine-tuning the ADME properties of more potent future derivatives but rendered this compound outside the range of useful potency. Finally, methoxy substitution on the thiophene (25d) was examined due to previous promising SAR trends,^59,63 but this compound showed no advantages in terms of LLE over CIQ. However, each of these derivatives exemplified that changes to the TIQ core were tolerated, and we hypothesized that other more hydrophilic substitution may follow a similar trend.

Table 4.

Optimization of A- and C-Ring Functionality for Thiophene Core^c

#	R1	C-ring	R2	pEC50 [95% CI] (max) (%)a		p(doubling conc.) (GluN2C/2D)b	sol. (μM)	cLogP	LLE (GluN2C/2D)
				GluN2C	GluN2D
CIQc (TIQ core)				5.3 (230)	5.3 (220)	4.9/4.5	8	5.6	−0.7/−1.1
25a	H			5.4 [5.3–5.5] (280)	5.4 [5.3–5.5] (350)	5.3/S.6	46	5.6	−0.3/0.0
25h	H			5.4 [5.3–5.4] (330)	5.3 [5.2–5.3] (340)	5.5/5.4	-	5.6	−0.1/−0.2
25b	H			5.3 [5.3–5.3] (290)	5.3 [5.2–5.4] (400)	5.3/5.6	-	5.4	−0.1/0.2
25c	H			4.9 [4.8–5.0] (290)	4.8 [4.6–4.9] (330)	4.8/4.9	-	3.6	1.2/1.3
25d	OMe			5.3 [5.2–5.4] (220)	5.2 [5.1–5.2] (220)	4.6/4.5	-	5.7	−1.1/−1.2

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding log-fitted pEC₅₀ value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. Data are between 10 and 12 oocytes from 2 frogs for each compound and receptor.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cPreviously published data for CIQ were included for comparison.

SAR of Additional TIQ Core Changes.

Several hydrophilic heterocycles were designed and synthesized to test this hypothesis (Table 5 and Scheme 3). The furan derivative, 25f, was the first compound in the series to show a simultaneous improvement in doubling concentration (p(doubling conc.) = 5.4–5.5) and decrease in cLogP (cLogP = 5.1) compared to CIQ. This led to a dramatic improvement in LLE (LLE = 0.3–0.4). Even more impressive was pyrrolopyrazine 25i, which exhibited excellent doubling concentrations (p(doubling conc.) = 5.8–5.9) while also lowering cLogP by 0.7 (cLogP = 4.9) compared to CIQ. This resulted in an approximate two log unit improvement in LLE (LLE = 1.0–1.1). It also maintained the aqueous solubility gains observed with 25a, displaying a 4.5-fold improvement over CIQ at 36 μM. However, other more hydrophilic derivatives such as 2-methylthiazole 25e (max = 100%) and N-methylpyrazole 25g (pEC₅₀ < 4.5) were both not tolerated. Unsubstituted phenyl derivative 36 (see the Supporting Information for synthesis) was also only weakly active (p(doubling conc.) = 5.0, LLE = −1.0). Replacement of the A-ring amide linker with a thioamide linker has previously been shown to improve potency > 10-fold in certain cases.⁶³ However, this modification was not tolerated in analogue 26 (max = 94–97%), which contains the tetrahydrothienopyridine scaffold. Nonetheless, compound 25i was an exciting discovery in identifying compounds with improved lipophilic efficiencies and other derivatives with this same pyrrolopyrazine core were subsequently developed.

Table 5.

Optimization of Heterocyclic C-Ring Derivatives^d

#	C-ring	X	pEC50 [95% CI] (max) (%)a		p(doubling conc.) (GluN2C/2D)b	sol. (μM)	cLogP	LLE (GluN2C/2D)
			GluN2C	GluN2D
CIQc		O	5.3 (230)	5.3 (220)	4.9/4.5	8	5.6	−0.7/−1.1
25f		O	5.2 [5.1–5.3] (340)	5.1 [5.0–5.3] (470)	5.4/5.5	-	5.1	0.3/0.4
25i		O	5.6 [5.5–5.7] (370)	5.5 [5.4–5.7] (370)	5.9/5.8	36	4.9	1.0/1.1
25e		O	ND (100)	ND (100)	-	-	4.8	-
25g		O	< 4.5 (210)	<4.5(220)	-	-	4.2	-
36		O	5.2 [5.1–5.3] (260)	5.1 [4.9–5.2] (300)	5.0/5.0	-	6.0	−1.0/−1.0
26		S	ND (97)	ND (94)	-	-	6.0	-

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding log-fitted pEC₅₀ value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. For compounds in which a pEC₅₀ value was not determined, maximum potentiation is reported as percent potentiation at 30 μM drug. Data are between 7 and 15 oocytes from 2 frogs for each compound and receptor for which an EC₅₀ value was determined and between 4 and 9 oocytes from 1 to 2 frogs for all other analogues.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cPreviously published data for CIQ were included for comparison.

^dND = not determined.

Pyrrolopyrazine- and Pyrrolopyrazinone-Based Analogues.

Based on previously established SAR within the series, a targeted sample of derivatives was designed and synthesized, keeping the pyrrole core consistent throughout (Table 6). First, based on previously described SAR,⁵⁹ the chlorine of compound 25i was substituted for a trifluoromethyl substituent to give compound 25j (Scheme 3). This compound improved the doubling concentrations (p(doubling conc.) = 5.6–6.1) into the nanomolar range while also displaying the highest aqueous solubility (58 μM) of any derivative in the series tested thus far. To examine whether the LLE boosts seen individually with the pyrrolopyrazine core and dihydroisoquinolinone core would synergize, a hybridized pyrrolopyrazinone core was designed and synthesized as described in Scheme 4. The resulting pyrrolopyrazinone (31a) displayed similar doubling concentrations (p(doubling conc.) = 5.9–6.1) and aqueous solubility (57 μM) to 25j. Fluorine was also well tolerated (EU-1180–453) in place of the trifluoromethyl group. Although a slight potency drop was observed (pEC₅₀ = 5.5), it maintained the improvements in LLE (LLE = 1.2) and doubling concentrations (p(doubling conc.) = 5.7) seen with 25i, while improving aqueous solubility nearly an order of magnitude (74 μM) compared to CIQ. EU-1180–453 is a free-flowing, white solid that is stable as a powder or a dimethyl sulfoxide (DMSO) stock for at least 30 days (longest time tested) at room temperature under regular atmosphere. Finally, the amide of 31a was converted to the thioamide to give 32, but this modification resulted in a complete loss of activity on the pyrrolopyrazinone core, similar to that observed for the tetrahydrothienopyridine core above.

Table 6.

Optimization of Potency and Aqueous Solubility for Pyrrolopyrazine- and Pyrrolopyrazinone-Based Derivatives^d

				pEC50 [95% CI] (max) (%)a
#	X	Y	R	GluN2C	GluN2D	p(doubling conc.) (GluN2C/2D)b	sol. (μM)	cLogP	LLE (GluN2C/2D)
CIQc (TIQcore)			Cl	5.3 (230)	5.3 (220)	4.9/4.5	8	5.6	−0.7/−1.1
25j	O		CF3	5.8 [5.7–5.9] (380)	5.7 [5.5–5.9] (290)	6.1/5.6	58	5.1	1.0/0.5
31a		O	CF3	5.9 [5.7–6.0] (300)	5.8 [5.6–6.0] (400)	5.9/6.1	57	5.2	0.7/0.9
EU-1180–453		O	F	5.5 [5.4–5.6] (350)	5.5 [5.4–5.6] (350)	5.7/5.7	74	4.5	1.2/1.2
32		S	CF3	ND (120)	ND (110)			5.4

^aFitted pEC₅₀ values are shown to have two significant figures when potentiation at 30 μM exceeded 130% of control; values in brackets are the 95% confidence interval for the corresponding log-fitted pEC₅₀ value; and values in parentheses are the fitted maximum response as a percentage of the initial glutamate (100 μM) and glycine (30 μM) current. For compounds in which a pEC₅₀ value was not determined, maximum potentiation is reported as percent potentiation at 30 μM drug. Data are from between 7 and 15 oocytes from 2 frogs for each compound and receptor.

^bp(doubling conc.) is the negative log of the doubling concentration.

^cPreviously published data for CIQ were included for comparison.

^dND = not determined.

Enantiomers.

Four compounds with promising profiles (25i, 25j, 31a, and EU-1180–453) were selected for separation via chiral HPLC to examine the behavior of the individual enantiomers. Upon separation and isolation, X-ray crystallography studies on (+)-EU-1180–453 determined its absolute configuration to be the R-(+)-enantiomer and allowed for absolute stereochemical assignment of the enantiomers.

Consistent with CIQ and its derivatives, the R-(+)-enantiomers were active, while the S-(−)-enantiomers were not. We selected compound R-(+)-EU-1180–453 for further study because of its attractive doubling concentrations (p(doubling conc.) = 5.8–5.9) and improved aqueous solubility (74 μM), exhibiting a >50:1 aqueous solubility to doubling concentration ratio compared to <1:1 for CIQ. In addition, R-(+)-EU-1180–453 had virtually no effect on GluN1/GluN2A and GluN1/GluN2B, or a variety of other receptors (Tables S5 and S6). Together, these properties make this a more attractive and useful compound than CIQ (Table 7).

To better understand the scope of actions for EU-1180–453, we evaluated whether either enantiomer could alter glutamate potency, in addition to their potentiation of the response to maximally effective concentrations of glutamate plus glycine. We measured the ratio of a response to a subsaturating concentration of glutamate in vehicle or EU-1180–453 enantiomer to that of a maximally effective concentration of glutamate at GluN1/GluN2D receptors. We did not detect any meaningful effect of S-(−)-EU-1180–453 on the ratio of the current response at any receptor combination, suggesting that this stereoisomer has no effect on glutamate potency. This is consistent with the lack of a detectable effect on the maximal response to co-agonists (Table 8). A similar result was found for 30 μM R-(+)-EU-1180–453 for GluN2A and GluN2B, which had no effect on the ratio of submaximal to maximal response (Table 8) or response to maximally effective agonist for GluN1/GluN2A (108 ± 2% of control, n = 6) and GluN1/GluN2B (109 ± 2% of control, n = 7). By contrast, R-(+)-EU- 1180–453 had strong effects on the ratio of response of submaximal to maximal concentrations of glutamate for GluN1/GluN2C and GluN1/GluN2D (Table 8), suggesting that this enantiomer may shift the glutamate potency in addition to its ability to potentiate the response to maximally effective concentrations of agonist. We subsequently recorded full concentration–effect curves for glutamate activation (in saturating 30 μM glycine) at both GluN1/GluN2C and GluN1/GluN2D in the absence and presence of R-(+)-EU-1180–453. We obtained fitted pEC₅₀ values (with non-overlapping 95% confidence intervals) of 6.1 [6.0–6.2] and 6.5 [6.4–6.6] for GluN1/GluN2C (Hill slopes 1.2–1.4, n = 15, 13), and 6.3 [6.2–6.3] and 6.6 [6.5–6.7] for GluN1/GluN2D (Hill slopes 1.1–1.5, n = 16–22) in the absence and presence of R-(+)-EU-1180–453, respectively. These data suggest that even larger potentiation will be observed for subsaturating concentrations of glutamate at GluN2C and GluN2D, as will occur at extrasynaptic receptors.⁷⁷

Table 8.

Single Glutamate Concentration Screen for Changes in Glutamate Potency R-(+)-EU-1180–453^a

	I30 μM/Icontrol (mean ± SEM, %) [low glutamate concentration/low glycine concentration, μM]
#	GluN2A	GluN2B	GluN2C N = 13	GluN2D N = 22
vehicle	40 ± 4 [3, 30]	84 ± 1 [3, 30]	54 ± 1 [0.5, 30]	66 ± 2 [0.5, 30]
30 μM S-(−)-EU-1180-453	45 ± 4 [3, 30] n = 11	85 ± 9 [3, 30] n = 6	57 ± 2 [0.5, 30] n = 13	72 ± 2* [0.5, 30] n = 7
Vehicle	46 ± 5 [3, 30–100]	79 ± 1 [3, 30–100]	19 ± 3 [0.3, 10]	29 ± 2 [0.3, 10]
30 μMR-(+)-EU-1180-453	53 ± 2 [3, 30–100] n = 28	77 ± 1 [3, 30–100] n = 22	48 ± 4* [0.3, 10] n = 13	55 ± 6* [0.3, 10] n = 16

^aData mean ± standard error for the relative current response at 0.5–3 μM glutamate compared to maximally active (30–100 μM) glutamate. 30 μM glycine was present in all solutions.

^*p < 0.05, paired t-test between drug and vehicle.

We also found that R-(+)-EU-1180–453 only potentiates activated NMDARs that have bound both co-agonists and does not remove the requirement of either co-agonist. We observed that 30 μM R-(+)-EU-1180–453 had virtually no effect on GluN1/GluN2D receptors in 10 μM glycine plus 50 μM racemic 2-amino-5-phosphonopentanoic acid (DL-APV), a selective competitive antagonist of the NMDAR glutamate binding site. This produced 2.5% of the response to a maximally effective concentration of glutamate plus glycine (n = 8 oocytes), which we interpret to reflect an enhanced glutamate potency that increases the small response to contaminant glutamate. Similarly, 30 μM R-(+)-EU-1180–453 in 10 μM glutamate plus 10 μM 7-chlorokynurenic acid (7-CKA), a selective competitive antagonist of the NMDAR glycine binding site, produced 0.4% of the response to maximally effective concentration of glutamate and glycine (n = 8 oocytes).

Off-Target and Pharmacokinetic Profile.

Racemic compounds 25i, 31a, and EU-1180–453 were further examined for their behavior against common off-target receptors. First, their specificity against other ion channels in the brain were examined via two-electrode voltage clamp recordings of Xenopus oocytes expressing AMPA, kainate, nicotinic acetylcholine, serotonin, GABA, glycine, and ATP receptors.⁵⁹ Current responses were tested with saturating concentrations of agonist in both the absence of drug and presence of 10 μM 25i, 31a, or EU-1180–453 (ca. 5–10 times the EC₅₀). Only one receptor–ligand combination (α4β2-nACh/31a) exhibited >50% inhibition, and none exhibited >75% inhibition (Table S5).

Racemic compounds 25i, 31a, and EU-1180–453 were also submitted to the National Institute of Mental Health Psychoactive Drug Screening Program (PDSP, https://pdsp.unc.edu/ims/investigator/web/) to examine the activity of both the pyrrolopyrazine and pyrrolopyrazinone cores at additional off-target receptors (Table S6). The primary binding screen examined inhibition of ligand binding at 10 μM test compound, and if the drug exhibited >50% inhibition, a secondary binding screen was run to determine K_i. Compounds 25i, 31a, and EU-1180–453 showed >50% inhibition at an average of only 3 of the 45 off-target receptors examined, and all K_i values were in the micromolar range, suggesting reasonable selectivity for the GluN2C- and GluN2D-containing NMDA receptors.

Racemic⁷⁸ EU-1180–453 was also dosed in mice to determine its pharmacokinetic behavior. EU-1180–453 was dosed i.p. in C57Bl/6 mice (n = 15, 3 per time point) at 10 mg/kg in vehicle consisting of 50% PEG400 in water, and samples were evaluated via LC/MS-MS at 0.25, 0.5, 1, 2, and 4 h (see Experimental Procedures). Concentrations in brain (962 ± 210 ng/mL, 2.5 ± 0.4 μM) and plasma (695 ± 111 ng/mL, 1.83 ± 0.29 μM) peaked at 0.25 h and steadily declined, resulting in excellent brain exposure (brain:plasma_AUC_LAST = 1.4 ± 0.5) and an acceptable plasma half-life (t_1/2 = 1.07 ± 0.04 h). CIQ has previously been reported as brain-penetrant but suffers from high plasma protein binding leading to low free fraction.⁶³ We therefore evaluated the plasma protein binding (PPB) of EU-1180–453 using equilibrium dialysis and found it to be 98.7% bound to rat plasma protein at 1 μM of drug. This is a greater than 10-fold decrease compared to CIQ, for which we observed >99.9% bound. A comparison of pharmacokinetic data between EU-1180–453 and CIQ can be seen in Table 9.⁶¹

Table 9.

Plasma Pharmacokinetics Comparison Between EU-1180–453 and CIQ

#	dose (mg/kg)a	Cmax (ng/mL)	Cmax (μM)	t1/2 (h)	PPB, fubb	[brain]: [plasma]c	cLogP	LLE (GluN2C/2D)
CIQ	20	2090 ± 20	2.16 ± 0.03	1.35 ± 0.12	<0.1	6.7 ± 2.2	5.6	−0.7/−1.1
EU-1180-453	10	695 ± 111	1.83 ± 0.29	1.07 ± 0.04	1.3	1.4 ± 0.5	4.5	1.2/1.2

^aCompounds were dosed i.p. in either 1:4:5 dimethylacetamide:PEG400:(5% dextrose in water) (CIQ, n = 3/group) or 1:1 PEG400:water (EU-1180–453, n = 3/group).

^bRat plasma protein binding reported as percent unbound (f_ub ) at 1 μM.

^cBrain and plasma concentrations in ng/g and ng/mL, respectively, at C_max.

CONCLUSIONS

Although CIQ is a useful in vitro tool compound for the study of GluN2C- and GluN2D-containing receptors, its in vivo and in vitro use is limited due to modest potency and potentiation, low aqueous solubility, and high lipophilicity. We have developed second-generation GluN2C- and GluN2D-selective PAMs that address each of these concerns. For example, racemic EU-1180–453 is a more potent potentiator (EC₅₀ ~3 μM) that shows log unit improvements in lipophilic efficiency (ΔLLE = 1.9–2.3), doubling concentration (Δp(doubling conc.) = 0.8–1.2), aqueous solubility (8–74 μM), and a log unit decrease in cLogP (ΔcLogP = 1.1) compared to CIQ. It is brain-penetrant, exhibits a reasonable half-life, and shows minimal off-target activity. The active enantiomer, R-(+)-EU-1180–453, exhibits ca. 1–2 μM doubling concentrations due to 4-fold potentiation and results in a doubling concentration to aqueous solubility ratio of >50:1. Limitations at this time center around potency, as there is a question of whether the given combination of in vitro potency, free fraction, and C_max,brain can drive measurable pharmacological activity in vivo. These in vivo experiments are currently ongoing in our lab, and the results will be reported in future publications. Overall, this work has resulted in second-generation in vitro tools to study positive modulation of GluN2C- and GluN2D-containing NMDARs and is an important step toward future in vivo work.

EXPERIMENTAL PROCEDURES

Biology.

Unfertilized Xenopus laevis oocytes were purchased from Ecocyte (Austin, TX). The oocytes were injected with mRNA to express recombinant rat GluN1/GluN2A, GluN1/GluN2B, GluN1/GluN2C, and GluN1/GluN2D, and two-electrode voltage clamp recordings were performed. Drs. S. Heinemann (Salk Institute), S. Nakanishi (Kyoto University), and P. Seeburg (University of Heidelberg) provided the cDNAs for rat GluN1–1a (GenBank accession numbers U11418 and U08261, referred to as GluN1 henceforth), GluN2A (D13211), GluN2B (U11419), GluN2C (M91563), and GluN2D (D13213). GluN2C and GluN2D were altered according to the literature.²⁵ Isolation of oocytes, synthesis of cRNA, and injections of cRNA were each done according to the literature.⁷⁹ Oocytes were placed in a perfusion chamber and continuously washed during TEVC recordings with a solution consisting of the following (in mM): 90 NaCl, 0.5 BaCl, 0.005 ethylenediaminetetraacetic acid (EDTA), 1.0 KCl, and 10 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at pH 7.4 and 23 °C. Glass electrodes were pulled from thin-walled glass capillaries (tip resistance, 0.5–2.5 MΩ) and filled with 0.3–3.0 M KCl, while the oocyte membrane potential was held constant at −40 mV via an OC-725C amplifier (Warner Instruments Co.). Each compound was brought up in 20 mM DMSO and diluted with recording solution containing 30 μM glycine and 100 μM glutamate to the target concentration. To prevent the current increase typically seen during experiments with oocytes expressing GluN1/GluN2A receptors, some oocytes were either injected with 20 nL of 100 mM K-BAPTA (potassium 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) or pretreated with 50 μM BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester) for 10 min. Compounds that potentiated GluN2A- and GluN2B-containing receptors were not studied further.

Every test compound was recorded at 5–7 concentrations in 4–19 oocytes from 1 to 3 different frogs at GluN2C- and GluN2D-containing NMDA receptors and in 4 to 17 oocytes from 1 to 4 frogs at GluN2A- and GluN2B-containing receptors. The potentiation of certain test compounds at a concentration of 10 μM was averaged and reported as I_10μM/I_control (mean ± SEM, %), where I is the current. For test compounds with potentiation that exceeded 125% at 30 μM, an average EC₅₀ value (the half-maximal effective concentration of potentiator) was determined by fitting the following equation

$$\text{response}=(100-\max )/\left(1+{\left([\text{concentration}]/{\text{EC}}_{50}\right)}^N\right)+\text{max}$$ (1)

to individual concentration–response curves normalized to the current in the absence of the potentiator (100%), where N is the Hill slope and max is the maximal response predicted for saturating concentration of potentiator. This is converted to the negative log of the value through the following

$${\text{pEC}}_{50}=-{\log }_{10}\left({\text{EC}}_{50}\right)$$ (2)

The concentration that gave a 2-fold increase in the current response was determined by rearranging eq 1 to yield

$$\text{doubling concentration}={\text{EC}}_{50}{((100-\max )/(200-\max )-1)}^{1/N}$$ (3)

where N is the Hill slope. This is also converted to the negative log of the value through the following

$$p(\text{doubling conc.})=-{\log }_{10}(\text{doubling concentration})$$ (4)

The lipophilic ligand efficiency (LLE) equation used in this paper is a modified version of the metric that was recently reviewed.⁸⁰ The negative log of the potency (pEC₅₀) is replaced by the negative log of the doubling concentration to normalize for positive modulation, while Log D is replaced by cLogP to give

$$\text{LLE}=p(\text{doubling conc.})-\text{cLogP}$$ (5)

IACUC-approved protocols and animal welfare regulations outlined in the “Guide for the Care and Use of Laboratory Animals” were both followed during pharmacokinetic evaluation of compound EU-1180–453. In vivo analysis was performed by Pharmaron (Irvine, CA). Briefly, a group (n = 15) of fed, male C57BL/6 mice approximately 6–8 weeks of age were injected IP with 10 mg/kg (10 mL/kg, 1 mg/mL) of drug using 50% PEG400 in water as a vehicle. Samples were collected from the blood and brain at 0.25, 0.5, 1, 2, and 4 h after administration (3 mice per time point) following CO₂ anesthesia. Collection from the brain was performed as follows: the mouse is terminally anesthetized via increasing concentration of CO₂ and as much blood is removed as possible via cardiac puncture. The cardiac puncture is done by opening the chest cavity to expose the heart, cutting an incision in the right auricle using surgical scissors and finally injecting a saline solution (~10 mL) slowly into the left ventricle via syringe. The mouse is placed head down at a 45° angle to facilitate blood removal. After perfusion, the skull is opened and the brain is removed. The brain is washed with saline, dried with surgical gauze, placed in tared tubes, and stored at −75 °C before analysis. For plasma sample preparation at 0.25–2 h time points, 15 μL of blank solution, 30 μL of plasma sample, and 150 μL of acetonitrile were added sequentially for protein precipitation. After centrifugation, 20 μL of the supernatant and 80 μL of CB were combined, and thus the final compound concentration in plasma (ng/mL) was corrected by multiplying by 5. For plasma sample preparation at the 4 h time point, 15 μL of blank solution, 30 μL of plasma sample, and 150 μL of acetonitrile were added sequentially for protein precipitation. For all brain sample preparation, 15 μL of blank solution, 30 μL of brain samples, and 150 μL of acetonitrile were added sequentially for protein precipitation. Brain samples were prepared by adding brain (g) to deionized water (mL) in a 1:4 ratio for homogenization. The mixtures were then vortexed for 30 s and subsequently centrifuged (~4000 rpm) for 15 min. The supernatant was diluted 3-fold with water, and a 2 μL aliquot of the diluted supernatant was injected into a Shimadzu LC-30A LC-MS/MS system with Phenomenex 2.6 μ PFP 100A column (30 mm × 2.1 mm) using verapamil as an internal standard. A gradient from 95% water (0.1% formic acid) to 95% ACN (0.1% formic acid) was run over 2 min at a flow rate of 0.6 mL/min. Brain and blood samples were collected at 15, 30, 60, 120, and 240 min from three C57Bl/6 mice at each time point.

