Novel Analogues of (R)-5-(Methylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (Sumanirole) Provide Clues to Dopamine D2/D3 Receptor Agonist Selectivity.

Novel 1-, 5-, and 8-substituted analogues of sumanirole (1), a dopamine D2/D3 receptor (D2R/D3R) agonist, were synthesized. Binding affinities at both D2R and D3R were higher when determined in competition with the agonist radioligand [(3)H]7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT) than with the antagonist radioligand [(3)H]N-methylspiperone. Although 1 was confirmed as a D2R-preferential agonist, its selectivity in binding and functional studies was lower than previously reported. All analogues were determined to be D2R/D3R agonists in both GoBRET and mitogenesis functional assays. Loss of efficacy was detected for the N-1-substituted analogues at D3R. In contrast, the N-5-alkyl-substituted analogues, and notably the n-butyl-arylamides (22b and 22c), all showed improved affinity at D2R over 1 with neither a loss of efficacy nor an increase in selectivity. Computational modeling provided a structural basis for the D2R selectivity of 1, illustrating how subtle differences in the highly homologous orthosteric binding site (OBS) differentially affect D2R/D3R affinity and functional efficacy.

Introduction

class="Chemical">Dopamine signaling is mediated by five G protein-coupled receptors (<class="Chemical">span class="Gene">GPCRs). These receptors are divided into two subfamilies on the basis of sequence similarity and pharmacological profiles. D1-like receptors (D1R and D5R) are coupled to Gαs and Gαolf that activate adenylyl cyclase-mediated cAMP production, whereas D2-like receptors (D2R, D3R, and D4R) are coupled to Gαi/o/z, and inhibit adenylyl cyclase-mediated cAMP production.[1] These receptors also recruit arrestin, which can signal independently of G proteins.[1]

The class="Chemical">dopamine D2-like receptor family has long been considered to be an important therapeutic target for the treatment of a variety of <class="Chemical">span class="Disease">neuropsychiatric disorders. Clinically used antipsychotics (e.g., haloperidol) are well-known D2-like receptor antagonists; this includes second-generation agents that have reduced extrapyramidal side effects and are thus termed “atypical” antipsychotics (e.g., quetiapine, olanzapine, and clozapine).[2−4] Further, D2-like receptor agonists (e.g., pergolide, bromocriptine, talipexole, pramipexole, ropinirole, and cabergoline) have been used to treat symptoms of Parkinson’s disease as well as associated dyskinesias and have a variety of potential neuroprotective properties.[5]

Nevertheless, medications that target class="Gene">D2-like receptors are associated with adverse side effects that reduce quality of life and medication compliance. This may in part be due to their inability to distinguish between D2-like receptor subtypes, eclass="Chemical">specially between the highly homologous <class="Chemical">span class="Gene">D2R and D3R, which are expressed differentially in the brain and mediate distinct physiological and behavioral processes. D2Rs are highly expressed in the dorsal striatum and are also found at significant levels in substantia nigra, ventral tegmental area, nucleus accumbens, olfactory tubercle, hypothalamus, amygdala, cortex, and hippocampus.[1,6] D2Rs contribute to the control of locomotion, learning, memory, and the rewarding response to addictive drugs.[7] In contrast, D3Rs are primarily expressed in the ventral striatum, including the nucleus accumbens,[6] and are a target of interest in addiction pharmacotherapy.[8−14]

The development of subtype-selective ligands at these class="Gene">D2-like receptors has been challenging. Although substantial effort has led to the development of <class="Chemical">span class="Chemical">D3R- and D4R-selective ligands,[9,15−17] significantly less progress has been made toward the development of D2R-selective compounds. Novel D2R-selective ligands can be useful as tools to probe the roles of D2-like receptor subtypes in vivo and could potentially lead to new pharmacotherapeutics for the treatment of a variety of disorders (e.g., antagonists for schizophrenia, partial or full agonists for Parkinson’s disease, hyperprolactinemia, and restless legs syndrome).

class="Chemical">Sumanirole ((R)-5,6-dihydro-5-(methylamino)-4H-<class="Chemical">span class="Chemical">imidazo[4,5,1-ij]quinolin-2(1H)-one (Z)-2-butenedioate, 1, U-95666E, PNU-95666E; Figure ) was reported previously to be a D2R-selective metabolite of (R)-5,6-dihydro-N,N-dimethyl-4H-imidazo[4,5,1-ij]quinolin-5-amine,[18,19] one compound in a series of imidazoquinolinones with varying dopaminergic and serotonergic activities.[20,21] A detailed pharmacological analysis of 1 described this compound as a dopaminergic agonist with >200-fold higher affinity for D2R than for D3R based on radioligand binding studies.[22] As such, 1 has been used in a variety of studies seeking to disentangle the roles of D2R and D3R signaling in vivo (e.g., see refs (23−26)). Compound 1 was evaluated in clinical trials for the treatment of Parkinson’s disease and restless legs syndrome but is not clinically approved.[27−31]

Figure 1

Structure of sumanirole (1).

Structure of <span class="Chemical">sumanirole (1).

The orthosteric binding site (OBS)—the site in which class="Chemical">dopamine binds to induce receptor signaling—is virtually identical in <class="Chemical">span class="Gene">D2R and D3R.[32] It is unclear how 1, which presumably binds in the OBS, is highly selective for D2R over D3R. Therefore, the goal of this study is to further examine the binding profile of 1 and to begin to elucidate the molecular determinants that confer subtype selectivity through chemical modification of the parent molecule at positions 1, 5, and 8.

Highly class="Chemical">D3R-selective compounds have been discovered using a “bivalent” design, which includes a high-affinity primary pharmacophore (PP), such as <class="Chemical">span class="Chemical">4-phenylpiperazine, connected to an extended aryl amide functional group that occupies a secondary receptor binding pocket (SBP) to enhance subtype selectivity.[32−34] Previously, we used a “synthon” approach to define the role of the primary and secondary pharmacophores (PP and SP, respectively) in D3R subtype selectivity and efficacy.[33,34] In this study, we take a similar approach using 1 as the PP, adding structural complexity through alkylation at the 1 and N-5 positions, adding a CN to the 8 position, and creating the first bivalent analogues of 1 by extending an aryl amide from its N-5 position with a butyl linking chain.

Synthetic strategies for 1 have been described previously.[18,35−40] In the present study, we extend this strategy to a series of analogues for which we develop structure–activity relationships (SAR) for both binding and receptor activation at class="Gene">D2R and <class="Chemical">span class="Chemical">D3R comparatively. We identify fully efficacious analogues and D2R-preferential ligands with extended aryl amide pharmacophores. Finally, using molecular modeling and simulations with 1 and selected analogues, we begin to explore the molecular interactions between these ligands at D2R and D3R to investigate the structural basis of D2R over D3R selectivity and efficacy.

Chemistry

For the synthesis of class="Chemical">12a, 12b, and 13, we adopted a strategy similar to the one used for the synthesis of compound 1(33) and depicted in Scheme . <class="Chemical">span class="Chemical">d-Phenylalanine (2a) or its 3-Br (2b) analogue was reacted with methyl chloroformate or benzyl chloroformate to form 3a and 3b, respectively, which were then coupled with methoxyamine in the presence of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide (EDC) to give 4a and 4b, respectively. Cyclization resulted by treating 4a and 4b with bis(trifluoroacetoxy)iodobenzene to afford 5a and 5b in good yields. Deprotection of 5a was implemented with Pd/C-catalyzed hydrogenolysis to afford 6a, which was reduced with borane to give 7a.[20] It was observed that 6a did not dissolve well in THF; thus, the reaction times were longer for 6a for reduction completion (5 days). Selective protection of the basic amines in 7a and 7b were effected by treating with N-(benzyloxycarbonyloxy)succinimide at −40 °C to afford 8a and 8b, which were treated with phosgene, followed by methoxyamine, to produce 9a and 9b, respectively. Compounds 9a and 9b were reacted with bis(trifluoroacetoxy)iodobenzene to provide the tricyclic products 10a and 10b. Removal of the carbobenzoxy (CBz) protecting group and cleavage of the N-methoxy group in 10a by means of hydrogenolysis over Pd(OH)2/C (Pearlman’s catalyst) gave product 12a.[20] Conversion of the 3-Br in 10b to 3-CN was conducted by Pd-catalyzed cross-coupling of 10b with Zn(CN)2 to give 11b, which was then transformed to the final product 12b by hydrogenolysis over Pearlman’s catalyst. Reductive amination of 12a with propionaldehyde and sodium triacetoxyborohydride afforded 13.[20]

Scheme 1

Synthesis of Analogues 12a, 12b, and 13

Reagents and conditions: (a) ClCO2CH3 or CBzCl, aq NaOH, THF/H2O, 2 h; (b) CH3ONH2, EDC, CH2Cl2, 24 h; (c) PhI(O2CCF3)2, CF3CO2H, CH2Cl2, 0 °C, 1 h; (d) H2 (50 psi), Pd/C (10%), EtOH; (e) BH3·Me2S, THF, reflux; (f) N-(benzyloxycarbonyloxy)succinimide, toluene, −40 °C, 30 min; (g) i. COCl2, Et3N, THF; ii. CH3ONH2; (h) PhI(O2CCF3)2, CHCl3, −5 °C; (i) Zn(CN)2, Pd(PPh3)4, DMF; (j) H2 (50 psi), Pd(OH)2/C, EtOH; (k) CH3CH2CHO, NaBH(OAc)3, THF.

Reagents and conditions: (a) Clclass="Chemical">CO2CH3 or <class="Chemical">span class="Chemical">CBzCl, aq NaOH, THF/H2O, 2 h; (b) CH3ONH2, EDC, CH2Cl2, 24 h; (c) PhI(O2CCF3)2, CF3CO2H, CH2Cl2, 0 °C, 1 h; (d) H2 (50 psi), Pd/C (10%), EtOH; (e) BH3·Me2S, THF, reflux; (f) N-(benzyloxycarbonyloxy)succinimide, toluene, −40 °C, 30 min; (g) i. COCl2, Et3N, THF; ii. CH3ONH2; (h) PhI(O2CCF3)2, CHCl3, −5 °C; (i) Zn(CN)2, Pd(PPh3)4, DMF; (j) H2 (50 psi), Pd(OH)2/C, EtOH; (k) CH3CH2CHO, NaBH(OAc)3, THF.

Compounds 14a, class="Chemical">14b, and 15 were prepared from 13. For 14a, reaction of 13 with <class="Chemical">span class="Chemical">benzylchloroformate primarily and unexpectedly protected the 1-position amide nitrogen rather than the secondary amine. Reaction with 1-bromopropane (Scheme ) gave the N,N-dipropylamine intermediate, which upon hydrogenolysis of the CBz protecting group gave 14a.1H NMR and optical rotation data corresponded with this previously reported structure.[20] Direct alkylation of 13 in DMF with K2CO3 gave a mixture of 14b and 15. The ratio of the two products (14b:15) depended on the reaction temperature, reaction time, and the ratio of 13 to 1-bromopropane. We found only product 14b was formed if the reaction temperature was kept at 40 °C or below, but the reaction was slow. The yield of product 15 could be improved by increasing the ratio of 1-bromopropane to 13, increasing the temperature, and prolonging the reaction time (details in Experimental Methods). Compound 14c was prepared using the same alkylation procedure as described for 14b, starting with compound 1.

Scheme 2

Synthesis of Analogues 14a–c and 15

Reagents and conditions: (a) i. CBz-Cl, Et3N, THF; ii. n-PrBr, K2CO3, DMF, heat; iii. H2, 10% Pd/C, EtOH; (b) n-PrBr, K2CO3, DMF, heat.

