Structural manipulation of aporphines via C10 nitrogenation leads to the identification of new 5-HT_7AR ligands

Abstract

Aporphine alkaloids containing a C10 nitrogen motif were synthesized and evaluated for affinity at 5-HT_1AR, 5-HT_2AR, 5-HT₆R and 5-HT_7AR. Three series of racemic aporphines were investigated: 1,2,10-trisubstituted, C10 N-monosubstituted and compounds containing a C10 benzofused aminothiazole moiety. The 1,2,10-trisubstituted series of compounds as a group displayed modest selectivity for 5-HT_7AR and also had moderate 5-HT_7AR affinity. Compounds from the C10 N-monosubstituted series generally lacked affinity for 5-HT_2AR and 5-HT₆R and showed strong affinity for 5-HT_1A or 5-HT_7AR. Compounds in this series that contained an N6-methyl group were up to 27-fold selective for 5-HT_7AR over 5-HT_1AR, whereas compounds with an N6-propyl substituent showed a reversal in this selectivity. The C10 benzofused aminothiazole analogues showed a similar binding profile as the C10 N-monosubstituted series i.e. strong affinity for 5-HT_1AR or 5-HT_7AR, with selectivity between the two receptors being similarly influenced by N6-methyl or N6-propyl substituents. Compounds 29 and 34a exhibit high 5-HT_7AR affinity, excellent selectivity versus dopamine receptors and function as antagonists in 5-HT_7AR cAMP-based assays. Compounds 29 and 34a have been identified as new lead molecules for further tool and pharmaceutical optimization.

Graphical Abstract

1. Introduction

The neurotransmitter serotonin (5-HT) is involved in numerous physiological functions. Seven 5-HT receptors types are known (5-HT₁R – 5-HT₇R) of which all except 5-HT₃R (a ligand-gated ion channel) are metabotropic G-protein coupled receptors (GPCRs). In all, there are at least 14 5-HT receptors owing to the occurrence of sub-types for some of the receptors. The 5-HT₇R is the most recently described serotonergic receptor [1–3] and it is involved in various pathophysiological functions such as regulation of circadian rhythm, body temperature and sleep. [4–6] There are four known 5-HT₇R isoforms: 5-HT_7AR, 5-HT_7BR and 5-HT_7CR isoforms are found in rats whereas 5-HT_7AR, 5-HT_7BR and 5-HT_7DR are present in humans [7]. The 5-HT_7AR is a full-length receptor comprising 445 amino acid residues, while the 5-HT_7BR is a truncated variant with 432 amino acid residues. The 5-HT_7DR is a distinct isoform containing 479 amino acid residues. The 5-HT_7AR is the most abundant of the human isoforms. 5-HT₇Rs signal via G_αs; agonist activation of 5-HT₇Rs leads to adenylate cyclase activation and cAMP production. [8]

5-HT₇R antagonists appear to be useful agents to remediate neuropsychiatric conditions including obsessive-compulsive disorder, schizophrenia, anxiety, depression and substance abuse. [9–12] Agonists of the 5-HT₇R are able to restore deficient locomotory activity and may be useful as therapeutics for spinal cord injury, amyotrophic lateral sclerosis and sleep apnea. The role of 5-HT₇R in the regulation of nociception, mitogenesis, gastrointestinal disorders, cardiopulmonary disorders and cognition has been less clear as both agonists and antagonists have shown positive outcomes in animal studies.[11]

A number of scaffolds have yielded potent 5-HT₇R ligands.[9] Among these are the so-called “long chain” arylpiperazines (e.g. 1, Fig. 1), arylsulfonamides (e.g. 2), biarylalkylamines (e.g. 3), azepines (e.g. 4) and aminotriazines (e.g. 5). However, selectivity of 5-HT₇R ligands especially versus the closely related 5-HT_1A receptor has been challenging. The 5-HT₇R antagonist JNJ-18038683 (4) is currently undergoing clinical trials for the treatment of cognitive impairment in bipolar disorder.[9] JNJ-1803863 is the only selective 5-HT₇R ligand that has been evaluated in clinical trials. The discovery of new potent and selective 5-HT₇R ligands promises to expand our armamentarium of useful pharmacological tools and drugs pertinent to various neuropsychiatric disorders. Thus, the identification of new 5-HT₇R ligands remains an area of current interest.

Figure 1.

Structures of typical 5-HT₇R ligands with “long chain” arylpiperazine (1), arylsulfonamide (2), biarylalkylamine (3), azepine (4, JNJ-18038683) and aminotriazine (5) scaffolds.

Aporphine alkaloids are known to have affinity across a range of serotonergic and dopaminergic receptors. In particular, structure-affinity relationship studies on aporphines have resulted in the identification of potent and selective ligands for serotonin 5-HT_1AR [13–16] and dopamine D₂R [17, 18] as well as compounds with interesting multi-receptor profiles [19–22]. In contrast to their evaluation as 5-HT_1A receptor ligands, the evaluation of aporphines as 5-HT₇R ligands has been little studied. In that regard, Johannson et al have described a number of atropisomeric 11-phenyl aporphines and 1,11-methylene aporphines (e.g. compounds 6 and 7, Fig. 2) with 5-HT₇R receptor potency and selectivity versus the 5-HT_1AR. [23, 24] Recently we conducted an SAR study on the aporphine alkaloid N- methyllaurotetanine and identified a number of compounds with moderate 5-HT₇R affinity and selectivity. (e.g. compound 8, Figure 2). [25]

Figure 2.

Structures of aporphine 5-HT7 receptor ligands

We regard the aporphine template as a privileged scaffold for the discovery of new CNS receptor targeting agents and envisage that structural modification of the aporphine core may lead to ligands that selectively target individual or multiple CNS receptors. Although numerous SAR studies have been performed on aporphines in relation to their affinity and activity at CNS receptors, there have been no previous SAR studies examining the effect of a C-10 nitrogen substituent in this context. Thus, in this exploratory SAR study, we were curious to determine the extent to which C10 nitrogen substituents would impact affinity and selectivity of the scaffold across a range of serotonin receptors, including 5-HT₇R. In this manuscript we describe the synthesis and pharmacological evaluation of novel aporphines that bear a C10 nitrogen substituent group at a subset of 5-HT receptors which has led to the identification of novel selective 5-HT₇R ligands. In addition, we describe computational docking experiments that shed light on the observed 5-HT₇R affinity of the newly identified ligands.

2. Results and discussion

Synthesis

As mentioned earlier in this study we sought to examine compounds that contain 1,2,10-trisubstituted and 10-substituted patterns on the racemic aporphine core. Compounds with 9,10- and 10,11- benzo-fused aminothiazole moieties were also targeted for synthesis and evaluation as these represented cyclized C10 nitrogen motifs. Additionally, we were interested in examining the impacts of N-methyl and N-propyl variations on 5-HT₇R affinity and selectivity of the aporphines as such N-substituent groups have featured prominently in the selectivity of aporphines at other CNS receptor targets. [17, 18, 26, 27]

The synthesis of compounds bearing 1,2,10-trisubstituted and 10-monosubstituted patterns required the preparation of the corresponding C10 aniline substrates. These aniline precursors were prepared as we have previously described (in a study evaluating the activity of C10 nitro-, amide- and aniline-substituted aporphine analogues at dopamine receptors).[28] Synthesis of the 1,2,10-trisubstituted analogues was accomplished by treatment of the aniline precursors 9a-e with various carboxylic anhydrides, methanesulfonic anhydride and potassium cyanate to give the corresponding amides (11-14), sulfonamides (15-18) and urea analogues (19-21) respectively (Scheme 1). Reductive methylation of aniline precursors gave the N,N-dimethylated analogues (22-24). In a similar fashion to synthesis of the 1,2,10-trisubstituted analogues, aniline precursors 10a and 10b were transformed into the corresponding 10-monosubstituted amide, sulfonamide, urea and N,N-dimethylated analogues 25–26, 27–28, 29–30 and 31–32 respectively.

