1-BENZYLSPIRO[PIPERIDINE-4,1’-PYRIDO[3,4-B]INDOLE] ‘CO-POTENTIATORS’ FOR MINIMAL FUNCTION CFTR MUTANTS

Jung-Ho Son; Puay-Wah Phuan; Jie S Zhu; Elena Lipman; Amy Cheung; Ka Yi Tsui; Dean J Tantillo; Alan S Verkman; Peter M Haggie; Mark J Kurth

doi:10.1016/j.ejmech.2020.112888

. Author manuscript; available in PMC: 2022 Jan 1.

Published in final edited form as: Eur J Med Chem. 2020 Oct 8;209:112888. doi: 10.1016/j.ejmech.2020.112888

1-BENZYLSPIRO[PIPERIDINE-4,1’-PYRIDO[3,4-B]INDOLE] ‘CO-POTENTIATORS’ FOR MINIMAL FUNCTION CFTR MUTANTS

Jung-Ho Son ^†,¹, Puay-Wah Phuan ^‡,¹, Jie S Zhu ^†,², Elena Lipman ^†, Amy Cheung ^†, Ka Yi Tsui ^†, Dean J Tantillo ^†, Alan S Verkman ^‡, Peter M Haggie ^‡,^*, Mark J Kurth ^†,^*

PMCID: PMC7744356 NIHMSID: NIHMS1636025 PMID: 33092904

Abstract

We previously identified a spiro[piperidine-4,1-pyrido[3,4-b]indole] class of co-potentiators that function in synergy with existing CFTR potentiators such as VX-770 or GLGP1837 to restore channel activity of a defined subset of minimal function cystic fibrosis transmembrane conductance regulator (CFTR) mutants. Here, structure-activity studies were conducted to improve their potency over the previously identified compound, 20 (originally termed CP-A01). Targeted synthesis of 37 spiro[piperidine-4,1-pyrido[3,4-b]indoles] was generally accomplished using versatile two or three step reaction protocols with each step having high efficiency. Structure-activity relationship studies established that analog 2i, with 6’-methoxyindole and 2,4,5-trifluorobenzyl substituents, had the greatest potency for activation of N1303K-CFTR, with EC₅₀ ~600 nM representing an ~17-fold improvement over the original compound identified in a small molecule screen.

Keywords: Cystic Fibrosis, CFTR, Modulator, Potentiator, N1303K-CFTR, c.3700A>G

Graphical Abstract

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INTRODUCTION:

Cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapy to restore defective folding and channel gating of CFTR mutants has been a major recent focus in cystic fibrosis (CF) drug development. CFTR modulators include ‘potentiators’, which restore channel gating, and ‘correctors’, which rescue misfolded CFTR to improve cell surface presentation [1]. Recently, a ‘triple combination’ therapy containing two correctors (tezacaftor / VX-661 and elexacaftor / VX-445) and ivacaftor (VX-770) was approved for CF subjects with at least one F508del-CFTR mutation [2–4]. With available potentiators and correctors it is anticipated that up to 90 % of CF subjects may have an efficacious CFTR modulator therapy [5].

There remains an unmet need to develop therapeutics for the remaining ~10% of CF subjects [5, 6]. We have described a novel approach that involves two potentiators, with the second potentiator called a ‘co-potentiator’, that function in synergy to increase chloride conductance for CFTR mutations found mainly in nucleotide binding domain 2 [7–9]. Recent high-throughput screening in cells expressing N1303K-CFTR identified four novel co-potentiator scaffolds, including spiro[piperidine-4,1-pyrido[3,4-b]indoles] and pyrazoloquinolines, some having sub-micromolar potency for activation of N1303K-CFTR when used together with VX-770 [9]. Herein, synthetic chemistry and structure-activity relationship studies were done to advance the spiro[piperidine-4,1-pyrido[3,4-b]indole] class of co-potentiators.

RESULTS:

Chemistry: General synthesis of spiro[piperidine-4,1-pyrido[3,4-b]indoles].

A synthetic scheme to enable investigation of co-potentiator structure-activity relationships (target analogs 1-6) was devised as outlined in Scheme #1 for the efficient synthesis and modification of the spiro[piperidine-4,1’-pyrido[3,4-b]indole] scaffold (9). Adapting technology developed by Mokrosz [10], commercial tryptamines (8) were condensed with N-alkylated (11 → 12; R²-X in CH₂Cl₂ + K₂CO₃) [11, 12] piperidin-4-ones (12 where n = 1) to directly deliver the targeted spiro[piperidine-4,1’-pyrido[3,4-b]indole] 9 by a classical Pictet–Spengler reaction [13, 14]. However, when preparing R² analogs of 9, it proved more efficient to prepare unalkylated analog 10 [acetic acid-mediated condensation of tryptamine 8 with piperidin-4-one (11; n = 1)] and then subsequently perform selective N¹-alkylation of 10 (R²-X in CH₂Cl₂ + K₂CO₃) to give 9. While the tryptamines employed in this chemistry were commercially available, these starting materials can be readily prepared in two steps by the Büchi/Mak method [15, 16] — 1H-indole nitro-olefination followed by exhaustive reduction with LiAlH₄ (7 → 8).

Scheme #1. — Synthetic route to spiro[piperidine-4,1’-pyrido[3,4-b]indole] analogs 1–6.

Figure 1 shows the synthesized analogs of the original hit spiro[piperidine-4,1’-pyrido[3,4-b]indole] 20 that were prepared in six rounds of synthetic study (synthesis rounds r1 through r6). The co-potentiator activities (tested in an N1303K-CFTR expressing FRT cell model) of the synthesized analogs of 20 (10 μM/V_MAX 100%) are summarized at the end of the chemistry description in Table 3, with further description of biological activity provided below (in the Biology: Characterization of spiro[piperidine-4,1-pyrido[3,4-b]indoles] co-potentiators section).

Table 3.

Bioassay results.

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Biology: Characterization of spiro[piperidine-4,1-pyrido[3,4-b]indoles] co-potentiators

The synthetic work began by preparing aryl analogs 1a-j (synthesis round r1) to investigate the benzylic aryl moiety by appropriately N¹-alkylating spiro[piperidine-4,1’-pyrido[3,4-b]indole] 10. Of these first ten compounds, nine (1b-j) had a better EC₅₀ than 20 and two (1c and 1j) showed improved V_max (Table 3). The EC₅₀ and V_MAX of the N¹-3-chloro-2,4-difluorobenzyl analog 1j (4.5 μM/120%) motivated us to next prepare a series of fluorobenzyl-analogs (2a-i) to examine the influence of fluoro-substitution patterns on co-potentiator activity. These round r2 targets (Figure 1) were again prepared by selectively N¹-alkylation of spiro[piperidine-4,1’-pyrido[3,4-b]indole] 10. Increasing the number of fluorine atoms on the benzyl moiety (e.g., 2ac/2f/2h with two fluorines versus 2d/2g/2i with three and 2e with five fluorines) improved the activity of these spirocycles. It was also noted that the positioning of the fluorine atoms on the benzyl moiety was important for maintaining both EC₅₀ and V_MAX (Table 3: for example, compare 2a versus 2b and 2c).

With results from synthetic rounds r1 and r2 in hand, the next objective (synthesis round r3, Figure 2) was to more broadly probe the requisite features of the N¹-R³-substituent by preparation of analogs 3a-h. Notably, deletion of R³ (i.e., 3a where R³ = H) gave an inactive compound and lengthening the N¹—aryl tether [−N(CH₂)nR³ from n = 1 (1a) to n = 2 (3c) or n = 3 (3d)] greatly reduced activity. Similarly, adding a methyl substituent to the CH₂ tether in 1a (12.6 μM/V_MAX 54%), giving 3b, also reduced activity (3b: 20 μM/V_MAX 100%). Replacing the phenyl moiety of 1a with a furan ring (3e: 16.4 μM/V_MAX 67%) reduced activity, while replacing the phenyl moiety of 1a with a pyridyl ring to electronically mimic the perfluorophenyl moiety of compound 2e was better tolerated with the nitrogen at position two (3f: 11.5 μM/V_MAX 107%), but poorly tolerated with the nitrogen at position three (3g: 17 μM/V_MAX 56%).

Having thoroughly investigated the N1-substituent in these spiro[piperidine-4,1’-pyrido[3,4-b]indoles], attention was next turned to a probe of the bisheterocyclic spiropiperidine moiety – synthesis round r4 (Figure 1) targeting analogs 4a-d. This work began with the preparation of spiroazepane, spiropyrrolidine, and spiropiperidine analogs where the piperidine ring of 2a was transformed into an azepine (4a), pyrrolidine (4b), and N-offset piperidine (4c) rings. These three analogs of 2a were prepared by reaction of 2-(5-methoxy-1H-indol-3-yl)ethan-1-amine (8 where R¹ = methoxy at C5) with the appropriate ketoamine (12), directly giving the targeted 2a analogs 4a-c. Interestingly, all three of these analogs were inactive. We next prepared piperidine ring-opened analogs truncated analog 4d.

Conformational analysis was done to investigate possible mechanisms to explain the differences between active compounds 2a, 2c and 2e, and inactive compounds 4a-4c. We began with conformational searching using Spartan10 (Wavefunction Inc., Irvine, CA). For the more flexible structures 4a and 4c, multiple runs of conformational search were performed and the results pooled. The conformational search runs were systematic and used the Merck Molecular Force Field (MMFF). All resulting conformers were subjected to single point energy calculations using PCM(chloroform)-B3LYP/6–31+G(d,p) [17–20]. Geometry optimizations were then performed on all conformers within 4 kcal/mol of the conformer with the lowest electronic energy, with the D3(BJ) dispersion correction [21, 22] included. All quantum chemical calculations were performed using Gaussian16 (Gaussian Inc., Wallingford, CT). An implicit chloroform solvent was chosen for these calculations to simulate the polarity inside a general protein binding site [23]. Since 2a, 2c, 2e, 4a-4c contain fluorine atoms, the 6–31+G(d,p) basis set might not be sufficient for computing accurate energies; consequently, we also calculated energies for relevant conformers with PCM-(chloroform)-B3LYP-D3(BJ)/6–311+G(2d,2p). B3LYP/6–311+G(2d,2p) has been reported as a standard method for use with high electron affinity atoms, i.e., O and F [24].

Our conformational analysis suggested a trend distinguishing between active 2a, 2c, and 2e, and inactive 4a-4c. Figure 2 shows the optimized geometries of the lowest energy conformers B3LYP/6–31+G(d,p) for each structure. Most of the energetically relevant conformers of the active co-potentiators (i.e., 2a, 2c, and 2e) have their benzene ring (Ring i) exposed for interaction, which we define as the “Open” form (Figure 2). Assuming that the CFTR binding site accommodates the Open form better and has residues in the vicinity to interact with the phenyl group of the co-potentiators, the Open conformers would allow for various interactions between the π-system and the CFTR interior. In contrast, most of the energetically relevant conformers for 4a-4c have one face of Ring i interacting with the rest of the molecule, precluding protein interactions with it; we refer to these conformers as “Closed” (Figure 2).

Figure 3 shows the superposition of all optimized conformers within 4 kcal/mol of the lowest energy conformer for 2a, 2c, 2e, and 4a-4c. We aligned the conformers and calculated their RMSD values using an RMSD calculating tool from VMD [25]. In this case, we considered conformers to be unique if they have an RMSD value greater than 0.05 relative to other conformers. We then calculated the Boltzmann weighted averages for the Open and Closed conformers at room temperature. This allows us to determine the population of Open vs. Closed conformers for each structure, assuming all are in equilibrium [26]. Table 1 summarizes the Boltzmann weighted averages for 2a, 2c, 2e, and 4a-4c. The results of this analysis emphasize the difference in conformational preferences for active and inactive structures

Table 1.

Predicted Boltzmann distributions for Open vs. Closed forms for 2a, 2c, 2e, and 4a–4c at (PCM)chloroform-B3LYP-D3(BJ)//6–31+G(d,p) (left) and (PCM)chloroform-B3LYP-D3(BJ)//6–331+G(2d,2p) (right).

	Free Energy at PCM-(chloroform)-B3LYP-D3(BJ)//6–31+G(d,p)		Electronic Energy at PCM-(chloroform)-B3LYP-D3(BJ)//6–311 +G(2d,2p)
Structures	Boltzmann Weight for Open form	Boltzmann Weight for Closed Form	Boltzmann Weight for Open form	Boltzmann Weight for Closed Form
2a	99.5%	0.5%	89.6%	10.4%
2c	98.7%	1.3%	96.7%	3.3%
2e	98.9%	1.1%	94.5%	5.5%
4a	3.7%	96.3%	1.7%	98.3%
4b	6.1%	93.9%	7.7%	92.3%
4c	2.9%	97.1%	1.7%	98.3%

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We also considered that these structures contain amines, which can be protonated in biological environments. Therefore, we performed conformational analysis on various protonated states for each structure as well. However, there were no consistent differences in conformational preferences for active and inactive structures. This analysis is complicated by the ability for amines to hydrogen bond with each other within each molecule (see Supporting Information for details), which may or may not occur in biologically relevant environs.

