The significance of chirality in drug design and synthesis of bitopic ligands as D₃ receptor (D₃R) selective agonists

Abstract

Due to the large degree of homology among dopamine D₂-like receptors, discovering ligands capable of discriminating between the D₂, D₃ and D₄ receptor subtypes remains a significant challenge. Previous work has exemplified the use of bitopic ligands as a powerful strategy in achieving subtype selectivity for agonists and antagonists alike. Inspired by the potential for chemical modification of the D₃ preferential agonists (+)-PD128,907 (1) and PF592,379 (2), we synthesized bitopic structures to further improve their D₃R selectivity. We found the (2S,5S) conformation of scaffold 2 resulted in a privileged architecture with increased affinity and selectivity for the D₃R. In addition, a cyclopropyl moiety incorporated into the linker and full resolution of the chiral centers resulted in lead compound 53 and the eutomer 53a that demonstrate significantly higher D₃R binding selectivities than the reference compounds. Moreover, the favorable metabolic stability in rat liver microsomes supports future studies in in vivo models of dopamine system dysregulation.

Graphical Abstract

INTRODUCTION

Dopamine D₃ receptors (D₃R) belong to the same D₂-like receptor family as D₂R and D₄R subtypes, sharing with them a high level of homology.^1–6 Hypo- or hyper-alteration of D₃R functionality with consequent changes in brain plasticity is associated with multiple neuropsychiatric disorders.^7–9 D₃R have recently been targeted for therapeutic potential, especially directed toward substance use disorders, due to their discrete localization in the CNS.¹⁰ However, the development of small drug-like molecules able to activate (agonists and partial agonists) or block (antagonists) the signaling pathways regulated by these receptors, namely Gi/o-protein-mediated inhibition of adenylyl cyclase or β-arrestin recruitment, has remained a challenge.¹

The quest toward developing high affinity and selective D₃R antagonists has been one of the successful focuses of our research laboratory, as well as for multiple other academic labs and pharmaceutical companies, over the last decade.^{3, 8, 11–13} D₃R antagonists have been designed and pharmacologically evaluated, mainly for treatment of schizophrenia, psychosis, and substance use disorders.^{9, 14–16} Little success has been achieved in advancing these compounds towards large clinical trials or FDA approval. For example, due to the high receptor expression in both CNS and periphery, most of the antipsychotic drugs acting as D₂-like antagonists or partial agonists show significant side effects, including: cardiovascular hypertension,^17–18 and metabolic syndrome¹⁹ associated with the blockade of dopaminergic receptors expressed in the β-pancreatic islets.²⁰ Similarly, D₃R selective antagonists have been extensively assessed preclinically for the treatment of psychostimulant use disorders.^21–25 However, increases in blood pressure observed in dog models,²⁶ when combined with cocaine, halted the development of GSK598,809 for this patient population.²⁶ Nevertheless, we have recently demonstrated that new generations of D₃R antagonists may have potential in the treatment of opioid use disorders,¹¹ significantly reducing self-administration of opioids in animal models, without detectable abuse liability.^27-29

Various D₃R agonists have been developed for the treatment of Parkinson’s disease (PD) and its locomotor associated disorders.³⁰ Despite extensive efforts over the years, the design of D₂-like receptor selective agonists has proven to be an elusive challenge.^{2, 9, 31} As mentioned above, this is likely due to the high degree of homology between the receptors’ orthosteric binding sites (OBS) recognized by the endogenous agonist dopamine (DA) to initiate physiological signaling activation. Indeed, most of the agonists currently used in therapies or as tools for in vitro and in vivo drug development (i.e. Pramipexol, 7-OH-DPAT, ergoline derivatives, (+)-PD128,907 (1)) show very limited D₃R selectivity,⁸ with D₃R over D₂R selectivity of ~10-fold. This could not only partially explain the cross-activation side effects observed in in vivo studies but could also limit validation of studies where it is difficult to discern the effects from activation of similarly co-expressed receptor subtypes.

Additionally, assessing agonist affinity and selectivity in binding studies could be challenging and radioligand-probe dependent, as we have discussed previously.³² In fact, some of the high binding selectivities reported in the past for promising D₃R agonists often do not directly translate into functional studies; this may be a consequence of the radiotracer probes and assay conditions used in radioligand competition experiments.

With the goal of developing a new generation of improved D₃R selective agonists, we have applied a well-established bitopic molecular approach to our drug-design.^{11–12, 33–36} We have previously demonstrated how combining a primary pharmacophore (PP) (recognizing the OBS) with a secondary pharmacophore (SP) (inspired by privileged structural synthons able to bind a secondary binding pocket (SBP) or an allosteric binding site (ABS)) can generate compounds with high subtype affinity, selectivity, as well as unique pharmacological behaviors (allosterism or biased agonism) as a consequence of highly specific ligand-induced receptor conformations.^{12, 35–36} Among the multiple possible reasons for designing drugs targeting SBP and ABS, as mentioned before, one of the most obvious is selectivity. It is well known that GPCR OBS within receptor subfamilies activated by the same endogenous ligands are highly conserved, making it very difficult to develop drugs that can discriminate among them. ABS, SBP, and receptor pockets accommodating linkers with diverse chemical structures, can be more specific for each protein, thus improving the odds of increased subtype selectivities, when specifically targeted in drug design. We have already demonstrated that the presence of specific SPs, and connecting linkers, is the key to achieving affinity, selectivity and efficacy modulation; while the nature of the primary pharmacophore remains responsible for the retention of efficacy.^{11–12, 36}

In particular, bitopic ligands presenting well known OBS antagonist or partial agonist PPs, such as (2,3-dichlorophenyl)piperazine (e.g., aripiprazole, PG648, PG1037, BAK2–66),^{3, 35, 37} 1-(3-chloro-5-ethyl-2-methoxyphenyl)piperazine (e.g., eticlopride, VK4–116),¹¹ 1-(2-chloro-3-ethylphenyl)piperazine (e.g., VK4–40),¹¹ 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile (e.g., SB269,652),^38–40 all maintained the functional profiles of their PPs, with a variety of allosteric pharmacological behaviors specifically induced by their linkers and SPs. Analogously, D₂R selective bitopic ligands built around the full agonist PP (e.g., sumanirole),³² retained full agonism functionality with unique biased functional selectivity.³⁵⁻³⁶

Inspired by these findings, we selected two D₃R preferential full-agonists as our main PP scaffolds for chemical modification guided by structure-activity relationship (SAR) studies. Compound 1 is probably the most widely used D₃R agonist for in vitro and in vivo studies. Despite its moderate subtype selectivity, and some preliminary SAR already reported,⁴¹ its structure showed multiple potential positions for additional synthetic modifications (e.g., phenolic alcohol and morpholine ring - Figure 1). In addition, compound PF592,379 (2)⁴² was reported by Pfizer Research and Development in the early 2000s. As part of their work directed toward a new generation of D₃R selective agonists, based on aminopyridine derivatives, they developed therapeutic agents for the treatment of multiple female sexual dysfunctions (i.e. female arousal disorder, hypoactive sexual desire disorder and female orgasmic disorder), male erectile and ejaculatory dysfunctions, and pain.^42–43 Despite the discontinuation of its development, 2 caught our attention due to multiple functional groups in its core PP moiety which could be readily alkylated to generate the desired bitopic analogues (Figure 2), increasing its structural complexity to study multiple aspects of receptor-drug interactions. A similar approach, in modifying morpholine and/or aminopyridine nuclei of 2, has been used by both Pfizer and Merck to generate libraries of molecules and SAR, which further informed our work and the choice of the structural synthons.^44–45 Moreover, the available extensive pharmacokinetic studies on 2,⁴⁶ particularly focused on absorption routes, bioavailability, metabolic pathways and elimination, in rats, dogs and humans, enhanced our interest and provided valuable information for drug design, and future potential characterization of our identified leads. Drug self-administration and drug discrimination studies in rats,^47–48 also confirmed the lack of abuse potential for 2, which is an important requirement in the development of compounds enhancing dopaminergic signaling with potential therapeutic applications. Finally, the complex but efficient synthetic scheme reported for 2, and its derivatives,^{42–43, 45} was an excellent starting point, allowing us to chemically intervene in some of the key steps to obtain different chirality and regiochemistry according to our synthetic needs. Extensive stereochemical modifications were not pursued with 1, which is commercially available as the D₃R eutomer in the (4aR,10bR) absolute configuration, and thus this stereochemistry was retained for initial drug design and synthesis.

Figure 1.

Bitopic drug design based on 1 as the PP scaffold. The PPs are highlighted in red, the linker portion of the new bitopic analogues is highlighted in green and all the different SPs are highlighted in blue. The SAR studies were focused on i) O-alkylation of the phenolic region of 1, ii) structural simplification of the PP via removal of the morpholine ring, iii) preparation of 15 and subsequent N-alkylation at the morpholine nitrogen.

Figure 2.

Bitopic drug design based on the PP scaffold 2. The PP is highlighted in magenta, the linker portions of the new bitopic analogues are highlighted in green, and all the different SPs are highlighted in blue. The structural modifications of the PP focused on N-n-dipropylation at the aniline nitrogen, preparation of the nor-diastereoisomers and subsequent N-alkylation at the morpholine nitrogen with different linkers and SPs. The linkers used in the SAR studies, to combine PP and SPs, included: i) n-butyl, ii) E-butenyl, iii) cis- and trans- (methyl)cyclopropyl-methyl, and iv) trans-(methyl)cyclopropyl-ethyl. When necessary, to further investigate SAR, all stereochemical combinations of diastereoisomers and/or enantiomers were prepared.

As depicted in Figure 1, the bitopic drug design based on scaffold 1 focused on i) O-alkylation of the phenolic oxygen of 1, ii) structural simplification of the PP via removal of the morpholine ring, iii) preparation of the nor-analogue 15 and subsequent iv) N-alkylation of the morpholine nitrogen. Analogously, the structural modifications based on scaffold 2 as the PP (Figure 2) were directed towards i) N,N-dipropylation at the aniline nitrogen, ii) preparation of the nor-diastereoisomers and subsequent iii)N-alkylation of the morpholine nitrogen with the desired linkers and SPs. The linkers used in the SAR studies, to connect PP and SPs, included: i) n-butyl,^{3, 35–36} ii) E-butenyl,^{3, 49} iii) cis- and trans- (methyl)cyclopropyl-methyl,^{12, 50} and iv) trans-(methyl)cyclopropyl-ethyl.^{12, 50} The choice of linkers and SPs, as privileged synthons and molecular fragments, is a result of previous research which highlighted their essential role in modulating pharmacological properties of new compounds in unique directions, otherwise not obtainable via canonical agonist-OBS interaction.^{11–12, 36–37, 44, 50–51} When necessary, all stereochemical combinations of diastereoisomers and/or enantiomers were prepared.

All the newly synthesized compounds were tested in radioligand competition binding studies for on-target affinities (D₂R, D₃R, D₄R). Selected leads were further evaluated for metabolic stability in rat liver microsomes and metabolite identification to assess their applicability in future in vivo studies, in rodents.

The objective of this work was to generate new, small molecule, D₃R agonists with improved affinity, subtype selectivity, and high structural complexity (presence of multiple chiral centers fully resolved). These molecules and the SAR described will serve as tools for molecular biology, computational chemistry, and in vivo and in vitro pharmacology, to disentangle questions about D₃R dopaminergic signaling, otherwise difficult to address with the currently available ligands.

RESULTS AND DISCUSSION

Chemistry

We were interested in the potential of scaffold 1 as the PP and proceeded to synthesize a series of bitopic compounds with various canonical aromatic SPs. As shown in Scheme 1A, we initially envisioned connecting the linker at the phenolic oxygen through a standard alkylation with N-(4-bromobutyl)phthalimide in the presence of excess potassium carbonate under reflux. Deprotection of the resulting phthalimide (3) yielded primary amine 4 which was used as a common intermediate to achieve compounds 5-10 via standard EDC-mediated amide coupling with the appropriate carboxylic acid. Incorporation of the dihydroquinolin-2(1H)-one motif as SP (11) was achieved via alkylation of the phenol with 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one under standard basic conditions with potassium carbonate at reflux (Scheme 1B).

Scheme 1.:

a) N-(4-Bromobutyl)phthalimide, KI, K₂CO₃, ACN, reflux; b) NH₂NH_2, EtOH, reflux; c) DIPEA, EDC, HOBt, ArCOOH, 0 °C to RT; d) 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one, KI, K₂CO₃, ACN, reflux.

We envisioned the linker could also connect to scaffold 1 at the morpholine nitrogen via reductive amination, effectively substituting the propyl side chain of 1 with the linker and SP of choice. Aldehyde 14 was thus synthesized from the commercially available dihydroquinolin-2(1H)-one through alkylation of the phenol with (4-bromobutoxy)(tert-butyl)dimethylsilane and potassium carbonate, followed by deprotection of the (tert-butyl)dimethylsilane (TBDMS) group in 12, and Dess-Martin oxidation of the resulting primary alcohol 13 to yield the precursor aldehyde 14 (Scheme 2A). We then proceeded to remove the propyl side chain of 1 by treatment with cyanogen bromide under exposure to microwave irradiation at elevated temperature and pressure (120 °C, 275 psi) to yield 15 (Scheme 2B). Compound 16 was readily prepared, starting from 14 and 15, via reductive amination with sodium triacetoxyborohydride (STAB) and catalytic acetic acid. Substitution at the N position in 16 was detrimental to the affinity of the 1 scaffold (Table 1) and therefore no more analogues were attempted.

Scheme 2.:

a) (4-bromobutoxy)(tert-butyl)dimethylsilane, KI, K₂CO₃, ACN, reflux; b) 1M TBAF, THF, RT; c) Dess-Martin periodinane (DMP), DCM, 0 °C to RT; d) i) BrCN, K₂CO₃, acetonitrile, microwave 120°C, 275 psi, 75W; ii) 37% HCl/H₂O e) cat. AcOH, Na(OAc)₃BH, 14.

Table 1.

Binding affinity data for compounds derived from 1 primary pharmacophore. Radioligand competition binding assays performed on HEK293 cells stably expressing hD₂R, hD₃R, and hD₄R in presence of [³H]-(R)-(+)-7-OH-DPAT.

vs. [3H]-(R)-(+)-7-OH-DPAT
Compound	Structure	D2R Ki ± SEM [nM]	D3R Ki ± SEM [nM]	D4R Ki ± SEM [nM]	D2R/D3R	D4R/D3R
Pramipexole		11.1 ± 0.532 (n=3)	1.32 ± 0.200 (n=5)	ND	8.41	ND
1		20.5 ± 2.87 (n=6)	1.69 ± 0.0892 (n=8)	26.6 ± 3.97 (n=4)	12.1	15.7
3		2,510 ± 553 (n=3)	1,190 ± 151 (n=3)	ND	2.11	ND
5		6,710 ± 741 (n=3)	806 ± 185 (n=5)	ND	8.33	ND
6		3,860 ± 975 (n=3)	613 ± 50.6 (n=3)	ND	6.30	ND
7		9,450 ± 3,130 (n=3)	6,010 ± 1,540 (n=4)	ND	1.57	ND
8		5,510 ± 182 (n=3)	1,330 ± 263 (n=3)	ND	4.14	ND
9		9,160 ± 276 (n=3)	2,130 ± 402 (n=3)	ND	4.30	ND
10		27,000 ± 11,400 (n=3)	8,660 ± 3,660 (n=3)	ND	3.12	ND
11		10,100 ± 3,590 (n=3)	1,590 ± 30.9 (n=3)	ND	6.35	ND
15		4,250 ± 1,320 (n=3)	403 ± 5.55 (n=3)	ND	10.5	ND
16		6,260 ± 179 (n=3)	11,300 ± 1,070 (n=3)	ND	0.554	ND
19a		245 ± 32 (n=3)	354 ± 45.1 (n=3)	ND	0.710	ND
19b		1,430 ± 210 (n=3)	1,770 ± 241 (n=3)	ND	0.808	ND
57		1.86 ± 0.591 (n=3)	40.9 ± 12.9 (n=3)	ND	0.0455	ND
58		0.844 ± 0.163 (n=3)	2.42 ± 0.340 (n=3)	ND	0.349	ND

Equilibrium dissociation constants (K_i) were derived from IC₅₀ values using the Cheng–Prusoff equation. Each K_i value represents the arithmetic mean ± S.E.M; n = number of independent experiments, each performed in triplicate. ND = Not Determined.

Intrigued by the prospect of testing structurally simplified analogues of 1, we proceeded to synthesize 19, where the morpholine ring was removed while retaining the oxygen and nitrogen heteroatoms in the same position to preserve their H-bond accepting activity (Scheme 3). Intermediate 17 was synthesized via a Neber rearrangement from the commercially available 6-methoxy-4-chromanone as reported in the literature.^52–53 Subsequent HBr assisted O-demethylation of 17 afforded 18, which was used to form compound 19 via reductive amination with N-(4-oxobutyl)-1H-indole-2-carboxamide.³⁶ The enantiomers of the resulting analogue (enantiomeric separation achieved via preparative chiral HPLC as described in the methods section) exhibited decreased affinity in radioligand binding studies relative to the parent compound (1), therefore no further analogues were synthesized.

Scheme 3.:

a) 48% HBr in water, reflux; b) AcOH, Na(OAc)₃BH, N-(4-oxobutyl)-1H-indole-2-carboxamide,³⁶ DCE, RT.

Relaying our efforts to scaffold 2,^42–44 we began by examining substitution at the aniline nitrogen (Scheme 4A) where the aniline was dialkylated using propionaldehyde and STAB at reflux. Substitution at the aniline nitrogen showed a detrimental effect on the binding profile of pharmacophore 2 (Table 2) redirecting us to the morpholine nitrogen for further manipulations (Scheme 4B). Starting from the nor-21 mixture of diastereomers, achieved in a ~3:1 mixture of (2R,5S):(2S,5S) by reported synthetic methods,⁴² 22 was synthesized via traditional alkylation with 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one and potassium carbonate at reflux. Encouraged by the affinity of 22, the two diastereomers of nor-21, 23a and 23b, were separated via flash chromatography and used independently thereafter. Their diastereomeric purity was assessed via chiral analytical HPLC (Figure S1), meanwhile their absolute stereochemistry was confirmed via X-ray crystallography (Figure 3B) of 23a, and by comparison with the previously reported spectroscopic data.⁴² Starting from the two pure diastereomers, their respective bitopic compounds were synthesized in parallel. Compounds 24 and 26 were synthesized from their respective nor-21 diastereomer via alkylation, analogous to 22 (chiral HPLC chromatograms in Figure S2). Compounds 25 and 27 were constructed via reductive amination with N-(4-oxobutyl)-1H-indole-2-carboxamide³⁶ (chiral HPLC chromatograms in Figure S3).

Scheme 4.:

a) AcOH, Na(OAc)₃BH, propionaldehyde, ACN, reflux; b) Resolution via flash chromatography; c) 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one, KI, K₂CO₃, ACN, reflux; d) AcOH, Na(OAc)₃BH, N-(4-oxobutyl)-1H-indole-2-carboxamide,³⁶ DCE, RT.

Table 2.

