1-BENZYLSPIRO[PIPERIDINE-4,1′-PYRIDO[3,4-b]indole] ‘co-potentiators’ for minimal function CFTR mutants

Article abstract of DOI:10.1016/j.ejmech.2020.112888

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.

Full text of DOI:10.1016/j.ejmech.2020.112888

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
1-BENZYLSPIRO[PIPERIDINE-4,10-PYRIDO[3,4-b]indole] ‘co-
potentiators’ for minimal function CFTR mutants
,
,
,
Jung-Ho Son a 1, Puay-Wah Phuan b 1, Jie S. Zhu a 2, Elena Lipman a, Amy Cheung a,
Ka Yi Tsui a, Dean J. Tantillo a, Alan S. Verkman b, Peter M. Haggie b **, Mark J. Kurth a
,
, *
a Department of Chemistry, University of California, Davis, CA, 95616, USA
b Departments of Medicine & Physiology, University of California San Francisco, CA 94143, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
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 [piperi-
dine-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 60-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.
Received 19 June 2020
Received in revised form
19 August 2020
Accepted 23 September 2020
Available online xxx
Keywords:
Cystic fibrosis
CFTR
Modulator
Potentiator
N1303K-CFTR
c.3700A>G
© 2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
and elexacaftor/VX-445) and ivacaftor (VX-770) was approved for
CF subjects with at least one F508del-CFTR mutation [2e4]. With
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
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 potenti-
ator called a ‘co-potentiator’, that function in synergy to increase
chloride conductance for CFTR mutations found mainly in nucleo-
tide binding domain 2 [7e9]. 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.
Abbreviations: 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.
* Corresponding author. 1 Shields Avenue, University of California, Davis, CA
95616, USA
** Corresponding author. 513 Parnassus Ave, University of California, San Fran-
cisco, CA, 94143-0521, USA
E-mail addresses: peter.haggie@ucsf.edu (P.M. Haggie), mjkurth@ucdavis.edu
(M.J. Kurth).
1
The authors contributed equally to the study.
Current Affiliation: Department of Chemistry, Stanford University, 337 Campus
2
Drive, Standford, CA 94305.
https://doi.org/10.1016/j.ejmech.2020.112888
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: J.-H. Son, P.-W. Phuan, J.S. Zhu et al., 1-BENZYLSPIRO[PIPERIDINE-4,10-PYRIDO[3,4-b]indole] ‘co-potentiators’ for
minimal function CFTR mutants, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112888

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthetic route to spiro [piperidine-4,10-pyrido [3,4-b]indole] analogs 1e6.
Scheme 2. Synthesis of constrained analog 6.
2. Results
structure-activity relationships (target analogs 1e6) was devised
as outlined in Scheme 1 for the efficient synthesis and modification
of the spiro [piperidine-4,10-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 þ K2CO3) [11,12] piperidin-4-ones (12 where n ¼ 1) to
2.1. Chemistry: General synthesis of spiro[piperidine-4,1-pyrido
[3,4-b]indoles]
A synthetic scheme to enable investigation of co-potentiator
2

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
directly deliver the targeted spiro [piperidine-4,10-pyrido [3,4-b]
indole] 9 by a classical PicteteSpengler 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 ¼ 1)] and then subse-
quently perform selective N1-alkylation of 10 (R2-X in
CH2Cl2 þ 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] d 1H-indole nitro-olefination followed by exhaustive
reduction with LiAlH4 (7 / 8).
Fig. 1 shows the synthesized analogs of the original hit spiro
[piperidine-4,10-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"EC50": (EC50 10 uM/
Vmax 100%), where uM is micromolar using the greek symbol mu
With results from synthetic rounds r1 and r2 in hand, the next
objective (synthesis round r3, Fig. 2) was to more broadly probe the
requisite features of the N1-R3-substituent by preparation of ana-
logs 3a-h. Notably, deletion of R3 (i.e., 3a where R3 ¼ H) gave an
inactive compound and lengthening the N1daryl tether
[-N(CH2)nR3 from n ¼ 1 (1a) to n ¼ 2 (3c) or n ¼ 3 (3d)] greatly
reduced activity. Similarly, adding a methyl substituent to the CH2
tether in 1a (EC5012.6
tivity (3b: EC50 20 M/VMAX 100%). Replacing the phenyl moiety of
1a with a furan ring (3e: EC50 16.4 M/VMAX 67%) reduced activity,
mM/VMAX 54%), giving 3b, also reduced ac-
m
m
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: EC50 11.5
mM/VMAX 107%), but poorly tolerated with the nitrogen at position
three (3g: EC50 17 M/VMAX 56%).
m
Having thoroughly investigated the N1-substituent in these
spiro [piperidine-4,10-pyrido [3,4-b]indoles], attention was next
turned to a probe of the bisheterocyclic spiropiperidine moiety e
synthesis round r4 (Fig. 1) targeting analogs 4a-d. This work began
with the preparation of spiroazepane, spiropyrrolidine, and spi-
ropiperidine analogs where the piperidine ring of 2a was trans-
formed 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 ¼ methoxy at C5) with the appropriate ketoamine (12), directly
giving the targeted 2a analogs 4a-c. Interestingly, all three of these
analogs were inactive. As such, we prepared the piperidine ring-
opened analog 4d.
(10 mM/VMAX 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).
The synthetic work began by preparing aryl analogs 1a-j (syn-
thesis round r1) to investigate the benzylic aryl moiety by appro-
priately N1-alkylating spiro [piperidine-4,10-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 Vmax (Table 3). The EC50
and VMAX of the N1-3-chloro-2,4-difluorobenzyl analog 1j (EC504.5
m
M/120%) motivated us to next prepare a series of fluorobenzyl-
analogs (2a-i) to examine the influence of fluoro-substitution pat-
terns on co-potentiator activity. These round r2 targets (Fig. 1) were
again prepared by selective N1-alkylation of spiro [piperidine-4,10-
pyrido [3,4-b]indole] 10. Increasing the number of fluorine atoms
on the benzyl moiety (e.g., 2a-c/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 VMAX (Table 3: for example, compare 2a versus 2b
and 2c).
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 Spartan 10 (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/6-31þG
(d,p)
[17e20].
Geometry
Fig. 1. Structural variants 1e6 of the spiro [piperidine-4,10-pyrido [3,4-b]indole] scaffold.
3