With exploration of the spiropiperidinepyrido aspects of the spiro[piperidine-4,1’-pyrido[3,4-b]indole] scaffold in hand with synthesis rounds r1-r4, we next turned to synthesis round r5 where the indole moiety of scaffold 9 was probed with analogs 5a-e. Spiro[piperidine-4,1’-pyrido[3,4-b]indoles] 5a-d were prepared by reacting the appropriate tryptamine 8 with 1-(2,4-difluorobenzyl)piperidin-4-one (12 in Scheme #1 where n = 1 and R² = 2,4-difluorobenzyl). Whereas 5b performed in a comparable manner to 20, 5a (8.2 μM/100%) showed mild improvement, and 5c (2.5 μM/89%) and 5d (2.4 μM/100%) were approximately 4–fold more potent. Finally, we prepared indole N-methylated analog 5e by employing 2-(5-methoxy-1-methyl-1H-indol-3-yl)ethan-1-amine in place of 2-(5-methoxy-1H-indol-3-yl)ethan-1-amine in a reaction with 1-(2,4-difluorobenzyl)piperidin-4-one (8 → 9 in Scheme #1). Compound 5e, however, was inactive In light of the structure-activity insights gained with synthetic rounds r1-r5, one final synthesis round (r6) was undertaken where targeting constrained spiropiperidinepyrido analog 14 became a high priority in an attempt to address the question of whether there is a conformational requirement for activity vis-à-vis the N¹-(benzyl) moiety. To address this issue, we set out to prepare 14, a constrained analog of compound 2f. C-Alkylation of diethyl acetamidomalonate with 4-(bromomethyl)-1,2-difluorobenzene followed by ester hydrolysis/decarboxylation (Scheme #2) set the stage for a subsequent Pictet–Spengler reaction to delivered tetrahydroisoquinoline-3-carboxylic acid 13 [27]. Unfortunately, all attempts at the Pictet-Spengler failed – an apparent consequence of the electron deficient nature of the difluorophenyl ring. In light of this outcome, our constrained analog study was modified to instead target constrained analog 6 (the nor-fluoro analog of 14). Commercially available 3-amino-4-phenylbutanoic acid (15) successfully participated in a Pictet–Spengler reaction to give 16 and subsequent esterification delivered methyl 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)acetate (17). Michael addition to methyl acrylate delivered bis-ester intermediate 18, which then underwent Dieckmann cyclization/decarboxylation to give ketone 19. A second Pictet–Spengler reaction [28] of 2-(5-methoxy-1H-indol-3-yl)ethan-1-amine (8 where R¹ = methoxy at C5) with 19 gave the spiro[piperidine-4,1’-pyrido[3,4-b]indole] 6b as the sole product – i.e., spiro[piperidine-4,1’-pyrido[3,4-b]indole] 6a was not obtained.

Scheme #2. — Synthesis of constrained analog 6.

To confirm the product is indeed 6b, we performed quantum chemical ¹H and ¹³C NMR calculations on both 6a and 6b (Figure 4). Both 6a and 6b were optimized using SMD(chloroform)-B3LYP-D3(BJ)//6–31+G(d,p) [21, 22, 29]. Using the gauge-including atomic orbital (GIAO) [30] method, chemical shift calculations were performed with SMD(chloroform)-mPW1PW91/6–311+G(2d,p) [31] on conformers that are within 4 kcal/mol relative to the lowest energy conformer. The chemical shifts of these energetically relevant conformers were then averaged using Boltzmann distributions. This is a common procedure for computational NMR studies [30, 32, 33]. Linear scaling (using slope= −1.0533, intercept= 186.524) and (slope= −1.0936, intercept= 31.802) (cheshirenmr.info) was applied to computed isotropic shieldings to arrive at the ¹³C and ¹H shifts, respectively. Due to the similarity in the calculated ¹H shifts, coupling constants (Hz) were also calculated using the same methods to confirm the identity of some diagnostic ¹H peaks (see SI for detail).

Table 2 shows the calculated chemical shifts for 6a and 6b and their deviations from the experimental shifts. Deviations within 6 ppm for ¹³C and 0.3 ppm for ¹H shifts are considered acceptable [30, 32, 33]. Although the ¹³C chemical shifts for 6a and 6b are quite similar, the computed chemical shift at position C13 of 6a deviates greatly from the experimental value, whereas all ¹³C shifts for 6b are within the acceptable deviation range. Similarly, the deviations between the calculated ¹H shifts for 6b and the experimental shifts are within the accepted range, but the calculated chemical shifts at positions H15, H16, and H18 for 6a have deviations greater than 0.3 ppm from the experimental shifts. Both ¹³C and ¹H chemical shifts for 6b have lower mean absolute deviations (MAD) compared to those for 6a as well. We also compared the free energies of the lowest energy conformers of 6a and 6b; that for 6a is ~2.3 kcal/mol higher than that for 6b (Figure 4), consistent with formation of the thermodynamic product during synthesis.

Table 2.

Calculated ¹³C and ¹H chemical shifts for 6a and 6b, and their deviation from experiments.

Atom Label	Exp. ¹³C δ (p.p.m)	Calculated 6a shift	Dev.	Calculate d 6b shift	Dev.	Exp. ¹H δ (p.p.m)	Calculated 6a shift	Dev.	Calculated 6b shift	Dev.
1	128.10	129.08	0.98	130.04	1.94	---	---	---	---	---
2	130.74	130.14	0.60	129.69	1.05	---	---	---	---	---
3	111.62	109.53	2.09	109.80	1.82	7.06	7.19	0.14	6.79	0.26
4	100.46	103.28	2.82	102.43	1.97	6.78	6.72	0.06	6.60	0.18
5	153.98	153.72	0.26	153.66	0.32	---	---	---	---	---
6	108.47	103.35	5.12	103.45	5.02	6.94	6.92	0.02	6.92	0.02
7	111.46	109.05	2.41	105.97	5.49	---	---	---	---	---
8	140.49	142.89	2.40	143.23	2.74	---	---	---	---	---
9	23.26	24.11	0.85	24.08	0.82	2.71	2.52	0.19	2.55	0.16
						2.71	2.59	0.12	2.62	0.09
10	43.76	39.56	4.21	40.94	2.82	3.08	2.94	0.14	3.07	0.01
						3.08	2.98	0.10	3.21	0.13
11	52.91	53.93	1.02	53.82	0.91	---	---	---	---	---
12	26.37	34.96	1.41	37.20	0.83	2.00	2.02	0.02	2.25	0.25
						1.88	1.79	0.08	1.72	0.15
13	50.40	43.27	7.13	48.26	2.13	2.94	3.06	0.12	2.90	0.04
						2.62	2.38	0.24	2.66	0.04
14	39.28	40.98	1.70	38.35	0.93	2.25	2.29	0.04	2.04	0.21
						1.88	1.53	0.34	1.88	0.15
15	56.05	52.83	3.22	56.32	0.27	2.88	3.26	0.38	2.76	0.12
16	36.64	29.75	5.89	35.51	0.13	2.62	2.41	0.21	2.38	0.24
						2.71	4.02	1.31	2.46	0.25
17	133.42	135.31	1.89	136.74	3.31	---	---	---	---	---
18	57.58	56.39	1.19	56.81	0.77	3.52	3.69	0.17	3.50	0.00
						3.86	4.22	0.36	3.86	0.02
19	133.80	135.63	1.83	136.99	3.19	---	---	---	---	---
20	126.02	126.24	0.22	126.36	0.34	7.06	7.15	0.09	7.24	0.18
21	125.81	125.98	0.17	125.82	0.01	7.11	7.10	0.01	7.14	0.03
22	126.40	126.37	0.02	126.86	0.46	7.14	7.12	0.02	7.18	0.04
23	127.67	128.94	1.27	129.60	1.93	7.06	7.16	0.10	6.99	0.06
24	51.35	53.17	1.82	53.14	1.79	3.86	3.67	0.19	3.56	0.30
MAD			2.11		1.71			0.19		0.12

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Abbreviation: MAD, mean absolute deviation; and Dev., deviation. Deviations of less than 6 ppm for ¹³C and less than 0.3 ppm for ¹H shifts are considered acceptable [30, 32, 33]. Chemical shifts exceeding the accepted deviations are bolded above. Coupling constants (J) were also calculated to confirm the identity of complex proton peaks (see SI for details).

The lowest energy conformer of 6b resembles more closely the Closed conformers described above, but that of 6a resembles the Open, i.e., where Ring i is available for interactions (Figure 5). Considering all conformers that are within 4 kcal/mol of the lowest energy conformers, Boltzmann distributions of open and closed forms of 6a and 6b were calculated (Figure 5). For 6a, ~99.3% of conformers are predicted to be Open, while only 23.2% of conformers are predicted to be Open for 6b. Therefore, we expect 6b to have low activity at best, if our hypothesis that conformational bias is related to activity is correct. As predicted, 6b was inactive when tested experimentally.

Figure 5. — Lowest energy conformers optimized at SMD(chloroform)6–31+G(d,p) with D3(BJ) for 6a and 6b respectively (top); the distance between centroids of *Rings i* and ii and *Rings i* and *iii* were measured. Superimposed images of energetically relevant conformers for 6a and 6b (bottom); 6a has more open form conformers in equilibrium.

The structural determinants for activity of compound classes 1-6 are summarized in Figure 6. On the indole aryl 6-member ring (R¹), 6’-methoxy (2i) was most potent, whereas substituents such as methoxy at 7’ position (5d) showed decreased activity. Replacement of 6’-methoxy with bromo (5b) led to an inactive compound, although a chloro (5c) substitution was tolerated. At the indole nitrogen position (R²), hydrogen (2i) was required as methylation (5e) led to inactivity. For the benzyl moiety on the terminal piperidine (R³), we found 2,4,5-trifluorobenzyl (2i) to be best, di-halo-substituted moieties such as 2,4-dichlorobenzyl (1d) and 3-chloro-5-fluorobenzyl (1e) showed decreased activity, and methylene-linked heterocycles such as 2-furyl (3e), 2-pyridyl (3f), or 3-pyridyl (3g) were active, but less potent. Likewise, at R³, lack of substitution on the terminal piperidine (3a), or compounds where the linked ring system is extended by longer linkers (3c and 3d) were also inactive. Finally, in terms of the bis-heterocyclic spiro-fused ring system, a piperidine ring (as in 2a, 2i and other compounds) was significantly more potent than an azepine (4a), and a pyrrolidine (4b), N-offset piperidine (4c) or ring-opened piperidine (4d) abolished activity.

Additional biological studies were performed for most potent compounds 2i and 2e. Copotentiator efficacy was initially determined by short-circuit current measurements in FRT cells expressing N1303K-CFTR in the presence of a transepithelial chloride gradient and with permeabilization of the basolateral cell membrane such that measured current directly reports CFTR channel activity. Representative data in Figure 7A show small increases in CFTR activity following addition of the cAMP agonist forskolin and the potentiator VX-770, followed by concentration-dependent increases in current following addition of the co-potentiators 2i (left) and 2e (middle), with EC₅₀ values 0.6 ± 0.2 and 2.1 ± 0.3 μM, respectively (right). We previously defined potentiators as either class I compounds, including VX-770 and GLPG1837 [34], or class II compounds such as the arylsulphonamidepyrrolopyridine and spiro[piperidine-4,1-pyrido[3,4-b]indole] co-potentiators that probably bind at distinct sites on CFTR [9]. Indeed, structural and pharmacological studies have provided evidence that VX-770 and GLPG1837 bind to the same site on CFTR [35, 36]. Thus, as expected from our prior studies [9], N1303KCFTR-expressing FRT cells treated with forskolin and GLPG1837 had similar 2i co-potentiator EC₅₀ of 0.8 ± 0.2 μM (Fig. 7B).

Figure 7. — Short-circuit current measurement of mutant CFTR activation by spiro[piperidine-4,1-pyrido[3,4-b]indoles]. *A. (left)* Short-circuit current data in FRT cells expressing N1303K-CFTR in response to 20 μM forskolin (fsk), 5 μM VX-770, indicated concentration of 2i and 2e, and 10 μM CFTR_inh-172. *(right)* Summary of concentration-dependence data (n=3, mean ± S.E.M.). B. Measurements done as in A, but with 20 μM GLPG1837 instead of VX-770. C. Short-circuit current in FRT cells expressing I1234del-CFTR in response to 20 μM forskolin, 5 μM VX-770, indicated concentration of 2i, and 10 μM CFTR_inh-172. D. Short-circuit current in FRT cells expressing R347P-CFTR in response to 20 μM forskolin, 5 μM VX-770, 20 μM 2i and 10 μM CFTR_inh-172. In all studies, cells were corrected with 3 μM VX-661 for 18–24 hours prior to measurement. All traces are representative of three replicates.

Compound 2i was also tested on a second minimal function CFTR mutation, I1234del-CFTR, which is generated by the c.3700A>G mutation that results in deletion of 6 amino acids from the CFTR polypeptide (p.Ile1234_Arg1239del-CFTR, hereafter termed I1234del-CFTR) due to introduction of a cryptic splice site in the CFTR transcript [37, 38]. As seen in Figure 7C, 2i further activates I1234del-CFTR following forskolin and VX-770 to increase channel activity with EC₅₀ of 0.7 ± 0.2 μM, similar to that found with N1303K-CFTR. As expected, the R347P-CFTR mutant, which is mildly responsive to VX-770 but not responsive to arylsulphonamidepyrrolopyridine co-potentiators [9], was not activated by 2i indicating that alternative mechanisms (such as increased cAMP signaling) are not responsible for activation of the N1303K- and I1234del-CFTR mutants (Fig. 7D).

The efficacy of 2i was also tested in 16HBE14o- human bronchial epithelial cell models in which the endogenous CFTR gene was edited to contain the N1303K mutation (16HBEge-N1303K) or the I1234del mutation (16HBEge-I1234del) [38, 39]. As shown in N1303K- (Fig. 8A) and I1234del-CFTR (Fig. 8B) expressing 16HBE14o- cells, addition of forskolin and then VX-770 produced limited channel activity. However, subsequent addition of 2i produced CFTR_inh-172-inhibitable responses of 16.1 ± 0.4 μA/cm² in N1303K- (Fig. 8A) and 6.1 ± 0.1 μA/cm² in I1234del-CFTR (Fig. 8B) expressing cells, approximately 6-fold and 2-fold greater that that produced by VX-770 alone.