The maximum aqueous solubility for compounds CIQ, 25a, 25i, 25j, 31a, and EU-1180–453 was determined using a BMG Labtech Nephelostar nephelometer (Offenburg, Germany) according to manufacturer’s instructions. Compound powder (1–3 mg) was taken up in DMSO (Sigma-Aldrich) to a 20 mM stock solution and serially diluted (~10 μL) to 15.0, 10.0, 5.00, 2.50, 1.25, and 0.625 μM stock solutions. These, along with a DMSO blank, were distributed in triplicate to black, clear-bottom 96-well plates in 3 μL increments and subsequently diluted 100-fold using the aqueous oocyte recording solution (pH 7.4) described above. DMSO content was ≤1.0% (v/v). Each solution was shaken gently for 30 min before submission to the nephelometer.

Receptor binding profiles, K_i determinations, and hERG activity of compounds 25i, 31a, and EU-1180–453 were generously provided by the National Institute of Mental Health’s Psychoactive Drug Screening Program, Contract #HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP is Directed by Bryan L. Roth at the University of North Carolina at Chapel Hill and Project Officer Jamie Driscoll at NIMH, Bethesda, MD. Radioligand binding was measured in the presence of 10 μM test compound. A K_i value was determined for each receptor at which test compound showed >50% inhibition. For experimental details, refer to the PDSP website https://pdsp.unc.edu/ims/investigator/web/.

Compounds 25i, 31a, and EU-1180–453 were also tested at 10 μM for actions at AMPA (GluA1–4), kainate (GluK2), GluN3 (GluN3A-B), nicotinic acetylcholine (α1β2γδ, α4β2, α7), serotonin (5-HT_3A), GABA_A (α₁β₂γ_2S), GABA_C (ρ1), glycine (α1), and purinergic (P_2X human) receptors expressed in Xenopus oocytes. cRNA encoding the receptor subunits was synthesized from linear template cDNA using the mMessage mMachine kit (Ambion). Oocytes were injected with 1–5 ng cRNA in a 50 nL volume and incubated in Barth’s solution at 15 °C for 2–5 days prior to recording. α1β2γδ nAChR subunits were injected at a 1:1:1:1 ratio, while α4β2 and α7 nAChR subunits were injected at a 1:1 ratio. The cDNAs encoding GABA_C, glycine, and serotonin subunits were provided by Dr. D. Weiss (University of Texas Health Science Center at San Antonio). cDNAs encoding nicotinic acetylcholine receptor subunits were provided by Drs. R. Papke (University of Florida) and S. Heinemann (Salk Institute). cDNAs encoding the purinergic receptors were provided by Dr. R. Hume (University of Michigan). GABA_A receptors were activated by 100 μM GABA, and GABA_C receptors were activated by 100 μM GABA. Acetylcholine was used at the indicated concentrations (in μM) to activate the nicotinic acetylcholine receptors: α1β2γδ (1), α4β2 (10), α7(300). 100 μM glycine was used to activate the glycine receptor, 100 μM serotonin was used to activate 5-HT_3A receptor, and 9 μM ATP was used to activate the purinergic receptors. Responses in the presence of test compound were expressed as a percent of control.

Plasma protein binding was determined by equilibrium dialysis using the Rapid Equilibrium Dialysis RED device (Pierce, Catalog #89810), according to manufacturer’s protocol. Compounds were prepared as 100 μM DMSO stocks and spiked into 1 mL of rat plasma (Bioreclamations) to make a final concentration of 1 μM. Plasma (300 μL) was dispensed into wells separated by an 8 KDa permeable cellulose membrane from wells containing 100 mM potassium phosphate, pH 7.4 (500 μL). The compound was tested in triplicate. The RED device was sealed, and the system was allowed to reach equilibrium for 6 h in a 37 °C incubator with gentle agitation at 100 rpm. After incubation, plasma samples were prepared by transferring 10 μL from plasma wells to 90 μL of fresh 100 mM potassium phosphate, pH 7.4, and buffer samples were prepared by transferring 90 μL from buffer wells to 10 μL of naïve plasma. In addition, a reference sample without equilibration was prepared in triplicate by mixing 10 μL of plasma containing 1 μM compound with 90 μL of buffer to determine compound recovery from the assay. Two volumes of 1:1 acetonitrile:methanol spiked with the internal standard phenytoin (0.2 μg/mL) were added to reference and samples. Precipitation of plasma protein binding was allowed for 15 min before the reference and samples were centrifuge clarified. Supernatant (10 μL) was used for LC/MS/MS analyses. Compounds were quantified on an API4000 MS/MS System (Applied Biosystems, Concord, Ontario, Canada) interfaced with an Agilent 1100 Series HPLC as previously described.⁸¹

Chemistry.

General Experimental.

All starting materials were purchased from commercial sources and used directly without further purification. Purification by flash column chromatography was done using a Teledyne ISCO Combiflash Companion instrument using Teledyne Redisep normal phase columns. ¹H and ¹³C NMR spectra were recorded on a Mercury-300, VNMR-400, INOVA-400, INOVA-500, or INOVA-600 NMR spectrometer. Chemical shifts were reported in ppm and referenced to the residual deuterated solvent. Reactions were monitored by thin-layer chromatography on pre-coated aluminum plates (silica gel 60 F254, 0.25 mm) or liquid chromatography–mass spectrometry (LCMS) on an Agilent Technologies 1200 series instrument. High-resolution mass spectra were recorded on a VG 70-S Nier Johnson or JEOL instrument by the Emory University Mass Spectroscopy Center. Purity was established via LCMS (Varian) in at least two solvent systems (MeOH:water/ACN:water or MeOH:water/MeOH:water) unless otherwise noted. All compounds described were >95% pure by LCMS besides one compound (12k) that was >85% pure. Optical rotations were established using a PerkinElmer 314 instrument.

General Procedure for α-Chloro Ketones (Procedure I).

Methyl ester (1 equiv) was dissolved in DCE (1.0 M), and 1 M tin(IV) chloride in DCM (5 equiv) was added dropwise for 10 min at room temperature. 2-Chloroacetyl chloride (4 equiv) was then added at room temperature, and the resulting mixture was heated to reflux for 90 min. Upon completion, the reaction was cooled to room temperature and quenched carefully with 1 M HCl. The aqueous layer was extracted with DCM, washed with saturated NaHCO₃ (2×) and brine, dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Biaryl Ketones (Procedure II).

To a suspension of potassium iodide (1.2 equiv) and potassium carbonate (2 equiv) in acetone (0.17 M) were added α-chloro ketone (1 equiv) and phenol (1.2 equiv). The resulting mixture was then heated open to atmosphere at reflux for 4 h. Upon completion, the reaction was cooled to room temperature and solvent was removed in vacuo. The resulting residue was then dissolved in EtOAc and washed with water. The organic layer was then extracted with EtOAc, dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Eneamides (Procedure III).

An appropriate microwave vial was charged with ketone (1 equiv), primary amine (1.2 equiv), tetraisopropoxytitanium (3 equiv), and DCE (0.2 M). The solution was stirred for 10 min before sodium triacetoxyborohydride (3 equiv) was added, and the mixture was irradiated at 120 °C for 30 min. Upon completion, the reaction was cooled to room temperature, diluted with DCM, and washed with 1 M HCl. The organic layer was extracted with DCM, washed with NaHCO₃, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound as a mixture of E/Z isomers, the mass was confirmed via LCMS, and carried forward without further characterization.

General Procedure for Final 1,4-Dihydroisoquinolin-3(2H)-one Compounds (Procedure IV-A).

A 10 mL microwave vial was charged with eneamide (1 equiv), platinum(IV) oxide (10 mol %), and ethanol. The vial was then purged with hydrogen, evacuated (3×) and reacted at room temperature under hydrogen for 6–24 h, and monitored via LCMS to ensure no dehalogenation occurred. Upon first sight of dehalogenation via LCMS, the reaction was diluted with MeOH, filtered through a plug of celite washing with MeOH, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Final 1,4-Dihydroisoquinolin-3(2H)-one Compounds (Procedure IV-B).

Eneamide (1 equiv) was dissolved in ethanol, 10% Pd/C (0.15 equiv) was added, and the mixture was hydrogenated at 50 psi overnight. The reaction mixture was filtered through a pad of celite, washed with methanol, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for α-Chloro Amides (Procedure V).

Primary amine (1 equiv) was dissolved in dry DCM, and triethylamine (2 equiv) was added. The solution was brought to 0 °C, 2-chloroacetyl chloride (1.2 equiv) was added, and the mixture was reacted at room temperature for 1 h, when deemed complete by LCMS. The reaction was then quenched with 1 M HCl, extracted into DCM, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Alkylation of Alkyl Halides Using Cesium Carbonate (Procedure VI).

Phenol (1.2 equiv) was dissolved in acetonitrile and cesium carbonate (4 equiv) was added. This mixture was stirred at room temperature for 2 h before α-chloroamide (1 equiv) in acetonitrile was added and allowed to react at room temperature overnight. The reaction was then quenched with saturated ammonium chloride, and the product extracted into EtOAc, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Alkylation of Alkyl Halide Using Sodium Hydride (Procedure VII).

60% Sodium hydride dispersion in mineral oil (1.4–3 equiv) was added to a solution of alcohol (1.2 equiv) in dry THF or DMF (ca. 0.4–0.8 M) at 0 °C under argon. After 30 min, alkyl halide in dry THF (~0.4–0.8 M) was added dropwise, and the mixture was stirred at room temperature overnight. Upon reaction completion, excess sodium hydride was quenched with water, poured over ice, and 1 M HCl was added. The organic layer was extracted with ethyl acetate (3×), and the organic layers were combined, washed with brine (1×), dried over Na₂SO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Bischler–Napieralski Cyclization (Procedure VIII).

Acetamide (1 equiv) was dissolved in dry acetonitrile (~0.05 M), and POCl₃ (3 equiv) was added. This was reacted at reflux overnight when the reaction was deemed complete via thin-layer chromatography (TLC). The reaction was cooled to room temperature and concentrated in vacuo to afford the crude compound. The crude imine was dissolved in dry MeOH and cooled to 0 °C before sodium borohydride (3 equiv) was added in ~100 mg portions. The mixture was stirred at room temperature for 10 min. The reaction was then concentrated and subsequently partitioned between EtOAc and H₂O. The product was then extracted into EtOAc, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Tetrahydroisoquinolines (Procedure IX).

Secondary amine (1 equiv) was dissolved in dry DCM, triethylamine (2 equiv) was added, and the solution was brought to 0 °C before acid chloride (1.2 equiv) was added dropwise. The mixture was stirred at room temperature for 1 h. The reaction was then quenched with 1 M HCl, and the organic layer was extracted into DCM, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for S_NAr Reactions (Procedure X).

Primary alcohol (1 equiv) was dissolved in dry THF or DMF (~0.2 M), and 60% sodium hydride dispersion in mineral oil (2–3 equiv) was added. This mixture was stirred for 30 min at room temperature before heteroaryl halide (1.5 equiv) was added. The reaction was stirred at 70 °C overnight and then cooled to room temperature, quenched carefully with water, and diluted with both EtOAc and water. The aqueous layer was extracted with EtOAc, the organic layers were combined, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Nitro Alkenes (Procedure XI).

Aldehyde (1 equiv) was dissolved in dry nitromethane (~1 M), and to this solution butylamine (0.12 equiv), acetic acid (0.2 equiv), and 4 Å molecular sieves (~10 wt %) were added. The solution was brought to reflux for 30 min before the mixture was concentrated in vacuo, and the crude material was purified via flash column chromatography to afford the title compound.

General Procedure for Ethylamines (Procedure XII).

Sulfuric acid (2.23 equiv) was added dropwise to a solution of lithium aluminum hydride (4.46 equiv) in THF (~0.15 M) at 0 °C and stirred for 20 min. Nitroalkene (1 equiv) dissolved in dry THF (~1.5 M) was then added dropwise at 0 °C and stirred for 10 min. The mixture was then heated to reflux for 5 min, cooled to 0 °C, and quenched carefully with iPrOH (6 equiv) and NaOH (9 equiv). The resulting suspension was then filtered and concentrated in vacuo to afford the crude title compound that was carried forward without further purification.

General Procedure for Carboxylic Acid Coupling to Final Compounds (Procedure XIII).

Carboxylic acid (1.1 equiv) was dissolved in dry DCM and cooled to 0 °C. EDCI (1.2 equiv) and DMAP (1.2 equiv) were then added and stirred for 2 h before the free amine (1 equiv) was added, and the mixture was stirred at room temperature overnight. The reaction was then quenched with deionized water, and the organic layer was extracted into DCM, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Thioamides (Procedure XIV).

Amide (1 equiv) in a microwave vial was dissolved in toluene and Lawesson’s reagent (1.5 equiv) was added. This mixture was allowed to react in microwave at 150 °C for 20 min. The reaction was then diluted with DCM; washed with saturated NaHCO₃, water, and brine; dried over MgSO₄; and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for α-Chloro Ketones Using DBN (Procedure XV).

Methyl ester (1 equiv) was dissolved in PhMe (0.5 M) and DBN (0.15 equiv) was added followed by chloroacetyl chloride (1.2 equiv). The mixture was heated to 115 °C and reacted for 4 h. The reaction was then diluted with DCM, and the organic layer was washed with 1 M HCL followed by 1 M NaOH. The organic layer was then dried over MgSO₄ and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Biaryl Ketones (Procedure XVI).

To a suspension of potassium iodide (1.2 equiv) and potassium carbonate (2 equiv) in acetone (0.3 M) were added α-chloro ketone (1 equiv) and phenol (1.2 equiv). The resulting mixture was then stirred open to atmosphere at room temperature overnight. Upon completion, the solvent was removed in vacuo and the resulting residue was dissolved in EtOAc and washed with water. The aqueous layer was then extracted with EtOAc, and all organic layers were combined, dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

General Procedure for Tertiary Eneamides (Procedure XVII).

An appropriate microwave vial was charged with ketone (1 equiv), primary amine (1.2 equiv), tetraisopropoxytitanium (3 equiv), and DCE (0.2 M), and the solution was stirred for 10 min before sodium triacetoxyborohydride (3 equiv) was added. The mixture was irradiated at 120 °C for 2 h. Upon completion, the reaction was cooled to room temperature, diluted with DCM, and washed with 1 M HCl. The organic layer was extracted with DCM, washed with NaHCO₃, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound as a mixture of E/Z isomers, mass was confirmed via LCMS, and it was carried forward without further characterization.

General Procedure for Final Pyrrolopyrazinone Compounds (Procedure XVIII).

Eneamide (1 equiv) was dissolved in EtOAc, and 10% Pd/C (0.15 equiv) was added. The flask was purged with hydrogen from a balloon (3×), and the mixture was hydrogenated (1 atm) overnight. The reaction mixture was filtered through a pad of celite, washed with methanol, and concentrated in vacuo. The crude product was purified via flash column chromatography to afford the title compound.

Methyl 2-(3-Isopropoxyphenyl)acetate (8a).

Methyl 2-(3-hydroxyphenyl)acetate (7.83 g, 47.1 mmol) was dissolved in THF (196 mL), and isopropanol (5.40 mL, 70.7 mmol), triphenylphosphine (18.5 g, 70.7 mmol), and 40% diethyl azodicarboxylate in toluene (32.2 mL, 70.7 mmol) were added and allowed to react overnight at room temperature. The crude product was purified via flash column chromatography (ISCO, Redisep 120 g column, 0–60% EtOAc/hexanes gradient) to afford the title compound as a clear oil (8.19 g, 83%). R_f (1:1 EtOAC/Hex): 0.88; ¹H NMR (400 MHz, CDCl₃) δ 7.22 (t, J = 8.1 Hz, 1H), 6.88–6.76 (m, 3H), 4.55 (hept, J = 6.1 Hz, 1H), 3.69 (s, 3H), 3.59 (s, 2H), 1.34 (d, J = 6.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 171.9, 158.0, 135.3, 129.5, 121.3, 116.8, 114.3, 69.7, 52.0, 41.2, 22.0. HRMS calcd for C₁₂H₁₇O₃, 209.11722 [M + H]⁺; found 209.11720 [M + H]⁺.

Methyl 2-(3-Isobutoxyphenyl)acetate (8b).

Methyl 2-(3-hydroxyphenyl)acetate (2.50 g, 15.0 mmol) was dissolved in dry DMF (46 mL) and potassium carbonate (8.32 g, 60.2 mmol) was added. This mixture was stirred at room temperature for 2 h before 1-iodo-2-methylpropane (3.70 mL, 30.1 mmol) was added. The resulting mixture was allowed to react at 60 °C overnight, after which the reaction was deemed complete via TLC. The product was then extracted into EtOAc, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–20% EtOAc/hexanes gradient) to afford the title compound as a clear oil (1.64 g, 49%). R_f (1:1 EtOAC/Hex): 0.91; ¹H NMR (400 MHz, CDCl₃) δ 7.25–7.20 (m, 1H), 6.88–6.77 (m, 3H), 3.72 (d, J = 6.5 Hz, 2H), 3.70 (s, 3H), 3.60 (s, 2H), 2.16–2.01 (m, 1H), 1.03 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 172.0, 159.4, 135.3, 129.5, 121.3, 115.5, 113.2, 74.3, 52.1, 41.2, 28.3, 19.3. HRMS calcd for C₁₃H₁₉O₃ , 223.13287 [M + H]⁺; found 223.13264 [M + H]⁺.

Methyl 2-(2-(2-Chloroacetyl)-5-isopropoxyphenyl)acetate (9a).

General procedure I was followed using 8a (8.10 g, 38.9 mmol), 1 M tin(IV) chloride in DCM (194 mL, 194 mmol), and 2-chloroacetyl chloride (12.5 mL, 156 mmol) in DCE (39 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 80 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a white solid (5.20 g, 47%). R_f (1:1 EtOAC/Hex): 0.81; ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J = 8.7 Hz, 1H), 6.82 (dd, J = 8.7, 2.6 Hz, 1H), 6.75 (d, J = 2.6 Hz, 1H), 4.63 (hept, J = 5.8 Hz, 3H), 4.63 (s, 2H), 3.91 (s, 2H), 3.68 (s, 3H), 1.35 (d, J = 6.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.6, 171.6, 161.4, 138.6, 132.3, 125.9, 120.5, 113.1, 70.2, 51.95, 47.2, 40.7, 21.9. HRMS calcd for C₁₄H₁₈O₄ Cl, 285.08881 [M + H]⁺; found 285.08867 [M + H]⁺.

Methyl 2-(2-(2-Chloroacetyl)-5-isobutoxyphenyl)acetate (9b).

General procedure I was followed using 8b (1.08 g, 4.86 mmol), 1 M tin(IV) chloride in DCM (24.3 mL, 24.3 mmol), and 2-chloroacetyl chloride (1.56 mL, 19.4 mmol) in DCE (4.9 mL). The crude product was purified via flash column chromatography (ISCO,Redisep 80 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a white solid (0.70 g, 48%). R_f (1:1 EtOAC/Hex): 0.88; ¹H NMR (400 MHz, CDCl₃) δ 7.74 (d, J = 8.7 Hz, 1H), 6.85 (dd, J = 8.7, 2.6 Hz, 1H), 6.79 (d, J = 2.6 Hz, 1H), 4.63 (s, 2H), 3.92 (s, 2H), 3.77 (d, J = 6.5 Hz, 2H), 3.69 (s, 3H), 2.08 (hept, J = 6.7 Hz, 1H), 1.01 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 191.7, 171.6, 162.6, 138.5, 132.2, 126.1, 119.6, 112.4, 74.5, 52.0, 47.2, 40.7, 28.1, 19.1. HRMS calcd for C₁₅H₂₀O₄Cl, 299.10446 [M + H]⁺; found 299.10431 [M + H]⁺.

Methyl 2-(2-(2-Chloroacetyl)-5-methoxyphenyl)acetate (9c).

General procedure I was followed using methyl 2-(3-methoxyphenyl)-acetate (8c) (2.23 mL, 13.9 mmol), 1 M tin(IV) chloride in DCM (69.4 mL, 69.4 mmol), and 2-chloroacetyl chloride (4.45 mL, 55.5 mmol) in DCE (14 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a white solid (1.65 g, 46%). R_f (1:1 EtOAC/Hex): 0.66; ¹H NMR (399 MHz, CDCl₃) δ 7.75 (d, J = 8.7 Hz, 1H), 6.86 (dd, J = 8.7, 2.6 Hz, 1H), 6.78 (d, J = 2.6 Hz, 1H), 4.63 (s, 2H), 3.92 (s, 2H), 3.85 (s, 3H), 3.69 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 191.8, 171.5, 162.8, 138.6, 132.1, 126.6, 119.1, 112.0, 55.5, 51.9, 47.1, 40.6. HRMS calcd for C₁₂H₁₄O₄Cl, 257.05751 [M + H]⁺; found 257.05751 [M + H]⁺.