Reagents and conditions: (a) i. class="Chemical">CBz-Cl, <class="Chemical">span class="Chemical">Et3N, THF; ii. n-PrBr, K2CO3, DMF, heat; iii. H2, 10% Pd/C, EtOH; (b) n-PrBr, K2CO3, DMF, heat.

Compounds 18a and 18b were synthesized according to Scheme . Protection of the basic class="Chemical">nitrogen on 1 with the <class="Chemical">span class="Chemical">CBz group using N-(benzyloxycarbonyloxy)succinimide afforded intermediate 16, which was treated with either NaH or K2CO3 followed by reaction with 1-bromopropane or 1-bromobutane to give 17a and 17b, respectively. Deprotection of the CBz group on 17a and 17b by Pd/C-catalyzed hydrogenolysis provided products 18a and 18b, respectively.

Scheme 3

Synthesis of Analogues 18a and 18b

Reagents and conditions: (a) N-(benzyloxycarbonyloxy)succinimide, THF, −40 °C to rt, 16 h; (b) NaH or K2CO3, RBr, THF, rt; (c) H2 (50 psi), Pd/C (10%), EtOH, 5 h.

Reagents and conditions: (a) class="Chemical">N-(benzyloxycarbonyloxy)succinimide, <class="Chemical">span class="Chemical">THF, −40 °C to rt, 16 h; (b) NaH or K2CO3, RBr, THF, rt; (c) H2 (50 psi), Pd/C (10%), EtOH, 5 h.

Scheme outlines the synthetic strategy used for the synthesis of the class="Chemical">aryl amide butyl-substituted derivatives 22a–c. Compounds 20a, 20b, 21a, and 21b were synthesized according to the earlier published procedure.[41] Compound 20c was synthesized via the <class="Chemical">span class="Chemical">acid chloride and was converted to 21c with Ph3P and CBr4. Coupling of 1 with 21a–c under basic conditions afforded the desired compounds 22a–c.

Scheme 4

Synthesis of Analogues 22a–c

Reagents and conditions: (a) i. SOCl2; ii. NH2(CH2)4OH, 0 °C to rt; (b) i. CDI, THF, rt; ii. NH2(CH2)4OH; 0 °C to rt; (c) Ph3P, CBr4, CH3CN; (d) 1, K2CO3, DMF, 60–65 °C, 3 h.

Reagents and conditions: (a) i. class="Chemical">SOCl2; ii. N<class="Chemical">span class="Chemical">H2(CH2)4OH, 0 °C to rt; (b) i. CDI, THF, rt; ii. NH2(CH2)4OH; 0 °C to rt; (c) Ph3P, CBr4, CH3CN; (d) 1, K2CO3, DMF, 60–65 °C, 3 h.

Results and Discussion

D2R and D3R Binding and Functional Data

Compound 1 was reported previously to be a highly selective class="Gene">D2R agonist[22] and thus should bind preferentially to the high affinity (active) conformation of <class="Chemical">span class="Gene">D2R. We determined the dissociation constant (Ki) values of 1 and its analogues as well as the prototypical D2-like agonists dopamine, 7-OH-DPAT, and quinpirole using both agonist and antagonist tracer ligands (Table ). Not surprisingly, the absolute Ki values for these agonists at both D2R and D3R were lower (i.e., higher affinities) when determined in competition with the agonist radioligand [3H]7-OH-DPAT compared to the antagonist radioligand [3H]N-methylspiperone. This is consistent with previous results demonstrating substantial probe sensitivity with dopamine receptor agonists, which more readily compete with agonist radioligands than antagonist radioligands.[42]

Table 1

In Vitro Radioligand Competition Binding at hD2R and hD3R

			[3H]N-methylspiperone competitiona			[3H]7-OH-DPAT competitiona
			hD2R	hD3R		hD2R	hD3R
compound	structure	cLogP	Ki ± SEM (nM)	Ki ± SEM (nM)	D3/D2	Ki ± SEM (nM)	Ki ± SEM (nM)	D3/D2
dopamine		0.17	3,690 ± 845	293 ± 92.0	0.08	8.73 ± 1.11	7.58 ± 2.12	0.87
7-OH-DPAT		4.0	143 ± 15.2	1.75 ± 0.355	0.01	2.27 ± 0.211	1.49 ± 0.393	0.66
quinpirole		0.27	2,950 ± 410	29.3 ± 2.66	0.01	5.56 ± 0.396	8.01 ± 1.75	1.4
1	R1=R2=H, R3=CH3	1.3	16,300 ± 2,930	6,330 ± 653	0.39	17.1 ± 2.03	546 ± 142	32
12ac	R1=R2=H, R3=H	1.0	12,100 ± 789	11,800 ± 3,660	0.98	114 ± 18.5	3,390 ± 724	30
12b	R1=R2=H, R3=CH3, 8-CN	1.2	N.T.b	27,200 ± 2,600		5,120 ± 307	N.T.b
13c	R1=R2=H, R3 = n-Pr	2.4	2,430 ± 468	410 ± 41.9	0.17	2.78 ± 0.273	25.5 ± 2.59	9.2
14ac	R1=H, R2=R3 = n-Pr	4.0	N.T.b	N.T.b		11.4 ± 1.05	27.1 ± 2.33	2.4
14b	R1=R2 = n-Pr, R3=H		868 ± 111	47.9 ± 9.39	0.06	8.08 ± 2.28	21.7 ± 1.28	2.7
14c	R1=H, R2 = n-Pr, R3=CH3	2.9	1,180 ± 312	290 ± 49.8	0.25	12.5 ± 2.54	104 ± 9.24	8.3
15	R1=R2= R3 = n-Pr	5.1	146 ± 1.48	6.05 ± 1.61	0.04	2.59 ± 0.177	3.39 ± 0.313	1.3
18a	R1 = n-Pr, R2=H, R3=CH3	2.4	751 ± 27.8	187 ± 14.0	0.25	13.2 ± 3.26	99.5 ± 1.19	7.5
18b	R1 = n-Bu, R2=H, R3=CH3	3.0	361 ± 38.9	10.3 ± 2.40	0.03	12.8 ± 2.39	142 ± 8.65	11
22a	R1=H, R2=A, R3=CH3	3.7	14,700 ± 4,890	2,610 ± 588	0.18	15.5 ± 1.32	256 ± 51.5	17
22b	R1=H, R2=B, R3=CH3	5.0	4,230 ± 1,680	337 ± 19.8	0.08	5.78 ± 0.418	76.9 ± 6.39	13
22c	R1=H, R2=C, R3=CH3	3.7	3,700 ± 777	1,090 ± 208	0.29	5.47 ± 1.11	12.5 ± 3.13	2.3

Each Ki value represents data from at least three independent experiments with each performed in triplicate. Binding assays are described in detail in the Experimental Methods.

N.T. = not tested.

Compound previously reported by Moon et al.[20]

Determined with ChemBioDraw Ultra 14.0.

Each Ki value represents <span class="Chemical">data from at least three independent experiments with each performed in triplicate. Binding assays are described in detail in the Experimental Methods.

Compound 1 was previously reported to be 215-fold selective for class="Gene">D2R over <class="Chemical">span class="Chemical">D3R.[22] Notably, the previous analysis relied upon an agonist radiotracer at D2R ([3H]U-86170)[42] ([3H]14a) but an antagonist radiotracer at D3R ([3H]spiperone), thereby potentially biasing the selectivity ratio toward D2R. We discovered that when using the antagonist [3H]N-methylspiperone as the radioligand, 1 displayed very low affinity for D2R and D3R (Ki ≥ 16 and 6.3 μM, respectively) and was slightly D3R-selective (∼2.5-fold). When the agonist [3H]7-OH-DPAT was used as the radiotracer, 1 displayed high affinity at D2R (Ki = 17.1 nM), a slightly higher Ki value than reported (Ki ≈ 9 nM) using [3H]14a).[18,22] In our assay, using [3H]7-OH-DPAT as the radiotracer, the affinity of 1 for D3R was higher (Ki = 546 nM) than previously reported (Ki = 1940 nM using [3H]spiperone).[22] Thus, using [3H]7-OH-DPAT at both receptors, we determined 1 to be 32-fold D2R-selective over D3R rather than >200-fold as previously reported.[22]

Like the parent compound 1, its analogues displayed binding affinities at class="Gene">D2R that were 28- to 955-fold higher when using [<class="Chemical">span class="Chemical">3H]7-OH-DPAT as compared to [3H]N-methylspiperone as the competitive radioligand. Binding to D3R exhibited the same probe-dependent pattern but with a smaller magnitude difference in calculated Ki, increasing <40-fold when using [3H]7-OH-DPAT as compared to [3H]N-methylspiperone as the competitive radioligand.

Because 1 and its analogues appeared to be agonists at both class="Gene">D2R and <class="Chemical">span class="Chemical">D3R based on their binding profiles—i.e., higher affinity in competition with agonist as compared to antagonist radiotracers—we focused on structure–activity relationships (SAR) at D2R and D3R using the agonist [3H]7-OH-DPAT as the tracer ligand. Removing the N-CH3 (12a) reduced affinities at both D2R and D3R by ∼6-fold. The addition of an 8-CN group (12b) nearly abolished affinity at D2R, so further modification at this position was not pursued.

By replacing the N-5-CH3 group in 1 with an n-propyl group (13), both class="Gene">D2R and <class="Chemical">span class="Chemical">D3R binding affinities improved (Ki = 2.78 and 25.5 nM, respectively). Whereas the N,N-di-n-propyl analogue, 14a, demonstrated similar affinity at D3R to 13, the D2R affinity was decreased ∼4-fold. The n-propylation at position 1 (14b) had little effect on D2R and D3R binding affinities, and addition of the N-5-n-propyl to 1 (14c) maintained a similar D2R affinity (Ki = 12.5 nM) but led to a small increase in D3R affinity, rendering this analogue less D2R selective (∼8-fold) than the parent compound (32-fold).

Global N-n-propyl substitutions at positions 1 and 5 (15) uniformly improved both class="Gene">D2R and <class="Chemical">span class="Chemical">D3R binding affinities. Of note, this analogue displayed similarly high affinities (Ki ∼ 3 nM) for D2R and D3R. Alkylation at the position 1 imidazo-nitrogen (18a, b) did not affect binding affinities at D2R as compared to the parent molecule. However, D3R affinities improved ∼3–5-fold. Addition of the butyl-linked arylamide (22a–c), used frequently in the 4-phenylpiperazine class of D3R-selective antagonists/partial agonists, resulted in D2R-selective agonists with high affinities (Ki = 5–15 nM). The discovery that all three of these analogues retained high binding affinities and D2R selectivities suggests that these molecules may not access the secondary binding pocket in the same way that the 4-phenylpiperazine-based D3R-selective antagonists/partial agonists do[12,33,43]and implies an important role of the 5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one PP in positioning the rest of the molecule in D2R.

To evaluate the functional activity of 1 and its analogues at class="Gene">D2R or <class="Chemical">span class="Chemical">D3R, these compounds were evaluated in two different in vitro functional assays (Table ). A bioluminescence resonance energy transfer (BRET)-based assay was used to measure agonist-induced activation of GαoA by either D2R or D3R. In this assay, receptor activation leads to the separation of GαoA-91-Rluc8 and complemented mVenus-Gβ1γ2 and a reduction in BRET.[33] In addition, a mitogenesis assay was used to characterize dose–response curves for receptor-mediated incorporation of [3H]thymidine in cells expressing the recombinant D2R or D3R.

