Novel Bivalent Ligands Based on the Sumanirole ... - ACS Publications

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Novel Bivalent Ligands Based on the Sumanirole Pharmacophore Reveal Dopamine D2 Receptor (D2R) Biased Agonism Alessandro Bonifazi,†,§ Hideaki Yano,†,§ Michael P. Ellenberger,† Ludovic Muller,‡ Vivek Kumar,† Mu-Fa Zou,† Ning Sheng Cai,† Adrian M. Guerrero,† Amina S. Woods,‡ Lei Shi,† and Amy Hauck Newman*,† †

Molecular Targets and Medications Discovery Branch, National Institute on Drug AbuseIntramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States ‡ Structural Biology Unit, National Institute on Drug AbuseIntramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore, Maryland 21224, United States S Supporting Information *

ABSTRACT: The development of bivalent ligands has attracted interest as a way to potentially improve the selectivity and/or affinity for a specific receptor subtype. The ability to bind two distinct receptor binding sites simultaneously can allow the selective activation of specific G-protein dependent or β-arrestin-mediated cascade pathways. Herein, we developed an extended SAR study using sumanirole (1) as the primary pharmacophore. We found that substitutions in the N-1- and/or N-5-positions, physiochemical properties of those substituents, and secondary aromatic pharmacophores can enhance agonist efficacy for the cAMP inhibition mediated by Gi/o-proteins, while reducing or suppressing potency and efficacy toward β-arrestin recruitment. Compound 19 was identified as a new lead for its selective D2 G-protein biased agonism with an EC50 in the subnanomolar range. Structure−activity correlations were observed between substitutions in positions N-1 and/or N-5 of 1 and the capacity of the new bivalent compounds to selectively activate G-proteins versus β-arrestin recruitment in D2R-BRET functional assays.

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INTRODUCTION Dopamine (DA) receptors are G protein-coupled receptors (GPCRs) having the characteristic seven transmembrane helical segments. The five DA receptor subtypes are classified into two families, the D1-like and D2-like receptors, based on their sequence and signaling pathway similarities. The D2-like receptor family includes DA D2 receptor (D2R), D3, and D4 receptors (D3R and D4R, respectively), which couple to Gi/o/z proteins, inhibiting adenylyl cyclase-mediated cAMP production. In addition, arrestin recruitment by these receptors is associated with receptor internalization and desensitization as well as G protein independent kinase signaling pathways.1 Functional selectivity (“bias profile”) is defined as the ability of GPCR ligands to differentially modulate canonical G-protein activation pathways or G-protein-independent pathways that involve different proteins such as β-arrestins.2,3 Several neurological disorders, such as schizophrenia, bipolar disorder, Parkinson’s disease, and restless legs syndrome, are strictly related to hypoactivation or hyperactivation of specific This article not subject to U.S. Copyright. Published 2017 by the American Chemical Society

DA transmission pathways, in particular those involving the D2like subtypes.4 Indeed, many widely prescribed medications activate or block D2-like receptors.1,5 In particular, most of the currently FDA-approved typical or “atypical” (with reduced extrapyramidal side effects) antipsychotic drugs (APDs) behave as D2R antagonists or partial agonists.6,7 However, despite showing efficacy in the treatment of positive symptoms, most of the current APDs lack significant effectiveness with regard to negative and/or cognitive symptoms.8 Further, D2-like receptor agonists are used to treat Parkinson’s disease and associated dyskinesias or are frequently prescribed for their potential neuroprotective properties.5 Nevertheless, medications targeting D2-like receptors are associated with motor, metabolic, cardiovascular, and potential emotional negative side effects.7,9,10 This may in part be due to the lack of drugs able to selectively discriminate between the D2-like receptor Received: December 20, 2016 Published: March 16, 2017 2890

DOI: 10.1021/acs.jmedchem.6b01875 J. Med. Chem. 2017, 60, 2890−2907

Journal of Medicinal Chemistry

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Figure 1. Design of new predicted D2R agonists or partial agonists using a bivalent molecular approach, inspired by the classic D3R selective antagonist PP.

which specific D2R pathways need to be selectively targeted to obtain new pharmacotherapies as well as which ones might be associated with side effects. Sumanirole ((R)-5,6-dihydro-5-(methylamino)-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (Z)-2-butenedioate; 1, U-95666E, PNU-95666E (Figure 1)) was reported to be a highly selective D2R full agonist that was originally developed for the treatment of Parkinson’s disease and restless legs syndrome.25,26 Despite being fully pharmacologically characterized in a variety of in vivo studies27−29 and evaluated in clinical trials, it has never been clinically approved.30−34 However, this parent molecule remains a valuable scaffold for lead optimization. The orthosteric binding site (OBS), the site in which DA, and presumably 1, binds to induce receptor activation, is highly similar in both D2R and D3R. In a recent study, using an agonist radiotracer to label both human D2R and D3R in radioligand competition binding assays, we observed how, despite its high D2R affinity and agonist potency, 1 showed modest selectivity for the D2R over the D3R.35 To determine differences in binding modes of 1 at the OBS of D2R and D3R, and to elucidate the molecular requirements that confer subtype selectivity, extensive SAR and modeling studies were performed as well.35 Because novel D2R-selective ligands can be useful as pharmacological tools, in this work, starting from previously obtained compounds (Figure 1) with highest affinity and selectivity for D2R, we extend SAR investigation using a bivalent approach to potentially bind simultaneously the OBS and secondary binding pocket (SBP) within the D2R. The bivalent design has been used successfully, over the past decade, to synthesize highly selective D3R antagonists.36−38 A bivalent compound combines both a primary pharmacophore (PP), for binding in the OBS, and an often aromatic secondary pharmacophore (SP), able to recognize a SBP via linkers of appropriate length.39−42 For example, the 2,3-dichlorophenylpiperazine is a well-known and widely used PP to produce high

subtypes, which, despite mediating different physiological processes in distinct brain regions, show high levels of structural homology in their binding sites.11 Thus, the need for the identification of new ligands with increased D2-like subtype selectivity and potentially reduced side effects remains an important objective in drug design. In addition to the receptor subtype selectivity, the development of ligands able to selectively activate canonical or noncanonical signaling pathways within the same receptor subtype poses a further challenge. The D2R subtype is one of the main targets at which the therapeutic relevance of functional selectivity is being identified. Structure−activity relationship (SAR) studies are being developed to highlight molecular determinants necessary for D2R functional selectivity,12−15 and biased agonism at the D2R has been suggested to underlie antipsychotic efficacy.16 For example, the efficacy of the FDA-approved atypical antipsychotic aripiprazole had been thought to be related to its behavior as a canonical antagonist at D2R.17−19 However, recent studies show partial agonism12,20−22 and bias toward the G protein pathway over β-arrestin recruitment.23,24 Thus, modification of the aripiprazole scaffold, in an effort to develop ligands that display bias between G protein signaling and β-arrestin recruitment, would address the D2R signaling discrimination among G-protein and/or βarrestin pathways which may be directly involved with the therapeutic actions of antipsychotic drugs.12,20 Unlike what has been already reported regarding the selective activation of D2R β-arrestin pathways, recent studies have highlighted new interesting structural requirements to obtain D2R biased agonism toward the cAMP inhibition mediated by Gi/o-proteins.14 Starting from these findings, we designed a new series of D2R preferential agonists able to selectively inhibit cAMP accumulation mediated by Gi/o-proteins signaling, dissecting the necessary structural requirements for their bias profile, and providing new pharmacological tools to understand 2891



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Figure 2. Novel compounds and their templates.

Scheme 1a

a

(a) NaH, THF, RT; (b) K2CO3, acetone, reflux.

the SP molecular determinants, additional bivalent molecules presenting the phenylbutoxyl moiety or the 7-hydroxy-3,4dihydroquinolin-2(1H)-one scaffold, inspired by aripiprazole, in both the N-1 and N-5 positions were designed and synthesized. The starting synthesis and evaluation of compounds having a simple nondecorated phenyl SP was important to generate initial SAR and fully understand how these analogues behaved when compared to substituted or more extended secondary pharmacophores. To determine the optimal distance between the two pharmacophores, polymethylene or polyethylene glycol

affinity and D3R subtype selectivity when linked to a properly substituted arylbutylamide.43,44 In this study, the N-5-CH3 or the N-5-n-propyl sumanirole scaffolds, playing the PP role essential for the recognition of the D2R OBS, were alkylated in both N-1 or N-5 positions with several SPs (Figures 1 and 2). The 2-benzofuranylbutylamide and 2-indolylbutylamide bivalent derivatives inserted in position N-5 (compounds 18 and 19, respectively) were prepared and evaluated for their binding affinities, as reported in the previous study.35 To extend the SAR and further explore 2892



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Scheme 2a

a

(a) KOH, DCM, p-TsCl; (b) NaH, THF, RT; (c) K2CO3, acetone, reflux.

Scheme 3a

a

(a) KOH, CHCl3, p-TsCl; (b) KI, acetone, reflux; (c) K2CO3, DMF, RT; (d) K2CO3, acetone, reflux.

Compounds 15 and 16 were prepared in an analogous way according to Scheme 2. Highly hydrophilic di- or triethylene glycol linkers were used with the aim of increasing the polarity of the final compounds without affecting the distances between the two aromatic pharmacophores. Compound 31 was prepared starting from triethylene glycol (29) in the presence of 4-methylbenzenesulfonyl chloride, transforming the hydroxyl functional groups into good leaving groups, allowing for the monosubstitution of 23, yielding intermediate 33. Compound 32 was obtained in the same way, starting from the commercially available 1-bromo-2-(2-bromoethoxy)ethane (30). Finally, the N-1 selective alkylation of 1 was performed as described above. To further explore the structural requirements of the second aromatic pharmacophore, the phenyl group was replaced with 3,4-dihydroquinolin-2(1H)-one. Compound 17 was synthesized as depicted in Scheme 3. The two hydroxyl groups of the commercially available tetraethylene glycol (35) were converted into the corresponding highly reactive di-iodo derivative 36 via tosylation in the presence of 4-methylbenzenesulfonyl chloride and subsequent treatment with a large excess of KI in acetone. Then, 36 was reacted with 7-hydroxy-3,4-dihydroquinolin-2(1H)-one (34) under basic conditions to afford 37, which then was stirred at reflux in acetone and potassium carbonate with 1 to afford the desired 17. The analogues 11 and 12, presenting 4 or 12 methylene units in the linker chain, respectively, were prepared following the same procedure described for 17, and reported in Scheme 4, using 1,4-

(which significantly decreases the molecule’s lipophilicity) linker chains of varying lengths were used to prepare all the desired compounds, in all the possible template combinations as summarized in Figure 2. The goal behind the design of D2R bivalent probes was not only to bind both the OBS and SBP, enhancing subtype affinity and selectivity but primarily to obtain novel agonists with a decoration theme from biased ligands that may lead to the selective activation of specific signaling pathways. Thus, in addition to being subjected to radioligand binding assays, the most relevant D2-preferential ligands, together with a few compounds selected from the previous study,35 were tested for their agonist potency in five different bioluminescence resonance energy transfer (BRET) functional studies.