Figure 8. — Activity of 2i in human airway epithelial cell cultures. A. Short-circuit current in gene-edited 16HBE14o- cells expressing N1303K-CFTR. B. Short-circuit current data in gene-edited 16HBE14o- cells expressing I1234del-CFTR. C. Summary of changes in short-circuit current (ΔIsc; mean ± S.E.M., n = 3, *P < 0.05). Concentrations: 20 μM forskolin, 5 μM VX-770, 10 μM 2i, and 10 μM CFTR_inh-172. 16HBE14o- cell models expressing I1234del-CFTR were corrected with 18 μM VX-661 and 3 μM VX-445 for 18–24 hours prior to measurement.

CONCLUSIONS:

This study extends our prior work that identified a novel class of CFTR modulators, termed co-potentiators, that act in synergy with existing potentiators such as VX-770 and GLPG1837 to increase chloride channel function of CFTR mutants [7–9]. The objective of this study was to optimize spiro[piperidine-4,1-pyrido[3,4-b]indoles] co-potentiators previously identified in a high-throughput screen [9]. A straightforward, high yielding synthetic strategy was developed to investigate structure-activity relationships. In total, 37 spiro[piperidine-4,1-pyrido[3,4-b]indoles] were synthesized to investigate the consequences of altering the indole and benzyl moieties. Analogs of the original spiro[piperidine-4,1’-pyrido[3,4-b]indole] compound 20 were prepared in six rounds of synthetic studies (synthesis rounds r1 through r6) starting from commercial tryptamines (8). These were condensed with N-alkylated piperidin-4-ones (12) to directly deliver the targeted spiro[piperidine-4,1’-pyrido[3,4-b]indole] 9 by a classical Pictet–Spengler reaction. When preparing R² analogs of 9, it proved more efficient to prepare unalkylated analog 10 [acetic acid-mediated condensation of tryptamine 8 with piperidin-4-one (11)] and then subsequently perform selective N¹-alkylation of 10 (R²-X in CH₂Cl₂ + K₂CO₃) to give 9.

It is estimated that up to 90% of CF subjects will benefit from available CFTR modulators including Trikafta [5], with ~10% of CF subjects not benefitted. For co-potentiators, we have demonstrated efficacy for several NBD2 mutants including the missense mutant N1303K, the c.3700A>G splicing mutant, and truncated CFTR protein generated by the premature termination codon (PTC) W1282X [7–9]. In addition, albeit to a lesser degree, channel activity of the VX-770-responsive G551D mutation can be further augmented by co-potentiators [7]. Co-potentiators may be beneficial for some PTCs if sufficient transcript is present, or in combination with future read-through or other therapies [40].

In summary, spiro[piperidine-4,1-pyrido[3,4-b]indoles] represent an evolvable CFTR co-potentiator scaffold with nanomolar potency that in synergy with existing potentiators activates certain CF-causing CFTR mutations. The expansion of spiro[piperidine-4,1-pyrido[3,4-b]indole] structure activity relationships may yield insight into the binding site of co-potentiators through computational or structural approaches [35, 36], as well as subsequent rational compound optimization.

MATERIALS AND METHODS:

General Experimental:

All compounds described in this manuscript have ≥95% purity. The analytical method used to determine purity was ¹H NMR (see the accompanying Supporting Information file which provides the ¹H- and ¹³C-NMR for the thirty-seven compounds assayed) and HPLC/HRMS. For HRMS analysis, samples were analyzed by flow-injection analysis into a Thermo Fisher Scientific LTQ Orbitrap (San Jose, CA) operated in the centroided mode. Samples were injected into a mixture of 50% MeOH/H₂O and 0.1% formic acid at a flow of 0.2 mL/min. Source parameters were 5.5kV spray voltage, capillary temperature of 275 °C and sheath gas setting of 20. Spectral data were acquired at a resolution setting of 100,000 FWHM with the lockmass feature, which typically results in a mass accuracy <2ppm.

Cell culture:

Fischer Rat Thyroid (FRT) cells transfected to stably express N1303K-CFTR, I1234del-CFTR and R347P-CFTR were cultured as described [8, 9]. Gene-edited 16HBE14o-cells expressing N1303K-CFTR were provided by CFFT lab and were cultured as described [39]. Gene-edited 16HBE14o- cells expressing I1234del-CFTR were a generous gift of Dr. Christine Bear (The Hospital for Sick Children, Toronto, Canada) and were cultured as described [38].

Short-circuit current measurement.

Short-circuit current measurements were made essentially as described in prior studies [8, 9]. In brief, measurements were made using a DVC-1000 voltage clamp (World Precision Instruments Inc., Sarasota, FL), cells were cultured on Snapwell clear permeable supports (Corning), and experiments were performed using HCO₃⁻_-buffered solutions at 37 °C. For studies using FRT cells the basolateral membrane was permeabilized with 250 μg/ml amphotericin B and experiments were done with a chloride gradient. For 16HBE14o- gene-edited cells experiments were done with a chloride gradient using Hepes-buffered solutions.

Statistical analysis.

Data are presented as mean ± S.E.M. Comparisons between two groups were performed using the unpaired Student’s t-test. P < 0.05 was considered as statistically significant.

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (8 → 9):

Tryptamine (8, 0.6 mmol) was mixed with glacial acetic acid (3 mL) in a 10 mL vial and stirred with a magnetic stirrer. Corresponding ketone (12, 0.5 mmol) was added and the vial was sealed with a plastic cap. The vial was heated at 100 °C for 16 h in an oil bath. After cooling, the solution was diluted with water (~20 mL) and neutralized by adding 4 M HCl. The product was extracted with dichloromethane, and the organic solution was washed with water, brine and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5 % MeOH/DCM).

General experimental for the synthesis of N-alkylated piperidin-4-one analogs (11 → 12):

4-Piperidone hydrochloride (11, 5 mmol) was mixed with 25 mL of dichloromethane in an Erlenmeyer flask. Small amount of methanol (5 drops) was added and benzyl bromide (2.5 mmol) and potassium carbonate (5 mmol) was added. The mixture was stirred at RT for 16h. Water was added to the reaction mixture and the product was extracted with dichloromethane. The extracted organic solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5 % MeOH/DCM).

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (10 → 9):

Compound 3a (0.5 mmol) was mixed with 5 mL of dichloromethane in a small vial. Small amount of methanol (1 drop) was added for better solubility, and benzyl bromide (0.5 mmol) and potassium carbonate (1.5 mmol) was added. The vial was capped, and the mixture was stirred at RT for 16h. Water was added to the reaction mixture and the product was extracted with dichloromethane. The extracted organic solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5 % MeOH/DCM).

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (8 → 10):

5-methoxytryptamine (8, 25 mmol) was mixed with glacial acetic acid (20 mL) in a round bottom flask. 4-piperidone hydrochloride (11, 25 mmol) was added and the solution was heated at 100 °C for 16 h in an oil bath with stirring. The solution was cooled to RT and diluted with water (100 mL) and neutralized with 4 M NaOH. Tan precipitate formed upon standing within an hour. The precipitate was filtered and washed with water and air dried. Yield = 54%.

General experimental for the synthesis of tryptamines (7 → 8):

1-Dimethylamino-2-nitroethylene (3 mmol) was mixed in TFA (4 mL) in a vial. Substituted indole (7, 3.6 mmol) was dissolved in dichloromethane (3 mL) separately and added. The mixture was stirred at RT for 2 h and the solution was diluted with dichloromethane. The organic solution was washed with water, brine and dried over magnesium sulfate. Solvent was removed in vacuo and purified by using flash column chromatography (50 % EtOAc/hexane). LiAlH₄ (12 mmol) was mixed with THF (75 mL) and cooled at −78 °C and stirred. The product from the previous step dissolved in small amount of THF was added dropwise and the mixture was stirred overnight with slowly warming to RT. The reaction mixture was cooled in an ice bath and quenched with water and 4 M NaOH solution. Organic solvent was removed in vacuo and the product was extracted with dichloromethane. The extracted solution was washed with water, brine, and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was directly used.

Synthesis of 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)acetic acid (16):

L-β-homophenylalanine hydrochloride (1 g, 4.6 mmol) was mixed with concentrated HCl (10 mL) in a round bottom flask and formaldehyde (37% aqueous solution, 4 mL, 49 mmol) was added. The mixture was refluxed at 100 °C for 3h with stirring. After cooling, solvent was removed in vacuo to obtain off-white solid in quantitative yield, which was used for the next step without purification.

Synthesis of methyl 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)acetate (17):

16 (1g, 4.3 mmol) was placed in a round bottom flask. Trimethylchlorosilane 1.26 mL was added dropwise with stirring, then methanol (20 mL) was added and stirred for 2 h. Solvent was removed in vacuo and dried in vacuum. The solid product was used for the next step without purification.

Synthesis of methyl 3-(3-(2-methoxy-2-oxoethyl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoate (18):

17 (0.62g, 3 mmol) was dissolved in dichloromethane (30 mL) and trifluoroacetic acid (3 mL) was added and stirred briefly. Solvent was removed in vacuo and the resulting oil was extracted with ethyl acetate. The organic solution was washed with saturated sodium bicarbonate solution, and the solvent was removed in vacuo. To the resulting oil, methyl acrylate (10 mL) was added and refluxed at 80 °C overnight. Solvent was removed in vacuo, and the product was used without purification for the next step.

Synthesis of 1,3,4,6,11,11a-hexahydro-2H-pyrido[1,2-b]isoquinolin-2-one (19):

Lithium diisopropylamide (LDA, 1M solution in THF/hexane, 10 mL) was cooled at −78 °C under N₂ atmosphere. Crude product of 18 was dissolved in anhydrous THF (8 mL) under N₂ atmosphere and added to the LDA solution by syringe. The mixture solution was stirred at −78 °C for 1 h. Concentrated HCl (0.8 mL) was added to the solution and brought to RT. Solvent was removed in vacuo then H₂O (25 mL) and concentrated HCl (25 mL) was added, and the solution was refluxed at 100 °C overnight. After cooling, the solution was basified by adding solid K₂CO₃ portion wise. The product was extracted with diethyl ether and the organic layer was washed with water, brine and dried with magnesium sulfate. Crude product (220 mg) was used for the next step without purification.

Synthesis of (2R,11aS)-6’-methoxy-1,2’,3,3’,4,4’,6,9’,11,11a-decahydrospiro[pyrido[1,2-b]isoquinoline-2,1’-pyrido[3,4-b]indole] (6b): Crude 19 (220 mg, 1.1 mmol) was placed in a glass vial and 5-methoxytryptamine (250 mg, 1.3 mmol) was added and glacial acetic acid (3 mL) was added. The vial was capped and heated at 100 °C overnight. After cooling, the solution was diluted with water (~20 mL) and neutralized by adding 4 M HCl. The product was extracted with dichloromethane, and the organic solution was washed with water, brine and dried over magnesium sulfate. The solvent was removed in vacuo, and the product was purified by using flash column chromatography (2.5 % MeOH/DCM).

1-Benzyl-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1a).

Yield = 152 mg (84 %).¹H NMR (400 MHz, CDCl₃) δ 8.30 (d, J = 35.1 Hz, 1H), 7.43 (d, J = 7.2 Hz, 2H), 7.39 – 7.27 (m, 3H), 7.22 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 6.82 (dt, J = 8.9, 1.8 Hz, 1H), 3.87 (s, 3H), 3.69 (s, 2H), 3.15 (t, J = 5.6 Hz, 2H), 2.96 – 2.77 (m, 2H), 2.77 – 2.53 (m, 4H), 2.25 (td, J = 13.4, 4.4 Hz, 2H), 1.79 (d, J = 13.7 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.99, 140.62, 136.72, 130.69, 129.67, 128.46, 127.64, 127.60, 111.62, 111.44, 108.42, 100.50, 63.09, 56.07, 50.52, 48.80, 39.16, 35.86, 23.23. HRMS (ESI) m/z for C₂₃H₂₈N₃O [M+H]. calcd 362.2232, found 362.2229.

1-(2-Fluoro-4-nitrobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1b).

Yield = 119 mg (56 %).¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.57 (td, J = 5.2, 2.8 Hz, 2H), 7.32 – 7.18 (m, 2H), 6.95 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.7, 2.5 Hz, 1H), 3.86 (d, J = 11.3 Hz, 5H), 3.14 (t, J = 5.7 Hz, 2H), 2.69 (t, J = 5.6 Hz, 2H), 2.64 – 2.54 (m, 4H), 2.01 (ddd, J = 13.8, 10.3, 6.5 Hz, 2H), 1.79 – 1.71 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 162.37, 159.88, 154.03, 150.11 (d, J_C-F = 8.4 Hz), 140.70, 132.64 (d, J_C-F = 7.8 Hz), 130.61, 130.06 (d, J_C-F = 3.7 Hz), 127.72, 119.34 (d, J_C-F = 20.7 Hz), 112.21 (d, J_C-F = 26.4 Hz), 111.51 (d, J_C-F = 7.6 Hz), 108.51, 100.53, 58.88, 56.06, 50.58, 48.83, 39.07, 36.37, 23.15. HRMS (ESI) m/z for C₂₃H₂₆FN₄O₃ [M+H]. calcd 425.1989, found 425.1978.

1-(5-Chloro-2-nitrobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1c).