Table 3
Bioassay results.
4

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 2. Optimized geometries of the lowest energy conformers of 2a, 2c, 2e and 4ae4c.Distances (Å) are measured from the centroid of Ring i, to those of Ring ii and Ring iii. The
lowest energy conformers for 4aec preferred a close from conformation, where one face of Ring i is blocked from potential interactions with the protein interior.
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 Gaussian 16 (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 6-31þG (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)/6-311þG (2d, 2p). B3LYP/6-311þG (2d, 2p) has been re-
ported 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. Fig. 2 shows the
optimized geometries of the lowest energy conformers at B3LYP/6-
31þG (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 (Fig. 2). Assuming that the CFTR binding site
accommodates the Open form better and has residues in the vi-
cinity to interact with the phenyl group of the co-potentiators, the
Open conformers would allow for various interactions between the
of this analysis emphasize the difference in conformational pref-
erences for active and inactive structures.
We also considered that these structures contain amines, which
can be protonated in biological environments. Therefore, we per-
formed conformational analysis on various protonated states for
each structure as well. However, there were no consistent differ-
ences in conformational preferences for active and inactive struc-
tures. This analysis is complicated by the ability for amines to
hydrogen bond with each other within each molecule (see Sup-
porting Information for details), which may or may not occur in
biologically relevant environs.
With exploration of the spiropiperidinepyrido aspects of the
spiro [piperidine-4,10-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-4,10-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 ¼ 1 and
R2 ¼ 2,4-difluorobenzyl). Whereas 5b performed in a comparable
manner to 20, 5a (EC50 8.2 mM/100%) showed mild improvement,
and 5c (EC50 2.5
mM/89%) and 5d (EC50 2.4 mM/100%) were
approximately 4efold 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
p
-system and the CFTR interior. In contrast, most of the energeti-
cally relevant conformers for 4a-4c have one face of Ring i inter-
acting with the rest of the molecule, precluding protein interactions
with it; we refer to these conformers as “Closed” (Fig. 2).
Fig. 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 con-
formers 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
high priority in an attempt to address the question of whether there
1
ꢀ
is a conformational requirement for activity vis-a-vis the N -(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 hy-
drolysis/decarboxylation (Scheme 2) set the stage for a subsequent
PicteteSpengler reaction to delivered tetrahydroisoquinoline-3-
carboxylic acid 13 [27]. Unfortunately, all attempts at the Pictet-
Spengler failed
e an apparent consequence of the electron
5

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 3. Superposition of optimized energetically relevant conformers for 2a, 2c, 2e, 4a-4c.
Table 1
Predicted Boltzmann distributions for Open vs. Closed forms for 2a, 2c, 2e, and 4ae4c at (PCM)chloroform-B3LYP-D3(BJ)//6-31þG (d,p) (left) and (PCM)chloroform-B3LYP-
D3(BJ)//6e331þG (2d,2p) (right).
Structures
Free Energy at PCM-(chloroform)-B3LYP-D3(BJ)//6-31þG (d,p)
Electronic Energy at PCM-(chloroform)-B3LYP-D3(BJ)//6-311þG (2d,2p)
Boltzmann Weight for Open form
Boltzmann Weight for Closed Form
Boltzmann Weight for Open form
Boltzmann Weight for Closed Form
2a
2c
2e
4a
4b
4c
99.5%
98.7%
98.9%
3.7%
6.1%
2.9%
0.5%
1.3%
1.1%
96.3%
93.9%
97.1%
89.6%
96.7%
94.5%
1.7%
7.7%
1.7%
10.4%
3.3%
5.5%
98.3%
92.3%
98.3%
deficient nature of the difluorophenyl ring. In light of this outcome,
our constrained analog study was modified to instead target con-
strained analog 6 (the nor-fluoro analog of 14). Commercially avail-
able 3-amino-4-phenylbutanoic acid (15) successfully participated in
a PicteteSpengler 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 PicteteSpengler reaction [28] of 2-(5-
methoxy-1H-indol-3-yl)ethan-1-amine (8 where R1 ¼ methoxy at
C5) with 19 gave the spiro [piperidine-4,10-pyrido [3,4-b]indole] 6b
as the sole product e i.e., spiro [piperidine-4,10-pyrido [3,4-b]indole]
6a was not obtained.
To confirm the product is indeed 6b, we performed quantum
chemical 1H and 13C NMR calculations on both 6a and 6b (Fig. 4).
Both 6a and 6b were optimized using SMD(chloroform)-B3LYP-
D3(BJ)//6-31þG (d,p) [21,22,29]. Using the gauge-including
atomic orbital (GIAO) [30] method, chemical shift calculations
were performed with SMD(chloroform)-mPW1PW91/6-311þG
(2d,p) [31] on conformers that are within 4 kcal/mol relative to
the lowest energy conformer. The chemical shifts of these ener-
getically relevant conformers were then averaged using Boltzmann
distributions. This is a common procedure for computational NMR
intercept ¼ 186.524) and (slope ¼ ꢀ1.0936, intercept ¼ 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 calcu-
lated 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 (Fig. 4), consistent with formation of the
thermodynamic product during synthesis.
The lowest energy conformer of 6b resembles more closely the
Closed conformers described above, but that of 6a resembles the
studies [30,32,33]. Linear scaling (using slope
¼ ꢀ1.0533,
6