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CHEMISTRY

The synthesis of compounds 4−10 is described in Scheme 1. A series of dibromo alkanes with lengths ranging from 4 to 12 methylene units were conjugated at first with 4-phenylbutanol (23) in the presence of 1 equiv of sodium hydride in order to obtain selectively the monoalkylated intermediates 24−28. Subsequently, 1 and its analogue 2 (Figure 1), prepared as previously described,45,46 were selectively alkylated at the N-1 position with the monobromo intermediates in the presence of potassium carbonate. The starting materials were thus converted into the desired final compounds in good yields and with regiospecificity. 2893



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Scheme 4a

a

(a) K2CO3, DMF, RT; (b) K2CO3, acetone, reflux.

Scheme 5a

a

(a) H2, Pd(OH)2, 50 psi, 1 h; (b) K2CO3, DMF, 60 °C; H2, Pd(OH)2, 50 psi, 48 h.

Scheme 6a

4-aminobutanol, CDI, THF, RT; (b) Ph3P, CBr4, ACN; (c) N-(benzyloxycarbonyloxy)succinimide, THF, −40 °C, 3 h; (d) 1-bromopropane, K2CO3, acetone, reflux; (e) H2, Pd(OH)2, 50 psi, 2 h; (f) 45, K2CO3, DMF, 60 °C.

a

14, and 20, to confirm the proposed structures and highlight the consistent fragmentation pattern associated with the selective substitution in position N-1 or N-5 (MS/MS spectra, Supporting Information, Figure S1). Moreover, NMR studies showed characteristic peaks with high similarity in chemical shifts across the series, confirming all structures assigned to the different regioisomers. To confirm the observation of the preferential alkylation in the amide NH position, the N-5 substituted regiosiomer 22 was prepared in a selective way, maintaining the amide function protected as reported in Scheme 5. Starting from diprotected 40,45 the Cbz protecting group was removed by hydrogenation in the presence of palladium hydroxide at a pressure of 50 psi, shaking in the Parr apparatus for 1 h. Under this kinetically

dibromobutane and 1,12-dibromododecane as building blocks for the linker preparation before the two-step alkylations. High resolution mass spectroscopy (HRMS) by electrospray in positive ion mode analysis was performed for 11, 14, 20, and 22 (Supporting Information, Table S1). The desired regioselective conjugation of the N-1 position was also confirmed by tandem mass spectrometry (MS/MS) analysis of 11 (Supporting Information, Figure S1). The MS/MS spectrum of the final product showed a fragmentation pattern consistent with the proposed structure and, in particular, the observed loss of the −NH2CH3 (protonated −NHCH3) fragment, highlighting how the reaction conditions allowed only alkylation of the amide NH. Similarly, MS/MS analyses were performed for all the couples of regioisomers 11 and 22, 2894



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Scheme 7a

a

K2CO3, DMF, 60 °C; (b) CH3CH2CHO, NaBH(O2CCH3)3, DCE.

Table 1. In Vitro Radioligand Competition Binding at hD2R and hD3R

a Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng−Prusoff equation.60 Each Ki value represents the arithmetic mean ± SEM of at least three independent experiments, each performed in duplicate. bDetermined with ChemDraw Professional 15.0. cPreviously reported in refs 35 and 59.

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Table 2. D2-Mediated cAMP Inhibition and β-Arrestin2 Recruitmentc

a Cannot be determined (often >5 μM). bValues between −10 and 0. cPotency (expressed as EC50 or IC50) and efficacy values (%, normalized to quinpirole Emax or Imax) for hD2R expressed in HEK293 cells. The values represent the arithmetic mean ± SEM of at least three independent experiments, each performed in triplicate. Antagonist mode dose−response experiments were performed in presence of 1 μM quinpirole. Emax and Imax values for EC50 = ND or IC50 = ND cannot be determined and the numbers shown are based on the highest or lowest drug induced BRET at 100 μM (highest concentration tested) of the compounds.

analogue 21 was prepared following the same procedure, starting from the previously reported 51 35 as a key intermediate. Finally, to complete the SAR studies and synthesize all the desired regioisomers, the indolylbutylamide pharmacophore was inserted, by nucleophilic substitution, in position N-1 in combination with both N-n-propyl or N,N-ndipropyl substituents in N-5, yielding 13 and 14 as described in Scheme 7.

controlled reaction condition, the cleavage of the Cbz was selectively achieved without affecting the methoxyl group protecting the N-1 position. Intermediate 41 was then alkylated in the presence of 38. The nucleophilic substitution involving the basic secondary amine in position N-5 required slightly stronger conditions, using potassium carbonate in DMF to yield 42. Finally, the cleavage of the methoxyl function was achieved by hydrogenation at a pressure of 50 psi, with palladium hydroxide as the catalyst, for 48 h. NMR studies performed on 22 showed considerably different chemical shifts of the most significant 1H and 13C peaks when compared to N1 substituted analogue 11 (singlet at 2.50 ppm and double doublets at 3.02 and 4.02 ppm for 11; singlet at 2.39 and double doublet at 4.18 for 22), supporting once more all of the assigned structures. Compound 20 was prepared as presented in Scheme 6. 1HIndole-2-carboxylic acid (43) was reacted with 4-aminobutan-1ol, using 1,1′-carbonyldiimidazole (CDI) as a coupling agent. The hydroxyl group was replaced with bromine in the presence of tetrabromomethane and triphenylphosphine in acetonitrile to afford 45.35,47 Selective protection of the basic secondary amine of 2 was effected with N-(benzyloxycarbonyloxy)succinimide at −40 °C. The n-propyl substituent was inserted in the N-1 position, and the cleavage of the Cbz protecting group was effected by hydrogenation with Pd/C to yield the desired intermediate, 50. The last coupling step was performed using potassium carbonate in DMF and heating the reaction at 60 °C for 12 h to afford 20. The structure of 20 was confirmed with the analysis of the molecular fragmentation induced by MS/MS (Supporting Information, Figure S1). The N-methyl

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RESULTS AND DISCUSSION Radioligand Binding. The affinity (Ki) values of 1 and its analogues, as well as the classic D2-like agonists DA and (−)-quinpirole and the partial agonist aripiprazole, were determined using agonist radioligand binding assays (Table 1). In particular, the absolute Ki values for these predicted agonists at both D2R and D3R were determined in competition with the agonist [3H]-(R)-(+)-7-OH-DPAT as opposed to the radiolabeled antagonist (e.g., [3H]N-methylspiperone) since it has been established that DA receptor agonists more readily compete against radiolabeled agonist,48 allowing the accurate evaluation of the affinity for the receptors’ active state. Indeed, we recently observed how 1 and its analogues showed higher affinities when determined in competition with the agonist [3H]-(R)-(+)-7-OH-DPAT compared to the antagonist [3H]Nmethylspiperone.35 The previously synthesized and characterized agonists35 2, 3, 18, and 19 are reported in Table 1 as reference compounds for a more complete understanding on how different secondary aromatic pharmacophores and regioisomers modulate D2R binding. On the basis of their binding affinities and pharmacological functional profiles, the 2896



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Table 3. D2-Mediated Gi Activation and Go Activationc

Cannot be determined (often >5 μM). bValues between −10 and 0. cPotency (expressed as EC50 or IC50) and efficacy values (%, normalized to quinpirole Emax or Imax) for hD2R expressed in HEK293 cells.c The values represent the arithmetic mean ± SEM of at least three independent experiments, each performed in triplicate. Antagonist mode dose−response experiments were performed in presence of 1 μM quinpirole. Emax and Imax values for EC50 = ND or IC50 = ND cannot be determined and the numbers shown are based on the highest or lowest drug induced BRET at 100 μM (highest concentration tested) of the compounds. a

crucial for the recognition and modulation of the D2R affinity and that alkylation in this position with bulky groups is well tolerated without affecting the ability of the PP to access the OBS. High lipophilicity might play a crucial role in molecular recognition of binding sites, interfering with the key interactions within the receptor pockets and potentially altering bioavailability. For this reason, 15 and 16 (analogues of 7 and 8) bearing much more hydrophilic diethylene or triethylene glycol linkers (see cLogP values in Table 1), respectively, were synthesized and tested. As expected, both compounds showed D2R affinities comparable to other molecules within the same series (D2R Ki = 111 and 34.7 nM, respectively), but the D3R affinities decreased about 3−5-fold. These findings highlight the idea that different linkers are not just simple connectors between two pharmacophores, but can be modulators of pharmacological profiles and, in this particular case, the higher polarity seems to preferentially lead the compounds toward increased D2R selectivity. To extend the SAR studies with a larger group of secondary aromatic pharmacophores, the 7-hydroxy-3,4-dihydroquinolin2(1H)-one moiety, inspired by the high affinity D2R partial agonist/antagonist and FDA-approved atypical antipsychotic, aripiprazole, was introduced in both the N-1 (connected by butyl, dodecanyl, and tetraethylene glycol chains) and N-5 (connected by butyl chain) positions. Aripiprazole shows very high affinity for both D2R and D3R (D2 Ki = 0.48 nM; D3 Ki = 3.57 nM), and large series of its derivatives have been reported to show D2R biased agonist profiles.19,12 Both 17 and 12, endowed with tetraethylene glycol and dodecanyl linkers, showed low affinities for both D2R (Ki = 306 and 284 nM,