Scheme 1. Reagents and conditions:

(a) corresponding anhydride, triethylamine, DCM, 2h-14h, rt, 37–73%; (b) methanesulfonic anhydride, triethylamine, DCM, rt, 14h, 33–48%; (c) potassium cyanate, acetic acid, water, 1h, rt, 30–86%; (d) formaldehyde, NaBH(OAc)₃, DCM, rt, 18h, 30–63%

Compounds 10a and 10b were reacted with potassium thiocyanate and bromine to afford the C10/C11 benzofused aminothiazole derivatives 33a and 33b and their respective C9/C10 regioisomers 34a and 34b (Scheme 2). In each case, the pair of regioisomers was present in an approximately 1:1 ratio after the reaction (as determined by ¹H NMR). The tedious purification of each pair of regioisomers was accomplished by prep TLC. The 9/10 regioisomers were slightly more polar than their corresponding 10/11 congeners.

Scheme 2. Reagents and conditions:

KSCN, Br₂, acetic acid, 0 °C - rt, 12h, 30–35%

Biological Evaluation

Table 1 summarizes data from evaluation of compounds in the 1,2,10-trisubstituted series of compounds. As a group, the analogues in Table 1 showed low affinity for the 5-HT receptors evaluated. The highest affinity observed at the 5-HT_1AR was for compound 16 (385 nM). Compounds with C1 methoxy groups in tandem with C10 methanesulfonamide or urea groups were poorly tolerated for affinity at the 5-HT_1AR (i.e. compounds 15 and 19 – no affinity in a primary assay). Compounds with a C10 butanamide group (e.g. 11, 12b, 13, 14) or a C10 methanesulfonamide group (e.g. 16-18) show slightly better affinity for the 5-HT_1AR than compounds with a C10 urea motif (e.g. 19-21). A similar situation ensued at the 5-HT_2AR as was evident from the lack of affinity of compounds 15 and 19 for the 5-HT_2AR. For compounds with C10 amide, sulfonamide or urea groups, C1 allyl or cyclopropylmethyl groups are better tolerated than C1 propargyloxy groups (e.g. compare 13 versus 14; 16 versus 18; 20 versus 21). Whereas a C1 methyl group was not beneficial for 5-HT_1AR and 5-HT_2AR affinity, affinity for the 5-HT₆R was maintained (e.g. compound 15, 86 nM at 5-HT₆R; no affinity at 5-HT_1AR and 5-HT_2AR). Although the affinities of the compounds for the 5-HT_7AR was moderate, in most cases individual compounds displayed higher affinity for the 5-HT_7AR than for the other receptors tested indicating selectivity of the group of compounds on a whole for the 5-HT_7AR.

Table 1.

Affinity of 1,2,10-trisubstituted analogues at 5-HT receptors

				Ki (nM)a
Cmpd. #	R1	R2	R3	5-HT1ARb	5-HT2ARc	5-HT6Rd	5-HT7ARe
9c		H	H	naf	110±14	132±17	104±13
11	Me		H	469±61	417±54	356±46	67±8.6
12a			H	1540±200	974±130	1500±190	636±82
12b			H	429±55	665±86	322±42	395±51
13			H	463±60	443±57	198±26	188±24
14			H	661±85	1038±130	354±46	455±59
15	Me		H	na	na	86±11	89±11
16			H	385±50	433±56	505±65	114±15
17			H	403±52	668±86	590±76	42±5.4
18			H	511±66	1331±170	218±28	31±4.0
19	Me		H	na	na	415±54	289±37
20			H	1280±170	857±110	527±68	353±46
21			H	911±120	na	410±53	123±16
22	Me	Me	Me	1570±200	295±38	127±16	73±9.4
23		Me	Me	1440±190	878±110	329±42	752±97
24		Me	Me	na	318±41	102±13	309±40
8-OH DPAT				0.8 ±0.01
Clozapine					3.0±0.4	4.0±0.5	11.4±1.5

^aExperiments carried out in triplicate;

^b[3H]WAY100635 used as radioligand;

^c[3H]Ketanserin used as radioligand;

^d[3H]LSD used as radioligand;

^e[3H]LSD used as radioligand;

^fna – not active (compounds displayed < 50% inhibition in a primary assay)

Data for evaluation of the C10 monosubstituted analogues are compiled in Table 2. As a group the compounds display stronger affinity for 5-HT_1AR and 5-HT_7AR than for 5-HT_2AR and 5-HT₆R. In fact, compound 31 was the only compound in this series to display affinity for 5-HT_2AR and 5-HT₆R (this being albeit fairly weak affinity). In this series, the compounds with a C10 amide motif seem to prefer binding to 5-HT_1AR over 5-HT_7AR (e.g. 25 and 26). The N-propyl-containing compound 25 showed good 5-HT_1AR affinity (K_i = 21 nM) and was selective over all other 5-HT receptors tested (no affinity at other 5-HT receptors). The C10 sulfonamide-containing compound 28 which also bears an N-propyl group, also showed selectivity for 5-HT_1AR over 5-HT_7AR; the C10 urea-containing N-propyl analogue 30 similarly displayed 5-HT_1AR versus 5-HT_7AR selectivity. Overall, it appears that irrespective of the C10 functionality, an N-propyl group is more favored for binding to the 5-HT_1AR than 5-HT_7AR in this series (e.g. compare the N-propyl analogues 25, 26, 28, 30 and 32 with the N-methyl analogues 27, 29 and 31). The compound with the highest 5-HT_7AR affinity in this series was compound 29 (K_i = 4.5 nM); 29 is 10- fold selective for 5-HT_7AR versus 5-HT_1AR and is among the most potent aporphinoid 5-HT₇R ligands identified to date.

Table 2.

Affinity of 10-monosubstituted analogues at 5-HT receptors

				Ki (nM)a
Cmpd #	R1	R2	R3	5-HT1ARb	5-HT2ARc	5-HT6Rd	5-HT7ARd	clogPe
10b	Pr	H	H	30±3.8	naf	na	63±8.2	3.6
25	Pr		H	21±2.7	na	na	na	3.8
26	Pr		H	15±2.0	na	na	311±40	4.9
27	Me		H	133±17	na	na	27±3.5	2.6
28	Pr		H	23±3.0	na	na	199±26	3.6
29	Me		H	49±6.3	na	na	4.5±0.6	2.8
30	Pr		H	236±30	na	na	na	3.8
31	Me	Me	Me	458±59	566±73	1588±200	17±2.2	3.9
32	Pr	Me	Me	256±33	na	na	984±130	5.0
8-OH DPAT				0.8 ±0.01
Clozapine					3.0±0.4	4.0±0.5	11.4±1.5

^aExperiments carried out in triplicate;

^b[3H]WAY100635 used as radioligand;

^c[3H]Ketanserin used as radioligand;

^d[3H]LSD used as radioligand;

^ecalculated with ChemDraw Professional v. 16.0.1.4;

^fna – not active (compounds displayed < 50% inhibition in a primary assay)

Data for evaluation of the benzofused aminothiazole analogues is shown in Table 3. As with the 10-monosubstituted derivatives, the aminothiazole analogues showed stronger affinity for 5-HT_1AR and 5-HT_7AR than for 5-HT_2AR and 5-HT₆R. The compounds all lacked affinity for the 5-HT₆R. Another similarity in the SAR of the benzofused aminothiazole series with the C10-monosubstituted series is that an N6-methyl moiety confers 5-HT_7AR selectivity versus 5-HT_1AR, whereas an N6-propyl moiety is preferred for 5-HT_1AR selectivity. Thus, the N-methyl analogues 33a and 34a are approximately 19- and 4-fold selective respectively for 5-HT_7AR, whereas the N-propyl analogues 33b and 34b are roughly 2-and 17-fold more selective respectively for the 5-HT_1AR.