Yield = 128 mg (58 %).¹H NMR (400 MHz, CDCl₃) δ 7.90 (s, 1H), 7.83 (d, J = 8.6 Hz, 1H), 7.68 (d, J = 2.3 Hz, 1H), 7.39 (dd, J = 8.6, 2.3 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.8, 2.4 Hz, 1H), 3.87 (d, J = 2.6 Hz, 5H), 3.16 (t, J = 5.7 Hz, 2H), 2.70 (t, J = 5.7 Hz, 2H), 2.67 – 2.61 (m, 4H), 2.08 – 1.98 (m, 2H), 1.78 (d, J = 13.5 Hz, 2H), 1.29 (s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 154.05, 147.99, 140.57, 138.85, 136.69, 130.80, 130.62, 127.99, 127.72, 125.99, 111.55, 111.52, 108.55, 100.54, 59.06, 56.07, 50.59, 48.95, 39.08, 36.39, 23.11. HRMS (ESI) m/z for C₂₃H₂₆ClN₄O₃ [M+H]. calcd 441.1693, found 441.1685.

1-(2,4-Dichlorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1d).

Yield = 156 mg (72 %).¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 2.2 Hz, 1H), 7.27 – 7.15 (m, 2H), 6.97 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.7, 2.4 Hz, 1H), 3.88 (s, 3H), 3.68 (s, 2H), 3.17 (t, J = 5.6 Hz, 2H), 2.88 – 2.52 (m, 6H), 2.28 – 2.00 (m, 2H), 1.80 (d, J = 14.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 154.05, 140.85, 135.06, 134.79, 133.21, 131.70, 130.57, 129.27, 127.77, 126.95, 111.49, 111.47, 108.59, 100.56, 59.02, 56.08, 50.58, 48.93, 39.12, 36.44, 23.22. HRMS (ESI) m/z for C₂₃H₂₆Cl₂N₃O [M+H]. calcd 430.1453, found 430.1447.

1-(3-Chloro-5-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1e).

Yield = 187 mg (91 %) ¹H NMR (800 MHz, CDCl₃) δ 8.09 (s, 1H), 7.25 – 7.15 (m, 2H), 7.08 – 7.00 (m, 2H), 6.98 (d, J = 2.7 Hz, 1H), 6.84 (dd, J = 8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.54 (s, 2H), 3.15 (t, J = 5.7 Hz, 2H), 2.71 (q, J = 5.2 Hz, 4H), 2.61 – 2.46 (m, 2H), 2.10 (td, J = 13.4, 4.2 Hz, 2H), 1.83 – 1.69 (m, 2H). ¹³C NMR (201 MHz, CDCl₃) δ 163.35, 162.12, 154.01, 142.79 (d, J_C-F = 7.8 Hz), 140.84, 134.76 (d, J_C-F = 10.4 Hz), 130.65, 127.76, 124.82 (d, J_C-F = 2.9 Hz), 114.87 (d, J_C-F = 24.9 Hz), 114.27 (d, J_C-F = 21.4 Hz), 111.52 (d, J_C-F = 18.3 Hz), 108.56, 100.57, 62.25, 56.07, 50.58, 48.91, 39.09, 36.33, 23.21. HRMS (ESI) m/z for C₂₃H₂₆ClFN₃O [M+H]. calcd 414.1748, found 414.1740.

1-(3-Chloro-2,6-difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1f).

Yield = 183 mg (85 %) ¹H NMR (400 MHz, CDCl₃) δ 8.34 (s, 1H), 7.30 (h, J = 5.4 Hz, 1H), 7.16 (d, J = 8.8 Hz, 1H), 6.96 (d, J = 2.6 Hz, 1H), 6.87 – 6.72 (m, 2H), 3.88 (s, 3H), 3.76 (s, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.79 (d, J = 11.4 Hz, 2H), 2.73 – 2.56 (m, 4H), 2.10 (td, J = 13.3, 4.7 Hz, 2H), 1.77 (d, J = 12.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 160.35 (dd, J_C-F = 248.7, 7.0 Hz), 157.24 (dd, J_C-F = 250.2, 8.4 Hz), 153.93, 140.88, 130.71, 129.69 (d, J_C-F = 9.5 Hz), 127.70, 116.55 (dd, J_C-F = 19.1, 4.0 Hz), 114.91 (t, J_C-F = 20.2 Hz), 111.83 (dd, J_C-F = 24.6, 4.0 Hz), 111.63, 111.33, 108.38, 100.52, 56.06, 50.44, 49.43, 48.31, 39.10, 36.18, 23.20. HRMS (ESI) m/z for C₂₃H₂₅ClF₂N₃O [M+H]. calcd 432.1654, found 432.1655.

1-(2-Chlorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1g).

Yield = 126 mg (64 %) ¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.54 (dd, J = 7.4, 2.3 Hz, 1H), 7.41 (dd, J = 7.5, 1.9 Hz, 1H), 7.26 (qd, J = 7.4, 6.6, 2.1 Hz, 2H), 7.22 – 7.13 (m, 1H), 6.98 (d, J = 2.7 Hz, 1H), 6.84 (dd, J = 8.8, 2.6 Hz, 1H), 3.89 (s, 3H), 3.74 (s, 2H), 3.17 (t, J = 5.7 Hz, 2H), 2.82 (dt, J = 12.1, 3.7 Hz, 2H), 2.74 – 2.58 (m, 4H), 2.14 (td, J = 13.1, 4.6 Hz, 2H), 1.80 (dd, J = 13.9, 2.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.00, 140.97, 135.92, 134.57, 131.10, 130.64, 129.58, 128.37, 127.75, 126.67, 111.57, 111.40, 108.45, 100.56, 59.64, 56.10, 50.65, 48.97, 39.16, 36.35, 23.22. HRMS (ESI) m/z for C₂₃H₂₇ClN₃O [M+H]. calcd 396.1843, found 396.1836.

1-(4-Bromo-3-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1h).

Yield = 155 mg (68 %) ¹H NMR (400 MHz, CDCl₃) δ 8.00 (s, 1H), 7.48 (dd, J = 8.2, 7.1 Hz, 1H), 7.21 (dd, J = 9.1, 2.9 Hz, 2H), 7.07 – 6.93 (m, 2H), 6.85 (dd, J = 8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.54 (s, 2H), 3.17 (t, J = 5.7 Hz, 2H), 2.79 – 2.65 (m, 4H), 2.63 – 2.44 (m, 2H), 2.10 (td, J = 13.2, 12.8, 4.4 Hz, 2H), 1.88 – 1.70 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 160.33, 157.87, 154.04, 140.89, 140.84 (d, J_C-F = 6.6 Hz), 133.17, 130.64, 127.80, 125.78 (d, J_C-F = 3.3 Hz), 116.90 (d, J_C-F = 22.0 Hz), 111.51 (d, J_C-F = 4.4 Hz), 108.62, 107.20 (d, J_C-F = 20.9 Hz), 100.61, 62.20, 56.10, 50.62, 48.92, 39.11, 36.42, 23.23. HRMS (ESI) m/z for C₂₃H₂₆BrFN₃O [M+H]. calcd 458.1243, found 458.1239.

1-(4-Bromo-2-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1i).

Yield = 190 mg (83 %) ¹H NMR (400 MHz, CDCl₃) δ 8.59 (s, 1H), 7.24 (t, J = 8.1 Hz, 1H), 7.15 (dd, J = 7.9, 2.6 Hz, 3H), 6.98 (d, J = 2.6 Hz, 1H), 6.81 (dd, J = 8.7, 2.5 Hz, 1H), 3.88 (s, 3H), 3.60 (s, 2H), 3.14 (t, J = 5.7 Hz, 2H), 2.72 (q, J = 7.2, 5.7 Hz, 4H), 2.59 (td, J = 12.1, 2.5 Hz, 2H), 2.10 (td, J = 13.5, 4.6 Hz, 2H), 1.69 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 162.50, 160.00, 153.93, 140.87, 132.96 (d, J_C-F = 5.1 Hz), 130.73, 127.72, 127.29 (d, J_C-F = 3.7 Hz), 123.77 (d, J_C-F = 15.0 Hz), 121.33 (d, J_C-F = 9.5 Hz), 119.03 (d, J_C-F = 25.3 Hz), 111.47 (d, J_C-F = 17.2 Hz), 108.44, 100.56, 56.08, 55.35, 50.50, 48.72, 39.13, 36.03, 23.25. HRMS (ESI) m/z for C₂₃H₂₆BrFN₃O [M+H]. calcd 458.1243, found 458.1228.

1-(3-Chloro-2,4-difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1j).

Yield = 124 mg (58 %) ¹H NMR (400 MHz, CDCl₃) δ 8.10 (s, 1H), 7.36 – 7.24 (m, 1H), 7.17 (s, 1H), 7.01 – 6.87 (m, 2H), 6.83 (dd, J = 8.7, 2.5 Hz, 1H), 3.88 (s, 3H), 3.65 (s, 2H), 3.15 (t, J = 5.7 Hz, 2H), 2.80 – 2.66 (m, 4H), 2.60 (td, J = 11.9, 2.6 Hz, 2H), 2.10 (td, J = 13.4, 4.6 Hz, 2H), 1.87 – 1.67 (m, 2H).¹³C NMR (101 MHz, CDCl₃) δ 158.08 (dd, J_C-F = 250.1, 2.9 Hz), 157.44 (d, J_C-F = 251.6, 2.9 Hz), 154.01, 140.77, 130.62, 129.34 (dd, J_C-F = 9.0, 5.7 Hz), 127.74, 121.95 (dd, J_C-F = 15.0, 4.0 Hz), 111.50, 111.46, 111.44 (dd, J_C-F = 20.6, 4.4 Hz), 109.77 (t, J_C-F = 21.3 Hz), 108.58, 100.56, 56.06, 55.41, 50.51, 48.65, 39.10, 36.27, 23.21.HRMS (ESI) m/z for C₂₃H₂₅ClF₂N₃O [M+H]. calcd 432.1654, found 432.1646.

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2a).

Yield = 138 mg (70 %) ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 7.37 (q, J = 7.9 Hz, 1H), 7.18 (d, J = 8.7 Hz, 1H), 6.94 (s, 1H), 6.82 (ddt, J = 14.4, 8.8, 5.4 Hz, 3H), 3.85 (s, 3H), 3.61 (s, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.75 (d, J = 11.4 Hz, 2H), 2.67 (t, J = 5.8 Hz, 2H), 2.57 (t, J = 11.7 Hz, 2H), 2.07 (td, J = 13.2, 4.3 Hz, 2H), 1.79 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ, 161.14 – 154.80 (m), 140.93 – 128.43 (m), 120.82 (dd, J = 14.8, 3.9 Hz), 111.57 – 108.50 (m), 103.92 – 22.26 (m). ¹³C NMR (101 MHz, CDCl₃) δ 163.05 (dd, J_C-F = 77.9, 11.9 Hz), 160.58 (dd, J_C-F = 79.0, 12.0 Hz), 154.90, 154.04, 140.83, 132.40 (dd, J_C-F = 9.6, 6.2 Hz), 130.53, 128.52, 127.75, 120.82 (dd, J_C-F = 14.8, 3.9 Hz), 111.04 (dd, J_C-F = 20.9, 3.7 Hz), 108.60, 103.56 (t, J_C-F = 25.8 Hz), 100.54, 56.72, 56.05, 55.16, 51.90, 50.54, 48.64, 40.07, 39.10, 37.28, 36.37, 23.22, 22.36. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2036.

1-(2,5-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2b).

Yield = 173 mg (87 %) ¹H NMR (400 MHz, CDCl₃) δ 8.12 – 7.83 (m, 1H), 7.19 (d, J = 8.8 Hz, 2H), 7.11 – 6.90 (m, 3H), 6.86 (d, J = 8.8 Hz, 1H), 3.90 (s, 3H), 3.64 (s, 2H), 3.16 (d, J = 5.8 Hz, 2H), 2.91 – 2.68 (m, 4H), 2.62 (t, J = 11.8 Hz, 2H), 2.24 – 1.98 (m, 2H), 1.81 (d, J = 13.6 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 159.22 (dd, J_C-F = 140.6, 2.3 Hz), 156.81 (dd, J_C-F = 140.5, 2.3 Hz), 154.04, 140.95, 130.65, 127.81, 127.28 (dd, J_C-F = 17.0, 7.3 Hz), 117.38 (dd, J_C-F = 24.1, 4.9 Hz), 116.26 (dd, J_C-F = 25.2, 8.5 Hz), 114.99 (dd, J_C-F = 24.2, 8.5 Hz), 111.57, 111.45, 108.59, 100.61, 56.08, 55.32, 50.58, 48.84, 39.11, 36.43, 23.23. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2031.

1-(3,5-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2c).

Yield = 113 mg (57 %) ¹H NMR (800 MHz, CDCl₃) δ 7.85 (s, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.00 – 6.92 (m, 3H), 6.84 (dd, J = 8.7, 2.4 Hz, 1H), 6.73 (tt, J = 8.9, 2.4 Hz, 1H), 3.88 (s, 3H), 3.58 (s, 2H), 3.16 (t, J = 5.7 Hz, 2H), 2.78 – 2.66 (m, 4H), 2.57 (td, J = 12.0, 2.4 Hz, 2H), 2.12 (td, J = 13.3, 4.3 Hz, 2H), 1.80 (dd, J = 14.1, 2.6 Hz, 2H). ¹³C NMR (201 MHz, CDCl₃) δ 163.66 (d, J_C-F = 12.9 Hz), 162.43 (d, J_C-F = 12.5 Hz), 154.08, 140.74, 130.55, 127.74, 111.52, 111.51 (dd, J_C-F = 20.9, 4.0 Hz), 111.49, 108.64, 102.48 (t, J_C-F = 25.5 Hz), 100.52, 62.34, 56.06, 50.54, 48.88, 39.09, 36.39, 23.21. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2030.