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 4. Structures of 6a and 6b. Based on quantum calculations at SMD(chloroform)-B3LYP-D3(BJ)/6-31þG (d,p). 6b is 2.33 kcal/mol more thermodynamically favorable than 6a.
Table 2
Calculated13C and 1H chemical shifts for 6a and 6b, and their deviation from experiments.
Atom Label Exp. 13 (p.p.m) Calculated 6a shift Dev. Calculated 6b shift Dev. Exp. 1H
C d d (p.p.m) Calculated 6a shift Dev. Calculated 6b shift Dev.
1
2
3
4
5
6
7
8
9
128.10
130.74
111.62
100.46
153.98
108.47
111.46
140.49
23.26
129.08
130.14
109.53
103.28
153.72
103.35
109.05
142.89
24.11
0.98 130.04
0.60 129.69
2.09 109.80
2.82 102.43
0.26 153.66
5.12 103.45
2.41 105.97
2.40 143.23
0.85 24.08
1.94
1.05
1.82 7.06
1.97 6.78
0.32
5.02 6.94
5.49
2.74
0.82 2.71
2.71
2.82 3.08
3.08
0.91
0.83 2.00
1.88
2.13 2.94
2.62
0.93 2.25
1.88
0.27 2.88
0.13 2.62
2.71
e
e
e
e
e
e
e
e
e
e
7.19
6.72
e
0.14 6.79
0.06 6.60
0.26
0.18
e
e
e
e
6.92
e
0.02 6.92
e
0.02
e
e
e
e
e
e
e
e
2.52
2.59
2.94
2.98
e
0.19 2.55
0.12 2.62
0.14 3.07
0.10 3.21
0.16
0.09
0.01
0.13
e
10
43.76
39.56
4.21 40.94
11
12
52.91
26.37
53.93
34.96
1.02 53.82
1.41 37.20
e
e
e
2.02
1.79
3.06
2.38
2.29
1.53
3.26
2.41
4.02
e
0.02 2.25
0.08 1.72
0.12 2.90
0.24 2.66
0.04 2.04
0.34 1.88
0.38 2.76
0.21 2.38
1.31 2.46
0.25
0.15
0.04
0.04
0.21
0.15
0.12
0.24
0.25
e
13
14
50.40
39.28
43.27
40.98
7.13 48.26
1.70 38.35
15
16
56.05
36.64
52.83
29.75
3.22 56.32
5.89 35.51
17
18
133.42
57.58
135.31
56.39
1.89 136.74
1.19 56.81
3.31
0.77 3.52
3.86
e
e
e
3.69
4.22
e
0.17 3.50
0.36 3.86
0.00 0.02
19
20
21
22
23
24
MAD
133.80
126.02
125.81
126.40
127.67
51.35
135.63
126.24
125.98
126.37
128.94
53.17
1.83 136.99
0.22 126.36
0.17 125.82
0.02 126.86
1.27 129.60
1.82 53.14
2.11
3.19
e
e
e
e
0.34 7.06
0.01 7.11
0.46 7.14
1.93 7.06
1.79 3.86
1.71
7.15
7.10
7.12
7.16
3.67
0.09 7.24
0.01 7.14
0.02 7.18
0.10 6.99
0.19 3.56
0.19
0.18
0.03
0.04
0.06
0.30
0.12
*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).
Open conformer, i.e., where Ring i is available for interactions
(Fig. 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 (Fig. 5). For 6a, ~99.3% of
conformers are predicted to be Open, while only 23.2% of con-
formers 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.
(R1), 60-methoxy (2i) was most potent, whereas substituents such
as methoxy at 70 position (5d) showed decreased activity.
Replacement of 60-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
The structural determinants for activity of compound classes
1e6 are summarized in Fig. 6. On the indole aryl 6-member ring
7

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 5. Lowest energy conformers optimized at SMD(chloroform)6-31þG (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.
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 M, respectively (right). We previously defined po-
m
tentiators as either class I compounds, including VX-770 and
GLPG1837 [34], or class II compounds such as the arylsulphona-
midepyrrolopyridine 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], N1303K-CFTR-
expressing FRT cells treated with forskolin and GLPG1837 had
Fig. 6. Structural determinants of spiro [piperidine-4,10-pyrido [3,4-b]indole] scaffold.
similar 2i co-potentiator EC50 of 0.8 0.2 mM (Fig. 7B).
See text for explanation.
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
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 com-
pounds) was significantly more potent than an azepine (4a), and a
pyrrolidine (4b), N-offset piperidine (4c) or ring-opened piperidine
(4d) abolished activity.
activity with EC50 of 0.7
0.2 mM, similar to that found with
N1303K-CFTR. As expected, the R347P-CFTR mutant, which is
mildly responsive to VX-770 but not responsive to arylsulphona-
midepyrrolopyridine 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).
2.2. Biology: Characterization of spiro[piperidine-4,1-pyrido[3,4-b]
indole] co-potentiators
Additional biological studies were performed for the most
potent compounds 2i and 2e. Co-potentiator efficacy was initially
determined by short-circuit current measurements in FRT cells
expressing N1303K-CFTR in the presence of a transepithelial chlo-
ride gradient and with permeabilization of the basolateral cell
membrane such that measured current directly reports CFTR
channel activity. Representative data in Fig. 7A show small in-
creases in CFTR activity following addition of the cAMP agonist
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 pro-
duced CFTRinh-172-inhibitable responses of 16.1 0.4 m
A/cm2 in
8