Table 2

In Vitro Agonist Activity at hD2R and hD3R

	hD2R Go BRET		hD3R Go BRET		hD2R mitogenesis		hD3R mitogenesis
compound	EC50 ± SEM (nM)	% DA max ± SEM	EC50 ± SEM (nM)	% DA max ± SEM	EC50 ± SEM (nM)	% DA max ± SEM	EC50 ± SEM (nM)	% DA max ± SEM
dopamine	5.0 ± 1.4	100	1.8 ± 0.2	100	7.7 ± 0.8	100 ± 3.0	2.7 ± 0.7	106 ± 2.1
quinpirole	N.T.b	N.T.b	N.T.b	N.T.b	8.2 ± 1.1	99 ± 1.2	3.3 ± 0.7	102 ± 2.9
1	60.1 ± 13	96.9 ± 2.5	186 ± 49	101 ± 6.0	64.7 ± 2.6	100 ± 2.2	669 ± 88	157 ± 1.6
12ac	697 ± 190	92.0 ± 2.3	1890 ± 450	85.2 ± 5.9	684 ± 7.2	95.3 ± 2.6	3400 ± 480	139 ± 5.8
13c	16.0 ± 3.6	101 ± 2.7	18.3 ± 4.4	94.5 ± 3.3	1.0 ± 0.10	97.3 ± 5.8	0.81 ± 0.05	107 ± 3.0
14ac	2.53 ± 0.58	109 ± 6.3	21.8 ± 2.9	86.9 ± 2.6	N.T.b	N.T.b	N.T.b	N.T.b
14b	68.0 ± 3.1	85.6 ± 5.0	1423 ± 4.4	64.5 ± 5.2	13.0 ± 0.5	92.1 ± 4.2	56.1 ± 7.2	52.0 ± 4.1
14c	29.0 ± 6.6	80.0 ± 3.6	30.7 ± 6.4	58.6 ± 2.2	18.1 ± 0.5	91.6 ± 5.0	148 ± 4.0	85.4 ± 9.2
15	33.9 ± 8.2	88.2 ± 3.5	51.9 ± 7.9	53.5 ± 8.0	13.7 ± 36	108 ± 2.7	3.7 ± 1.4	47.0 ± 7.3
18a	38.7 ± 22	95.8 ± 3.9	53.7 ± 18	74.5 ± 5.1	549 ± 36	92.0 ± 6.4	178 ± 38	58.0 ± 3.5
18b	30.5 ± 14	84.8 ± 7.2	25.6 ± 7.5	40.4 ± 8.3	83.0 ± 18	56.0 ± 3.6	22.0 ± 4	28.0 ± 1.6
22a	43.3 ± 11	103 ± 3.0	115 ± 24	88.1 ± 4.2	19.5 ± 3.5	96.4 ± 4.9	19.6 ± 6.1	97.2 ± 2.8
22b	22.7 ± 5.4	107 ± 2.5	94.3 ± 23	86.9 ± 2.6	27.7 ± 3.5	106 ± 2.7	53.0 ± 16	103 ± 4.6
22c	109 ± 18	104 ± 3.2	125 ± 43	95.3 ± 3.1	102 ± 3.7	90.1 ± 9.9	11.9 ± 4.1	105 ± 2.9

Each EC50 value represents data from at least three independent experiments. Functional assays are described in detail in the Experimental Methods.

N.T. = not tested.

Compound previously reported by Moon et al.[20] DA = dopamine.

Each EC50 value represents <span class="Chemical">data from at least three independent experiments. Functional assays are described in detail in the Experimental Methods.

Compound previously reported by Moon et al.[20] <span class="Chemical">DA = <class="Chemical">span class="Chemical">dopamine.

Overall, 1 and its analogues displayed agonist profiles at class="Gene">D2R and <class="Chemical">span class="Chemical">D3R in both the Go BRET and mitogenesis assays as compared to standard D2-like agonists dopamine and quinpirole. As expected for compounds with high efficacy, Go BRET and mitogenesis EC50 values were more similar to Ki values calculated in competition with the agonist radioligand [3H]7-OH-DPAT than to Ki values calculated in competition with the antagonist radioligand [3H]N-methylspiperone. These results are more pronounced with D2R (compared to D3R), an observation consistent with the substantial relative difference between the high- and low-affinity receptor populations of D2R and D3R.[44] Agonist binding at D2R can be robustly inhibited by nonhydrolyzable analogues of GTP, dramatically shifting apparent Ki values. In contrast, D3R agonist binding is relatively insensitive to nonhydrolyzable GTP analogues, maintaining substantial high-affinity binding in the absence of efficient G protein signaling.[45−47] The partial retention of high-affinity binding at D3R in conditions that favor low-affinity binding, i.e., in the [3H]N-methylspiperone binding assay, is a likely explanation for the less dramatic difference between [3H]7-OH-DPAT-derived and [3H]N-methylspiperone-derived Ki values at D3R in comparison to the very large differences seen at D2R.

In an assay of receptor-activated mitogenesis, McCall et al. (2005) reported an EC50 of 4.6 nM at class="Gene">D2R for 1, but no activity was observed in a <class="Chemical">span class="Chemical">D3R-mediated mitogenesis assay when tested using concentrations up to 1 μM, although the data were not shown.[22] In contrast, we report that 1 is a fully efficacious agonist in mitogenesis assays at both D2R and D3R (EC50 = 65 and 669 nM, respectively; Table ). Overall, our data for both Go BRET and mitogenesis suggest that 1 is a full agonist at both D2R and D3R and only modestly (3–10-fold) D2R-selective over D3R in these cell-based functional assays (Table ). It is notable that our binding and functional studies agree that, although 1 is certainly a preferential agonist for D2R over D3R, the degree of subtype selectivity may be more modest than previously suggested.

In addition, class="Gene">D2R binding selectivity over <class="Chemical">span class="Chemical">D3R for this series of ligands is more pronounced than D2R selectivity in the functional assays. The most potent and fully efficacious D2R agonist in this set was 14a (EC50 = 2.53 nM, Table ). Curiously, compounds 14b, 15, and 18a, which have an n-propyl group at the 1-position, each show modest but significantly decreased efficacy at D3R compared to D2R, which may suggest functional separability between subtypes. This decrease in efficacy was even more dramatic (40.4% at D3R vs 84.8% at D2R, Table ) with n-butyl-substituted 18b.

The analogues containing class="Chemical">N-5-butyl-arylamides (22a–c) diclass="Chemical">splayed full agonist activity. These functional groups have been used by a number of laboratories to make highly <class="Chemical">span class="Chemical">D3R-selective antagonists and low efficacy partial agonists.[9] On the basis of modeling derived from the D3R crystal structure, an extended aryl SP attached to a PP, such as the D2R/D3R partial agonist 2,3-dichlorophenylpiperazine via a butylamide linker, results in compounds in which the PP binds in the OBS and the SP binds in an SBP.[32,33] Interactions between the PP and OBS have been associated with functional activity, whereas interactions between the SP and the SBP, particularly with extracellular loop 1, typically dictate subtype selectivity.[33,48]

Under the hypothesis that 1 occupies the OBS of class="Gene">D2R or <class="Chemical">span class="Chemical">D3R in a manner similar to dopamine and the phenylpiperazine moiety of reported D3R-selective antagonists, it is likely that the agonist activity of 22a–c is mediated by interactions of their 5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one PP within the OBS. This also provides an explanation for previously reported D3R-selective agonists containing the 6-N-propyl-4,5,6,7-tetrahydro-1,3-benzothiazole-2,6-diamine of D3R agonist pramipexole as the PP and a butyl-arylamide as the SP.[49,50]

Structural Basis for the D2R over D3R Selectivity of Compound 1

To understand the structural basis of the class="Gene">D2R over <class="Chemical">span class="Chemical">D3R selectivity of compound 1, we used molecular modeling and simulations to compare the binding modes of compound 1 in D2R and D3R. Our docking results showed that in both D2R and D3R, the protonated amine N-5 forms a salt bridge with the side chain of conserved Asp3.32, and the aromatic moiety contacts residues Phe6.51, Phe6.52, and His6.55 of transmembrane 6 (TM6) in the OBS;[51] however, the orientation of the imidazolinone moiety is ambiguous. Top-scoring docking poses in either receptor did not show a clear preference for the carbonyl O of the imidazolinone moiety to point either up toward the second extracellular loop (EL2) (“up” pose) or down toward the intracellular side of the transmembrane domain (“down” pose).

To further evaluate the orientational preference of the class="Chemical">imidazolinone moiety, we carried out molecular dynamics (MD) simulations starting from either the “up” or the “down” pose for both <class="Chemical">span class="Gene">D2R and D3R (see Experimental Methods for details). The simulations indicated that the “up” poses in D2R and D3R converged: the N-1 forms a hydrogen bond with the side chain of Ser5.42, whereas the carbonyl O of the imidazolinone moiety forms a hydrogen bond with the side chain of His6.55 (Figure a,b). By contrast, the “down” poses in D2R and D3R showed divergent interactions with the receptors: the N-1 interacts with Ser1935.42 in D2R, whereas it interacts with Ser1965.46 in D3R instead (Figure c,d). By calculating the ligand–receptor binding energy using the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) approach for the frames along each MD trajectory, we found that the “down” pose has more favorable binding energy than the “up” pose in D2R; however, the reverse preference was marginally observed in D3R (Figure e). Furthermore, the “down” pose in D2R has more favorable energy than either “up” or “down” poses in D3R (Figure e), consistent with the higher affinity of compound 1 for D2R over D3R.

Figure 2

Predicted binding modes of 1 in D2R and D3R. (a–d) The “up” poses of 1 in D2R (a) and D3R (b) converged, forming ligand–receptor interactions with Asp3.32, Ser5.42, Phe6.51, Phe6.52, and His6.55, whereas the “down” poses in D2R (c) and D3R (d) showed divergent interactions. The ligand is shown as sticks in green for the “up” pose and in magenta for the “down” pose. The different conformations of the EL2 and the N-terminal segment of TM5 between D2R and D3R (cyan) likely result from the divergent amino acid residues within this region (cyan sticks) and may contribute to the differential binding modes in D2R and D3R. TMs 6 and 7 are not shown for clarity. (e) The average and standard deviation of MM/GBSA receptor–ligand binding energy values from the last 60 ns of MD trajectories are shown as a barplot for the “up” and “down” poses in D2R and D3R. In D2R, the “down” pose has lower binding energy values than the “up” pose, whereas in D3R, the reverse preference is observed. The “down” pose in D2R has lower binding energy values than the “up” pose in D3R, consistent with the selectivity of 1 for D2R over D3R.

Predicted binding modes of 1 in class="Gene">D2R and <class="Chemical">span class="Chemical">D3R. (a–d) The “up” poses of 1 in D2R (a) and D3R (b) converged, forming ligand–receptor interactions with Asp3.32, Ser5.42, Phe6.51, Phe6.52, and His6.55, whereas the “down” poses in D2R (c) and D3R (d) showed divergent interactions. The ligand is shown as sticks in green for the “up” pose and in magenta for the “down” pose. The different conformations of the EL2 and the N-terminal segment of TM5 between D2R and D3R (cyan) likely result from the divergent amino acid residues within this region (cyan sticks) and may contribute to the differential binding modes in D2R and D3R. TMs 6 and 7 are not shown for clarity. (e) The average and standard deviation of MM/GBSA receptor–ligand binding energy values from the last 60 ns of MD trajectories are shown as a barplot for the “up” and “down” poses in D2R and D3R. In D2R, the “down” pose has lower binding energy values than the “up” pose, whereas in D3R, the reverse preference is observed. The “down” pose in D2R has lower binding energy values than the “up” pose in D3R, consistent with the selectivity of 1 for D2R over D3R.