6’-Methoxy-1-(3,4,5-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2d).

Yield = 159 mg (77 %).¹H NMR (400 MHz, CDCl₃) δ 7.76 (s, 1H), 7.23 (d, J = 8.6 Hz, 1H), 7.04 (t, J = 7.5 Hz, 2H), 6.96 (d, J = 2.4 Hz, 1H), 6.84 (dt, J = 8.7, 1.8 Hz, 1H), 3.87 (s, 3H), 3.52 (s, 2H), 3.16 (t, J = 5.7 Hz, 2H), 2.71 (q, J = 5.3 Hz, 4H), 2.56 (t, J = 11.8 Hz, 2H), 2.10 (td, J = 13.2, 4.3 Hz, 2H), 1.81 (d, J = 13.7 Hz, 2H). ¹³C NMR (201 MHz, CDCl₃) δ 154.07, 151.14 (ddd, J_C-F = 249.5, 10.1, 3.7 Hz), 140.68, 139.34 – 138.02 (m), 135.07, 130.58, 127.74, 112.54 (dd, J_C-F = 17.1, 3.7 Hz), 111.53, 111.50, 108.64, 100.53, 61.90, 56.05, 50.55, 48.79, 39.08, 36.34, 23.19. HRMS (ESI) m/z for C₂₃H₂₄F₃N₃O [M+H]. calcd 416.1950, found 416.1939.

6’-Methoxy-1-((perfluorophenyl)methyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2e).

Yield = 202 mg (90 %).¹H NMR (400 MHz, CDCl₃) δ 7.73 (s, 1H), 7.19 (d, J = 8.7 Hz, 1H), 6.95 (t, J = 1.8 Hz, 1H), 6.83 (dt, J = 8.7, 1.9 Hz, 1H), 3.87 (d, J = 1.4 Hz, 3H), 3.79 (d, J = 2.4 Hz, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.76 (d, J = 11.1 Hz, 2H), 2.72 – 2.61 (m, 4H), 2.06 (td, J = 13.1, 4.5 Hz, 2H), 1.80 (d, J = 13.6 Hz, 2H). ¹³C NMR (201 MHz, CDCl₃) δ 154.03, 145.63 (d, J_C-F = 248.0 Hz), 140.70, 140.59 (d, J_C-F = 254.2 Hz), 137.39 (d, J_C-F = 251.6 Hz), 130.54, 127.73, ,111.47, 111.46, 110.75 (t, J_C-F = 18.7 Hz), 108.66, 100.50, 56.01, 50.26, 48.92, 48.07, 39.04, 36.34, 23.20. HRMS (ESI) m/z for C₂₃H₂₃F₅N₃O [M+H]. calcd 452.1761, found 452.1753.

1-(3,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2f).

Yield = 88 mg (44 %).¹H NMR (400 MHz, CDCl₃) δ 8.15 – 7.91 (m, 1H), 7.25 (dt, J = 16.3, 7.7 Hz, 2H), 7.17 – 7.03 (m, 2H), 6.97 (s, 1H), 6.84 (dd, J = 8.8, 2.4 Hz, 1H), 3.88 (s, 3H), 3.55 (s, 2H), 3.16 (t, J = 5.7 Hz, 2H), 2.72 (q, J = 8.2, 6.0 Hz, 4H), 2.55 (t, J = 11.1 Hz, 2H), 2.11 (td, J = 13.5, 4.8 Hz, 2H), 1.81 (s, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.05, δ 151.15 (dd, J_C-F = 78.7, 12.7 Hz), 148.69 (dd, J_C-F = 78.1, 12.5 Hz), 140.79, 135.47, 130.62, 127.76, 124.91, 117.80 (d, J_C-F = 17.2 Hz), 116.90 (d, J_C-F = 17.2 Hz), 111.52, 111.48, 108.59, 100.57, 62.14, 56.07, 50.62, 48.80, 39.10, 36.31, 23.21. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2032.

6’-Methoxy-1-(2,3,4-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2g).

Yield = 200 mg (96 %) ¹H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.18 (d, J = 8.8 Hz, 1H), 7.15 – 7.03 (m, 1H), 6.99 – 6.86 (m, 2H), 6.85 – 6.67 (m, 1H), 3.87 (d, J = 2.9 Hz, 3H), 3.68 – 3.59 (m, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.77 – 2.65 (m, 4H), 2.65 – 2.50 (m, 2H), 2.16 – 1.98 (m, 2H), 1.85 – 1.71 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.99, 150.40 (ddd, J_C-F = 250.2, 9.9, 2.6 Hz), 150.20 (d, J_C-F = 250.4, 9.5, 3.0 Hz), 140.80, 139.88 (dt, J_C-F = 251.8, 15.6 Hz), 130.65, 127.72, 124.92 (ddd, J_C-F = 8.5, 6.3, 3.2 Hz), 122.42 (dd, J_C-F = 12.1, 2.8 Hz), 111.71 (dd, J_C-F = 17.1, 3.9 Hz), 111.51, 111.42, 108.52, 100.53, 56.05, 55.14, 50.49, 48.58, 39.08, 36.21, 23.20. HRMS (ESI) m/z for C₂₃H₂₄F₃N₃O [M+H]. calcd 416.1950, found 416.1941.

1-(2,3-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2h).

Yield = 184 mg (93 %) ¹H NMR (400 MHz, CDCl₃) δ 7.85 (s, 1H), 7.20 (d, J = 8.8 Hz, 2H), 7.14 – 7.02 (m, 2H), 6.96 (d, J = 2.4 Hz, 1H), 6.83 (dd, J = 8.8, 2.4 Hz, 1H), 3.88 (s, 3H), 3.70 (d, J = 1.7 Hz, 2H), 3.15 (t, J = 5.7 Hz, 2H), 2.85 – 2.73 (m, 2H), 2.70 (t, J = 5.7 Hz, 2H), 2.60 (td, J = 11.9, 2.7 Hz, 2H), 2.16 – 1.99 (m, 2H), 1.80 (dq, J = 14.1, 2.9 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.06, 151.26 (dd, J_C-F = 119.9, 12.8 Hz), 148.79 (dd, J_C-F = 119.9, 12.8 Hz), 140.86, 130.54, 127.77, 127.62 (d, J_C-F = 11.4 Hz), 126.18 (t, J_C-F = 3.5 Hz), 123.68 (dd, J_C-F = 6.6, 4.8 Hz), 116.10, 115.93, 111.47, 108.62, 100.55, 56.06, 55.41, 50.52, 48.74, 39.10, 36.43, 23.21. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2037.

6’-Methoxy-1-(2,4,5-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2i).

Yield = 193 mg (93 %) ¹H NMR (400 MHz, CDCl₃) δ 8.06 (s, 1H), 7.30 (ddd, J = 10.7, 8.9, 6.6 Hz, 1H), 7.19 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 2.4 Hz, 1H), 6.95 – 6.86 (m, 1H), 6.84 (dd, J = 8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.59 (d, J = 1.3 Hz, 2H), 3.16 (t, J = 5.7 Hz, 2H), 2.81 – 2.66 (m, 4H), 2.60 (dd, J = 23.9, 2.7 Hz, 2H), 2.10 (td, J = 13.7, 4.6 Hz, 2H), 1.80 (dd, J = 14.1, 2.7 Hz, 2H).¹³C NMR (101 MHz, CDCl₃) δ 156.20 (ddd, J_C-F = 247.5, 10.1, 2.2 Hz), 154.03, 149.1 (ddd, J_C-F = 251.7, 14.9, 14.0), 146.8 (ddd, J_C-F = 245.3, 12.5, 4.0), 140.79, 130.64, 127.76, 121.72 (dt, J_C-F = 16.9, 4.6 Hz), 118.72 (dd, J_C-F = 18.9, 5.7 Hz), 111.52, 111.47, 108.60, 105.31 (dd, J_C-F = 28.6, 20.5 Hz), 100.57, 56.06, 54.76, 50.52, 48.70, 39.09, 36.34, 23.21. HRMS (ESI) m/z for C₂₃H₂₄F₃N₃O [M+H]. calcd 416.1950, found 416.1940.

6’-Methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3a).

¹H NMR (400 MHz, MeOD) δ 7.18 (d, J = 8.6 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H), 6.72 (dd, J = 8.7, 2.5 Hz, 1H), 3.80 (s, 3H), 3.11 – 2.94 (m, 4H), 2.84 (dt, J = 12.7, 3.9 Hz, 2H), 2.66 (t, J = 5.7 Hz, 2H), 1.96 (td, J = 13.5, 4.7 Hz, 2H), 1.72 (d, J = 12.8 Hz, 2H). ¹³C NMR (101 MHz, MeOD) δ 153.53, 140.40, 131.32, 127.35, 110.98, 110.44, 106.88, 99.80, 54.91, 51.11, 40.53, 38.51, 35.15, 21.97. HRMS (ESI) m/z for C₁₆H₂₂N₃O [M+H]. calcd 272.1763, found 272.1755.

6’-Methoxy-1-(1-phenylethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3b).

Yield = 44 mg (36 %)¹H NMR (800 MHz, MeOD) δ 7.45 – 7.42 (m, 2H), 7.39 (t, J = 7.7 Hz, 2H), 7.34 – 7.30 (m, 1H), 7.21 (d, J = 8.8 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.76 (dd, J = 8.7, 2.4 Hz, 1H), 3.81 (s, 3H), 3.21 (td, J = 6.0, 2.8 Hz, 2H), 3.18 – 3.15 (m, 1H), 2.82 (s, 1H), 2.80 – 2.76 (m, 2H), 2.65 (s, 1H), 2.56 (s, 1H), 2.32 (ddd, J = 14.4, 12.8, 4.5 Hz, 1H), 2.21 (ddd, J = 14.3, 12.9, 4.4 Hz, 1H), 2.05 (s, 1H), 1.99 – 1.94 (m, 1H), 1.85 (dd, J = 14.5, 2.9 Hz, 1H), 1.53 (d, J = 6.8 Hz, 3H). ¹³C NMR (201 MHz, MeOD) δ 153.78, 137.50, 131.54, 128.28, 127.67, 127.45, 126.96, 116.74, 111.28, 111.16, 106.76, 99.71, 65.04, 54.85, 45.50, 45.38, 38.49, 34.03, 20.81, 18.26. HRMS (ESI) m/z for C₂₄H₃₀N₃O [M+H]. calcd 376.2389, found 376.2389.

6’-Methoxy-1-phenethyl-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3c).

Yield = 50 mg (40 %)¹H NMR (800 MHz, MeOD) δ 7.32 (t, J = 7.5 Hz, 2H), 7.28 (d, J = 7.4 Hz, 2H), 7.24 – 7.11 (m, 2H), 6.92 (d, J = 2.5 Hz, 1H), 6.76 (dd, J = 8.7, 2.5 Hz, 1H), 3.81 (s, 3H), 3.17 (t, J = 5.8 Hz, 2H), 3.00 (d, J = 11.9 Hz, 2H), 2.92 (dd, J = 10.7, 6.0 Hz, 2H), 2.85 (dd, J = 10.8, 6.0 Hz, 2H), 2.80 – 2.61 (m, 4H), 2.20 (td, J = 13.8, 4.4 Hz, 2H), 1.89 (d, J = 14.1 Hz, 2H). ¹³C NMR (201 MHz, MeOD) δ 153.67, 139.20, 138.53, 131.46, 128.34, 128.25, 127.17, 126.05, 111.18, 110.89, 107.14, 99.77, 59.60, 54.89, 51.27, 47.91, 38.56, 33.94, 32.27, 21.47. HRMS (ESI) m/z for C₂₄H₃₀N₃O [M+H]. calcd 376.2389, found 376.2387.

6’-Methoxy-1-(3-phenylpropyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3d).

Yield = 21 mg (16 %)¹H NMR (400 MHz, MeOD) δ 7.38 – 7.23 (m, 4H), 7.20 (dd, J = 8.3, 5.1 Hz, 2H), 6.97 – 6.86 (m, 1H), 6.75 (dt, J = 8.7, 1.7 Hz, 1H), 3.81 (s, 3H), 3.17 (t, J = 5.8 Hz, 2H), 3.01 (d, J = 12.0 Hz, 2H), 2.73 (dt, J = 14.8, 6.8 Hz, 8H), 2.21 (td, J = 13.9, 4.3 Hz, 2H), 2.06 – 1.85 (m, 4H). ¹³C NMR (101 MHz, MeOD) δ 153.71, 141.28, 138.32, 131.47, 128.11, 128.06, 127.16, 125.72, 111.15, 110.95, 107.23, 99.74, 57.14, 54.87, 51.12, 47.93, 38.56, 33.74, 32.98, 27.41, 21.48. HRMS (ESI) m/z for C₂₅H₃₂N₃O [M+H]. calcd 390.2545, found 390.2572.

1-(Furan-2-ylmethyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3e).