Table 3.

Affinity of aminothiazole analogues at 5-HT receptors

		Ki (nM)a
Cmpd #	R1	5-HT1ARb	5-HT2ARc	5-HT6Rd	5-HT7ARd	clogPe
33a	Me	187±24	1040±130	naf	9.9±1.3	3.5
33b	Pr	93±12	na	na	150±19	4.5
34a	Me	23±3.0	na	na	6.5±0.8	3.5
34b	Pr	7.5±0.97	na	na	132±17	4.5
8-OH DPAT		0.8 ±0.01
Clozapine			3.0±0.4	4.0±0.5	11.4±1.5

^aExperiments carried out in triplicate;

^b[3H]WAY100635 used as radioligand;

^c[3H]Ketanserin used as radioligand;

^d[3H]LSD used as radioligand;

^ecalculated with ChemDraw Professional v. 16.0.1.4;

^fna – not active (compounds displayed < 50% inhibition in a primary assay)

The compounds with the highest 5-HT_7AR and 5-HT_1A affinity (29, 33a, 34a and 34b) were further profiled for affinity at dopamine receptors (D₁R - D₅R). Table 4 shows these results. Compound 29 lacked affinity for all receptors except D₃R, where low affinity was seen (K_i = 2460 nM). Compound 33a showed low affinity for D₁R, D₂R, D₃R and D₄R (K_i = 1150, 1740, 381 and 481 nM respectively) and had no affinity for D₅R. Compound 34a lacked affinity for all dopamine receptors. Similar dopamine receptor binding studies on the 5-HT_1AR selective compound 34b revealed a lack of affinity for D₁R, D₂R, D₄R and D₅R; low affinity (K_i = 2110 nM) was observed for D₃R

Table 4.

Affinity of 29, 33a, 34a and 34b at dopamine receptors

	Ki (nM)a
Cmpd #	D1Rb	D2Rc	D3Rc	D4Rc	D5R
29	nad	na	2460±320	na	na
33a	115 0±150	1740±220	381±49	481±62	na
34a	na	na	na	na	na
34b	na	na	2110±270	na	na
(+)-butaclamol	4.30±0.55
Haloperidol		5.58±0.72
Nemonapride			1.18±0.15	0.86±0.11

^aExperiments carried out in triplicate;

^b[3H]SCH23390 used as radioligand;

^c[3H]N-Methylspiperone used as radioligand;

^dna – not active (compounds displayed < 50% inhibition in a primary assay)

Compounds 29 and 34a were evaluated for 5-HT₇R functional activity using Hit Hunter® agonist and antagonist cAMP assays at Eurofins Pharma Discovery Services. Data from these assays is provided in Table 5. Both compounds were found to be 5-HT₇R antagonists with EC₅₀ values of 0.125 and 0.26 μM respectively for 29 and 34a, which was comparable to the positive control spiperone; the compounds lacked agonist activity in this assay.

Table 5.

Functional activity of compounds 29 and 34a in cAMP assays

Compound	Assay mode	Agonista EC50(μM)	Antagonista IC50 (μM)
29	agonist	>10	-
29	antagonist	-	0.125
34a	agonist	>10	-
34a	antagonist	-	0.26
Serotonin	agonist	0.07	-
Spiperone.HCl	antagonist	-	0.31

^aExperiments performed in duplicate

Docking studies

Computational docking simulations were performed in order to rationalize the high measured 5-HT₇R binding affinities for compounds 29, 33a and 34a. In this context, we investigated the docked ligand poses and identified key receptor-ligand interactions that influence binding to the 5-HT₇R and provide a deeper appreciation of the observed high affinity towards this receptor system. A homology model of the 5-HT₇R was generated from the high-resolution crystal structure of the human serotonin 5-HT_1B G protein-coupled receptor with pdb code 4IAQ [29]. This approach involved utilization of the Schrödinger Prime Structure Prediction and Glide software modules and manual intervention to support the generation of known key receptor-ligand interactions. The model for the 5-HT₇R structure, therefore, comprised suitable backbone and side-chain orientations within the binding site. The docking simulations of compounds 29, 33a and 34a into the 5-HT₇R binding site exploited the Schrödinger Induced Fit and Glide methodologies in Standard Precision (SP) mode. Using this docking protocol, the Glidescore scoring function was used to give an estimate of the ligand binding affinities for the highest ranked poses of compounds 29, 33a and 34a in the 5-HT₇R target. The ligand binding poses are depicted in Figure 3.

Figure 3.

Docked poses of A. compound 29 (with binding energy of −8.9 kcal/mol), B. compound 33a (with binding energy of −8.7 kcal/mol) and C. compound 34a (with binding energy of −10.0 kcal/mol) shown by green carbon atoms in the serotonin 5-HT7 receptor target shown by secondary structure elements and grey carbon atoms for select residues. Key quaternary N – Asp salt bridges are depicted by the pink dashed lines, H-bonding interactions by the yellow dashed lines, π-π stacking by the blue dashed lines and corresponding π-cation interactions by the green dashed lines.

Compounds 29 and 34a give very similar binding poses in the serotonin 5-HT₇R binding pocket as shown in Figures 3A and C. The bound structures with these ligands comprise the key salt bridge between the quaternary N atom and Asp162, hydrogen bonding interactions to the Ser243 side chain and Ala247 backbone and π-stacking interactions between the ligand aromatic rings and receptor Phe343 and Phe352 residues. Compound 34a gives a better predicted binding energy (−10.0 kcal/mol) compared to 29 (−8.9 kcal/mol), due to a slightly stronger protonated N-Asp salt bridge and better π-π interactions involving the fused thiazole ring in 34a.

For compound 33a, in order to accommodate the modified position of the fused thiazole ring, the best binding pose identified by induced fit docking involves a refined 5-HT7 receptor structure with a rotated Asp162 side-chain orientation. As depicted in Figure 3B, key receptor-ligand interactions include the quaternary N – Asp162 salt bridge, H-bonds to Ser234 and Ser243 and a π-cation interaction between the same quaternary N and Phe352. As a consequence of the modified binding pose, the binding energy for compound 33a (−8.7 kcal/mol) is worse than that for 34a (−10.0kcal/mol) primarily due to the lack of complementary π-π interactions involving the ligand aromatic rings in 33a.

3. Conclusions

In this study, we sought to evaluate the extent to which C10 nitrogen substituents on the aporphine core impact affinity and selectivity across a range of 5-HT receptors, including 5-HT_7AR. Aporphines with 1,2,10-trisubstituted and 10-substituted substitution patterns as well as ring D aminothiazole moieties were investigated.

We found that affinities of the 1,2,10-trisubstituted analogues across the four receptors evaluated (5-HT_1AR, 5-HT_2AR, 5-HT₆R and 5-HT_7AR) was low to moderate; C10 N-substitution here did not result in pronounced high affinity and selectivity for any of the 5-HT receptors, although there was generally a slight preference for 5-HT₇R binding.

In contrast, the C10 monosubstituted group of compounds as well as the benzofused aminothiazole group of compounds were found to selectively target 5-HT_1AR and 5-HT_7AR and largely lacked affinity for 5-HT_2AR and 5-HT₆R. Taken together with the results from the trisubstituted series, it would appear that C1 and C2 alkoxy substituents on the aporphine core are not required for affinity to 5-HT_7AR and 5-HT_1AR. Furthermore, the SAR results indicate that in general, an N6-methyl substituent engenders 5-HT_7AR selectivity over 5-HT_1AR whereas this selectivity is reversed with an N6-propyl substituent.