Binding affinity data for compounds derived from 2 primary pharmacophore. Radioligand competition binding assays performed on HEK293 cells stably expressing hD₂R and hD₃R, in presence of [³H]-(R)-(+)-7-OH-DPAT.

vs. [3H]-(R)-(+)-7-OH-DPAT
Compound	Structure	D2R Ki ± SEM [nM]	D3R Ki ± SEM [nM]	D4R Ki ± SEM [nM]	D2R/D3R	D4R/D3R
2		1,740 ± 348 (n=5)	185 ± 20.5 (n=8)	292 ± 22.2 (n=3)	9.41	1.58
20		8,240 ± 1,670 (n=3)	21,100 ± 2,310 (n=5)	>100,000	0.391	>4.74
22		113 ± 22.7 (n=4)	173 ± 48.2 (n=5)	3,220 ± 842 (n=3)	0.653	18.6
23a		7,100 ± 402 (n=3)	1,520 ± 183 (n=3)	ND	4.67	ND
23b		2,340 ± 458 (n=3)	424 ± 32.4 (n=3)	ND	5.52	ND
24		2,660 ± 370 (n=3)	24,200 ± 4,500 (n=3)	ND	0.110	ND
26		34.6 ± 4.86 (n=3)	31.2 ± 8.72 (n=3)	ND	1.11	ND
25		5,220 ± 735 (n=3)	6,470 ± 675 (n=3)	ND	0.807	ND
27		134 ± 21.6 (n=4)	5.96 ± 0.477 (n=4)	357 ± 71.8 (n=3)	22.5	59.9
30		21,100 ± 2,100 (n=3)	73,000 ± 2,810 (n=3)	ND	0.289	ND
31		341±27.6 (n=3)	1,150±210 (n=3)	ND	0.297	ND
rac-trans-36		14,100 ± 1,790 (n=3)	66,200 ± 25,600 (n=3)	ND	0.213	ND
trans-37a		216 ± 32.6 (n=3)	1,190 ± 165 (n=3)	ND	0.182	ND
trans-37b		707 ± 19.8 (n=3)	404 ± 34 (n=3)	ND	1.75	ND
cis-38a		1,070 ± 40.9 (n=3)	2,150 ± 159 (n=3)	ND	0.498	ND
cis-38b		120 ± 16.6 (n=3)	276 ± 30 (n=4)	ND	0.435	ND
cis-39a		11,300 ± 2,280 (n=3)	26,200 ± 2,520 (n=3)	ND	0.431	ND
cis-39b		7,730 ± 2,070 (n=3)	32,900 ± 2,700 (n=3)	ND	0.235	ND
rac-trans-53		106 ± 7.94 (n=5)	2.84 ± 0.462 (n=5)	315 ± 16.2 (n=3)	37.3	111
53a		87.8±9.81 (n=3)	1.85±0.137 (n=3)	286 ± 23.9 (n=3)	47.5	155
53b		831±99.5 (n=3)	282±24 (n=3)	2,930 ± 719 (n=3)	2.95	10.4

Equilibrium dissociation constants (K_i) were derived from IC₅₀ values using the Cheng–Prusoff equation. Each K_i value represents the arithmetic mean ± S.E.M; n = number of independent experiments, each performed in triplicate. ND = Not Determined.

Figure 3.

A) X-Ray crystal structure of (−)-(1R,2S)-41a [C8(R)-C6(S)]; B) X-ray crystal structure of 23a. The assigned absolute configuration for (2R,5S)-23a [C8(R)-C11(S)], and its spectroscopic data matches with the literature.⁴²

Having confirmed the privileged stereochemistry of the morpholine ring (Table 2) with the compounds in Scheme 4, we proceeded to examine the effect of structural changes on the linker. Initial efforts were aimed at synthesizing compounds 30 and 31, the unsaturated analogues of 24 and 26, respectively. As shown in Scheme 5, allyl bromide was dimerized via cross olefin metathesis using Hoveyda-Grubbs catalyst 2^nd generation to form (E)-1,4-dibromobut-2-ene, 28, in 9:1 (E:Z) ratio.⁵⁴ Alkylation of 3,4-dihydroquinolin-2(1H)-one at the phenolic oxygen was accomplished with a slight excess of dibromobutene under gentle reflux conditions with potassium carbonate to yield 29. Lastly, 30 and 31 were achieved from alkylation of 23a and 23b with 29, respectively (chiral HPLC chromatograms in Figure S4). The E stereochemistry of the olefin in the linker was further confirmed through Nuclear Overhauser Effect (N.O.E.) analysis (Figures S5 and S6).

Scheme 5.:

a) HG-II Grubb’s catalyst, DCM; b) 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one, KI, K₂CO₃, ACN, reflux; c) 23a or 23b, K₂CO₃, ACN, reflux.

We were also interested in investigating the addition of a cyclopropyl ring to the linker. As reported in Scheme 6, we began by constructing a 4-carbon linker with a cyclopropyl ring separated by one methylene unit from both pharmacophores. Similarly to what has been previously described,^{12, 55–56} starting from the mixture of isomers of the ethyl 2-cyanocyclopropanecarboxylate, reduction with lithium aluminum hydride afforded amino alcohol 32, which was then subjected to EDC-mediated amide coupling with indole-2 carboxylic acid, yielding 33, establishing the SP in the desired position and preparing the alcohol necessary for the aldehyde intermediate. Previous efforts at accessing the separate cis and trans indole-cyclopropyl-alcohol intermediate in 33 have relied on distillation of the ethyl 2-cyanocyclopropanecarboxylate starting material to separate the cis and trans cyclopropyl moieties,^{12, 55–56} however we observed that the cis and trans 33 were readily separable by standard flash chromatography, allowing the use of the racemic ethyl 2-cyanocyclopropanecarboxylate starting material and circumventing less efficient distillation procedures. Having separated cis and trans cyclopropyl intermediates, we continued to prepare the desired aldehyde by Dess-Martin oxidation of the alcohol to achieve the exclusively trans-34 and cis-35 aldehydes. Reductive amination of the cis and trans aldehydes with 23a and 23b yielded four sets of diastereomers: 36, 37, 38, and 39. Compounds 36 and 39 have a fixed (2R,5S) stereochemistry at the morpholine ring, and both consist of two sets of enantiomers at the cyclopropyl ring: 36 the two trans-cyclopropyl enantiomers, 39 the two cis-cyclopropyl enantiomers. The same is true for compounds 37 and 38, however the morpholine ring in these is fixed with the (2S,5S) stereochemistry. To extend our study of the effect of the structural configuration of the linker on the affinity of these bitopic ligands, when possible, we proceeded to separate the enantiomeric pairs across the cyclopropyl ring for 37, 38, and 39. Using chiral preparative HPLC, these three diastereomeric sets were separated to yield the individual enantiomers. Six of the total eight possible enantiomers (one chiral center was fixed allowing for a total of eight enantiomers) were thus isolated (analytical chiral HPLC chromatograms in Figure S7). Compound 36, due to the challenges in achieving sufficient enantiomeric separation via preparative HPLC and being aware of the unfavorable conformation of its (2R,5S)-morpholine ring for binding pose, was tested as trans diastereomeric mixture (Figure S7). Due to the moderate affinities observed (Table 2), absolute stereochemistry of the individual enantiomers was not explored. Previous studies by our group had revealed that addition of the cyclopropyl ring to the linker may render the four-carbon length too short, and incorporation of an additional methylene unit between the cyclopropyl ring and SP yielded improved D₃R binding selectivities, and also produced intriguing pharmacological allosteric properties.¹² Consequently, the results from compounds 36, 37, 38, and 39 suggested that the homologated analogues of these compounds had great potential as lead targets.

Scheme 6.:

a) LiAlH₄, THF, 0 °C to RT; b) Indole-2-carboxylic acid, DIPEA, EDC, HOBt, 0 °C to RT; c) diastereoisomers separation via flash chromatography; d) Dess-Martin periodinane (DMP), DCM, 0 °C to RT; e) cat. AcOH, Na(OAc)₃BH, 23a or 23b, DCE; f) Chiral resolution via preparative chiral HPLC (AD-H column) or flash chromatography.

Binding data collected from compounds 24, 25, 26, 27, 30, and 31 had conclusively shown the (2S,5S) stereochemistry at the morpholine to be significantly privileged over the (2R,5S) stereochemistry. As a result, we were poised to optimize a new diastereoselective synthetic strategy that would access the desired (2S,5S) diastereomer, 23b, selectively, given the current synthetic strategy to access the nor-21 scaffold yielded a 3:1 ratio in favor of the undesired diastereomer.⁴² Rapid access to large amounts of the desired (2S,5S) diastereomer became particularly important as we endeavored to develop a new set of compounds containing a five carbon-length linker with a cyclopropyl ring. Thus, we adapted the diastereoselective synthetic scheme already reported for the synthesis of the (2R,5S)-diatereoisomer,⁴² toward obtaining the desired (2S,5S) absolute configuration.

Our new diastereoselective approach to access 23b (Scheme 8A) features the enantioselective reduction of α-chloroketone 2-chloro-1-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)ethan-1-one, synthesized in two steps from readily available materials,⁴² with (+)-DIP-Cl to yield 47 selectively in a 97.5:2.5 enantiomeric ratio as determined by analytical chiral HPLC (Figure S8). This reduction step allowed the installation of the right stereochemistry at the key chiral center in 23b. Compound 47 was then treated with potassium carbonate to yield epoxide 48 quantitatively, and then achieve 49 via epoxide opening with L-Alaninol ((S)-(+)-2-aminopropanol). Formation of the intermediate epoxide 48 was found to be crucial for the successful nucleophilic attack of the L-Alaninol amine, nucleophilic displacement of the chlorine did not occur under any conditions tested with 47 and L-Alaninol. With 49 successfully synthesized, both key chiral centers of 23b had been established with the desired S stereochemistry. Following Cbz protection of the secondary amine in 49 with Cbz-succinimide, the morpholine ring was closed under traditional Mitsunobu conditions to displace the less hindered primary alcohol and yield the fully constructed scaffold of 23b. Deprotection of the 2,5-dimethylpyrrole protecting group with hydroxylamine hydrochloride, followed by deprotection of the Cbz group via Pd(OH)₂/C catalyzed hydrogenation yielded 23b. With this rapid diastereoselective route, we could now proceed to synthesize the desired bitopic compounds with a five carbon cyclopropyl linker.

Scheme 8.:

a) (+)-DIP, DCM, −40 °C to RT; b) K₂CO₃, ACN, reflux; c) (S)-2-aminopropan-1-ol, toluene, reflux; d) N-(Benzyloxycarbonyloxy)succinimide, THF, −40 °C to RT; e) DIAD, PPh₃, THF, RT; f) hydroxylamine hydrochloride (NH₂OH^.HCl), EtOH, reflux; g) H₂ (50 psi), Pd/C; h) cat. AcOH, Na(OAc)₃BH, N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide,^{12, 36} DCE; i) Chiral resolution via preparative chiral HPLC-ADH; j) Chiral resolution via preparative chiral HPLC-ADH; k) Dess-Martin periodinane (DMP), DCM, 0 °C to RT; l) cat. AcOH, Na(OAc)₃BH

Our group had previously shown the trans conformation across the cyclopropyl ring to be favorable over the cis conformation for an array of bitopic compounds.¹² As a result, we began by synthesizing the racemic trans bitopic compound 53 (Scheme 8A). Starting with the trans aldehyde N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide, synthesized selectively with trans stereochemistry, from our previously reported procedure,³⁶ standard reductive amination conditions with 23b gave us access to 53 in good yields. We further separated the trans-53 racemic mixture with preparative chiral HPLC to isolate the two trans cyclopropyl enantiomers 53a and 53b (analytical chiral HPLC chromatograms in Figure S9).

The excellent affinity and selectivity exhibited by the 53a over 53b prompted us to determine the absolute stereochemistry of these two diastereoisomers. We identified the absolute stereochemistry of the (−)-(1R,2S)-41a intermediate, isolated via chiral HPLC resolution (analytical chiral HPLC chromatograms in Figure S10), in the synthesis of the linker (Scheme 7), by X-ray crystallography (Figure 3A). As a result we had HPLC and optical rotation information of the two trans enantiomers of alcohols (+)-(1S,2R)-43 and (−)-(1R,2S)-44 (analytical chiral HPLC chromatograms in Figure S11), and aldehydes (−)-(1S,2R)-45 and (+)-(1R,2S)-46 (Scheme 7). Separation of the two trans enantiomers could also be readily accomplished for the alcohol intermediate 54 (Scheme 8B), which was separated via preparative chiral HPLC into (+)-43 and (−)-44 (analytical chiral HPLC chromatograms in Figure S12), from which the absolute stereochemistry of both was determined comparing to reported analytical HPLC (Figure S12) and specific optical rotation, with the same compounds prepared as in Scheme 7. Knowing the absolute configuration of both enantiomers of alcohol 54 (Scheme 8B), (+)-43 and (−)-44 were converted to the corresponding aldehydes by Dess-Martin oxidation, followed by reductive amination with 23b to yield 53a,b, with the stereochemistry at all four chiral centers fully resolved and assigned (Scheme 8B). The use of analytical HPLC was essential at every step of this sequence to ensure the correct stereochemistry was assigned (Figures S13 and S14).

Scheme 7.:

a) CH₃NO₂, t-BuOLi, t-BuOH/THF, 0°C to RT; b) MsCl, TEA, DCM, 0°C to RT, followed by preparative chiral HPLC resolution; c) LAH, THF, 0°C to reflux; d) EDC, HOBt, DIPEA, THF, 0°C to RT; e) DMP, DCM, RT.

Further, we wished to investigate the importance of the PP in the activity observed by these bitopic molecules. Thus, as additional SAR investigations on the nature of the PP to induce agonist or antagonist profiles, we proceeded to synthesize bitopic molecules containing the same initial linker and SP used in scaffolds 1 and 2, with PPs widely characterized as D₃R selective antagonists.¹¹ As shown in Scheme 9, from standard alkylation of two substituted phenylpiperazines with 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one, we synthesized compounds 57 and 58, respectively.

Scheme 9.:

a) K₂CO₃, N,N-DMF, 90 °C.

Radioligand Binding Data

The affinities of the new compounds were tested by radioligand competition binding assays at hD₂R, hD₃R and hD₄R using the agonist [³H]-(R)-(+)-7-OH-DPAT as the radiotracer. We have previously demonstrated and established how competition against an agonist radioligand allows a more accurate determination of affinities for novel unlabeled D₂-like agonists.^{31–32, 57} Importantly, affinities of D₂-like agonists and partial agonists, when determined in competition against an agonist radioligand probe, reflect binding the receptors in their “active” state. The D₃R preferential agonists pramipexole, 1 and the diastereomeric mixture of 2 (synthesized as reported⁴²) were tested in parallel with the new bitopic analogues, and in the same assay conditions, to allow a direct comparison of affinities and receptor subtype selectivity.

SAR based on 1 as primary pharmacophore

Consistent with the literature, 1 showed low nanomolar affinity for D₃R (K_i = 1.69 nM), but only a moderate 12-fold selectivity over D₂R. A very similar profile was observed for pramipexole as well, (D₃R K_i = 1.69 nM; D₂R/D₃R = 8.41), confirming the need for more selective D₃R agonists as pharmacological tools for in vitro and in vivo studies. Our studies proved that every structural modification that was made to scaffold 1 markedly reduced or completely prevented binding at either D₂R and/or D₃R (Table 1). Indeed, the phenolic –OH is essential for high D₃R binding affinity, since all the O-alkylated derivatives completely lost affinity (D₃R K_is ranging from 613 nM to 11,300 nM) and subtype selectivity (D₂R/D₃R ratios <10-fold), independent from the structure of the SPs. Analogously, the tertiary basic center is also essential for high affinity at D₃R of 1. Indeed, the nor-analogue 15 showed >200-fold reduced affinity at both D₂R and D₃R, despite maintaining a similar 10-fold selectivity over D₂R. The replacement of the n-propyl chain, with the n-butyl linker and SP was also detrimental. Particularly, introducing the 3,4-dihydroquinolin-2(1H)-one, which is known to be a D₂-like privileged SP,³⁵ present in the structure of the D₂R partial agonist aripiprazole, resulted in complete loss of affinity (16; K_is >5,000 nM at both D₂R and D₃R) confirming that 1 is a privileged pharmacophore with structural features that cannot be altered to pursue more complex derivatives in its currently available geometrical configuration. Even the simplification of the scaffold, by removal of the morpholine ring, as depicted in the enantiomers 19a and 19b, induced >100–1,000-fold loss of affinity at both receptor subtypes, independently from the absolute configuration of the stereocenter. However, in the design of bitopic antagonists, alkylation of the phenyl-piperazine basic nitrogen with different eticlopride (known potent D₂/D₃ orthosteric antagonist) inspired PPs¹¹ seems to be better tolerated. Indeed, both compounds 57 and 58, sharing the same 3,4-dihydroquinolin-2(1H)-one SP, showed high affinities at both receptor subtypes (57: D₂R K_i = 1.86 nM, D₃R K_i = 40.9 nM; 58: D₂R K_i = 0.844 nM, D₃R K_i = 2.42 nM), and 57 presented 22-fold selectivity for D₂R over D₃R. This selectivity profile also confirmed that the 3,4-dihydroquinolin-2(1H)-one SP promotes better recognition of the D₂R, as observed in previously reported aripiprazole-based SARs.^{35, 58–59}

SAR based on 2 as the PP

Compound 2 was presented for the first time by Pfizer, as part of a new series of selective D₃R agonists, based on variously substituted aminopyridines, as potential therapeutic agents for the treatment of female sexual dysfunctions.⁴² Pfizer explored the structural requirements of the orthosteric PP scaffold of 2 in SAR studies based on regiochemical substitutions on the morpholine ring, modulation of the basic nature of the morpholine by converting it to cyclic amides, as well as introducing alkyl and aromatic groups in both aniline and morpholine nitrogen, respectively.^42–44 Compound 2 presents two chiral centers in the morpholine moiety, and Pfizer focused their initial work on the development of the (2R,5S)-enantiomer. In order to better evaluate the significance of the chirality on the morpholine ring and understand how the new extended bitopic molecules would change the stereochemical requirements for optimal recognition of the D₃R OBS and SBP, we prepared and tested all the possible diastereomeric combinations.

We synthesized and tested 2 as a 3:1 diastereomeric mixture of (2R,5S) and (2S,5S), following the synthetic schemes previously described.⁴² The affinity observed in our binding assays was consistent with those previously reported,⁴⁵ confirming a moderate D₃R affinity (K_i = 185 nM) and a preferential selectivity for D₃R over D₂R (9.4-fold D₃R selective). Although its D₃R affinity was not comparable to 1, 2 showed more versatility in terms of chemical modifications and its lower affinity and selectivity implied significant room for pharmacological improvements. In order to identify the positions that better tolerated substitutions, the affinities of 20 (presenting n-dipropyl substituents on the aniline nitrogen) and the two nor-diastereoisomers (2R,5S)-23a and (2S,5S)-23b were assessed and compared. As expected, alkylation of the aniline nitrogen resulted in complete loss of D₂-like affinity (K_i >5,000 nM and >10,000 nM for D₂R and D₃R, respectively). De-alkylation of the morpholine nitrogen also decreased affinity for both receptor subtype of ~3–9 fold, suggesting that a tertiary nitrogen is preferred. However, the first interesting observation was that among the two nor-analogues, the (2S,5S)-23b diastereoisomer was the one showing a slightly higher affinity for D₃R (K_i = 424 nM), giving a first indication of a preferential enantio-specificity. The importance of the morpholine ring chirality and its high enantio-specificity in binding D₃R was highlighted in a definitive manner by the affinity profiles of all the newly prepared bitopic analogues. Indeed, when comparing couples of diastereoisomers (2R,5S)-24 and (2S,5S)-26 (presenting the 3,4-dihydroquinolin-2(1H)-one SP), and (2R,5S)-25 and (2S,5S)-27 (presenting the 2-indole butylamide SP), we observed how the (2S,5S) stereochemistry is privileged for high D₃R affinity. Particularly, (2S,5S)-27 showed low nanomolar D₃R affinity (K_i = 5.96 nM), >1,000-fold higher when compared to its (2R,5S)-25 inactive diastereoisomer, and 30-fold higher when compared to the parent molecule 2. This observation not only confirmed the success of the bitopic molecular approach in achieving high affinity, but (2S,5S)-27 presented an improved D₃R subtype selectivity (22.5-fold selective over D₂R) and underscored the significance of the stereochemistry for the morpholine ring position-2 in order to obtain optimal binding interactions. Similarly, (2S,5S)-26 has a D₃R affinity 775-fold better with respect to its (2R,5S)-24 diastereoisomer, but an overall lack of subtype selectivity, consistent with the presence of 3,4-dihydroquinolin-2(1H)-one SP, which, as discussed above, improves D₂R subtype binding.