To further valiclass="Chemical">date the differential binding modes of 1 in <class="Chemical">span class="Gene">D2R and D3R, we carried out docking studies using the resulting models from our MD simulations (see Experimental Methods) for selected compound 1 analogues, compounds 13 and 15. As described above, the replacement of the N-5-CH3 group in 1 with an n-propyl group (13) improved binding affinities at both D2R and D3R while retaining some selectivity, albeit lower than that of 1 (Ki = 2.78 and 25.5 nM for D2R and D3R, respectively; D3R/D2R = 9.2-fold selectivity). The N-n-propyl substitutions at positions 1 and 5 (15), on the other hand, improved the affinity only at D3R but not D2R compared to 13 (Ki = 2.59 and 3.39 nM for D2R and D3R, respectively; D3R/D2R = 1.3-fold selectivity), resulting in the complete loss of D2R selectivity. Our docking results for 13, which retains some selectivity, showed the same preference for the “up” or ”down” orientation compared to 1 (Figure a,b). The improved affinities of 13 in both D2R and D3R can be attributed to the n-propyl group at position 5 inserting into a previously identified high affinity hydrophobic pocket at the interface of TMs 6 and 7 (Ptm67 pocket).[33] In contrast, the docking results for 15, which loses selectivity, showed that the “up” orientation is preferred in both D2R and D3R, i.e., the alkylations at positions 1 and 5 cause a switch in the orientational preference of the imidazolinone moiety in D2R (Figure c,d). The improved affinity of 15 compared to 13 in D3R can be attributed to the n-propyl groups at positions 1 and 5, making additional interactions with the pockets at the interface of TMs 3 and 5 and the interface of TMs 2 and 3 (Ptm23 pocket).[33] The lack of change in affinity of 15 compared to 13 in D2R is likely due to the competing effects of the unfavorable switch in the imidazolinone orientation from “down” to “up” and the favorable additional interactions formed by the n-propyl groups. Thus, the predicted binding poses of 13 and 15 in D2R and D3R establish SAR consistent with the experimental results and support the differential binding modes of their parent compound 1 in these two receptors.

Figure 3

Predicted binding modes of analogues 13 and 15 in D2R and D3R. For each compound, the largest cluster of poses is shown. Compound 13 (orange) binds with similar orientational preference as 1 in both D2R (a) and D3R (b), whereas 15 (slate blue) reverses to the “up” pose in D2R (c) as in D3R (d). The n-propyl group of 13 interacts with the Ptm67 pocket residues Trp6.48, Phe6.51, Thr7.39, and Tyr7.43. The additional n-propyl group of 15 interacts with the Ptm23 pocket residues Val2.61, Leu2.64, and Phe3.28.

Predicted binding modes of analogues 13 and 15 in class="Gene">D2R and <class="Chemical">span class="Chemical">D3R. For each compound, the largest cluster of poses is shown. Compound 13 (orange) binds with similar orientational preference as 1 in both D2R (a) and D3R (b), whereas 15 (slate blue) reverses to the “up” pose in D2R (c) as in D3R (d). The n-propyl group of 13 interacts with the Ptm67 pocket residues Trp6.48, Phe6.51, Thr7.39, and Tyr7.43. The additional n-propyl group of 15 interacts with the Ptm23 pocket residues Val2.61, Leu2.64, and Phe3.28.

We observed divergent conformations of class="Gene">EL2 and the extracellular portion of <class="Chemical">span class="Gene">TM5 that may contribute to the differential binding modes in D2R and D3R. Specifically, several divergent residues in and near EL2 and Ile2035.52 in D2R relative to Gly2025.52 in D3R likely modulate the proline kink (prokink) angles at Pro5.50 differentially,[52] resulting in a larger distance between the extracellular tips of TMs 3 and 5 in D2R compared to D3R; consequently, the subcavity enclosed by EL2 and the extracellular portion of TM5 accommodate the imidazolinone moiety of 1 differently in D2R and D3R (Figure c,d).

Conclusions

In the present study, we report the synthesis of parent compound 1 and a series of 1-, 5-, and 8-substituted analogues that were evaluated for binding and functional activity at class="Gene">D2R and <class="Chemical">span class="Chemical">D3R. Importantly, binding conditions—notably the use of agonist or antagonist radioligand probes—dramatically affect calculated binding affinities, especially for D2R, in turn altering calculated receptor selectivity ratios. Relatedly, compound 1 is considerably less D2R-selective in our binding and functional studies than previously reported.[22] In this series of 1 analogues, D3R affinity generally improved along with D2R affinity, resulting in analogues with higher D2R affinity but less D2R selectivity than the parent ligand. Modifications at different positions on the parent compound template had distinct effects. For example, simple alkyl substitutions at the N-1 position produced analogues with reduced efficacy at D3R and no significant subtype selectivity, suggesting that modification at this position differentially affects interactions at the OBS, which is highly homologous between D2R and D3R. Modification with n-butyl-arylamide linkers at the N-5 position, found in classic D3R-selective antagonists,[34] was well-tolerated and resulted in potent and relatively nonselective D2R full agonists with improved D2R binding affinity. This finding demonstrates that the butyl-arylamide does not universally differentiate binding at D2R and D3R, as has been observed in the 4-phenylpiperazine class of D3R-selective antagonists/partial agonists, and extends our hypothesis that binding of the PP in the OBS determines how the rest of the molecule binds to the receptor, affecting both efficacy and subtype selectivity.

On the basis of molecular modeling and simulation class="Chemical">data, 1 and the analogues described herein bind to and activate <class="Chemical">span class="Gene">D2R and D3R similarly. However, amino acid sequence differences between D2R and D3R in two regions, EL2 and the N-terminal segment of TM5, may dictate the subtype selectivity of 1 and the analogues reported in this study. The contributions of subtle differences in the binding mode of the PP in the OBS and additional contributions of the SP to subtype selectivity and efficacy will require further investigation. Moreover, in the absence of a crystal structure of the active state of either D2R or D3R, more extensive SAR studies will be required in the pursuit of highly D2R-selective agonists.

Experimental Methods

Synthesis

All chemicals and solvents were purchased from chemical suppliers unless otherwise stated and used without further purification. Dry class="Chemical">THF was freshly distilled from <class="Chemical">span class="Chemical">sodium benzophenone ketyl. All melting points were determined on a Thomas–Hoover melting point apparatus 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 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). Infrared spectra were recorded as a neat film on NaCl plates with a PerkinElmer Spectrum RX I FT-IR system. Microanalyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree with ±0.4% of calculated values. 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 CHCl3/CH3OH/NH4OH (CMA) in the percentage indicated where NH4OH is 1%. If not otherwise stated, all spectroscopic data and yields refer to the free base. On the basis of these analyses, all final compounds are >95% pure.

General Synthetic Procedure for 3a,b from 2a,b

A solution of class="Chemical">3-substituted d-phenylalanine and <class="Chemical">span class="Chemical">NaOH (1 equiv) in H2O (NaOH/H2O; 1 g/30 mL) and THF (phenylalanine/THF; 1 mmol/1.5 mL) was cooled to −15 °C, and a solution of methyl chloroformate or benzyl chloroformate (1.3 equiv) in THF (acid chloride/THF; 4 mmol/1 mL) was added dropwise. When one-half of the acid chloride had been added, a solution of NaOH (1.5 equiv) in H2O (NaOH/H2O; 1 g/2 mL) was added, and the addition continued. The reaction mixture was stirred at rt for an additional 2 h after the addition was completed, and it was then acidified with 10% aq HCl solution to pH 2. The mixture was extracted twice with Et2O, and the combined extracts were washed with brine, dried (MgSO4), and filtered. The solvent was removed under vacuum to leave the product as a clear oil. Compound 3a was used for the next step without purification.

(R)-2-((Methoxycarbonyl)amino)-3-(3-bromophenyl)propanoic acid (3b)

Compound 3b was prepared from 2b (5.01 g, 20.5 mmol) in 90% (5.54 g) as a clear <span class="Chemical">oil, which slowly solidified to a white solid after standing at rt. Mp 67–71 °C. 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 7.40 (m, 1H), 7.34 (s, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.11 (d, J = 7.6 Hz, 1H), 5.13 (d, J = 8.0 Hz, 1H), 4.66 (dd, J = 6.0, 5.6 Hz, 1H), 3.69 (s, 3H), 3.18 (dd, J = 13.8, 6.0 Hz, 1H), 3.06 (dd, J = 14.0, 6.4 Hz, 1H).

General Synthetic Procedure for 4a,b from 3a,b

To a solution of 3a,b in class="Chemical">CH2Cl2 was added an <class="Chemical">span class="Chemical">aq solution of Na2CO3 (0.65 eq, Na2CO3/H2O (1 g/1.7 mL). Methoxyamine hydrochloride (CH3ONH2·HCl; 1.15 equiv) and EDC (1.1 equiv) were then added, and the resulting mixture was stirred at rt for 24 h. The mixture was diluted with THF (to dissolve the precipitate), and the layers were separated. The aq layer was extracted with 1:1 THF/Et2O, and the combined organic extracts were washed with 10% aq HCl solution and a saturated NaHCO3 solution successively. The organic layer was dried (MgSO4) and concentrated to give the crude product 4a,b.

Benzyl (R)-[1-Methoxyamino-1-oxo-3-phenylpropan-2-yl]carbamate (4a)

Compound 4a was obtained as a white solid in 69% yield (22.5 g) from 2a (16.5 g) in 2 steps and purified by crystallization from <span class="Chemical">ethyl acetate. Mp 136–140 °C (dec). 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 8.51 (br s, 1H), 7.38–7.16 (m, 10H), 5.37 (br s, 1H), 5.07 (s, 2H), 4.23 (m, 1H), 3.61 (s, 3H), 3.08 (m, 2H).

Methyl (R)-[3-(3-Bromophenyl)-1-methoxyamino-1-oxopropan-2-yl]carbamate (4b)

Compound 4b was obtained as a white solid in 88% yield (5.28 g) from 3b (5.45 g, 18.0 mmol) and purified by crystallization from <span class="Chemical">ethyl acetate. Mp 143–145 °C. 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 9.21 (br, 1H), 7.36 (d, J = 1.2 Hz, 2H), 7.15 (m, 2H), 5.56 (br, 1H), 4.28 (m, 1H), 3.64 (s, 3H), 3.62 (s, 3H), 3.02 (m, 2H).

General Synthetic Procedure for 5a,b from 4a,b

A suspension of compound 4a,b in class="Chemical">CH2Cl2 (4a,b/<class="Chemical">span class="Chemical">CH2Cl2; 1 mmol/4 mL) was cooled in an ice bath, and CF3CO2H (2.7 equiv) was added. Bis(trifluoroacetoxy)iodobenzene (PhI(CF3CO2)2; 1 equiv) was added portionwise over 10 min at 0 °C, and the mixture was stirred at this temperature for 1 h. The mixture was washed with a 10% Na2CO3 solution and dried (MgSO4). Solvent was removed under vacuum to give the product as an amber oil. Purification by flash column chromatography, eluting with hexane/EtOAc (1:1) gave the desired product 5a,b.

Benzyl (R)-(1-Methoxy-2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)carbamate (5a)

Compound 5a was prepared from 4a (11.2 g, 34.2 mmol) and purified by column chromatography in 90% yield (10.2 g) as a light brown solid. Mp 59–62 °C. IR (<span class="Chemical">NaCl): 1694 cm–1. 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 7.38 (m, 5H), 7.33 (m, 1H), 7.22 (d, J = 8.0 Hz, 2H), 7.08 (dt, J = 7.6, 1.2 Hz, 1H), 5.84 (br, 1H), 5.15 (s, 2H), 4.41 (m, 1H), 3.93 (s, 3H), 3.47 (m, 1H), 2.84 (t, J = 14.8 Hz, 1H).