lead compounds, together with the novel derivatives with highest affinity, were further evaluated in this study using bioluminescence resonance energy transfer assays (BRET), with the aim to identify structural requirements necessary for functionally selective agonism (“biased agonism”). The new compounds were evaluated for their agonist and/or antagonist activities in five different BRET-based constructs (Tables 2 and 3): (i) adenylyl cyclase inhibition, (ii) β-arrestin recruitment, (iii) Gi protein activation, (iv) Go protein activation, and (v) GI protein recruitment. Using [3H]-(R)-(+)-7-OH-DPAT as the radiotracer, the affinity of 1 (Table 1) for D2R and D3R (Ki = 46.3 and 573 nM, respectively) was consistent with the recently reported data.35 By replacing the N-5-CH3 group in 1 with an n-propyl group (2), both D2R and D3R binding affinities improved (Ki = 2.78 and 25.5 nM for D2R and D3R, respectively), maintaining a similar D2R/D3R selectivity (9-fold). Global N-n-propyl substitutions at positions 1 and 5 (3) did not affect D2R binding affinity as compared to the parent molecule 2; however, the D3R affinity markedly increased by 7.5-fold. The observation of how alkylation at the position 1 imidazonitrogen was very well tolerated in terms of D2R affinity suggested the possibility to introduce and extend linkers, presenting a secondary aromatic pharmacophore. Independently from the presence of N-5-CH3 or N-5-n-propyl groups on the sumanirole moiety, all the new compounds with a simple phenylbutoxyl moiety connected in position N-1 by polymethylene linker chains of different lengths (from n = 4 to n = 12) showed very similar D2R binding affinities ranging from 23.8 to 124 nM, with a D2R/D3R selectivity ratio 8 > 9 > 16) while largely maintaining similar efficacy for cAMP inhibition. Also, this series of compounds shows decreases in potencies for both cAMP inhibition and β-arrestin2 recruitment. The same trend in potencies is observed when the phenyl group is replaced by 3,4-dihydro-quinolin-2-one (in the order of 11 > 12 = 17). On the other hand, substitution at the N-5 position with a short alkyl linker followed by various aromatic groups (18, 19, 22) comprises different characteristics in which the potency for cAMP inhibition and β-arrestin2 recruitment is consistently higher than 1, with the exception of 18 being similarly potent for cAMP inhibition. Next, owing to the high potency profile in both cAMP inhibition and β-arrestin2 recruitment, the effect of substitution with n-propyl groups at positions N-1 and N-5 in 3 was evaluated. When substituted with a linker followed by the 2indole amide at position N-1, efficacy is improved but potency is decreased for both cAMP inhibition and β-arrestin2 recruitment (e.g., 14). Interestingly, a similar efficacy level is observed in both assays for 11, another N-1 substituted ligand without propyl substituents (albeit 3,4-dihydro-quinolin-2-one instead of indole). At the same time, 14 shows an EC50 falling in between 3 and 11 for both assays, indicating the dipropyl substituent at the N-5 position contributes to the enhancement of potency. Indeed, the D2R affinity from binding results parallels the potency differences of these three ligands. In contrast, N-5 substitution of 3 with the butyl-2-indole-amide moiety exhibits much lower potency (20) in both assays. When compared to 3 and 19 (equivalent of 20 without the propyl groups at N-1 and N-5 positions), 20 not only shows significantly reduced potency for both assays, in terms of efficacy, but 20 shows an intriguing enhancement of functional selectivity toward cAMP inhibition in which only 5% efficacy is observed for β-arrestin2 recruitment with full agonist efficacy for cAMP inhibition. Together with the low efficacy of 3 (31%), a propyl substituent at N-1 position may play a role in suppressing β-arrestin2 recruitment. To assess the signaling bias between the cAMP inhibition and β-arrestin2 recruitment, bias indices were derived by two different methods: the operational model24,55,56 and the Emax− EC50 model15 (Table 4). Both methods integrate efficacy and potency differences into a single index. Notably, most of the numbers converge in the two independent analysis methods,

respectively) and D3R (Ki = 640 and 598 nM, respectively). Analogously, 11, with a butyl spacer between N-1 position and the 3,4-dihydroquinolin-2(1H)-one scaffold, showed a Ki of 338 nM, with a moderate 4-fold D2R-selectivity over D3R. Shifting the butyl aryl linker from N-1 position to N-5-CH3 secondary amine position afforded the regioisomer 22, presenting higher D2R selectivity (D2R/D3R = 9.5-fold) with 10-fold increased D2R affinity (Ki = 40 nM), essentially identical to the parent compound, 1. The binding data obtained for 22 were compared with the ones previously reported for 18 and 19, bearing 2benzofuranylbutyl and 2-indolylbutylamide secondary aromatic groups, respectively.35 The most interesting combination of D2R affinity and simultaneous D2R/D3R selectivity was observed for 19 (D2R Ki = 5.78 nM and 13.3-fold selectivity over D3R), suggesting the 2-indolylamide as the most promising secondary pharmacophore, which has previously been described as a privileged structure of the D2-like receptors.49,50 Compound 19 was modified by maintaining the butyl linked 2-indolylamide and introducing two n-propyl groups in both N-1 and N-5 positions at the same time. The obtained compound 20 was fully characterized and, despite a very high D2R affinity in the low nanomolar range (D2R Ki = 8.43 nM), it demonstrated significantly higher D3R affinity in the subnanomolar range (D3R Ki = 0.69 nM), with a consequent reversed 12-fold selectivity for the D3R over D2R. The same binding profile was confirmed with 21, differing from 20 by the presence of N-5-CH3 instead of N-5-n-propyl. Indeed, 21, despite a slightly reduced overall affinity (D2R Ki = 21.8 nM and D3R Ki = 2.45 nM), showed a similar 9-fold preferential selectivity toward D3R over D2R. Finally, when the 2-indolylbutylamide was inserted in position N-1, in combination with both N-5-n-propyl or N,N-5-n-dipropyl substituents (13 and 14, respectively), the binding results show an improved or similar D2R affinity (13 D2R Ki = 18.5 and 14 D2R Ki = 86.8) despite significantly lower D3R affinities. This interesting finding opens the possibility of identifying not only selective D2R ligands but also D3R agonists or partial agonists potentially endowed with bias functional profiles. Moreover, it highlights how the sumanirole scaffold, when substituted in different key positions, can achieve high discrimination and preferential recognition of D2R or D3R OBS, respectively. In particular, positions N-1 and N-5, if properly substituted with specific secondary pharmacophores, appear to be critical for achieving D2R or D3R selectivity, respectively. Additional binding studies were performed in the same cells used for the BRET assays, described in the subsequent functional assay sections, in order to determine Ki values for 1, 19, and 20 (Supporting Information, Table S2). Of note, similar affinities were obtained using either stably transfected D2R or transiently transfected D2R-BRET constructs, demonstrating that the transient transfection of the receptor and the G protein do not affect the ligand affinity or the overall Bmax (Supporting Information, Table S2). These data confirm that the Ki values were similar to the corresponding agonist potencies (EC50 values). Cellular Function Assays. cAMP Inhibition and βArrestin Recruitment. To evaluate whether the most interesting compounds with the highest D2R affinities were able to create key interactions within the D2R protein leading to a selective activation of specific signaling pathways (biased agonism), extensive BRET studies were performed in different cell constructs as reported in Tables 2 and 3 and Supporting Information, Table S3. 2898



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Table 4. Bias Factor Valuesa

a

(1 and 3), the N-5 position alkylated group (18, 19, and 22), and the combination compound (14). Lower potencies are observed for N-1 position substituted analogues (8, 9, 15, 17). In terms of efficacies, the relative differences among the ligands are much more pronounced compared to the cAMP inhibition results. While 1 and its N-5 propyl substituted analogue 2 show higher efficacies over prototypical agonist quinpirole, N-5 substituted series (18, 19, and 22) also maintains full or higher efficacy levels. N-1 and N-5 propyl substituted compound 3, the N-1 substituted group (particularly 8, 9, and 12), as well as the combination group (14 and 20), show partial activities less than 50% of quinpirole with generally low potencies. The partial agonist characteristics were then tested in antagonist mode with 1 μM quinpirole preactivation. As seen in Imax values, significant antagonism activities were confirmed for those showing partial agonism. The discrepancies of Imax between Gi1 activation and cAMP inhibition, in which cAMP inhibition shows lack of antagonist activities in antagonist mode, are likely due to the saturability of the biosensor proteins. Partial antagonist activity (by the ligands less than −100% Imax) probably allows residual cAMP molecules to saturate the sensitive biosensor proteins, masking the detection of antagonist activities. Meanwhile, Gi1 activation assay in antagonist mode clearly demonstrates antagonist activities of the partial agonists, indicating the advantage of the efficacy detection in Gi1 activation assay. When compared to Gi1 BRET, notably the overall potencies for GoA BRET, are higher by about 5-fold for most of the ligands. However, relative differences among the ligands for GoA BRET are similar regarding both the potency and efficacy (Table 3 right). In good agreement with Gi1 results, antagonist mode in GoA activation assay does show partial agonist activities converting into partial antagonist activities, particularly in the N-1 substituted series (8, 9, 16, 15, and 12) and the combination group (20 and 14). Finally, Gi1 activation results were also substantiated by another configuration of BRET that detects the coupling of G protein to the receptor (termed engagement BRET; Supporting Information, Table 2). Overall, the potencies are slightly lower than the Gi1 activation results, while the efficacies are also lower than the activation results. This could be due to the fusion proteins or the ratio between the amount of receptor and G protein expressed. Nevertheless, the relative potency and efficacy differences among the tested ligands are similar to the Gi1 activation results. In summary, the cellular function studies revealed distinct characteristics at N-1 and N-5 substitution. N-1 substitution results in compounds that are less potent partial agonists for both G protein and β-arrestin activities. N-5 substitution results in compounds that are more potent for both G protein and βarrestin activities while varying in Emax close to full for G protein and partial for β-arrestin2 coupling. In particular, 19 exhibits the most G protein biased profile. n-Propyl substitution also gives us an insight for the differential effect between N-1 and N-5 substitution. The comparison among the propyl substituent combination compounds (i.e., 3, 14, and 20) illustrates that, despite overall lower potencies, substitution of N-1 propyl with a linked 3,4-dihydro-quinolin-2-one (3−14) increases the efficacy for both G protein and β-arrestin activities while substitution of N-5 propyl with a linked 3,4-dihydroquinolin-2-one (3−20) shows a much reduced efficacy at βarrestin attributed to an intriguing enhancement of functional selectivity toward cAMP inhibition and further corroborating

The values above 0.5 are highlighted.

validating the accuracy of our obtained values. As expected, due to the high potency in the cAMP inhibition assay, 19 shows the most significant cAMP inhibition bias close to two logs of magnitude. Compound 20 also shows a moderate bias toward cAMP inhibition due to the very low efficacy in β-arrestin2 recruitment. On the contrary, N-1-substitution shows weak βarrestin2 recruitment bias despite their low efficacies (8 and 16). Lastly, the extent of antagonist activities were tested in antagonist mode in which 1 μM quinpirole is added before the test ligands to determine if they block agonist activity. Imax values (normalized to the BRET ratio of 1 μM quinpirole) are following the patterns of partiality in Emax by the compounds themselves. Meanwhile, the lack of antagonist activities in cAMP inhibition is consistent with overall high efficacies seen for most of the compounds tested. Gi and Go Activation. Beyond the cAMP and β-arrestin readouts, activation of specific Gα species within Gi-like proteins was further investigated for their biased activities. Gi1 and GoA, expressed in different degrees in different brain regions, are homologous but most discrete within Gi-like proteins. While both Gi1 and GoA are widely expressed throughout the brain, Go activity has been implicated more in striatal brain function.57 Detected by the BRET change due to the conformational change within the G protein heterotrimer,58 Gi1 activation follows a similar trend of relative potency differences among all the ligands, albeit the overall potency is substantially lower (Table 3 left). Particularly, higher potencies relative to 1 for Gi1 assay are observed for the N-1 n-propyl substituted analogues 2899



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the role of the N-1 propyl substituent in lowering efficacy for βarrestin activity.