Having identified the best stereochemical combination for the PP, we focused our attention on the nature of the linker portion of the molecules. We have published extensively on the role of the linkers as critical modulators of the pharmacological profiles of bitopic ligands.^{11–12, 35–37, 49} We assessed how linkers differing in length, rigidity and chirality can help improve binding affinities, receptor subtype selectivity, and enhance allosteric properties and biased agonism. Among multiple structural manipulations of linkers, introduction of rigid elements, such as a double bond or cyclopropyl ring are some of the most well-studied functionalizations used in developing SARs.⁵⁰ In this new series of analogues, the presence of a trans double bond resulted in a complete loss of binding at both D₂R and D₃R ((2R,5S)-30 and (2S,5S)-31). However, we were intrigued by assessing the potential of a cyclopropyl ring in the linker. The addition of either cis or trans cyclopropyl rings, introduced two extra chiral centers in the bitopic compounds, increasing the total number of the possible diastereoisomers, but allowing a detailed screening of the spatial requirements needed to maximize ligand-receptor interactions. Consistent with previous studies performed on D₃R antagonists, when the cyclopropyl was embedded in a butyl chain,¹² the linker was too short for optimal binding, and all the compounds, independent from the stereochemical combinations tested, resulted in moderate or poor D₃R affinity and selectivity. However, the resolved diastereoisomers rel-trans-(2S,5S)-37a and rel-trans-(2S,5S)-37b, despite their relatively low D₃R affinity, showed some indications that trans-cyclopropyl is the preferred binding configuration, and that among the two trans enantiomers there might be enantio-specificity towards receptor subtype recognition. Indeed, trans-(2S,5S)-37a showed a slightly preferential (~5-fold) affinity for D₂R (K_i = 216 nM), meanwhile trans-(2S,5S)-37b preferentially binds D₃R (K_i = 404 nM). Based on these observations, extending the cyclopropyl linker by one extra methylene unit, reaching the optimal linker length and regiochemistry,^{12, 36} successfully yielded rel-trans-(2S,5S)-53, which showed a significantly improved D₃R affinity (K_i = 2.84 nM) and selectivity over D₂R (D₂R/D₃R = 37.3).

To further disentangle the importance of the chirality of the cyclopropyl ring, its two enantiomers were resolved; compounds obtained presented four chiral centers with all the absolute configurations assigned. Binding studies on these analogues provided critical insight on how specific stereochemistry at the PP, as well as in the linker, are essential to achieve the highest affinities in this new series of compounds, and the highest D₃R selectivity observed for bitopic agonists. Compound 53a, presenting (2S,5S)-PP configuration and (1R,2S)-trans-cylopropyl stereochemistry, showed D₃R K_i = 1.85 nM and an unprecedented 47.5-fold selectivity for D₃R over D₂R (D₂R K_i = 87.8 nM). Instead, its (1S,2R)-trans-cyclopropyl diastereoisomer 53b, showed 152-fold reduced affinity for D₃R (K_i = 282 nM) and complete loss of selectivity (D₂R K_i = 831 nM, D₂R/D₃R = 2.95). Moreover, competition binding experiments for 53, 53a, 53b and 27 in the presence of antagonist [³H]-N-methylspiperone (similar assay conditions to previously described protocols)^{11, 13, 31} provided initial evidence of the new compounds’ agonist profile. Indeed, their affinities were significantly reduced for all D₂-like receptor subtypes, and their D₃R selectivity increased, (53: D₂R K_i = 8,280 ± 462 nM (n=3), D₃R K_i = 34 ± 2.23 nM (n=3), D₄R K_i = 6,650 ± 662 nM (n=3), D₂R/D₃R = 244, D₄R/D₃R = 196; 53a: D₂R K_i = 1,610 ± 94 nM (n=3), D₃R K_i = 5.36 ± 0.522 nM (n=3), D₄R K_i = 1,520 ± 109 nM (n=3), D₂R/D₃R = 300, D₄R/D₃R = 284; 53b: D₂R K_i = 70,600 ± 15,900 nM (n=3), D₃R K_i = 2,900 ± 463 nM (n=3), D₄R K_i = 62,700 ± 25,600 nM (n=3), D₂R/D₃R = 24, D₄R/D₃R = 22; 27: D₂R K_i = 21,000 ± 1,160 nM (n=3), D₃R K_i = 182 ± 28.3 nM (n=3), D₄R K_i = 16,800 ± 3,470 nM (n=3), D₂R/D₃R = 115, D₄R/D₃R = 92) when compared to the K_i obtained in competition versus agonist [³H]-(R)-(+)-7-OH-DPAT (Table 2). This is consistent with our extensive experimental observations^{32, 35–36} on how agonists cannot compete in the same way with antagonist radiotracers, thus, as we highlighted, the need for accurate assay conditions to determine affinity and receptor subtype selectivities, which could be otherwise under- and over-estimated, respectively. Moreover, these data seem to suggest that while D₂R and D₃R adopt different antagonist-bound conformations, facilitating the development of selective antagonists, the higher similarity of their agonist-bound states is reflected in the inherent difficulty of developing subtype selective agonists.

Phase I metabolic stability of 53 in rat liver microsomes and metabolites identification.

Compound 53 presented affinity and selectivity profiles very similar to the eutomer 53a, thus it was selected for metabolic stability evaluation, and to be considered as a candidate for future in vivo studies. Compound 53 showed moderate stability to Phase I metabolism in rat liver microsomes fortified with NADPH with 53±1% intact remaining at 60 minutes, post incubation (Figure 4A). The compound shows complete stability controls without NADPH suggesting specific CYP-dependent metabolism. Lastly, the metabolites formed due to Phase I metabolism were identified using high resolution mass spectrometry and are reported in Figure 4B.

Figure 4.

(A) Metabolic Stability of 53. The compound shows moderate instability to Phase I metabolism in rat liver microsomes fortified with NADPH. Compound 53 shows complete stability in negative control without NADPH. (B) Metabolite Identification of 53 following phase I metabolic stability using high resolution mass spectrometry. The major phase I metabolite was determined to be the hydroxylated product (m/z=450.2498, *Note the position assigned to the hydroxyl group is arbitrary). Minor metabolites were identified to be N-dealkylation (m/z= 194.1288) and the oxidation (m/z=432.2293) products.

CONCLUSIONS

Herein we demonstrated how SAR studies based on well-defined stereochemistry led to the identification of a new high affinity and selectivity D₃R agonist 53a. In particular, we observed that: i) 1 is a unique compound, and in its current structural conformations cannot be altered or simplified to pursue more complex derivatives, ii) a complete inversion of configuration at the chiral center in position-2 is essential to successfully develop new bitopic ligands based on 2 ((2S,5S)- is the only morpholine ring configuration tolerated, for optimal D₃R binding, by all the bitopic analogues studied); iii) cyclopropyl linkers optimized in their length, relative regiochemical positions of PP and SP, relative and absolute stereochemistry ((1R,2S) is the privileged cyclopropyl configuration) can dramatically modulate pharmacological profiles.

The high molecular complexity of 53a, combined with its fully resolved tridimensional structure, and unprecedented D₃R agonist affinity/selectivity, will undoubtedly aid computational studies to better understand D₃R ligand-receptor interactions, as well as underscore potential biased agonism as a consequence of specific receptor conformations.

Moreover, due to the large degree of homogeneity among the D₂-like family of dopamine receptors and consequent lack of selective agonists, 53 and the eutomer 53a, presenting the highest D₃R to D₂R binding selectivity reported for agonists, to date, may have the potential to become the main pharmacological reference tools for future D₃R in vitro and/or in vivo studies, taking into account the promising metabolic stability of 53 in rat liver microsomes.

EXPERIMENTAL METHODS

Chemistry

All chemicals and solvents were purchased from chemical suppliers unless otherwise stated and used without further purification. All melting points were determined on an OptiMelt automated melting point system and are uncorrected. The ¹H and ¹³C NMR spectra were recorded on a Varian Mercury Plus 400 instrument. Proton chemical shifts are reported as parts per million (δ ppm) relative to tetramethylsilane (0.00 ppm) as an internal standard, or to deuterated solvents. Coupling constants are measured in Hz. Chemical shifts for ¹³C NMR spectra are reported as parts per million (δ ppm) relative to deuterated CHCl₃ or deuterated MeOH (CDCl₃ 77.5 ppm, CD₃OD 49.3 ppm). Chemical shifts, multiplicities and coupling constants (J) have been reported and calculated using Vnmrj Agilent-NMR 400MR or MNova 9.0 software. Gas chromatography-mass spectrometry (GC/MS) data were acquired (where obtainable) using an Agilent Technologies (Santa Clara, CA) 7890B GC equipped with an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. × 0.25 μm film thickness) and a 5977B mass-selective ion detector in electron-impact mode. Ultrapure grade helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port and transfer line temperatures were 250 and 280 °C, respectively, and the oven temperature gradient used was as follows: the initial temperature (70 °C) was held for 1 min and then increased to 300 °C at 20 °C/min and maintained at 300 °C for 4 min, total run time 16.5 min. Column chromatography was performed using a Teledyne Isco CombiFlash RF flash chromatography system, or a Teledyne Isco EZ-Prep chromatography system. Preparative thin layer chromatography was performed on Analtech silica gel plates (1000 μm). Preparative chiral HPLC was performed using a Teledyne Isco EZ-Prep chromatography system, or an Agilent Technologies HP series 1200 system, with DAD (Diode Array Detector) and ELS detectors. HPLC analysis was performed using an Agilent Technologies 1260 Infinity system coupled with DAD (Diode Array Detector). Separation of the analyte, purity and enantiomeric/diastereomeric excess determinations were achieved at 40 ^oC using the methods reported in each detailed reaction description. Preparative and analytical HPLC columns were purchased from Daicel corporation or Phenomenex. HPLC methods and conditions are reported in the descriptions of the chemical reactions where they were applied. Microanalyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree with ± 0.4% of calculated values. HRMS (mass error within 5 ppm) and MS/MS fragmentation analysis were performed on a LTQ-Orbitrap Velos (Thermo-Scientific, San Jose, CA) coupled with an ESI source in positive ion mode to confirm the assigned structures and regiochemistry. Optical rotations were determined using a Jasco DIP-370 polarimeter. Unless otherwise stated, all the test compounds were evaluated to be >95% pure on the basis of combustion analysis, NMR, GC-MS, and HPLC-DAD. The detailed analytical results are reported in the characterization of each final compound and summarized in the supplementary table S1.

2-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)isoindoline-1,3-dione (3).

To a solution of (4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-ol (1) (0.50 g, 1.7 mmol) in acetonitrile (AcOH,10 mL), K₂CO₃ (4.8 g, 17 mmol) was added followed by 2-(4-bromobutyl)isoindoline-1,3-dione (0.65 g, 8.2 mmol) and KI (5.4 mg, 0.053 mmol). The reaction mixture was stirred under an inert atmosphere at reflux overnight. The reaction mixture was cooled, filtered, and the solvent was removed in vacuo. The crude material was purified via flash column chromatography, eluting with 5% DMA (CH₂Cl₂: MeOH: NH₄OH 95:5:0.5) to afford the product as a white solid, in 58% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.88 – 7.80 (m, 2H), 7.74 – 7.67 (m, 2H), 6.91 (s, 1H), 6.74 – 6.63 (m, 2H), 4.47 (dd, J = 10.7, 3.7 Hz, 1H), 4.39 (d, J = 9.2 Hz, 1H), 4.11 – 4.02 (m, 1H), 3.96 – 3.80 (m, 5H), 3.75 (t, J = 6.8 Hz, 2H), 2.89 (d, J = 11.5 Hz, 1H), 2.68 (td, J = 11.2, 10.4, 6.0 Hz, 1H), 2.57 – 2.40 (m, 2H), 1.90 – 1.81 (m, 2H), 1.59 – 1.54 (m, 3H), 1.48 (dq, J = 15.1, 6.9 Hz, 1H), 0.95 – 0.86 (m, 3H). The free base was converted into the corresponding oxalate salt. CHN Anal (C₂₆H₃₀N₂O₅ ^. H₂C₂O₄ ^. 0.5 H₂O) calculated C 61.19, H 6.05, N 5.10; found C 60.87, H 5.78, N 5.00. m.p. 158–159 °C

4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butan-1-amine (4).

To a solution of 3 (0.5 mg, 1.1 mmol) in EtOH (50 mL), hydrazine (36 mg,14 mL, 1.1 mmol) was added under an inert atmosphere and was stirred at reflux overnight. The reaction mixture was cooled to room temperature and the solvent was removed in vacuo. The product was resuspended in DCM and the organic fraction was extracted (3X) with 75 mL portions of aqueous K₂CO₃, which after drying over anhydrous MgSO₄, was concentrated in vacuo to afford the crude product as a white solid, in 89% yield. ¹H NMR (400 MHz, CDCl₃) δ 6.93 (d, J = 2.7 Hz, 1H), 6.76 – 6.65 (m, 2H), 4.48 (dd, J = 10.8, 3.7 Hz, 1H), 4.41 (d, J = 9.2 Hz, 1H), 4.07 (dd, J = 11.4, 3.3 Hz, 1H), 3.99 – 3.81 (m, 4H), 2.90 (d, J = 11.7 Hz, 1H), 2.79 – 2.70 (m, 2H), 2.67 (dd, J = 10.4, 6.2 Hz, 1H), 2.58 – 2.41 (m, 2H), 2.33 – 2.21 (m, 1H), 1.78 (p, J = 6.6 Hz, 2H), 1.48 (ddd, J = 29.8, 16.3, 6.9 Hz, 4H), 0.91 (t, J = 7.4 Hz, 3H).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)-9H-fluorene-2-carboxamide (5).

To a solution of 9H-fluorene-2-carboxylic acid (72 mg, 0.34 mmol) in DCM (10 mL) under an inert atmosphere at 0°C, EDC (76 mg, 0.39 mmol) and HOBt (53 mg, 0.39 mmol) were added. After 30 minutes of vigorous stirring, 4 (0.10 g, 0.31 mmol) and DIPEA (0.075 mL, 0.42 mmol) were added to the reaction mixture. The reaction stirred overnight gradually warming to RT. The solvent was evaporated and the compound was purified via flash chromatography, eluting with EtOAc/hexanes (9:1) to give 68 mg product as a tan colored powder, in 43% yield. 1H NMR (400 MHz, CDCl₃) δ 7.93 (s, 1H), 7.86 – 7.71 (m, 3H), 7.57 (d, J = 7.3 Hz, 1H), 7.38–7.26 (m, 2H), 6.99 (s, 1H), 6.72 (q, J = 9.1 Hz, 2H), 6.38 (s, 1H), 4.48 (dd, J = 10.8, 3.6 Hz, 1H), 4.39 (d, J = 9.2 Hz, 1H), 4.04–4.00 (m, 3H), 3.88 (m, 4H), 3.56 (q, J = 6.4 Hz, 2H), 2.92 – 2.80 (m, 1H), 2.67 (s, 1H), 2.55 – 2.38 (m, 2H), 2.27 (d, J = 14.2 Hz, 1H), 1.86 (m, 4H), 1.57 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 167.73, 153.02, 147.26, 144.69, 144.00, 143.38, 140.70, 133.00, 127.55, 126.93, 125.71, 125.15, 123.71, 122.73, 120.49, 119.65, 116.43, 116.04, 110.48, 75.54, 68.16, 67.24, 66.00, 59.44, 55.57, 51.99, 39.75, 36.86, 26.86, 26.46, 19.07, 11.82. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 17.073 min, purity >99%, de >95% (absorbance at 280 nm). HRMS (ESI) C₃₂H₃₆O₄N₂ + H⁺ calculated 513.27478, found 513.27432 (−0.9ppm). CHN Anal (C₃₂H₃₆N₂O₄ ^. 0.75 H₂O) calculated C 73.05, H 7.18, N 5.32; found C 73.11, H 7.03, N 5.17. m.p. 165–166 °C$;{\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +38.83 (0.065 g/100 mL in DCM).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)-1H-indole-2-carboxamide (6).

The compound was prepared following the same procedure described for 5, starting from 1H-indole-2-carboxylic acid (55 mg, 0.34 mmol). The compound was purified via flash chromatography, eluting with EtOAc/hexanes (9:1) to give 0.10 g product as a white solid, in 69% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.11 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 8.3 Hz, 1H), 7.26 (m, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.95 (s, 1H), 6.79 – 6.67 (m, 3H), 6.35 (s, 1H), 4.49 (dd, J = 10.8, 3.7 Hz, 1H), 4.40 (d, J = 9.2 Hz, 1H), 4.15 – 3.96 (m, 3H), 3.89 (q, J = 11.3, 10.9 Hz, 2H), 3.61 – 3.52 (m, 2H), 2.89 (d, J = 11.7 Hz, 1H), 2.67 (d, J = 15.1 Hz, 1H), 2.58 – 2.41 (m, 2H), 2.27 (s, 1H), 1.90 – 1.80 (m, 3H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.45, 152.97, 147.33, 136.03, 130.79, 127.71, 124.41, 122.75, 121.90, 120.61, 116.46, 116.07, 111.79, 110.44, 101.64, 75.55, 68.08, 67.25, 66.02, 59.43, 55.60, 52.01, 39.30, 26.75, 26.55, 19.07, 11.82. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 29.973 min, purity >95%, de >90% (absorbance at 230 nm). HRMS (ESI) C₂₇H₃₃O₄N₃ + H⁺ calculated 464.25438, found 464.25347 (−1.9ppm). CHN Anal (C₂₇H₃₃N₃O₄ ^. 0.25 H₂O) calculated C 69.28, H 7.21, N 8.98; found C 69.40, H 7.33, N 9.07. m.p. 143–144 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{24}$: +35.4 (0.130 g/100 mL in DCM).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)benzofuran-2-carboxamide (7).

The compound was prepared following the same procedure described for 5, starting from benzofuran-2-carboxylic acid (55 mg, 0.34 mmol). The compound was purified via flash chromatography, eluting with EtOAc/hexanes (9:1) to give a 0.13 g of the product as a yellow oil, in 86% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.66 (d, J = 7.8 Hz, 1H), 7.49 – 7.35 (m, 3H), 7.33 – 7.23 (m, 1H), 6.95 (s, 1H), 6.82 (s, 1H), 6.79 – 6.66 (m, 2H), 4.48 (dd, J = 11.0, 3.7 Hz, 1H), 4.39 (d, J = 9.2 Hz, 1H), 4.15 – 4.02 (m, 2H), 4.01 – 3.91 (m, 2H), 3.91 – 3.81 (m, 2H), 3.56 (q, J = 6.2 Hz, 2H), 2.89 (d, J = 11.6 Hz, 1H), 2.68 (dd, J = 17.8, 10.9 Hz, 1H), 2.57 – 2.41 (m, 2H), 1.85 (s, 2H), 1.56 (s, 3H), 1.47 (s, 1H), 1.26 (t, J = 7.1 Hz, 1H), 0.91 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.87, 154.67, 153.08, 148.85, 147.24, 127.65, 126.69, 123.59, 122.68, 122.64, 116.40, 116.03, 111.71, 110.36, 110.16, 75.57, 68.02, 67.26, 66.01, 59.43, 55.61, 52.02, 38.97, 26.78, 26.45, 19.07,11.84. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 17.984 min, purity >99%, de >95% (absorbance at 273 nm). HRMS (ESI) C₂₇H₃₂O₅N₂ + H⁺ calculated 465.23840, found 465.23812 (−0.6ppm). CHN Anal (C₂₇H₃₂N₂O₅) calculated C 69.81, H 6.94, N 6.03; found C 70.09, H 7.12, N 6.01. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{24}$: +45.3 (0.225 g/100 mL in DCM).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)benzo[d]thiazole-2-carboxamide (8).