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 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 M forskolin (fsk), 5 M VX-770, indicated concentration of 2i and 2e, and 10 M CFTRinh-172. (right) Summary of concentration-dependence data
(n ¼ 3, mean S.E.M.). B. Measurements done as in A, but with 20 M GLPG1837 instead of VX-770. C. Short-circuit current in FRT cells expressing I1234del-CFTR in response to
20 M forskolin, 5 M VX-770, indicated concentration of 2i, and 10 M CFTRinh-172. D. Short-circuit current in FRT cells expressing R347P-CFTR in response to 20 M forskolin,
M VX-770, 20 M 2i and 10 M CFTRinh-172. In all studies, cells were corrected with 3 M VX-661 for 18e24 h prior to measurement. All traces are representative of three
replicates.
m
m
m
m
m
m
m
m
5
m
m
m
m
Fig. 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 ( M forskolin, 5 M VX-
Isc; mean S.E.M., n ¼ 3, *P < 0.05). Concentrations: 20
770, 10 M 2i, and 10 M CFTRinh-172.16HBE14o-cell models expressing I1234del-CFTR were corrected with 18 M VX-661 and 3 M VX-445 for 18e24 h prior to measurement.
D
m
m
m
m
m
m
N1303K- (Figs. 8A) and 6.1 0.1
m
A/cm2 in I1234del-CFTR (Fig. 8B)
3. Conclusions
expressing cells, approximately 6-fold and 2-fold greater that
produced by VX-770 alone.
This study extends our prior work that identified a novel class of
CFTR modulators, termed co-potentiators, that act in synergy with
9

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
existing potentiators such as VX-770 and GLPG1837 to increase
chloride channel function of CFTR mutants [7e9]. The objective of
this study was to optimize spiro [piperidine-4,1-pyrido [3,4-b]in-
doles] 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-4,10-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-4,10-
pyrido [3,4-b]indole] 9 by a classical PicteteSpengler 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 þ 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 [7e9]. In addition, albeit to a lesser degree, channel ac-
tivity of the VX-770-responsive G551D mutation can be further
augmented by co-potentiators [7]. Co-potentiators may be benefi-
cial for some PTCs if sufficient transcript is present, or in combi-
nation with future read-through or other therapies [40].
clamp (World Precision Instruments Inc., Sarasota, FL), cells were
cultured on Snapwell clear permeable supports (Corning), and
experiments were performed using HCOꢀ3 -buffered solutions at
37 ꢂC. For studies using FRT cells the basolateral membrane was
permeabilized with 250 mg/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. Com-
parisons 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′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indoles] (8 / 9): Trypt-
amine (8, 0.6 mmol) was mixed with glacial acetic acid (3 mL) in a
10 mL vial and stirred with a magnetic stirrer. Corresponding ke-
tone (12, 0.5 mmol) was added and the vial was sealed with a
plastic cap. The vial was heated at 100 ꢂC 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 16 h. 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′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indoles] (10 / 9): Com-
pound 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 16 h. 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).
In summary, spiro [piperidine-4,1-pyrido [3,4-b]indoles] repre-
sent 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 computa-
tional or structural approaches [35,36], as well as subsequent
rational compound optimization.
4. 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 cen-
troided 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 parame-
ters were 5.5 kV spray voltage, capillary temperature of 275 ꢂC and
sheath gas setting of 20. Spectral data were acquired at a resolution
setting of 100,000 FWHM with the lockmass feature, which typi-
cally results in a mass accuracy <2 ppm.
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].
General experimental for the synthesis of 2′,3′,4′,9′-tetrahy-
drospiro[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 ꢂC 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 ¼ 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 dichloro-
methane. 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/hex-
ane). LiAlH4 (12 mmol) was mixed with THF (75 mL) and cooled
at ꢀ78 ꢂC and stirred. The product from the previous step dissolved
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
10