Overall, our study provides valuable SAR information by revealing that the inclusion of a C10 nitrogen substituent group on the ring A unsubstituted aporphine core, allows for selective binding of the scaffold to 5-HT₇R and 5-HT_1AR. Compounds 29 and 34a were found to function as 5-HT₇R antagonists in assays that measured cAMP production. These compounds do not display any appreciable affinity for dopamine receptors, have moderate selectivity versus 5-HT_1AR and represent good starting points from which to further optimize C10 nitrogenated aporphines as selective 5-HT₇R antagonists. The impact of the configuration of the chiral center of the aporphines on affinity and selectivity will need to be investigated in future. Optimization of the ligands for 5-HT₇R affinity is expected to be challenging given the 5-HT_1AR affinity of the series, but is nevertheless a promising dimension for future studies.

4. Experimental

4.1. Synthetic experimental procedures

General Procedures:

All glass apparatus was oven-dried prior to use. HRESIMS spectra were obtained using an Agilent 6520 Q-TOF instrument. ¹H NMR and ¹³C NMR spectra were recorded using a Bruker DPX-500 spectrometer (operating at 500 and 600 MHz for ¹H; 125 and 150 MHz, respectively, for ¹³C) using CDCl₃ as solvent. Tetramethylsilane (δ 0.00 ppm) served as an internal standard in ¹H NMR and ¹³C NMR unless stated otherwise. Chemical shift (δ 0.00 ppm) values are reported in parts per million and coupling constants in Hertz (Hz). Splitting patterns are described as singlet (s), doublet (d), triplet (t), and multiplet (m). Reactions were monitored by TLC with Whatman Flexible TLC silica gel G/UV 254 precoated plates (0.25 mm). TLC plates were visualized in UV light (254 nm) and by staining with phosphomolybdate spray reagent, vanillin or iodine. Flash column chromatography was performed with silica gel 60 (EMD Chemicals, 230–400 mesh, 0.04–0.063 mm particle size). Preparative thin layer chromatography was performed with silica gel GF plates (Analtech, catalog # 02003). All chemicals and reagents were obtained from Sigma-Aldrich and Fischer Scientific (USA) in reagent grade and were used without further purification. Yields reported are after purification.

General procedure for synthesis of amide analogues:

To a stirred solution of the corresponding aniline (0.2 mmol) in DCM (10 mL) was added the respective anhydride (0.4 mmol) and the resulting reaction mixture was stirred at rt for 2 h under nitrogen atmosphere. After completion of the reaction (monitored by TLC) the reaction mixture was concentrated in-vacuo and a solution of NaOH (0.2 g) in methanol (10 mL) was added and stirred for another 30 min at the same temperature. The reaction mixture was concentrated in-vacuo and the obtained crude mass was dissolved in water and extracted with DCM (3×10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated in-vacuo to give crude product, which was purified by preparative TLC purification eluting in 5% MeOH/DCM.

N-(1,2-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)butyramide (11)

37% yield. Yellow oil. ¹H NMR (600 MHz, CDCl₃): δ 8.25 (d, J = 8.6 Hz, 1H), 7.66 (s, 1H), 7.28 (s, 1H), 7.18 – 7.13 (m, 1H), 6.54 (s, 1H), 3.81 (s, 3H), 3.56 (s, 3H), 3.12 – 3.05 (m, 1H), 3.02 (m, 1H), 2.99 – 2.92 (m, 2H), 2.61 (m, 1H), 2.54 (m, 1H), 2.48 – 2.41 (m, 4H), 2.29 (t, J = 7.4 Hz, 2H), 1.74 – 1.67 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 171.4, 152.0, 144.8, 137.6, 136.9, 128.9, 128.8, 128.1, 127.5, 126.6, 119.0, 117.9, 110.8, 62.2, 60.2, 55.8, 53.3, 44.0, 39.8, 35.2, 29.2, 19.2, 13.8 ppm. HRESIMS m/z 381.2173 [M+H]⁺ (calcd. for C₂₃H₂₈N₂O₃, 381.2177)

N-(1-(cyclopropylmethoxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)acetamide (12a).

48% yield. Dark yellow oil. ¹H NMR (600 MHz, CDCl₃): δ 8.29 (d, J = 8.6 Hz, 1H), 7.62 – 7.51 (m, 1H), 7.45 (s, 1H), 7.20 (d, J = 8.4 Hz, 1H), 6.45 (s, 1H), 3.72 (s, 3H), 3.55 (m, 1H), 3.25 (m, 1H), 3.18 – 3.07 (m, 2H), 3.07 – 2.99 (m, 1H), 2.98 – 2.91 (m, 1H), 2.64 – 2.52 (m, 3H), 2.48 (s, 3H), 2.05 (s, 3H), 0.98 (m, 1H), 0.34 – 0.27 (m, 2H), 0.03 – −0.07 (m, 2H) ppm.¹³C NMR (125 MHz, CDCl₃): δ 168.3, 152.4, 143.9, 136.9, 136.9, 129.6, 128.4, 128.4, 128.0, 127.1, 118.9, 117.8, 110.8, 77.8, 62.2, 55.8, 53.2, 43.4, 34.8, 29.7, 24.8, 11.0, 3.4, 3.1 ppm. HRESIMS m/z 393.2179 [M+H]⁺ (calcd. for C₂₄H₂₈N₂O_3, 393.2170)

N-(1-(cyclopropylmethoxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)butyramide (12b)

40% yield. White solid. Melting point: 89.1 °C – 90.1 °C. ¹H NMR (500 MHz, CDCl₃): δ 8.31 (d, J = 8.6 Hz, 1H), 7.57 (s, 1H), 7.20 (s, 1H), 7.11 (dd, J = 8.5, 1.8 Hz, 1H), 6.46 (s, 1H), 3.72 (s, 3H), 3.56 (m, 1H), 3.25 (m, 1H), 3.08 – 2.98 (m, 1H), 2.98 – 2.87 (m, 3H), 2.55 (m, 1H), 2.48 (m, 1H), 2.43 – 2.36 (m, 4H), 2.23 (t, J = 7.4 Hz, 2H), 1.65 (m, 2H), 1.03 – 0.94 (m, 1H), 0.89 (t, J = 7.4 Hz, 3H), 0.30 (m, 2H), −0.02 (m, 2H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 171.4, 152.1, 143.7, 137.4, 136.8, 129.6, 128.5, 128.4, 127.2, 127.1, 118.9, 117.6, 110.8, 77.8, 62.2, 55.8, 53.3, 43.9, 39.8, 35.2, 29.1, 19.1, 13.8, 11.0, 3.4, 3.0 ppm. HRESIMS m/z 421.2490. [M+H]⁺ (calcd. for C₂₆H₃₃N₂O₃, 421.2486)

N-(1-(allyloxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)butyramide (13)

55% yield. Brown solid. Melting point: 99.1 °C – 99.6 °C. ¹H NMR (500 MHz, CDCl₃): δ 8.27 (d, J = 8.5 Hz, 1H), 7.65 (s, 1H), 7.18–7.10 (m, 2H), 6.53 (s, 1H), 5.88 (m, 1H), 5.16 (m, 1H), 5.05 (m, 1H), 4.28 (m, 1H), 4.09 (m, 1H), 3.79 (s, 3H), 3.10 (s, 1H), 3.05 – 2.93 (m, 3H), 2.61 (m, 1H), 2.54 (m, 1H), 2.45 (m, 4H), 2.29 (t, J = 7.4 Hz, 2H), 1.71 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃) δ 171.3, 152.1, 143.3, 137.5, 136.9, 134.2, 129.3, 128.7, 128.7, 128.2, 127.0, 118.9, 117.6, 117.5, 110.8, 73.7, 62.2, 55.9, 53.3, 43.9, 39.8, 35.1, 29.1, 19.2, 13.8 ppm. HRESIMS m/z 407.2329 [M+H]⁺ (calcd. for C₂₅H₃₀N₂O₃, 407.2177)