Methyl (R)-(6-Bromo-1-methoxy-2-oxo-1,2,3,4-tetrahydroquinolin-3-yl)carbamate (5b)

Compound 5b was prepared from 4b (5.18 g, 15.7 mmol) and purified by column chromatography in 88% yield (5.57 g) as a light brown solid. Mp 97–99.5 °C. 1H NMR (400 MHz, <span class="Chemical">CDCl3) δ 7.45 (d, J = 8.0 Hz, 1H), 7.38 (s, 1H), 7.10 (d, J = 8.0 Hz, 1H), 5.78 (br, 1H), 4.40 (m, 1H), 3.93 (s, 3H), 3.76 (s, 3H), 3.39 (m, 1H), 2.83 (t, J = 10.8 Hz, 1H).

(R)-3-Amino-1-methoxy-3,4-dihydroquinolin-2(1H)-one (6a)

Compound 5a (4.00 g) was dissolved in class="Chemical">ethanol (80 mL) in a Parr bottle, and 10% <class="Chemical">span class="Chemical">Pd/C (400 mg) was added. The mixture was hydrogenolyzed (with an initial pressure of 50 psi) until its completion (the reaction was monitored by TLC). The mixture was then filtered over Celite, and the filtrate was concentrated under vacuum to give 6a, which was used in the next step without further purification. Mp 183–185 °C. 1H NMR (400 MHz, CDCl3) 7.31 (m, 1H), 7.17 (d, J = 8.4 Hz, 2H), 7.03 (dt, J = 7.4, 7.4 Hz, 1H), 3.89 (s, 3H), 3.60 (dd, J = 6.4, 6.0 Hz, 1H), 3.06 (dd, J = 6.4, 6.0 Hz, 1H), 2.81 (t, J = 14.2 Hz, 1H), 2.04 (br s, 2H). GC-MS (EI) m/z 192 (M+).

General Synthetic Procedure for 7a,b from 5b or 6a

A solution or suspension of 5b or 6a (4 mL of class="Chemical">THF/mmol) in <class="Chemical">span class="Chemical">THF was cooled to 0 °C, and borane-methyl sulfide (BH3·Me2S; 10.0 M, 6 equiv) was added slowly. The mixture was allowed to warm to rt and stirred for 2.5 h. The mixture was heated to reflux for 48 h (solution) or 5 days (suspension). The resulting clear solution was then cooled to 0 °C and quenched slowly with 10% HCl solution (: hydrogen evolution). The mixture was heated to reflux again for 1.5 h, cooled to 0 °C, and basified (pH >10) with 12 N NaOH solution. The mixture was extracted twice with Et2O, and the combined extracts were washed with brine, dried (MgSO4), and concentrated to give a clear oil (7b) or a dark oil (7a), which was carried on to the next reaction without further purification.

(R)-1,2,3,4-Tetrahydroquinolin-3-amine (7a)[20]

1H NMR (400 MHz, <span class="Chemical">CDCl3) δ 6.98 (m, 2H), 6.63 (m, 1H), 6.49 (d, J = 10.4 Hz, 1H), 3.83 (br, 1H), 3.36 (m, 2H), 3.03 (m, 2H), 2.78 (dd, J = 14.8, 6.4 Hz, 1H), 1.68 (br, 2H). GC-MS (EI) m/z 148 (M+).

(R)-6-Bromo-N-methyl-1,2,3,4-tetrahydroquinolin-3-amine (7b)

GC-MS (EI) m/z 240 (M+), 242 (M+).

General Synthetic Procedure for 8a,b from 7a,b

A solution of 7a,b in class="Chemical">toluene (0.6 mL of <class="Chemical">span class="Chemical">toluene/mmol) was stirred at −40 °C while N-(benzyloxycarbonyloxy)succinimide (1.15 equiv) in toluene (1.5 mL of toluene/mmol) was added slowly. The mixture was stirred at this temperature for 30 min after the addition and quenched with a 10% NaHCO3 solution. The mixture was then allowed to warm to 0 °C, and MeOH was added. The resulting mixture was stirred at rt overnight and extracted with EtOAc. The combined extracts were dried (MgSO4) and concentrated to afford the crude product, which was purified by flash column chromatography to give the desired product 8a,b.

Benzyl (R)-(1,2,3,4-Tetrahydroquinolin-3-yl)carbamate (8a)

Compound 8a was prepared from 7a (410 mg, 2.77 mmol) and purified by column chromatography, eluting with <span class="Chemical">hexane/ethyl acetate (2:1) as a solid in 80% yield (626 mg). Mp 76–78.5 °C. 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 7.39–7.28 (m, 5H), 6.97 (dd, J = 8.8, 2.0 Hz, 2H), 6.64 (dt, J = 8.8, 0.8 Hz, 1H), 6.50 (dd, J = 8.8, 0.8 Hz, 1H), 5.16 (s, 2H), 4.57 (m, 1H), 3.30 (d, J = 6.8 Hz, 2H), 3.02–2.80 (m, 4H). GC-MS (EI) m/z 282 (M+).

Benzyl (R)-(6-Bromo-1,2,3,4-tetrahydroquinolin-3-yl)(methyl)carbamate (8b)

Compound 8b was prepared from 5b (5.50 g, 16.7 mmol) in 2 steps and purified by column chromatography, eluting with <span class="Chemical">hexane/ethyl acetate (2:1) in 64% yield (4.07 g). 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 7.36–7.26 (m, 8H), 5.11 (s, 2H), 4.44 (m, 1H), 3.85 (m, 1H), 3.25 (m, 2H), 2.93–2.81 (m, 2H), 2.88 (s, 3H).

General Synthetic Procedure for 9a,b from 8a,b

A solution of 8a,b and class="Chemical">Et3N (3 equiv) in dry <class="Chemical">span class="Chemical">THF (4 mL THF/mmol) was added dropwise to a solution of phosgene (1.07 equiv) in THF (8 mL of THF/mmol) at 0 °C. After 1 h, CH3ONH2·HCl (2 equiv) and Et3N (3 equiv) were added, and the mixture was stirred at rt for 2 days. The mixture was diluted with Et2O and washed with HO and brine. The organic layer was dried (MgSO4) and concentrated to give crude product 9a,b.

Benzyl (R)-(1-(Methoxycarbamoyl)-1,2,3,4-tetrahydroquinolin-3-yl)carbamate (9a)

Compound 9a was prepared from 8a (1.63 g, 6.97 mmol) and purified by column chromatography, eluting with class="Chemical">hexane/ethyl acetate (1:2) as a solid in 74% yield (1.56 g). Mp 107–108.5 °C. IR (<class="Chemical">span class="Chemical">NaCl): 1694 cm–1. 1H NMR (400 MHz, CDCl3) δ 7.86 (br, 1H), 7.40 (m, 1H), 7.31 (m, 5H), 7.19 (m, 1H), 7.16–7.04 (m, 2H), 5.06 (br s, 3H), 4.19 (m, 1H), 3.74 (m, 2H), 3.70 (s, 3H), 3.07 (m, 1H), 2.71 (m, 1H).

Benzyl (R)-(6-Bromo-1-(methoxycarbamoyl)-1,2,3,4-tetrahydroquinolin-3-yl)(methyl)carbamate (9b)

Compound 9b was prepared from 8b (3.18 g, 8.48 mmol) and purified by flash column chromatography (<span class="Chemical">hexane/ethyl acetate; 1:2) as a white solid in 94% yield (3.58 g). Mp 104–106 °C (dec). 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 7.64 (br, 1H), 7.38–7.26 (m, 8H), 5.14 (s, 2H), 4.44 (m, 1H), 3.86 (m, 1H), 3.77–3.72 (m, 1H), 3.74 (s, 3H), 2.93–2.81 (m, 2H), 2.88(s, 3H).

General Synthetic Procedure for 10a,b from 9a,b

A solution of 9a,b in class="Chemical">CHCl3 (7.5 mL <class="Chemical">span class="Chemical">CHCl3/mmol) was cooled to −5 °C in an ice-salt bath. PhI(CF3CO2)2 (1.2 equiv) was added, and the mixture was stirred at −5 to 0 °C for 4 h and then at rt for 2 h. The reaction mixture was washed with a 10% Na2CO3 solution, back-extracting the aq layer with Et2O. The combined organic layers were dried (MgSO4) and concentrated to give a brown oil. Purification by flash column chromatography, eluting with hexane/EtOAc (40:60) afforded the product 10a,b.

Benzyl (R)-(1-Methoxy-2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)carbamate (10a)

Compound 10a was prepared from 9a (6.30 g, 17.7 mmol) and purified by column chromatography as a light brown solid in 82% yield (5.17 g). class="Gene">Mp 70–73 °C (dec). [α]D24 −13.5 (c 0.43, <class="Chemical">span class="Chemical">MeOH). 1H NMR (400 MHz, CDCl3) δ 7.35 (m, 5H), 7.03 (t, J = 7.8 Hz, 1H), 6.98 (d, J = 7.6 Hz, 1H), 6.88 (d, J = 7.6 Hz, 1H), 5.07 (s, 2H), 4.87 (br, 1H), 4.54 (m, 1H), 4.08 (s, 3H), 4.01 (m, 1H), 3.83 (dd, J = 12.0, 3.6 Hz, 1H), 3.11 (m, 1H), 2.90 (m, 1H).

Benzyl (R)-(8-Bromo-1-methoxy-2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)(methyl)carbamate (10b)

Compound 10b was prepared from 9b (3.40 g, 7.59 mmol) and purified by column chromatography as a light brown solid in 73% yield (2.48 g). Mp 168–170 °C (dec). 1H NMR (400 MHz, <span class="Chemical">CDCl3) δ 7.38 (m, 5H), 6.94 (s, 1H), 6.86 (s, 1H), 5.14 (s, 2H), 4.53 (m, 1H), 4.12 (m, 1H), 4.08 (s, 3H), 3.74 (m, 1H), 3.17 (m, 1H), 3.02–2.90 (m, 1H), 2.95 (s, 3H).

Benzyl (R)-(8-Cyano-1-methoxy-2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)(methyl)carbamate (11b)

To a solution of 10b (2.38 g, 5.30 mmol) in class="Chemical">DMF (20 mL) was added <class="Chemical">span class="Chemical">Zn(CN)2 (1.24 g, 10.6 mmol) and Pd(PPh3)4 (0.30 g, 5 mol %) under argon. The reaction mixture was heated to 80 °C overnight. DMF was removed under vacuum, and the mixture was filtered. The filtrate was diluted with H2O and extracted 3 times with EtOAc. The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by flash column chromatography, eluting with hexane/EtOAc (40:60) to afford 11b in 63% yield (1.31 g) as a white solid. Mp 174–176 °C (dec). 1H NMR (400 MHz, CDCl3) δ 7.36 (m, 5H), 7.24 (s, 1H), 7.21 (s, 1H), 5.17 (s, 2H), 4.58 (m, 1H), 4.16–4.09 (m, 1H), 4.10 (s, 3H), 3.74 (m, 1H), 3.17 (m, 1H), 3.02–2.90 (m, 1H), 2.95 (s, 3H).

General Synthetic Procedure for 12a,b from 10a, 11b

A mixture of 10a or class="Chemical">11b and <class="Chemical">span class="Chemical">Pd(OH)2/C (20%, 4.0 g of 10a or 11b/1.0 g of catalyst) in absolute ethanol (20 mL/1.0 g of 10a or 11b) in a Parr bottle was hydrogenolyzed (50 psi). After the reaction was completed (∼20 h, monitored by TLC), the mixture was filtered through Celite. Removal of the solvent afforded the crude product, which was purified by flash chromatography, eluting with CMA to give 12a,b.