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
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
39.16, 35.86, 23.23. HRMS (ESI) m/z for C23H28N3O [MþH]. calcd
362.2232, found 362.2229.
1-(2-Fluoro-4-nitrobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1b).
7.92 (s, 1H), 7.57
Yield ¼ 119 mg (56%).1H NMR (400 MHz, CDCl3)
d
(td, J ¼ 5.2, 2.8 Hz, 2H), 7.32e7.18 (m, 2H), 6.95 (d, J ¼ 2.4 Hz, 1H),
6.82 (dd, J ¼ 8.7, 2.5 Hz, 1H), 3.86 (d, J ¼ 11.3 Hz, 5H), 3.14 (t,
J ¼ 5.7 Hz, 2H), 2.69 (t, J ¼ 5.6 Hz, 2H), 2.64e2.54 (m, 4H), 2.01 (ddd,
J ¼ 13.8, 10.3, 6.5 Hz, 2H), 1.79e1.71 (m, 2H). 13C NMR (101 MHz,
(16): L-b-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 ꢂC for 3 h 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.
CDCl3)
d
162.37, 159.88, 154.03, 150.11 (d, JC-F ¼ 8.4 Hz), 140.70,
132.64 (d, JC-F ¼ 7.8 Hz), 130.61, 130.06 (d, JC-F ¼ 3.7 Hz), 127.72,
119.34 (d, JC-F ¼ 20.7 Hz), 112.21 (d, JC-F ¼ 26.4 Hz), 111.51 (d, JC-
¼ 7.6 Hz), 108.51, 100.53, 58.88, 56.06, 50.58, 48.83, 39.07, 36.37,
F
23.15. HRMS (ESI) m/z for C23H26FN4O3 [MþH]. calcd 425.1989,
found 425.1978.
Synthesis of methyl 2-(1,2,3,4-tetrahydroisoquinolin-3-yl)
acetate (17): 16 (1 g, 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 (0.62 g,
3 mmol) was dissolved in dichloromethane (30 mL) and trifluoro-
acetic 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 ꢂC 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]iso-
quinolin-2-one (19): Lithium diisopropylamide (LDA, 1 M solution
in THF/hexane, 10 mL) was cooled at ꢀ78 ꢂC 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 ꢂC 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 ꢂC 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.
1-(5-Chloro-2-nitrobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1c).
7.90 (s, 1H), 7.83
Yield ¼ 128 mg (58%).1H NMR (400 MHz, CDCl3)
d
(d, J ¼ 8.6 Hz, 1H), 7.68 (d, J ¼ 2.3 Hz, 1H), 7.39 (dd, J ¼ 8.6, 2.3 Hz,
1H), 7.23 (d, J ¼ 8.7 Hz, 1H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.83 (dd, J ¼ 8.8,
2.4 Hz, 1H), 3.87 (d, J ¼ 2.6 Hz, 5H), 3.16 (t, J ¼ 5.7 Hz, 2H), 2.70 (t,
J ¼ 5.7 Hz, 2H), 2.67e2.61 (m, 4H), 2.08e1.98 (m, 2H), 1.78 (d,
J ¼ 13.5 Hz, 2H), 1.29 (s, 1H). 13C NMR (101 MHz, CDCl3)
d 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 [MþH]. calcd
441.1693, found 441.1685.
1-(2,4-Dichlorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1d).
7.92 (s, 1H), 7.47
Yield ¼ 156 mg (72%).1H NMR (400 MHz, CDCl3)
d
(d, J ¼ 8.3 Hz, 1H), 7.41 (d, J ¼ 2.2 Hz, 1H), 7.27e7.15 (m, 2H), 6.97 (d,
J ¼ 2.4 Hz,1H), 6.83 (dd, J ¼ 8.7, 2.4 Hz,1H), 3.88 (s, 3H), 3.68 (s, 2H),
3.17 (t, J ¼ 5.6 Hz, 2H), 2.88e2.52 (m, 6H), 2.28e2.00 (m, 2H), 1.80
(d, J ¼ 14.3 Hz, 3H). 13C NMR (101 MHz, CDCl3)
d 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 [MþH]. calcd 430.1453, found
430.1447.
1-(3-Chloro-5-fluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1e).
8.09 (s, 1H),
Yield ¼ 187 mg (91%) 1H NMR (800 MHz, CDCl3)
d
7.25e7.15 (m, 2H), 7.08e7.00 (m, 2H), 6.98 (d, J ¼ 2.7 Hz, 1H), 6.84
(dd, J ¼ 8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.54 (s, 2H), 3.15 (t, J ¼ 5.7 Hz,
2H), 2.71 (q, J ¼ 5.2 Hz, 4H), 2.61e2.46 (m, 2H), 2.10 (td, J ¼ 13.4,
4.2 Hz, 2H), 1.83e1.69 (m, 2H). 13C NMR (201 MHz, CDCl3)
d 163.35,
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 ꢂC 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 so-
lution 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 ¼ 152 mg (84%).1H NMR
162.12, 154.01, 142.79 (d, JC-F ¼ 7.8 Hz), 140.84, 134.76 (d, JC-
¼ 10.4 Hz), 130.65, 127.76, 124.82 (d, JC-F ¼ 2.9 Hz), 114.87 (d, JC-
F
¼ 24.9 Hz), 114.27 (d, JC-F ¼ 21.4 Hz), 111.52 (d, JC-F ¼ 18.3 Hz),
F
108.56, 100.57, 62.25, 56.07, 50.58, 48.91, 39.09, 36.33, 23.21. HRMS
(ESI) m/z for C23H26ClFN3O [MþH]. calcd 414.1748, found 414.1740.
1-(3-Chloro-2,6-difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetra-
hydrospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1f).
8.34 (s, 1H), 7.30
Yield ¼ 183 mg (85%) 1H NMR (400 MHz, CDCl3)
d
(h, J ¼ 5.4 Hz, 1H), 7.16 (d, J ¼ 8.8 Hz, 1H), 6.96 (d, J ¼ 2.6 Hz, 1H),
6.87e6.72 (m, 2H), 3.88 (s, 3H), 3.76 (s, 2H), 3.13 (t, J ¼ 5.7 Hz, 2H),
2.79 (d, J ¼ 11.4 Hz, 2H), 2.73e2.56 (m, 4H), 2.10 (td, J ¼ 13.3, 4.7 Hz,
2H), 1.77 (d, J ¼ 12.8 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 160.35
(dd, JC-F ¼ 248.7, 7.0 Hz), 157.24 (dd, JC-F ¼ 250.2, 8.4 Hz), 153.93,
(400 MHz, CDCl3)
d
8.30 (d, J ¼ 35.1 Hz, 1H), 7.43 (d, J ¼ 7.2 Hz, 2H),
140.88, 130.71, 129.69 (d, JC-F ¼ 9.5 Hz), 127.70, 116.55 (dd, JC-
7.39e7.27 (m, 3H), 7.22 (d, J ¼ 8.7 Hz, 1H), 6.96 (d, J ¼ 2.5 Hz, 1H),
6.82 (dt, J ¼ 8.9, 1.8 Hz, 1H), 3.87 (s, 3H), 3.69 (s, 2H), 3.15 (t,
J ¼ 5.6 Hz, 2H), 2.96e2.77 (m, 2H), 2.77e2.53 (m, 4H), 2.25 (td,
J ¼ 13.4, 4.4 Hz, 2H), 1.79 (d, J ¼ 13.7 Hz, 2H). 13C NMR (101 MHz,
¼ 19.1, 4.0 Hz), 114.91 (t, JC-F ¼ 20.2 Hz), 111.83 (dd, JC-F ¼ 24.6,
F
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 [MþH]. calcd
432.1654, found 432.1655.
CDCl3)
d
153.99, 140.62, 136.72, 130.69, 129.67, 128.46, 127.64,
1-(2-Chlorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahydrospiro
[piperidine-4,1′-pyrido[3,4-b]indole] (1g). Yield ¼ 126 mg (64%)
127.60, 111.62, 111.44, 108.42, 100.50, 63.09, 56.07, 50.52, 48.80,
11