Methyl 2-(2-(2-Chloroacetyl)-4,5-dimethoxyphenyl)acetate (9d).

General procedure I was followed using methyl 2-(3,4-dimethoxyphenyl)-acetate (8d) (2.50 g, 11.9 mmol), 1 M tin(IV) chloride in DCM (59.5 mL, 59.5 mmol), and 2-chloroacetyl chloride (3.81 mL, 47.6 mmol) in DCE (12 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–55% EtOAc/hexanes gradient) to afford the title compound as a white solid (1.13 g, 33%). R_f (1:1 EtOAC/Hex): 0.45; ¹H NMR (400 MHz, CDCl₃) δ 7.23 (s, 1H), 6.74 (s, 1H), 4.63 (s, 2H), 3.92 (s, 3H), 3.91 (s, 3H), 3.88 (s, 2H), 3.68 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 191.9, 171.8, 152.4, 147.4, 130.5, 126.0, 115.6, 112.7, 56.2, 56.0, 52.0, 47.2, 40.0. HRMS calcd for C₁₃H₁₆O₅Cl, 287.06808 [M + H]⁺; found 287.06795 [M + H]⁺.

Methyl 2-(5-Isopropoxy-2-(2-(4-methoxyphenoxy)acetyl)-phenyl)acetate (10a).

General procedure II was followed using 9a (332 mg, 1.17 mmol), potassium iodide (232 mg, 1.40 mmol), 4-methoxyphenol (174 mg, 1.40 mmol), and potassium carbonate (322 mg, 2.33 mmol) in acetone (7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a white solid (289 mg, 67%). R_f (1:1 EtOAC/Hex): 0.73; ¹H NMR (500 MHz, CDCl₃) δ 7.83 (d, J = 8.7 Hz, 1H), 6.92–6.74 (m, 6H), 5.11 (s, 2H), 4.65 (hept, J = 6.0 Hz, 1H), 3.94 (s, 2H), 3.75 (s, 3H), 3.67 (s, 3H), 1.36 (d, J = 6.1 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 195.5, 171.8, 161.3, 154.3, 152.3, 138.32, 131.8, 126.3, 120.4, 115.9, 114.6, 113.1, 76.8, 72.1, 70.2, 55.7, 52.0, 40.5, 21.9. HRMS calcd for C₂₁H₂₅O₆, 373.17043 [M + H]⁺; found 373.17143 [M + H]⁺.

Methyl 2-(5-Isobutoxy-2-(2-(4-methoxyphenoxy)acetyl)phenyl)-acetate (10b).

General procedure II was followed using 9b (700 mg, 2.34 mmol), potassium iodide (467 mg, 2.81 mmol), 4-methox-yphenol (349 mg, 2.81 mmol), and potassium carbonate (648 mg, 4.69 mmol) in acetone (14 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a white solid (469 mg, 52%). R_f (1:1 EtOAC/Hex): 0.90; ¹H NMR (500 MHz, CDCl₃) δ 7.83 (d, J = 8.7 Hz, 1H), 6.90–6.73 (m, 6H), 5.11 (s, 2H), 3.94 (s, 2H), 3.78 (d, J = 6.6 Hz, 2H), 3.75 (s, 3H), 3.67 (s, 3H), 2.10 (hept, J = 6.5 Hz, 1H), 1.03 (d, J = 6.7 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 195.6, 171.8, 162.5, 154.3, 152.3, 138.3, 131.8, 126.5, 119.5, 114.6, 112.4, 74.6, 72.1, 55.7, 52.0, 40.5, 28.2, 19.2. HRMS calcd for C₂₂H₂₇O₆, 387.18022 [M + H]⁺; found 387.18013 [M + H]⁺.

Methyl 2-(5-Methoxy-2-(2-(4-methoxyphenoxy)acetyl)phenyl)-acetate (10c).

General procedure II was followed using 9c (10.2 g, 39.7 mmol), potassium iodide (7.92 g, 47.7 mmol), 4-methoxyphenol (5.92 g, 47.7 mmol), and potassium carbonate (11.0 g, 79.0 mmol) in acetone (238 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 120 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white solid (7.23 g, 53%). R_f (1:1 EtOAC/Hex): 0.59; ¹H NMR (400 MHz, CDCl₃) δ 7.83 (d, J = 8.7 Hz, 1H), 6.88–6.77 (m, 6H), 5.10 (s, 2H), 3.94 (s, 2H), 3.85 (s, 3H), 3.74 (s, 3H), 3.66 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 195.6, 171.7, 162.7, 154.3, 152.2, 138.2, 131.7, 126.8, 119.0, 115.8, 114.6, 112.0, 72.0, 55.6, 55.5, 51.9, 40.5. HRMS calcd for C₁₉H₂₁O₆, 345.13326 [M + H]⁺; found 345.13321 [M + H]⁺.

Methyl 2-(4,5-Dimethoxy-2-(2-(4-methoxyphenoxy)acetyl)-phenyl)acetate (10d).

General procedure II was followed using 9d (1.08 g, 3.77 mmol), potassium iodide (750 mg, 4.52 mmol), 4-methoxyphenol (561 mg, 4.52 mmol), and potassium carbonate (1.04 g, 7.53 mmol) in acetone (23 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 20–90% EtOAc/hexanes gradient) to afford the title compound as a white solid (503 mg, 36%). R_f (1:1 EtOAC/Hex): 0.45; ¹H NMR (500 MHz, CDCl₃) δ 7.36 (s, 1H), 6.89–6.78 (m, 4H), 6.75 (s, 1H), 5.08 (s, 2H), 3.93 (s, 3H), 3.91 (s, 3H), 3.90 (s, 2H), 3.75 (s, 3H), 3.67 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 196.1, 172.0, 154.4, 152.3, 152.2, 147.4, 130.2, 126.4, 115.9, 115.5, 114.7, 112.6, 72.6, 56.3, 56.1, 55.7, 52.0, 39.9. HRMS calcd for C₂₀H₂₃O₇, 375.14383 [M + H]⁺; found 375.14387 [M + H]⁺.

(E/Z)-2-(3-Chlorobenzyl)-6-isopropoxy-1-((4-methoxyphenoxy)-methylene)-1,4-dihydroisoquinolin-3(2H)-one (11a).

General procedure III was followed using compound 10a (148 mg, 0.397 mmol), 3-chlorobenzylamine (58 μL, 0.48 mmol), tetraisopropoxytitanium (362 μL, 1.19 mmol), and sodium triacetoxyborohydride (253 mg, 1.19 mmol) in DCE (2.0 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and an orange oil (130 mg, 71%). HRMS calcd for C₂₇H₂₇O₄NCl, 464.16231 [M + H]⁺; found 464.16224 [M + H]⁺.

(E/Z)-6-Isopropoxy-1-((4-methoxyphenoxy)methylene)-2-(3-(trifluoromethyl)benzyl)-1,4-dihydroisoquinolin-3(2H)-one (11b).

General procedure III was followed using compound 10a (75 mg, 0.22 mmol), 3-(trifluoromethyl)benzylamine (37 μL, 0.26 mmol), tetraisopropoxytitanium (198 μL, 0.653 mmol), and sodium triacetoxyborohydride (138 mg, 0.653 mmol) in DCE (1.1 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a yellow oil (45 mg, 45%). HRMS calcd for C₂₈H₂₉O₄NF₃, 500.20432 [M + H]⁺; found 500.20464 [M + H]⁺.

(E/Z)-2-(3-Fluorobenzyl)-6-isopropoxy-1-((4-methoxyphenoxy)-methylene)-1,4-dihydroisoquinolin-3(2H)-one (11c).

General procedure III was followed using compound 10a (50 mg, 0.13 mmol), 3-fluorobenzylamine (18 μL, 0.16 mmol), tetraisopropoxytitanium (122 μL, 0.403 mmol), and sodium triacetoxyborohydride (85 mg, 0.40 mmol) in DCE (0.7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and an orange oil (35 mg, 58%). HRMS calcd for C₂₇H₂₇O₄NF, 448.19186 [M + H]⁺; found 448.19096 [M + H]⁺.

(E/Z)-2-(3-Chlorobenzyl)-6-isobutoxy-1-((4-methoxyphenoxy)-methylene)-1,4-dihydroisoquinolin-3(2H)-one (11d).

General procedure III was followed using compound 10b (500 mg, 1.29 mmol), 3-chlorobenzylamine (190 μL, 1.55 mmol), tetraisopropoxytitanium (1.18 mL, 3.88 mmol), and sodium triacetoxyborohydride (823 mg, 3.88 mmol) in DCE (6.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–25% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and an orange oil (450 mg, 73%). HRMS calcd for C₂₈H₂₉O₄NCl, 478.17796 [M + H]⁺; found 478.17837 [M + H]⁺.

(E/Z)-2-(3-Chlorobenzyl)-6-methoxy-1-((4-methoxyphenoxy)-methylene)-1,4-dihydroisoquinolin-3(2H)-one (11e).

General procedure III was followed using compound 10c (100 mg, 0.290 mmol), 3-chlorobenzylamine (43 μL, 0.35 mmol), tetraisopropoxytitanium (260 μL, 0.87 mmol), and sodium triacetoxyborohydride (185 mg, 0.871 mmol) in DCE (1.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as an ~1:2 mixture of E/Z isomers and an orange oil (74 mg, 59%). HRMS calcd for C₂₅H₂₃O₄NCl, 436.13101 [M + H]⁺; found 436.13017 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 2.68 min). Method 2: 85–95% ACN in water over 6 min at 1 mL/min (retention time = 2.32 min).

(E/Z)-6-Methoxy-1-((4-methoxyphenoxy)methylene)-2-(4,4,4-trifluorobutyl)-1,2-dihydroisoquinolin-3(4H)-one (11f).

General procedure III was followed using compound 10c (100 mg, 0.290 mmol), 4,4,4-trifluorobutan-1-amine (44 mg, 0.35 mmol), tetraisopropoxytitanium (260 μL, 0.87 mmol), and sodium triacetoxyborohydride (185 mg, 0.871 mmol) in DCE (1.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and an orange oil (42 mg, 34%). HRMS calcd for C₂₂H₂₃O₄NF₃, 422.15737 [M + H]⁺; found 422.15750 [M + H]⁺.

(E/Z)-6-Methoxy-1-((4-methoxyphenoxy)methylene)-2-(2,2,2-trifluoroethyl)-1,2-dihydroisoquinolin-3(4H)-one (11g).

General procedure III was followed using compound 10c (150 mg, 0.436 mmol), 2,2,2-trifluoroethanamine (69 μL, 0.87 mmol, 2 equiv), tetraisopropoxytitanium (400 μL, 1.3 mmol), and sodium triacetoxyborohydride (277 mg, 1.31 mmol) in DCE (2 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a yellow oil (33 mg, 19%). HRMS calcd for C₂₀H₁₉O₄NF₃, 394.12607 [M + H]⁺; found 394.12617 [M + H]⁺.

E/Z)-6-Methoxy-1-((4-methoxyphenoxy)methylene)-2-propyl-1,4-dihydroisoquinolin-3(2H)-one (11h).

General procedure III was followed using compound 10c (75 mg, 0.22 mmol), propylamine (21 μL, 0.26 mmol), tetraisopropoxytitanium (198 μL, 0.653 mmol), and sodium triacetoxyborohydride (138 mg, 0.653 mmol) in DCE (1.1 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–40% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a brown oil (31 mg, 40%). HRMS calcd for C₂₁H₂₈O₄N, 358.20128 [M + H]⁺; found 358.20118 [M + H]⁺.

(E/Z)-2-(2-(1H-Indol-3-yl)ethyl)-6-methoxy-1-((4-methoxyphenoxy)methylene)-1,4-dihydroisoquinolin-3(2H)-one (11i).

General procedure III was followed using compound 10c (75 mg, 0.22 mmol), tryptamine (42 mg, 0.26 mmol), tetraisopropoxytitanium (198 μL, 0.653 mmol), and sodium triacetoxyborohydride (138 mg, 0.653 mmol) in DCE (1.1 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a red oil (20 mg, 40%). HRMS calcd for C₂₈H₂₇O₄N₂, 455.19653 [M + H]⁺; found 455.19586 [M + H]⁺.

(E/Z)-6-Methoxy-2-(2-(2-methoxyethoxy)ethyl)-1-((4-methoxyphenoxy)methylene)-1,4-dihydroisoquinolin-3(2H)-one (11j).

General procedure III was followed using compound 10c (60 mg, 0.18 mmol), 2-(2-methoxyethoxy)ethanamine (25 mg, 0.21 mmol), tetraisopropoxytitanium (159 μL, 0.524 mmol), and sodium triacetoxyborohydride (111 mg, 0.524 mmol) in DCE (0.9 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a red oil (14 mg, 19%). HRMS calcd for C₂₃H₂₈O₆N, 414.19111 [M + H]⁺; found 414.19038 [M + H]⁺.

(E/Z)-2-(3-(Dimethylamino)propyl)-6-methoxy-1-((4-methoxyphenoxy)methylene)-1,4-dihydroisoquinolin-3(2H)-one (11k).

General procedure III was followed using compound 10c (50 mg, 0.15 mmol), 3-(dimethylamino)-1-propylamine (37 μL, 0.29 mmol, 2 equiv), tetraisopropoxytitanium (132 μL, 0.436 mmol), and sodium triacetoxyborohydride (92 mg, 0.44 mmol) in DCE (0.7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–10% DCM/(1% NH₄OH in MeOH) gradient) to afford the title compound as a mixture of E/Z isomers and a red oil (13 mg, 23%). HRMS calcd for C₂₃H₂₉O₄N₂, 397.21218 [M + H]⁺; found 397.21243 [M + H]⁺.

(E/Z)-6-Methoxy-1-((4-methoxyphenoxy)methylene)-2-(prop-2-yn-1-yl)-1,4-dihydroisoquinolin-3(2H)-one (11l).

General procedure III was followed using compound 10c (200 mg, 0.581 mmol), propargylamine (112 μL, 1.74 mmol), tetraisopropoxytitanium (495 μL, 1.74 mmol), and sodium triacetoxyborohydride (369 mg, 1.74 mmol) in DCE (3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a single E/Z isomer and a white solid (160 mg, 80%). HRMS calcd for C₂₁H₂₀O₄N, 350.13868 [M + H]⁺; found 350.13837 [M + H]⁺.

2-((1H-1,2,3-Triazol-4-yl)methyl)-6-methoxy-1-((4-methoxyphenoxy)methylene)-1,4-dihydroisoquinolin-3(2H)-one (11m).

37% Formaldehyde in H₂O (0.17 mL, 2.2 mmol, 10 equiv) and acetic acid (0.018 mL, 0.32 mmol, 1.5 equiv) were dissolved in THF (0.2 mL) and stirred for 15 min. Sodium azide (21 mg, 0.32 mmol, 1.5 equiv) and 11l (75 mg, 0.22 mmol, 1 equiv) dissolved in THF (0.2 mL) were then added in succession and stirred for 10 min. Then, sodium ascorbate (9.0 mg, 0.045 mmol, 0.2 equiv) was added, followed by an aqueous CuSO₄ solution (200 mg/mL, 0.05 equiv), and the reaction was stirred at room temperature for 24 h. The reaction was then concentrated before being dissolved in EtOAc and washed with 1 M NaOH. The organic layer was extracted with EtOAc, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–10% DCM/(1% NH₄OH in MeOH) gradient) to afford the title compound as a single E/Z isomer and a yellow oil (24 mg, 29%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.46; ¹H NMR (399 MHz, CDCl₃) δ 7.51 (s, 1H), 7.20 (d, J = 8.5 Hz, 1H), 6.99–6.95 (m, 2H), 6.88–6.83 (m, 3H), 6.75 (dd, J = 8.5, 2.6 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.53 (s, 1H), 5.23 (s, 2H), 3.79 (s, 3H), 3.78 (s, 3H), 3.68 (s, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 169.5, 159.5, 156.0, 150.7, 132.1, 130.8, 130.8, 123.8, 123.7, 121.9, 117.8, 114.9, 114.8, 113.2, 112.1, 55.7, 55.4, 40.0, 39.0. HRMS calcd for C₂₁H₂₁O₄N₄, 393.15573 [M + H]⁺; found 393.15487 [M + H]⁺.

(E/Z)-2-(3-Chlorobenzyl)-6,7-dimethoxy-1-((4-methoxyphenoxy)methylene)-1,4-dihydroisoquinolin-3(2H)-one (11n).

General procedure III was followed using compound 10d (200 mg, 0.534 mmol), 3-chlorobenzylamine (78 μL, 0.64 mmol), tetraisopropoxytitanium (486 μL, 1.60 mmol), and sodium triacetoxyborohydride (340 mg, 1.60 mmol) in DCE (2.7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a white solid (150 mg, 59%). HRMS calcd for C₂₆H₂₅O₅NCl, 466.14158 [M + H]⁺; found 466.14082 [M + H]⁺.

2-(3-Chlorobenzyl)-6-isopropoxy-1-((4-methoxyphenoxy)-methyl)-1,4-dihydroisoquinolin-3(2H)-one (12a).

General procedure IV-A was followed using compound 11a (150 mg, 0.323 mmol) and PtO₂ (19 mg, 0.046 mmol) in ethanol (2.6 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as an orange oil (15 mg, 10%). R_f (1:1 EtOAc/Hex): 0.59; ¹H NMR (500 MHz, CDCl₃) δ 7.22–7.15 (m, 3H), 7.09–7.05 (m, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.81–6.69 (m, 6H), 5.46 (d, J = 15.4 Hz, 1H), 4.63–4.59 (m, 1H), 4.54 (hept, J = 6.1 Hz, 1H), 4.37 (d, J = 15.4 Hz, 1H), 4.08–3.99 (m, 2H), 3.91 (d, J = 19.3 Hz, 1H), 3.75 (s, 3H), 3.64 (d, J = 19.3 Hz, 1H), 1.33 (d, J = 6.1 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 169.7, 157.8, 154.3, 152.2, 139.2, 134.5, 134.1, 130.0, 128.0, 127.6, 127.1, 125.8, 124.3, 115.4, 114.7, 114.4, 114.3, 71.7, 70.0, 59.9, 55.7, 48.6, 37.7, 22.1. HRMS calcd for C₂₇H₂₉O₄NCl, 466.17796 [M + H]⁺; found 466.17791 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 90–95% MeOH in water over 5 min at 1 mL/min (retention time = 2.56 min). Method 2: 85–95% ACN in water over 6 min at 1 mL/min (retention time = 3.00 min).

6-Isopropoxy-1-((4-methoxyphenoxy)methyl)-2-(3-(trifluoromethyl)benzyl)-1,4-dihydroisoquinolin-3(2H)-one (12b).

General procedure IV-B was followed using compound 11b (45 mg, 0.090 mmol) and 10% Pd/C (14 mg, 0.014 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a clear oil (32 mg, 71%). R_f (1:1 EtOAc/Hex): 0.65; ¹H NMR (500 MHz, CDCl₃) δ 7.51–7.44 (m, 2H), 7.39–7.34 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 6.82–6.77 (m, 2H), 6.76–6.73 (m, 1H), 6.73–6.66 (m, 3H), 5.52 (d, J = 15.6 Hz, 1H), 4.65–4.59 (m, 1H), 4.58–4.51 (m, 1H), 4.48 (d, J = 15.5 Hz, 1H), 4.10–4.00 (m, 2H), 3.93 (d, J = 19.2 Hz, 1H), 3.75 (s, 3H), 3.66 (d, J = 19.3 Hz, 1H), 1.33 (d, J = 6.1 Hz, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 169.8, 157.9, 154.3, 152.1, 138.2, 134.0, 130.9, 130.9 (q, J = 32.0 Hz, ²J), 129.2, 127.1, 124.5 (q, J = 3.8 Hz, ³J), 124.3 (q, J = 3.9 Hz, ³J), 124.2, 124.0 (q, J = 272.5 Hz, ¹J), 115.4, 114.7, 114.5, 114.3, 71.7, 70.0, 60.1, 55.7, 48.9, 37.7, 22.0. HRMS calcd for C₂₈H₂₉O₄NF₃, 500.20432 [M + H]⁺; found 500.20333 [M + H]⁺ Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 3.54 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 1.05 min).

2-(3-Fluorobenzyl)-6-isopropoxy-1-((4-methoxyphenoxy)-methyl)-1,4-dihydroisoquinolin-3(2H)-one (12c).

General procedure IV-B was followed using compound 11c (104 mg, 0.232 mmol) and 10% Pd/C (37 mg, 0.035 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (67 mg, 64%). R_f (1:1 EtOAc/Hex): 0.57; ¹H NMR (400 MHz, CDCl₃) δ 7.25–7.19 (m, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 7.7 Hz, 1H), 6.94–6.88 (m, 2H), 6.81–6.69 (m, 6H), 5.49 (d, J = 15.5 Hz, 1H), 4.65–4.60 (m, 1H), 4.54 (hept, J = 6.0 Hz, 1H), 4.39 (d, J = 15.5 Hz, 1H), 4.10–3.99 (m, 2H), 3.92 (d, J = 19.2 Hz, 1H), 3.75 (s, 3H), 3.65 (d, J = 19.3 Hz, 1H), 1.34 (d, J = 6.1 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 169.7, 163.0 (d, J = 246.5 Hz, ¹J), 157.8, 154.2, 152.2, 139.7 (d, J = 7.0 Hz, ³J), 134.1, 130.1 (d, J = 8.2 Hz, ³J), 127.1, 124.3, 123.2 (d, J = 2.8 Hz, ⁴J), 115.4, 114.6, 114.6 (d, J = 21.6 Hz, ²J), 114.4, 114.3 (d, J = 21.3 Hz, ²J), 114.2, 71.6, 69.9, 59.9, 55.7, 48.6, 37.6, 22.0. HRMS calcd for C₂₇H₂₉O₄NF, 450.20751 [M + H]⁺; found 450.20731 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 90–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.54 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 1.01 min).

2-(3-Chlorobenzyl)-6-isobutoxy-1-((4-methoxyphenoxy)methyl)-1,4-dihydroisoquinolin-3(2H)-one (12d).

General procedure IV-A was followed using compound 11d (200 mg, 0.418 mmol) and PtO₂ (24 mg, 0.059 mmol) in ethanol (3.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a red foam (82 mg, 54%). R_f (1:1 EtOAc/Hex): 0.76; ¹H NMR (400 MHz, CDCl₃) δ 7.22–7.14 (m, 3H), 7.09–7.03 (m, 2H), 6.81–6.69 (m, 6H), 5.46 (d, J = 15.5 Hz, 1H), 4.62 (t, J = 5.0 Hz, 1H), 4.37 (d, J = 15.5 Hz, 1H), 4.09–3.99 (m, 2H), 3.94 (d, J = 19.3 Hz, 1H), 3.75 (s, 3H), 3.71 (d, J = 6.5 Hz, 2H), 3.66 (d, J = 19.3 Hz, 1H), 2.08 (hept, J = 6.9 Hz, 1H), 1.02 (d, J = 6.7 Hz, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 169.7, 159.1, 154.2, 152.2, 139.2, 134.5, 134.0, 129.9, 127.9, 127.6, 127.0, 125.7, 124.3, 115.4, 114.7, 113.3, 113.0, 74.5, 71.6, 59.9, 55.7, 48.6, 37.6, 28.2, 19.2. HRMS calcd for C₂₈H₃₁O₄NCl, 480.19361 [M + H]⁺; found 480.19404 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 2.05 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.86 min).