N-(2-methoxy-6-methyl-1-(prop-2-yn-1-yloxy)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)butyramide (14)

52% yield. Yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 8.27 (d, J = 8.6 Hz, 1H), 7.67 (s, 1H), 7.19 (s, 1H), 7.14 (d, J = 8.5 Hz, 1H), 6.54 (s, 1H), 4.46 (dd, J = 15.0, 2.3 Hz, 1H), 4.33 (dd, J = 15.0, 2.4 Hz, 1H), 3.80 (s, 3H), 3.16 – 3.06 (m, 1H), 3.05 – 2.95 (m, 3H), 2.62 (m, 1H), 2.55 (m, 1H), 2.51 – 2.43 (m, 4H), 2.31 – 2.25 (m, 3H), 1.72 (m, 2H), 0.95 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 171.3, 152.0, 142.3, 137.5, 137.1, 129.4, 128.0, 127.3, 127.2, 118.9, 117.8, 110.9, 79.2, 74.9, 62.1, 59.6, 55.9, 53.2, 43.8, 39.8, 35.1, 29.0, 19.1, 13.8 ppm. HRESIMS m/z 405.2173 [M+H]⁺ (calcd. for C₂₅H₂₈N₂O₃, 405.2177)

N-(6-propyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)acetamide (25)

73% yield. Off-white solid. Melting point: 99.8 °C – 100.3 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.56 (d, J = 8.4 Hz, 1H), 7.51 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.30 (m, 1H), 7.22 (t, J = 11.5 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 7.5 Hz, 1H), 3.44 (m, 1H), 3.18 – 3.09 (m, 2H), 3.07 (m, 1H), 2.87 (m, 1H), 2.72 – 2.67 (m, 1H), 2.63 (m, 1H), 2.52 – 2.40 (m, 2H), 2.11 (s, 3H), 1.63 – 1.48 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm.¹³C NMR (125 MHz, CDCl₃): δ 168.4, 137.3, 136.1, 133.6, 133.3, 130.4, 127.7, 127.0, 124.3, 121.6, 119.5, 119.4, 118.7, 59.3, 56.2, 49.4, 33.9, 28.9, 24.7, 18.9, 12.0 ppm. HRESIMS m/z 329.1964 [M+H]⁺ (calcd. for C₂₁H₂₅N₂O, 321.1961)

N-(6-propyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)butyramide (26)

50% yield. Pale yellow solid. Melting point: 104.9 °C – 106.5 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.66 (d, J = 7.4 Hz, 2H), 7.51 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 7.0 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.07 (d, J = 7.4 Hz, 1H), 3.61 – 3.50 (m, 1H), 3.31 – 3.15 (m, 3H), 3.01 – 2.92 (m, 1H), 2.84 – 2.71 (m, 2H), 2.66 – 2.52 (m, 2H), 2.37 (t, J = 7.4 Hz, 2H), 1.80 (m, 2H), 1.74 – 1.59 (m, 2H), 1.05 (t, J = 7.3 Hz, 3H), 1.00 (t, J = 7.3 Hz, 3H) ppm.¹³C NMR (150 MHz, CDCl₃): δ 171.5, 137.3, 136.2, 133.5, 133.2, 133.1, 130.4, 127.7, 126.9, 124.4, 121.5, 119.6, 118.6, 61.9, 53.5, 53.4, 43.9, 39.7, 34.2, 29.7, 28.9, 19.1, 13.8 ppm. HRESIMS m/z 349.2279 [M+H]⁺ (calcd. for C₂₃H₂₉N₂0, 349.2274)

General procedure for preparation of methanesulfonamide analogues:

To a stirred solution of corresponding aniline (0.1 mmol) in DCM (10 mL), methanesulfonic anhydride (0.25 mmol) and triethylamine (0.3 mmol) were added. The resulting mixture was stirred at rt for 12h under nitrogen atmosphere. After completion of the reaction, the reaction mixture was concentrated in-vacuo and a solution of NaOH (0.2 g) in methanol (10 mL) was added to the flask and stirring was continued for another 30 min. The reaction mixture was concentrated in-vacuo, dissolved in water and extracted with DCM (3×10 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated in-vacuo. The crude product thus obtained was purified using preparative TLC eluting in 5% MeOH/DCM to give respective sulfonamide analogues.

N-(1,2-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (15)

45% yield. Off-white solid. Melting point: 153.0 °C – 154.6 °C. ¹H NMR (600 MHz, CDCl₃): δ 8.26 (d, J = 8.5 Hz, 1H), 7.09 (s, 1H), 7.05 – 7.02 (m, 1H), 6.55 (s, 1H), 3.80 (s, 3H), 3.59 (s, 3H), 3.13 – 3.04 (m, 1H), 3.03 – 2.93 (m, 6H), 2.64 – 2.59 (m, 1H), 2.52 (m, 1H), 2.48 – 2.41 (m, 4H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 152.0, 144.9, 138.3, 135.6, 129.7, 129.3, 128.8, 127.3, 126.1, 119.6, 118.7, 111.3, 62.0, 60.2, 55.8, 53.2, 43.9, 39.6, 35.1, 29.1 ppm. HRESIMS m/z 389.1529 [M+H]⁺ (calcd. for C₂₀H₂₅N₂O₄S, 389.4895)

N-(1-(cyclopropylmethoxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (16)

46% yield. Brown solid. Melting point: 140.3 °C – 141.5 °C. ¹H NMR (500 MHz, CDCl₃): δ 8.41 (d, J = 8.5 Hz, 1H), 7.18 (s, 1H), 7.08 (s, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.53 (s, 1H), 3.78 (s, 3H), 3.63 (m, 1H), 3.34 (m, 1H), 3.16 – 3.06 (m, 2H), 3.06 – 2.97 (m, 5H), 2.63 (m, 2H), 2.58 – 2.44 (m, 4H), 1.08 – 0.99 (m, 1H), 0.37 (d, J = 8.1 Hz, 2H), 0.10 – −0.03 (m, 2H) ppm. ¹³C NMR (125 MHz, CDCl₃) δ 152.2, 143.8, 137.9, 135.4, 130.4, 129.7, 128.7, 128.1, 126.6, 119.4, 118.4, 111.1, 77.9, 62.0, 55.8, 53.5, 53.2, 39.6, 35.0, 11.0, 3.4, 3.1 ppm.

N-(1-(allyloxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (17)

33% yield. Light-yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 8.30 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.5, 2.3 Hz, 1H), 6.55 (s, 1H), 5.88 (ddt, J = 16.4, 10.4, 5.9 Hz, 1H), 5.17 (dd, J = 17.2, 1.5 Hz, 1H), 5.06 (dd, J = 10.4, 1.2 Hz, 1H), 4.31 (dd, J = 12.0, 5.9 Hz, 1H), 4.12 (dd, J = 12.0, 5.9 Hz, 1H), 3.80 (s, 3H), 3.14 – 3.07 (m, 1H), 3.07 – 2.95 (m, 6H), 2.63 (dd, J = 16.3, 3.1 Hz, 1H), 2.55 – 2.44 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 152.2, 143.5, 138.2, 135.5, 134.2, 130.1, 129.4, 128.8, 127.1, 126.5, 119.4, 118.4, 117.6, 111.3, 73.8, 62.0, 55.9, 53.2, 43.8, 39.6, 35.0, 29.0 ppm. HRESIMS m/z 415.1690 [M+H]+ (calcd. for C₂₂H₂₆N₂O₄S, 415.1686)

N-(2-methoxy-6-methyl-1-(prop-2-yn-1-yloxy)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (18)