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
1H NMR (400 MHz, CDCl3)
d
8.13 (s,1H), 7.54 (dd, J ¼ 7.4, 2.3 Hz,1H),
1-(2,5-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
7.41 (dd, J ¼ 7.5, 1.9 Hz, 1H), 7.26 (qd, J ¼ 7.4, 6.6, 2.1 Hz, 2H),
7.22e7.13 (m, 1H), 6.98 (d, J ¼ 2.7 Hz, 1H), 6.84 (dd, J ¼ 8.8, 2.6 Hz,
1H), 3.89 (s, 3H), 3.74 (s, 2H), 3.17 (t, J ¼ 5.7 Hz, 2H), 2.82 (dt, J ¼ 12.1,
3.7 Hz, 2H), 2.74e2.58 (m, 4H), 2.14 (td, J ¼ 13.1, 4.6 Hz, 2H), 1.80
(2b).
Yield ¼ 173 mg (87%) 1H NMR (400 MHz, CDCl3)
d
8.12e7.83 (m,
1H), 7.19 (d, J ¼ 8.8 Hz, 2H), 7.11e6.90 (m, 3H), 6.86 (d, J ¼ 8.8 Hz,
1H), 3.90 (s, 3H), 3.64 (s, 2H), 3.16 (d, J ¼ 5.8 Hz, 2H), 2.91e2.68 (m,
4H), 2.62 (t, J ¼ 11.8 Hz, 2H), 2.24e1.98 (m, 2H), 1.81 (d, J ¼ 13.6 Hz,
(dd, J ¼ 13.9, 2.4 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 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 [MþH]. calcd 396.1843,
found 396.1836.
2H). 13C NMR (101 MHz, CDCl3)
d
159.22 (dd, JC-F ¼ 140.6, 2.3 Hz),
156.81 (dd, JC-F ¼ 140.5, 2.3 Hz), 154.04, 140.95, 130.65, 127.81,
127.28 (dd, JC-F ¼ 17.0, 7.3 Hz), 117.38 (dd, JC-F ¼ 24.1, 4.9 Hz), 116.26
(dd, JC-F ¼ 25.2, 8.5 Hz),114.99 (dd, JC-F ¼ 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 [MþH]. calcd 398.2044, found 398.2031.
1-(3,5-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
1-(4-Bromo-3-fluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1h).
Yield ¼ 155 mg (68%) 1H NMR (400 MHz, CDCl3)
d
8.00 (s, 1H), 7.48
(dd, J ¼ 8.2, 7.1 Hz, 1H), 7.21 (dd, J ¼ 9.1, 2.9 Hz, 2H), 7.07e6.93 (m,
2H), 6.85 (dd, J ¼ 8.7, 2.5 Hz, 1H), 3.89 (s, 3H), 3.54 (s, 2H), 3.17 (t,
J ¼ 5.7 Hz, 2H), 2.79e2.65 (m, 4H), 2.63e2.44 (m, 2H), 2.10 (td,
J ¼ 13.2, 12.8, 4.4 Hz, 2H), 1.88e1.70 (m, 2H). 13C NMR (101 MHz,
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2c).
Yield ¼ 113 mg (57%) 1H NMR (800 MHz, CDCl3)
d
7.85 (s, 1H), 7.24
(d, J ¼ 8.7 Hz, 1H), 7.00e6.92 (m, 3H), 6.84 (dd, J ¼ 8.7, 2.4 Hz, 1H),
6.73 (tt, J ¼ 8.9, 2.4 Hz, 1H), 3.88 (s, 3H), 3.58 (s, 2H), 3.16 (t,
J ¼ 5.7 Hz, 2H), 2.78e2.66 (m, 4H), 2.57 (td, J ¼ 12.0, 2.4 Hz, 2H), 2.12
(td, J ¼ 13.3, 4.3 Hz, 2H), 1.80 (dd, J ¼ 14.1, 2.6 Hz, 2H). 13C NMR
CDCl3)
d
160.33, 157.87, 154.04, 140.89, 140.84 (d, JC-F ¼ 6.6 Hz),
133.17, 130.64, 127.80, 125.78 (d, JC-F ¼ 3.3 Hz), 116.90 (d, JC-
¼ 22.0 Hz), 111.51 (d, JC-F ¼ 4.4 Hz), 108.62, 107.20 (d, JC-
(201 MHz, CDCl3)
¼ 12.5 Hz), 154.08, 140.74, 130.55, 127.74, 111.52, 111.51 (dd, JC-
¼ 20.9, 4.0 Hz), 111.49, 108.64, 102.48 (t, JC-F ¼ 25.5 Hz), 100.52,
d
163.66 (d, JC-F ¼ 12.9 Hz), 162.43 (d, JC-
F
¼ 20.9 Hz), 100.61, 62.20, 56.10, 50.62, 48.92, 39.11, 36.42, 23.23.
F
F
HRMS (ESI) m/z for C23H26BrFN3O [MþH]. calcd 458.1243, found
F
458.1239.
62.34, 56.06, 50.54, 48.88, 39.09, 36.39, 23.21. HRMS (ESI) m/z for
1-(4-Bromo-2-fluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
C
23H26F2N3O [MþH]. calcd 398.2044, found 398.2030.
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1i).
6′-Methoxy-1-(3,4,5-trifluorobenzyl)-2′,3′,4′,9′-tetrahy-
Yield ¼ 190 mg (83%) 1H NMR (400 MHz, CDCl3)
d
8.59 (s, 1H), 7.24
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
Yield ¼ 159 mg (77%).1H NMR (400 MHz, CDCl3)
(2d).
(t, J ¼ 8.1 Hz,1H), 7.15 (dd, J ¼ 7.9, 2.6 Hz, 3H), 6.98 (d, J ¼ 2.6 Hz,1H),
6.81 (dd, J ¼ 8.7, 2.5 Hz, 1H), 3.88 (s, 3H), 3.60 (s, 2H), 3.14 (t,
J ¼ 5.7 Hz, 2H), 2.72 (q, J ¼ 7.2, 5.7 Hz, 4H), 2.59 (td, J ¼ 12.1, 2.5 Hz,
2H), 2.10 (td, J ¼ 13.5, 4.6 Hz, 2H), 1.69 (s, 2H). 13C NMR (101 MHz,
d
7.76 (s, 1H), 7.23
(d, J ¼ 8.6 Hz,1H), 7.04 (t, J ¼ 7.5 Hz, 2H), 6.96 (d, J ¼ 2.4 Hz,1H), 6.84
(dt, J ¼ 8.7, 1.8 Hz, 1H), 3.87 (s, 3H), 3.52 (s, 2H), 3.16 (t, J ¼ 5.7 Hz,
2H), 2.71 (q, J ¼ 5.3 Hz, 4H), 2.56 (t, J ¼ 11.8 Hz, 2H), 2.10 (td, J ¼ 13.2,
4.3 Hz, 2H), 1.81 (d, J ¼ 13.7 Hz, 2H). 13C NMR (201 MHz, CDCl3)
CDCl3)
d
162.50, 160.00, 153.93, 140.87, 132.96 (d, JC-F ¼ 5.1 Hz),
130.73, 127.72, 127.29 (d, JC-F ¼ 3.7 Hz), 123.77 (d, JC-F ¼ 15.0 Hz),