The compound was prepared following the same procedure described for 5, starting from benzo(d)thiazole-2-carboxylic (43 mg, 0.24 mmol). The compound was purified via flash chromatography, eluting with EtOAc/hexanes (9:1) to give 83 mg of the product as a clear wax, in 79% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.93 (dd, J = 26.8, 8.1 Hz, 2H), 7.45 (dt, J = 23.7, 7.4 Hz, 2H), 6.86 (d, J = 2.9 Hz, 1H), 6.71 – 6.57 (m, 2H), 4.45 – 4.29 (m, 2H), 4.03 – 3.72 (m, 5H), 3.48 (s, 2H), 3.35 – 3.26 (m, 1H), 2.83 (d, J = 11.7 Hz, 1H), 2.70 – 2.55 (m, 1H), 2.42 (dtd, J = 24.0, 11.9, 10.8, 3.6 Hz, 2H), 2.26 – 2.16 (m, 1H), 1.84 (m, 3H), 1.50 (dt, J = 11.3, 6.2 Hz, 1H), 1.44 – 1.33 (m, 1H), 0.83 (t, J = 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.06, 159.89, 153.11, 152.88, 147.21, 137.11, 126.70, 126.57, 124.24, 122.64, 122.36, 116.37, 116.08, 110.32, 75.59, 67.92, 67.26, 66.01, 59.45, 55.61, 52.04, 39.48, 26.74, 26.35, 19.09, 11.83. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 + 0.1% DEA, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm; RT 16.477 min, purity >99%, de >95% (absorbance at 280 nm). HRMS (ESI) C₂₆H₃₁O₄N₃S + H⁺ calculated 482.21080, found 482.21006 (−1.5ppm). CHN Anal (C₂₆H₃₁N₃O₄S) calculated C 64.84, H 6.49, N 8.73; found C 64.64, H 6.51, N 8.44. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{24}$: +37.3 (0.215 g/100 mL in DCM).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)benzo[d]thiazole-5-carboxamide (9)

The compound was prepared following the same procedure described for 5, starting from benzo[d]thiazole-5-carboxylic acid (84 mg, 0.47 mmol). The compound was purified via flash chromatography, eluting with EtOAc/hexanes (9:1) to give 100 mg of the product as a clear viscous oil, in 44% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.06 (d, J = 1.4 Hz, 1H), 8.47 (s, 1H), 8.00 (d, J = 8.4 Hz, 1 H), 7.88 (dd, J = 8.7, 1.8 Hz, 1H), 6.98–6.90 (m, 1H), 6.70 (q, J = 8.8 Hz, 2H), 6.46 (s, 1H), 4,52–4.43 (m, 1H), 4.39 (d, J = 9.2 Hz, 1H), 4.09–3.95 (m, 2H), 3.94–3.8 (m, 2H), 3.58 (q, J = 6.3, 2H), 2.89 (d, J = 11.7 Hz, 1H), 2.68 (td, J = 11.1, 6.1 Hz, 1H), 2.55–2.39 (m, 2H), 2.31–2.20 (m, 1H), 1.85 (d, J = 9.4 Hz, 4H), 1.66 (s, 1H), 1.60–1.39 (m, 2H), 0.91 (t, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 167.16, 155.34, 153.31, 153.16, 147.44, 136.85, 133.52, 124.51, 122.87, 122.25, 122.01, 116.60, 116.24, 110.55, 75.74, 67.44, 66.18, 59.60, 55.78, 52.20, 40.05, 26.95, 26.63, 19.26, 12.01. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 + 0.1% DEA, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 25.177 min, purity >95%, de >90% (absorbance at 230 nm). HRMS (ESI) C₂₆H₃₁O₄N₃S + H⁺ calculated 482.21080, found 482.20892 (−3.9ppm). CHN Anal (C₂₆H₃₁N₃O₄S) calculated C 64.84, H 6.49, N 8.73; found C 64.93, H 6.77, N 8.46. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{26}$: +41.7 (0.255 g/100 mL in DCM).

N-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (10)

The compound was prepared following the same procedure described for 5, starting from 1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (77 mg, 0.47 mmol). The compound was purified via flash chromatography, eluting with 2% DMA (CH₂Cl₂:MeOH 98:2 + 0.1% NH₄OH) to give 90 mg of the product as a clear viscous oil, in 41% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.38 (d, J = 4.8 Hz, 1H) 7.95 (dd, J = 8.0, 1.5 Hz, 1H), 7.16–7.02 (m, 2H), 6.91 (d, J = 2.8 Hz, 1H), 6.83 (d, J = 0.9 Hz, 1H), 6.75–6.60 (m, 2H), 4.46 (dd, J = 10.8, 3.7 Hz, 1H), 4.38 (d, J = 9.2 Hz, 1H), 4.03 (dd, J = 11.5, 3.5 Hz, 1H), 3.98–3.78 (m, 4H), 3.51 (q, J = 6.2 Hz, 2H), 2.88 (d, J = 11.7 Hz, 1H), 2.76–2.57 (m, 1H), 2.53–2.40 (m, 3H), 2.30–2.18 (m, 1H), 1.80 (dt, J = 12.8, 6.4 Hz, 4H), 1.61–1.36 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 161.43, 161.35, 153.09, 147.92, 147.40, 145.65, 131.62, 130.85, 122.71, 120.41, 110.64, 101.61, 75.58, 68.31, 67.27, 65.96, 59.56, 55.72, 52.08, 39.45, 26.86, 26.37, 19.03, 11.90. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 + 0.1% DEA, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 35.552 min, purity >95%, de >90% (absorbance at 230 nm). HRMS (ESI) C₂₆H₃₂O₄N₄ + H⁺ calculated 465.24963, found 465.24713 (−5.2ppm). CHN Anal (C₂₆H₃₂N₄O₄) calculated C 67.22, H 6.94, N 12.06; found C 67.39, H 7.10, N 11.89. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{26}$: +38.2 (0.211 g/100 mL in DCM).

7-(4-(((4aR,10bR)-4-propyl-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-yl)oxy)butoxy)-3,4-dihydroquinolin-2(1H)-one (11)

The compound was prepared following the same procedure described for 3, starting from 1 (0.150 g, 0.5 mmol) and 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (0.158 g, 0.5 mmol). The compound was purified via flash chromatography, eluting with 5% DMA (CH₂Cl₂:MeOH 98:2 + 0.1% NH₄OH) to give 100 mg of the product as a clear viscous oil, in 43% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.59 (s, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.94 (s, 1H), 6.77–6.65 (m, 2H) 6.52 (d, J = 8.3 Hz, 1H), 6.28 (s, 1H), 4.48 (d J = 10.6 Hz, 1H), 4.41 (d, J = 9.2 Hz, 1H), 4.08 (d, J = 11.4 Hz, 1H), 4.02–3.75 (m, 6H), 2.89 (t, J = 7.9 Hz, 3H), 2.73–2.64 (m, 1H), 2.61 (t, J = 7.5 Hz, 2H), 2.57–2.44 (m, 2H), 2.33–2.22 (m, 1H), 1.99–1.86 (m, 4H), 1.53–1.46 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.42, 158.79, 153.38, 147.36, 138.20, 128.86, 122.82, 116.56, 116.20, 115.93, 110.50, 108.84, 102.32, 75.84, 68.17, 67.94, 67.50, 66.19, 59.64, 55.79, 52.21, 31.29, 26.19, 26.13, 24.79, 19.27, 12.01. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 + 0.1% DEA, flow rate 1mL/min), total run time 40 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm; RT 24.783 min, purity >95%, de >90% (absorbance at 214 nm). HRMS (ESI) C₂₇H₃₄O₅N₂ + H⁺ found 467.25295 (−2.3ppm). CHN Anal (C₂₇H₃₄N₂O₅) calculated C 69.51, H 7.35, N 6.00; found C 69.77, H 7.55, N 5.84. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{26}$: +25.5 (0.335 g/100 mL in DCM).

7-(4-((tert-butyldimethylsilyl)oxy)butoxy)-3,4-dihydroquinolin-2(1H)-one (12)

To a stirring solution of 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (0.5 g, 3.1 mmol) and K₂CO₃ (10 eq.) in ethanol (20 mL), (4-bromobutoxy)(tert-butyl)dimethylsilane (0.82 g, 3.1 mmol) was added portion-wise, and the mixture stirred at reflux overnight. The solvent was evaporated, the residue dissolved with DCM, and washed with water and brine. The organic phase was dried over MgSO₄, filtered, and evaporated in vacuo to afford the crude product. The compound was purified via flash chromatography, eluting with EtOAc/hexanes (5:5) to give 0.91 g of product as colorless viscous oil, in 84% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.75 (s, 1H), 7.04 (d, J = 8.3 Hz, 1H) 6.52 (d, J = 8.1 Hz, 1H), 6.29 (d, J = 3.3 Hz, 1H), 3.67 (br s, 2H), 2.89 (br s, 2H), 2.61 (br s, 2H), 1.82 (br s, 2H), 1.67 (br s, 2H), 0.90 (q, J = 2.2 Hz, 9H), 0.05 (q, J = 2.2 Hz, 6H).

7-(4-hydroxybutoxy)-3,4-dihydroquinolin-2(1H)-one (13)

A solution of 12 (0.91 g, 2.6 mmol) and 1M TBAF (2 eq.) in THF (30 mL) was stirred at room temperature for 4 hours. The reaction mixture was washed with water, dried over MgSO₄, filtered, and evaporated in vacuo to afford 0.5 g of the desired product as colorless viscous oil, in 82% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.04 (d, J = 8.2 Hz, 1H), 6.53 (dd, J = 8.3, 2.5 Hz, 1H), 6.36 (d, J = 2.4 Hz, 1H), 3.98 (t, J = 6.2 Hz, 2H), 3.69 (t, J = 6.4 Hz, 2H), 2.88 (dd, J = 8.5, 6.5 Hz, 2H), 2.64–2.56 (m, 2H), 2.33 (br s, 1H), 1.87 (tt, J = 8.2, 5.9 Hz, 2H), 1.78–1.68 (m, 2H).

4-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)butanal (14)

To solution of 13 (0.5 g, 2.1 mmol) in DCM (10 mL) at 0 ^oC, Dess-Martin periodinane (1 g, 2.4 mmol) was added portion-wise. The reaction mixture was stirred at 0 ^oC for 1 hour, then allowed to warm to room temperature and washed with 2N NaHCO₃ water solution. The organic phase was dried over MgSO₄, filtered, and evaporated in vacuo to afford the crude product. The compound was purified via flash chromatography, eluting with EtOAc/hexanes (3:7) to give 0.27 g of product as colorless viscous oil, in 55% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.84 (s, 1H), 8.02 (br s, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.50 (dd, J = 8.3, 2.4 Hz, 1H), 6.30 (d, J = 2.5 Hz, 1H), 3.97 (t, J = 6.0 Hz, 2H), 2.89 (dd, J = 8.4, 6.6 Hz, 2H), 2.70–2.57 (m, 4H), 2.16–2.05 (m, 2H).

(4aR,10bR)-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-ol (15)

To a stirred solution of 1 (0.23 g, 0.92 mmol) in 1M BrCN (0.29 g, 2.8 mmol) in acetonitrile, K₂CO₃ (0.38 g, 2.8 mmol) was added. The solution was stirred for 40 minutes in a microwave at 120°C, 275 psi, 75W. The reaction was analyzed with gas chromatography to confirm the intermediate cyanate was formed. The reaction mixture was filtered and concentrated in vacuo. A solution of 5 mL of concentrated HCl in 5mL of H₂O was made and added to the dry crude material. The reaction solution was stirred at reflux overnight. The reaction solution was concentrated in vacuo. A solution of ammonia in methanol was added until a pH of 8 was reached. The solution mixture was concentrated in vacuo and was purified via flash column chromatography, eluting with 5% DMA (CH₂Cl₂: MeOH: NH₄OH 95:5:0.5) to afford 60 mg of the product as a tan viscous oil in 31% yield. ¹H NMR (400 MHz, CDCl₃) δ 6.85 (d, J = 1.2 Hz, 1H), 6.67 (d, J = 1.5 Hz, 2H), 4.32 (d, J = 9.2 Hz, 1H), 4.16–4.03 (m, 2H), 3.99–3.82 (m, 2H), 3.20–2.97 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 149.69, 147.61, 122.92, 116.98, 116.10, 111.71, 76.40, 68.10, 67.97, 54.39, 46.30. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 flow rate 1mL/min), total run time 70 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm; RT 40.879 min, purity >95%, de >90% (absorbance at 230 nm). GC/MS (EI) RT 10.360 min, 207.1 m/z.

7-(4-((4aR,10bR)-9-hydroxy-2,3,4a,10b-tetrahydro-4H,5H-chromeno[4,3-b][1,4]oxazin-4-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (16)

To a stirred solution of (4aR,10bR)-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazin-9-ol (15) (0.060 g, 0.29 mmol) in 1,2-dichloroethane (10mL) was added N-(4-oxobutyl)-1H-indole-2-carboxamide (0.068 g, 0.29 mmol) and catalytic amounts of AcOH. Na(OAc)₃BH (0.093 g, 0.44 mmol) was added after solution stirred for 1 hour. Solution was left at room temperature and allowed to stir overnight. The solution mixture was concentrated in vacuo and was purified twice via flash column chromatography, eluting with 10% DMA (CH₂Cl₂: MeOH: NH₄OH 95:5:0.5) and EtOAc/hexanes (6:4), respectively. The partially purified product was purified again with a preparative TLC eluting with 5% DMA (CH₂Cl₂: MeOH: NH₄OH 95:5:0.5) to afford 10 mg of the product as a white solid in 8.1% yield, after all the combined purification steps. ¹H NMR(400 MHz, CDCl₃ + CD₃OD) δ 7.04 (d, J = 8.3 Hz, 1H), 6.83 (d, J = 2.7 Hz, 1H), 6.69–6.60 (m, 2H), 6.52 (dd, J = 8.3, 2.5 Hz, 1H), 6.34 (d, J = 2.4 Hz, 1H), 4.47 (dd, J = 10.7, 3.6 Hz, 1H), 4.40 (d, J = 9.2 Hz, 1H), 4.08—4.03 (m, 1H), 3.95 (t, J = 6.0 Hz, 2H), 3.91–3.81 (m, 2H), 2.95–2.85 (m, 3H), 2.85–2.76 (m, 1H), 2.64–2.42 (m, 5H), 1.86–1.61 (m, 4H); ¹³C NMR (101 MHz, CDCl₃ + CD₃OD) δ 172.19, 158.61, 150.57, 146.59, 138.09, 128.76, 122.57, 116.49, 116.11, 115.93, 111.63, 108.89, 102.30, 75.7 m7, 67.89, 67.29, 65.85, 59.71, 53.34, 52.08, 31.06, 27.20, 24.58, 22.66; HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (80:20 flow rate 1mL/min), total run time 70 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm; RT 27.262 min, purity >99%, de >95% (absorbance at 214 nm). HRMS (ESI) C₂₄H₂₈O₅N₂ + H⁺ calculated 425.20710, found 425.20788 (1.8ppm). m.p. 185–186 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +43.3 (0.060 g/100 mL in MeOH).

3-amino-6-hydroxychroman-4-one (18)

3-amino-6-methoxychroman-4-one hydrochloride (17) (0.94 g, 4.1 mmol) was dissolved in 48% hydrobromic acid (23 mL) and stirred at 120 °C for 8 hours and 60 °C overnight. The reaction mixture was cooled to room temperature and neutralized with 7N ammonia solution in methanol until a pH of 8 was reached. The solution mixture was concentrated in vacuo and filtered on a silica plug, washing with EtOAc/hexanes (9:1) to give 0.27 g of product as a yellow viscous oil, in 37% yield, which was used in the next step without further purification. GC/MS (EI) RT 9.203 min, 179.0 m/z.

N-(4-((6-hydroxy-4-oxochroman-3-yl)amino)butyl)-1H-indole-2-carboxamide (19)

To a stirred solution of 3-amino-6-hydroxychroman-4-one (18) (0.12 g, 0.70 mmol) in 1–2 dichloroethane (10mL) was added N-(4-oxobutyl)-1H-indole-2-carboxamide (0.15 g, 0.70 mmol) and catalytic amounts of AcOH. Na(OAc)₃BH (0.21 g, 1.0 mmol) was added after solution stirred for 1 hour. Solution was left at room temperature and allowed to stir overnight. The solution mixture was concentrated in vacuo and was purified via flash column chromatography, eluting with EtOAc/hexanes (7:3). HRMS (ESI) C₂₂H₂₃O₄N₃ + H⁺ calculated 394.17613, found 394.17640 (0.6ppm). The racemic mixture was purified via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (70:30, flow rate 20 mL/min) to yield two different fractions (6 mg and 4 mg of yellow solid, respectively), enriched of the corresponding enantiomers. 19a: ¹H NMR (400 MHz, CDCl₃ + CD₃CN) δ 9.27 (s, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.24–7.19 (m, 2H), 7.07 (t, J = 7.5 Hz, 1H), 6.98 (dd, J = 8.9, 3.1 Hz, 1H), 6.85–6.77 (m, 2H), 6.71 (s, 1H), 4.45 (dd, J = 11.1, 5.3 Hz, 1H), 4.04 (t, J = 11.4 Hz, 1H), 3.57 (dd, J = 11.7, 5.3 Hz, 1H), 3.44 (q, J = 6.5 Hz, 2H), 2.73 (ddt, J = 23.7, 11.7, 5.8 Hz, 2H), 1.63 (dq, J = 28.0, 7.3 Hz, 4H); ¹³C NMR (101 MHz, CDCl₃ + CD₃CN) δ 161.59, 155.67, 152.87, 150.95, 136.10, 131.02, 127.68, 124.72, 124.21, 121.85, 120.45, 119.44, 118.74, 111.85, 111.22, 102.05, 70.80, 60.62, 47.94, 39.30, 27.45, 27.23, 27.17, 26.29. 19b: ¹H NMR (400 MHz, CDCl₃ + CD₃CN) δ 9.29 (s, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H), 7.27–7.21 (m, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.00 (dd, J = 8.9, 3.1 Hz, 1H), 6.84–6.79 (m, 2H), 6.67 (s, 1H), 4.47 (dd, J = 11.1, 5.3 Hz, 1H), 4.06 (t, J = 11.4 Hz, 1H), 3.58 (dd, J = 11.7, 5.3 Hz, 1H), 3.47 (q, J = 6.5 Hz, 2H) 2.84–2.61 (m, 2H), 1.66 (dp, J = 29.1, 7.2 Hz, 4H); ¹³C NMR (101 MHz, CDCl₃ + CD₃CN) δ 192.85, 161.72, 155.81, 150.93, 136.19, 130.98, 127.74, 124.84, 124.37, 121.93, 120.58, 119.92, 119.56, 118.87, 111.34, 107.91, 102.14, 70.87, 60.71, 48.02, 39.43, 27.52, 27.26. Analytical HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30 flow rate 1mL/min), total run time 90 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm; enantiomer 19a: RT 48.546 min, purity >90%, er 70:30 (absorbance at 214 nm); enantiomer 19b: RT 54.171 min, purity >90%, er 90:10 (absorbance at 214 nm). [α] values for compounds 19a and 19b were not determined.