2-(3-Chlorobenzyl)-6-methoxy-1-((4-methoxyphenoxy)methyl)-1,2-dihydroisoquinolin-3(4H)-one (12e).

General procedure IV-A was followed using compound 11e (113 mg, 0.259 mmol) and PtO₂ (8.4 mg, 0.037 mmol) in ethanol (2 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a clear oil (63 mg, 56%). R_f (1:1 EtOAc/Hex): 0.59; ¹H NMR (400 MHz, CDCl₃) δ 7.22–7.11 (m, 3H), 7.09–7.02 (m, 2H), 6.82–6.67 (m, 6H), 5.44 (d, J = 15.5 Hz, 1H), 4.62 (t, J = 5.0 Hz, 1H), 4.37 (d, J = 15.5 Hz, 1H), 4.08–3.99 (m, 2H), 3.94 (d, J = 19.1 Hz, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.66 (d, J = 19.2 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 169.6, 159.4, 154.2, 152.1, 139.2, 134.4, 134.1, 129.9, 127.8, 127.6, 127.1, 125.7, 124.6, 115.4, 114.6, 112.8, 112.3, 71.5, 59.9, 55.7, 55.3, 48.6, 37.6. HRMS calcd for C₂₅H₂₅O₄NCl, 438.14666 [M + H]⁺; found 438.14644 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.83 min).

6-Methoxy-1-((4-methoxyphenoxy)methyl)-2-(4,4,4-trifluorobutyl)-1,2-dihydroisoquinolin-3(4H)-one (12f).

General procedure IV-B was followed using compound 11f (79 mg, 0.19 mmol) and 10% Pd/C (30 mg, 0.028 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a clear oil (18 mg, 23%). R_f (1:1 EtOAc/Hex): 0.47; ¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J = 8.4 Hz, 1H), 6.83 (d, J = 2.6 Hz, 1H), 6.82–6.69 (m, 5H), 4.68 (t, J = 5.4 Hz, 1H), 4.09–3.98 (m, 2H), 3.81 (d, J = 19.3 Hz, 1H), 3.81 (s, 3H), 3.74 (s, 3H), 3.56 (d, J = 19.3 Hz, 1H), 3.37–3.26 (m, 2H), 2.18–2.05 (m, 2H), 1.94–1.83 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 169.5, 159.6, 154.3, 152.2, 134.2, 127.1, 127.0 (q, J = 276.2 Hz, ¹J), 124.6, 115.4, 114.7, 112.9, 112.4, 71.7, 61.2, 55.7, 55.4, 45.7, 37.7, 31.3 (q, J = 29.1 Hz, ²J), 20.5 (q, J = 2.6 Hz, ³J). HRMS calcd for C₂₂H₂₅O₄NF₃, 424.17302 [M + H]⁺; found 424.17256 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 6 min at 1 mL/min (retention time = 4.69 min). Method 2: 85–95% ACN in water over 6 min at 1 mL/min (retention time = 1.48 min).

6-Methoxy-1-((4-methoxyphenoxy)methyl)-2-(2,2,2-trifluoroethyl)-1,2-dihydroisoquinolin-3(4H)-one (12g).

General procedure IV-B was followed using compound 11g (33 mg, 0.084 mmol) and 10% Pd/C (13 mg, 0.013 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–30% EtOAc/hexanes gradient) to afford the title compound as a clear oil (8.8 mg, 27%). R_f (1:1 EtOAc/Hex): 0.60; ¹H NMR (400 MHz, CDCl₃) δ 7.19 (d, J = 8.4 Hz, 1H), 6.84 (dd, J = 8.6, 2.8 Hz, 1H), 6.81–6.72 (m, 4H), 6.72 (d, J = 2.3 Hz, 1H), 5.07–4.96 (m, 1H), 4.94 (dd, J = 7.6, 4.0 Hz, 1H), 4.09 (dd, J = 9.7, 3.8 Hz, 1H), 3.99 (dd, J = 9.7, 7.8 Hz, 1H), 3.96–3.82 (m, 2H), 3.81 (s, 3H), 3.75 (s, 3H), 3.68 (d, J = 19.6 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 169.9, 159.7, 154.4, 151.9, 133.6, 127.3, 126.7 (q, J = 273.5 Hz, ¹J), 123.6, 115.3, 114.7, 113.1, 112.6, 72.1, 61.3, 55.7, 55.4, 46.3 (q, J = 33.6 Hz, ²J), 37.4. HRMS calcd for C₂₀H₂₁O₄NF₃, 396.14172 [M + H]⁺; found 396.14143 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 10 min at 1 mL/min (retention time = 1.21 min). Method 2: 75–95% MeOH in water over 6 min at 1 mL/min (retention time = 3.78 min).

6-Methoxy-1-((4-methoxyphenoxy)methyl)-2-propyl-1,4-dihydroisoquinolin-3(2H)-one (12h).

General procedure IV-B was followed using compound 11h (31 mg, 0.088 mmol) and 10% Pd/C (14 mg, 0.013 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a clear oil (15 mg, 48%). R_f (1:1 EtOAc/Hex): 0.28; ¹H NMR (500 MHz, CDCl₃) δ 7.19 (d, J = 8.4 Hz, 1H), 6.83–6.68 (m, 6H), 4.68 (t, J = 5.3 Hz, 1H), 4.16–4.00 (m, 3H), 3.84 (d, J = 19.2 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.56 (d, J = 19.2 Hz, 1H), 3.14–3.06 (m, 1H), 1.70–1.55 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.2, 159.4, 154.2, 152.4, 134.5, 127.4, 125.1, 115.5, 114.7, 112.7, 112.3, 71.4, 60.9, 55.7, 55.4, 48.3, 37.8, 21.0, 11.4.NMR (126 MHz, CDCl₃) δ 169.3, 159.4, 154.2, 152.3, 134.3, 127.1, 124.6, 115.4, 114.7, 112.8, 112.2, 71.5, 63.1, 55.7, 55.4, 37.3, 34.7. HRMS calcd for C₂₁H₂₆O₄N, 356.18563 [M + H]⁺; found 356.18553 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.35 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 0.81 min).

2-(2-(1H-Indol-3-yl)ethyl)-6-methoxy-1-((4-methoxyphenoxy)-methyl)-1,4-dihydroisoquinolin-3(2H)-one (12i).

A modified version of general procedure IV-B was followed using compound 11i (21 mg, 0.046 mmol), 10% Pd/C (7.3 mg, 0.0069 mmol), and AcOH (catalytic) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as a clear oil (10 mg, 45%). R_f (1:1 EtOAc/Hex): 0.17; ¹H NMR (500 MHz, CDCl₃) δ 7.88 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 7.3 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.89–6.83 (m, 2H), 6.78–6.62 (m, 6H), 4.42–4.33 (m, 2H), 3.94–3.89 (m, 2H), 3.84 (d, J = 19.2 Hz, 1H), 3.81 (s, 3H), 3.73 (s, 3H), 3.58 (d, J = 19.0 Hz, 1H), 3.53–3.43 (m, 1H), 3.08 (t, J = 7.2 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 169.3, 159.3, 154.1, 152.3, 136.2, 134.4, 127.3, 127.0, 125.2, 122.2, 122.0, 119.4, 118.8, 115.4, 114.6, 113.1, 112.6, 112.1, 111.1, 71.4, 61.9, 55.7, 55.4, 48.0, 37.9, 23.7. HRMS calcd for C₂₈H₂₉O₄N₂, 457.21218 [M + H]⁺; found 457.21230 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 2.29 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.94 min).

6-Methoxy-2-(2-(2-methoxyethoxy)ethyl)-1-((4-methoxyphenoxy)methyl)-1,4-dihydroisoquinolin-3(2H)-one (12j).

General procedure IV-B was followed using compound 11j (16 mg, 0.039 mmol) and 10% Pd/C (6.2 mg, 0.0059 mmol) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a red oil (10 mg, 83%). R_f (1:1 EtOAc/Hex): 0.44; ¹H NMR (500 MHz, CDCl₃) δ 7.16 (d, J = 8.4 Hz, 1H), 6.83–6.66 (m, 6H), 4.93 (t, J = 5.0 Hz, 1H), 4.18–4.01 (m, 3H), 3.84 (d, J = 18.2 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.73–3.68 (m, 1H), 3.65–3.59 (m, 1H), 3.59–3.51 (m, 3H), 3.51–3.45 (m, 1H), 3.44–3.40 (m, 2H), 3.31 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.6, 159.3, 154.1, 152.5, 134.4, 127.2, 125.4, 115.4, 114.6, 112.6, 112.2, 71.8, 71.4, 70.4, 70.0, 62.3, 59.0, 55.7, 55.4, 46.9, 37.8. HRMS calcd for C₂₃H₃₀O₆N, 416.20676 [M + H]⁺; found 416.20700 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 6 min at 1 mL/min (retention time = 3.58 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.86 min).

2-(3-(Dimethylamino)propyl)-6-methoxy-1-((4-methoxyphenoxy)methyl)-1,4-dihydroisoquinolin-3(2H)-one (12k).

A modified version of general procedure IV-B was followed using compound 11k (16 mg, 0.039 mmol), 10% Pd/C (6.2 mg, 0.0059 mmol), and AcOH (catalytic) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a red oil (10 mg, 83%). R_f (1:1 EtOAc/Hex): 0.40; ¹H NMR (500 MHz, CDCl₃) δ 7.16 (d, J = 8.4 Hz, 1H), 6.83–6.66 (m, 6H), 4.93 (t, J = 5.0 Hz, 1H), 4.18–4.01 (m, 3H), 3.84 (d, J = 18.2 Hz, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.73–3.68 (m, 1H), 3.65–3.59 (m, 1H), 3.59–3.51 (m, 3H), 3.51–3.45 (m, 1H), 3.44–3.40 (m, 2H), 3.31 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.6, 159.3, 154.1, 152.5, 134.4, 127.2, 125.4, 115.4, 114.6, 112.6, 112.2, 71.8, 71.4, 70.4, 70.0, 62.3, 59.0, 55.7, 55.4, 46.9, 37.8. HRMS calcd for C₂₃H₃₀O₄N₂, 399.22783 [M + H]⁺; found 399.22743 [M + H]⁺. Purity was established to be 85% pure using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 50–95% MeOH in water over 6 min at 1 mL/min (retention time = 5.56 min). Method 2: 75–95% MeOH in water over 6 min at 1 mL/min (retention time = 1.18 min).

2-((1H-1,2,3-Triazol-4-yl)methyl)-6-methoxy-1-((4-methoxyphenoxy)methyl)-1,4-dihydroisoquinolin-3(2H)-one (12l).

A modified version of general procedure IV-B was followed using compound 11m (22 mg, 0.056 mmol), 10% Pd/C (8.9 mg, 0.0083 mmol), and AcOH (catalytic) in ethanol (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (15 mg, 68%). R_f (9:1 DCM/MeOH): 0.43; ¹H NMR (400 MHz, CDCl₃) δ 7.66 (s, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.80–6.65 (m, 6H), 5.27 (d, J = 15.3 Hz, 1H), 4.87 (t, J = 4.8 Hz, 1H), 4.68 (d, J = 15.2 Hz, 1H), 4.16–4.00 (m, 2H), 3.91 (d, J = 19.3 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 3H), 3.60 (d, J = 19.4 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 170.1, 159.4, 154.2, 152.2, 133.7, 127.1, 124.3, 115.4, 114.6, 112.9, 112.2, 71.6, 61.1, 55.7, 55.4, 41.1, 37.5. HRMS calcd for C₂₁H₂₃O₄N₄, 395.17138 [M + H]⁺; found 395.17077 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 6 min at 1 mL/min (retention time = 1.78 min). Method 2: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.53 min).

2-(3-Chlorobenzyl)-6,7-dimethoxy-1-((4-methoxyphenoxy)-methyl)-1,4-dihydroisoquinolin-3(2H)-one (12m).

General procedure IV-A was followed using compound 11n (136 mg, 0.292 mmol) and PtO₂ (17 mg, 0.041 mmol) in ethanol (2.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a clear oil (30 mg, 22%). R_f (1:1 EtOAc/Hex): 0.28; ¹H NMR (300 MHz, CDCl₃) δ 7.24–7.16 (m, 3H), 7.12–7.06 (m, 1H), 6.82–6.63 (m, 6H), 5.49 (d, J = 15.5 Hz, 1H), 4.59 (t, J = 5.1 Hz, 1H), 4.37 (d, J = 15.5 Hz, 1H), 4.08–4.03 (m, 2H), 3.97–3.92 (m, 1H), 3.90 (s, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.75 (s, 3H), 3.73–3.59 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 169.8, 154.3, 152.2, 149.0, 147.8, 139.2, 134.5, 130.0, 127.9, 127.6, 125.8, 124.7, 124.2, 115.4, 114.7, 110.2, 109.0, 71.5, 60.1, 56.1, 56.0, 55.7, 48.5, 36.9. HRMS calcd for C₂₆H₂₇O₅NCl, 468.15723 [M + H]⁺; found 468.15734 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.45 min). Method 2: 85–95% ACN in water over 6 min at 1 mL/min (retention time = 1.34 min).

2-(3,4-Dimethoxyphenyl)ethanamine (14).

General procedure V was followed using compound 3,4-dimethoxyphenethylamine (13) (5.59 mL, 33.1 mmol), triethylamine (9.23 mL, 66.2 mmol), and 2-chloroacetyl chloride (3.16 mL, 39.7 mmol) in dry DCM (120 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 10–80% EtOAc/hexanes gradient) to afford the title compound as a white solid (6.32 g, 74%). R_f (1:1 EtOAc/Hex): 0.56; ¹H NMR (300 MHz, CDCl₃) δ 6.82 (d, J = 8.0 Hz, 1H), 6.77–6.69 (m, 2H), 6.64 (s, 0H), 4.02 (s, 2H), 3.87 (s, 3H), 3.86 (s, 3H), 3.53 (q, J = 6.9 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 165.7, 149.0, 147.8, 130.8, 120.6, 111.8, 111.3, 55.9, 42.7, 41.1, 35.1. HRMS calcd for C₁₂H₁₇O₃NCl, 258.08915 [M + H]⁺; found 258.08876 [M + H]⁺.

2-(Benzyloxy)-N-(3,4-dimethoxyphenethyl)acetamide (15a).

General procedure VII was followed using compound 14 (2.97 g, 11.5 mmol), benzyl alcohol (1.41 g, 13.0 mmol), and 60% sodium hydride dispersion in mineral oil (0.61 g, 15 mmol) in THF (28 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 30–100% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (2.7 g, 70%). R_f (1:1 EtOAC/Hex): 0.19; ¹H NMR (400 MHz, CDCl₃) δ 7.40–7.20 (m, 4H), 6.85–6.65 (m, 4H), 4.49 (s, 2H), 3.97 (s, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 3.54 (q, J = 6.8 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) δ 171.1, 169.4, 149.0, 147.6, 136.8, 131.1, 128.5, 128.1, 127.8, 120.6, 111.7, 111.2, 73.4, 69.5, 55.8, 40.0, 35.2. HRMS calcd for C₁₉H₂₄O₄N, 330.16998 [M + H]⁺; found 330.16945 [M + H]⁺.

N-(3,4-Dimethoxyphenethyl)-2-((tetrahydro-2H-pyran-4-yl)oxy)-acetamide (15b).

General procedure VII was followed using compound 14 (0.20 g, 0.78 mmol), 4-hydroxytetrahydropyran (95 mg, 0.93 mmol), and 60% sodium hydride dispersion in mineral oil (43 mg, 1.1 mmol) in THF (3.9 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–10% MeOH/DCM gradient) to afford the title compound as a white solid (0.19 g, 75%). R_f (9:1 DCM/MeOH): 0.62; ¹H NMR (500 MHz, chloroform-d) δ 6.79 (d, J = 7.9 Hz, 1H), 6.75–6.70 (m, 2H), 6.62 (s, 1H), 3.93 (s, 2H), 3.90–3.83 (m, 8H), 3.53 (q, J = 6.9 Hz, 2H), 3.47 (tt, J = 8.7, 4.0 Hz, 1H), 3.39 (ddd, J = 12.0, 9.7, 2.7 Hz, 2H), 2.78 (t, J = 7.1 Hz, 2H), 1.85–1.78 (m, 2H), 1.56–1.45 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 169.7, 149.1, 147.8, 131.1, 120.6, 111.8, 111.3, 75.0, 67.3, 65.3, 55.9, 55.9, 39.9, 35.2, 32.1. HRMS calcd for C₁₇H₂₆O₅N, 324.18055 [M + H]⁺; found 324.18056 [M + H]⁺.

N-(3,4-Dimethoxyphenethyl)-2-(pyridine-3-yloxy)acetamide (15c).

General procedure VI was followed using compound 14 (0.20 g, 0.78 mmol), 3-hydroxypyridine (89 mg, 0.93 mmol), and cesium carbonate (1.0 g, 3.1 mmol) in acetonitrile (4.6 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–10% MeOH/DCM gradient) to afford the title compound as a white solid (0.15 g, 60%). R_f (9:1 DCM/MeOH): 0.49; ¹H NMR (500 MHz, CDCl₃) δ 8.28–8.23 (m, 2H), 7.24–7.18 (m, 1H), 7.14–7.08 (m, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.70–6.63 (m, 3H), 4.47 (s, 2H), 3.83 (s, 3H), 3.81 (s, 3H), 3.57 (q, J = 6.9 Hz, 2H), 2.78 (t, J = 7.0 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 167.2, 153.4, 149.1, 147.8, 143.4, 138.3, 130.9, 124.0, 120.9, 120.7, 111.8, 111.4, 67.5, 55.9, 55.8, 40.2, 35.2. HRMS calcd for C₁₇H₂₁O₄N₂, 317.14958 [M + H]⁺; found 317.14920 [M + H]⁺.

N-(3,4-Dimethoxyphenethyl)-2-((4-methoxybenzyl)oxy)-acetamide (15d).

General procedure VII was followed using compound 14 (18.8 g, 73.0 mmol), 4-methoxybenzyl alcohol (12.1 g, 87.5 mmol), and 60% sodium hydride dispersion in mineral oil (4.38 g, 110. Mmol) in THF (196 mL). The crude material was purified via flash column chromatography (ISCO, Redisep 320 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as an orange solid (20.1 g, 74%). R_f (1:1 EtOAc/Hex): 0.67; ¹H NMR (500 MHz, CDCl₃) δ 7.22 (t, J = 7.8 Hz, 1H), 7.19–7.13 (m, 2H), 6.91–6.84 (m, 2H), 6.81–6.73 (m, 3H), 6.65 (s, 1H), 4.43 (s, 2H), 3.94 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H), 3.54 (q, J = 7.0 Hz, 2H), 2.80 (t, J = 7.1 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 169.6, 159.9, 159.6, 140.3, 129.6, 129.5, 128.9, 121.1, 114.4, 114.0, 112.0, 73.2, 69.3, 55.3, 55.2, 39.8, 35.7. HRMS calcd for C₂₀H₂₆O₅N, 360.18055 [M + H]⁺; found 360.18023 [M + H]⁺.

1-((Benzyloxy)methyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (16a).

General procedure VIII was followed using compound 15a (2.67 g, 8.11 mmol), phosphorous oxychloride (2.27 mL, 24.3 mmol), and sodium borohydride (0.64 g, 24 mmol) in acetonitrile (~40 mL) and MeOH (~10 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 10–90% EtOAc/hexanes gradient) to afford the title compound as a white foam (1.6 g, 62% over two steps). R_f (1:1 EtOAC/Hex): 0.29; ¹H NMR (300 MHz, CDCl₃) δ 7.38–7.26 (m, 5H), 6.58 (s, 2H), 4.68–4.50 (m, 2H), 4.17 (dd, J = 8.2, 3.9 Hz, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.75–3.62 (m, 2H), 3.25–3.10 (m, 1H), 3.04–2.90 (m, 1H), 2.74 (t, J = 5.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 147.6, 147.1, 138.0, 128.4, 127.8, 127.7, 127.7, 126.6, 111.8, 109.2, 73.3, 72.9, 55.9, 55.8, 55.0, 40.0, 29.0. HRMS calcd for C₁₉H₂₄O₃N, 314.17507 [M + H]⁺; found 314.17453 [M + H]⁺.

6,7-Dimethoxy-1-(((tetrahydro-2H-pyran-4-yl)oxy)methyl)-1,2,3,4-tetrahydroisoquinoline (16b).

General procedure VIII was followed using compound 15b (187 mg, 0.578 mmol), phosphorous oxychloride (0.27 mL, 2.9 mmol), and sodium borohydride (66 mg, 1.7 mmol) in acetonitrile (9.2 mL) and MeOH (2.4 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–10% MeOH/DCM gradient) to afford the title compound as a clear oil (29 mg, 16%). R_f (9:1 DCM/MeOH): 0.44; ¹H NMR (399 MHz, chloroform-d) δ 6.62 (s, 1H), 6.58 (s, 1H), 4.08 (dd, J = 8.8, 3.9 Hz, 1H), 3.98–3.87 (m, 2H), 3.83 (s, 3H), 3.82 (s, 3H), 3.72 (dd, J = 9.2, 4.0 Hz, 1H), 3.63–3.49 (m, 2H), 3.48–3.37 (m, 2H), 3.18 (dt, J = 11.7, 5.7 Hz, 1H), 2.96 (dt, J = 11.8, 5.9 Hz, 1H), 2.73 (t, J = 5.8 Hz, 2H), 2.40 (s, 1H), 1.97–1.85 (m, 2H), 1.69–1.52 (m, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 147.6, 147.0, 127.9, 127.2, 111.9, 109.3, 74.4, 70.9, 65.6, 56.0(0), 55.9(9), 55.8, 55.2, 40.2, 32.6, 32.3, 29.3. HRMS calcd for C₁₇H₂₆O₄N, 308.18563 [M + H]⁺; found 308.18552 [M + H]⁺.

6,7-Dimethoxy-1-((pyridin-3-yloxy)methyl)-1,2,3,4-tetrahydroisoquinoline (16c).