121.33 (d, JC-F ¼ 9.5 Hz), 119.03 (d, JC-F ¼ 25.3 Hz), 111.47 (d, JC-
d
154.07, 151.14 (ddd, JC-F
¼
249.5, 10.1, 3.7 Hz), 140.68,
139.34e138.02 (m), 135.07, 130.58, 127.74, 112.54 (dd, JC-F ¼ 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 [MþH]. calcd
416.1950, found 416.1939.
¼ 17.2 Hz), 108.44, 100.56, 56.08, 55.35, 50.50, 48.72, 39.13, 36.03,
F
23.25. HRMS (ESI) m/z for C23H26BrFN3O [MþH]. calcd 458.1243,
found 458.1228.
1-(3-Chloro-2,4-difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetra-
6′-Methoxy-1-((perfluorophenyl)methyl)-2′,3′,4′,9′-tetrahy-
hydrospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(1j).
8.10 (s, 1H),
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2e)¶.
7.73 (s, 1H), 7.19
Yield ¼ 124 mg (58%) 1H NMR (400 MHz, CDCl3)
d
Yield ¼ 202 mg (90%).1H NMR (400 MHz, CDCl3)
d
7.36e7.24 (m, 1H), 7.17 (s, 1H), 7.01e6.87 (m, 2H), 6.83 (dd, J ¼ 8.7,
2.5 Hz, 1H), 3.88 (s, 3H), 3.65 (s, 2H), 3.15 (t, J ¼ 5.7 Hz, 2H),
2.80e2.66 (m, 4H), 2.60 (td, J ¼ 11.9, 2.6 Hz, 2H), 2.10 (td, J ¼ 13.4,
(d, J ¼ 8.7 Hz, 1H), 6.95 (t, J ¼ 1.8 Hz,1H), 6.83 (dt, J ¼ 8.7, 1.9 Hz,1H),
3.87 (d, J ¼ 1.4 Hz, 3H), 3.79 (d, J ¼ 2.4 Hz, 2H), 3.13 (t, J ¼ 5.7 Hz,
2H), 2.76 (d, J ¼ 11.1 Hz, 2H), 2.72e2.61 (m, 4H), 2.06 (td, J ¼ 13.1,
4.5 Hz, 2H), 1.80 (d, J ¼ 13.6 Hz, 2H). 13C NMR (201 MHz, CDCl3)
4.6 Hz, 2H), 1.87e1.67 (m, 2H).13C NMR (101 MHz, CDCl3)
d 158.08
(dd, JC-F ¼ 250.1, 2.9 Hz), 157.44 (d, JC-F ¼ 251.6, 2.9 Hz), 154.01,
d
154.03, 145.63 (d, JC-F ¼ 248.0 Hz), 140.70, 140.59 (d, JC-
¼ 254.2 Hz), 137.39 (d, JC-F ¼ 251.6 Hz), 130.54, 127.73,111.47,
140.77, 130.62, 129.34 (dd, JC-F ¼ 9.0, 5.7 Hz), 127.74, 121.95 (dd, JC-
F
¼ 15.0, 4.0 Hz),111.50,111.46,111.44 (dd, JC-F ¼ 20.6, 4.4 Hz),109.77
111.46, 110.75 (t, JC-F ¼ 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 [MþH].
calcd 452.1761, found 452.1753.
F
(t, JC-F ¼ 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 [MþH]. calcd
432.1654, found 432.1646.
1-(3,4-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole] (2f). Yield ¼ 88 mg
1-(2,4-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2a).
7.85 (s, 1H), 7.37
(44%).1H NMR (400 MHz, CDCl3)
d 8.15e7.91 (m, 1H), 7.25 (dt,
Yield ¼ 138 mg (70%) 1H NMR (400 MHz, CDCl3)
d
J ¼ 16.3, 7.7 Hz, 2H), 7.17e7.03 (m, 2H), 6.97 (s, 1H), 6.84 (dd, J ¼ 8.8,
2.4 Hz, 1H), 3.88 (s, 3H), 3.55 (s, 2H), 3.16 (t, J ¼ 5.7 Hz, 2H), 2.72 (q,
J ¼ 8.2, 6.0 Hz, 4H), 2.55 (t, J ¼ 11.1 Hz, 2H), 2.11 (td, J ¼ 13.5, 4.8 Hz,
(q, J ¼ 7.9 Hz, 1H), 7.18 (d, J ¼ 8.7 Hz, 1H), 6.94 (s, 1H), 6.82 (ddt,
J ¼ 14.4, 8.8, 5.4 Hz, 3H), 3.85 (s, 3H), 3.61 (s, 2H), 3.13 (t, J ¼ 5.7 Hz,
2H), 2.75 (d, J ¼ 11.4 Hz, 2H), 2.67 (t, J ¼ 5.8 Hz, 2H), 2.57 (t,
J ¼ 11.7 Hz, 2H), 2.07 (td, J ¼ 13.2, 4.3 Hz, 2H), 1.79 (s, 2H). 13C NMR
2H),1.81 (s, 2H). 13C NMR (101 MHz, CDCl3)
d 154.05, d 151.15 (dd, JC-
¼ 78.7, 12.7 Hz), 148.69 (dd, JC-F ¼ 78.1, 12.5 Hz), 140.79, 135.47,
F
(101 MHz, CDCl3)
d
, 161.14e154.80 (m), 140.93e128.43 (m), 120.82
130.62, 127.76, 124.91, 117.80 (d, JC-F ¼ 17.2 Hz), 116.90 (d, JC-
(dd, J ¼ 14.8, 3.9 Hz), 111.57e108.50 (m), 103.92e22.26 (m). 13C
¼ 17.2 Hz), 111.52, 111.48, 108.59, 100.57, 62.14, 56.07, 50.62, 48.80,
F
NMR (101 MHz, CDCl3)
d
163.05 (dd, JC-F ¼ 77.9, 11.9 Hz), 160.58 (dd,
39.10, 36.31, 23.21. HRMS (ESI) m/z for C23H26F2N3O [MþH]. calcd
JC-F ¼ 79.0, 12.0 Hz), 154.90, 154.04, 140.83, 132.40 (dd, JC-F ¼ 9.6,
6.2 Hz), 130.53, 128.52, 127.75, 120.82 (dd, JC-F ¼ 14.8, 3.9 Hz), 111.04
(dd, JC-F ¼ 20.9, 3.7 Hz), 108.60, 103.56 (t, JC-F ¼ 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 [MþH]. calcd
398.2044, found 398.2036.
398.2044, found 398.2032.
6′-Methoxy-1-(2,3,4-trifluorobenzyl)-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2g)¶.
8.24 (s, 1H), 7.18
Yield ¼ 200 mg (96%) 1H NMR (400 MHz, CDCl3)
d
(d, J ¼ 8.8 Hz, 1H), 7.15e7.03 (m, 1H), 6.99e6.86 (m, 2H), 6.85e6.67
(m, 1H), 3.87 (d, J ¼ 2.9 Hz, 3H), 3.68e3.59 (m, 2H), 3.13 (t,
12