33% yield. Yellow oil. ¹H NMR (500 MHz, CDCl₃): δ 8.29 (d, J = 8.5 Hz, 1H), 7.10 (d, J = 1.9 Hz, 1H), 7.05 (dd, J = 8.5, 2.2 Hz, 1H), 6.55 (s, 1H), 4.50 (m, 1H), 4.37 (m, 1H), 3.80 (s, 3H), 3.12 – 3.04 (m, 1H), 3.02 – 2.92 (m, 6H), 2.62 (m, 1H), 2.49 (m, 5H), 2.26 (t, J = 2.4 Hz, 1H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 152.0, 142.3, 138.3, 135.7, 130.1, 129.6, 129.3, 127.4, 126.9, 119.5, 118.7, 111.2, 79.1, 75.01, 62.0, 59.8, 55.9, 53.2, 44.0, 39.5, 35.1, 29.1 ppm. HRESIMS m/z 413.1532 [M+H]+ (calcd. for C₂₂H₂₄N₂O₄S, 413.1530)

N-(6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (27)

48% yield. Light brown oil. ¹H NMR (500 MHz, CDCl₃): δ 7.61 (d, J = 8.1 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.21 – 7.17 (m, 1H), 7.12 – 7.07 (m, J = 8.5 Hz, 2H), 7.02 (d, J = 7.6 Hz, 1H), 3.18 (m, 2H), 3.14 – 3.02 (m, 2H), 2.99 (s, 3H), 2.74 – 2.67 (m, 1H), 2.63 (m, 1H), 2.56 – 2.47 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 135.9, 135.0, 132.6, 132.1, 131.7, 130.6, 127.2, 126.2, 124.1, 120.7, 119.5, 118.6, 60.8, 52.4, 42.9, 38.5, 33.0, 27.8 ppm. HRESIMS m/z 329.1322 [M+H^]+ (calcd. for C₁₈H₂₁N₂O₂S, 329.1318)

N-(6-propyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)methanesulfonamide (28)

38% yield. Dark brown oil. ¹H NMR (500 MHz, CDCl₃): δ 7.61 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 7.7 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 7.10 (s, 1H), 7.07 (dd, J = 8.3, 2.1 Hz, 1H), 7.01 (d, J = 7.6 Hz, 1H), 3.39 (dd, J = 14.0, 3.8 Hz, 1H), 3.17 – 3.05 (m, 3H), 3.00 (s, 3H), 2.86 (m, 1H), 2.69 (m, 1H), 2.59 (m, 1H), 2.47 – 2.35 (m, 2H), 1.63 – 1.48 (m, 2H), 0.91 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 136.2, 134.9, 133.0, 131.9, 130.9, 127.7, 127.2, 126.0, 124.1, 120.7, 119.4, 118.6, 58.2, 55.4, 48.4, 38.5, 33.2, 28.1, 18.3, 11.1 ppm. HRESIMS m/z 357.1636 [M+H]⁺ (calcd. for C₂₀H₂₅N₂O₂S, 357.1631)

General procedure for synthesis of urea analogues:

To a magnetically stirred solution of amine (100 mg, 1 equiv.) in 10% acetic acid solution (10 mL) (v/v), a solution of potassium cyanate (2 equiv.) in water (5 mL) was added. The reaction was stirred at rt for 2h. After completion of the reaction (monitored by TLC), the reaction was quenched with saturated sodium bicarbonate and extracted with EtOAc (3×15 mL). The combined organic layer was dried over Na₂SO₄, filtered, concentrated under reduced pressure and the crude product thus obtained was purified using preparative TLC, eluting in 5% MeOH/DCM to give the respective urea analogues.

1-(1,2-dimethoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)urea(19):

40 % yield. Brown solid. Melting point: 208 °C – 209 °C. ¹H NMR (600 MHz, CDCl₃): δ 8.15 (d, J = 8.5 Hz, 1H), 7.53 (s, 1H), 7.22 (s, 1H), 7.13 (d, J = 8.3 Hz, 1H), 6.50 (s, 1H), 5.12 (bs, 2H), 3.77 (s, 3H), 3.53 (s, 3H), 3.15 – 3.06 (m, 1H), 3.00 (m, 2H), 2.93 (m, 1H), 2.59 (m, 1H), 2.53 (m, 1H), 2.45 (m, 4H). ¹³C NMR (150 MHz, CDCl₃): δ 156.8, 152.2, 144.8, 137.9, 137.1, 129.1, 128.3, 127.2, 126.6, 126.3, 119.5, 118.8, 110.8, 62.0, 60.2, 55.8, 53.0, 43.3, 34.7, 28.5 ppm. HRESIMS m/z 354.1811 [M+H]+ (calcd. for C₂₀H₂₄N₃O₃, 354.4295)

1-(1-(cyclopropylmethoxy)-2-methoxy-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)urea (20):

30 % yield. Brown oil. ¹H NMR (600 MHz, CDCl₃): δ 8.26 (d, J = 8.5 Hz, 1H), 8.22 (s, 1H), 7.28 – 7.22 (m, 1H), 6.46 (s, 1H), 5.48 (bs, 2H), 3.75 (s, 3H), 3.57 – 3.52 (m, 1H), 3.34 – 3.28 (m, 1H), 3.22 – 3.08 (m, 3H), 2.94 (m, 1H), 2.62 (m, 3H), 2.54 (s, 3H), 1.01 (m, 1H), 0.34 (d, J = 6.5 Hz, 2H), 0.07 – −0.04 (m, 2H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 157.1, 152.6, 143.7, 138.49, 136.1, 129.5, 128.6, 127.2, 126.6, 124.6, 118.7, 118.0, 110.4, 77.8, 61.9, 55.8, 52.8, 42.4, 34.1, 27.7, 11.0, 3.4, 3.2 ppm. HRESIMS m/z 394.2123 [M+H]⁺ (calcd. for C₂₃H₂₈N₃O₃, 394.4945).

1-(2-methoxy-6-methyl-1-(prop-2-yn-1-yloxy)-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)urea (21):

30 % yield. Light brown solid. Melting point: 172.4 – 172.8 °C. ¹H NMR (600 MHz, Acetone): δ 8.25 (d, J = 8.6 Hz, 1H), 8.09 (s, 1H), 7.55 (s, 1H), 7.32 (d, J = 8.3 Hz, 1H), 6.70 (s, 1H), 5.44 (bs, 2H), 4.60 (m, 1H), 4.44 (m, 1H), 3.86 (s, 3H), 3.08 – 3.04 (m, 2H), 2.98 (m, 1H), 2.87 – 2.83 (m, 2H), 2.64 (m, 1H), 2.47 (s, 3H), 2.41 (m, 2H) ppm. ¹³C NMR (150 MHz, Acetone): δ 155.7, 152.1, 142.1, 139.9, 137.5, 129.6, 128.9, 127.7, 127.4, 125.6, 117.3, 116.1, 110.8, 79.5, 75.3, 62.6, 59.6, 59.1, 55.3, 53.1, 43.4, 35.3 ppm. HRESIMS m/z 378.1819 [M+H]+ (calcd. for C₂₂H₂₄N₃O₃, 378.4515).