with gradient elution, starting with 10% (organic) linearly increasing to 60% up to 5 min, maintaining at 60% (5−15 min), and re-equlibrating to 10%. The total run time for each analyte was 20 min. Unless otherwise stated, all the compounds were evaluated to be >95% pure on the basis of combustion analysis, NMR, GC-MS, HPLC-DAD, HRMS (using a ESI source in positive mode with a mass error less than 5 ppm), and MS/MS fragmentation analysis (LTQ-Orbitrap Velos Thermo-Scientific, San Jose, CA) to confirm the assigned structures and regiochemistry. (R)-1-(4-(4-Phenylbutoxy)butyl)-5-(propylamino)-5,6-dihydro-4Himidazo[4,5,1-ij]quinolin-2(1H)-one (4). 24 (0.23 g; 0.0008 mol) was added to a solution of 246 (0.20 g; 0.0008 mol) and K2CO3 (0.6 g; 0.0045 mol) in acetone (15 mL). The reaction mixture was stirred at reflux overnight and filtered, and the solvent was evaporated. The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product as a colorless oil in 57% yield. The free base was converted into the oxalate salt (white solid). 1H NMR (400 MHz, DMSO-d6) δ 0.90 (t, J = 7.2 Hz, 3H), 1.46−1.73 (m, 10H), 2.54 (t, J = 7.4 Hz, 2H), 2.95−3.04 (m, 4H), 3.22 (d, J = 16.8 Hz, 2H), 3.32−3.39 (m, 4H), 3.79−3.81 (m, 2H), 4.11 (t, J = 7.0 Hz, 1H), 6.82−6.97 (m, 3H), 7.10 (dd, J = 7.2, 3.8 Hz, 3H), 7.24 (t, J = 7.4 Hz, 2H), ppm. 13C NMR (100 MHz, CD3OD) δ 9.85, 19.29, 25.13, 26.37, 26.45, 27.90, 28.89, 35.22, 39.59, 40.76, 51.38, 69.78, 70.32, 106.78, 107.74, 114.00, 119.69, 119.88, 122.02, 122.04, 125.21, 125.30, 127.80, 127.87, 128.00, 142.27, 153.49, 165.21, ppm. HPLC-DAD (Rt: 5.476 min; purity 90.32%); mp 108−109 °C; [α]23 D −14.8 (MeOH, c 0.29). Anal. (C27H37N3O2·1.25H2C2O4·0.25H2O·0.25NH4OH) C, H, N. (R)-5-(Methylamino)-1-(4-(4-phenylbutoxy)butyl)-5,6-dihydro4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (5). The reaction followed the procedure described for 4 starting from 145 (0.20 g; 0.0009 mol) and 24 (0.28 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product in a 43% yield. 1H NMR (400 MHz, CDCl3) δ 1.56−1.85 (m, 8H), 2.54 (s, 3H), 2.61 (m, 2H), 2.77 (dd, J = 7.6, 7.6 Hz, 1H), 3.05 (dd, J = 4.0, 4.4 Hz, 1H), 3.38−3.44 (m, 5H), 3.63 (dd, J = 7.2, 7.2 Hz, 1H), 3.85−3.90 (m, 2H), 4.05 (dd, J = 3.6, 3.6 Hz, 1H), 6.83 (t, J = 8.2 Hz, 2H), 6.96 (t, J = 7.6 Hz, 1H), 7.17 (m, 3H), 7.26 (t, J = 7.6 Hz, 2H), ppm. The free base was converted into the oxalate salt (white solid). 13 C NMR (100 MHz, CD3OD) δ 25.11, 26.28, 26.48, 27.86, 28.87, 30.49, 35.20, 39.28, 40.77, 52.31, 69.77, 70.32, 106.85, 113.74, 119.96, 122.09, 125.11, 125.28, 127.86, 127.98, 142.26, 153.55, 164.42, ppm; mp 126−127 °C; [α]23 D −5.6 (MeOH, c 0.07). Anal. (C25H33N3O2· 1.25H2C2O4·0.5H2O) C, H, N. (R)-1-(6-(4-Phenylbutoxy)hexyl)-5-(propylamino)-5,6-dihydro4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (6). The reaction followed the procedure described for 4 starting from 246 (0.20 g; 0.0008 mol) and 25 (0.30 g; 0.0008 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product as a colorless oil in 32% yield. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.2 Hz, 3H), 1.35−1.85 (m, 14H), 2.63−2.80 (m, 6H), 3.08 (dd, J = 4.0, 4.0 Hz, 1H), 3.29 (m, 1H), 3.36−3.39 (m, 4H), 3.56 (dd, J = 8.0, 8.0 Hz, 1H), 3.82−3.93 (m, 3H), 4.09 (m, 1H), 6.82 (dd, J = 7.6, 7.6 Hz, 2H), 6.97 (t, J = 7.6 Hz, 1H), 7.14−7.18 (m, 3H), 7.26 (t, J = 7.8 Hz, 2H), ppm. The free base was converted into the oxalate salt (white solid). 13C NMR (100 MHz, CD3OD) δ 9.84, 19.39, 25.49, 26.15, 26.60, 27.83, 28.16, 28.83, 29.13, 35.18, 39.71, 40.91, 70.23, 70.27, 106.77, 114.06, 119.86, 122.03, 125.21, 127.85, 127.88, 127.97, 142.28, 153.54, 165.41, ppm. HPLC-DAD (Rt: 6.000 min; purity 96.26%); mp 155−156 °C; [α]23 D −3.1 (MeOH, c 0.10). Anal. (C29H41N3O2· 1.5H2C2O4·0.25NH4OH) C, H, N. (R)-5-(Methylamino)-1-(6-(4-phenylbutoxy)hexyl)-5,6-dihydro4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (7). The reaction followed the procedure described for 4 starting from 145 (0.20 g; 0.0009 mol) and 25 (0.30 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product in a 43% yield. 1H NMR (400 MHz, CDCl3) δ 1.37−1.73 (m, 12H), 2.54 (s, 3H), 2.60−2.63 (m, 2H), 2.78 (dd, J = 7.2, 7.6 Hz, 1H), 3.06 (dd, J = 4.0, 4.0 Hz, 1H), 3.22 (m, 1H), 3.35−3.41 (m, 4H), 3.63 (dd, J = 7.2, 6.8 Hz, 1H), 3.81−3.86 (m, 2H), 4.05 (dd, J = 4.0, 4.0 Hz,

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CONCLUSION The current studies have revealed distinct pharmacological profiles at N-1 and/or N-5 substitution and led to the identification of new potent and efficacious D2R biased agonists. The combined affinities and functional profiles of the new bivalent compounds allowed us to classify the different templates into two macro-families according to their regiochemistry. In detail, substitutions in the N-5 position increase D2R subtype affinity (Ki) and enhance potencies (EC50), in a selective manner, toward the D2R G-protein signaling (leading to cAMP inhibition), with 19 showing close to 2 orders of magnitude difference. In contrast, when focusing on the efficacies with which the new bivalent molecules activate the specific D2R functional pathways, it is remarkable how N-1 substitutions lead to decreased affinities (Ki) and potencies (EC50) but allow near full activation (Emax) of only Gi/o signaling pathways (i.e., 11, 16, 20). While 19 exhibits the most compelling case of G protein bias among the novel analogues tested with a low nanomolar affinity for the D2R subtype and picomolar EC50 potency for cAMP inhibition, to a lesser extent, G protein bias was also observed with compound 20. Overall, 19 is a good candidate for further evaluation in in vivo animal models for D2R-mediated pathologies such as psychomotor function. Future and planned animal studies with 19 and 20 compared to β-arrestin biased agonists will reveal mechanistically related behaviors12 and the interplay between G-protein- and β-arrestin-mediated signaling in D2R-related physiological effects.

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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. Coupling constants are measured in Hz. Chemical shifts for 13 C 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). Gas chromatography−mass spectrometry (GC/MS) data were acquired (where obtainable) using an Agilent Technologies (Santa Clara, CA) 6890N 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 5973 mass-selective ion detector in electronimpact 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 (100 °C) was held for 3 min and then increased to 295 °C at 15 °C/min over 13 min, and finally maintained at 295 °C for 10 min. All column chromatography was performed using silica gel (Merck, 230−400 mesh, 60 Å) or preparative thin layer chromatography (silica gel, Analtech, 1000 μm). The eluting solvent system CMA refers to CHCl3/CH3OH/NH4OH in the percentage indicated where NH4OH is usually 1%. If not otherwise stated, all spectroscopic data and yields refer to the free base. Microanalyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree with ±0.4% of calculated values. HPLC analysis was performed using an Agilent system coupled with UV−vis/DAD (diode array detector). Separation of the analyte was achieved at 40 °C using an Agilent Poroshell 120 EC-C18 (4.6 mm × 50 mm, 2.7 μm) column. The mobile phase used (0.5 mL/min flow rate) was composed of 0.1% acetic acid in H2O and acetonitrile 2900