5-((5S)-5-methyl-4-propylmorpholin-2-yl)-N,N-dipropylpyridin-2-amine (20)

To a solution of 2 diastereomeric mixture⁴² (50 mg, 0.21 mmol) in acetonitrile (10 mL), propionaldehyde (49 mg, 0.84 mmol) was added, followed by catalytic amount of glacial acetic acid. The reaction mixture was stirred at reflux for 2 hours, cooled to room temperature, followed by portion-wise addition of sodium triacetoxyborohydride (178 mg, 0.84 mmol). The mixture was stirred at reflux overnight. The reaction was monitored via GC/MS until complete consumption of the starting material. The solvent was evaporated in vacuo to give a crude product, which was purified via flash chromatography, eluting with 5% DMA (CH₂Cl₂:MeOH 95:5 + 0.5% NH₄OH). The pure compound was obtained as diastereomeric mixture, as a yellow oil, in 22% yield (15 mg) ¹H NMR (400 MHz, CDCl₃) δ 8.12 (s, 1H), 7.44 (d, J = 9.2 Hz, 1H), 6.40 (d, J = 8.9 Hz, 1H), 4.48 (br s, 1H), 3.93 (br s, 1H), 3.77 (d, J = 11.3 Hz, 1H), 3.38 (t, J = 7.7 Hz, 4H), 2.93 (br s, 1H), 2.62 (br s, 2H), 2.43 (br s, 2H), 1.60 (h, J = 7.7 Hz, 6H) 1.13 (br s, 3H), 0.97–0.82 (m, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 158.08, 147.95, 146.78,135.68, 129.00, 126.10, 105.33, 77.36, 56.53, 53.02, 51.43, 50.75, 29.86, 20.93, 12.00, 11.61. GC/MS (EI) RT 11.905 min, 319.3 m/z. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (95:5 flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 250–273 nm, RT 5.907 min and 6.267 min, purity >95%, dr 1:1 (absorbance at 254 nm). HRMS (ESI) C₁₉H₃₃ON₃ + H⁺ calculated 320.26964, found 320.26896 (−2.1ppm).

7-(4-((5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)butoxy)-3,4-dihydroquinolin-2(1H)-one (22)

To a solution of 21 (65:35 diastereomeric mixture, 50 mg, 0.26 mmol) in acetonitrile (5 mL), was added K₂C₂O₃ (10 eq.), followed by 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (92.6 mg, 0.31 mmol). The reaction mixture was stirred at reflux overnight. The solvent was evaporated in vacuo, and the crude residue purified via flash chromatography, eluting with 10% CMA (CHCl₃:MeOH 9:1 + 0.1% NH₄OH). The pure product was obtained as 2:1 diastereomeric mixture, as yellow oil, in 28% yield (30 mg). ¹H NMR (400 MHz, CDCl₃) δ 8.94 (s, 1H, 33% isomer), 8.65 (s, 1H, 66% isomer), 8.12 (s, 1H, 33% isomer), 8.03 (s, 1H, 66% isomer), 7.50–7.38 (m, 1H), 7.02 (d, J 8.3 Hz, 1H) 6.49 (td, J = 10.8, 8.8, 5.9 Hz, 1H), 6.36–6.32 (m, 1H), 4.75 (s, 2H, 33% isomer), 4.62 (s, 2H, 66% isomer), 4.46 (t, J = 10.0 Hz, 1H), 4.00–3.91 (m, 3H, 66% isomer), 3.86–3.77 (m, 3H, 33% isomer), 3.69 (d, J = 11.6 Hz, 1H, 33% isomer), 3.38 (t, J = 10.8 Hz, 1H, 66% isomer), 2.98–2.76 (m, 4H), 2.71–2.48 (m, 3H), 2.47–2.37 (m, 2H), 2.29 (dt, J = 13.1, 6.7 Hz, 2H, 33% isomer), 2.19 (t, J = 11.0 Hz, 2H, 66% isomer), 1.91–1.69 (m, 4H), 1.08 (d, J = 6.6 Hz, 3H, 33% isomer), 1.01 (d, J = 6.2 Hz, 3H, 66% isomer), ¹³C NMR (101 MHz, CDCl₃) δ 172.14, 171.97, 158.75, 158.50, 158.46, 146.46, 146.35, 138.44, 138.40, 136.95, 136.41, 128.79, 128.76, 126.08, 126.05, 115.96, 115.91, 109.15, 108.63, 108.47, 102.45, 102.24, 76.65, 75.88, 73.48, 71.58, 68.18, 67.97, 58.60, 55.04, 54.15, 53.14, 53.02, 52.21, 31.27, 31.24, 27.36, 27.35, 24.75, 23.60, 22.17, 15.09, 8.96. HPLC method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30 + 0.1% DEA, flow rate 1mL/min), total run time 50 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 22.568 min and 27.336 min, purity >95%, dr 65:35 (absorbance at 230 nm). HPLC method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30 + 0.1% DEA, flow rate 1mL/min), total run time 50 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 16.751 min and 29.776 min, purity >95%, dr 35:65 (absorbance at 230 nm). HRMS (ESI) C₂₃H₃₀O₃N₄ + H⁺ calculated 411.23907, found 411.23863 (−1.0ppm). CHN Anal (C₂₃H₃₀N₄O₃ ^. 0.4 CH₂Cl₂^. 0.2 CH₃OH ) calculated C 62.87, H 7.06, N 12.43; found C 63.06, H 6.83, N 12.09.

5-((2R,5S)-5-methylmorpholin-2-yl)pyridin-2-amine (23a) and 5-((2S,5S)-5-methylmorpholin-2-yl)pyridin-2-amine (23b)

The diastereomeric mixture (65:35 ratio of (2R,5S):(2S,5S)) was separated via flash chromatography, eluting with 5% to 10% DMA (CH₂Cl₂:MeOH + 0.5% NH₄OH). GC/MS (EI) RT 9.494 min and 9.540 min, 193.1 m/z. The (2R,5S)-diastereoisomer eluted first as a white solid. All the spectroscopic data were consistent with the reported ones.^{42 1}H NMR (400 MHz, CD₃OD) δ 7.91 (d, J = 2.3 Hz, 1H), 7.51 (dd, J = 8.7, 2.3, 1H), 6.61 (d, J = 8.6 Hz, 1H), 4.33 (dd, J = 10.7, 2.5 Hz, 1H), 3.95 (dd, J = 11.4, 3.3 Hz, 1H), 3.37–3.33 (m, 2H), 3.11–2.92 (m, 1H), 2.83 (dd, J = 12.7, 10.7 Hz, 1H), 1.07 (d, J = 6.5 Hz, 3H). HPLC method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (85:15 flow rate 1mL/min), total run time 50 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 19.193 min, purity >99%, de >99% (absorbance at 230 nm). HPLC method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (90:10 flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 42.578 min, purity >95%, de >95% (absorbance at 230 nm). CHN Anal (C₁₀H₁₅N₃O ^. 0.1 H₂O) calculated C 61.58, H 7.86, N 21.54; found C 61.35, H 7.46, N 21.31. The (2S,5S)-diastereoisomer eluted second as a pale yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 7.88 (d, J = 2.3 Hz, 1H), 7.49 (dd, J = 8.5, 2.2 Hz, 1H), 6.58 (d, J = 8.6 Hz, 1H), 4.35 (dd, J = 10.0, 2.9 Hz, 1H), 3.84 (dd, J = 11.5, 3.0 Hz, 1H), 3.74–3.63 (m, 1H), 3.11–2.91 (m, 2H), 2.73 (dd, J = 13.2, 3.0 Hz, 1H), 1.33 (d, J = 7.1 Hz, 3H). HPLC method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (85:15 flow rate 1mL/min), total run time 50 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 20.228 min, purity >95%, de >99% (absorbance at 230 nm). HPLC method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (90:10 flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 38.246 min, purity >99%, de >99% (max absorbance at 230 nm). CHN Anal (C₁₀H₁₅N₃O ^. 0.1 H₂O) calculated C 61.58, H 7.86, N 21.54; found C 61.71, H 7.61, N 21.33.

7-(4-((2R,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)butoxy)-3,4-dihydroquinolin-2(1H)-one (24)

The desired product was prepared following the same procedure described for 22, starting from 23a (30 mg, 0.16 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.04 (s, 1H), 7.77 (s, 1H), 7.45 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.49 (t, J = 9.4 Hz, 2H), 6.25 (s, 1H), 4.54 (s, 2H), 4.45 (d, J = 10.3 Hz, 1H), 3.98–3.89 (m, 2H), 3.83 (d, J = 11.3 Hz, 1H), 3.39 (t, J = 10.8 Hz, 1H), 2.99–2.83 (m, 4H), 2.61 (t, J = 7.5 Hz, 2H), 2.47–2.37 (m, 1H), 2.29 (dt, J = 12.9, 6.8 Hz, 1H), 2.19 (t, J = 11.1 Hz, 1H), 1.88–1.57 (m, 4H), 1.02 (d, J = 6.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 171.50, 158.77, 158.41, 146.39, 138.29, 136.45, 128.86, 126.15, 116.03, 108.54, 108.47, 102.43, 76.64, 73.50, 68.00, 58.58, 55.06, 53.12, 31.27, 27.32, 24.78, 22.15, 15.10. HPLC method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 28.163 min, purity >95%, de >99% (absorbance at 230 nm). HPLC method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 33.735 min, purity >99%, de >99% (absorbance at 230 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₃₀O₃N₄ + H⁺ calculated 411.23907, found 411.23818 (−0.2ppm). CHN Anal (C₂₃H₃₀N₄O₃ ^. 3 H₂C₂O₄ ^. 0.75 H₂O) calculated C 50.18, H 5.45, N 8.07; found C 50.09, H 5.64, N 8.46. m.p. 178–179 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +24.8 (0.295 g/100 mL in MeOH).

N-(4-((2R,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)butyl)-1H-indole-2-carboxamide (25)

To a solution of 23a (60 mg, 0.31 mmol) in 1,2-dichloroethane (10 mL), N-(4-oxobutyl)-1H-indole-2-carboxamide³⁶ (71 mg, 0.31 mmol) was added, followed by catalytic amount of glacial acetic acid. The reaction mixture was stirred at room temperature for 2 hours, followed by portion-wise addition of sodium triacetoxyborohydride (178 mg, 0.84 mmol). The reaction was stirred at room temperature overnight, the solvent was evaporated in vacuo to give a crude product, which was purified via flash chromatography, eluting with 15% DMA (CH₂Cl₂:MeOH 85:15 + 0.1% NH₄OH). The pure compound was obtained as 62 mg of colorless viscous oil, in 47% yield. ¹H NMR(400 MHz, CDCl₃) δ 9.31 (s, 1H), 8.03 (d, J = 2.2 Hz, 1H), 7.64 (dd, J = 8.0, 1.0 Hz, 1H), 7.47–7.40 (m, 2H), 7.31–7.26 (m, 1H), 7.14 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.82 (dd, J = 2.2, 0.9 Hz, 1H), 6.47–6.44 (m, 1H), 6.32 (s, 1H), 4.55 (s, 2H), 4.46 (dd, J = 10.5, 2.3 Hz, 1H), 3.83 (dd, J =11.3, 3.4 Hz, 1H), 3.53–3.49 (m, 2H), 3.43–3.37 (m, 1H), 2.97–2.80 (m, 2H), 2.42 (ddd, J = 10.0, 6.3, 3.5 Hz, 1H), 2.26 (ddd, J = 12.9, 7.7, 5.6 Hz, 1H), 2.18 (dd, J = 11.7, 10.5 Hz, 1H), 1.58 (qd, J = 8.0, 3.6 Hz, 4H), 1.02 (d, J = 6.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 161.73, 158.29, 146.17, 136.52, 136.31, 130.93, 127.79, 126.01, 124.64, 122.04, 120.83, 112.06, 108.56, 101.89, 76.60, 73.41, 58.25, 53.12, 51.01, 27.95, 23.29, 15.09. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30 to 50:50, flow rate 1mL/min), total run time 120 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 80.465 min, purity >99%, de >99% (absorbance at 230 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₂₉O₂N₅ + H⁺ found 408.23917 (−0.6 ppm). CHN Anal (C₂₃H₂₉N₅O₂ ^. 2 H₂C₂O₄ ^. 0.85 H₂O) calculated C 53.79, H 5.80, N 11.62; found C 54.19, H 6.05, N 11.22. m.p. 176–177 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +30.8 (0.055 g/100 mL in MeOH).

7-(4-((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)butoxy)-3,4-dihydroquinolin-2(1H)-one (26)

The desired product was prepared following the same procedure described for 22, starting from 23b (30 mg, 0.16 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.99 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 6.50 (dd, J = 12.5, 8.0 Hz, 2H), 6.31 (s, 1H), 5.27 (br s, 2H), 4.47 (t, J = 6.3 Hz, 1H), 3.96 (t, J = 6.5 Hz, 2H), 3.85 (d, J = 11.1 Hz, 1H), 3.72 (d, J = 11.2 Hz, 1H), 2.89 (t, J = 7.5 Hz, 3H), 2.61 (dt, J = 7.9, 4.7 Hz, 4H), 2.50 (hept, J = 7.1, 6.2 Hz, 2H), 1.87–1.75 (m, 2H), 1.67 (q, J = 7.4 Hz, 2H), 1.11 (d, J = 6.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 172.19, 158.77, 158.24, 144.39, 138.18, 137.62, 128.86, 125.74, 115.93, 109.36, 109.13, 102.31, 75.62, 71.72, 68.09, 54.05, 52.78, 51.78, 31.19, 27.31, 24.70, 23.34, 8.65. HPLC method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 35.369 min, purity >95%, de >90% (absorbance at 230 nm). HPLC method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λabsorbance signals measured in the range of 210–280 nm, RT 18.507 min, purity 94.2%, de 88.4% (absorbance at 230.4 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₃₀O₃N₄ + H⁺ calculated 411.23907, found 411.23798 (−1.6ppm). CHN Anal (C₂₃H₃₀N₄O₃ ^. 2 H₂C₂O₄ ^. 2 H₂O) calculated C 51.75, H 6.11, N 8.94; found C 51.58, H 5.94, N 8.77. Salt was highly hygroscopic therefore melting point could not be determined; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: −1.37 (0.085 g/100 mL in MeOH).

N-(4-((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)butyl)-1H-indole-2-carboxamide (27)

The desired product was prepared following the same procedure described for 25, starting from 23b (30 mg, 0.16 mmol) The pure compound was obtained as 43 mg of colorless viscous oil, in 63% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.28 (s, 1H), 8.06 (d, J = 2.3 Hz, 1H), 7.64 (dd, J = 8.1, 1.0 Hz, 1H), 7.48–7.40 (m, 2H), 7.28 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H), 7.14 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.83 (d, J = 1.5 Hz, 1H), 6.51–6.44 (m, 1H), 6.41 (s, 1H), 4.51 (br s, 2H), 4.47 (dd, J = 9.4, 3.6 Hz, 1H), 3.87 (dd, J = 11.2, 2.8 Hz, 1H), 3.74 (dd, J = 11.2, 2.1 Hz, 1H), 3.52 (q, J = 6.6 Hz, 2H), 2.92–2.83 (m, 1H), 2.66–2.52 (m, 2H), 2.49 (t, J = 7.1 Hz, 2H), 1.65 (dq, J = 37.1, 7.5 Hz, 4H), 1.10 (d, J = 6.6, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 161.69, 158.20, 146.44, 136.64, 136.27, 131.03, 127.82, 126.27, 124.64, 122.05, 120.85, 112.02, 108.52, 101.88, 76.14, 72.18, 53.85, 53.09, 51.57, 39.65, 27.75, 24.52, 8.24. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30 to 50:50, flow rate 1mL/min), total run time 120 min, multiple DAD λ absorbance signals measured in the range of 230–280 nm, RT 89.625 min, purity >99%, de >99% (absorbance at 230 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₂₉O₂N₅ + H⁺ found 408.23994 (1.3 ppm). CHN Anal (C₂₃H₂₉N₅O₂ ^. 2 H₂C₂O₄ ^. 0.5 H₂O) calculated C 54.36, H 5.74, N 11.74; found C 54.45, H 5.85, N 11.86. m.p. 132–133 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: −7.27 (0.055 g/100 mL in MeOH).

(E)-1,4-dibromobut-2-ene (28)

To a two neck round bottom flask was added Hoveyda-Grubbs catalyst 2^nd generation (25.0 mg, 0.04 mmol) and subsequently purged with argon. Degassed dichloromethane (6 mL) were added via syringe to the flask to form a green solution of the catalyst. To the solution was added freshly distilled allyl bromide⁶⁰ (5.0 mL, 52.1 mmol), gas evolution from the reaction was observed and within a minute the reaction mixture had become brown in color. The reaction mixture was allowed to stir for approximately 10 more minutes after which the solvent was removed under reduced pressure and the product purified by standard flash chromatography with hexanes/EtOAc (95:5) to yield 1.70 g of 1,4-dibromobut-2-ene as a white crystal, in 15% yield, and in 9:1 ratio of the E:Z product. ¹H NMR (400 MHz, CDCl₃) δ 5.98 (br s, 2H), 4.03–3.90 (m, 4H).

(E)-7-((4-bromobut-2-en-1-yl)oxy)-3,4-dihydroquinolin-2(1H)-one (29)

(E)-1,4-dibromobut-2-ene (28) (0.200 g, 0.94 mmol) and 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (0.155 g, 5.55 mmol) were dissolved in acetonitrile (5 mL) with gentle heating, to the reaction mixture was added anhydrous K₂CO₃ (1.29 g, 9.40 mmol) and a catalytic amount of KI (approx. 25.0 mg). The reaction was subsequently refluxed for 1.5 hours and then allowed to stir overnight at room temperature. The solvent was then removed under reduced pressure and the crude material purified via flash chromatography, eluting with hexanes/EtOAc (75:25), to yield 0.060 g of the product as a white solid, in 22% yield. ¹H NMR (400 MHz, CDCl₃) δ 8.77 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.52 (d, J = 8.2 Hz, 1H), 6.39 (s, 1H), 6.13–6.01 (m, 1H), 6.02–5.89 (m, 1H), 4.52 (s, 1H), 3.98 (d, J = 5.6 Hz, 2H), 2.96–2.84 (m, 2H), 2.70–2.55 (m, 2H).

7-(((E)-4-((2R,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)but-2-en-1-yl)oxy)-3,4-dihydroquinolin-2(1H)-one (30)

To a round bottom flask were added 29 (0.0342 g, 0.115 mmol) and 23a (0.0220 g, 0.110 mmol) and subsequently dissolved with acetonitrile (5 mL). To the solution was added anhydrous K₂CO₃ (0.150 g, 1.20 mmol) and the reaction was set to reflux for 50 minutes and then allowed to return to room temperature to be stirred overnight. The solvent was removed under reduced pressure and the crude mixture purified via flash chromatography eluting with 5% DMA (DCM:MeOH 95:5 + 0.5% NH₄OH), to yield 0.0365 g of the desired product, as a white solid, in 78% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.81 (s, 1H), 7.38 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.5 Hz, 1H), 6.53 (t, J = 7.9 Hz, 2H), 6.45 (s, 1H), 5.86 (br s, 2H), 4.54 (s, 2H), 4.37 (d, J = 10.5 Hz, 1H), 3.81 (d, J = 11.5 Hz, 1H), 3.56 (d, J = 14.0 Hz, 1H), 3.37 (d, J = 11.0 Hz, 1H), 2.95 (dd, J = 13.9, 7.3 Hz, 1H), 2.82 (d, J = 9.6 Hz, 3H), 2.51 (t, J = 7.8 Hz, 2H), 2.43 (s, 1H), 2.15 (t, J = 11.3 Hz, 1H), 1.05 (d, J = 6.0 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 174.05, 160.69, 159.26, 146.18, 139.73, 137.84, 131.45, 129.87, 129.54, 125.80, 117.41, 111.43, 110.45, 109.89, 103.78, 77.39, 73.85, 68.79, 59.19, 56.12, 56.08, 31.90, 25.39, 14.68. HPLC Method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 31.234 min, purity >95%, de >99% (absorbance at 214 nm). HPLC Method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 31.721 min, purity >99%, de >99% (absorbance at 214 nm). HPLC Method C: column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 130 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 46.609 min, purity >95%, de >99% (max absorbance at 214 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₂₈O₃N₄ + H⁺ calculated 409.22342, found 409.22293 (−1.1 ppm). CHN Anal (C₂₃H₂₈N₄O₃ ^. 3 H₂C₂O₄ ^. 2.5 H₂O) calculated C 48.13, H 5.43, N 7.74; found C 47.96, H 5.31, N 7.42. m.p. 123–124 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +20.0 (0.080 g/100 mL in MeOH).