1-(6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)urea (29)

83% yield. White solid. Melting point: 125.6 °C – 126.9 °C. ¹H NMR (500 MHz, CDCl₃): δ 7.56 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 7.5 Hz, 1H), 7.34 (bs, 1H), 7.26 (s, 1H), 7.20 (m, 2H), 7.04 (d, J = 7.5 Hz, 1H), 5.12 (bs, 2H), 3.25 – 3.16 (m, 2H), 3.08 (m, 2H), 2.74 (m, 1H), 2.65 (m, 1H), 2.54 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 156.7, 137.9, 136.0, 133.1, 133.0, 132.5, 130.0, 127.7, 127.2, 124.6, 121.5, 120.6, 119.9, 61.7, 53.3, 43.5, 33.8, 28.6 ppm. HRESIMS m/z 294.1604 [M+H]⁺ (calcd. for C₁₈H₁₉N₃O, 294.1601)

1-(6-propyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-yl)urea (30)

86% yield. Brown Solid. Melting point: 114.5 °C – 114.9 °C. ¹H NMR (600 MHz, Acetone-d₆) δ 8.26 (dd, J = 2.2, 1.0 Hz, 1H), 8.20 (ddd, J = 8.6, 2.5, 0.9 Hz, 1H), 8.05 (d, J = 8.6 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.34 (t, J = 7.7 Hz, 1H), 7.23 (d, J = 7.6 Hz, 1H), 3.60 – 3.55 (m, 1H), 3.44 (m, 1H), 3.24 (m, 1H), 3.08 (m, 1H), 3.02 (m, 1H), 2.90 (bs, 2H), 2.79 (m, 1H), 2.62 (m, 1H), 2.50 – 2.42 (m, 2H), 1.69 – 1.56 (m, 2H), 0.99 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (125 MHz, Acetone-d₆): δ 147.7, 141.8, 138.4, 136.5, 135.6, 132.5, 130.8, 128.0, 125.6, 124.3, 123.9, 123.3, 59.9, 56.8, 50.0, 34.6, 30.1, 20.5, 12.2 ppm. HRESIMS m/z 322.1922 [M+H]⁺ (calcd. for C₂₀H₂₄N₃O, 322.4315)

General procedure for synthesis of N,N-dimethylated analogues:

To a stirred solution of the corresponding aniline (0.1 mmol) in DCM (10 mL) was added formaldehyde (37% aqueous solution, 5 μL, 0.1 mmol) and the reaction mixture was stirred at room temperature for 1h. NaBH(OAc)₃ (200 mg, 1 mmol) was added and the resulting reaction mixture was stirred at rt for 12h. After completion of the reaction (monitored by TLC) the reaction mixture was quenched with saturated sodium bicarbonate solution and extracted with DCM (3×15 mL). The combined organic layer was dried over Na₂SO₄, filtered and concentrated in-vacuo. The residue thus obtained was purified using preparative TLC eluting in 5% MeOH/DCM to give the N-methylated analogues.

1,2-dimethoxy-N,N,6-trimethyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-amine (22)

30% yield. Light purple solid. Melting point: 150.1 °C – 150.9 °C. ¹H NMR (600 MHz, CDCl₃): δ 8.20 (d, J = 8.8 Hz, 1H), 6.62 (dd, J = 8.8, 2.7 Hz, 1H), 6.54 (d, J = 2.5 Hz, 1H), 6.46 (s, 1H), 3.80 (s, 3H), 3.58 (s, 3H), 3.15 – 3.07 (m, 1H), 3.05 – 2.95 (m, d H), 2.94 (s, 6H), 2.65 – 2.57 (m, 2H), 2.50–2.52 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 152.0, 149.6, 144.2, 137.7, 129.4, 128.5, 127.5, 127.0, 120.6, 111.3, 110.9, 109.5, 62.6, 60.0, 55.8, 53.4, 44.1, 40.4, 35.9, 29.3 ppm. HRESIMS m/z 339.2065 [M+H]+ (calcd. for C₂₁H₂₇N₂O₂, 339.2067)

1-(cyclopropylmethoxy)-2-methoxy-N,N,6-trimethyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-amine (23)

40% yield. Orange oil. ¹H NMR (500 MHz, CDCl₃): δ 8.24 (d, J = 8.8 Hz, 1H), 6.53 (dd, J = 8.8, 2.7 Hz, 1H), 6.44 (d, J = 2.6 Hz, 1H), 6.35 (s, 1H), 3.69 (s, 3H), 3.48 (m, 1H), 3.27 (m, 1H), 3.03 – 2.96 (m, 1H), 2.90 – 2.86 (m, 3H), 2.84 (s, 6H), 2.53 – 2.45 (m, 2H), 2.39 (s, 3H), 2.39 – 2.33 (m, 1H), 1.07 – 0.98 (m, 1H), 0.34 – 0.27 (m, 2H), 0.03 – −0.04 (m, 2H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 152.1, 149.5, 143.1, 137.5, 129.9, 128.3, 127.9, 126.9, 121.0, 111.2, 110.7, 109.5, 77.4, 62.6, 55.8, 53.4, 44.0, 40.4, 35.9, 29.2, 11.1, 3.3, 3.1 ppm. HRESIMS m/z 379.2378 [M+H]+ (calcd. for C₂₄H₃₁N₂O₂, 379.2380)

1-(allyloxy)-2-methoxy-N,N,6-trimethyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-amine (24)

48% yield. Brown oil. ¹H NMR (600 MHz, CDCl₃): δ 8.22 (d, J = 8.8 Hz, 1H), 6.59 (dd, J = 8.8, 2.6 Hz, 1H), 6.53 (d, J = 2.2 Hz, 1H), 6.45 (s, 1H), 5.94 (ddt, J = 10.7, 5.8 Hz, 1H), 5.19 (dd, J = 17.2, 1.4 Hz, 1H), 5.06 (d, J = 10.4 Hz, 1H), 4.25 (dd, J = 12.0, 5.9 Hz, 1H), 4.14 (dd, J = 12.0, 5.7 Hz, 1H), 3.78 (s, 3H), 3.14 – 3.06 (m, 1H), 3.03 – 2.95 (m, 3H), 2.92 (s, 6H), 2.58 – 2.62 (m, 2H), 2.48 (m, 4H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 152.2, 149.6, 142.9, 137.4, 134.6, 129.7, 128.3, 127.9, 126.6, 120.7, 117.2, 111.3, 110.8, 109.6, 73.4, 62.5, 55.8, 53.3, 43.8, 40.4, 35.7, 29.0 ppm. HRESIMS m/z 365.2222 [M+H]+ (calcd. for C₂₃H₂₉N₂O₂, 365.2224)

N,N,6-trimethyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-amine (31)

63% yield. Brown oil. ¹H NMR (500 MHz, CDCl₃): δ 7.52 (d, J = 8.6 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.12 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.5 Hz, 1H), 6.61 (dd, J = 8.6, 2.6 Hz, 1H), 6.54 (d, J = 2.2 Hz, 1H), 3.17 – 3.08 (m, 2H), 3.07 – 3.02 (m, 1H), 3.02 – 2.96 (m, 1H), 2.91 (s, 6H), 2.69 – 2.65 (m, 1H), 2.63 (m, 1H), 2.50 (s, 3H), 2.46 (m, 1H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 150.1, 136.4, 134.1, 133.3, 132.6, 126.8, 126.3, 124.8, 123.1, 120.6, 111.9, 111.5, 62.4, 53.6, 44.2, 40.6, 35.0, 29.2 ppm. HRESIMS m/z 279.1861 [M+H]⁺ (calcd. for C₁₉H₂₃N₂, 279.1856)

N,N-dimethyl-6-propyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinolin-10-amine (32)

62%. Light brown solid. Melting point: 164.5 °C – 165.8 °C. ¹H NMR (500 MHz, CDCl₃): δ 7.63 (d, J = 8.6 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.00 (d, J = 7.5 Hz, 1H), 6.73 (dd, J = 8.6, 2.6 Hz, 1H), 6.66 (d, J = 2.4 Hz, 1H), 3.59 – 3.51 (m, 1H), 3.29 – 3.22 (m, 1H), 3.20 – 3.12 (m, 2H), 3.02 (s, 6H), 3.01 – 2.95 (m, 1H), 2.80 (s, 1H), 2.76 (s, 1H), 2.58 (m, 2H), 1.77 – 1.58 (m, 2H), 1.01 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 150.1, 136.6, 134.3, 133.6, 133.1, 126.7, 126.3, 124.8, 123.2, 120.6, 111.9, 111.5, 59.6, 56.4, 49.4, 40.6, 34.9, 29.2, 19.2, 12.2 ppm. HRESIMS m/z 307.2168 [M+H]⁺ (calcd. for C₂₁H₂₇N₂, 307.2169)

General Procedure for synthesis of benzofused aminothiazole analogues - compounds 33–34 (a-b)

To a magnetically stirred solution of respective aniline (100 mg, 1 equiv.) in acetic acid was added potassium thiocyanate (2 equiv.) at 0 °C. This was followed by addition of a solution of bromine (1.2 equiv, vol?.) in acetic acid (1 mL) in a dropwise manner. The resulting mixture was allowed to warm to rt and stirred at the same temperature for 12h. After completion of the reaction, the reaction mixture was concentrated in-vacuo, treated with saturated sodium bicarbonate solution and extracted with EtOAc (3×20 mL). The combined organic layer was dried using anhydrous Na₂SO₄ and concentrated in-vacuo. The crude compounds thus obtained were purified by preparative TLC using 5% MeOH/DCM.