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1H), 6.83 (dd, J = 7.2, 7.6 Hz, 2H), 6.97 (t, J = 7.8 Hz, 1H), 7.16 (d, J = 7.6 Hz, 3H), 7.26 (t, J = 7.6 Hz, 2H), ppm. The free base was converted into the oxalate salt (white solid). 13C NMR (100 MHz, CD3OD) δ 25.50, 26.14, 26.29, 27.83, 28.15, 28.84, 29.14, 30.53, 35.18, 39.27, 40.91, 52.34, 70.24, 70.28, 106.88, 113.56, 119.95, 122.15, 125.07, 125.28, 127.85, 127.97, 142.27, 153.59, 164.76, ppm. HPLC-DAD (Rt: 5.733 min; purity 98.00%); mp 119−121 °C; [α]23 D −7.0 (MeOH, c 0.06). Anal. (C27H37N3O2·1.6H2C2O4·0.4NH4OH) C, H, N. (R)-5-(Methylamino)-1-(8-(4-phenylbutoxy)octyl)-5,6-dihydro4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (8). The reaction followed the procedure described for 4 starting from 145 (0.20 g; 0.0009 mol) and 26 (0.34 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product in a 24% yield. 1H NMR (400 MHz, CDCl3) δ 1.30−1.76 (m, 16H), 2.54 (s, 3H), 2.63 (t, J = 7.4 Hz, 2H), 2.78 (dd, J = 7.6, 7.6 Hz, 1H), 3.06 (dd, J = 4.0, 4.0 Hz, 1H), 3.22 (m, 1H), 3.35−3.42 (m, 4H), 3.64 (dd, J = 7.6, 7.2 Hz, 1H), 3.81−3.86 (m, 2H), 4.05 (dd, J = 4.0, 4.0 Hz, 1H), 6.83 (dd, J = 7.2, 7.6 Hz, 2H), 6.98 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.2 Hz, 3H), 7.26 (m, 2H), ppm. 13C NMR (100 MHz, CDCl3) δ 26.13, 26.83, 28.09, 28.68, 29.26, 29.34, 29.41, 29.70, 29.74, 31.17, 34.15, 35.74, 41.39, 42.87, 53.29, 70.67, 70.94, 105.70, 117.19, 119.62, 121.06, 125.65, 126.14, 127.75, 128.25, 128.42, 142.52, 153.55, ppm. The free base was converted into the oxalate salt (white solid); mp 133−134 °C; [α]23 D −7.1 (MeOH, c 0.45). Anal. (C29H41N3O2· H2C2O4·0.5H2O) C, H, N. (R)-5-(Methylamino)-1-(10-(4-phenylbutoxy)decyl)-5,6-dihydro4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (9). The reaction followed the procedure described for 4 starting from 145 (0.20 g; 0.0009 mol) and 27 (0.33 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product in a 24% yield. 1H NMR (400 MHz, CDCl3) δ 1.26−1.86 (m, 20H), 2.55 (s, 3H), 2.60−2.63 (m, 2H), 2.82 (dd, J = 8.0, 8.0 Hz, 1H), 3.08 (dd, J = 4.2, 4.2 Hz, 1H), 3.22 (m, 1H), 3.37−3.41 (m, 4H), 3.66 (t, J = 9.6 Hz, 1H), 3.81−3.86 (m, 2H), 4.07 (dd, J = 4.2, 4.2 Hz, 1H), 6.83 (dd, J = 7.6 Hz, 8.0 Hz, 2H), 6.99 (t, J = 7.8 Hz, 1H), 7.15−7.19 (m, 3H), 7.28 (m, 2H), ppm. 13C NMR (100 MHz, CDCl3) δ 26.16, 26.87, 28.09, 28.68, 29.26, 29.29, 29.42, 29.43, 29.52, 29.75, 31.03, 34.03, 35.75, 41.41, 42.74, 53.28, 53.76, 70.67, 71.00, 105.75, 117.20, 119.61, 121.08, 125.65, 128.24, 128.42, 142.53, 153.53, ppm. The free base was converted into the oxalate salt (white solid); mp 114−115 °C; [α]23 D − 2.8 (MeOH, c 0.15). Anal. (C31H45N3O2·1.5H2C2O4· 1.25H2O) C, H, N. (R)-5-(Methylamino)-1-(12-(4-phenylbutoxy)dodecyl)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (10). The reaction followed the procedure described for 4 starting from 145 (0.20 g; 0.0009 mol) and 28 (0.39 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 1% CMA to give the pure product in a 24% yield. 1H NMR (400 MHz, CDCl3) δ 1.24−1.87 (m, 24H), 2.55 (s, 3H), 2.61−2.65 (t, J = 7.4 Hz, 2H), 2.79 (dd, J = 8.0, 8.0 Hz, 1H), 3.07 (dd, 4.0, 4.0 Hz, 1H), 3.24 (m, 1H), 3.38−3.41 (m, 4H), 3.65 (dd, J = 7.2, 6.8 Hz, 1H), 3.81−3.86 (m, 2H), 4.07 (dd, J = 4.6, 4.6 Hz, 1H), 6.84 (dd, J = 7.6, 8.0 Hz, 2H), 6.99 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 3H), 7.25 (m, 2H), ppm. The free base was converted into the oxalate salt (white solid). 13C NMR (100 MHz, CD3OD) δ 25.85, 26.29, 26.38, 27.89, 28.21, 28.86, 28.95, 29.11, 29.20, 29.23, 29.28, 29.30, 30.51, 35.22, 39.28, 40.98, 52.31, 70.23, 70.47, 106.85, 113.68, 119.96, 122.12, 125.08, 125.30, 127.87, 127.93, 127.99, 142,29, 153.57, 164.77, ppm; mp 142−143 °C; [α]23 D −0.9 (MeOH, c 0.23). Anal. (C33H49N3O2·H2C2O4·2H2O) C, H, N. (R)-5-(Methylamino)-1-(4-((2-oxo-1,2,3,4-tetrahydroquinolin-7yl)oxy)butyl)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (11). The reaction followed the procedure described for 4, starting from 145 (0.10 g; 0.0005 mol) and 38 (0.15 g; 0.0005 mol). The crude compound was purified via flash chromatography, eluting with 5% CMA to give the pure product as a yellow oil in 38% yield. 1H NMR (400 MHz, CDCl3) δ 1.78−1.80 (m, 2H), 1.81−1.94 (m, 2H), 2.50 (s, 3H), 2.56 (t, J = 12.0 Hz, 2H), 2.75 (dd, J = 7.6, 7.6 Hz, 1H), 2.84 (t, J = 7.4 Hz, 2H), 3.02 (dd, J = 4.0, 4.0 Hz, 1H), 3.19 (m, 1H), 3.63 (dd, J = 7.2, 7.2 Hz, 1H), 3.88−3.95 (m, 4H), 4.02 (dd, J = 3.6, 3.2 Hz, 1H),

6.37 (s, 1H), 6.44 (d, J = 8.4 Hz, 1H), 6.82 (t, J = 7.6 Hz, 2H), 6.96 (t, J = 8.6 Hz, 2H), 9.03 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 24.57, 25.13, 26.30, 31.05, 31.07, 34.09, 40.82, 42.89, 53.23, 67.34, 102.12, 105.72, 108.76, 115.74, 117.32, 119.82, 121.17, 126.15, 127.57, 128.61, 138.16, 153.63, 158.45, 171.57, ppm. The free base was converted into the maleate salt (white solid). HRMS (ESI) calculated 421.2234, found 421.2241 (M + H+); mp 144−145 °C (dec); [α]23 D −6.7 (MeOH, c 0.18). Anal. (C24H28N4O3·H4C4O4·H2O) C, H, N. (R)-5-(Methylamino)-1-(12-((2-oxo-1,2,3,4-tetrahydroquinolin-7yl)oxy)dodecyl)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (12). The reaction followed the procedure described for 4 starting from 145 (0.10 g; 0.0005 mol) and 39 (0.20 g; 0.0005 mol). The crude compound was purified via flash chromatography, eluting with 5% CMA to give the pure product as a yellow oil in 34% yield. 1H NMR (400 MHz, CDCl3) δ 1.24−1.43 (m, 18H), 1.69−1.76 (m, 2H), 2.53 (s, 3H), 2.58 (t, J = 7.4 Hz, 2H), 2.76−2.88 (m, 3H), 3.06 (dd, J = 4.0, 4.0 Hz, 1H), 3.23 (m, 1H), 3.56−3.67 (m, 2H), 3.77−3.89 (m, 3H), 4.03 (dd, J = 4.0, 4.2 Hz, 1H), 6.36 (s, 1H), 6.48 (d, J = 8.4 Hz, 1H), 6.82 (dd, J = 7.2, 7.2 Hz, 2H), 7.00 (m, 2H), 8.54 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 24.57, 25.97, 26.82, 28.64, 29.20, 29.22, 29.80, 29.39, 29.41, 29.44, 30.95, 31.09, 33.96, 41.39, 42.74, 53.24, 68.16, 102.25, 105.71, 108.64, 115.53, 117.07, 119.61, 121.06, 126.12, 127.76, 128.52, 138.19, 153.54, 158.76, 171.87, ppm. The free base was converted into the oxalate salt (white solid); mp 147−148 °C; [α]23 D −6.5 (MeOH, c 0.20). Anal. (C32H44N4O3·1.25H2C2O4·1.25H2O) C, H, N. (R)-N-(4-(2-Oxo-5-(propylamino)-5,6-dihydro-4H-imidazo[4,5,1ij]quinolin-1(2H)-yl)butyl)-1H-indole-2-carboxamide (13). A solution of 246 (0.08 g; 0.0003 mol), 45 (0.10 g; 0.0003 mmol), and K2CO3 (0.09 g; 0.0007 mmol) in DMF (2 mL) was stirred overnight at 60 °C, filtered, and evaporated to yield a crude product. The crude compound was purified via preparative TLC, eluting with 10% CMA to give the pure product as a white solid in 14% yield. 1H NMR (400 MHz, CDCl3) δ 0.92 (t, J = 7.4 Hz, 3H), 1.50 (m, 2H), 1.70 (t, J = 6.8 Hz, 2H), 1.89 (t, J = 7.0 Hz, 2H), 2.67−2.81 (m, 3H), 3.08 (dd, J = 4.4, 4.0 Hz, 1H), 3.32 (m, 1H), 3.54−3.63 (m, 3H), 3.95 (dt, J = 6.8, 6.8 Hz, 2H), 4.12 (dd, J = 3.2, 3.6 Hz, 1H), 6.86 (dd, J = 7.2, 7.6 Hz, 2H), 6.97 (d, J = 7.6 Hz, 1H), 7.00 (m, 1H), 7.15−7.09 (m, 2H), 7.26 (m, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 9.76 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 11.71, 23.42, 25.66, 26.59, 31.52, 39.55, 40.74, 43.68, 49.32, 51.57, 102.79, 105.71, 111.71, 117.76, 119.98, 120.41, 121.36, 121.98, 124.23, 126.21, 127.34, 127.73, 131.02, 136.33, 153.89, 161.81, ppm; mp 201−204 °C (dec); [α]23 D −0.3 (CHCl3, c 0.40). Anal. (C26H31N5O2·0.5H2O) C, H, N. (R)-N-(4-(5-(Dipropylamino)-2-oxo-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-1(2H)-yl)butyl)-1H-indole-2-carboxamide (14). The reaction followed the procedure described for 13 starting from 52 (0.04 g; 0.0001 mol). The crude compound was purified via preparative TLC, eluting with 10% CMA to give the pure product a white solid in 17% yield. 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.4 Hz, 6H), 1.46 (m, 4H), 1.70 (m, 2H), 1.90 (m, 2H), 2.46−2.60 (m, 4H), 2.82−2.94 (m, 2H), 3.31 (m, 1H), 3.48 (t, J = 11.4 Hz, 1H), 3.57 (q, J = 6.4 Hz, 2H), 3.96 (t, J = 6.6 Hz, 2H), 4.20 (dd, J = 4.4, 4.8 Hz, 1H), 6.80 (d, J = 7.6 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H), 6.96−7.01 (m, 2H), 7.09−7.14 (m and br s, 2H), 7.26 (dt, J = 4.0 Hz, 3.6 Hz, 1H), 7.42 (d, J = 8.0, 1H), 7.64 (d, J = 8.0 Hz, 1H), 9.62 (br s, 1H). 13 C NMR (100 MHz, CDCl3) δ 11.68, 22.32, 25.58, 26.57, 26.87, 39.57, 40.68, 52.82, 54.68, 102.80, 105.42, 111.88, 119.31, 119.84, 120.42, 121.15, 121.99, 124.24, 126.25, 127.33, 127.75, 131.01, 136.30, 153.89, 161.78, ppm. HRMS (ESI) calculated 488.3020, found 488.3016 (M + H+); mp 197−199 °C; [α]23 D −5.7 (MeOH, c 0.07). Anal. (C29H37N5O2·0.15CHCl3) C, H, N. (R)-5-(Methylamino)-1-(2-(2-(4-phenylbutoxy)ethoxy)ethyl)-5,6dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (15). 32 (0.27 g; 0.0009 mol) was added portionwise to a solution of 145 (0.20 g; 0.0009 mol) and K2CO3 (0.62 g; 0.005 mol) in acetone (50 mL). The reaction mixture was stirred at reflux overnight and filtered, and the solvent was evaporated. The crude compound was purified via flash chromatography, eluting with 10% CMA to give the pure product as a yellow oil in 49% yield. 1H NMR (400 MHz, CDCl3) δ 1.58−1.69 (m, 2901