(R)-5-Amino-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (12a)[20]

Compound class="Chemical">12a was prepared from 10a (6.34 g, 18 mmol) and purified by column chromatography (10% <class="Chemical">span class="Chemical">CMA) as an oil in 73% yield (2.48 g). [α]D23 −16.9 (c 0.52, MeOH). 1H NMR (400 MHz, CDCl3) δ 9.82 (br s, 1H), 7.00–6.91 (m, 2H), 6.83 (d, J = 7.2 Hz, 1H), 4.06 (dd, J = 16.0, 4.0 Hz, 1H), 3.62 (dd, J = 12.0, 7.2 Hz, 1H), 3.33 (m, 1H), 3.06 (dd, J = 11.8, 4.4 Hz, 1H), 2.71 (dd, J = 16.0, 6.6 Hz, 1H), 1.67 (br s, 2H). 13C NMR (100 MHz, CDCl3) δ 155.15, 127.56, 126.45, 120.84, 120.03, 117.72, 107.46, 51.57, 42.96, 30.89. GC-MS (EI) m/z 189 (M+). Anal. (C10H11N3O·H2O) C, H, N.

(R)-5-(Methylamino)-2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-8-carbonitrile (12b)

Compound 12b was prepared from class="Chemical">11b (1.00 g, 2.64 mmol) and purified by column chromatography (10% <class="Chemical">span class="Chemical">CMA) as an oil in 85% yield (0.49 g). 1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 7.21 (s, 1H), 3.98 (ddd, J = 12.4, 8.0, 0.8 Hz, 1H), 3.71 (dd, J = 12.4, 6.4 Hz, 1H), 3.28 (m, 1H), 3.06 (dd, J = 16.0, 4.0 Hz, 1H), 2.80 (dd, J = 16.0, 7.2 Hz, 1H), 2.53 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 150.09, 125.99, 125.29, 124.49, 119.51, 118.35, 108.49, 104.72, 52.40, 42.70, 34.06, 30.39. GC-MS (EI) m/z 228 (M+). Anal. (C12H12N4O·1/4H2O) C, H, N.

(R)-5-(Propylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (13)[20]

To a solution of class="Chemical">12a (230 mg, 1.20 mmol) in <class="Chemical">span class="Chemical">THF (5 mL) was added propionaldehyde (84 mg, 1.44 mmol), NaBH(OAc)3 (381 mg, 1.80 mmol), and a catalytic amount of HOAc (2–3 drops), and the mixture was stirred at rt overnight. The reaction mixture was basified with a minimum volume of saturated Na2CO3 solution. H2O and solvent were then removed under vacuum. The residue was further dried under high vacuum, and CHCl3 (20 mL) was added. The mixture was filtered, and the solid was washed with CHCl3 (3 × 20 mL). The filtrate was concentrated to give crude product 13, which was purified by column chromatography, eluting with 15% CMA to afford pure product (200 mg) in 71% yield. [α]D24 −15.6 (c 1.1, MeOH). 1H NMR (400 MHz, CDCl3) δ 9.94 (br s, 1H), 7.00–6.91 (m, 2H), 6.86 (d, J = 7.2 Hz, 1H), 4.10 (dd, J = 12.0, 4.0 Hz, 1H), 3.62 (dd, J = 12.0, 7.2 Hz, 1H), 3.33 (m, 1H), 3.06 (dd, J = 16.0, 4.4 Hz, 1H), 2.82–2.66 (m, 3H), 1.67 (br s, 1H), 1.51 (m, 2H), 0.93 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 155.15, 127.28, 126.24, 121.52, 119.86, 117.54, 107.54, 51.57, 49.25, 43.25, 31.35, 23.37, 11.70. GC-MS (EI) m/z 231 (M+). Anal. (C13H17N3O·1/2H2O) C, H, N.

(R)-5-(Dipropylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (14a)[20]

class="Chemical">Benzyl chloroformate (639 mg, 3.75 mmol) in dry <class="Chemical">span class="Chemical">THF (2 mL) was added dropwise at 0 °C under argon to a solution of 13 (787 mg, 3.41 mmol) and Et3N (1.38 g, 13.6 mmol) in dry THF (10 mL). The reaction was warmed to rt for 3 h after the addition. The mixture was diluted with H2O, and the two layers were separated. The aqueous layer was extracted with CHCl3. The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by column chromatography (eluting with CHCl3/MeOH; 93:7) to afford benzyl (R)-2-oxo-5-(propylamino)-5,6-dihydro-4H-imidazo[4,5,1-ij]quinoline-1(2H)-carboxylate (853 mg, 69%). 1H NMR (400 MHz, CDCl3) δ 7.55–7.50 (m, 3H), 7.42–7.31 (m, 3H), 7.01 (t, J = 7.8 Hz, 2H), 6.96 (m, 1H), 5.48 (s, 2H), 3.99 (ddd, J = 12.8, 4.0, 1.2 Hz, 1H), 3.60 (dd, J = 12.8, 7.0 Hz, 1H), 3.28 (m, 1H), 3.04 (dd, J = 16.0, 4.0 Hz, 1H), 2.80–2.61 (m, 3H), 1.56–1.43 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H). To a solution of this CBz-protected intermediate (683 mg, 1.87 mmol) in DMF (10 mL) were added K2CO3 (516 mg, 3.74 mmol) and n-PrBr (460 mg, 3.74 mmol), and the mixture was heated to 65 °C and stirred overnight. The mixture was then cooled to rt and filtered. The filtrate was concentrated. The residue was diluted with H2O (10 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were dried (MgSO4) and concentrated. The residue was purified by column chromatography (eluting with CHCl3/MeOH; 93:7) to provide benzyl (R)-5-(dipropylamino)-2-oxo-5,6-dihydro-4H-imidazo[4,5,1-ij]quinoline-1(2H)-carboxylate (356 mg) in 46% yield. 1H NMR (400 MHz, CDCl3) δ 7.54–7.50 (m, 3H), 7.42–7.31 (m, 3H), 7.02–6.94 (m, 2H), 5.48 (s, 2H), 4.14 (ddd, J = 12.0, 4.8, 0.8 Hz, 1H), 3.41 (t, J = 11.8 Hz, 1H), 3.24 (m, 1H), 2.96–2.81 (m, 2H), 2.60–2.44 (m, 4H), 1.52–1.40 (m, 4H), 0.89 (t, J = 7.2 Hz, 6H). This intermediate (330 mg, 0.81 mmol) was dissolved in EtOH (10 mL) in a Parr bottle, and Pd/C (10%, 50 mg) was added. The mixture was hydrogenolyzed at an initial pressure of 50 psi for 5 h. The mixture was then filtered over Celite. The filtrate was concentrated, and the residue was purified by column chromatography (eluting with 10% CMA) to give pure product 14a (179 mg) in 81% yield. [α]D22 −4.25 (c 0.6, CHCl3). 1H NMR (400 MHz, CDCl3) δ 10.20 (br, 1H), 6.98–6.91 (m, 2H), 6.85 (d, J = 6.8 Hz, 1H), 4.18 (ddd, J = 11.6, 4.0, 0.8 Hz, 1H), 3.46 (t, J = 11.4 Hz, 1H), 3.31 (m, 1H), 2.97–2.83 (m, 2H), 2.60–2.46 (m, 4H), 1.45 (m, 4H), 0.90 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 155.26, 127.33, 126.27, 121.32, 119.66, 119.11, 107.35, 54.74, 52.81, 40.39, 26.74, 22.30, 11.69. GC-MS (EI) m/z 273 (M+). Anal. (C16H23N3O·0.25 H2O) C, H, N.

(R)-5-(Propylamino)-1-propyl-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (14b)

Compound 13 (60 mg, 0.26 mmol) was dissolved in class="Chemical">DMF (2 mL), and to the solution was added <class="Chemical">span class="Chemical">1-bromopropane (62 mg, 0.52 mmol) and K2CO3 (72 mg, 0.52 mmol). The mixture was heated to 40 °C for 48 h. The mixture was filtered after cooling to rt, and the filtrate was concentrated under vacuum. The residue was purified by preparative TLC, eluting with 5% CMA to afford 14b (55 mg, 78%) as an oil. [α]D24 −14.4 (c 0.55, MeOH). 1H NMR (400 MHz, CDCl3) δ 6.98 (dd, J = 8.0, 7.6 Hz, 1H), 6.85 (dd, J = 7.6, 0.8 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.10 (ddd, J = 12.0, 4.4, 1.2 Hz, 1H), 3.82 (m, 2H), 3.55 (dd, J = 12.0, 8.0 Hz, 1H), 3.28 (m, 1H), 3.05 (dd, J = 15.6, 4.4 Hz, 1H), 2.72 (m, 3H), 1.78 (m, 2H), 1.50 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H), 0.92 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 153.57, 127.82, 126.24, 120.98, 119.55, 117.44, 105.63, 51.61, 49.29, 43.53, 42.93, 31.70, 23.44, 21.97, 11.68, 11.36. GC-MS (EI) m/z 273 (M+). Anal. (C16H23N3O·1/4H2O) C, H, N.

(R)-5-[Methyl(propyl)amino]-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (14c)

Compound class="Chemical">14c was synthesized from <class="Chemical">span class="Chemical">1 and 1-bromopropane in 73% yield using the same procedure as for 14b. [α]D25 −6.6 (c 0.29, MeOH). 1H NMR (400 MHz, CDCl3) δ 10.0 (s, 1H), 6.94 (m, 2H), 6.85 (d, J = 7.2 Hz, 1H), 4.22 (ddd, J = 12.0, 4.4, 1.2 Hz, 1H), 3.53 (dd, J = 12.0 6.6 Hz, 1H), 3.11 (m, 1H), 2.95 (m, 2H), 2.57 (m, 2H), 2.42 (s, 3H), 1.54 (m, 2H), 0.91 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 155.06, 127.35, 126.12, 121.39, 119.69, 118.63, 107.35, 57.40, 56.11, 39.98, 38.22, 27.10, 20.93, 11.70. GC-MS (EI) m/z 245 (M+). Anal. (C14H19N3O·1/2H2O) C, H, N.

(R)-5-(Dipropylamino)-1-propyl-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (15)

To the solution of 13 (52 mg, 0.23 mmol) in class="Chemical">DMF (2 mL) was added <class="Chemical">span class="Chemical">1-bromopropane (138 mg, 1.13 mmol) and K2CO3 (128 mg, 0.92 mmol). The mixture was heated to 65 °C overnight. The mixture was then filtered after cooling to rt, and the filtrate was concentrated under vacuum. The residue was purified by preparative TLC, eluting with 5% CMA to afford 15 (53 mg, 76%). [α]D25 −1.4 (c 0.70, MeOH). 1H NMR (400 MHz, CDCl3) δ 6.97 (dd, J = 7.6, 7.2 Hz, 1H), 6.85 (dd, J = 7.2, 0.8 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 4.17 (ddd, J = 11.6, 4.2, 0.8 Hz, 1H), 3.82 (t, J = 7.4 Hz, 2H), 3.45 (t, J = 11.6 Hz, 1H), 3.29 (m, 1H), 2.90 (m, 2H), 2.53 (m, 4H), 1.78 (m, 2H), 1.62 (br s, 1H), 1.45 (m, 2H), 0.97 (t, J = 7.6 Hz, 3H), 0.89 (t, J = 7.6 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 153.58, 127.78, 126.28, 120.76, 119.43, 118.96, 105.38, 54.65, 52.81, 42.91, 40.47, 27.02, 22.30, 21.96, 11.68, 11.35. GC-MS (EI) m/z 315(M+). Anal. (C19H29N3O) C, H, N.