7-methyl-6a,7,8,9-tetrahydro-6H-benzo[de]thiazolo[4’,5’:5,6]benzo[1,2-g]quinolin-2-amine (33a):

30% yield. Orange solid. Melting point: 215 °C – 216 °C. ¹H NMR (500 MHz, CDCl₃): δ 7.65 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.22 – 7.16 (m, 1H), 6.99 (d, J = 7.3 Hz, 1H), 5.30 (bs, 2H), 3.29 (d, J = 10.4 Hz, 1H), 3.20 – 3.12 (m, 1H), 3.08 – 2.97 (m, 2H), 2.78 (m, 1H), 2.70 (m, 1H), 2.61 – 2.47 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 165.6, 151.8, 133.6, 133.5, 132.4, 131.8, 128.5, 128.1, 127.6, 127.1, 122.3, 121.8, 118.2, 61.6, 53.4, 44.1, 34.2, 29.0 ppm. HRESIMS m/z 308.1219 [M+H]⁺ (calcd. for C₁₈H₁₈N₃S, 308.1216)

6-methyl-5,6,6a,7-tetrahydro-4H-benzo[de]thiazolo[5’,4’:4,5]benzo[1,2-g]quinolin-10-amine (33b):

32% yield. Light yellow oil. ¹H NMR (600 MHz, CDCl₃): δ 7.80 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.32 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 5.86 (s, 2H), 3.41 (d, J = 11.8 Hz, 1H), 3.13 (dd, J = 13.8, 4.1 Hz, 2H), 3.11 – 3.03 (m, 1H), 2.88 (m, 1H), 2.67 (m, 2H), 2.49 – 2.38 (m, 2H), 1.55 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 166.8, 151.5, 134.1, 134.0, 133.9, 133.7, 130.6, 129.5, 127.6, 126.8, 121.6, 118.4, 116.2, 59.5, 56.3, 49.3, 34.5, 29.1, 19.2, 12.2 ppm. HRESIMS m/z 336.1582 [M+H]⁺ (calcd. for C₂₀H₂₂N₃S, 336.1529)

7-propyl-6a,7,8,9-tetrahydro-6H-benzo[de]thiazolo[4’,5’:5,6]benzo[1,2-g]quinolin-2-amine (34a):

35% yield. Brownish-white solid. Melting point: 198.1 °C – 199.5 °C. ¹H NMR (500 MHz, CDCl₃): δ 7.84 (s, 1H), 7.45 (d, J = 7.7 Hz, 1H), 7.34 (s, 1H), 7.17 (t, J = 7.6 Hz, 1H), 6.99 (d, J = 7.5 Hz, 1H), 5.50 (bs, 2H), 3.17 (m, 3H), 3.03 (m, 1H), 2.69 (m, 2H), 2.52 – 2.49 (m, 4H) ppm. ¹³C NMR (125 MHz, CDCl₃): δ 166.4, 151.6, 133.9, 133.6, 133.5, 133.5, 130.9, 129.4, 127.6, 127.0, 121.6, 118.6, 116.3, 62.2, 53.4, 44.0, 34.6, 29.1 ppm. HRESIMS m/z 308.1219 [M+H]⁺ (calcd. for C₁₈H₁₈N₃S, 308.1216)

6-propyl-5,6,6a,7-tetrahydro-4H-benzo[de]thiazolo[5’,4’:4,5]benzo[1,2-g]quinolin-10-amine (34b):

35% yield. Off-yellow solid. Melting point: 156.6 °C – 157.5 °C. ¹H NMR (600 MHz, CDCl₃): δ 7.80 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.32 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 7.5 Hz, 1H), 5.86 (s, 2H), 3.41 (d, J = 11.8 Hz, 1H), 3.17 – 3.11 (m, 2H), 3.07 (m, 1H), 2.88 (m, 1H), 2.66 (m, 2H), 2.49 – 2.38 (m, 2H), 1.55 (m, 2H), 0.90 (t, J = 7.4 Hz, 3H) ppm. ¹³C NMR (150 MHz, CDCl₃): δ 166.8, 151.5, 134.1, 134.0, 133.9, 133.7, 130.6, 129.5, 127.6, 126.8, 121.6, 118.4, 116.2, 59.5, 56.3, 49.3, 34.5, 29.1, 19.2, 12.2 ppm. HRESIMS m/z 336.1534 [M+H]⁺ (calcd. for C₂₀H₂₂N₃S, 336.1529)

4.2. Biological assay procedures

4.2.1. Receptor binding assays

All receptor binding assays were performed by the Psychoactive Drug Screening Program (PDSP). Complete details of the assays performed may be found online in the PDSP assay protocol book. (http://pdsp.med.unc.edu/PDSP%20Protocols%20II%202013-03-28.pdf).

4.2.2. 5-HT₇R Hit Hunter® cAMP assays

Cell Handling

cAMP Hunter cell lines were expanded from freezer stocks according to standard procedures. Cells were seeded in a total volume of 20 μL in white-walled 384-well microplates and incubated at 37 °C for the appropriate time prior to testing. cAMP modulation was determined using the DiscoverX Hit Hunter cAMP XS+ assay

Gs agonist format

For agonist determination, cells were incubated with sample to induce response. Media was aspirated from cells and replaced with 15 μL 2:1 HBSS/10 mM Hepes: cAMP XS + Ab reagent. Intermediate dilution of sample stocks was performed to generate 4X sample in assay buffer. 5 μL of 4X sample was added to cells and incubated at 37 °C or room temperature for 30 or 60 minutes. Vehicle concentration was 1%.

Gs antagonist format

For antagonist determination, cells were pre-incubated with sample followed by agonist challenge at the EC₈₀ concentration. Media was aspirated from cells and replaced with 10 μL 1:1 HBSS/HEPES:cAMP XS + Ab reagent. 5 μL of 4X compound was added to the cells and incubated at 37 °C or room temperature for 30 minutes. 5 μL of 4X EC₈₀ agonist was added to the cells and incubated at 37 °C or room temperature for 30 or 60 minutes

Signal detection

After appropriate compound incubation, assay signal was generated through incubation with 20 μL cAMP XS + ED/CL lysis cocktail for one hour followed by incubation with 20 μL cAMP XS + EA reagent for three hours at room temperature. Microplates were read following signal generation with a Perkin-Elmer Envision TM instrument for chemiluminescent signal detection.

Data analysis

Compound activity was analyzed using CBIS data analysis suite (ChemInnovation, CA). For Gs agonist mode assays, percentage activity is calculated using the following formula: %Activity = 100% × (mean RLU of test sample – mean RLU of vehicle control)/(mean RLU of MAX control – mean RLU of vehicle control). For Gs antagonist mode, percentage inhibition is calculated using the following formula: %Inhibition =100%×(1-(mean RLU of test sample – mean RLU of vehicle control)/(mean RLU of EC80 control – mean RLU of vehicle control)).