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4H), 2.54 (s, 3H), 2.60−2.65 (m, 4H), 2.76 (dd, J = 7.6, 8.0 Hz, 1H), 3.03 (dd, J = 3.8, 4.0 Hz, 1H), 3.21 (m, 1H), 3.41−3.68 (m, 6H), 3.76 (t, J = 8.2 Hz, 2H), 4.05 (m, 2H), 6.84 (m, 1H), 6.92 (m, 2H), 7.16 (m, 3H), 7.27 (m, 2H), ppm. The free base was converted into the oxalate salt (white solid). 13C NMR (100 MHz, CD3OD) δ 26.29, 27.74, 28.79, 30.49, 35.16, 39.26, 41.14, 52.26, 65.49, 68.67, 69.75, 70.11, 70.71, 107.74, 113.44, 119.97, 122.02, 125.01, 125.33, 127.90, 128.02, 128.35, 142.28, 153.59, 164.76, ppm; mp 80 °C (dec); [α]23 D −13.3 (MeOH, c 0.15). Anal. (C25H33N3O3·H2C2O4·H2O) C, H, N. (R)-5-(Methylamino)-1-(2-(2-(2-(4-phenylbutoxy)ethoxy)ethoxy)ethyl)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (16). The reaction followed the procedure described for 15 starting from 33 (0.40 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 15% CMA to give the pure product as a yellow oil in 44% yield. 1H NMR (400 MHz, CDCl3) δ 1.60−1.69 (m, 4H), 2.55 (s, 3H), 2.61 (t, J = 7.4 Hz, 2H), 2.84 (m, 1H), 2.93 (m, 1H), 3.06 (dd, J = 3.8, 3.6 Hz, 1H), 3.23 (m, 1H), 3.45−3.84 (m, 12H), 4.05 (m, 2H), 4.15 (m, 1H), 6.86 (m, 1H), 6.95 (m, 2H), 7.16 (m, 3H), 2.24 (m, 2H), ppm. The free base was converted into the oxalate salt (highly hygroscopic white solid). 13C NMR (100 MHz, CD3OD) δ 26.03, 27.82, 28.85, 30.51, 35.19, 39.28, 41.13, 52.27, 68.64, 69.72, 70.09, 70.13, 70.18, 70.62, 107.76, 113.50, 119.69, 122.02, 125.03, 125.31, 127.88, 128.00, 128.34, 142.30, 153.58, 164.90, ppm. HPLC-DAD (Rt: 5.468 min; purity 94.08%); mp 85 °C (dec); [α]23 D +20.0 (MeOH, c 0.08). Anal. (C27H37N3O4·2H2C2O4·1/3H2O· 1/3NH4OH) C, H, N. (R)-5-(Methylamino)-1-(2-(2-(2-(2-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)ethoxy)ethoxy)ethoxy)ethyl)-5,6-dihydro-4Himidazo[4,5,1-ij]quinolin-2(1H)-one (17). The reaction followed the procedure described for 16 starting from 37 (0.15 g; 0.00003 mol). The crude compound was purified via flash chromatography, eluting with 7% CMA to give the pure product as a yellow oil in 30% yield. 1H NMR (400 MHz, CDCl3) δ 1.83 (br s, 1H), 2.56 (s, 3H), 2.84 (m, 3H), 3.03 (dd, J = 4.0, 4.0 Hz, 1H), 3.25 (m, 1H), 3.59−3.68 (m, 12H), 3.74−3.81 (m, 4H), 4.02−4.08 (m, 4H), 6.42 (s, 1H), 6.49 (d, J = 8.0 Hz, 1H), 6.84 (t, J = 8.0 Hz, 1H), 6.91−7.01 (m, 3H), 8.30 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 24.57, 31.05, 33.90, 41.39, 42.64, 53.16, 67.68, 69.25, 69.62, 70.51, 70.61, 70.66, 70.81, 102.56, 106.63, 108.67, 116.01, 116.87, 119.86, 121.23, 125.90, 128.09, 128.53, 138.30, 153.61, 158.34, 171.46, ppm. The free base was converted into the oxalate salt (white solid); mp 97−98 °C; [α]23 D −1.1 (MeOH, c 0.10). Anal. (C28H36N4O6·2H2C2O4·2H2O) C, H, N. (R)-N-(4-((2-Oxo-1-propyl-1,2,5,6-tetrahydro-4H-imidazo[4,5,1ij]quinolin-5-yl) (propyl)amino)butyl)-1,4-dihydroquinoline-2-carboxamide (20). A solution of 50 (0.08 g; 0.0003 mol), 45 (0.12 g; 0.0004 mol), and K2CO3 (0.33 g; 0.002 mol) in DMF (10 mL) was stirred overnight at 60 °C, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with 5% CMA to give the pure product as a yellow oil in 12% yield. 1H NMR (400 MHz, CDCl3) δ 0.86 (t, J = 7.4 Hz, 3H), 0.95 (t, J = 7.4 Hz, 3H), 1.24−1.89 (m, 8H), 2.49−2.62 (m, 3H), 2.87−2.94 (m, 2H), 3.15 (m, 1H), 3.29 (m, 1H), 3.49−3.52 (m, 3H), 3.73−3.83 (m, 2H), 4.14 (dd, J = 3.2, 3.2 Hz, 1H), 6.32 (br s, 1H), 6.79−6.85 (m, 2H), 6.96 (t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.29 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.0 Hz, 1H), 9.31 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 11.33, 11.68, 21.96, 22.05, 26.38, 26.95, 27.47, 39.43, 40.64, 42.93, 50.40, 52.68, 54.49, 101.77, 105.32, 105.48, 111.86, 118.71, 119.47, 120.58, 120.86, 121.89, 124.37, 126.24, 127.75, 130.86, 136.12, 153.60, 161.51, ppm. HPLC-DAD (Rt: 5.318 min; purity 96.95%); [α]23 D +5.5 (MeOH, c 0.09). HRMS (C29H37N5O2 + H+) (ESI) calculated 488.3020, found 488.3033 (M + H+). (R)-N-(4-(Methyl(2-oxo-1-propyl-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)amino)butyl)-1H-indole-2-carboxamide (21). The reaction followed the procedure described for 20 starting from 5135 (0.24 g; 0.0009 mol). The crude compound was purified via flash chromatography, eluting with 5% CMA to give the pure product as colorless oil in 18% yield. 1H NMR (400 MHz, CDCl3) δ 0.95 (t, J = 7.6 Hz, 3H), 1.53−1.67 (m, 4H), 1.77 (q, J = 7.2 Hz, 2H), 2.34 (s, 3H), 2.54−2.63 (m, 2H), 2.91 (m, 2H), 3.14 (m, 1H), 3.47−3.58 (m,

3H), 3.83 (t, J = 7.4 Hz, 2H), 4.13 (dd, J = 4.0, 4.0 Hz, 1H), 6.82 (t, J = 6.6 Hz, 2H), 6.90−6.98 (m, 3H), 7.09 (t, J = 7.4 Hz, 1H), 7.23 (m, 1H), 7.45 (d, J = 8.4 Hz, 1H), 7.59 (d, J = 8.0 Hz, 1H), 10.04 (br s, 1H), ppm. 13C NMR (100 MHz, CDCl3) δ 11.46, 22.09, 25.14, 27.25, 27.41, 38.24, 39.54, 40.42, 43.09, 53.77, 57.25, 102.39, 105.75, 112.14, 118.35, 119.61, 120.51, 121.13, 121.93, 124.28, 126.30, 127.76, 131.18, 136.55, 153.73, 162.00, ppm; [α]22 D +23.1 (CHCl3, c 0.16). Anal. (C27H33N5O2·1.5H2O·0.25NH4OH) C, H, N. (R)-5-(Methyl(4-((2-oxo-1,2,3,4-tetrahydroquinolin-7-yl)oxy)butyl)amino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (22). A suspension of 42 (0.13 g; 0.0003 mol) and 10% Pd(OH)2/C (0.10g) in ethanol (15 mL) was charged at a pressure of 50 psi with H2 and shaken in a Parr apparatus for 48 h. The resulting mixture was filtered through Celite, evaporated, and purified via flash chromatography, eluting with 5% CMA to give the pure product as a white solid in 85% yield. 1H NMR (400 MHz, CDCl3) δ 1.64−1.68 (m, 2H), 1.74−1.81 (m, 2H), 2.39 (s, 3H), 2.56−2.68 (m, 3H), 2.83−2.92 (m, 4H), 3.19 (m, 1H), 3.54 (t, J = 11.2 Hz, 1H), 3.70 (dd, J = 6.8, 7.2 Hz, 1H), 3.92 (t, J = 6.0 Hz, 2H), 4.18 (dd, J = 4.0, 4.0 Hz, 1H), 6.44 (s, 1H), 6.47 (d, J = 8.4 Hz, 1H), 6.82 (d, J = 7.6 Hz, 1H), 6.88−6.95 (m, 2H), 6.98 (d, J = 8.0 Hz, 1H), 9.23 (br s, 1H), 10.11 (br s, 1H), ppm. 13 C NMR (100 MHz, CDCl3) δ 24.05, 24.39, 26.72, 26.81, 30.91, 37.96, 39.95, 50.26, 53.82, 57.15, 58.04, 67.87, 102.55, 107.30, 108.73, 115.73, 118.39, 119.69, 121.48, 126.09, 127.26, 128.47, 138.15, 155.70, 158.51, 172.43, ppm. HRMS (ESI) calculated 421.2234, found 421.2228 (M + H+); mp 85 °C (dec); [α]23 D −3.8 (MeOH, c 0.08). Anal. (C24H28N4O3·2/3CHCl3·CH3OH) C, H, N. 4-(4-Bromobutoxy)butyl)benzene (24). 1,4-Dibromobutane (1.50 g; 0.007 mol) was added dropwise to a solution of 23 (1.00 g; 0.007 mol) and sodium hydride (0.35 g; 0.014 mol) in THF (50 mL). The solution was stirred at reflux for 4 h and then at room temperature overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic phase was dried with Na2SO4, filtered, and evaporated to give the crude product, which was purified via flash chromatography, eluting with hexane:EtOAc (99.5:0.5) to give the pure product as a colorless oil in 45% yield. 1H NMR (400 MHz, CDCl3) δ 1.57−1.74 (m, 6H), 1.91−1.98 (m, 2H), 2.63 (t, J = 7.4 Hz, 2H), 3.39−3.46 (m, 6H), 7.15−7.19 (m, 3H), 7.26−7.27 (m, 2H), ppm. GC-MS (EI) m/z 284.1 (M+). 4-((6-Bromohexyl)oxy)butyl)benzene (25). The reaction followed the procedure described for 24 starting from 1,6-dibromohexane (1.50 g; 0.006 mol). The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (99.5:0.5) to give the pure product as a colorless oil in 40% yield. 1H NMR (400 MHz, CDCl3) δ 1.35−1.70 (m, 10H), 1.71−1.90 (m, 2H), 2.64 (t, J = 7.4 Hz, 2H), 3.37−3.44 (m, 6H), 7.16−7.19 (m, 3H), 7.27−7.29 (m, 2H), ppm. GC-MS (EI) m/z 312.1 (M+). 4-((8-Bromooctyl)oxy)butyl)benzene (26). The reaction followed the procedure described for 24 starting from 1,8-dibromooctane (1.63 g; 0.006 mol). The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (98:2) to give the pure product as a colorless oil in 58% yield. 1H NMR (400 MHz, CDCl3) δ 1.31−1.71 (m, 14H), 1.82−1.88 (m, 2H), 2.63 (t, J = 7.4 Hz, 2H), 3.36−3.43 (m, 6H), 7.17−7.19 (m, 3H), 7.26−7.29 (m, 2H), ppm. GC-MS (EI) m/z 340.2 (M+). 4-((10-Bromodecyl)oxy)butyl)benzene (27). The reaction followed the procedure described for 24 starting from 1,10-dibromodecane (1.80 g; 0.006 mol). The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (98:2) to give the pure product as a colorless oil in 45% yield. 1H NMR (400 MHz, CDCl3) δ 1.30−1.68 (m, 18H), 1.82−1.87 (m, 2H), 2.63 (t, J = 7.4 Hz, 2H), 3.36−3.43 (m, 6H), 7.17−7.19 (m, 3H), 7.27−7.29 (m, 2H), ppm. GC-MS (EI) m/z 368.2 (M+). 4-((12-Bromododecyl)oxy)butyl)benzene (28). The reaction followed the procedure described for 24 starting from 1,12dibromododecane (2.20 g; 0.007 mol). The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (98:2) to give the pure product as a colorless oil in 42% yield. 1H NMR (400 MHz, CDCl3) δ 1.27−1.71 (m, 22H), 1.82−1.89 (m, 2H), 2.64 (t, J = 2902