Yield = 23 mg (18 %)¹H NMR (400 MHz, CDCl₃) δ 8.13 (s, 1H), 7.41 (s, 1H), 7.20 (dd, J = 8.8, 1.3 Hz, 1H), 6.92 (t, J = 1.8 Hz, 1H), 6.80 (dt, J = 8.7, 1.9 Hz, 1H), 6.37 – 6.30 (m, 2H), 3.84 (d, J = 1.4 Hz, 3H), 3.70 (s, 2H), 3.11 (t, J = 5.7 Hz, 2H), 2.84 (d, J = 11.6 Hz, 2H), 2.71 – 2.64 (m, 4H), 2.24 (td, J = 13.5, 4.2 Hz, 2H), 1.80 (d, J = 13.8 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 154.13, 150.38, 142.80, 140.42, 130.74, 127.70, 111.74, 111.62, 110.48, 110.06, 108.63, 100.60, 56.17, 54.84, 50.47, 48.66, 39.29, 35.91, 23.33. HRMS (ESI) m/z for C₂₁H₂₆N₃O₂ [M+H]. calcd 352.2025, found 352.2024.

6’-Methoxy-1-(pyridin-2-ylmethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3f).

Yield = 118 mg (65 %).¹H NMR (400 MHz, CDCl₃) δ 8.59 (d, J = 4.9 Hz, 1H), 8.34 (s, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 7.17 (d, J = 8.3 Hz, 2H), 6.93 (s, 1H), 6.78 (dd, J = 8.7, 2.6 Hz, 1H), 3.84 (s, 3H), 3.76 (s, 2H), 3.12 (t, J = 5.7 Hz, 2H), 2.77 (d, J = 11.4 Hz, 2H), 2.71 – 2.58 (m, 4H), 2.15 (td, J = 13.4, 12.8, 4.3 Hz, 2H), 1.75 (d, J = 13.6 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 158.30, 153.99, 149.46, 140.95, 136.54, 130.73, 127.70, 123.53, 122.28, 111.65, 111.37, 108.38, 100.51, 64.61, 56.10, 50.63, 49.14, 39.18, 36.19, 23.26. HRMS (ESI) m/z for C₂₂H₂₇N₄O [M+H]. calcd 363.2185, found 363.2188.

6’-Methoxy-1-(pyridin-3-ylmethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3g).

Yield = 61 mg (34 %).¹H NMR (400 MHz, MeOD) δ 8.53 (s, 1H), 8.44 (d, J = 4.9 Hz, 1H), 7.80 (dd, J = 7.8, 2.2 Hz, 1H), 7.39 (dd, J = 7.9, 5.0 Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 6.88 (d, J = 2.3 Hz, 1H), 6.70 (dt, J = 8.8, 1.8 Hz, 1H), 3.78 (s, 3H), 3.58 (s, 2H), 3.02 (t, J = 5.7 Hz, 2H), 2.64 (t, J = 5.6 Hz, 4H), 2.46 (t, J = 12.1 Hz, 2H), 2.06 (tt, J = 14.1, 7.0 Hz, 2H), 1.69 (d, J = 13.7 Hz, 2H). ¹³C NMR (101 MHz, MeOD) δ 153.55, 149.62, 147.56, 139.80, 137.96, 134.14, 131.35, 127.32, 123.72, 111.09, 110.52, 107.03, 99.77, 59.46, 54.91, 50.90, 48.08, 38.43, 34.75, 21.82. HRMS (ESI) m/z for C₂₂H₂₇N₄O [M+H]. calcd 363.2185, found 363.2180.

1-(4-(tert-Butyl)benzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3h).

Yield = 111 mg (53 %). ¹H NMR (400 MHz, CDCl₃) δ 8.52 (br, 1H), 7.32 (s, 4H), 7.22 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.8, 2.6 Hz, 1H), 3.88 (s, 3H), 3.63 (s, 2H), 3.15 (t, J = 5.7 Hz, 2H), 2.82 (d, J = 11.6 Hz, 2H), 2.71 (t, J = 5.7 Hz, 2H), 2.57 (td, J = 12.1, 2.4 Hz, 2H), 2.20 (td, J = 13.4, 4.4 Hz, 2H), 1.79 (s, 2H), 1.34 (s, 9H). ¹³C NMR (101 MHz, CDCl₃) δ 153.97, 150.32, 140.87, 134.19, 130.71, 129.29, 127.67, 125.27, 111.58, 111.39, 108.34, 100.53, 62.95, 56.09, 50.63, 48.87, 39.17, 36.03, 34.51, 31.40, 23.27. HRMS (ESI) m/z for C₂₇H₃₆N₃O [M+H]. calcd 418.2858, found 418.2849.

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[azepane-4,1’-pyrido[3,4-b]indole] (4a).

Yield = 56 mg (76 %)¹H NMR (400 MHz, MeOD) δ 7.48 – 7.31 (m, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.09 – 6.92 (m, 2H), 6.91 (t, J = 1.8 Hz, 1H), 6.75 (dt, J = 8.9, 1.9 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 2H), 3.15 (dp, J = 24.7, 6.3, 5.8 Hz, 2H), 2.75 (h, J = 6.8 Hz, 6H), 2.09 (dtd, J = 44.9, 14.8, 7.3 Hz, 3H), 1.86 (dd, J = 13.8, 6.4 Hz, 1H), 1.72 (p, J = 5.9 Hz, 2H). ¹³C NMR (101 MHz, MeOD) δ 163.37 (dd, J_C-F = 93.9, 12.1 Hz), 160.91 (dd, J_C-F = 94.0, 12.2 Hz), 153.70, 138.27, 132.81 (dd, J_C-F = 9.7, 6.1 Hz), 131.21, 127.05, 121.39 (dd, J_C-F = 14.6, 3.7 Hz), 111.36, 110.90 (dd, J_C-F = 21.2, 3.7 Hz), 110.89, 105.10, 103.44 (t, J_C-F = 26.0 Hz), 99.84, 57.47, 55.62, 54.89, 53.95, 50.44, 38.60, 38.56, 36.75, 24.83, 20.59. HRMS (ESI) m/z for C₂₄H₂₈F₂N₃O [M+H]. calcd 412.2200, found 412.2193.

1’-(2,4-Difluorobenzyl)-6-methoxy-2,3,4,9-tetrahydrospiro[pyrido[3,4-b]indole-1,3’-pyrrolidine] (4b).

Yield = 83 mg (43 %) ¹H NMR (800 MHz, CDCl₃) δ 8.87 (s, 1H), 7.33 (td, J = 8.6, 6.6 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 6.94 (d, J = 2.6 Hz, 1H), 6.89 – 6.84 (m, 2H), 6.83 (dd, J = 8.7, 2.6 Hz, 1H), 3.87 (s, 3H), 3.79 – 3.73 (m, 2H), 3.26 (dt, J = 13.0, 4.6 Hz, 1H), 3.18 – 3.07 (m, 3H), 2.78 – 2.68 (m, 2H), 2.64 (q, J = 9.0 Hz, 1H), 2.53 (d, J = 8.9 Hz, 1H), 2.34 (dt, J = 13.3, 8.5 Hz, 1H), 2.14 (ddd, J = 13.0, 9.2, 3.1 Hz, 1H). ¹³C NMR (201 MHz, CDCl₃) δ 162.58 (dd, J_C-F = 208.3, 11.7 Hz), 161.34 (dd, J_C-F = 208.7, 12.1 Hz), 153.96, 140.73, 132.11–132.03 (m), 130.58, 127.26, 120.86, 111.63, 111.30, 111.21 (dd, J_C-F = 21.3, 3.7 Hz), 106.03, 104.09 (t, J_C-F = 25.7 Hz), 100.49, 65.57, 59.95, 56.05, 52.59, 52.48, 41.80, 39.15, 22.30. HRMS (ESI) m/z for C₂₂H₂₄F₂N₃O [M+H]. calcd 384.1887, found 384.1894.

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-3,1’-pyrido[3,4-b]indole] (4c).

Yield = 85 mg (43 %) ¹H NMR (400 MHz, CDCl₃) δ 9.65 (s, 1H), 7.31 – 7.27 (m, 1H), 7.22 (d, J = 6.7 Hz, 1H), 6.96 (d, J = 2.2 Hz, 1H), 6.90 – 6.76 (m, 3H), 3.88 (s, 3H), 3.60 (q, J = 12.9 Hz, 2H), 3.47 – 3.17 (m, 2H), 3.00 (s, 1H), 2.92 – 2.61 (m, 3H), 2.43 – 2.05 (m, 3H), 1.96 – 1.67 (m, 2H), 1.58 (s, 1H). ¹³C NMR (101 MHz, CDCl₃) δ 163.37 (dd, J_C-F = 84.4, 12.1 Hz), 160.90 (dd, J_C-F = 84.5, 12.3 Hz), 153.79, 139.99, 132.55 (dd, J_C-F = 9.5, 6.2 Hz), 130.43, 127.18, 120.51 (dd, J_C-F = 14.3, 3.7 Hz), 111.74, 111.31, 111.13 (dd, J_C-F = 20.9, 3.7 Hz), 106.14, 104.29 (t, J_C-F = 25.9 Hz), 100.22, 63.38, 56.48, 56.07, 53.34, 52.17, 38.90, 36.31, 22.64, 22.09. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2040.

1-Benzyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)piperidin-4-amine (4d).

Yield = 105 mg (53 %)¹H NMR (400 MHz, CDCl₃) δ 8.37 (s, 1H), 7.34 (q, J = 7.8 Hz, 1H), 7.30 – 7.18 (m, 1H), 7.01 (s, 1H), 6.83 (dt, J = 32.9, 9.1 Hz, 3H), 3.88 (s, 3H), 3.53 (s, 2H), 2.98 (dq, J = 11.1, 6.8 Hz, 4H), 2.84 (d, J = 11.2 Hz, 2H), 2.50 (tt, J = 10.0, 4.3 Hz, 1H), 2.07 (t, J = 11.5 Hz, 2H), 1.86 (d, J = 12.4 Hz, 2H), 1.54 – 1.24 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 162.87 (dd, J_C-F = 77.0, 12.3 Hz), 160.41 (dd, J_C-F = 78.0, 11.8 Hz), 153.88, 132.19 (dd, J_C-F = 9.5, 6.2 Hz), 131.61, 127.85, 122.84, 120.95 (dd, J_C-F = 15.0, 3.7 Hz), 113.56, 112.16, 111.91, 110.96 (dd, J_C-F = 20.9, 3.7 Hz), 103.48 (t, J_C-F = 25.7 Hz), 100.75, 55.97, 54.82, 54.65, 52.15, 46.76, 32.66, 26.02. HRMS (ESI) m/z for C₂₃H₂₈F₂N₃O [M+H]. calcd 400.2200, found 400.2188.

1-(2,4-Difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5a).

Yield = 103 mg (56 %).¹H NMR (400 MHz, CDCl₃) δ 8.94 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.43 – 7.25 (m, 2H), 7.13 (hept, J = 8.4, 7.5 Hz, 2H), 6.84 – 6.61 (m, 2H), 3.66 (s, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.86 – 2.69 (m, 4H), 2.69 – 2.52 (m, 2H), 2.19 (dt, J = 13.0, 7.5 Hz, 2H), 1.73 (d, J = 13.7 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.22 (dd, J_C-F = 91.1, 11.9 Hz), 160.74 (dd, J_C-F = 91.6, 11.9 Hz), 139.72, 135.68, 132.93 (dd, J_C-F = 9.5, 5.9 Hz), 127.30, 121.56, 119.77 (dd, J_C-F = 15.0, 3.7 Hz), 119.18, 118.12, 111.21 (dd, J_C-F = 21.3, 3.7 Hz), 110.99, 108.50, 103.78 (t, J_C-F = 25.7 Hz), 55.07, 50.44, 48.50, 39.14, 35.77, 23.15. HRMS (ESI) m/z for C₂₂H₂₄F₂N₃ [M+H]. calcd 368.1938, found 368.1935.

6’-Bromo-1-(2,4-difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5b).

Yield = 51 mg (54 %) ¹H NMR (400 MHz, CDCl₃) δ 8.83 (s, 1H), 7.60 (d, J = 2.1 Hz, 1H), 7.36 (td, J = 8.7, 6.6 Hz, 1H), 7.19 (dd, J = 8.6, 2.0 Hz, 1H), 7.12 (d, J = 8.5 Hz, 1H), 6.83 – 6.68 (m, 2H), 3.65 (s, 2H), 3.11 (t, J = 5.7 Hz, 2H), 2.77 (dd, J = 8.4, 2.9 Hz, 2H), 2.70 – 2.57 (m, 4H), 2.14 (td, J = 13.3, 4.6 Hz, 2H), 1.72 (dd, J = 14.1, 2.4 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.19 (dd, J_C-F = 93.7, 11.9 Hz), 160.72 (dd, J_C-F = 94.1, 11.9 Hz), 141.14, 134.23, 132.81 (dd, J_C-F = 9.7, 5.7 Hz), 129.14, 124.23, 120.79, 119.76 (dd, J_C-F = 14.9, 3.9 Hz), 112.38, 112.31, 111.19 (dd, J_C-F = 21.1, 3.9 Hz), 108.39, 103.80 (t, J_C-F = 25.9 Hz), 55.12, 50.39, 48.46, 38.99, 35.76, 22.99. HRMS (ESI) m/z for C₂₂H₂₃BrF₂N₃ [M+H]. calcd 446.1043, found 446.1036.

6’-Chloro-1-(2,4-difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5c).