General procedure VIII was followed using compound 15c (0.20 g, 0.64 mmol), phosphorous oxychloride (0.30 mL, 3.2 mmol), and sodium borohydride (72 mg, 1.9 mmol) in acetonitrile (7.4 mL) and MeOH (1.9 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–20% MeOH/DCM gradient) to afford the title compound as a white solid (0.12 g, 60%). R_f (8:2 DCM/MeOH): 0.66; ¹H NMR (500 MHz, CDCl₃) δ 8.34 (dd, J = 2.6, 0.9 Hz, 1H), 8.21 (dd, J = 4.1, 1.9 Hz, 1H), 7.25–7.16 (m, 2H), 6.65 (s, 1H), 6.61 (s, 1H), 4.36 (dd, J = 7.9, 4.6 Hz, 1H), 4.21–4.12 (m, 2H), 3.84 (s, 4H), 3.82 (s, 3H), 3.19 (ddd, J = 12.2, 6.8, 5.4 Hz, 1H), 3.03 (dt, J = 12.1, 5.5 Hz, 1H), 2.75 (q, J = 5.3 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 148.0, 147.3, 142.4, 138.1, 128.3, 126.0, 123.9, 121.2, 112.1, 109.5, 71.2, 56.1, 55.8, 54.6, 39.8, 29.2. HRMS calcd for C₁₇H₂₁O₃N₂, 301.15467 [M + H]⁺; found 301.15442 [M + H]⁺.

(3-Chlorophenyl)(1-(hydroxymethyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methanone (17a).

General procedure IX was followed using compound 16a (1.56 g, 4.98 mmol), triethylamine (1.39 mL, 9.96 mmol), and 3-chlorobenzoyl chloride (0.77 mL, 6.0 mmol) in DCM (80 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 10–90% EtOAc/hexanes gradient) to afford the title compound as a white foam (1.4 g) that was carried forward without further characterization. This material (1.4 g, 3.0 mmol) was dissolved in a 2:3 mixture of chloroform (40 mL) and methane sulfonic acid (60 mL) and allowed to react at room temperature for 6 h. The reaction was then poured over ice, dilute with EtOAc, and quenched with saturated NaHCO₃. The organic layer was then washed with brine (3×), dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 10–90% EtOAc/hexanes gradient) to afford the title compound as a white foam (0.48 g, 44% over two steps). R_f (1:1 EtOAC/Hex): 0.07; ¹H NMR (400 MHz, CDCl₃) δ 7.60–6.97 (m, 4H), 6.78–6.38 (m, 2H), 5.75 (dd, J = 8.6, 3.7 Hz, 0.5H), 4.83–4.74 (m, 0.5H), 4.08 (dd, J = 11.6, 3.9 Hz, 1H), 3.93 (dd, J = 11.5, 8.9 Hz, 1H), 3.89–3.70 (m, 6H), 3.67 (dd, J = 11.8, 4.2 Hz, 0.5H), 3.57 (td, J = 13.5, 12.9, 4.0 Hz, 1H), 3.23 (td, J = 13.0, 4.2 Hz, 0.5H), 3.06 (td, J = 14.1, 12.2, 6.0 Hz, 0.5H), 2.92 (ddd, J = 17.1, 11.9, 5.8 Hz, 1H), 2.69 (t, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 171.1, 170.4, 148.3, 148.2, 147.8, 147.4, 138.0, 137.6, 134.6, 134.3, 130.1, 130.0, 129.7, 129.6, 129.6, 128.4, 128.0, 127.8, 127.0, 126.6, 125.7, 125.7, 124.9, 124.4, 124.3, 111.7, 111.3, 110.0, 109.5, 66.5, 65.8, 64.6, 64.6, 59.6, 56.0, 56.0, 55.9, 55.2, 42.1, 35.5, 28.8, 27.6, 15.3. HRMS calcd for C₁₉H₂₁O₄NCl, 362.11536 [M + H]⁺; found 362.11479 [M + H]⁺.

(1-(Hydroxymethyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (17b).

In step 1, a modified version of general procedure VIII was followed, in which the material was refluxed for only 1 h instead of overnight and only one equivalent of POCl₃ was used due to the loss of the PMB-protecting group and conversion to the alkyl chloride upon continued heating. This proceeded using compound 15d (11.1 g, 30.8 mmol), phosphorous oxychloride (2.87 mL, 30.8 mmol), and sodium borohydride (3.50 g, 92.5 mmol) in acetonitrile (493 mL) and MeOH (123 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–15% MeOH/DCM gradient) to afford crude 6,7-dimethoxy-1-(((4-methoxybenzyl)oxy)-methyl)-1,2,3,4-tetrahydroisoquinoline as a clear oil (4.71 g) that was carried forward without further characterization. General procedure IX was followed for step 2 using the above compound (4.71 g, 13.7 mmol), triethylamine (3.82 mL, 27.4 mmol), and 3-(trifluoromethyl)-benzoyl chloride (2.48 mL, 16.4 mmol) in DCM (214 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 80 g column, 0–100% EtOAc/hexanes gradient) to afford impure (6,7-dimethoxy-1-(((4-methoxybenzyl)oxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone as a clear oil (2.77 g) that was carried forward without further characterization. Finally, this compound (2.77 g, 5.37 mmol) was hydrogenated at 50 psi using 10% Pd/C (0.57 g, 0.54 mmol) in MeOH (32 mL) overnight. The reaction mixture was filtered through a pad of celite, washed with methanol, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white foam (1.01 g, 8% over three steps). ¹H NMR (500 MHz, CDCl₃) δ 7.84–7.54 (m, 4H), 6.76–6.37 (m, 2H), 5.77 (dd, J = 8.8, 3.6 Hz, 0.8H), 4.88–4.71 (m, 0.2H), 4.14–4.07 (m, 1H), 4.02–3.93 (m, 1H), 3.88 (s, 2.5H), 3.86 (s, 3.5H), 3.79–3.70 (m, 2H), 3.64–3.55 (m, 0.8H), 3.31–3.21 (m, 0.2H), 3.09 (ddd, J = 17.7, 12.5, 6.5 Hz, 0.2H), 2.94 (ddd, J = 17.0, 11.9, 5.7 Hz, 0.8H), 2.74–2.66 (m, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 171.4, 148.4, 148.1, 136.7, 130.2, 129.3, 126.8, 125.8, 124.3, 123.9, 111.5, 110.1, 66.9, 56.1, 55.9, 55.5, 42.3, 28.7. (Unable to discern between fluorine splitting and minor peaks; only major peaks reported; major CF₃ peak not reported.) HRMS calcd for C₂₀H₂₁O₄NF₃, 396.14172 [M + H]⁺; found 369.14133 [M + H]⁺.

(3-Chlorophenyl)(6,7-dimethoxy-1-(((5-methoxythiophen-2-yl)-oxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)methanone (18a).

Compound 17a (100 mg, 0.276 mmol), 2-iodo-5-methoxythiophene (73 mg, 0.30 mmol), 1,10-phenanthroline (10 mg, 0.055 mmol), copper(I) iodide (11 mg, 0.055 mmol), and cesium carbonate (0.18 g, 0.55 mmol) were charged to a 10 mL microwave vial. The vial was capped, purged with argon, evacuated three times, and toluene (0.5 mL) was added. The vial was heated to 80 °C and allowed to react for 24 h. Upon completion, the reaction was cooled to room temperature, filtered through a pad of celite, washed with EtOAc, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 10–90% EtOAc/hexanes gradient) to afford the title compound as a white foam (28 mg, 21%). R_f (1:1 EtOAC/Hex): 0.47; ¹H NMR (400 MHz, CDCl₃) δ 7.58–7.28 (m, 4H), 6.79–6.41 (m, 2H), 5.92 (dd, J = 13.0, 4.5 Hz, 1H), 5.77 (s, 1H), 5.05 (dd, J = 9.0, 3.2 Hz, 0.5H), 4.88 (dd, J = 13.1, 5.4 Hz, 0.5H), 4.38 (d, J = 5.1 Hz, 1H), 4.18 (t, J = 10.0 Hz, 0.5H) 4.01 (dd, J = 10.5, 4.0 Hz, 0.5H), 3.95–3.72 (m, 9H), 3.62 (td, J = 13.1, 3.6 Hz, 1H), 3.24 (td, J = 12.9, 3.7 Hz, 0.5H), 3.09 (td, J = 16.8, 5.9 Hz, 0.5H), 2.86 (ddd, J = 15.3, 13.1, 5.9 Hz, 0.5H), 2.76 (dd, J = 16.2, 3.0 Hz, 0.5H), 2.68 (d, J = 15.4 Hz, 0.5H). ¹³C NMR (101 MHz, CDCl₃) δ 170.1, 169.4, 168.4, 155.1, 155.0, 152.7, 152.0, 148.7, 148.5, 148.3, 147.9, 147.9, 147.6, 139.9, 137.9, 134.7, 134.6, 134.4, 130.0, 130.0, 129.9, 129.8, 129.8, 129.6, 127.7, 127.1, 126.8, 126.7, 126.1, 125.5, 124.7, 124.6, 124.3, 123.1, 116.4, 111.8, 111.4, 111.3, 109.9, 109.8, 109.5, 103.4, 102.8, 100.7, 77.2, 76.2, 74.8, 68.8, 65.9, 60.5, 60.4, 57.0, 56.0, 55.9, 51.8, 51.5, 51.1, 42.7, 42.2, 35.5, 28.9, 28.8, 27.6, 15.3. HRMS calcd for C₂₄H₂₅O₅NClS, 474.11365 [M + H]⁺; found 474.11362 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.74 min). Method 2: 75–95% ACN in water over 5 min at 1 mL/min (retention time = 3.56 min).

(6,7-Dimethoxy-1-((pyridin-2-yloxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (18b).

General procedure X was followed using compound 17b (25 mg, 0.063 mmol), 2-fluoropyrimidine (8.2 μL, 0.095 mmol), and 60% sodium hydride dispersion in mineral oil (4.6 mg, 0.19 mmol) in THF (0.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white foam (8.0 mg, 27%). R_f (1:1 EtOAC/Hex): 0.40; ¹H NMR (600 MHz, CDCl₃) δ 8.08 (d, J = 3.7 Hz, 0.5H), 7.91 (d, J = 4.1 Hz, 0.5H), 7.67 (s, 0.5H), 7.59 (d, J = 7.6 Hz, 0.5H), 7.52 (t, J = 7.1 Hz, 1.5H), 7.45 (t, J = 8.4 Hz, 1H), 7.40–7.35 (m, 1H), 7.33 (t, J = 7.6 Hz, 0.5H), 6.86–6.76 (m, 1.5H), 6.71 (d, J = 8.3 Hz, 0.5H), 6.66 (d, J = 8.3 Hz, 0.5H), 6.61 (s, 0.5H), 6.54 (s, 1H), 6.06–6.01 (m, 0.5H), 5.14–5.09 (m, 0.5H), 4.89–4.78 (m, 1H), 4.63 (dd, J = 11.2, 4.2 Hz, 0.5H), 4.50 (t, J = 10.8 Hz, 0.5H), 4.39 (dd, J = 11.8, 3.4 Hz, 0.5H), 3.83 (s, 1.5H), 3.81 (s, 1.5H), 3.79 (s, 1.5H), 3.75 (s, 1.5H), 3.71–3.63 (m, 0.5H), 3.63–3.57 (m, 0.5H), 3.29 (td, J = 12.9, 4.2 Hz, 0.5H), 3.06 (ddd, J = 17.7, 12.6, 6.2 Hz, 0.5H), 2.83–2.70 (m, 1H), 2.60 (d, J = 15.2 Hz, 0.5H). ¹³C NMR (151 MHz, CDCl₃) δ 170.0, 169.3, 163.4, 162.9, 148.7, 148.3, 147.9, 147.6, 146.6, 146.5, 139.0, 137.3, 137.0, 131.2, 130.9, 130.9, 130.6, 130.5, 129.8, 129.2, 128.7, 127.0, 126.2, 126.1, 126.0, 125.8, 124.7, 124.7, 123.9, 123.5, 117.3, 117.2, 111.8, 111.4, 111.4, 111.1, 110.4, 109.8, 66.8, 66.6, 57.0, 56.1, 55.9, 51.7, 42.0, 35.5, 28.9, 27.7. (Unable to discern between fluorine splitting and minor peaks, all peaks reported as seen.) HRMS calcd for C₂₅H₂₄O₄N₂F₃, 473.16827 [M + H]⁺; found 473.16871 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 50–95% MeOH in water over 6 min at 1 mL/min (retention time = 4.27 min). Method 2: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.10 min).

(6,7-Dimethoxy-1-((pyrimidin-2-yloxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (18c).

General procedure X was followed using compound 17b (25 mg, 0.063 mmol), 2-chloropyrimidine (11 mg, 0.095 mmol), and 60% sodium hydride dispersion in mineral oil (3.1 mg, 0.13 mmol) in DMF (0.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a clear oil (14 mg, 47%). R_f (9:1 DCM/MeOH): 0.67; ¹H NMR (600 MHz, CDCl₃) δ 8.45 (d, J = 4.5 Hz, 1H), 8.37 (d, J = 4.6 Hz, 1H), 7.66–7.29 (m, 4H), 6.90–6.82 (m, 1.5H), 6.62 (s, 0.5H), 6.54 (d, J = 7.0 Hz, 1H), 6.07–6.02 (m, 0.5H), 5.11 (dd, J = 9.2, 3.3 Hz, 0.5H), 4.87 (dd, J = 11.4, 4.2 Hz, 0.5H), 4.81 (dd, J = 13.1, 6.0 Hz, 0.5H), 4.71 (dd, J = 11.3, 6.8 Hz, 0.5H), 4.61–4.54 (m, 0.5H), 4.45 (dd, J = 11.8, 3.8 Hz, 0.5H), 3.84 (s, 1.5H), 3.82 (s, 1.5H), 3.79 (s, 1.5H), 3.76 (s, 1.5H), 3.72–3.67 (m, 1H), 3.34–3.27 (m, 0.5H), 3.06 (ddd, J = 17.5, 12.5, 6.3 Hz, 0.5H), 2.81 (ddd, J = 17.4, 10.8, 6.9 Hz, 0.5H), 2.77–2.71 (m, 0.5H), 2.65 (d, J = 16.1 Hz, 0.5H). ¹³C NMR (151 MHz, CDCl₃) δ 170.0, 169.4, 165.0, 164.6, 159.3, 148.8, 148.3, 147.9, 147.7, 137.2, 136.9, 131.2, 131.0, 130.9, 130.8, 130.7, 123.0, 129.2, 128.8, 127.2, 126.4, 126.3, 126.0, 125.9, 124.6, 124.5, 123.7, 123.5, 122.8, 121.0, 115.5, 115.3, 111.8, 111.4, 110.2, 109.8, 68.7, 67.9, 56.6, 56.1, 55.9, 51.2, 42.5, 35.6, 28.9, 27.7 (Unable to discern between fluorine splitting and minor peaks, all peaks reported as seen.) HRMS calcd for C₂₄H₂₃O₄N₃F₃, 474.16325 [M + H]⁺; found 474.16477 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 50–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.84 min). Method 2: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 0.82 min).

(6,7-Dimethoxy-1-(((tetrahydro-2H-pyran-4-yl)oxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (18d).

General procedure IX was followed using compound 16b (29 mg, 0.094 mmol), triethylamine (26 μL, 0.19 mmol), and 3-(trifluoromethyl)benzoyl chloride (17 μL, 0.11 mmol) in DCM (1.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white foam (45 mg, quant.). R_f (1:1 EtOAC/Hex): 0.62; ¹H NMR (500 MHz, chloroform-d) δ 7.85 (s, 0.5H), 7.71–7.45 (m, 3.5H), 6.79 (s, 0.5H), 6.64 (s, 0.5H), 6.60 (s, 0.5H), 6.41 (s, 0.5H), 5.77 (t, J = 5.5 Hz, 0.5H), 4.83 (ddd, J = 13.8, 11.3, 5.1 Hz, 1H), 3.95–3.83 (m, 7.5H), 3.77 (s, 1.5H), 3.74–3.53 (m, 2.5H), 3.51–3.36 (m, 2.5H), 3.22 (td, J = 12.7, 4.2 Hz, 0.5H), 3.09 (td, J = 14.4, 12.3, 6.2 Hz, 0.5H), 2.85 (ddd, J = 17.0, 11.7, 6.0 Hz, 0.5H), 2.75 (dd, J = 16.2, 3.4 Hz, 0.5H), 2.65 (d, J = 15.7 Hz, 0.5H), 1.93–1.82 (m, 2H), 1.65–1.49 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 170.2, 169.1, 148.6, 148.2, 147.8, 147.5, 137.4, 137.3, 131.3, 131.0, 130.8, 130.5, 129.9, 129.3, 129.0, 128.8, 126.8, 126.3, 126.2, 126.1, 125.8, 125.7, 125.0(8), 125.0(6), 124.4, 123.6, 111.8, 111.4, 110.3, 109.8, 75.0, 74.0, 70.0, 69.6, 65.5, 65.4, 65.3, 57.9, 56.0, 55.9, 52.0, 42.6, 35.4, 32.4, 32.3, 32.1, 32.0, 29.0, 27.6. (Unable to discern between fluorine splitting and minor peaks, all peaks reported as seen.) HRMS calcd for C₂₅H₂₉O₅NF₃, 480.19923 [M + H]⁺; found 480.19912 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 50–95% MeOH in water over 6 min at 1 mL/min (retention time = 3.62 min). Method 2: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 0.88 min).

(6,7-Dimethoxy-1-((pyridin-3-yloxy)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (18e).

General procedure IX was followed using compound 16c (25 mg, 0.082 mmol), triethylamine (23 μL, 0.16 mmol), and 3-(trifluoromethyl)-benzoyl chloride (15 μL, 0.10 mmol) in DCM (1.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white foam (17 mg, 44%). R_f (9:1 DCM/MeOH): 0.64; ¹H NMR (600 MHz, CDCl₃) δ 8.08 (d, J = 3.7 Hz, 0.5H), 7.91 (d, J = 4.1 Hz, 0.5H), 7.67 (s, 0.5H), 7.59 (d, J = 7.6 Hz, 0.5H), 7.52 (t, J = 7.1 Hz, 1.5H), 7.45 (t, J = 8.4 Hz, 1H), 7.40–7.35 (m, 1H), 7.33 (t, J = 7.6 Hz, 0.5H), 6.86–6.76 (m, 1.5H), 6.71 (d, J = 8.3 Hz, 0.5H), 6.66 (d, J = 8.3 Hz, 0.5H), 6.61 (s, 0.5H), 6.54 (s, 1H), 6.06–6.01 (m, 0.5H), 5.14–5.09 (m, 0.5H), 4.89–4.78 (m, 1H), 4.63 (dd, J = 11.2, 4.2 Hz, 0.5H), 4.50 (t, J = 10.8 Hz, 0.5H), 4.39 (dd, J = 11.8, 3.4 Hz, 0.5H), 3.83 (s, 1.5H), 3.81 (s, 1.5H), 3.79 (s, 1.5H), 3.75 (s, 1.5H), 3.71–3.63 (m, 0.5H), 3.63–3.57 (m, 0.5H), 3.29 (td, J = 12.9, 4.2 Hz, 0.5H), 3.06 (ddd, J = 17.7, 12.6, 6.2 Hz, 0.5H), 2.83–2.70 (m, 1H), 2.60 (d, J = 15.2 Hz, 0.5H). ¹³C NMR (151 MHz, CDCl₃) δ 170.0, 169.3, 163.4, 162.9, 148.7, 148.3, 147.9, 147.6, 146.6, 146.5, 139.0, 137.3, 137.0, 131.2, 130.9, 130.9, 130.6, 130.5, 129.8, 129.2, 128.7, 127.0, 126.2, 126.1, 126.0, 125.8, 124.7, 124.7, 123.9, 123.5, 117.3, 117.2, 111.8, 111.4, 111.4, 111.1, 110.4, 109.8, 66.8, 66.6, 57.0, 56.7, 55.9, 51.7, 42.0, 35.5, 28.9, 27.7. (Unable to discern between fluorine splitting and minor peaks, all peaks reported as seen.) HRMS calcd for C₂₆H₂₀O₄N₂F₃, 473.16827 [M + H]⁺; found 473.16919 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 50–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.59 min). Method 2: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 0.77 min).

3-(2-Nitrovinyl)thiophene (20a).

General procedure XI was followed using thiophene-3-carbaldehyde (19a) (3.00 g, 26.7 mmol), butylamine (0.32 mL, 3.2 mmol), acetic acid (0.31 mL, 5.4 mmol), and 4 Å molecular sieves (300 mg) in nitromethane (27 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a yellow solid (3.3 g, 79%). R_f (1:1 EtOAc/Hex): 0.58; ¹H NMR (500 MHz, CDCl₃) δ 8.01 (dd, J = 13.5, 0.5 Hz, 1H), 7.73 (ddd, J = 2.5, 1.3, 0.6 Hz, 1H), 7.49 (d, J = 13.5 Hz, 1H), 7.44 (ddd, J = 5.1, 2.9, 0.7 Hz, 1H), 7.28 (ddd, J = 5.1, 1.3, 0.5 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 136.7, 132.6, 132.5, 132.2, 128.2, 125.1. HRMS calcd for C₆H₆O₂NS, 156.01138 [M + H]⁺; found 156.01151 [M + H]⁺.

2-Methoxy-5-(2-nitrovinyl)thiophene (20b).

2-Methoxythiophene (1.06 g, 9.28 mmol) was dissolved in Et₂O (19 mL, 0.5 M), and n-butyllithium (5.57 mL, 13.9 mmol, 2.5 M in hexanes) was added dropwise under nitrogen at 0 °C. The mixture was then heated to reflux and allowed to react for 1 h. The reaction was then cooled to 0 °C and quenched carefully via dropwise addition of water before the organic layer was extracted with Et₂O, washed with brine (3×), dried over MgSO₄, and concentrated in vacuo to give the crude 5-methoxythiophene-2-carbaldehyde (19b) as a yellow oil (1.32 g, 100%) that was carried forward without further purification. General procedure XI was then followed using the crude material (1.32 g, 9.28 mmol), butylamine (0.110 mL, 1.11 mmol), acetic acid (0.106 mL, 1.86 mmol), and 4 Å molecular sieves (132 mg) in nitromethane (19 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a red solid (1.25 g over two steps, 73%). R_f (1:1 EtOAc/Hex): 0.66; ¹H NMR (399 MHz, CDCl₃) δ 8.02 (d, J = 13.1 Hz, 1H), 7.24 (d, J = 13.1 Hz, 2H), 7.18 (d, J = 4.2 Hz, 1H), 6.25 (d, J = 4.2 Hz, 1H), 3.97 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 172.2, 136.2, 133.7, 131.9, 120.4, 106.4, 60.6. HRMS calcd for C₇H₈O₃NS, 186.02194 [M + H]⁺; found 186.02186 [M + H]⁺.