Article

(R)-1-Methoxy-5-(methylamino)-5,6-dihydro-4H-imidazo[4,5,1ij]quinolin-2(1H)-one (41). A suspension of 4045 (0.50 g; 0.0014 mol) and 10% Pd(OH)2/C (0.10 g) in ethanol (25 mL) was charged at a pressure of 50 psi with H2 and shaken in a Parr apparatus for 1 h. The resulting mixture was filtered through Celite, evaporated, and purified via flash chromatography, eluting with MeOH:CHCl3 (15:85) to give the pure product as a yellow oil in 70% yield. 1H NMR (400 MHz, CDCl3) δ 2.52 (s, 3H), 2.76 (dd, J = 7.6, 8.0 Hz, 1H), 3.04 (dd, J = 4.0, 3.6 Hz, 1H), 3.20 (m, 1H), 3.63 (dd, J = 6.8, 7.2 Hz, 1H), 4.00 (dd, J = 3.8, 4.0 Hz, 1H), 4.10 (s, 3H), 6.86 (d, J = 2.8 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), ppm. (R)-1-Methoxy-5-(methyl(4-((2-oxo-1,2,3,4-tetrahydroquinolin-7yl)oxy)butyl)amino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin2(1H)-one (42). K2CO3 (0.89 g; 0.0065 mol) and 38 (0.57 g; 0.0019 mol) were added to a solution of 41 (0.30 g; 0.0013 mol) in DMF (15 mL). The reaction was stirred overnight at 60 °C, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with MeOH:CHCl3 (2:98) to give the pure product as a yellow oil in 40% yield. 1H NMR (400 MHz, CDCl3) δ 1.58−1.65 (m. 2H), 1.72−1.79 (m, 2H), 2.35 (s, 3H), 2.55−2.65 (m, 4H), 2.82−2.96 (m, 4H), 3.12−3.19 (m, 1H), 3.49 (t, J = 11.2 Hz, 1H), 3.89−3.95 (m, 2H), 4.03 (s, 3H), 4.14 (dd, J = 4.0, 4.0 Hz, 1H), 6.39 (s, 1H), 6.46 (d, J = 8.0 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.96−6.99 (m, 2H), 9.12 (br s, 1H), ppm. N-(4-Hydroxybutyl)-1H-indole-2-carboxamide (44).35 A solution of 43 (1.00 g; 0.006 mol), 4-aminobutan-1-ol (0.6 g; 0.006 mol), and 1,1′-carbonyldiimidazole (1.00 g; 0.006 mol) in THF (20 mL) and DMF (2 mL) was stirred at room temperature overnight. The solvent was evaporated and the crude compound was purified via flash chromatography, eluting with EtOAc to give the pure product as a colorless oil in 98% yield. 1H NMR (400 MHz, CDCl3) δ 1.54−1.68 (m, 4H), 2.97 (br s, 1H), 3.39 (t, J = 6.4 Hz, 2H), 3.59 (t, J = 5.8 Hz, 2H), 6.90 (s, 1H), 7.05 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.4 Hz, 1H), 10.0 (br s, 1H), ppm N-(4-Bromobutyl)-1H-indole-2-carboxamide (45).35 A solution of CBr4 (3.00 g; 0.009 mol), triphenylphosphine (2.40 g, 0.009 mol), and 44 in acetonitrile (25 mL) was stirred at room temperature overnight. The solution was diluted with 15% NaOH aqueous solution and extracted with EtOAc. The organic layer was dried with Na2SO4, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (1:4) to give the pure product as yellow solid in 38% yield. 1H NMR (400 MHz, CDCl3) δ 1.78−1.85 (m, 2H), 1.94−2.00 (m, 2H), 3.45 (t, J = 6.4 Hz, 2H), 3.53 (t, J = 6.6 Hz, 2H), 6.23 (br s, 1H), 6.83 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.28 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 9.49 (br s, 1H), ppm. Benzyl (R)-(2-Oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)(propyl)carbamate (46). Benzyl(2,5-dioxopyrrolidin-1yl)carbonate (0.25 g; 0.010 mol) was added to a solution of 245 (0.23 g; 0.010 mol) in THF (50 mL) at −40 °C. The reaction mixture was stirred for 3 h at −40 °C. The solution was diluted with 10% NaHCO3 aqueous solution and extracted with EtOAc. The organic layer was dried with Na2SO4, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (1:1) to give the pure product as yellow oil in 73% yield. 1H NMR (400 MHz, CDCl3) δ 0.86−0.96 (m, 3H), 1.51−1.69 (m, 2H), 2.93 (d, J = 11.2 Hz, 1H), 3.23−3.36 (m, 3H), 3.82−3.89 (m, 1H), 4.10−4.17 (m, 1H), 4.28−4.35 (m, 1H), 5.18 (br s, 2H), 6.84 (d, J = 6.8 Hz, 1H), 6.93−6.99 (m, 2H), 7.26−7.36 (m, 5H), 10.29 (br s, 1H), ppm. Benzyl (R)-(2-Oxo-1-propyl-1,2,5,6-tetrahydro-4H-imidazo[4,5,1ij]quinolin-5-yl)(propyl)carbamate (48). Potassium carbonate (0.50 g; 0.0035 mol) and 1-bromopropane (0.18 g; 0.0014 mol) were added to a solution of 46 (0.25 g; 0.0072 mol) in acetone (25 mL). The reaction mixture was stirred at reflux overnight, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with acetone:CHCl3 (5:95) to give the pure product as a colorless oil in 91% yield. 1H NMR (400 MHz, CDCl3) δ 0.86−0.97 (m, 6H), 1.53−1.63 (m, 2H), 1.72−1.81

7.4 Hz, 2H), 3.37−3.43 (m, 6H), 7.16−7.19 (m, 3H), 7.26−7.29 (m, 2H), ppm. GC-MS (EI) m/z 396.2 (M+). (Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl)bis(4-methylbenzenesulfonate) (31). KOH (6.72 g; 0.120 mol) was added to a solution of 29 (2 mL; 0.015 mol) and 4-methylbenzenesulfonyl chloride (5.72 g; 0.030 mol) in DCM (100 mL) at 0 °C. The reaction was stirred at 0 °C for 3 h. The solution was diluted with water and extracted with DCM. The organic layer was dried with Na2SO4, filtered, and evaporated to give the crude compound in 90% yield, which was used in the next step without further purification. 1H NMR (400 MHz, CDCl3) δ 2.44 (s, 6H), 3.65−3.67 (m, 8H), 4.13 (dd, J = 3.6, 4.8 Hz, 4H), 7.33 (d, J = 8.0 Hz, 4H), 7.79 (d, J = 8.4 Hz, 4H), ppm. 4-(2-(2-Bromoethoxy)ethoxy)butyl)benzene (32). 30 (2.00 g; 0.008 mol) was added portionwise to a stirred solution of 23 (1.26 g; 0.008 mol) and sodium hydride (0.43 g; 0.016 mol) in THF (100 mL). The reaction mixture was stirred at reflux for 3 h, after which the solution was diluted with water and extracted with EtOAc. The organic layer was dried with Na2SO4, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (85:15) to give the pure product as a yellow oil in 38% yield. 1H NMR (400 MHz, CDCl3) δ 1.64−69 (m, 4H), 2.64 (t, J = 7.4 Hz, 2H), 3.48−3.58 (m, 4H), 3.63−3.67 (m, 2H), 3.81−3.84 (m, 2H), 4.00 (dd, J = 2.0, 2.0 Hz, 1H), 4.18 (dd, J = 2.0, 2.0 Hz, 1H), 7.17−7.19 (m, 3H), 7.25−7.29 (m, 2H), ppm. 2-(2-(2-(4-Phenylbutoxy)ethoxy)ethoxy)ethyl 4-Methylbenzenesulfonate (33). The reaction followed the procedure described for 32 starting from 31 (7.2 g; 0.015 mol). The crude compound was then purified via flash chromatography, eluting with hexane:EtOAc (75:25) to give the pure product as a yellow oil in a 48% yield. 1H NMR (400 MHz, CDCl3) δ 1.61−1.68 (m, 4H), 2.44 (s, 3H), 2.62 (t, J = 7.4 Hz, 2H), 3.46 (t, J = 6.2 Hz, 2H), 3.53−3.61 (m, 8H), 3.67 (t, J = 4.8 Hz, 2H), 4.14 (dd, J = 4.4, 3.6 Hz, 2H), 7.15−7.18 (m, 3H), 7.24−7.28 (m, 2H), 7.33 (d, J = 7.6 Hz, 2H), 7.79 (dd, J = 1.6, 2.0 Hz), ppm. 1-Iodo-2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethane) (36). KOH (10.0 g; 0.180 mol) was added to a solution of 35 (5.00 g; 0.026 mol) and 4-methylbenzenesulfonyl chloride (10.0 g; 0.051 mol) in DCM (150 mL) at 0 °C. The reaction was stirred at 0 °C for 3 h, and the solvent was evaporated. The residue was dissolved with acetone (25 mL), KI (5.00 g; 0.03 mol) was added portionwise and the mixture was stirred at reflux overnight. The reaction was diluted with EtOAc, then washed with brine and 10% NaHSO3. The organic layer was dried with Na2SO4, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (8:2) to give the pure product as a brown oil in 85% yield. 1H NMR (400 MHz, CDCl3) δ 3.24 (t, J = 6.8 Hz, 4H), 3.65 (s, 8H), 3.74 (t, J = 7.2 Hz, 4H), ppm. 7-(2-(2-(2-(2-Iodoethoxy)ethoxy)ethoxy)ethoxy)-3,4-dihydroquinolin-2(1H)-one (37). 36 (2.50 g; 0.006 mol) was added to a solution of 34 (1.00 g; 0.006 mol) and K2CO3 (1.65 g; 0.012 mol) in acetone (30 mL) and stirred at reflux overnight. The resulting mixture was filtered, dissolved in EtOAc, and washed with 10% Na2S2O3 aqueous solution. The organic layer was dried with Na2SO4, filtered, and evaporated to yield a crude product. The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (5:5) to give the pure product as a yellow oil in 67% yield. 1H NMR (400 MHz, CDCl3) δ 2.58 (dd, J = 6.4, 7.2 Hz, 2H), 2.86 (m, 2H), 3.23 (dd, J = 5.2, 6.8 Hz, 2H), 3.65−3.73 (m, 10H), 3.81 (dd, J = 4.0, 4.4 Hz, 2H), 4.08 (t, J = 2.4 Hz, 2H), 6.37 (d, J = 2.4 Hz, 1H), 6.51 (dd, J = 2.8, 2.4 Hz, 1H), 7.00 (d, J = 8.8 Hz, 1H), 8.37 (br s, 1H), ppm. GCMS (EI) m/z 449.1 (M+). 7-((12-Bromododecyl)oxy)-3,4-dihydroquinolin-2(1H)-one (39). The reaction followed the procedure described for 37 starting from 1,12-dibromododecane (1.50 g; 0.0045 mol). The crude compound was purified via flash chromatography, eluting with hexane:EtOAc (7:3) to give the pure product as a yellow oil in 60% yield. 1H NMR (400 MHz, CDCl3) δ 1.25−1.44 (m, 16H), 1.71−1.88 (m, 4H), 2.60 (dd, J = 6.4, 6.8 Hz, 2H), 2.88 (t, J = 7.4 Hz, 2H), 3.39 (t, J = 7.0 Hz, 2H), 3.90 (t, J = 6.4 Hz), 6.27 (d, J = 2.4 Hz, 1H), 6.51 (dd, J = 2.4, 2.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.53 (br s, 1H), ppm. GC-MS (EI) m/z 409.2 (M+). 2903