Benzyl (R)-Methyl(2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)carbamate (16)

class="Chemical">N-(Benzyloxycarbonyloxy)succinimide (386 mg, 1.55 mmol) solution in <class="Chemical">span class="Chemical">THF (10 mL) was added dropwise to the solution of 1 (300 mg, 1.47 mmol) in dry THF (10 mL) at −40 °C under an argon atmosphere. The reaction mixture was slowly warmed to rt and stirred overnight. The reaction mixture was then quenched with 10% sat. NaHCO3 solution (20 mL) and extracted with EtOAc (3 × 10 mL). The organic layers were combined, dried, concentrated, and purified by column chromatography using 60% EtOAc/hexanes as eluent to provide 498 mg (91%) of oily material. 1H NMR (400 MHz, CDCl3) δ 10.23 (br, 1H), 7.36 (m, 5H), 6.95 (m, 2H), 6.84 (d, J = 7.8 Hz, 1H), 5.18 (s, 2H), 4.65 (m, 1H), 4.12 (dd, J = 11.6, 4.8 Hz, 1H), 3.71 (m, 1H), 3.13 (dd, J = 15.6, 11.2 Hz, 1H), 2.86 (s, 3H), 3.00–2.84 (m, 1H).

Benzyl (R)-Methyl(2-oxo-1-propyl-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)carbamate (17a)

class="Chemical">1-Bromopropane (90 mg, 0.73 mmol) was added to the reaction mixture of 16 (122 mg, 0.36 mmol) and <class="Chemical">span class="Chemical">K2CO3 (504 mg, 3.66 mmol) in acetone (5 mL) and stirred at reflux for 20 h. The reaction mixture was filtered, concentrated, and purified using flash chromatography with 10% acetone/CHCl3 as eluent to provide 138 mg (93%) of 17a. [α]D24 +41.4 (c 0.58, MeOH). 1H NMR (400 MHz, CDCl3) δ 7.36 (br, 5H), 6.99 (ddd, J = 7.6, 7.2, 0.8 Hz, 1H), 6.84 (m, 2H), 5.18 (s, 2H), 4.65 (br, 1H), 4.13 (dd, J = 12.0, 4.8 Hz, 1H), 3.82 (t, J = 7.2 Hz, 2H), 3.73 (m, 1H), 3.14 (dd, J = 15.6, 7.2 Hz, 1H), 2.94 (m, 4H), 1.78 (m, 2H), 0.97 (dt, J = 7.6, 0.8 Hz, 3H).

Benzyl (R)-Methyl(2-oxo-1-butyl-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)carbamate (17b)

class="Chemical">NaH (60% in <class="Chemical">span class="Chemical">mineral oil, 30 mg, 0.75 mmol) was washed with hexane (3 × 1 mL) and then suspended in dry THF. To this suspension was added 16 (168 mg, 0.5 mmol) in dry THF, and the mixture was stirred at rt for 30 min. 1-Bromobutane (137 mg, 1.0 mmol) was added, and the mixture was stirred at rt overnight. The mixture was quenched with H2O, extracted with CHCl3, dried (MgSO4), and concentrated. The residue was purified by column chromatography, eluting with hexane/ethyl acetate (1:1) to afford 115 mg (68%) of 17b. 1H NMR (400 MHz, CDCl3) δ 7.33 (br, 5H), 6.97 (m, 1H), 6.83 (m, 2H), 5.18 (s, 2H), 4.63 (br, 1H), 4.13 (dd, J = 12.0, 4.4 Hz, 1H), 3.85 (t, J = 7.6 Hz, 2H), 3.74 (m, 1H), 3.12 (dd, J = 15.6, 7.6 Hz, 1H), 2.93 (m, 4H), 1.74 (m, 2H), 1.40 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H).

(R)-5-(Methylamino)-1-propyl-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (18a)

Compound 17a (100 mg, 0.26 mmol) and class="Chemical">Pd/C (10%, 20 mg) in absolute <class="Chemical">span class="Chemical">EtOH (5 mL) was hydrogenolyzed (50 psi) for 5 h. TLC showed that the reaction was complete. The mixture was filtered over Celite. The filtrate was concentrated, and the residue was purified by preparative TLC, eluting with 5% CMA to provide 58 mg (90%) of 18a. [α]D25 −12.9 (c 1.00, MeOH). 1H NMR (400 MHz, CDCl3) δ 6.98 (dd, J = 7.6, 7.6 Hz, 1H), 6.85 (dd, J = 7.6, 0.8 Hz, 1H), 6.82 (dd, J = 7.6, 0.8 Hz, 1H), 4.06 (ddd, J = 12.0, 4.0, 0.8 Hz, 1H), 3.82 (m, 2H), 3.64 (dd, J = 12.0, 6.8 Hz, 1H), 3.23 (m, 1H), 3.07 (dd, J = 16.0, 4.0 Hz, 1H), 2.78 (dd, J = 16.0, 7.2 Hz, 1H), 2.54 (s, 3H),1.77 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 153.61, 127.83, 126.13, 121.06, 119.62, 117.13, 105.72, 53.30, 42.96, 42.84, 34.06, 31.11, 21.97, 11.37. GC-MS (EI) m/z 245 (M+). Anal. (C14H19N3O·1/4H2O) C, H, N.

(R)-5-(Methylamino)-1-butyl-5,6-dihydro-4H-imidazo[4,5,1-ij]quinolin-2(1H)-one (18b)

Compound 18b was synthesized from 17b in 92% yield using the same procedure as for 18a. [α]D24 −15.9 (c 0.58, class="Chemical">MeOH). 1H NMR (400 MHz, <class="Chemical">span class="Chemical">CDCl3) δ 6.98 (dd, J = 8.0, 7.6 Hz, 1H), 6.85 (dd, J = 7.6, 0.8 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 4.06 (ddd, J = 12.0, 4.0, 1.2 Hz, 1H), 3.85 (m, 2H), 3.63 (dd, J = 12.0, 7.2 Hz, 1H), 3.22 (m, 1H), 3.06 (dd, J = 16.0, 4.2 Hz, 1H), 2.79 (dd, J = 16.0, 7.2 Hz, 1H), 2.54 (s, 3H), 1.73 (m, 2H), 1.40 (m, 2H), 0.95 (t, J = 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 153.57, 127.78, 126.15, 121.04, 119.60, 117.17, 105.69, 53.30, 42.88, 41.10, 34.11, 31.16, 30.74, 20.09, 13.72. GC-MS (EI) m/z 259 (M+). Anal. (C15H21N3O·3/4H2O) C, H, N.

N-(4-Hydroxybutyl)-9H-fluorene-2-carboxamide (20c)

class="Chemical">Thionyl chloride (<class="Chemical">span class="Chemical">SOCl2, 2 mL/mmol) was added to fluorene-2-carboxylic acid (2.32 g, 11.03 mmol).[53] The solution was stirred at reflux for 3 h and concentrated in vacuo. Residual SOCl2 was removed by azeotropic distillation in dry benzene. The resulting solid was dissolved in CHCl3 (5 mL). To a stirred solution of the 1-amino-4-butanol (0.98 g, 11.0 mmol) in CHCl3 (20 mL) and 0.5 M aq sodium hydroxide (8 mL) cooled to 0 °C was added the acid chloride solution dropwise. The solution was stirred vigorously for 3 h at rt. The organic layer was separated, dried with Na2SO4, and concentrated in vacuo. The crude product (2.77 g, 89%) was used in the next step without purification.

N-(4-Bromobutyl)-9H-fluorene-2-carboxamide (21c)

To a suspension of compound 20c (1.50 g, 5.33 mmol) in class="Chemical">acetonitrile were added <class="Chemical">span class="Chemical">triphenylphosphine (2.80 g, 10.7 mmol) and carbon tetrabromide (3.54 g, 10.7 mmol). The yellow solution was stirred overnight at rt. Acetonitrile was evaporated, and the product was purified by column chromatography using 25% EtOAc/hexanes as eluent to give 0.46 g (25%) of 21c. 1H NMR (400 MHz, CDCl3) δ 7.97 (s, 1H), 7.83–7.76 (m, 3H), 7.58 (d, J = 7.6 Hz, 1H), 7.43–7.34 (m, 2H), 6.22 (br s, 1H), 3.94 (s, 2H), 3.56–3.47 (m, 4H), 2.03–1.96 (m, 2H), 1.86–1.79 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 167.99, 145.10, 144.16, 143.65, 140.80, 132.94, 127.84, 127.16, 125.79, 125.37, 123.95, 120.73, 119.90, 39.32, 37.05, 33.49, 30.23, 28.59.

(R)-N-(4-(Methyl(2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)amino)butyl)benzofuran-2-carboxamide (22a)

To a solution of 1 (247 mg, 1.22 mmol) in class="Chemical">DMF (5 mL) were added 21a (360 mg, 1.22 mmol) and <class="Chemical">span class="Chemical">K2CO3 (503 mg, 3.64 mmol), and the mixture was heated for 3 h at 60–65 °C. The solvent was evaporated, and the product was purified by column chromatography using 1% MeOH/CH2Cl2 as eluent to give 79 mg (16%) of 22a. Mp 77–78 °C. [α]D25 +10.66 (c 0.075, MeOH). 1H NMR (400 MHz, CDCl3) δ 10.31 (s, 1H), 7.64 (d, J = 7.6 Hz, 1H), 7.46–7.15 (m, 5H), 6.96–6.81 (m, 3H), 4.19 (dd, J = 11.8, 0.8 Hz, 1H), 3.57–3.47 (m, 3H), 3.26–3.19 (m, 1H), 2.98–2.88 (m, 2H), 2.67–2.59 (m, 2H), 2.40 (s, 3H), 1.74–1.70 (m, 2H), 1.65–1.60 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 159.02, 155.11, 154.72, 149.00, 127.70, 127.34, 126.77, 126.26, 123.67, 122.73, 121.49, 119.73, 118.44, 111.67, 110.27, 107.48, 57.48, 53.55, 39.92, 39.34, 38.19, 27.32, 26.86, 25.29. Anal. (C24H26N4O3·3H2O) for C, H, N.

(R)-N-(4-(Methyl(2-oxo-2,4,5,6-tetrahydro-1H-imidazo[4,5,1-ij]quinolin-5-yl)amino)butyl)-1H-indole-2-carboxamide (22b)

The same procedure employed for 22b was used for 22a (400 mg, 1.36 mmol). The product was eluted with 2% class="Chemical">MeOH/<class="Chemical">span class="Chemical">CH2Cl2 as eluent then repurified by column chromatography using 65% acetone/CHCl3 to give 115 mg (20%) of 22b. Mp 130–131 °C. [α]D25 +8.57 (c 0.075, MeOH). 1H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 10.18 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.06–7.02 (m, 2H), 6.92–6.83 (m, 3H), 6.76 (d, J = 6.8 Hz, 1H), 4.08 (dd, J = 11.8, 4.0 Hz, 1H), 3.48–3.41 (m, 3H), 3.10–3.03 (m, 1H), 2.86–2.74 (m, 2H), 2.53–2.41 (m, 2H), 2.27 (s, 3H), 1.61–1.56 (m, 2H), 1.52–1.47 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 162.26, 155.16, 136.72, 131.14, 127.67, 127.41, 126.21, 124.29, 121.90, 121.56, 120.49, 119.84, 118.57, 112.27, 107.46, 102.65, 57.34, 53.56, 40.25, 39.66, 38.18, 27.34, 26.73, 25.14. Anal. (C24H27N5O2·4H2O) for C, H, N.