2-Methyl-4-(2-nitrovinyl)thiazole (20c).

General procedure XI was followed using 2-methylthiazole-4-carbaldehyde (19c) (1.00 g, 7.86 mmol), butylamine (93 μL, 0.94 mmol), acetic acid (90 μL, 1.6 mmol), and 4 Å molecular sieves (100 mg) in nitromethane (8 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–80% EtOAc/hexanes gradient) to afford the title compound as a yellow solid (0.41 g, 31%). R_f (1:1 EtOAc/Hex): 0.70; ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 7.85 (s, 1H), 7.54 (s, 1H), 2.75 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.9, 146.9, 139.1, 130.5, 126.2, 19.4. HRMS calcd for C₆H₇O₂N₂S, 171.02227 [M + H]⁺; found 171.02191 [M + H]⁺.

3-(2-Nitrovinyl)furan (20d).

General procedure XI was followed using furan-3-carbaldehyde (19d) (1.80 mL, 20.8 mmol), butylamine (0.25 mL, 2.5 mmol), acetic acid (0.24 mL, 4.2 mmol), and 4 Å molecular sieves (200 mg) in nitromethane (21 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a yellow solid (2.9 g, quant.). R_f (1:1 EtOAc/Hex): 0.83; ¹H NMR (400 MHz, CDCl₃) δ 7.93 (d, J = 13.5 Hz, 1H), 7.86–7.81 (m, 1H), 7.56–7.48 (m, 1H), 7.39 (d, J = 13.5 Hz, 1H), 6.59–6.55 (m, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 147.3, 145.3, 136.7, 129.6, 118.2, 107.2. No ionization observed by HRMS.

1-Methyl-5-(2-nitrovinyl)-1H-pyrazole (20e).

General procedure XI was followed using 1-methyl-1H-pyrazole-5-carbaldehyde (19e) (2.00 g, 18.2 mmol), butylamine (0.22 mL, 2.2 mmol), acetic acid (0.21 mL, 3.6 mmol), and 4 Å molecular sieves (200 mg) in nitromethane (18 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a yellow solid (1.7 g, 62%). R_f (1:1 EtOAc/Hex): 0.65; ¹H NMR (400 MHz, CDCl₃) δ 7.94 (d, J = 13.4 Hz, 1H), 7.54 (d, J = 2.2 Hz, 1H), 7.51 (d, J = 13.4 Hz, 1H), 6.66 (d, J = 2.2 Hz, 1H), 4.02 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 139.4, 137.6, 133.2, 124.4, 107.5, 37.3. HRMS calcd for C₆H₈O₂N₃, 154.06110 [M + H]⁺; found 154.06109 [M +H]⁺.

2-(Thiophen-3-yl)ethan-1-amine (21a).

General procedure XII was followed using compound 20a (3.27 g, 21.1 mmol), sulfuric acid (2.51 mL, 47.0 mmol), and 2 M lithium aluminum hydride in THF (47.0 mL, 94.0 mmol) in THF (144 mL) to afford the title compound as a red oil (2.10 g). The crude material was confirmed via LCMS and carried forward without further purification.

2-(5-Methoxythiophen-2-yl)ethan-1-amine (21b).

General procedure XII was followed using compound 20b (1.19 g, 6.43 mmol), sulfuric acid (0.76 mL, 14 mmol), and 2 M lithium aluminum hydride in THF (14.3 mL, 28.7 mmol) in THF (39 mL) to afford the crude title compound as a pale yellow oil (1.01 g). The crude material was confirmed via LCMS and carried forward without further purification.

2-(2-Methylthiazol-4-yl)ethan-1-amine (21c).

General procedure XII was followed using compound 20c (411 mg, 2.42 mmol), sulfuric acid (0.29 mL, 5.4 mmol), and 1 M lithium aluminum hydride in THF (10.8 mL, 10.8 mmol) in THF (17 mL) to afford the title compound as a red oil (60 mg). The crude material was confirmed via LCMS and carried forward without further purification.

2-(Furan-3-yl)ethan-1-amine (21d).

General procedure XII was followed using compound 20d (1.45 g, 10.4 mmol), sulfuric acid (1.24 mL, 23.2 mmol), and 2 M lithium aluminum hydride in THF (23.2 mL, 46.5 mmol) in THF (71 mL) to afford the title compound as a yellow oil (0.89 g). The crude material was confirmed via LCMS and carried forward without further purification.

2-(1-Methyl-1H-pyrazol-5-yl)ethan-1-amine (21e).

General procedure XII was followed using compound 20e (1.60 g, 10.5 mmol), sulfuric acid (1.24 mL, 23.3 mmol), and 2 M lithium aluminum hydride in THF (23.3 mL, 46.6 mmol) in THF (71 mL) to afford the title compound as a pale yellow oil (0.80 g). The crude material was confirmed via LCMS and carried forward without further purification.

2-Chloro-N-(2-(thiophen-3-yl)ethyl)acetamide (22a).

General procedure V was followed using crude compound 21a (2.10 g, 16.5 mmol), triethylamine (3.34 mL, 33.0 mmol), and 2-chloroacetyl chloride (1.59 mL, 19.8 mmol) in DCM (60 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white solid (2.20 g, 51% over two steps). R_f (1:1 EtOAc/Hex): 0.47; ¹H NMR (500 MHz, CDCl₃) δ 7.30 (dd, J = 4.9, 2.9 Hz, 1H), 7.05–7.01 (m, 1H), 6.96 (dd, J = 4.9, 1.3 Hz, 1H), 6.65 (s, 1H), 4.02 (s, 2H), 3.56 (d, J = 6.1 Hz, 2H), 2.89 (t, J = 6.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 138.6, 127.9, 126.2, 121.6, 42.7, 40.2, 23.0. HRMS calcd for C₈H₁₁ONClS, 204.02444 [M + H]⁺; found 204.02490 [M + H]⁺.

2-Chloro-N-(2-(5-methoxythiophen-2-yl)ethyl)acetamide (22b).

General procedure V was followed using crude compound 21b (1.01 g, 6.43 mmol), triethylamine (1.79 mL, 12.9 mmol), and 2-chloroacetyl chloride (0.62 mL, 7.7 mmol) in DCM (23 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as an orange solid (0.92 g, 61% over two steps). R_f (1:1 EtOAc/Hex): 0.43; ¹H NMR (400 MHz, CDCl₃) δ 6.75 (s, 1H), 6.47–6.41 (m, 1H), 6.01 (d, J = 3.7 Hz, 1H), 4.04 (s, 2H), 3.85 (s, 3H), 3.51 (q, J = 6.5 Hz, 2H), 2.89 (t, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 166.0, 165.0, 126.6, 122.8, 103.2, 60.2, 42.6, 40.9, 30.1. HRMS calcd for C₉H₁₃O₂NClS, 234.03500 [M + H]⁺; found 234.03492 [M + H]⁺.

2-Chloro-N-(2-(2-methylthiazol-4-yl)ethyl)acetamide (22c).

General procedure V was followed using crude compound 21c (60 mg, 0.42 mmol), triethylamine (0.12 mL, 0.84 mmol), and 2-chloroacetyl chloride (34 μL, 0.42 mmol) in DCM (1.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (27 mg, 5% over two steps). R_f (1:1 EtOAc/Hex): 0.39; ¹H NMR (500 MHz, CDCl₃) δ 7.59 (s, 0H), 6.81 (s, 1H), 4.03 (s, 2H), 3.62 (q, J = 6.5 Hz, 2H), 2.93 (t, J = 6.3 Hz, 2H), 2.68 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 165.9, 153.5, 114.0, 42.8, 39.3, 30.4. HRMS calcd for C₈H₁₁ON₂ClS, 219.03534 [M + H]⁺; found 219.03522 [M + H]⁺.

2-Chloro-N-(2-(furan-3-yl)ethyl)acetamide (22d).

General procedure V was followed using crude compound 21d (0.860 g, 7.74 mmol), triethylamine (2.16 mL, 15.5 mmol), and 2-chloroacetyl chloride (0.74 mL, 9.3 mmol) in DCM (28 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as a red oil (0.84 g, 43% over two steps). R_f (1:1 EtOAc/Hex): 0.61; ¹H NMR (500 MHz, CDCl₃) δ 7.39 (t, J = 1.7 Hz, 1H), 7.31–7.26 (m, 1H), 6.66 (s, 1H), 6.30 (dd, J = 1.8, 0.9 Hz, 1H), 4.03 (s, 2H), 3.50 (q, J = 6.2 Hz, 2H), 2.68 (t, J = 6.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 143.4, 139.7, 121.3, 110.7, 42.7, 39.8, 24.8. HRMS calcd for C₈H₁₀O₂NClNa, 210.02923 [M + H]⁺; found 210.02875 [M + H]⁺.

2-Chloro-N-(2-(1-methyl-1H-pyrazol-5-yl)ethyl)acetamide (22e).

General procedure V was followed using crude compound 21e (0.78 g, 6.2 mmol), triethylamine (1.74 mL, 12.5 mmol), and 2-chloroacetyl chloride (0.60 mL, 7.5 mmol) in DCM (23 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as a yellow oil (0.86 g, 41% over two steps). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.43; ¹H NMR (500 MHz, CDCl₃) δ 7.40 (d, J = 1.9 Hz, 1H), 6.79 (bs, 1H), 6.07 (dt, J = 1.9, 0.6 Hz, 1H), 4.03 (s, 2H), 3.81 (s, 3H), 3.57 (dt, J = 7.1, 6.0 Hz, 3H), 2.88 (t, J = 7.1 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 166.1, 139.0, 138.5, 104.8, 42.6, 38.5, 36.2, 25.5. HRMS calcd for C₈H₁₃ON₃Cl, 202.07417 [M + H]⁺; found 202.07392 [M + H]⁺.

2-Chloro-N-(2-(thiophen-2-yl)ethyl)acetamide (22f).

General procedure V was followed using 2-(thiophen-2-yl)ethan-1-amine (21f) (2.00 g, 15.7 mmol), triethylamine (3.18 mL, 31.4 mmol), and 2-chloroacetyl chloride (1.51 mL, 18.9 mmol) in DCM (57 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–60% EtOAc/hexanes gradient) to afford the title compound as a red oil (2.64 g, 82%). R_f (1:1 EtOAc/Hex): 0.51; ¹H NMR (500 MHz, CDCl₃) δ 7.18 (dd, J = 5.2, 1.2 Hz, 1H), 6.95 (dd, J = 5.1, 3.4 Hz, 1H), 6.88–6.83 (m, 1H), 6.75 (s, 1H), 4.03 (s, 2H), 3.58 (q, J = 6.7 Hz, 2H), 3.07 (t, J = 6.7 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 165.9, 140.6, 127.2, 125.6, 124.2, 42.7, 41.1, 29.7. HRMS calcd for C₈H₁₀ONClNaS, 226.00638 [M + Na]⁺; found 226.00658 [M + Na]⁺.

N-(2-(1H-Pyrrol-1-yl)ethyl)-2-chloroacetamide (22g).

General procedure V was followed using 2-(1H-pyrrol-1-yl)ethanamine (21g) (1.00 g, 9.08 mmol), triethylamine (2.53 mL, 18.2 mmol), and 2-chloroacetyl chloride (0.87 mL, 11 mmol) in DCM (33 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as an orange solid (1.7 g, quant.). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.69; ¹H NMR (500 MHz, CDCl₃) δ 6.66 (t, J = 2.0 Hz, 2H), 6.19 (t, J = 2.0 Hz, 2H), 4.05 (t, J = 5.9 Hz, 2H), 4.03 (s, 2H), 3.62 (q, J = 5.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 120.6, 109.0, 48.6, 42.5, 41.3. HRMS calcd for C₈H₁₂ON₂Cl, 187.06327 [M + H]⁺; found 187.06360 [M + H]⁺.

2-(4-Methoxyphenoxy)-N-(2-(thiophen-3-yl)ethyl)acetamide (23a).

General procedure VI was followed using compound 22a (1.00 g, 4.91 mmol), 4-methoxyphenol (0.731 g, 5.89 mmol), and cesium carbonate (6.40 g, 19.6 mmol) in acetonitrile (15 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as a white solid (1.20 g, 84%). R_f (1:1 EtOAc/Hex): 0.40; ¹H NMR (500 MHz, CDCl₃) δ 7.28 (dd, J = 4.9, 3.0 Hz, 1H), 6.97–6.94 (m, 1H), 6.93 (dd, J = 4.9, 1.3 Hz, 1H), 6.87–6.77 (m, 4H), 6.66 (s, 1H), 4.42 (s, 2H), 3.77 (s, 3H), 3.60 (d, J = 6.1 Hz, 2H), 2.87 (t, J = 6.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 168.4, 154.7, 151.3, 138.8, 128.0, 126.1, 121.6, 115.6, 114.9, 68.1, 55.7, 39.3, 30.2. HRMS calcd for C₁₅H₁₈O₃NS, 292.10019 [M + H]⁺; found 292.10028 [M + H]⁺.

2-(4-Methoxyphenoxy)-N-(2-(5-methoxythiophen-2-yl)ethyl)-acetamide (23b).

General procedure VI was followed using compound 22b (0.89 g, 3.8 mmol), 4-methoxyphenol (0.57 g, 4.6 mmol) and cesium carbonate (4.96 g, 15.2 mmol) in acetonitrile (38 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white solid (0.97 g, 79%). R_f (1:1 EtOAc/Hex): 0.41; ¹H NMR (400 MHz, CDCl₃) δ 6.88–6.79 (m, 4H), 6.75 (bs, 1H), 6.37 (dt, J = 3.7, 0.8 Hz, 1H), 5.98 (d, J = 3.7 Hz, 1H), 4.43 (s, 2H), 3.84 (s, 3H), 3.77 (s, 3H), 3.55 (q, J = 6.6 Hz, 2H), 2.88 (t, J = 7.0 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 168.4, 164.9, 154.7, 151.3, 126.9, 122.7, 115.6, 114.8, 103.2, 68.1, 60.2, 55.7, 40.1, 30.3. HRMS calcd for C₁₆H₂₀O₄NS, 322.11076 [M + H]⁺; found 322.11032 [M + H]⁺.

2-(4-Methoxyphenoxy)-N-(2-(2-methylthiazol-4-yl)ethyl)-acetamide (23c).

General procedure VI was followed using compound 22c (27 mg, 0.12 mmol), 4-methoxyphenol (18 mg, 0.15 mmol), and cesium carbonate (161 mg, 0.494 mmol) in acetonitrile (0.4 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a clear oil (35 mg, 93%). R_f (1:1 EtOAc/Hex): 0.08; ¹H NMR (500 MHz, CDCl₃) δ 7.31 (s, 1H), 6.82 (s, 4H), 6.74 (s, 1H), 4.43 (s, 2H), 3.76 (s, 3H), 3.67 (q, J = 6.1 Hz, 2H), 2.93 (t, J = 6.8 Hz, 2H), 2.65 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.5, 166.1, 154.6, 153.6, 151.5, 115.6, 114.8, 113.9, 68.2, 55.7, 38.3, 30.9, 19.2. HRMS calcd for C₁₅H₁₉O₃N₂S, 307.11109 [M + H]⁺; found 307.11127 [M + H]⁺.

N-(2-(Furan-3-yl)ethyl)-2-(4-methoxyphenoxy)acetamide (23d).

General procedure VI was followed using compound 22d (0.837 g, 4.46 mmol), 4-methoxyphenol (0.665 g, 5.35 mmol), and cesium carbonate (5.81 g, 17.8 mmol) in acetonitrile (13 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as a clear oil (1.07 g, 87%). R_f (1:1 EtOAc/Hex): 0.18; ¹H NMR (500 MHz, CDCl₃) δ 7.37 (t, J = 1.7 Hz, 1H), 7.23–7.18 (m, 1H), 6.87–6.76 (m, 4H), 6.67 (bs, 0H), 6.27 (dd, J = 1.8, 0.9 Hz, 1H), 4.42 (s, 2H), 3.77 (s, 3H), 3.54 (q, J = 6.9 Hz, 2H), 2.66 (t, J = 6.9 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 168.4, 154.7, 151.3, 143.3, 139.6, 121.5, 115.6, 114.8, 110.7, 68.1, 55.7, 38.9, 25.0. HRMS calcd for C₁₅H₁₈O₄N, 276.12303 [M + H]⁺; found 276.12303 [M + H]⁺.

2-(4-Methoxyphenoxy)-N-(2-(1-methyl-1H-pyrazol-5-yl)ethyl)-acetamide (23e).

General procedure VI was followed using compound 22e (0.83 g, 4.1 mmol), 4-methoxyphenol (0.613 g, 4.94 mmol), and cesium carbonate (5.36 g, 16.5 mmol) in acetonitrile (24 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% EtOAc/hexanes gradient) to afford the title compound as a white solid (1.0 g, 84%). R_f (1:1 EtOAc/Hex): 0.76; ¹H NMR (399 MHz, CDCl₃) δ 7.39 (d, J = 1.8 Hz, 1H), 6.87–6.76 (m, 5H), 6.03 (d, J = 1.9 Hz, 1H), 4.43 (s, 2H), 3.81 (s, 3H), 3.77 (s, 3H), 3.61 (q, J = 6.9 Hz, 2H), 2.88 (t, J = 7.1 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃) δ 168.7, 154.8, 151.2, 139.1, 138.3, 115.6, 114.8, 104.7, 68.0, 55.7, 37.7, 36.2, 25.8. HRMS calcd for C₁₅H₂₀O₃N₃, 290.14992 [M + H]⁺; found 290.14994 [M + H]⁺.

2-(4-Methoxyphenoxy)-N-(2-(thiophen-2-yl)ethyl)acetamide (23f).

General procedure VI was followed using compound 22f (2.64 g, 13.0 mmol), 4-methoxyphenol (1.93 g, 15.6 mmol), and cesium carbonate (16.9 g, 51.8 mmol) in acetonitrile (39 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as an orange solid (3.26 g, 86%). R_f (1:1 EtOAc/Hex): 0.40; ¹H NMR (500 MHz, CDCl₃) δ 7.16 (dd, J = 5.1, 1.2 Hz, 1H), 6.93 (dd, J = 5.1, 3.4 Hz, 1H), 6.87–6.77 (m, 6H), 4.43 (s, 2H), 3.77 (s, 3H), 3.61 (q, J = 6.7 Hz, 2H), 3.07 (t, J = 6.8 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 168.5, 154.7, 151.3, 140.9, 127.1, 125.5, 124.1, 115.7, 114.8, 68.1, 55.7, 40.3, 29.9. HRMS calcd for C₃₀H₃₅O₆N₂S₂, 583.19361 [2M + H]⁺; found 583.19343 [2M + H]⁺.

N-(2-(1H-Pyrrol-1-yl)ethyl)-2-(4-methoxyphenoxy)acetamide (23g).

General procedure VI was followed using compound 22g (0.87 g, 4.7 mmol), 4-methoxyphenol (0.694 g, 5.59 mmol), and cesium carbonate (6.08 g, 18.7 mmol) in acetonitrile (28 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (1.1 g, 89%). R_f (1:1 EtOAc/Hex): 0.30; ¹H NMR (500 MHz, CDCl₃) δ 6.88–6.76 (m, 4H), 6.69 (s, 1H), 6.61 (t, J = 2.1 Hz, 2H), 6.17 (t, J = 2.1 Hz, 2H), 4.43 (s, 2H), 4.08–4.01 (m, 2H), 3.78 (s, 3H), 3.65 (q, J = 6.1 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 168.8, 154.8, 151.2, 120.6, 115.6, 114.8, 108.9, 68.0, 55.7, 48.9, 40.4. HRMS calcd for C₁₅H₁₉O₃N₂, 275.13902 [M + H]⁺; found 275.13913 [M + H]⁺.

7-((4-Methoxyphenoxy)methyl)-4,5,6,7-tetrahydrothieno[2,3-c]-pyridine (24a).

General procedure VIII was followed using compound 23a (1.17 g, 4.02 mmol), POCl₃ (1.87 mL, 20.1 mmol), and sodium borohydride (0.456 g, 12.1 mmol) in acetonitrile (60 mL) and then methanol (17 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as an orange oil (0.120 g, 11%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.63; ¹H NMR (500 MHz, CDCl₃) δ 7.16 (d, J = 5.1 Hz, 1H), 6.91–6.88 (m, 2H), 6.85–6.82 (m, 3H), 4.49–4.43 (m, 1H), 4.17–4.06 (m, 2H), 3.77 (s, 3H), 3.70 (dd, J = 20.3, 11.2 Hz, 1H), 3.31 (dt, J = 12.5, 4.8 Hz, 1H), 3.06 (ddd, J = 13.0, 8.4, 4.9 Hz, 1H), 2.80–2.63 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 154.1, 152.7, 135.5, 133.9, 127.5, 123.0, 115.7, 114.6, 72.2, 55.7, 54.3, 41.7, 26.7. HRMS calcd for C₁₅H₁₈O₂NS, 276.10528 [M + H]⁺; found 276.10526 [M + H]⁺.

2-Methoxy-4-((4-methoxyphenoxy)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (24b).

General procedure VII was followed using compound 23b (0.94 g, 2.9 mmol), POCl₃ (1.36 mL, 14.6 mmol), and sodium borohydride (0.33 g, 8.8 mmol) in acetonitrile (58 mL) and then methanol (15 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a red oil (0.17 g, 19%). R_f (EtOAc): 0.17; ¹H NMR (500 MHz, CDCl₃) δ 6.88–6.82 (m, 4H), 5.95 (s, 1H), 4.22–4.16 (m, 1H), 4.10 (dd, J = 9.2, 3.7 Hz, 1H), 4.02–3.94 (m, 1H), 3.83 (s, 3H), 3.76 (s, 3H), 3.28 (dt, J = 12.1, 5.2 Hz, 1H), 3.07 (ddd, J = 12.2, 7.4, 4.9 Hz, 1H), 2.76–2.59 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 163.9, 154.0, 152.9, 130.7, 122.2, 115.6, 114.7, 101.3, 71.2, 60.3, 55.7, 54.1, 41.4, 25.4. HRMS calcd for C₁₆H₂₀O₃NS, 306.11584 [M + H]⁺; found 306.11537 [M + H]⁺.

4-((4-Methoxyphenoxy)methyl)-2-methyl-4,5,6,7-tetrahydrothiazolo[5,4-c]pyridine (24c).

General procedure VIII was followed using compound 23c (35 mg, 0.11 mmol), POCl₃ (53 μL, 0.57 mmol), and sodium borohydride (13 mg, 0.34 mmol) in acetonitrile (1.8 mL) and then methanol (0.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as a clear oil (3.6 mg, 9%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.40; ¹H NMR (500 MHz, CDCl₃) δ 6.79 (s, 4H), 4.03 (t, J = 5.2 Hz, 2H), 3.73 (s, 3H), 3.12–2.92 (m, 6H), 2.65 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 154.3, 153.9, 152.8, 115.5, 114.6, 113.4, 67.5, 55.7, 48.7, 48.5, 31.4, 19.1. HRMS calcd for C₁₅H₁₉O₂N₂S, 291.11618 [M + H]⁺; found 291.11615 [M + H]⁺.