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times with 3 mL (3 × 1 mL/well) of ice-cold binding buffer. Then 65 μL of PerkinElmer MicroScint 20 scintillation cocktail was added to each well, and filters were counted using a PerkinElmer MicroBeta microplate counter (calculated efficiency: 41%). IC50 values for each compound were determined from dose−response curves, and Ki values were calculated using the Cheng−Prusoff equation;60 Kd values for [3H]-(R)-(+)-7-OH-DPAT (D2R, 2.93 nM; D3R, 1.49 nM) were determined via separate homologous competitive binding experiments. These analyses were performed using GraphPad Prism version 6.00 for Macintosh (GraphPad Software, San Diego, CA). Ki values were determined from at least three independent experiments and are reported as mean ± SEM. Bioluminescence Resonance Energy Transfer (BRET) Receptor Function Assays. BRET assay: Variations of bioluminescence resonance energy transfer assay (BRET) were performed to detect receptor ligand induced events for (i) adenylyl cyclase inhibition, (ii) Gi protein activation, (iii) Go protein activation, (iv) β-arrestin recruitment, and (v) Gi engagement. (i) Adenylyl cyclase inhibition assay uses a CAMYEL biosensor construct that contains RLuc and YFP, allowing detection of intracellular cAMP change.51 To study Gαi-like dependent inhibition activity, cells were prestimulated with 10 μM forskolin (Sigma) 10 min prior to sample reading. (ii−iii) Gi or Go protein activation assay uses RLuc-fused Gαi1 or GαoA protein subunit and GFP10-fused Gγ2 protein for resonance energy transfer (RET) pair. Flag-tagged receptor and untagged Gβ1 constructs were cotransfected. (iv) β-Arrestin recruitment assay uses RLuc-fused receptor and mVenus-fused β-arrestin2 for the RET pair. GRK2 was cotransfected to assist an enhanced phosphorylation required for the β-arrestin recruitment. (v) Gi protein engagement assay uses RLucfused receptor and mVenus-fused Gαi1 for the RET pair. As reported previously,61 cells were harvested, washed, and resuspended in phosphate-buffered saline (PBS). Approximately 200000 cells/well were distributed in 96-well plates, and 5 μM coelenterazine H (substrate for BRET1) or 5 μM coelenterazine 400a (substrate for BRET2) was added to each well. One minute after addition of coelenterazine, ligands were added to each well. In antagonist mode, ligands were added 15 min before the addition of 1 μM quinpirole. The fluorescence of the acceptor was quantified (for BRET1 excitation at 500 nm and emission at 540 nm for 1 s recording, for BRET2 excitation at 410 nm and emission at 510 nm for 1 s recording) in Mithras LB940 (Berthold Technologies, Bad Wildbad, Germany) to confirm the constant expression levels across experiments. In parallel, BRET signal from the same batch of cells was determined as the ratio of the light emitted by mVenus (510−540 nm) over RLuc (485 nm) for BRET1 and GFP10 (515 nm) over RLuc (370−450 nm) for BRET2. Results are calculated for the BRET change (BRET ratio for the corresponding drug minus BRET ratio in the absence of the drug). Data and statistical analysis were performed with Prism 5 (GraphPad Software). Bias Factor Analysis. Bias factors were calculated by the operational model24,55,56 and the Emax−EC50 model.15 Briefly, for the operational model, using the equation below, a transduction coefficient (R, below) was calculated for each ligand in cAMP and β-arrestin assays. The transduction coefficients in β-arrestin assay were then subtracted from cAMP assay for each ligand to yield bias factors shown in the table.

(m, 2H), 2.92 (d, J = 11.2 Hz, 1H), 3.21−3.36 (m, 3H), 3.78−3.87 (m, 3H), 4.13 (d, J = 11.6 Hz, 1H), 4.25−4.32 (m, 1H), 5.15 (br s, 2H), 6.80−6.82 (m, 2H), 6.97 (t, J = 7.6 Hz, 1H), 7.26−7.34 (m, 5H), ppm. (R)-1-Propyl-5-(propylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (50). A suspension of 48 (0.26 g; 0.0006 mol) and 10% Pd/C (0.10 g) in ethanol (15 mL) was charged at a pressure of 50 psi with H2 and shaken in a Parr apparatus overnight. The resulting mixture was filtered through Celite, evaporated, and purified via flash chromatography, eluting with MeOH:CHCl3 (4:96) to give the pure product as a yellow oil in 87% yield. 1H NMR (400 MHz, CDCl3) δ 0.88−0.94 (m, 6H), 1.52−1.58 (m, 2H), 1.71−1.76 (m, 2H), 2.70− 2.77 (m, 2H), 2.84 (dd, J = 8.8, 8.4 Hz, 1H), 3.09 (dd, J = 4.4, 4.0 Hz, 1H), 3.30−3.34 (m, 1H), 3.60 (dd, J = 8.0, 8.0 Hz, 1H), 3.75 (td, J = 1.6, 2.0, 1.6 Hz, 2H), 4.13 (dd, J = 3.6, 3.6 Hz, 1H), 6.78 (dd, J = 7.2, 8.0 Hz, 2H), 6.93 (t, J = 7.8 Hz, 1H), ppm. GC-MS (EI) m/z 273.2 (M+). (R)-5-(Dipropylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin2(1H)-one (52). A mixture of 2 45 (0.50 g; 0.0002 mol), propionaldehyde (0.16 g; 0.0003 mol), and sodium triacetoxyborohydride (0.70 g; 0.0003 mol) in DCE (10 mL) was added with catalytic amount of acetic acid (0.1 mL) and stirred overnight at room temperature. The solution was basified with 38% NH4OH aqueous solution (pH = 8) and extracted with DCM. The organic layer was dried with Na2SO4, filtered, and evaporated to yield the pure product without further purification in 98% yield. 1H NMR (400 MHz, CDCl3) δ 0.89 (t, J = 7.2 Hz, 6H), 1.43−1.48 (m, 4H), 2.47−2.58 (m, 4H), 2.90 (t, J = 7.0 Hz, 2H), 3.28−3.31 (m, 1H), 3.44 (t, J = 11.6 Hz, 1H), 4.16 (dd, J = 3.6 Hz, 4.4 Hz, 1H), 6.85 (dd, J = 4.4 Hz, 4.0 Hz, 2H), 6.95 (t, J = 7.6 Hz, 1H), 7.99 (br s, 1H). GC-MS (EI) m/z 273.1 (M+) Radioligand Binding Studies. Radioligand binding assays were conducted similarly to those previously described.35,59 HEK293 cells stably expressing human D2R or D3R 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 nonessential amino acids, 1× 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 premixed Earle’s Balanced Salt Solution (EBSS) with 5 mM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21 °C. The supernatant was removed, and the pellet was resuspended in 10 mL of hypotonic lysis buffer (5 mM MgCl2, 5 mM Tris, pH 7.4 at 4 °C) and centrifuged at 20000 rpm 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)-(+)-7OH-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, all test compounds were freshly dissolved in 30% DMSO and 70% H2O to a stock concentration of 1 mM or 100 μM. To assist the solubilization of free-base compounds, 10 μL of glacial acetic acid was added along with the DMSO (in place of 10 μL final H2O volume). Each test compound was then diluted into 11 halflog serial dilutions using 30% DMSO vehicle; final test concentrations ranged from 10 μM to 10 pM. Membranes were diluted in fresh binding buffer. Radioligand competition experiments were conducted in 96-well plates containing 300 μL of fresh binding buffer, 50 μL of diluted test compound, 100 μL of membranes (80 or 40 μg total protein for hD2R or hD3R, respectively), and 50 μL of radioligand diluted in binding buffer ([3H]-(R)-(+)-7-OH-DPAT: 1.5 nM final concentration for hD2, 0.5 nM final concentration for hD3, ARC, Saint Louis, MO). 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 and the reaction incubated for 90 min at room temperature. The reaction was terminated by filtration through a PerkinElmer 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

Y = basal +

Emax − basal 1 + [(1 + A /KA)R × A]n

For the Emax−EC50 model, using the equation below, a bias factor for each compound was calculated using results from cAMP and βarrestin assays. The bias factor of the reference compound quinpirole was then subtracted from the bias factor of each compound to yield the Δbias factor shown in the table.

⎛E ⎞ ⎛E ⎞ bias factor = log10⎜ max ⎟ − log10⎜ max ⎟ ⎝ EC50 ⎠cAMP ⎝ EC50 ⎠arrestin 2904


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* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01875. Elemental analyses, HPLC-DAD, HRMS results, MS/MS spectra, D2R-BRET, Ki, and Bmax determinations and D2R-mediated Gi engagement data (PDF) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: (443)-740-2887. Fax: (443)-740-2111. E-mail: [email protected]. ORCID

Amy Hauck Newman: 0000-0001-9065-4072 Author Contributions §

A.B. and H.Y. contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Support for this research was provided by the Intramural Research Program of the National Institute on Drug Abuse (A.B., H.Y., M.P.E., L.M., V.K., M.-F.Z., N.S.C., A.G., A.W., L.S., and A.H.N.) and a fellowship from the Japan Society for the Promotion of Science (H.Y.). A special thanks to J. Robert Lane (Monash University) for providing valuable advice and bias factor equations.

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ABBREVIATIONS USED DA, dopamine; GPCR, G-protein coupled receptor; APD, antipsychotic drug; SAR, structure−activity relationship; D2R, D2 dopamine receptor; D3R, D3 dopamine receptor; OBS, orthosteric binding site; SBP, secondary binding pocket; PP, primary pharmacophore; SP, secondary pharmacophore; BRET, bioluminescence resonance energy transfer; CMA, chloroform/methanol/ammonium hydroxide; HRMS, high resolution mass spectroscopy; HPLC-DAD, high pressure liquid chromatography-diode array detector

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REFERENCES

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