7-(((E)-4-((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)but-2-en-1-yl)oxy)-3,4-dihydroquinolin-2(1H)-one (31)

The desired product was prepared following the same procedure described for 30, starting from 23b (0.0200 g, 0.103 mmol). The crude material was purified via flash chromatography, eluting with 5% DMA (DCM:MeOH 95:5 + 0.5% NH₄OH), to yield 0.0379 g of the desired product as a white solid, in 90% yield. ¹H NMR (400 MHz, CD₃OD) δ 7.85 (s, 1H), 7.43 (dd, J = 8.7, 2.5 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.53 (t, J = 8.5 Hz, 2H), 6.45 (d, J = 2.5 Hz, 1H), 5.85 (br s, 2H), 4.52 (s, 2H), 4.37 (dt, J = 7.9, 3.4 Hz, 1H), 3.84 (d, J = 11.5 Hz, 1H), 3.70 (d, J = 10.6 Hz, 1H), 3.13 (q, J = 13.5 Hz, 2H), 2.91–2.77 (m, 3H), 2.59–2.45 (m, 4H), 1.10 (d, J = 4.4 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD) δ 173.99, 160.59, 159.32, 146.23, 139.70, 137.96, 131.19, 130.62, 129.56, 126.04, 117.38, 110.44, 109.91, 103.71, 77.04, 72.69, 68.90, 57.21, 52.86, 31.89, 25.37, 8.50. HPLC Method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 37.386 min, purity 92.6%, de 86.3% (absorbance at 214 nm). HPLC Method B: column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 19.762 min, purity 91.5%, de >83.4% (absorbance at 214.4 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₃H₂₈O₃N₄ + H⁺ calculated 409.22342, found 409.22282 (−2.8 ppm). CHN Anal (C₂₃H₂₈N₄O₃ ^. 2 H₂C₂O₄ ^. 2 H₂O) calculated C 51.92, H 5.81, N 8.97; found C 51.85, H 5.71, N 8.93. m.p. 110–111 °C; ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: −5.33 (0.075 g/100 mL in MeOH).

(2-(aminomethyl)cyclopropyl)methanol (32)

To a round bottom flask was added lithium aluminum hydride (LAH, 2.106 g, 55.5 mmol) and subsequently purged with argon. Anhydrous tetrahydrofuran (25 mL) was added to the flask to form a gray suspension and the solution was subsequently cooled to 0 °C in an ice bath. Ethyl 2-cyanocyclopropanecarboxylate¹² (2.57 g, 18.5 mmol) was then dissolved in 5 mL of anhydrous tetrahydrofuran and added dropwise to the solution of LAH via syringe. Upon complete addition of the ethyl 2-cyanocyclopropanecarboxylate, the reaction mixture was removed from the ice bath and allowed to reach room temperature and stirred with moderate heating for 2 h. The reaction mixture was then removed from the heating source and stirred at room temperature overnight. 3 mL of ice cold water were added to the reaction, followed by 3 mL of 15% NaOH which resulted in the formation of a white solid precipitate. An additional 15 mL of water were added to the reaction mixture and it was then stirred for 30 minutes. Excess MgSO₄ was added to the reaction mixture to absorb water and the suspension was stirred for additional 30 minutes. The suspension was then filtered over celite and the solvent removed under reduced pressure to yield 1.801 g of the desired product as a colorless oil, in 96% yield, which was carried on to the next step without further purification.

N-((2-(hydroxymethyl)cyclopropyl)methyl)-1H-indole-2-carboxamide (33)

Indole-2-carboxylic acid (3.220 g, 20.0 mmol) was dissolved in anhydrous tetrahydrofuran (50 mL) and cooled to 0 °C in an ice bath. To this solution was subsequently added 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC hydrochloride) (4.14 g, 21.6 mmol) and hydroxybenzotriazole (3.16 g, 23.4 mmol) in one portion and allowed to stir for 30 minutes at 0 °C. A solution of 32 (1.801 g, 17.8 mmol, 1.78 M) in DCM was added dropwise, followed by N,N-diisoproylethylamine (Hünig’s base or DIPEA) (4.4 mL, 25.2 mmol). The reaction mixture was then removed from the ice bath and allowed to reach room temperature and stirred overnight. The reaction mixture was quenched with the addition of 50 mL of saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (3 × 50 mL), the combined organic phases were dried with Na₂SO₄, concentrated under reduced pressure, and the crude was purified by flash chromatography, gradually increasing the mobile phase polarity up to hexanes/EtOAc (2:3) (EtOAc:Hex), followed by isocratic elution. The diastereoisomers 33b and 33a were separated with Rf:0.25 and Rf:0.20 respectively; 33b eluted first as a colorless oil (0.241 g, 5.5% yield); ¹H NMR (400 MHz, CD₃OD) δ 7.59 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.21 (t, J = 7.7 Hz, 1H), 7.09–6.99 (m, 2H), 3.96–3.87 (m, 1H), 3.73 (dd, J = 14.3, 4.8 Hz, 1H), 3.45 (t, J = 9.8 Hz, 1H), 3.23 (dd, J = 14.3, 7.0 Hz, 1H), 1.25 (br s, 2H), 0.82 (q, J = 7.5 Hz, 1H), 0.26 (d, J = 5.6 Hz, 1H); 33a eluted second as a colorless oil (0.117 g, 2.7% yield) and its spectroscopic data were consistent with the previously reported data;^{12 1}H NMR (400 MHz, CD₃OD) δ 7.56 (d, J = 7.9 Hz, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.17 (t, J = 7.7 Hz, 1H), 7.10–6.97 (m, 2H), 3.44 (dd, J = 11.6, 5.8 Hz, 1H), 3.31 (p, J = 5.4, 4.4 Hz, 2H), 3.23 (dd, J = 13.8, 6.4 Hz, 1H), 0.99 (d, J = 6.0 Hz, 2H), 0.50 (d, J = 6.7 Hz, 1H), 0.44 (d, J = 6.7 Hz, 1H).

trans-N-((2-formylcyclopropyl)methyl)-1H-indole-2-carboxamide (34)

The desired product was prepared following the same procedure described for 35 starting from 33a (0.117 g, 0.50 mmol) to yield 99.0 mg as a colorless viscous oil, in 81.7% yield. ¹H NMR (400 MHz, CDCl₃) δ 10.12 (s, 1H), 8.99 (dd, J = 3.8, 1.5 Hz, 1H), 7.59 (s, 1H), 7.52 (d, J = 8.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.13 (t, J = 7.8 Hz, 1H), 7.03–6.87 (m, 2H), 3.48 (dt, J = 12.7, 5.8 Hz, 1H), 3.24 (dt, J = 13.8, 6.5 Hz, 1H), 1.82 (s, 1H), 1.75 (s, 1H), 1,24 (d, J = 4.2 Hz, 1H), 1.05 (d, J = 6.6 Hz, 1H).

cis-N-((2-formylcyclopropyl)methyl)-1H-indole-2-carboxamide (35)

Compound 33b (0.241 g, 0.99 mmol) was dissolved in tetrahydrofuran (20 mL) and cooled to −78 °C under an argon atmosphere. To the solution was added Dess-Martin periodinane (0.636 g, 1.50 mmol) in one portion and the reaction was allowed to warm to room temperature. The reaction mixture was then stirred for 1.5 h, at which point the solvent was removed under reduced pressure and the crude mixture partially purified by flash chromatography, eluting with hexanes/EtOAc (7:3) to yield 45 mg of the desired product as a viscous colorless oil, in 18.8% yield, and used directly in the following step.

trans-N-((2-(((2R,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)methyl)-1H-indole-2-carboxamide (36)

Compound 36 was synthesized following the same procedure described for 39, starting from 23a (30.0 mg, 0.16 mmol) and 34 (37.5 mg, 0.16 mmol) to yield 36.6 mg of the final product, as a colorless oil, in 54.5% yield. The mixture was additionally purified via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 70:30 up to 50:50, flow rate 25 mL/min) to yield the desired product as a 1:1.6 mixture of the two diastereoisomers. ¹H NMR (400 MHz, CDCl₃) δ 9.33 (s, 1H), 8.00 (d, J = 16.4 Hz, 1H), 7.65 (d, J = 8.2 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.42–7.21 (m, 2H), 7.14 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 27.7 Hz, 1H), 6.43 (d, J = 59.1 Hz, 1H), 6.26 (dd, J = 18.3, 8.5 Hz, 1H), 4.45 (d, J = 10.6 Hz, 1H), 4.39 (s, 2H), 3.82 (t, J = 10.0 Hz, 1H), 3.73 (s, 1H, 38% isomer), 3.49 (s, 1H, 62% isomer), 3.40 (t, J = 11.4 Hz, 3H), 3.10 (t, J = 13.5 Hz, 1H, 62% isomer), 2.90 (d, J = 13.6 Hz, 1H, 38% isomer), 2.65 (dd, J = 13.2, 6.7 Hz, 1H, 38% isomer), 2.48 (s, 1H, 62% isomer), 2.30 (t, J = 11.0 Hz, 2H, 62% isomer), 2.09 (dd, J = 15.0, 8.4 Hz, 2H, 38% isomer), 1.21 (d, J = 5.9 Hz, 3H, 38% isomer), 1.01 (d, J = 6.0 Hz, 3H, 62% isomer), 0.92 (br s, 2H), 0.56 (d, J = 19.1 Hz, 1H), 0.42 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 161.58, 158.24, 146.41, 146.36, 136.34, 136.26, 130.91, 130.82, 127.86, 125.97, 124.65, 122.11, 120.85, 120.83, 112.06, 112.05, 108.34, 102.11, 102.00, 92.67, 73.37, 59.04, 58.90, 57.43, 57.33, 54.81, 43.71, 19.07, 17.42, 15.13, 15.01, 14.51, 14.21, 10.93, 9.11. HRMS (ESI) C₂₄H₂₉O₂N₅ + H⁺ calculated 420.23940, found 420.23979 (0.9ppm). Analytical HPLC for trans-36: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 170 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 66.064 min, purity >99% (absorbance at 230 nm).

trans-N-((2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)methyl)-1H-indole-2-carboxamide (37)

Compound 37 was synthesized following the same procedure described for 39, starting from the 23b (30.0 mg, 0.16 mmol) and 34 (37.6 mg, 0.16 mmol) to yield 35.0 mg of the final product as a colorless oil, in 52% yield. ¹H NMR (400 MHz, CDCl₃) δ 10.34 (s, 1H, 50% isomer), 10.05 (s, 1H), 50% isomer), 8.10 (s, 1H, 50% isomer), 7.88 (d, J = 6.7 Hz, 1H, 50% isomer), 7.67 (t, J = 8.0 Hz, 2H), 7.43 (d, J = 8.4 Hz, 1H), 7.32–7.19 (m, 2H), 7.11 (q, J = 10.1, 9.4 Hz, 2H), 6.24 (d, J = 8.6 Hz, 1H, 50% isomer), 6.03 (d, J = 8.6 Hz, 1H, 50% isomer), 5.05 (br s, 2H), 4.66 (d, J = 10.5 Hz, 1H, 50% isomer), 4.60 (d, J = 10.5 Hz, 1H, 50% isomer), 4.16–3.83 (m, 2H), 3.72–3.62 (m, 1H), 3.47–3.27 (m, 2H), 3.10 (d, J = 13.2 Hz, 1H, 50% isomer), 2.94 (dd, J = 22.9, 12.5 Hz, 1H), 2.77 (s, 1H, 50% isomer), 2.69–2.49 (m, 1H), 2.32 (t, J = 11.0 Hz, 1H, 50% isomer), 2.19 (d, J = 11.3 Hz, 1H, 50% isomer), 1.26 (d, J = 6.4 Hz, 3H, 50% isomer), 1.18 (d, J = 7.0 Hz, 3H, 50% isomer), 1.14–0.79 (m, 2H), 0.65 (s, 1H), 0.52 (s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 161,73, 161.70, 158.29, 144.49, 144.24, 137.05, 136.94, 136.70, 136.59, 131.31, 131.13, 127.86, 127.82, 124.53, 124.42, 124.29, 123.73, 122.24, 122.18, 120.64, 120.51, 112.03, 112.00, 109.04, 108.94, 104.76, 103.90, 74.58, 74.45, 70.14, 58.29, 57.59, 51.97, 51.85, 51.75, 51.46, 43.48, 43.42, 29.85, 22.46, 19.12, 18.23, 14.07, 13.52, 10.09, 9.44, 9.28, 9.11. HRMS (ESI) C₂₄H₂₉O₂N₅ + H⁺ calculated 420.23940, found 420.23976 (0.8ppm). The racemic mixture was purified via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 70:30 up to 50:50, flow rate 25 mL/min) to yield two different fractions corresponding to the two enantiomers 37a and 37b. Analytical HPLC for 37a: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 70 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 37.316 min, purity >95%, de >99% (absorbance at 230 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.22 (s, 1H), 8.07 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.43 (d, J = 8.6 Hz, 1H), 6.37 (s, 1H), 4.46 (d, J = 10.0 Hz, 1H), 4.40 (s, 2H), 3.87 (d, J = 11.2 Hz, 1H), 3.69 (d, J = 11.1 Hz, 1H), 3.39 (q, J = 7.6 Hz, 2H), 2.97 (d, J = 6.9 Hz, 1H), 2.69 (d, J = 12.0 Hz, 1H), 2.60 (t, J = 10.9 Hz, 1H), 2.39 (s, 2H), 1.26 (br s, 1H), 1.10–1.04 (m, 3H), 0.98 (br s, 1H), 0.57 (d, J = 6.7 Hz, 1H), 0.45 (t, J = 6.6 Hz, 1H). Analytical HPLC for 37b: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 70 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 42.160 min, purity 92.9%, de 89.4% (max absorbance at 230.4 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.28 (s, 1H), 8.06 (s, 1H), 7.65 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.30 (d, J = 7.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.83 (s, 1H), 6.46 (s, 1H), 6.32 (d, J = 8.4 Hz, 1H), 4.46 (d, J = 9.9 Hz, 1H), 4.36 (s, 2H), 3.87 (d, J = 11.3 Hz, 1H), 3.71 (d, J = 11.1 Hz, 1H), 3.46 (dt, J = 12.7, 5.9 Hz, 1H), 3.29 (dt, J = 13.8, 6.7 Hz, 1H), 2.99 (d, J = 7.3 Hz, 1H), 2.69 (d, J = 11.9 Hz, 1H), 2.65–2.53 (m, 2H), 2.19 (dd, J = 12.7, 7.4 Hz, 1H), 1.26 (s, 1H), 1.08 (d, J = 6.6 Hz, 3H), 0.94 (d, J = 6.8 Hz, 1H), 0.56 (dd, J = 8.6, 4.8 Hz, 1H), 0.51–0.42 (m, 1H). [α] values for compounds 37a and 37b were not determined.

cis-N-((2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)methyl)-1H-indole-2-carboxamide (38)

Compound 38 was synthesized following the same procedure described for 39, starting from 23b (11.0 mg, 0.06 mmol) and 35 (37.5 mg, 0.16 mmol) to yield 12.3 mg of the final product as a colorless oil, in 48% yield. HRMS (ESI) C₂₄H₂₉O₂N₅ + H⁺ calculated 420.23940, found 420.24047 (2.5ppm). The racemic mixture was purified via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 70:30 up to 50:50, flow rate 25 mL/min) to yield two different fractions corresponding to the two diastereoisomers 38a and 38b. Analytical HPLC for 38a: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 100 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 78.730 min, purity >95%, de >99% (absorbance at 230 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.17 (s, 1H), 7.98 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.59–7.35 (m, 3H), 7.29 (d, J = 7.5 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.82 (s, 1H), 6.43 (d, J = 8.5 Hz, 1H), 4.57 (d, J = 9.4 Hz, 1H), 4.39 (s, 2H), 4.31 (dt, J = 13.9, 6.7 Hz, 1H), 4.02 (d, J = 10.8 Hz, 1H), 3.81 (d, J = 11.4 Hz, 1H), 3.15 (s, 1H), 2.82 (ddd, J = 39.5, 26.2, 13.0 Hz, 3H), 2.51 (d, J = 12.1 Hz, 1H), 2.20 (t, J = 12.0 Hz, 1H), 1.26 (br s, 2H), 1.15 (d, J = 6.6 Hz, 3H), 0.94–0.83 (m, 1H), 0.22 (d, J = 5.4 Hz, 1H); Analytical HPLC for 38b: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 100 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 82.436 min, purity >99%, de >99% (absorbance at 230 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.19 (s, 1H), 8.08 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.46 (dd, J = 15.8, 8.4 Hz, 3H), 7.30 (d, J = 7.5 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.01 (s, 1H), 6.49 (d, J = 8.5 Hz, 1H), 4.63 (d, J = 10.5 Hz, 1H), 4.46 (s, 2H), 4.26 (dd, J = 14.4, 7.0 Hz, 1H), 3.97 (d, J = 12.0 Hz, 1H), 3.72 (d, J = 11.3 Hz, 1H), 2.98–2.83 (m, 3H), 2.78 (t, J = 12.1 Hz, 1H), 2.58 (t, J = 11.3 Hz, 1H), 2.21 (t, J = 11.9 Hz, 1H), 1.26 (br s, 1H), 1.17 (d, J = 6.8 Hz, 3H), 1.14 (br s, 1H), 0.87 (d, J = 5.8 Hz, 1H), 0.21 (d, J = 5.3 Hz, 1H). [α] values for compounds 38a and 38b were not determined.

cis-N-((2-(((2R,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)methyl)-1H-indole-2-carboxamide (39)

Compound 35 (37.5 mg, 0.155 mmol) was dissolved in 1,2-dichloroethane (6 mL) and to the mixture was added 23a (30.0 mg, 0.155 mmol). To the stirring solution was added catalytic amount of acetic acid and the reaction was allowed to stir for 30 minutes, after which sodium triacetoxyborohydride (50.0 mg, 0.23 mmol) was added in one portion, and the reaction mixture was allowed to stir at room temperature overnight. The solvent was removed under reduced pressure and the crude residue purified via flash chromatography, gradually increasing the polarity of the mobile phase, with two fractions of desired product, enriched with the respective diastereoisomer, eluting with 10% DMA (DCM:MeOH 9:1 + 0.5% NH₄OH) to yield colorless oils. The diastereoisomers were further purified via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 70:30 up to 50:50, flow rate 25 mL/min) to yield two different fractions corresponding to the two diastereoisomers 39a (5.7 mg; 8.7%) and 39b (7.6 mg; 12% yield). Analytical HPLC for 39a: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 90 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 38.626 min, purity >99%, de >99% (absorbance at 230 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.49 (s, 1H), 7.95 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 8.3 Hz, 1H), 7.29 (br s, 2H), 7.15 (s, 1H), 7.05 (s, 1H), 6.45 (d, J = 8.5 Hz, 1H), 4.64 (d, J = 13.5 Hz, 2H), 4.48 (s, 1H), 3.89 (d, J = 11.9 Hz, 1H), 3.56 (t, J = 11.3 Hz, 1H), 3.32 (d, J = 11.9 Hz, 1H), 2.68 (t, J = 12.2 Hz, 1H), 2.59 (t, J = 12.6 Hz, 1H), 2.47–2.28 (m, 2H), 2.18–2.05 (m, 2H), 1.13 (br s, 2H), 1.03–0.98 (m, 3H), 0.88 (br s, 1H), 0.24 (br s, 1H). HRMS (ESI) C₂₄H₂₉O₂N₅ + H⁺ calculated 420.23940, found 420.23983 (1ppm). Analytical HPLC for 39b: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (60:40, flow rate 1mL/min), total run time 90 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 48.157 min, purity >99%, de >99% (absorbance at 230 nm). ¹H NMR (400 MHz, CDCl₃) δ 9.38 (s, 1H), 7.90 (s, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.29 (d, J = 8.3 Hz, 2H), 7.18–7.10 (m, 1H), 6.90 (s, 1H), 6.84 (s, 1H), 6.33 (d, J = 8.4 Hz, 1H), 4.48–4.34 (m, 3H), 4.09–3.95 (m, 1H), 3.82 (t, J = 10.1 Hz, 2H), 3.49 (t, J = 10.9 Hz, 1H), 3.44–3.30 (m, 1H), 3.14 (t, J = 12.9 Hz, 1H), 2.96 (d, J = 10.6 Hz, 2H), 2.55–2.47 (m, 1H), 2.42–2.22 (m, 2H), 1.11 (d, J = 6.3 Hz, 3H), 1.01 (d, J = 6.2 Hz, 1H), 0.18 (q, J = 5.5 Hz, 1H); HRMS (ESI) C₂₄H₂₉O₂N₅ + H⁺ calculated 420.23940, found 420.23976 (0.8ppm). [α] values for compounds 39a and 39b were not determined.