7-((4-Methoxyphenoxy)methyl)-4,5,6,7-tetrahydrofuro[2,3-c]-pyridine (24d).

General procedure VIII was followed using compound 23d (1.02 g, 3.71 mmol), POCl₃ (1.73 mL, 18.5 mmol), and sodium borohydride (0.421 g, 11.1 mmol) in acetonitrile (59 mL) and then methanol (15 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–20% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as an orange oil (0.570 g, 59%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.59; ¹H NMR (500 MHz, CDCl₃) δ 7.31–7.26 (m, 1H), 6.91–6.79 (m, 4H), 6.26 (d, J = 1.9 Hz, 1H), 4.33–4.25 (m, 2H), 4.11–4.03 (m, 1H), 3.76 (s, 3H), 3.21 (dt, J = 12.7, 5.0 Hz, 1H), 2.98 (ddd, J = 12.6, 7.7, 4.9 Hz, 1H), 2.62–2.44 (m, 2H), 2.21 (bs, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 154.0, 152.9, 148.3, 141.1, 117.4, 115.7, 114.6, 110.4, 69.5, 55.7, 52.8, 41.6, 23.9. HRMS calcd for C₁₅H₁₈O₃N, 260.12812 [M + H]⁺; found 260.12799 [M + H]⁺.

4-((4-Methoxyphenoxy)methyl)-1-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine (24e).

General procedure VIII was followed using compound 23e (0.70 g, 2.4 mmol), POCl₃ (1.13 mL, 12.1 mmol), and sodium borohydride (0.275 g, 7.26 mmol) in acetonitrile (39 mL) and then methanol (10 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as a clear oil (0.25 g, 38%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.47; ¹H NMR (399 MHz, CDCl₃) δ 7.33 (s, 1H), 6.92–6.78 (m, 4H), 4.28 (dd, J = 8.5, 4.0 Hz, 1H), 4.11 (dd, J = 9.1, 4.0 Hz, 1H), 3.92 (t, J = 8.8 Hz, 1H), 3.76 (s, 3H), 3.76 (s, 3H), 3.32 (dt, J = 12.3, 5.0 Hz, 1H), 3.07–2.98 (m, 1H), 2.74–2.55 (m, 2H), 2.16 (bs, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 154.0, 152.9, 137.5, 134.3, 115.6, 115.2, 114.6, 71.6, 55.7, 51.2, 40.9, 35.6, 22.7. HRMS calcd. HRMS calcd for C₁₅H₂₀O₂N₃, 274.15500 [M + H]⁺; found 274.15504 [M + H]⁺.

4-((4-Methoxyphenoxy)methyl)-4,5,6,7-tetrahydrothieno[3,2-c]-pyridine (24f).

General procedure VIII was followed using compound 23f (1.00 g, 3.43 mmol), POCl₃ (1.60 mL, 17.2 mmol), and sodium borohydride (0.390 g, 10.3 mmol) in acetonitrile (60 mL) and then methanol (17 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as an orange oil (371 mg, 39%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.86; ¹H NMR (500 MHz, CDCl₃) δ 7.11 (d, J = 5.2 Hz, 1H), 6.91–6.80 (m, 5H), 4.39–4.33 (m, 1H), 4.19 (dd, J = 9.2, 3.8 Hz, 1H), 4.06–3.99 (m, 1H), 3.77 (s, 3H), 3.70 (dd, J = 21.8, 11.2 Hz, 1H), 3.37–3.28 (m, 1H), 3.14–3.05 (m, 1H), 2.97–2.80 (m, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 154.0, 152.9, 135.9, 133.7, 124.6, 122.4, 115.6, 114.7, 71.2, 55.7, 54.2, 41.3, 25.9. HRMS calcd for C₁₅H₁₈O₂NS, 276.10528 [M + H]⁺; found 276.10513 [M + H]⁺.

1-((4-Methoxyphenoxy)methyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]-pyrazine (24g).

General procedure VIII was followed using compound 23g (1.12 g, 4.08 mmol), POCl₃ (1.90 mL, 20.4 mmol), and sodium borohydride (0.463 g, 12.3 mmol) in acetonitrile (65 mL) and then methanol (16 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–10% (1% NH₄OH in MeOH)/DCM gradient) to afford the title compound as a white solid (0.777 g, 74%). R_f (90:10:0.1 DCM/MeOH/NH₄OH): 0.58; ¹H NMR (500 MHz, CDCl₃) δ 6.93–6.81 (m, 4H), 6.61 (t, J = 2.0 Hz, 1H), 6.18 (t, J = 3.0 Hz, 1H), 5.96 (dt, J = 3.3, 1.3 Hz, 1H), 4.46 (dd, J = 8.9, 3.3 Hz, 1H), 4.25 (dd, J = 9.2, 3.4 Hz, 1H), 4.06–3.93 (m, 3H), 3.78 (s, 3H), 3.37 (dt, J = 12.4, 4.0 Hz, 1H), 3.24–3.15 (m, 1H), 2.28 (s, 1H). ¹³C NMR (126 MHz, CDCl₃) δ 154.0, 152.8, 126.8, 119.5, 115.5, 114.7, 107.8, 102.8, 71.3, 55.7, 53.0, 45.6, 42.0. HRMS calcd for C₁₅H₁₉O₂N₂, 259.14410 [M + H]⁺; found 259.14418 [M + H]⁺.

(3-Chlorophenyl)(7-((4-methoxyphenoxy)methyl)-4,7-dihydrothieno[2,3-c]pyridin-6(5H)-yl)methanone (25a).

General procedure IX was followed using compound 24a (100 mg, 0.363 mmol), triethylamine (101 μL, 0.726 mmol), and 3-chlorobenzoyl chloride (56 μL, 0.44 mmol) in DCM (5.7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a white foam (50 mg, 33%). R_f (1:1 EtOAc/Hex): 0.69; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 8.05–8.01 (m, 0.05H), 7.96–7.92 (m, 0.05H), 7.63 (bs, 0.3H), 7.57–7.52 (m, 0.5H), 7.45–7.32 (m, 3.3H), 7.32–7.23 (m, 2H), 6.95–6.78 (m, 5.2H), 6.09 (t, J = 5.4 Hz, 0.6H), 5.31 (dd, J = 9.1, 4.3 Hz, 0.4H), 4.92 (dd, J = 13.3, 5.6 Hz, 0.4H), 4.36 (dd, J = 9.3, 5.2 Hz, 0.6H), 4.28 (dd, J = 9.3, 6.1 Hz, 0.6H), 4.19 (t, J = 9.6 Hz, 0.4H), 3.98 (dd, J = 9.9, 4.4 Hz, 0.4H), 3.89 (dd, J = 13.7, 4.8 Hz, 0.6H), 3.77 (s, 3H), 3.63–3.54 (m, 0.6H), 3.26–3.15 (m, 0.4H), 2.99 (ddd, J = 17.1, 12.4, 5.7 Hz, 0.4H), 2.83–2.74 (m, 1H), 2.71 (dd, J = 15.9, 3.1 Hz, 0.6H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 170.5, 169.6, 153.9, 153.8, 152.3, 151.8, 137.6, 137.4, 136.0, 134.7, 134.4, 134.1, 132.1, 130.3, 130.1, 130.0, 130.0, 128.2, 127.8, 127.3, 126.8, 126.7, 125.5, 124.8, 124.7, 124.6, 124.3, 69.9, 69.6, 55.7, 55.6, 50.7, 43.2, 35.9, 26.5, 25.4. HRMS calcd for C₂₂H₂₁O₃NClS, 414.09252 [M + H]⁺; found 414.09298 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.42 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.702 min).

(5-Chlorothiophen-2-yl)(7-((4-methoxyphenoxy)methyl)-4,7-dihydrothieno[2,3-c]pyridin-6(5H)-yl)methanone (25b).

General procedure XIII was followed using compound 24a (25 mg, 0.091 mmol), EDCI (17 mg, 0.11 mmol), DMAP (13 mg, 0.11 mmol), and 5-chlorothiophene-2-carboxylic acid (16 mg, 0.10 mmol) in DCM (0.4 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a clear oil (10 mg, 26%). R_f (1:1 EtOAc/Hex): 0.90; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.41 (dd, J = 15.3, 1.8 Hz, 0.7H), 7.30 (d, J = 5.1 Hz, 0.7H), 7.24 (d, J = 5.1 Hz, 0.3H), 7.08 (dd, J = 15.3, 1.9 Hz, 0.3H), 6.89–6.73 (m, 6H), 6.02 (t, J = 5.2 Hz, 0.3H), 5.47 (dd, J = 8.9, 4.2 Hz, 0.7H), 4.91 (dd, J = 13.3, 4.7 Hz, 0.7H), 4.27 (dd, J = 9.4, 4.9 Hz, 0.3H), 4.22–4.15 (m, 2H), 4.12 (dd, J = 13.6, 4.3 Hz, 0.3H), 3.77 (s, 2H), 3.76 (s, 1H), 3.05 (td, J = 13.1, 4.3 Hz, 0.7H), 2.89–2.73 (m, 2H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 163.8, 162.9, 154.0, 153.8, 152.1, 151.6, 136.6, 134.1, 132.0, 130.0, 129.8, 129.7, 129.4, 129.4, 129.3, 129.3, 129.3, 129.2, 129.2, 129.0, 128.0, 127.3, 126.7, 125.0, 124.7, 123.5, 121.8, 115.4, 114.8, 114.6, 114.4, 69.9, 55.7, 55.7, 54.6, 51.0, 42.1, 36.1, 26.6, 25.4. HRMS calcd for C₂₀H₁₉O₃NClS₂, 420.04894 [M + H]⁺; found 420.04910 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.68 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.76 min).

(E)-4,4,4-Trifluoro-1-(7-((4-methoxyphenoxy)methyl)-4,7-dihydrothieno[2,3-c]pyridin-6(5H)-yl)but-2-en-1-one (25c).

General procedure XIII was followed using compound 24a (25 mg, 0.091 mmol), EDCI (17 mg, 0.11 mmol), DMAP (13 mg, 0.11 mmol), and (E)-4,4,4-trifluorobut-2-enoic acid (14 mg, 0.10 mmol) in DCM (0.4 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a yellow oil (12 mg, 33%). R_f (1:1 EtOAc/Hex): 0.71; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.62 (d, J = 3.9 Hz, 0.5H), 7.28–7.24 (m, 1H), 7.15 (d, J = 3.8 Hz, 0.5H), 6.95–6.77 (m, 6H), 5.91 (t, J = 5.0 Hz, 0.5H), 5.81 (dd, J = 8.5, 5.0 Hz, 0.5H), 4.74 (dd, J = 13.4, 5.1 Hz, 0.5H), 4.47 (dd, J = 14.0, 4.8 Hz, 0.5H), 4.39–4.34 (m, 0.5H), 4.31–4.17 (m, 1.5H), 3.78 (s, 1.5H), 3.76 (s, 1.5H), 3.74–3.67 (m, 0.5H), 3.19 (td, J = 12.9, 3.5 Hz, 0.5H), 3.02–2.87 (m, 1H), 2.86–2.79 (m, 0.5H), 2.73 (dd, J = 16.4, 3.0 Hz, 0.5H). HRMS calcd for C₁₉H₁₉O₃NF₃S, 398.10323 [M + H]⁺; found 398.10325 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.00 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.64 min).

(3-Chlorophenyl)(2-methoxy-4-((4-methoxyphenoxy)methyl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (25d).

General procedure IX was followed using compound 24b (25 mg, 0.082 mmol), triethylamine (23 μL, 0.16 mmol), and 3-chlorobenzoyl chloride (13 μL, 0.10 mmol) in DCM (1.3 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a mixture of rotamers in solution and a yellow oil (13 mg, 36%). R_f (1:1 EtOAc/Hex): 0.63; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.65 (bs, 0.5H), 7.44–7.32 (m, 3.5H), 7.29–7.24 (m, 0.5H), 6.90–6.76 (m, 4.5H), 6.01 (s, 0.5H), 5.79 (t, J = 4.6 Hz, 0.5H), 5.79 (s, 0.5H), 5.03 (dd, J = 9.5, 3.0 Hz, 0.5H), 4.94 (dd, J = 13.2, 5.6 Hz, 0.5H), 4.37–4.26 (m, 1H), 4.10 (t, J = 9.8 Hz, 0.5H), 3.94–3.86 (m, 1H), 3.86 (s, 1.5H), 3.81 (s, 1.5H), 3.77 (s, 3H), 3.73–3.67 (m, 0.5H), 3.24 (td, J = 12.8, 4.2 Hz, 0.5H), 3.06–2.97 (m, 0.5H), 2.85–2.76 (m, 0.5H), 2.68 (dd, J = 16.2, 3.6 Hz, 0.5H), 2.63–2.57 (m, 0.5H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 172.5, 170.6, 169.7, 164.5, 164.5, 153.8, 153.7, 152.5, 152.0, 137.7, 137.6, 134.7, 134.2, 130.3, 130.0, 129.9, 129.8, 129.1, 127.9, 127.7, 126.8, 125.7, 124.7, 121.9, 119.7, 115.3, 114.8, 114.5, 114.4, 101.0, 100.8, 69.4, 68.2, 60.1, 60.1, 56.1, 55.7, 55.7, 51.0, 43.5, 36.1, 25.2, 24.1. HRMS calcd for C₂₃H₂₃O₄NClS, 444.10308 [M + H]⁺; found 444.10290 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.33 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.69 min).

(3-Chlorophenyl)(4-((4-methoxyphenoxy)methyl)-2-methyl-6,7-dihydrothiazolo[5,4-c]pyridin-5(4H)-yl)methanone (25e).

General procedure IX was followed using compound 24c (5.0 mg, 0.014 mmol), triethylamine (3.8 μL, 0.028 mmol), and 3-chlorobenzoyl chloride (2.1 μL, 0.017 mmol) in DCM (0.2 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a yellow oil (5.5 mg, 93%). R_f (1:1 EtOAc/Hex): 0.61; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.58 (s, 0.2H), 7.46–7.34 (m, 3H), 7.32–7.27 (m, 0.8H), 6.92–6.77 (m, 4H), 6.02–5.97 (m, 0.8H), 5.26–5.21 (m, 0.2H), 4.95 (dd, J = 13.3, 5.7 Hz, 0.2H), 4.34 (dd, J = 9.0, 4.6 Hz, 0.8H), 4.19–4.13 (m, 1H), 3.99–3.91 (m, 1H), 3.76 (s, 2H), 3.75 (s, 1H), 3.65–3.56 (m, 0.8H), 3.28–3.19 (m, 0.2H), 3.14–3.04 (m, 0.2H), 2.93–2.81 (m, 1.8H), 2.68 (s, 2H), 2.67 (s, 1H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 172.5, 170.4, 169.6, 165.8, 154.0, 153.9, 152.0, 151.6, 149.9, 148.0, 137.2, 137.1, 134.8, 134.5, 130.3, 130.3, 130.2, 130.2, 127.6, 127.0, 125.6, 125.2, 124.6, 123.7, 115.3, 114.9, 114.5, 114.5, 69.1, 68.9, 55.7, 54.4, 49.6, 43.4, 36.4, 27.6, 26.7, 19.5, 19.4. HRMS calcd for C₂₂H₂₂O₃N₂ClS, 429.10342 [M + H]⁺; found 429.10342 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.69 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.65 min).

(3-Chlorophenyl)(7-((4-methoxyphenoxy)methyl)-4,7-dihydrofuro[2,3-c]pyridin-6(5H)-yl)methanone (25f).

General procedure IX was followed using compound 24d (28 mg, 0.108 mmol), triethylamine (30 μL, 0.21 mmol), and 3-chlorobenzoyl chloride (17 μL, 0.13 mmol) in DCM (1.7 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a clear oil (19 mg, 44%). R_f (1:1 EtOAc/Hex): 0.44; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.62 (s, 0.3H), 7.44–7.32 (m, 4H), 7.28–7.24 (m, 0.7H), 6.86–6.77 (m, 4H), 6.35–6.30 (m, 1H), 5.86 (t, J = 4.1 Hz, 0.7H), 5.17 (dd, J = 8.8, 2.3 Hz, 0.3H), 4.89 (dd, J = 13.3, 5.3 Hz, 0.3H), 4.49 (dd, J = 10.1, 3.2 Hz, 0.7H), 4.40 (dd, J = 10.1, 4.2 Hz, 0.7H), 4.11 (t, J = 9.7 Hz, 0.3H), 4.05 (dd, J = 10.1, 3.2 Hz, 0.3H), 3.83 (dd, J = 13.7, 4.9 Hz, 0.7H), 3.77 (s, 1H), 3.77 (s, 2H), 3.75–3.68 (m, 0.7H), 3.25–3.13 (m, 0.3H), 2.88–2.78 (m, 0.3H), 2.67–2.58 (m, 0.7H), 2.58–2.45 (m, 1H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 170.9, 169.9, 153.8, 153.7, 152.5, 151.9, 145.7, 144.8, 142.6, 142.3, 137.7, 137.4, 134.6, 134.3, 130.2, 130.0, 129.9, 129.9, 127.8, 126.8, 125.6, 124.7, 118.5, 116.9, 115.3, 114.8, 114.5, 114.4, 110.5, 110.1, 68.5, 67.4, 55.7, 54.9, 50.1, 44.4, 36.7, 23.2, 22.0. HRMS calcd for C₂₂H₂₁O₄NCl, 398.11536 [M + H]⁺; found 398.11536 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.11 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.67 min).

(3-Chlorophenyl)(4-((4-methoxyphenoxy)methyl)-1-methyl-1,4,6,7-tetrahydro-5H-pyrazolo[4,3-c]pyridin-5-yl)methanone (25g).

General procedure IX was followed using compound 24e (49 mg, 0.18 mmol), triethylamine (50 μL, 0.36 mmol), and 3-chlorobenzoyl chloride (28 μL, 0.38 mmol) in DCM (2.8 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a white solid (71 mg, 96%). R_f (1:1 EtOAc/Hex): 0.64; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.62 (s, 0.5H), 7.47 (s, 0.5H), 7.44–7.31 (m, 3H), 7.29–7.23 (m, 0.5H), 7.18 (dd, J = 7.5, 3.9 Hz, 0.5H), 6.89–6.76 (m, 4H), 5.96 (d, J = 5.4 Hz, 0.5H), 5.18 (dd, J = 9.5, 4.0 Hz, 0.5H), 4.98 (dd, J = 13.4, 5.8 Hz, 0.5H), 4.26 (dd, J = 9.4, 5.4 Hz, 0.5H), 4.20 (dd, J = 9.4, 5.7 Hz, 0.5H), 4.04 (t, J = 9.8 Hz, 0.5H), 3.93 (dd, J = 13.9, 5.3 Hz, 0.5H), 3.84 (dd, J = 10.0, 4.1 Hz, 0.5H), 3.79 (s, 1.5H), 3.79 (s, 1.5H), 3.76 (s, 3H), 3.63–3.53 (m, 0.5H), 3.17 (td, J = 13.0, 4.3 Hz, 0.5H), 2.93 (ddd, J = 17.5, 12.0, 5.9 Hz, 0.5H), 2.79–2.68 (m, 1H), 2.65 (dd, J = 15.5, 3.5 Hz, 0.5H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 171.0, 169.9, 153.8, 153.7, 152.4, 151.9, 137.8, 137.6, 137.5, 135.9, 135.6, 134.9, 134.7, 134.3, 130.3, 130.1, 130.0, 130.0, 129.1, 128.3, 127.8, 126.7, 125.5, 125.4, 124.6, 115.3, 114.8, 114.5, 114.4, 113.8, 112.8, 69.4, 68.7, 55.7, 53.0, 47.8, 42.4, 36.1, 35.1, 22.5, 21.7, 21.4. HRMS calcd for C₂₂H₂₃O₃N₃Cl, 412.14225 [M + H]⁺; found 412.14237 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.53 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 0.82 min).

(3-Chlorophenyl)(4-((4-methoxyphenoxy)methyl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (25h).

General procedure IX was followed using compound 24f (50 mg, 0.18 mmol), triethylamine (51 μL, 0.36 mmol), and 3-chlorobenzoyl chloride (28 μL, 0.22 mmol) in DCM (2.8 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a white foam (49 mg, 65%). R_f (1:1 EtOAc/Hex): 0.77; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.67 (bs, 0.5H), 7.45–7.34 (m, 3H), 7.31–7.27 (m, 0.5H), 7.19 (dd, J = 16.2, 5.2 Hz, 1H), 6.99 (d, J = 5.2 Hz, 0.5H), 6.91–6.78 (m, 4H), 6.75 (d, J = 5.2 Hz, 0.5H), 5.99 (t, J = 4.8 Hz, 0.5H), 5.22 (dd, J = 9.6, 3.5 Hz, 0.5H), 4.99 (dd, J = 13.1, 5.4 Hz, 0.5H), 4.35 (qd, J = 9.8, 5.1 Hz, 1H), 4.11 (t, J = 9.8 Hz, 0.5H), 3.97–3.90 (m, 1H), 3.77 (s, 3H), 3.73–3.65 (m, 0.5H), 3.25 (td, J = 12.7, 4.0 Hz, 0.5H), 3.19–3.07 (m, 0.5H), 2.97–2.89 (m, 1H), 2.84 (dd, J = 16.0, 3.1 Hz, 0.5H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 170.7, 169.8, 153.8, 153.7, 152.5, 152.0, 137.7, 137.6, 136.3, 134.7, 134.3, 133.9, 132.3, 130.9, 130.3, 130.0, 129.9, 129.9, 128.0, 126.8, 125.7, 125.6, 125.0, 124.7, 123.9, 123.8, 69.5, 68.4, 56.2, 55.7, 51.1, 43.3, 36.1, 25.8, 24.8. HRMS calcd for C₄₄H₄₀O₆N₂Cl₂NaS₂, 849.15970 [2M + Na]⁺; found 849.16095 [2M + Na]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.33 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.69 min).

(3-Chlorophenyl)(1-((4-methoxyphenoxy)methyl)-3,4-dihydropyrrolo[1,2-a]pyrazin-2(1H)-yl)methanone (25i).