Yield = 134 mg (67 %).¹H NMR (400 MHz, CDCl₃) δ 8.86 – 8.49 (m, 1H), 7.43 (d, J = 9.3 Hz, 2H), 7.21 (d, J = 8.7 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.82 (q, J = 9.0 Hz, 2H), 3.69 (s, 2H), 3.11 (d, J = 5.8 Hz, 2H), 2.81 (d, J = 11.3 Hz, 2H), 2.67 (q, J = 8.5, 8.0 Hz, 4H), 2.18 (q, J = 8.0, 5.3 Hz, 2H), 1.76 (d, J = 13.6 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.25 (dd, J_C-F = 100.2, 12.4 Hz), 160.77 (dd, J_C-F = 100.6, 12.3 Hz), 141.11, 133.91, 132.90 (dd, J_C-F = 9.8, 5.8 Hz), 128.44, 124.90, 121.74, 119.48 (d, J_C-F = 15.4 Hz), 117.69, 111.85, 111.29 (d, J_C-F = 20.7 Hz), 108.51, 103.82 (t, J_C-F = 25.7 Hz), 54.96, 50.36, 48.43, 39.00, 35.72, 22.99. HRMS (ESI) m/z for C₂₂H₂₃ClF₂N₃ [M+H]. calcd 402.1549, found 402.1539.

1-(2,4-Difluorobenzyl)-7’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5d).

Yield = 90 mg (45 %).¹H NMR (400 MHz, CDCl₃) δ 8.24 (s, 1H), 7.48 – 7.32 (m, 2H), 6.94 – 6.68 (m, 4H), 3.82 (s, 3H), 3.67 (s, 2H), 3.13 (t, J = 5.7 Hz, 2H), 2.83 – 2.76 (m, 2H), 2.72 – 2.54 (m, 4H), 2.17 (td, J = 13.5, 13.1, 4.4 Hz, 2H), 1.77 (d, J = 13.7 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 163.17 (dd, J_C-F = 91.6, 11.9 Hz), 160.70 (dd, J_C-F = 92.1, 12.1 Hz), 156.28, 138.34, 136.25, 132.75 (dd, J_C-F = 9.5, 5.9 Hz), 121.77, 119.96 (dd, J_C-F = 14.8, 3.8 Hz), 118.65, 111.20 (dd, J_C-F = 20.8, 3.8 Hz), 108.93, 108.49, 103.74 (t, J_C-F = 25.7 Hz), 94.99, 55.78, 54.99, 50.38, 48.59, 39.15, 35.97, 23.14. HRMS (ESI) m/z for C₂₃H₂₆F₂N₃O [M+H]. calcd 398.2044, found 398.2033.

1-(2,4-Difluorobenzyl)-6’-methoxy-9’-methyl-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5e).

Yield = 47 mg (23 %).¹H NMR (400 MHz, CDCl₃) δ 7.45 (q, J = 8.1 Hz, 1H), 7.20 (d, J = 8.7 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.87 (dtd, J = 28.7, 9.4, 2.5 Hz, 3H), 3.89 (s, 6H), 3.65 (s, 2H), 3.10 (t, J = 5.7 Hz, 2H), 2.72 (tt, J = 18.0, 9.9 Hz, 6H), 2.52 – 2.22 (m, 2H), 1.72 (d, J = 13.7 Hz, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 162.63 (d, J_C-F = 11.9 Hz), 160.15 (d, J_C-F = 12.1 Hz), 153.93, 140.67, 132.74, 132.27 (br), 126.69, 120.93 (br), 111.39, 111.02 (d, J_C-F = 21.6 Hz), 109.59, 109.17, 103.62 (t, J_C-F = 25.7 Hz), 100.23, 56.10, 54.96, 51.63, 48.56, 38.81, 34.79, 32.32, 24.10. HRMS (ESI) m/z for C₂₄H₂₈F₃N₃O [M+H]. calcd 412.2200, found 412.2191.

(+/−)-(2R,11aS)-6’-methoxy-1,2’,3,3’,4,4’,6,9’,11,11a-decahydrospiro[pyrido[1,2-b]isoquinoline-2,1’-pyrido[3,4-b]indole] (6b).

¹H NMR (599 MHz, cdcl₃) δ 8.34 (d, J = 20.8 Hz, 1H), 7.14 (dq, J = 13.2, 6.7, 6.2 Hz, 2H), 7.11 – 6.99 (m, 3H), 6.94 (d, J = 2.6 Hz, 1H), 6.78 (dt, J = 8.7, 2.3 Hz, 1H), 3.86 (s, 4H), 3.52 (d, J = 15.3 Hz, 1H), 3.22 – 3.08 (m, 2H), 2.94 – 2.88 (m, 1H), 2.84 (tq, J = 8.2, 3.1, 2.6 Hz, 1H), 2.77 – 2.72 (m, 3H), 2.69 (t, J = 5.7 Hz, 2H), 2.29 – 2.19 (m, 1H), 2.00 (dt, J = 13.9, 3.1 Hz, 1H), 1.92 – 1.82 (m, 2H). ¹³C NMR (101 MHz, CDCl₃) δ 153.98, 140.49, 133.80, 133.42, 130.74, 128.10, 127.67, 126.40, 126.02, 125.81, 111.62, 111.46, 108.47, 100.46, 57.58, 56.05, 52.91, 51.35, 50.40, 43.76, 39.28, 36.37, 35.64, 23.26. HRMS (ESI) m/z for C₂₄H₂₈N₃O [M+H]. calcd 374.2232, found 374.2222.

Supplementary Material

NIHMS1636025-supplement-1.pdf^{(1.2MB, pdf)}

NIHMS1636025-supplement-2.xlsx^{(11.3KB, xlsx)}

HIGHLIGHTS:

Approximately 10% of cystic fibrosis subject remain without CFTR modulator therapy
Co-potentiators represent a novel class of CFTR modulators
Co-potentiators act in synergy with potentiators to activate rare CFTR mutants
Spiro[piperidine-4,1-pyrido[3,4-b]indole] co-potentiators were optimized by structure-activity studies
Spiro[piperidine-4,1-pyrido[3,4-b]indoles] with EC₅₀ down to 600 nM, an approximately 17-fold improvement, were identified

ACKNOWLEDGEMENTS:

This work was supported by the Cystic Fibrosis Foundation [HAGGIE19R2 and HAGGIE20XX0]; the National Institutes of Health [DK072517, DK099803, DK075302, EY13574]; and the UC Davis Tara K. Telford CF Fund [fellowship to J.S.Z.]. The authors thank Dr. Christine Bear (Hospital for Sick Children, Toronto, Canada) for provision of the gene edited 16HBE14o- cells expressing I1234del-CFTR, Drs. Martin Mense and Hillary Valley (Cystic Fibrosis Foundation Therapeutics Laboratory) for provision of the gene edited 16HBE14o-cells expressing N1303K-CFTR, and Drs. Andras Rab and Jeong Hong (Emory University) for provision of the FRT cell model expressing R347P-CFTR. The authors also thank Dr. Marc Anderson (San Francisco State University) for helpful discussions.

ABBREVIATIONS USED:

CFTR: cystic fibrosis transmembrane conductance regulator
DMF: dimethylformamide
DMSO: dimethyl sulfoxide
FRT: Fischer Rat Thyroid
HPLC: high-performance liquid chromatography
HRMS: high-resolution mass spectrometry
MAD: mean absolute deviation
MMFF: Merck Molecular Force Field
PBS: phosphate-buffered saline
PTCs: premature termination codons
RT: room temperature
TLC: thin layer chromatography

Footnotes

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Supporting Information: ¹H and ¹³C NMR data (PDF) for all assayed compounds, molecular formula strings (CSV), mol2 files of all energetically relevant conformers, Boltzmann distribution analyses of 2a, 2c, 2e, 4a-4c, and 6a-6b, and the computed H¹ coupling constants (J) for 6a and 6b. The Supporting Information is available free of charge on the ACS Publications website at DOI: ####.

Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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12.Abd Rabo M, Mahmoud M and Pagenkopf BL (2010) Synthesis of 5-azaindoles via a cycloaddition reaction between nitriles and donor-acceptor cyclopropanes. Org. Letts 12:3168–3171. [DOI] [PubMed] [Google Scholar]
13.Pictet A and Spengler T (1911) Uber die bildung von isochinolin-derivaten durch einwirkung von methylal auf phenyl-athylamin, phenyl-alanin und tyrosin. Berichte der Deutschen Chemischen Gesellschaft 44:2030–2036. [Google Scholar]
14.Cox ED and Cook JM (1995) The Pictet-Spengler condensation: a new direction for an old reaction. Chem. Rev 95:1797–1842. [Google Scholar]
15.Buchi G and Mak C-P (1977) Nitro olefination of indoles and some substituted benzenes with 1-dimethylamino-2-nitroethylene. J. Org. Chem 42:1784–1786. [Google Scholar]
16.Ranjan P, Ojeda GM, Sharma UK and Van der Eycken EV (2019) Metal-free dearomatization: Direct access to spiroindol(en)ines in batch and continuous-flow. Chem. Eur. J 25:2442–2446. [DOI] [PubMed] [Google Scholar]
17.Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Phys. Chem 98:5648–5652. [Google Scholar]
18.Lee CJ, Wang W and Parr RG (1988) Development of Colle-Selvatti correlation-energy formula into a function of the electron density. Phys. Rev. B: Condens. Matter Maeter. Phys 37:785–789. [DOI] [PubMed] [Google Scholar]
19.Miehlich B, Savin A, Stoll H and Preuss H (1989) Results obtained with the correlation energy density functionals of Becke and Lee, Yang and Parr. Chem. Phys. Lett 157:200–206. [Google Scholar]
20.Cammi R, Mennucci B and Tomasi J, In computational chemistry; Reviews of current trends, in In computational chemistry; Reviews of current trends., Leszczynski J, Editor. 2003, World Scientific Publishing Co. Pte. Ltd.: Singapore. [Google Scholar]
21.Grimme S, Antony J, Ehrlich S and Krieg H (2010) A consistent and accurante ab initio parameterization of density functional dispersion correction (DFT-D) fir the 94 elements H-Pu. J. Chem. Phys 132:154104. [DOI] [PubMed] [Google Scholar]
22.Grimme S, Ehrlich S and Goerigk L (2011) Effect of damping function in dispersion corrected density functional theory. J. Comp. Chem 132:1456. [DOI] [PubMed] [Google Scholar]
23.Lee JK and Tantillo DJ (2003) Computational insights into the mechanism of orotidine monophosphate decarboxylase. Adv. Phys. Org. Chem 38:183–218. [Google Scholar]
24.Jursic BS (1996) Computation of electronic affinities of O and F atoms, and energy profile of F-H2 reaction by densiy functional theory. J. Chem. Phys 104:4151. [Google Scholar]
25.Humphrey W, Dalke A and Schulten K (1996) VMD - Visual Molecular Dynamics. J. Mol. Graphics 14:33–38. [DOI] [PubMed] [Google Scholar]
26.Sharp K and Matschinsky F (2015) Translation of Ludwig Boltzmann’s paper “on the relationship between second fundamental theorem of the mechanical theory of heat and probability calculations regarding teh conditions for thermal equilibrium” Sitzungberichte der Kaiserlichen Akademie der Wissenschaften. Mathematisch-Naturwissen Classe. Abt II, LXXVI 1877, pp 373–435 Entropy 17:1971–2009. [Google Scholar]
27.Xu X, Ge R, Li L, Wang J, Lu X, Xue S, Chen X, Li Z and Bian J (2018) Exploring the tetrahydroisoquinoline thiohydantoin scaffold blockade of the androgen receptor as potent anti-prostate cancer agents. Eur. J. Med. Chem 143:1325–1344. [DOI] [PubMed] [Google Scholar]
28.Harrison JR, O’Brien P, Porter DW and Smith NM (1999) Studies towards the preparation of spartine-like diamines for asymetric synthesis. J. Chem. Soc 24:3623–3631. [Google Scholar]
29.Marenich AV and Truhlar DG (2009) Universal solvation model based on solute electron density and on a continuum model of solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113:6378–6396. [DOI] [PubMed] [Google Scholar]
30.Grimblat N and Sarotti AM (2016) Computational chemistry to the rescue: modern toolboxes for the assignment of complex molecules by GIAO NMR calculations. Chem. Eur. J 22:12246–12261. [DOI] [PubMed] [Google Scholar]
31.Adamo C and Barone V (1998) Exchange functionals with improved long-range behavior and adiabetic connection methods without adjustable parameters: The mPW and mPW1PW models. J. Chem, Phys 108:664–675. [Google Scholar]
32.Lodewyk MW, Siebert MR and Tantillo DJ (2012) Computational prediction of 1H and 13C chemical shifts: a useful tool for natural products, mechanistic, and synthetic organic chemistry. Chem. Rev 112:1839–1862. [DOI] [PubMed] [Google Scholar]
33.Willoughby PH, Jansma MJ and Hoye TR (2014) A guide to small-molecule structure assignment through computation of (1H and 13C) NMR chemical shifts. Nat. Protoc 9:643–660. [DOI] [PubMed] [Google Scholar]
34.Van der Plas SE, Kelgtermans H, De Munck T, Martina SLX, Dropsit S, Quinton E, De Blieck A, Joannesse C, Tomaskovic L, Jans M, Christophe T, Van der Aar E, Borgonovi M, Nelles L, Gees M, Stouten PFW, Van Der Schueren J, Mammoliti O, Conrath K and Andres MJ (2018) Discovery of N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-carboxamide (GLPG1837), a novel potentiator which can open class III mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels to a high extent. J. Med. Chem 61:1425–1435. [DOI] [PubMed] [Google Scholar]
35.Yeh HI, Qui L, Sohma Y, Conrath K, Zou X and Hwang TC (2019) Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770. J. Gen. Physiol 151:912–928. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Liu F, Zhang Z, Levit A, Levring J, Touhara KK, Shoichet BK and Chen J (2019) Structural identification of a hotspot on CFTR for potentiation. Science 364:1184–1188. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Molinski SV, Gonska T, Huan LJ, Baskin B, Janahi IA, Ray PN and Bear CE (2014) Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234V) informs of strategies for future medical intervention. Genetics Med. 16:625–632. [DOI] [PubMed] [Google Scholar]
38.Molinski SV, Ahmadi S, Ip W, Ouyang H, Villella A, Miller JP, Lee P-S, Kulleperuma K, Du K, Di Paola M, Eckford PDW, Laselva O, Huan LJ, Wellhauser L, Li E, Ray PN, Pomes R, Moraes T, Gonska T, Ratjen F and Bear CE (2017) Orkambi and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue. EMBO Mol. Med 9:1224–1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Valley HC, Bukis KM, Bell A, Cheng Y, Wong E, Jordan NJ, Allaire NE, Sivachenko A, Liang F, Bihler H, Thomas PJ, Mahlou J and Mense M (2018) Isogenic cell models of cystic-fibrosis-causing variants in natively expressing pulmonary epithelial cells. J. Cyst. Fibros 18:476–483. [DOI] [PubMed] [Google Scholar]
40.Sharma J, Keeling KM and Rowe SM (2020) Pharmacological approaches fror targeting cystic fibrosis nonsense mutations. Eur. J. Med. Chem 299:epub. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