(R)-N-(4-(Methyl(2-oxo-1,2,5,6-tetrahydro-4H-imidazo[4,5,1-ij]quinolin-5-yl)amino)butyl)-9H-fluorene-2-carboxamide (22c)

The same procedure employed for 22c was used for 22a (561 mg, 1.63 mmol). The product was eluted with 3% class="Chemical">MeOH/<class="Chemical">span class="Chemical">CH2Cl2 as eluent and then repurified by column chromatography using 65% acetone/CHCl3 to give 184 mg (24%) of 22c. Mp 103–104 °C. [α]D25 +8.69 (c 0.115, MeOH). 1H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 7.94 (s, 1H), 7.79–7.23 (m, 7H), 7.14–7.11 (m, 1H), 6.89–6.72 (m, 3H), 4.05 (dd, J = 11.6, 4.0 Hz), 3.75 (s, 2H), 3.45 (dd, J = 12.4, 6.2 Hz), 3.88–3.33 (m, 1H), 3.10–3.03 (m, 1H), 2.84–2.70 (m, 2H), 2.57–2.45 (m, 2H), 2.27 (s, 3H), 1.66–1.60 (m, 2H), 1.55–1.50 (m, 2H). 13C NMR (100 MHz, CDCl3) δ 168.05, 154.97, 144.50, 143.88, 143.21, 140.55, 133.07, 127.46, 127.16, 126.83, 126.19, 125.82, 125.05, 123.86, 121.35, 120.37, 119.56, 119.46, 118.27, 107.35, 57.28, 53.49, 39.93 (d, J = 10.3 Hz), 37.97, 36.74, 30.89, 27.21, 26.51, 25.19. Anal. (C29H30N4O2·3H2O) C, H, N.

Radioligand Binding Studies

class="Gene">Radioligand binding assays were conducted similarly to methods previously described.[12,54−56] Briefly, <class="Chemical">span class="CellLine">HEK293 cells were stably transfected with either human D2R or human D3R in our laboratory. These cells 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 of hygromycin (Life Technologies, Grand Island, NY) and stored 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) without calcium and with 5 μM 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 20,000 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, and membranes were diluted to 500 μg/mL. For [3H]N-methylspiperone binding studies, the binding buffer (EBSS with calcium) was made from 8.7 g/L of Earle’s Balanced Salts without phenol red (US Biological, Salem, MA) and 2.2 g/L of sodium bicarbonate at pH 7.4; 500 μg/mL membranes were stored at −80 °C for later use. For [3H]7-OH-DPAT binding studies, membranes were harvested fresh; the binding buffer was made from 50 mM Tris, 10 mM MgCl2, and 1 mM EDTA at pH 7.4.

Test compounds were freshly dissolved the class="Chemical">day of the assay in 30% <class="Chemical">span class="Chemical">DMSO and 70% H2O to a stock concentration of 1 mM or 100 μM. For assisting the solubilization of free-base compounds, 10 μL of glacial HOAc was added (in place of 10 μL final H2O volume) along with 100% DMSO initially; the solution was briefly sonicated, and then the solution was brought up to 1 mM or 100 μM final concentration by adding H2O. Each test compound was then diluted into 13 half-log serial dilutions using 30% DMSO vehicle; the final test concentrations ranged from 100 μM to 10 pM.

Membranes were diluted in fresh binding buffer to a 10× concentration: the final concentration of membranes was 10 μg total protein for class="Chemical">[3H]N-methylspiperone binding at <class="Chemical">span class="Gene">D2R or D3R, and 40 or 20 μg total protein for [3H]7-OH-DPAT binding at D2R or D3R, respectively. Radioligands were diluted in binding buffer to a final concentration of 0.4 nM ([3H]N-methylspiperone, PerkinElmer), 1.0 nM ([3H]7-OH-DPAT, ARC, St. Louis, MO) for D2R, or 0.5 nM ([3H]7-OH-DPAT) for D3R. Radioligand competition binding experiments were conducted for 60 min at rt in glass tubes containing 300 μL of fresh appropriate binding buffer containing with 0.2 mM sodium metabisulfite, 50 μL of diluted test compound, 100 μL of membranes ([3H]N-methylspiperone: 10 μg of total protein for D2R or D3R; [3H]7-OH-DPAT: 40 or 20 μg total protein for D2R or D3R, respectively), and 50 μL of radioligand for a final reaction volume of 500 μL diluted in binding buffer ([3H]N-methylspiperone: 0.4 nM final concentration, PerkinElmer, Waltham, MA; [3H]7-OH-DPAT: 1.0 and 0.5 nM final concentration for hD2 and hD3, respectively, ARC, St. Louis, MO). Nonspecific binding was determined in the presence of 10 μM butaclamol (Sigma-Aldrich, St. Louis, MO), and total binding was determined with 30% DMSO vehicle. All compound dilution concentrations were run in triplicate. The reaction was incubated for 1 h at rt. The reactions were terminated by filtration through Whatman GF/B filters, presoaked for 1 h in 0.5% polyethylenimine, using a Brandel R48 filtering manifold (Brandel Instruments, Gaithersburg, MD), and washed. The filters were washed 3 times with 3 mL/wash of ice-cold binding buffer. Filters were transferred to scintillation vials and incubated with 3 mL of CytoScint liquid scintillation cocktail (MP Biomedicals, Solon, OH), and vials were counted using a PerkinElmer Tri-Carb 2910 TR liquid scintillation counter (Waltham, MA).

For both class="Gene">radioligands, one- and two-site models were compared in GraphPad Prism (GraphPad Software, San Diego, CA). One-site binding models were preferred over two-site binding models in an extra sum-of-squares F test; thus, individual IC50 values were determined for each compound via nonlinear regression using only a one-site competition model of dose–reclass="Chemical">sponse curves in GraphPad Prism. Each IC50 value was converted to Ki values using the Cheng–Prusoff equation;[57] Kd values for <class="Chemical">span class="Chemical">[3H]N-methylspiperone (D2R: 0.133 nM, D3R: 0.265 nM) and [3H]7-OH-DPAT (D2R: 2.24 nM, D3R: 1.30 nM) were determined via separate saturation binding curves. Reported Ki values were determined from at least three independent experiments and are reported as mean ± SEM.

BRET-Based Go BRET Assay

The BRET-based Go activation assay was described previously.[33] Briefly, class="CellLine">HEK293T cells were transiently transfected with pcDNA3.1 vectors carrying <class="Chemical">span class="Gene">D2R or D3R, GαoA fused to Renilla luciferase 8 (Rluc8) within in α-helical domain, Gβ1 fused to V1 (the N-terminal split of mVenus; residues 1–155) at its N-terminus, and Gγ2 fused to V2 (the C-terminal split of mVenus; residues 156–240) using polyethylenimine (Polysciences, Inc.). Transfected cells were maintained in culture with DMEM (GIBCO) supplemented with 10% FBS, and transfection media was replaced with fresh media after ∼24 h. Experiments were performed ∼48 h after transfection.

Transfected cells were then washed, harvested, and resuspended in class="Chemical">PBS supplemented with 5 mM <class="Chemical">span class="Chemical">glucose and distributed in 96-well black/white plates (Wallac, PerkinElmer Life and Analytical Sciences). Cells were then incubated with coelenterazine H (5 μM) (Dalton Pharma Services), and after 8 min, compounds were added with final concentrations ranging from 10 pM to 100 μM. After 2 min, the BRET[1] signal was measured using a Pherastar FS (BMG Labtech) and was calculated as the ratio of the light emitted by mVenus (510–540 nm) over that emitted by RLuc8 (485 nm). Data were normalized to vehicle (0%) and dopamine (100%), and nonlinear regression analysis was performed using the sigmoidal dose–response function in GraphPad Prism to generate EC50 values. Data are expressed as a percentage of the maximum dopamine-stimulated response as mean ± SEM.

Mitogenesis Assays

class="Species">Chinese hamster ovary (<class="Chemical">span class="Gene">CHOp) cells expressing the human D2Rs or D3Rs were maintained in α-MEM with 10% FBS, 0.05% pen-strep, and 400 μg/mL of G418. To measure D2 receptor-mediated stimulation of mitogenesis, CHOp-D2 cells were seeded in 96-well plates at a concentration of 5,000 cells/well. The cells were incubated at 37 °C in α-MEM with 10% FBS. After 48–72 h, the cells were rinsed twice with serum-free αMEM and incubated for 24 h at 37 °C. Serial dilutions of test compounds were made in serum-free αMEM using a Biomek robotics workstation. For agonists, the medium was removed and replaced with 100 μL of test compound in serum-free α-MEM. After another 24 h incubation at 37 °C, 0.25 μCi of [3H]thymidine in αMEM supplemented with 10% FCS was added to each well, and the plates were further incubated for 2 h at 37 °C. The cells were trypsinized by the addition of a 10× trypsin solution (1% trypsin in calcium–magnesium-free phosphate-buffered saline); cells were filtered, and radioactivity in cells was determined by scintillation spectrometry.

Identical methods were used to measure class="Chemical">[3H]thymidine incorporation in <class="Chemical">span class="CellLine">CHOp-D3 cells, except that cells were incubated with agonists for 16 h before the assay was terminated. Time-response curves indicated that incubation times longer than 16 h resulted in increased background and agonist EC50 values (i.e., decreased potency) in CHOp-D3 cells (data not shown).

class="Chemical">Data were normalized to <class="Chemical">span class="Chemical">dopamine (100%), and nonlinear regression analysis was performed using the sigmoidal dose–response function in GraphPad Prism to generate EC50 values. Data are expressed as a percentage of the maximum dopamine-stimulated response as mean ± SEM.

Molecular Modeling and Simulations

The binding modes of compound 1 in class="Gene">D2R and <class="Chemical">span class="Chemical">D3R were predicted by computational docking and molecular dynamics (MD) simulations. The ligand was docked to equilibrated models of D2R and D3R, which were built based on the D3R crystal structure.[32,33,48] The N-terminal segment was predicted de novo, and a truncated poly-Gly segment was replaced for ICL3. Docking was performed using an induced-fit docking protocol in the Schrödinger software (release 2013–3; Schrödinger, LLC: New York, NY). For both “up” and “down” orientations of the imidazolinone moiety, the best IFDScore docking pose was selected to perform the MD simulations. The binding modes of 13 and 15 in D2R and D3R were predicted by docking to the representative frames from the MD simulations of 1 in D2R and D3R.

The MD simulations were performed in the explicit class="Chemical">water–POPC <class="Chemical">span class="Chemical">lipid bilayer solvent environment using Desmond Molecular Dynamics System (version 3.8; D. E. Shaw Research, New York, NY) with the CHARMM36 protein force field,[58−60] the CHARMM36 lipid force field,[61] and TIP3P water model. The ligand parameters were obtained from the GAAMP server[62] with the initial force field based on CGenFF with ParamChem.[63] The protonation state of compound 1 at pH 7.0 was predicted by the Epik program in the Schrödinger software. The system charges were neutralized, and a solvent concentration of 0.15 M NaCl was added. The Na+ binding site at the highly conserved Asp2.50 is known to collapse upon receptor activation.[64] Because compound 1 is an agonist, the active-state-like conformation of the receptor was modeled without a Na+ ion bound at this site. The system was initially minimized and equilibrated with restraints on the ligand heavy atoms and protein backbone atoms followed by a production stage of 600 ns with all atoms unrestrained. For D2R, a second set of trajectories (600 ns for each pose) was collected and reached convergence with the first set.

The MM/GBSA ligand–receptor binding energy was calculated using CHARMM[65] (version c36a2) with the GBSW implicit solvent model.[66] For each frame being considered, the protein and ligand components were extracted and then minimized with restraints on all heavy atoms except for the side chains within 4 Å of the ligand before the energies were calculated.