General procedure IX was followed using compound 24g (50 mg, 0.19 mmol), triethylamine (54 μL, 0.39 mmol), and 3-chlorobenzoyl chloride (30 μL, 0.23 mmol) in DCM (3.0 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a white foam (67 mg, 87%). R_f (1:1 EtOAc/Hex): 0.58; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 7.69 (bs, 0.7H), 7.48–7.31 (m, 3H), 7.26–7.22 (m, 0.3H), 6.88–6.74 (m, 4H), 6.72–6.68 (m, 0.7H), 6.67–6.62 (m, 0.3H), 6.24 (t, J = 3.1 Hz, 0.3H), 6.21 (t, J = 3.1 Hz, 0.7H), 6.17 (t, J = 4.9 Hz, 0.3H), 6.12 (d, J = 3.4 Hz, 0.3H), 5.94 (d, J = 2.4 Hz, 0.7H), 5.44 (dd, J = 10.2, 3.8 Hz, 0.7H), 4.97 (dd, J = 13.7, 3.9 Hz, 0.7H), 4.40 (dd, J = 9.8, 4.2 Hz, 0.3H), 4.28 (dd, J = 9.8, 5.8 Hz, 0.3H), 4.20–4.07 (m, 2H), 4.04–3.84 (m, 2H), 3.77 (s, 3H), 3.51–3.42 (m, 0.7H). ¹³C NMR (151 MHz, CDCl₃, probe temp: −25 °C) δ 170.7, 169.5, 153.8, 153.7, 152.5, 151.9, 137.2, 136.8, 134.8, 134.3, 130.4, 130.2, 130.1, 129.9, 128.1, 126.9, 125.9, 124.8, 124.6, 123.2, 120.2, 119.5, 115.4, 114.8, 114.5, 114.4, 108.8, 108.6, 105.0, 104.8, 70.2, 68.7, 55.7, 54.3, 49.4, 45.0, 44.3, 43.5, 35.9. HRMS calcd for C₂₂H₂₂O₃N₂Cl, 397.13135 [M + H]⁺; found 397.13144 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.87 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 0.96 min).

(1-((4-Methoxyphenoxy)methyl)-3,4-dihydropyrrolo[1,2-a]-pyrazin-2(1H)-yl)(3-(trifluoromethyl)phenyl)methanone (25j).

General procedure IX was followed using compound 24g (100 mg, 0.387 mmol), triethylamine (108 μL, 0.774 mmol), and 3-chlorobenzoyl chloride (70 μL, 0.46 mmol) in DCM (6.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as rotamers in solution and a white foam (0.10 g, 62%). R_f (1:1 EtOAc/Hex): 0.60; ¹H NMR (600 MHz, CDCl₃, probe temp: −25 °C) δ 8.01 (bs, 0.7H), 7.80 (d, J = 7.2 Hz, 0.7H), 7.72 (d, J = 7.6 Hz, 1H), 7.65 (s, 0.3H), 7.63–7.54 (m, 1.3H), 6.89–6.75 (m, 4H), 6.74–6.70 (m, 0.7H), 6.66 (m, 0.3H), 6.28–6.17 (m, 1H), 6.14 (d, J = 3.3 Hz, 0.3H), 5.95 (d, J = 2.5 Hz, 0.7H), 5.42 (dd, J = 10.4, 3.8 Hz, 0.7H), 5.00 (dd, J = 13.7, 4.0 Hz, 0.7H), 4.43 (dd, J = 9.9, 4.2 Hz, 0.3H), 4.34–4.29 (m, 0.3H), 4.23–4.09 (m, 2H), 4.06–3.95 (m, 1H), 3.89 (dd, J = 10.1, 4.0 Hz, 1H), 3.77 (s, 3H), 3.50 (td, J = 13.5, 4.1 Hz, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 170.8, 169.5, 153.9, 153.7, 152.5, 151.8, 136.3, 136.0, 131.2, 130.8, 130.6, 130.0, 129.6, 129.1, 126.9, 125.1, 124.7, 124.6, 123.8, 123.0, 122.9, 120.3, 119.5, 115.4, 114.8, 114.5, 114.4, 108.9, 108.6, 105.1, 104.9, 70.2, 68.6, 55.7, 54.3, 49.5, 44.9, 44.3, 43.5, 35.9. HRMS calcd for C₂₃H₂₂O₃N₂F₃, 431.15770 [M + H]⁺; found 431.15762 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.90 min). Method 2: 95% MeOH in water over 3 min at 1 mL/min (retention time = 0.90 min).

(3-Chlorophenyl)(4-((4-methoxyphenoxy)methyl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanethione (26).

General procedure XIV was followed using compound 25a (48 mg, 0.12 mmol) and Lawesson’s reagent (80 mg, 0.20 mmol) in toluene (1.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–40% EtOAc/hexanes gradient) to afford the title compound as a clear oil (11 mg, 18%). R_f (1:1 EtOAc/Hex): 0.77; ¹H NMR (600 MHz, CDCl₃) δ 7.24–7.15 (m, 3H), 7.06–7.01 (m, 2H), 6.83–6.77 (m, 3H), 6.76–6.71 (m, 3H), 6.49 (d, J = 15.2 Hz, 1H), 4.91 (dd, J = 6.9, 4.7 Hz, 1H), 4.82 (d, J = 15.2 Hz, 1H), 4.53 (d, J = 19.6 Hz, 1H), 4.28 (d, J = 19.6 Hz, 1H), 4.11–4.03 (m, 2H), 3.81 (s, 3H), 3.76 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 199.4, 159.9, 154.4, 151.8, 137.2, 134.6, 133.4, 130.1, 127.9, 127.7, 126.7, 125.5, 123.1, 115.5, 114.7, 113.3, 111.6, 70.6, 62.7, 56.2, 55.7, 55.4, 47.7. HRMS calcd for C₂₅H₂₅O₃NClS, 454.12382 [M + H]⁺; found 454.12380 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.84 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.41 min).

Methyl 2-(1H-Pyrrol-1-yl)acetate (27).

Glycine methyl ester hydrochloride (26) (5.00 g, 39.8 mmol, 1 equiv) and sodium acetate (4.90 g, 59.7 mmol, 1.5 equiv) were dissolved in a 2:1 (v/v) mixture of acetic acid (50 mL) and water (25 mL). 2,5-Dimethoxytetrahydrofuran (5.66 mL, 43.8 mmol, 1.1 equiv) was then added, and the resulting mixture was heated to 100 °C for 4 h before cooling to room temperature. The crude mixture was poured into water, washed with EtOAc, and the aqueous phase was neutralized with solid Na₂CO₃. This was then extracted with EtOAc, washed with brine, dried over MgSO₄, and concentrated in vacuo. The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–50% EtOAc/hexanes gradient) to afford the title compound as a clear oil (3.76 g, 68%). R_f (1:1 EtOAC/Hex): 0.68; ¹H NMR (399 MHz, CDCl₃) δ 6.70 (t, J = 2.1 Hz, 2H), 6.24 (t, J = 2.1 Hz, 2H), 4.67 (s, 2H), 3.79 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 169.3, 121.8, 109.1, 52.5, 50.7. HRMS calcd for C₇H₁₀O₂N, 140.07061 [M + H]⁺; found 140.07063 [M + H]⁺.

Methyl 2-(2-(2-Chloroacetyl)-1H-pyrrol-1-yl)acetate (28).

General procedure XV was followed using 27 (6.80 g, 48.9 mmol), DBN (0.91 g, 7.3 mmol), and 2-chloroacetyl chloride (4.70 mL, 58.6 mmol) in PhMe (82 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 40 g column, 0–100% EtOAc/hexanes gradient) to afford the title compound as a white solid (8.8 g, 83%). R_f (1:1 EtOAC/Hex): 0.60; ¹H NMR (500 MHz, CDCl₃) δ 7.06 (dd, J = 4.2, 1.6 Hz, 1H), 6.92 (dd, J = 2.4, 1.7 Hz, 1H), 6.26 (dd, J = 4.2, 2.6 Hz, 1H), 5.03 (s, 2H), 4.51 (s, 2H), 3.76 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 181.6, 168.8, 132.3, 127.7, 120.4, 109.5, 51.0, 45.5. HRMS calcd for C₉H₁₃O₃NCl, 216.04220 [M + H]⁺; found 216.04225 [M + H]⁺.

Methyl 2-(2-(2-(4-Methoxyphenoxy)acetyl)-1H-pyrrol-1-yl)-acetate (29).

General procedure XVI was followed using 28 (0.84 g, 3.9 mmol), potassium iodide (0.77 g, 4.6 mmol), 4-methoxyphenol (0.94 g, 4.6 mmol), and potassium carbonate (2.14 g, 15.5 mmol) in acetone (19 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 24 g column, 0–70% EtOAc/hexanes gradient) to afford the title compound as a white solid (1.6 g, quant.). R_f (1:1 EtOAC/Hex): 0.76; ¹H NMR (399 MHz, CDCl₃) δ 7.17 (dd, J = 4.1, 1.6 Hz, 1H), 6.90 (dd, J = 2.5, 1.5 Hz, 1H), 6.89–6.76 (m, 4H), 6.26 (dd, J = 4.2, 2.6 Hz, 1H), 5.05 (s, 2H), 5.00 (s, 2H), 3.75 (s, 3H), 3.74 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 185.5, 168.9, 154.2, 152.3, 131.6, 127.8, 119.6, 115.7, 114.6, 109.4, 70.8, 55.7, 52.4, 51.0. HRMS calcd for C₁₆H₁₈O₅N, 304.11795 [M + H]⁺; found 304.11780 [M + H]⁺.

1-((4-Methoxyphenoxy)methylene)-2-(3-(trifluoromethyl)-benzyl)-1,2-dihydropyrrolo[1,2-a]pyrazin-3(4H)-one (30a).

This procedure was run at a concentration of 1 M due to incomplete conversion of the starting material to product. General procedure XVII was followed using compound 29 (0.20 g, 0.66 mmol), 3-(trifluoromethyl)benzylamine (113 μL, 0.790 mmol), tetraisopropoxytitanium (0.58 mL, 2.0 mmol), and sodium triacetoxyborohydride (0.42 g, 2.0 mmol) in DCE (0.8 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 12 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a white solid (89 mg, 32%). HRMS calcd for C₂₃H₂₀O₃N₂F₃, 429.14205 [M + H]⁺; found 429.14178 [M + H]⁺.

2-(3-Fluorobenzyl)-1-((4-methoxyphenoxy)methylene)-1,2-dihydropyrrolo[1,2-a]pyrazin-3(4H)-one (30b).

This procedure was run at a concentration of 1 M due to incomplete conversion of the starting material to product. General procedure XVII was followed using compound 29 (50 mg, 0.16 mmol), 3-fluorobenzylamine (28 μL, 0.20 mmol), tetraisopropoxytitanium (0.15 mL, 0.49 mmol), and sodium triacetoxyborohydride (0.10 g, 0.49 mmol) in DCE (0.16 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a mixture of E/Z isomers and a white solid (24 mg, 38%). HRMS calcd for C₂₂H₂₀O₃N₂F, 379.14525 [M + H]⁺; found 379.14551 [M + H]⁺.

1-((4-Methoxyphenoxy)methyl)-2-(3-(trifluoromethyl)benzyl)-1,2-dihydropyrrolo[1,2-a]pyrazin-3(4H)-one (31a).

General procedure XVIII was followed using compound 30a (55 mg, 0.13 mmol) and Pd/C (20 mg, 0.019 mmol) in EtOAc (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a white foam (20 mg, 36%). R_f (1:1 EtOAc/Hex): 0.62; ¹H NMR (500 MHz, CDCl₃) δ 7.54–7.50 (m, 2H), 7.45 (d, J = 7.7 Hz, 1H), 7.42–7.37 (m, 1H), 6.82–6.75 (m, 2H), 6.71–6.66 (m, 2H), 6.65 (dd, J = 2.7, 1.6 Hz, 1H), 6.22 (dd, J = 3.5, 2.8 Hz, 1H), 6.00 (dd, J = 3.5, 1.5 Hz, 1H), 5.44 (d, J = 15.4 Hz, 1H), 4.87 (d, J = 17.0 Hz, 1H), 4.83 (dd, J = 4.5, 3.3 Hz, 1H), 4.77 (d, J = 17.0 Hz, 1H), 4.49 (d, J = 15.4 Hz, 1H), 4.15 (dd, J = 9.7, 3.2 Hz, 1H), 4.04 (dd, J = 9.7, 4.7 Hz, 1H), 3.75 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.3, 154.5, 152.0, 137.4, 131.2, 131.1 (q, J = 31.7 Hz, ²J), 129.35, 124.80 (q, J = 3.8 Hz, ³J), 124.6 (q, J = 3.8 Hz, ³J), 124.0, 123.9 (q, J = 272.5 Hz, ¹J), 118.9, 115.6, 114.7, 109.6, 103.9, 72.4, 55.7, 55.0, 49.0, 48.6. HRMS calcd for C₂₃H₂₂O₃N₂F₃, 431.15770 [M + H]⁺; found 431.15735 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.82 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 0.87 min).

2-(3-Fluorobenzyl)-1-((4-methoxyphenoxy)methyl)-1,2-dihydropyrrolo[1,2-a]pyrazin-3(4H)-one (EU-1180–453).

General procedure XVIII was followed using compound 30b (24 mg, 0.063 mmol) and Pd/C (10 mg, 0.010 mmol) in EtOAc (5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–35% EtOAc/hexanes gradient) to afford the title compound as a white solid (14 mg, 58%). R_f (1:1 EtOAc/Hex): 0.68; mp 87–88 °C, ¹H NMR (400 MHz, CDCl₃) δ 7.30–7.20 (m, 1H), 7.03 (d, J = 7.9 Hz, 1H), 7.00–6.90 (m, 2H), 6.83–6.74 (m, 2H), 6.73–6.65 (m, 2H), 6.64 (dd, J = 2.7, 1.6 Hz, 1H), 6.22 (dd, J = 3.3, 2.8 Hz, 1H), 5.99 (dd, J = 3.5, 1.5 Hz, 1H), 5.43 (d, J = 15.4 Hz, 1H), 4.86 (d, J = 17.0 Hz, 1H), 4.82 (d, J = 3.7 Hz, 2H), 4.75 (d, J = 17.0 Hz, 1H), 4.37 (d, J = 15.4 Hz, 1H), 4.15 (dd, J = 9.7, 3.2 Hz, 1H), 4.02 (dd, J = 9.7, 4.5 Hz, 1H), 3.75 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 166.2, 163.0(d, J = 246.9 Hz, ¹J), 154.3, 152.0, 138.7 (d, J = 7.1 Hz, ³J), 130.33 (d, J = 8.2 Hz, ³J), 124.1, 123.5 (d, J = 2.9 Hz, ⁴J), 118.8, 115.6, 114.9 (d, J = 21.9 Hz, ²J), 114.7 (d, J = 21.2 Hz, ²J), 114.6, 109.5, 103.8, 72.2, 55.7, 54.6, 49.0, 48.2. HRMS calcd for C₂₂H₂₂O₃N₂F, 381.16090 [M + H]⁺; found 381.16132 [M + H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 1.41 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 0.80 min).

1-((4-Ethoxyphenoxy)methyl)-2-(3-(trifluoromethyl)benzyl)-1,2-dihydropyrrolo[1,2-a]pyrazine-3(4H)-thione (32).

General procedure XIV was followed using compound 31a (13 mg, 0.030 mmol) and Lawesson’s reagent (18 mg, 0.045 mmol) in toluene (0.5 mL). The crude product was purified via flash column chromatography (ISCO, Redisep 4 g column, 0–20% EtOAc/hexanes gradient) to afford the title compound as a yellow oil (4.3 mg, 32%). R_f (1:1 EtOAc/Hex): 0.83; ¹H NMR (399 MHz, CDCl₃) δ 7.57–7.48 (m, 2H), 7.43–7.37 (m, 2H), 6.84–6.69 (m, 4H), 6.67 (dd, J = 2.7, 1.6 Hz, 1H), 6.40 (d, J = 15.3 Hz, 1H), 6.22 (dd, J = 3.5, 2.8 Hz, 1H), 6.00 (dd, J = 3.5, 1.4 Hz, 1H), 5.44 (d, J = 18.0 Hz, 1H), 5.15 (d, J = 18.0 Hz, 1H), 5.07 (dd, J = 6.0, 3.4 Hz, 1H), 4.93 (d, J = 15.3 Hz, 1H), 4.17 (dd, J = 10.0, 3.4 Hz, 1H), 4.10 (dd, J = 10.0, 6.1 Hz, 1H), 3.76 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 195.2, 154.6, 151.7, 135.7, 131.2 (q, J = 32.5 Hz, ²J), 130.8, 124.9 (q, J = 3.7 Hz, ³J), 124.7 (q, J = 3.7 Hz, ³J), 124.4 (q, J = 271.8 Hz, ¹J), 122.8, 118.7, 115.6, 114.7, 109.4, 104.1, 77.2, 72.0, 58.2, 56.6, 55.7. HRMS calcd for C₂₃H₂₁O₂N₂F₃S, 445.12031 [M − H]⁺; found 445.12034 [M − H]⁺. Purity was established using an Agilent pump on a Zorbax XBD-C18 column (4.6 mm × 50 mm, 3.5 μm). Method 1: 75–95% MeOH in water over 3 min at 1 mL/min (retention time = 2.59 min). Method 2: 85–95% MeOH in water over 5 min at 1 mL/min (retention time = 1.17 min).

Separation of 25i Enantiomers.

Semipreparative separation of enantiomers from racemic 25i (0.020 g) was done using a ChiralPak AD-H (30 mm × 250 mm) with the following conditions: 20 mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 90% hexanes/10% IPA over 90 min to afford R-(+)-25i, t_R 59.2 min; S-(−)-25i, t_R 70.7 min. The enantiomeric excess (ee) was determined using an Agilent pump on a ChiralPak OD-H column (4.6 mm × 150 mm, 5 μm) with the following conditions: 1 mL/min flow rate, 10 μL injection volume, 90% hexanes/10% IPA. R-(+)-25i: t_R 31.5 min; >99% ee; ${[\alpha ]}_{\text{D}}^{20}=+172\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$. S-(−)-25i: t_R 41.4 min, >99% ee; ${[\alpha ]}_{\text{D}}^{20}=-173\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$.

Separation of 25j Enantiomers.

Semipreparative separation of 25j enantiomers from racemic 25j (0.020 g) was done using a ChiralPak AD-H (30 mm × 250 mm) with the following conditions: 20 mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 90% hexanes/10% IPA over 60 min to afford R-(+)-25j, t_R 41.4 min; S-(−)-25j, t_R 50.5 min. The enantiomeric excess (ee) was determined using an Agilent pump on a ChiralPak OD-H column (4.6 mm × 150 mm, 5 μm) with the following conditions: 1 mL/min flow rate, 10 μL injection volume, 90% hexanes/10% IPA. R-(+)-25j: t_R 27.1 min; >99% ee; ${[\alpha ]}_{\text{D}}^{20}=+148\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$. S-(–)-25j: t_R 32.4 min, >99% ee; ${[\alpha ]}_{\text{D}}^{20}=-149\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$.

Separation of 31a Enantiomers.

Semipreparative separation of 31a enantiomers from racemic 31a (0.020 g) was done using a ChiralPak AD-H (30 mm × 250 mm) with the following conditions: 20 mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 90% hexanes/10% IPA over 60 min to afford S-(−)-31a, t_R 33.8 min; R-(+)-31a, t_R 47.2 min. The enantiomeric excess (ee) was determined using an Agilent pump on a ChiralPak OD-H column (4.6 mm × 150 mm, 5 μm) with the following conditions: 1 mL/min flow rate, 10 μL injection volume, 90% hexanes/10% IPA. S-(−)-31a: t_R 21.4 min;>99% ee; ${[\alpha ]}_{\text{D}}^{20}=-45\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$. R-(+)-31a: t_R 30.1 min, >99% ee; ${[\alpha ]}_{\text{D}}^{20}=+45\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$.

Separation of EU-1180–453 Enantiomers.

Semipreparative separation of EU-1180–453 enantiomers from racemic EU-1180–453 (0.020 g) was done using a ChiralPak AD-H (30 mm × 250 mm) with the following conditions: 20 mL/min flow rate, 10 mL injection volume (2 mg/1 mL), 75% hexanes/25% IPA over 60 min to afford S-(−)-EU-1180–453, t_R 24.3 min; R-(+)-EU-1180–453, t_R 37.5 min. The enantiomeric excess (ee) was determined using an Agilent pump on a ChiralPak AD-H column (4.6 mm × 150 mm, 5 μm) with the following conditions: 1 mL/min flow rate, 10 μL injection volume, 80% hexanes/20% IPA. S-(−)-EU-1180–453: t_R 14.5 min; >99% ee; ${[\alpha ]}_{\text{D}}^{20}=-43\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$. R-(+)-EU-1180–453: t_R 22.1 min, >99% ee; ${[\alpha ]}_{\text{D}}^{20}=+44\left(\text{c }0.10,{\text{ dry CHCl}}_3\right)$.

ABBREVIATIONS

AMPA

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

ACN

acetonitrile

ATP

adenosine triphosphate

AMPAR

AMPA receptor

AUC

area under the curve

ABD

agonist binding domain

ATD

amino terminal domain

BAPTA-AM

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetraacetoxymethyl ester

CTD

carboxy terminal domain

CNS

central nervous system

CIQ

(3-chlorophenyl)-(6,7-dimethoxy-1-((4-methoxyphenoxy)methyl)-3,4-dihydroi-soquinolin-2(1H)-yl)methanone

DNA

deoxyribonucleic acid

DBN

1,5-diazabicyclo[4.3.0]non-5-ene

DCE

dichloroethy-lene

DCM

dichloromethane

DMAP

4-dimethylaminopyridine

DMSO

dimethyl sulfoxide

EtOAc

ethyl acetate

EDCI

1-ethyl-(3-dimethylaminopropyl)-carbonyldiimide hydrochloride

EDTA

Ethylenediaminetetraacetic acid

FDA

Food and Drug Administration

GABA

γ-aminobutyric acid

HPLC

high-performance liquid chromatography

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IACUC

Institutional Animal Care and Use Committee

IP

intraperitoneal

iGluR

ionotropic glutamate receptor

LCMS

liquid chromatography–mass spectrometry

LLE

lipophilic ligand efficiency

NAM

negative allosteric modulator

TCN

N-(4-(2-benzoyl-hydrazine-1-carbonyl)benzyl)-3-chloro-4-fluorobenzenesulfonamide

NMDA

N-methyl-d-aspartic acid

NMDAR

NMDA receptor

NIMH

National Institute for Mental Health

NMR

nuclear magnetic resonance

PFP

pentafluorophenyl

PAM

positive allosteric modulator

PEG

poly(ethylene glycol)

K-BAPTA

potassium 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

PS

pregnenolone sulfate

PDSP

Psychoactive Drug Screening Program

RNA

ribonucleic acid

RPM

rotations per minute

SEM

standard error of the mean

SAR

structure–activity relationship

THF

tetrahydrofuran

TEVC

two-electrode voltage clamp

TIQ

tetrahydroisoquinoline

TMD

transmembrane domain