Enantiomeric resolution of Racemic Nitro-Olefin Intermediate (±)-41³⁶

Chiral HPLC separation was performed on the racemic mixture, synthesized as previously reported,³⁶ using an Agilent system coupled with UV-Vis/DAD (Diode Array Detector). Separation of the analyte, purity and enantiomeric excess determinations were achieved at 40 °C using Chiralcel OZ-H (Daicel Corporation CPI Company) column (20mm x 250 mmL, 5 μm). The mobile phase used (10 mL/min flow rate) was composed of 10% 2-propanol in hexanes with isocratic elution. The total run time was 60 min, eluting first (−)-(1R,2S)-41a ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: −224.76 (0.860 g/100 mL in MeOH), followed by (+)-(1S,2R)-41b ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: +208.94 (0.760 g/100 mL in MeOH) with a separation time between peaks of approximately 2 minutes. Analytical HPLC for (−)-(1R,2S)-41a: column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (90:10), total run time 40 min, RT 21.021 min, purity 96.1%, ee 92.3% (absorbance at 254 nm). Analytical HPLC for (+)-(1S,2R)-41b: column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (90:10), total run time 40 min, RT 27.677 min, purity 97.1%, ee 94.2% (absorbance at 254 nm). Spectroscopic data for both enantiomers were identical to the racemic mixture.³⁶

(2-(2-aminoethyl)cyclopropyl))methanol ((1R,2S)-42)

Compound was prepared following the synthetic procedure described for (1S,2R)-43, starting from (−)-(1R,2S)-41a.

N-(2-(2-(hydroxymethyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide ((+)-(1S,2R)-43)

Compound was prepared following the synthetic procedure described for (−)-(1R,2S)-44, starting from (1R,2S)-42 (0.130 g, 1.10 mmol). Upon reaction completion, solvent was evaporated under vacuo and the crude material was purified by Combiflash chromatography, eluting in 75% EtOAc/Hexanes to afford the desired product as a pale viscous yellow oil (0.065g / 22% yield). ¹H NMR (400 MHz, CDCl3) 9.58 δ (s, 1H), 7.61 (dd, J = 8.4, 0.8 Hz, 1H), 7.40 (dd, J = 8.4, 0.8 Hz, 1H), 7.27−7.23 (m, 1H), 7.12−7.08 (m, 1H), 7.06 (bt, J = 5.2 Hz, 1H), 6.97 (dd, J = 2.0, 0.8 Hz, 1H),3.98−3.88 (m, 2H), 3.38−3.32 (m, 1H), 3.19 (bs, 1H), 1.99−1.93 (m, 2H), 1.12−1.03 (m, 1H), 0.97−0.90 (m, 1H), 0.71−0.65 (m, 1H), 0.43−0.34 (m, 2H); ¹³C NMR (100 MHz, CDCl3) δ 162.1, 136.3, 130.9, 127.6, 124.4, 121.9, 120.5, 112.0, 102.5, 67.1, 39.8, 34.0, 20.9, 16.0, 9.0. GC−MS (EI) m/z 258.1 (M+). ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: +42.258 (0.310 g/100 mL in MeOH). Analytical HPLC column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 70:30), total run time 60 min, RT 19.654 min, ee 88.5% (absorbance at 254.4 nm).

(2-(2-aminoethyl)cyclopropyl)methanol ((1S,2R)-43)

Lithium aluminum hydride (0.205 g, 5.40 mmol), as a mineral oil dispersion, was added to a vacuum purged round bottom flask, cooled on 0°C ice bath, slowly suspended with the dropwise addition of anhydrous THF (10 mL) and stirred vigorously for 1 hour. A solution of (+)-(1S,2R)-41b (0.33 g, 1.80 mmol) in anhydrous THF (10 mL) was then added dropwise to the stirring suspension over 15 minutes. Upon completion of the addition, reaction flask was removed from the ice bath and allowed to gradually warm to room temperature overnight with constant stirring. Upon completion, reaction was again cooled to 0°C on ice bath and subsequently quenched with the dropwise addition of a 1:1 mixture of MeOH/2N aqueous NaOH. Precipitated solids were then filtered and washed repeatedly with ethyl acetate, collecting and concentrating the resultant filtrate under vacuo to afford the crude amino alcohol product without further purification (0.189 g / 92% yield).

N-(2-(2-(hydroxymethyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide ((−)-(1R,2S)-44)

To a well stirring solution of indole-2-carboxylic acid (0.307g, 1.9 mmol) dissolved in anhydrous THF (10 mL), cooled to 0°C on ice bath, and under inert atmosphere, was added EDC hydrochloride (0.416g, 2.10 mmol) and HOBt (0.281 g, 2.00 mmol) together as a single portion. Reaction mixture was subsequently removed from ice and allowed to stir, warming to room temperature over the following hour. (1S,2R)-43 (0.200 g, 1.70 mmol) was dissolved in anhydrous THF (15 mL) and then added dropwise into the vigorously stirring solution. N,N-Diisopropylethylamine (0.297 g, 0.401 mL, 2.30 mmol) was then added via syringe as a single portion and the reaction allowed to proceed at room temperature over the following 4 hours. Upon reaction completion, solvent was evaporated under vacuo and the crude material was purified by Combiflash chromatography, eluting in 75% EtOAc/Hexanes to afford the desired product as a pale viscous yellow oil (0.075g / 16.7% yield). ¹H NMR (400 MHz, CDCl3) 9.58 δ (s, 1H), 7.61 (dd, J = 8.4, 0.8 Hz, 1H), 7.40 (dd, J = 8.4, 0.8 Hz, 1H), 7.27−7.23 (m, 1H), 7.12−7.08 (m, 1H), 7.06 (bt, J = 5.2 Hz, 1H), 6.97 (dd, J = 2.0, 0.8 Hz, 1H),3.98−3.88 (m, 2H), 3.38−3.32 (m, 1H), 3.19 (bs, 1H), 1.99−1.93 (m, 2H), 1.12−1.03 (m, 1H), 0.97−0.90 (m, 1H), 0.71−0.65 (m, 1H), 0.43−0.34 (m, 2H); ¹³C NMR (100 MHz, CDCl3) δ 162.1, 136.3, 130.9, 127.6, 124.4, 121.9, 120.5, 112.0, 102.5, 67.1, 39.8, 34.0, 20.9, 16.0, 9.0. GC−MS (EI) m/z 258.1 (M+). ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: −38.378 (0.185 g/100 mL in MeOH). Analytical HPLC column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 70:30), total run time 40 min, RT 26.135 min, ee 88.5% (absorbance at 254 nm).

N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide ((−)-(1S,2R)-45)

Compound was prepared following the synthetic procedure described for (+)-(1R,2S)-46, starting from (+)-(1S,2R)-43 (0.065 g, 0.26 mmol). Crude material was purified by Combiflash chromatography, eluting in 50% EtOAc/Hexanes to afford the desired product as a pale white solid (0.45 g / 68% yield). ¹H NMR (400 MHz, CDCl3) δ 9.54 (s, 1H), 9.08 (d, J = 5.2 Hz, 1H), 7.64 (dd, J = 8.4, 0.8 Hz, 1H), 7.44 (dd, J = 8.4, 0.8 Hz, 1H), 7.29 (dd, J = 7.0, 0.8 Hz, 1H), 7.15−7.11 (m, 1H),6.85 (m, 1H), 6.46 (bt, J = 5.2 Hz, 1H), 3.59 (dd, J = 12.8, 7.0 Hz, 2H), 1.76−1.68 (m, 3H), 1.58−1.53 (m, 1H), 1.37−1.32 (m, 1H), 1.02−0.98 (m, 1H); ¹³C NMR (100 MHz, CDCl3) δ 200.7, 161.8, 136.3, 130.5, 127.6, 124.6, 121.9, 120.7, 112.0, 102.0, 39.3, 32.8, 29.9, 20.2, 14.6. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: −30.352 (0.425 g/100 mL in MeOH).

N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide ((+)-(1R,2S)-46)

To a well stirring solution of (−)-(1R,2S)-44 (0.075 g, 0.29 mmol) in anhydrous DCM (10 mL) and under inert atmosphere, was added Dess-Martin Periodinane (0.142 g, 0.33 mmol) as a single portion. Reaction was then allowed to proceed at room temperature over the following 1.5 hours with vigorous stirring. Upon completion, reaction mixture was diluted with additional DCM (50 mL) and decanted to separatory funnel, wherein the organic phase was washed repeatedly with 10 mL portions of an aqueous 10% NaHCO₃ solution. Organic phase was then dried over anhydrous Na₂SO₄, filtered and concentrated under vacuo to afford the crude product as a viscous red oil. Crude material was subsequently purified by Combiflash chromatography, eluting in 50% EtOAc/Hexanes to afford the desired product as a pale white solid (0.035 g / 47% yield). ¹H NMR (400 MHz, CDCl3) δ 9.54 (s, 1H), 9.08 (d, J = 5.2 Hz, 1H), 7.64 (dd, J = 8.4, 0.8 Hz, 1H), 7.44 (dd, J = 8.4, 0.8 Hz, 1H), 7.29 (dd, J = 7.0, 0.8 Hz, 1H), 7.15−7.11 (m, 1H),6.85 (m, 1H), 6.46 (bt, J = 5.2 Hz, 1H), 3.59 (dd, J = 12.8, 7.0 Hz, 2H), 1.76−1.68 (m, 3H), 1.58−1.53 (m, 1H), 1.37−1.32 (m, 1H), 1.02−0.98 (m, 1H); ¹³C NMR (100 MHz, CDCl3) δ 200.7, 161.8, 136.3, 130.5, 127.6, 124.6, 121.9, 120.7, 112.0, 102.0, 39.3, 32.8, 29.9, 20.2, 14.6. ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: +22.58 (0.124 g/100 mL in MeOH).

(S)-2-chloro-1-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)ethan-1-ol (47)

In a round bottom flask covered in aluminum foil, (+)-B-Chlorodiisopinocampheylborane ((+)-DIP-Cl) (3.37 g, 10.5 mmol) was weighed quickly and with limited exposure to light, dissolved with tBuOMe/THF (3:7, 10 mL), placed under an argon atmosphere and subsequently cooled to −40° C in a dry ice/acetonitrile bath. Compound 2-chloro-1-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)ethan-1-one⁴² (2.06 g, 8.2 mmol) was then dissolved in THF (4 mL) and added dropwise to the solution of (+)-DIP-Cl. The reaction was allowed to stir overnight, slowly warming to room temperature. The reaction completion was monitored via thin layer chromatography (TLC) and GC/MS, then 30 mL of saturated NH₄Cl aqueous solution were added to the reaction mixture and allowed to stir for 1 hour at room temperature. The organic phase was collected, and the aqueous layer was extracted with EtOAc (3 × 25 mL). The combined organic layers were washed with brine, dried with Na₂SO₄, filtered and the solvent was removed under reduced pressure. The crude residue was purified via flash chromatography, eluting with hexanes/EtOAc (75:25), to yield 2.07 g of the desired compound as a yellow oil, in 78.6% yield. Spectroscopic data was concurrent with the previously reported ones.⁴² GC/MS (EI) RT 10.785 min, 250.1 m/z. HPLC column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 80:20, flow rate 1mL/min), total run time 50 min, RT 18.163 min, purity >95%, ee >99% (absorbance at 230 nm). ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: +23.3 (0.645 g/100 mL in CHCl₃). Enantiomeric excess and absolute configuration were confirmed via HPLC comparison (same HPLC methods described above) with the (R)-enantiomer (RT 13.056 min, purity 94.7%, ee 90.3%), re-synthesized, as standard, using (−)-DIP-Cl as previously reported.⁴² ${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{23}$: −21.1 (0.620 g/100 mL in CHCl₃)

(S)-2-(2,5-dimethyl-1H-pyrrol-1-yl)-5-(oxiran-2-yl)pyridine (48)

Compound 47 (2.06 g, 0.0082 moles) was dissolved in acetonitrile (200 mL), anhydrous K₂CO₃ was added to the solution, (21.3 g, 0.154 moles) and it was stirred overnight at reflux. The reaction mixture was allowed to cool to room temperature and the suspension was filtered. The solvent was removed under reduced pressure to yield 1.59 g of the desired compound as a brown oil, which was used in the next step without further purification. GC/MS (EI) RT 9.672 min, 214.1 m/z.

(S)-2-(((S)-2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)-2-hydroxyethyl)amino)propan-1-ol (49)

Compound 48 (1.59g, 7.39 mmol) was dissolved in 150 mL of toluene and the solution was stirred to homogeneity. (S)-2-aminopropan-1-ol (2.77g, 36.9 mmol) was added to the solution and the reaction mixture was then heated to reflux and stirred overnight. The reaction was cooled to room temperature and the solvent removed under reduced pressure. The crude residue was dissolved in DCM (30 mL) and washed with water (5 mL). The organic phase was dried with Na₂SO₄, filtered, and evaporated. The crude material was purified via flash chromatography with gradient elution, increasing the polarity of the mobile phase up to 20% DMA (DCM:MeOH + 0.5% NH₄OH), to yield 450 mg of the desired compound as an off-white solid, in 21% yield. ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 8.58 (s, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 5.89 (s, 2H), 4.81 (d, J = 9.5 Hz, 1H), 3.66 (d, J = 10.8 Hz, 1H), 3.4 (t, J = 9.4 Hz, 1H), 2.99 (d, J = 12.1 Hz, 1H), 2.87 (dt, J = 21.7, 10.3 Hz, 2H), 2.11 (s, 6H), 1.47 (d, J = 6.6 Hz, 3H).

Benzyl ((S)-2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)-2-hydroxyethyl)((S)-1-hydroxypropan-2-yl)carbamate (50)

Compound 49 (0.450 g, 1.55 mmol) was dissolved in 25 mL of anhydrous tetrahydrofuran and subsequently cooled to −40° C with a cryo-cooler. In a separate vial, a solution of N-(Benzyloxycarbonyloxy)succinimide in anhydrous tetrahydrofuran (0.465 g, 1.87, 0.2 M) was prepared and transferred dropwise into the reaction flask. Upon completion of the N-(Benzyloxycarbonyloxy)succinimide solution addition, the cryo-cooler was powered off to allow the reaction mixture to slowly reach room temperature. Following overnight stirring at room temperature, an additional equivalent of N-(Benzyloxycarbonyloxy)succinimide (0.386 g, 1.55 mmol) was added to the reaction mixture and the reaction stirred at room temperature for an additional 2 h. The reaction was quenched with the addition of saturated NaHCO₃ aqueous solution (25 mL) and methanol (20 mL) and stirred at room temperature for 1 hr. The organic solvents were removed under reduced pressure and the remaining aqueous layer was extracted with DCM (3 × 30 mL). The combined organic layers were dried with Na₂SO₄, filtered and evaporated under reduced pressure. The crude material was partially purified via flash chromatography, gradually increasing the mobile phase polarity up to hexanes/EtOAc (4:6), to yield 505 mg of the desired product as a colorless oil, in 76.9% yield.

benzyl (2S,5S)-2-(6-(2,5-dimethyl-1H-pyrrol-1-yl)pyridin-3-yl)-5-methylmorpholine-4-carboxylate (51)

Triphenylphosphine (TPP) (0.38 g, 1.44 mmol) and 50 (0.500 g, 1.20 mmol) were dissolved in toluene (20 mL). The solution was stirred at room temperature while diisopropyl azodicarboxylate (DIAD) (0.28 mL, 1.44 mmol) was added dropwise. The reaction mixture was then stirred for 20 h at room temperature. The reaction mixture was evaporated under reduced pressure and the crude residue was purified via flash chromatography, gradually increasing the mobile phase polarity up to hexanes/EtOAc (8:2), to yield 363 mg of the desired product as a colorless viscous oil, in 74.6% yield. ¹H NMR (400 MHz, CDCl₃ + CD₃OD) δ 8.61 (d, J = 12.4 Hz, 1H), 7.86 (d, J = 8.3 Hz, 1H), 7.38 (br s, 5H), 7.23 (d, J = 8.5 Hz, 1H), 5.90 (s, 2H), 5.19 (d, J = 9.8 Hz, 2H), 5.11–4.96 (m, 1H), 4.37–3.99 (m, 2H), 3.89 (d, J = 15.6 Hz, 2H), 3.32–2.96 (m, 1H), 2.12 (s, 6H), 1.32 (d, J = 5.8 Hz, 3H).

benzyl (2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholine-4-carboxylate (52)

Compound 51 (0.363 g, 0.895 mmol) was dissolved in 10 mL of ethanol, hydroxylamine hydrochloride (0.522 g, 7.52 mmol) was then added to the solution in one portion, and the reaction mixture was heated to reflux. After 4 h, additional 8 equivalents of hydroxylamine hydrochloride were added to the flask and the reaction mixture was allowed to stir at reflux overnight. The solvent was removed under reduced pressure, the crude mixture was dissolved in DCM, and washed with saturated NaHCO₃ aqueous solution. The organic phase was dried with Na₂SO₄, filtered, and evaporated. The crude mixture was purified via flash chromatography, eluting with 2% DMA (DCM:MeOH +1% NH₄OH), to yield desired product as a purple oil, in quantitative yield. ¹H NMR (400 MHz, CDCl₃) δ 8.05 (d, J = 9.8 Hz, 1H), 7.47 (d, J = 8.6 Hz, 1H), 7.35 (d, J = 9.4 Hz, 5H), 6.49 (d, J = 8.5 Hz, 1H), 5.16 (d, J = 10.8 Hz, 2H), 5.09–5.01 (m, 1H), 4.51 (s, 2H), 4.38–4.11 (m, 2H), 3.81 (d, J = 16.1 Hz, 2H), 3.06 (dt, J = 25.5, 12.3 Hz, 1H), 1.32 (d, J = 6.2 Hz, 3H).