NIHMS1636025-supplement-1.pdf^{(1.2MB, pdf)}

NIHMS1636025-supplement-2.xlsx^{(11.3KB, xlsx)}

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[R11] 11.Bridges TM, Brady AE, Kennedy JP, Daniels RN, Miller NR, Kim K, Breininger ML, Gentry PR, Brogan JT, Jones CK, Conn PJ and Lindsley CW (2008) Synthesis and SAR of analogues of the M1 allosteric agonist TBPB. Part I: Exploration of alternative benzyl and privileged structure moieties. Bioorg. Med. Chem. Lett 18:5439–5442. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R12] 12.Abd Rabo M, Mahmoud M and Pagenkopf BL (2010) Synthesis of 5-azaindoles via a cycloaddition reaction between nitriles and donor-acceptor cyclopropanes. Org. Letts 12:3168–3171. [DOI] [PubMed] [Google Scholar]

[R13] 13.Pictet A and Spengler T (1911) Uber die bildung von isochinolin-derivaten durch einwirkung von methylal auf phenyl-athylamin, phenyl-alanin und tyrosin. Berichte der Deutschen Chemischen Gesellschaft 44:2030–2036. [Google Scholar]

[R14] 14.Cox ED and Cook JM (1995) The Pictet-Spengler condensation: a new direction for an old reaction. Chem. Rev 95:1797–1842. [Google Scholar]

[R15] 15.Buchi G and Mak C-P (1977) Nitro olefination of indoles and some substituted benzenes with 1-dimethylamino-2-nitroethylene. J. Org. Chem 42:1784–1786. [Google Scholar]

[R16] 16.Ranjan P, Ojeda GM, Sharma UK and Van der Eycken EV (2019) Metal-free dearomatization: Direct access to spiroindol(en)ines in batch and continuous-flow. Chem. Eur. J 25:2442–2446. [DOI] [PubMed] [Google Scholar]

[R17] 17.Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J. Phys. Chem 98:5648–5652. [Google Scholar]

[R18] 18.Lee CJ, Wang W and Parr RG (1988) Development of Colle-Selvatti correlation-energy formula into a function of the electron density. Phys. Rev. B: Condens. Matter Maeter. Phys 37:785–789. [DOI] [PubMed] [Google Scholar]

[R19] 19.Miehlich B, Savin A, Stoll H and Preuss H (1989) Results obtained with the correlation energy density functionals of Becke and Lee, Yang and Parr. Chem. Phys. Lett 157:200–206. [Google Scholar]

[R20] 20.Cammi R, Mennucci B and Tomasi J, In computational chemistry; Reviews of current trends, in In computational chemistry; Reviews of current trends., Leszczynski J, Editor. 2003, World Scientific Publishing Co. Pte. Ltd.: Singapore. [Google Scholar]

[R21] 21.Grimme S, Antony J, Ehrlich S and Krieg H (2010) A consistent and accurante ab initio parameterization of density functional dispersion correction (DFT-D) fir the 94 elements H-Pu. J. Chem. Phys 132:154104. [DOI] [PubMed] [Google Scholar]

[R22] 22.Grimme S, Ehrlich S and Goerigk L (2011) Effect of damping function in dispersion corrected density functional theory. J. Comp. Chem 132:1456. [DOI] [PubMed] [Google Scholar]

[R23] 23.Lee JK and Tantillo DJ (2003) Computational insights into the mechanism of orotidine monophosphate decarboxylase. Adv. Phys. Org. Chem 38:183–218. [Google Scholar]

[R24] 24.Jursic BS (1996) Computation of electronic affinities of O and F atoms, and energy profile of F-H2 reaction by densiy functional theory. J. Chem. Phys 104:4151. [Google Scholar]

[R25] 25.Humphrey W, Dalke A and Schulten K (1996) VMD - Visual Molecular Dynamics. J. Mol. Graphics 14:33–38. [DOI] [PubMed] [Google Scholar]

[R26] 26.Sharp K and Matschinsky F (2015) Translation of Ludwig Boltzmann’s paper “on the relationship between second fundamental theorem of the mechanical theory of heat and probability calculations regarding teh conditions for thermal equilibrium” Sitzungberichte der Kaiserlichen Akademie der Wissenschaften. Mathematisch-Naturwissen Classe. Abt II, LXXVI 1877, pp 373–435 Entropy 17:1971–2009. [Google Scholar]

[R27] 27.Xu X, Ge R, Li L, Wang J, Lu X, Xue S, Chen X, Li Z and Bian J (2018) Exploring the tetrahydroisoquinoline thiohydantoin scaffold blockade of the androgen receptor as potent anti-prostate cancer agents. Eur. J. Med. Chem 143:1325–1344. [DOI] [PubMed] [Google Scholar]

[R28] 28.Harrison JR, O’Brien P, Porter DW and Smith NM (1999) Studies towards the preparation of spartine-like diamines for asymetric synthesis. J. Chem. Soc 24:3623–3631. [Google Scholar]

[R29] 29.Marenich AV and Truhlar DG (2009) Universal solvation model based on solute electron density and on a continuum model of solvent defined by the bulk dielectric constant and atomic surface tensions. J. Phys. Chem. B 113:6378–6396. [DOI] [PubMed] [Google Scholar]

[R30] 30.Grimblat N and Sarotti AM (2016) Computational chemistry to the rescue: modern toolboxes for the assignment of complex molecules by GIAO NMR calculations. Chem. Eur. J 22:12246–12261. [DOI] [PubMed] [Google Scholar]

[R31] 31.Adamo C and Barone V (1998) Exchange functionals with improved long-range behavior and adiabetic connection methods without adjustable parameters: The mPW and mPW1PW models. J. Chem, Phys 108:664–675. [Google Scholar]

[R32] 32.Lodewyk MW, Siebert MR and Tantillo DJ (2012) Computational prediction of 1H and 13C chemical shifts: a useful tool for natural products, mechanistic, and synthetic organic chemistry. Chem. Rev 112:1839–1862. [DOI] [PubMed] [Google Scholar]

[R33] 33.Willoughby PH, Jansma MJ and Hoye TR (2014) A guide to small-molecule structure assignment through computation of (1H and 13C) NMR chemical shifts. Nat. Protoc 9:643–660. [DOI] [PubMed] [Google Scholar]

[R34] 34.Van der Plas SE, Kelgtermans H, De Munck T, Martina SLX, Dropsit S, Quinton E, De Blieck A, Joannesse C, Tomaskovic L, Jans M, Christophe T, Van der Aar E, Borgonovi M, Nelles L, Gees M, Stouten PFW, Van Der Schueren J, Mammoliti O, Conrath K and Andres MJ (2018) Discovery of N-(3-carbamoyl-5,5,7,7-tetramethyl-5,7-dihydro-4H-thieno[2,3-c]pyran-2-yl)-1H-pyrazole-5-carboxamide (GLPG1837), a novel potentiator which can open class III mutant cystic fibrosis transmembrane conductance regulator (CFTR) channels to a high extent. J. Med. Chem 61:1425–1435. [DOI] [PubMed] [Google Scholar]

[R35] 35.Yeh HI, Qui L, Sohma Y, Conrath K, Zou X and Hwang TC (2019) Identifying the molecular target sites for CFTR potentiators GLPG1837 and VX-770. J. Gen. Physiol 151:912–928. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R36] 36.Liu F, Zhang Z, Levit A, Levring J, Touhara KK, Shoichet BK and Chen J (2019) Structural identification of a hotspot on CFTR for potentiation. Science 364:1184–1188. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R37] 37.Molinski SV, Gonska T, Huan LJ, Baskin B, Janahi IA, Ray PN and Bear CE (2014) Genetic, cell biological, and clinical interrogation of the CFTR mutation c.3700 A>G (p.Ile1234V) informs of strategies for future medical intervention. Genetics Med. 16:625–632. [DOI] [PubMed] [Google Scholar]

[R38] 38.Molinski SV, Ahmadi S, Ip W, Ouyang H, Villella A, Miller JP, Lee P-S, Kulleperuma K, Du K, Di Paola M, Eckford PDW, Laselva O, Huan LJ, Wellhauser L, Li E, Ray PN, Pomes R, Moraes T, Gonska T, Ratjen F and Bear CE (2017) Orkambi and amplifier co-therapy improves function from a rare CFTR mutation in gene-edited cells and patient tissue. EMBO Mol. Med 9:1224–1243. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R39] 39.Valley HC, Bukis KM, Bell A, Cheng Y, Wong E, Jordan NJ, Allaire NE, Sivachenko A, Liang F, Bihler H, Thomas PJ, Mahlou J and Mense M (2018) Isogenic cell models of cystic-fibrosis-causing variants in natively expressing pulmonary epithelial cells. J. Cyst. Fibros 18:476–483. [DOI] [PubMed] [Google Scholar]

[R40] 40.Sharma J, Keeling KM and Rowe SM (2020) Pharmacological approaches fror targeting cystic fibrosis nonsense mutations. Eur. J. Med. Chem 299:epub. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

1-BENZYLSPIRO[PIPERIDINE-4,1’-PYRIDO[3,4-B]INDOLE] ‘CO-POTENTIATORS’ FOR MINIMAL FUNCTION CFTR MUTANTS

Jung-Ho Son

Puay-Wah Phuan

Jie S Zhu

Elena Lipman

Amy Cheung

Ka Yi Tsui

Dean J Tantillo

Alan S Verkman

Peter M Haggie

Mark J Kurth

Abstract

Graphical Abstract

INTRODUCTION:

RESULTS:

Chemistry: General synthesis of spiro[piperidine-4,1-pyrido[3,4-b]indoles].

Scheme #1.

Figure 1.

Table 3.

Figure 2.

Figure 3.

Table 1.

Scheme #2.

Figure 4.

Table 2.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

CONCLUSIONS:

MATERIALS AND METHODS:

General Experimental:

Cell culture:

Short-circuit current measurement.

Statistical analysis.

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (8 → 9):

General experimental for the synthesis of N-alkylated piperidin-4-one analogs (11 → 12):

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (10 → 9):

General experimental for the synthesis of 2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indoles] (8 → 10):

General experimental for the synthesis of tryptamines (7 → 8):

Synthesis of 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)acetic acid (16):

Synthesis of methyl 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)acetate (17):

Synthesis of methyl 3-(3-(2-methoxy-2-oxoethyl)-3,4-dihydroisoquinolin-2(1H)-yl)propanoate (18):

Synthesis of 1,3,4,6,11,11a-hexahydro-2H-pyrido[1,2-b]isoquinolin-2-one (19):

1-Benzyl-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1a).

1-(2-Fluoro-4-nitrobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1b).

1-(5-Chloro-2-nitrobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1c).

1-(2,4-Dichlorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1d).

1-(3-Chloro-5-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1e).

1-(3-Chloro-2,6-difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1f).

1-(2-Chlorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1g).

1-(4-Bromo-3-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1h).

1-(4-Bromo-2-fluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1i).

1-(3-Chloro-2,4-difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (1j).

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2a).

1-(2,5-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2b).

1-(3,5-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2c).

6’-Methoxy-1-(3,4,5-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2d).

6’-Methoxy-1-((perfluorophenyl)methyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2e).

1-(3,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2f).

6’-Methoxy-1-(2,3,4-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2g).

1-(2,3-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2h).

6’-Methoxy-1-(2,4,5-trifluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (2i).

6’-Methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3a).

6’-Methoxy-1-(1-phenylethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3b).

6’-Methoxy-1-phenethyl-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3c).

6’-Methoxy-1-(3-phenylpropyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3d).

1-(Furan-2-ylmethyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3e).

6’-Methoxy-1-(pyridin-2-ylmethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3f).

6’-Methoxy-1-(pyridin-3-ylmethyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3g).

1-(4-(tert-Butyl)benzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (3h).

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[azepane-4,1’-pyrido[3,4-b]indole] (4a).

1’-(2,4-Difluorobenzyl)-6-methoxy-2,3,4,9-tetrahydrospiro[pyrido[3,4-b]indole-1,3’-pyrrolidine] (4b).

1-(2,4-Difluorobenzyl)-6’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-3,1’-pyrido[3,4-b]indole] (4c).

1-Benzyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)piperidin-4-amine (4d).

1-(2,4-Difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5a).

6’-Bromo-1-(2,4-difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5b).

6’-Chloro-1-(2,4-difluorobenzyl)-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5c).

1-(2,4-Difluorobenzyl)-7’-methoxy-2’,3’,4’,9’-tetrahydrospiro[piperidine-4,1’-pyrido[3,4-b]indole] (5d).