5-((2S,5S)-5-methylmorpholin-2-yl)pyridin-2-amine (23b)

Compound 52 (0.320 g, 0.977 mmol) was dissolved in 30 mL of ethanol and added to a hydrogenation flask, followed by the addition of Pearlman’s catalyst (Pd(OH)₂/C 20 wt. %, 0.350 g, 0.498 mmol). The reaction mixture was charged and shaken in a Parr apparatus under 50 psi of hydrogen gas atmosphere. The reaction was allowed to proceed for 1 hr. The reaction mixture was subsequently filtered through a wet celite plug, rinsed with ethanol, and evaporated to yield 75 mg of the desired product as a white solid, in 39.7% yield. All the spectroscopic and HPLC data were consistent with the ones described for 23b. GC/MS (EI) RT 9.533 min, 207.0 m/z. HPLC column Chiralcel OD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (90:10 flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 31.552 min, purity 94.5%, de >99% (absorbance at 230.4 nm). Diastereomeric excess was determined via HPLC comparison (same HPLC method described above) with the (R,S)-diastereoisomer 23a previously described (RT 37.332 min, purity >99, ee >99%).

trans-N-(2-(2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide (53)

N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide^{12, 36} was added to a solution of 23b (0.075g, 0.388 mmol) in 1,2-dichloroethane (50 mL). Catalytic amount of glacial acetic acid was added to the stirring solution, and the reaction mixture was allowed to stir for 30 minutes at room temperature before adding sodium triacetoxyborohydride (0.123 g, 0.582 mmol) in one portion. The reaction was stirred for an additional 30 minutes at room temperature, after which the solvent was removed under reduced pressure. The crude mixture was purified via flash chromatography, gradually increasing the mobile phase polarity from 100% DCM to 10% DMA (DCM:MeOH + 0.5% NH₄OH)) to afford 150 mg of the desired product as a 50:50 diastereomeric mixture, as an off-white solid, in 89.2% yield. ¹H NMR (400 MHz, CDCl₃) δ 9.61 (s, 1H, 50% isomer), 9.57 (s, 1H, 50% isomer), 8.06 (d, J = 8.7 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H), 7.32–7.23 (m, 2H), 7.13 (t, J = 7.6 Hz, 1H), 6.94 (s, 1H, 50% isomer), 6.87 (s, 1H, 50% isomer), 6.47 (d, J = 8.4 Hz, 1H), 4.62 (br s, 2H), 4.50 (d, J = 10.0 Hz, 1H), 3.94 (t, J = 11.6 Hz, 1H), 3.72 (t, J = 9.1 Hz, 1H), 3.66–3.47 (m, 2H), 3.03 (d, J = 31.6 Hz, 1H), 2.75 (d, J = 12.5 Hz, 1H), 2.60 (dt, J = 21.5, 11.0, Hz, 1H), 2.47 (dd, J = 12.6, 6.2 Hz, 1H, 50% isomer), 2.34 (t, J = 9.7 Hz, 1H, 50% isomer), 2.22 (dd, J = 12.7, 7.5 Hz, 1H, 50% isomer), 2.10 (s, 1H, 50% isomer), 1.70–1.49 (m, 2H), 1.11 (dd, J = 1.6, 6.66, 3H), 0.71 (d, J = 36.6 Hz, 2H), 0.43 (t, J = 7.0 Hz, 2H); ¹³C NMR (400 MHz, CDCl₃) δ 161.75, 161.72, 158.28, 158.25, 146.23, 146.15, 136.75, 136.43, 131.20, 131.10, 127.82, 126.13, 124.60, 124.54, 122.02, 120.81, 120.76, 112.10, 108.66, 108.59, 102.45, 102.05, 75.89, 75.55, 71.93, 71.58, 58.84, 53.15, 52.82, 52.17, 51.91, 39.85, 39.82, 33.86, 33.72, 22.21, 16.50, 16.18, 15.37, 11.52, 10.78, 8.63, 8.53. HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (50:50 flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 21.873 min and 36.176, purity >95%, dr 50:50 (absorbance at 230 nm). The free base was converted into the corresponding oxalate salt. HRMS (ESI) C₂₅H₃₁O₂N₅ + H⁺ calculated 434.25505, found 434.25604 (2.3ppm). CHN Anal (C₂₅H₃₁N₅O₂ ^. 2 H₂C₂O₄ ^. H₂O) calculated C 55.15, H 5.90, N 11.09; found C 55.55, H 5.75, N 11.12. About 50 mg the oxalate salt were free-based and the diastereomeric mixture was separated via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 40:60 up to 50:50, flow rate 20 mL/min) to yield two different fractions corresponding to the two diastereoisomers (53a) N-(2-((1R,2S)-2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide and (53b) N-(2-((1S,2R)-2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5 methylmorpholino)methyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide. Analytical HPLC for 53a: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (50:50, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 21.362 min, purity >99%, de >99% (absorbance at 230 nm). m.p. 83–84 °C with prior decomposition;${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: +17.8 (0.045 g/100 mL in MeOH). Analytical HPLC for 53b: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (50:50, flow rate 1mL/min), total run time 70 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 35.338 min, purity >95%, de >99% (absorbance at 230 nm). m.p. 63–64 °C with prior decomposition;${\left[\mathrm{\alpha }\right]}_{\mathrm{D}}^{25}$: −14.3 (0.06 g/100 mL in MeOH).

(−)-N-(2-((1R,2S)-2-(hydroxymethyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide (44) and (+)-N-(2-((1S,2R)-2-(hydroxymethyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide (43).

Racemic mixture trans-54 was re-synthesized as previously described.³⁶ The racemic mixture was separated via preparative chiral HPLC (Chiralpak AD-H 21mm x 250mm; 5μm), eluting with n-hexane:2-propanol (from 90:10 up to 80:20, flow rate 18 mL/min) to yield two different fractions corresponding to the two enantiomers (−)-44 and (+)-43. Analytical HPLC for (−)-44, method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 80:20, flow rate 1mL/min), total run time 100 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 35.599 min, purity 88.8%, ee 78.5% (absorbance at 214 nm); method B: column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 47.728 min, purity 89.5%, ee 79% (max absorbance at 214 nm). The results of the HPLC analysis performed on OZ-H column, matched with the analysis performed on the same (−)-(1R,2S)-44 resolved as described in scheme 7. Spectroscopic data and optical rotation were consistent with the ones reported above for the same (−)-(1R,2S)-44. Analytical HPLC for (+)-43, method A: column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 80:20, flow rate 1mL/min), total run time 100 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 40.379 min, purity >95%, ee >99% (absorbance at 214 nm); method B: column Chiralcel OZ-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (from 90:10 up to 70:30, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 33.440 min, purity >95%, ee >99% (absorbance at 214 nm). The results of the HPLC analysis performed on OZ-H column, matched with the analysis performed on the same (+)-(1S,2R)-43 resolved as described in scheme 7. Spectroscopic data and optical rotation were consistent with the ones reported above for the same (+)-(1S,2R)-43.

N-(2-((1S,2R)-2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide (45)

Compound 45 was synthesized following the same procedure described for 35, starting from (+)-43 (62.20 mg, 0.24 mmol). Spectroscopic data and optical rotation were consistent with the ones reported above for the same (−)-(1S,2R)-45.

N-(2-((1R,2S)-2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide (46)

Compound 46 was synthesized following the same procedure described for 35, starting from (−)-44 (60 mg, 0.23 mmol). Spectroscopic data and optical rotation were consistent with the ones reported above for the same (+)-(1R,2S)-46.

N-(2-((1S,2R)-2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide (53b)

Compound 53b was synthesized following the same procedure described for 53, starting from 45 (3.20 mg, 0.02 mmol). Spectroscopic data were consistent with the ones obtained for 53 and HPLC analyses, using 53b as standard, were consistent to the results previously obtained, and reported above, for 53b, confirming the assigned absolute configuration. Analytical HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (50:50, flow rate 1mL/min), total run time 60 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 35.991 min, purity >99%, ee >99% (absorbance at 230 nm).

N-(2-((1R,2S)-2-(((2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino)methyl)cyclopropyl)ethyl)-1H-indole-2-carboxamide (53a)

Compound 53a was synthesized following the same procedure described for 53, starting from 46 (40 mg, 0.16 mmol). Spectroscopic data were consistent with the ones obtained for 53 and HPLC analyses, using 53a as standard, were consistent to the results previously obtained, and reported above, obtained for 53a, confirming the assigned absolute configuration. Analytical HPLC column Chiralpak AD-H (4.6mm x 250mm; particle size 5μm), elution with n-hexane:2-propanol (50:50, flow rate 1mL/min), total run time 45 min, multiple DAD λ absorbance signals measured in the range of 210–280 nm, RT 20.626 min, purity >99%, ee >99% (absorbance at 230 nm).

7-(4-(4-(3-chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (57)

K₂CO₃ (2.173g, 15.7 mmol) was added to a solution of 1-(3-chloro-5-ethyl-2-methoxyphenyl)piperazine¹¹ (0.50g, 1.97 mmol) in N,N-dimethylformamide, followed by 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (0.70g, 2.37 mmol), at room temperature. The reaction mixture was warmed to 90 °C and stirred for 6h. The reaction mixture was diluted with ice-cold water and extracted with chloroform. The combined organic phase was washed with ice-cold water (4 X 200 mL), dried over Na₂SO₄, filtered, and evaporated. The crude compound was purified by flash chromatography, eluting with 20% acetone in chloroform, to obtain the desired product in 67 % yield. ¹H NMR (400 MHz, CDCl₃) δ 7.82 (brs, 1H), 7.04 (d, J = 8.2 Hz, 1H), 6.84 (s, 1H), 6.61 (s, 1H), 6.52 (d, J = 8.3 Hz, 1H), 6.30 (s, 1H), 3.97 (dd, J = 13.2, 6.6 Hz, 2H), 3.81 (d, J = 22.7 Hz, 3H), 3.14 (s, 3H), 3.00 – 2.83 (m, 3H), 2.74 – 2.38 (m, 8H), 1.99 – 1.50 (m, 6H), 1.21 (dd, J = 18.6, 11.0 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.38, 158.65, 146.37, 146.16, 140.77, 138.04, 128.67, 128.20, 122.16, 116.65, 115.72, 108.61, 102.09, 67.91, 58.99, 58.26, 53.75, 50.27, 31.09, 29.37, 28.45, 27.25, 25.73, 24.59, 23.40, 15.38. The free base was converted into the corresponding hydrochloride salt, which was precipitated from acetone. HRMS (ESI) C₂₆H₃₄O₃N₃Cl + H⁺ calculated 472.23615, found 472.23603 (−1.4ppm). M.p. 193−194 °C, Anal (C₂₆H₃₄ClN₃O₃^.HCl^.1.25H₂O) calculated C 58.81, H 7.12, N 7.91; found C 58.71, H 6.66, N 7.52.

7-(4-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (58)

The compound was synthesized according to the same method described for 57, starting form 1-(2-chloro-3-ethylphenyl)piperazine¹¹ (0.50g, 2.23 mmol) and 7-(4-bromobutoxy)-3,4-dihydroquinolin-2(1H)-one (0.80g, 2.67 mmol). The desired product was obtained in 74 % yield. ¹H NMR (400 MHz, CDCl₃) δ 7.68 (s, 1H), 7.16 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 8.1 Hz, 1H), 6.94 (t, J = 8.1 Hz, 2H), 6.53 (d, J = 8.1 Hz, 1H), 6.30 (s, 1H), 3.97 (t, J = 6.1 Hz, 2H), 3.07 (s, 3H), 2.90 (t, J = 7.4 Hz, 2H), 2.84 – 2.71 (m, 2H), 2.63 (dd, J = 16.9, 9.3 Hz, 5H), 2.56 – 2.38 (m, 2H), 1.83 (dd, J = 14.0, 7.1 Hz, 2H), 1.77 – 1.66 (m, 2H), 1.62 (s, 2H), 1.23 (t, J = 7.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 171.70, 158.67, 149.62, 143.20, 138.11, 128.68, 126.86, 123.93, 117.97, 115.67, 108.77, 108.67, 102.18, 67.93, 58.25, 53.46, 51.56, 31.08, 27.45, 27.29, 24.58, 23.44, 14.08. The free base was converted into the corresponding hydrochloride salt, which was precipitated from acetone. M.p. 197−198 °C, Anal (C₂₅H₃₂ClN₃O₂^.HCl^.0.5H₂O) calculated C 61.60, H 6.69, N 8.62; found C 61.60, H 7.03, N 8.62.

Radioligand Binding Studies

Radioligand binding assays were conducted similarly to previously described.^{31, 36} HEK293 cells stably expressing human D_2LR or D₃R or D_4.4 were grown in a 50:50 mix of DMEM and Ham’s F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1X antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, NY) and kept in an incubator at 37 °C and 5% CO₂. Upon reaching 80–90% confluence, cells were harvested using pre-mixed Earle’s Balanced Salt Solution (EBSS) with 5 mM EDTA (Life Technologies) and centrifuged at 3,000 rpm for 10 min at 21 °C. The supernatant was removed, and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl₂, 5 mM Tris, pH 7.4 at 4 °C) and centrifuged at 14,500 rpm (~25,000 g) for 30 min at 4 °C. The pellet was then resuspended in fresh binding buffer. A Bradford protein assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration. For [³H]-(R)-(+)-7-OH-DPAT binding studies, membranes were harvested fresh; the binding buffer was made from 50 mM Tris, 10 mM MgCl₂, 1 mM EDTA, pH 7.4. On test day, each test compound was diluted into half-log serial dilutions using 30% DMSO vehicle. When it was necessary to assist solubilization of the drugs at the highest tested concentration, 0.1% acetic acid (final concentration v/v) was added alongside the vehicle. Membranes were diluted in fresh binding buffer. Radioligand competition experiments were conducted in 96-well plates containing 300 μl fresh binding buffer, 50 μl of diluted test compound, 100 μl of membranes (40–80 μg/well, 20–40 μg/well, and 30–60 μg/well total protein for hD_2LR, hD₃R, and hD_4.4R respectively), and 50 μl of radioligand diluted in binding buffer ([³H]-(R)-(+)-7-OH-DPAT: 1.5 nM final concentration for hD_2L, 0.5 nM final concentration for hD₃, and 3 nM final concentration for hD_4.4 ARC, Saint Louis, MO). Aliquots of [³H]-(R)-(+)-7-OH-DPAT solution were also quantified accurately to determine how much radioactivity was added, taking in account the experimentally determined counter efficiency. Nonspecific binding was determined using 10 μM (+)-butaclamol (Sigma-Aldrich, St. Louis, MO) and total binding was determined with 30% DMSO vehicle. All compound dilutions were tested in duplicate or triplicate and the reaction incubated for 90 min at room temperature. The reaction was terminated by filtration through Perkin Elmer Uni-Filter-96 GF/B, presoaked for 90 min in 0.5% polyethylenimine, using a Brandel 96-Well Plates Harvester Manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed 3 times with 3 mL (3 × 1 mL/well) of ice-cold binding buffer. 65 μL Perkin Elmer MicroScint 20 Scintillation Cocktail was added to each well and filters were counted using a Perkin Elmer MicroBeta Microplate Counter. IC₅₀ values for each compound were determined from dose-response curves and K_i values were calculated using the Cheng-Prusoff equation;⁶¹ K_d values for [³H]-(R)-(+)-7-OH-DPAT were determined via separate homologous competitive binding experiments. When a complete inhibition couldn’t be achieved at the highest tested concentrations, K_i values have been extrapolated by constraining the bottom of the dose-response curves (= 0% residual specific binding) in the non-linear regression analysis. These analyses were performed using GraphPad Prism version 6.00 for Macintosh (GraphPad Software, San Diego, CA). All the results were rounded to the third significant figure. K_i values were determined from at least 3 independent experiments and are reported as mean ± SEM.

Phase I Metabolism in Rat Liver Microsomes

For phase I metabolism, the reactions were carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system (1.3 mM NADPH, 3.3 mM glucose 6-phosphate, 3.3 mM MgCl₂, 0.4 U/mL glucose-6-phosphate dehydrogenase, 50 μM sodium citrate). Reactions in triplicate were initiated by addition of the liver microsomes to the incubation mixture (compound final concentration was 10 μM; 0.5 mg/mL microsomes). Compound disappearance was monitored via LC/MS/MS. Chromatographic analysis was performed using an Accela™ ultra high-performance system consisting of an analytical pump, and an autosampler coupled with TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). Separation of the analyte from potentially interfering material was achieved at ambient temperature using Agilent Eclipse Plus column (100 × 2.1mm i.d.) packed with a 1.8 μm C18 stationary phase. The mobile phase used was composed of 0.1% Formic Acid in Acetonitrile and 0.1% Formic Acid in water with gradient elution, starting with 10% (organic) linearly increasing to 99% up to 2.5 min, and re-equilibrating to 10% by 2.7 min. The total run time for each analyte was 5.0 min.

Metabolite identification (MET-ID) was performed on a Dionex ultra high-performance LC system coupled with Q Exactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham MA). Separation was achieved using Agilent Eclipse Plus column (100 × 2.1mm i.d; maintained at 35^oC) packed with a 1.8 μm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Pumps were operated at a flow rate of 0.3 mL/min for 7 min using gradient elution. The mass spectrometer controlled by Xcalibur software 4.0.27.13 (Thermo Scientific) was operated with a HESI ion source in positive ionization mode. Metabolites were identified in the full-scan mode (from m/z 50 to 1600) by comparing t = 0 samples with t = 60 min samples and structures were proposed based on the accurate mass information.

X-ray crystal data on compounds (−)-(1R,2S)-41a and (2R,5S)-23a

Single-crystal X-ray diffraction data on compound (−)-(1R,2S)-41a was collected using Cu Kα radiation and a Bruker Photon 100 CMOS area detector. Single-crystal X-ray diffraction data on compound (2R,5S)-23a was collected using Mo Kα radiation and a Bruker APEX II area detector. The crystals were prepared for data collection by coating with high viscosity microscope oil. The oil-coated crystal was mounted on a micro-mesh mount (MiteGen, Inc.) and transferred to the diffractometer. The structures were solved by direct methods and refined by full-matrix least squares on F² values using the programs found in the SHELXL suite (Bruker, SHELXL v2014.7, 2014, Bruker AXS Inc., Madison, WI). Corrections were applied for Lorentz, polarization, and absorption effects. Parameters refined included atomic coordinates and anisotropic thermal parameters for all non-hydrogen atoms. The H atoms were included using a riding model. Complete information on data collection and refinement is available in the supplemental material.

The 0.331 × 0.105 × 0.020 mm³ crystal of (−)-(1R,2S)-41a was orthorhombic in space group P2₁2₁2₁, with unit cell dimensions a = 5.6918(2) Å, b = 6.2010(2) Å, c = 26.4447(10) Å, α = 90°, β = 90°, and γ = 90°. Data was collected at 150K. Data was 98.13% complete to 67.7° θ (~0.83 Å) with an average redundancy of 5.52. The final anisotropic full matrix least-squares refinement on F² with 120 variables converged at R₁ = 3.49%, for the observed data and wR2 = 9.45% for all data.

The 0.294 × 0.099 × 0.054 mm³ crystal of (2R,5S)-23a was monoclinic in space group C2, with unit cell dimensions a = 30.3306(18) Å, b = 5.4531(3) Å, c = 12.7310(7) Å, α = 90°, β = 99.926(2)°, and γ = 90°. Data was collected at 293K. Data was 99.6% complete to 25.242° θ (~0.83 Å) with an average redundancy of 2.71. The final anisotropic full matrix least-squares refinement on F² with 261 variables converged at R₁ = 4.26%, for the observed data and wR2 = 10.42% for all data.

ABBREVIATIONS USED

DA

dopamine

CNS

central nervous system

OBS

orthosteric binding site

SBP

secondary binding pocket

PP

primary pharmacophore

SP

secondary pharmacophore

EDC

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide

DMP

Dess-Martin periodinane

TBDMS

(tert-butyl)dimethylsilane

STAB

sodium triacetoxyborohydride

HPLC

high pressure liquid chromatography

DIP-Cl

B-Chlorodiisopinocampheylborane

7-OH-DPAT

7-Hydroxy-N,N-dipropyl-2-aminotetralin