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
J ¼ 5.7 Hz, 2H), 2.77e2.65 (m, 4H), 2.65e2.50 (m, 2H), 2.16e1.98 (m,
51.27, 47.91, 38.56, 33.94, 32.27, 21.47. HRMS (ESI) m/z for
2H), 1.85e1.71 (m, 2H). 13C NMR (101 MHz, CDCl3)
d
153.99, 150.40
C
24H30N3O [MþH]. calcd 376.2389, found 376.2387.
(ddd, JC-F ¼ 250.2, 9.9, 2.6 Hz), 150.20 (d, JC-F ¼ 250.4, 9.5, 3.0 Hz),
140.80, 139.88 (dt, JC-F ¼ 251.8, 15.6 Hz), 130.65, 127.72, 124.92 (ddd,
JC-F ¼ 8.5, 6.3, 3.2 Hz), 122.42 (dd, JC-F ¼ 12.1, 2.8 Hz), 111.71 (dd, JC-
6′-Methoxy-1-(3-phenylpropyl)-2′,3′,4′,9′-tetrahydrospiro
[piperidine-4,1′-pyrido[3,4-b]indole] (3d). Yield ¼ 21 mg (16%)1H
NMR (400 MHz, MeOD)
d
7.38e7.23 (m, 4H), 7.20 (dd, J ¼ 8.3, 5.1 Hz,
¼ 17.1, 3.9 Hz), 111.51, 111.42, 108.52, 100.53, 56.05, 55.14, 50.49,
2H), 6.97e6.86 (m, 1H), 6.75 (dt, J ¼ 8.7, 1.7 Hz, 1H), 3.81 (s, 3H), 3.17
(t, J ¼ 5.8 Hz, 2H), 3.01 (d, J ¼ 12.0 Hz, 2H), 2.73 (dt, J ¼ 14.8, 6.8 Hz,
8H), 2.21 (td, J ¼ 13.9, 4.3 Hz, 2H), 2.06e1.85 (m, 4H). 13C NMR
F
48.58, 39.08, 36.21, 23.20. HRMS (ESI) m/z for C23H24F3N3O [MþH].
calcd 416.1950, found 416.1941.
1-(2,3-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
(101 MHz, MeOD) d 153.71, 141.28, 138.32, 131.47, 128.11, 128.06,
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2h).
7.85 (s, 1H), 7.20
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
[MþH]. calcd 390.2545, found 390.2572.
Yield ¼ 184 mg (93%) 1H NMR (400 MHz, CDCl3)
d
(d, J ¼ 8.8 Hz, 2H), 7.14e7.02 (m, 2H), 6.96 (d, J ¼ 2.4 Hz, 1H), 6.83
(dd, J ¼ 8.8, 2.4 Hz, 1H), 3.88 (s, 3H), 3.70 (d, J ¼ 1.7 Hz, 2H), 3.15 (t,
J ¼ 5.7 Hz, 2H), 2.85e2.73 (m, 2H), 2.70 (t, J ¼ 5.7 Hz, 2H), 2.60 (td,
J ¼ 11.9, 2.7 Hz, 2H), 2.16e1.99 (m, 2H), 1.80 (dq, J ¼ 14.1, 2.9 Hz, 2H).
1-(Furan-2-ylmethyl)-6′-methoxy-2′,3′,4′,9′-tetrahydrospiro
[piperidine-4,1′-pyrido[3,4-b]indole] (3e). Yield ¼ 23 mg (18%)1H
NMR (400 MHz, CDCl3)
d
8.13 (s, 1H), 7.41 (s, 1H), 7.20 (dd, J ¼ 8.8,
13C NMR (101 MHz, CDCl3)
d
154.06, 151.26 (dd, JC-F ¼ 119.9,
1.3 Hz, 1H), 6.92 (t, J ¼ 1.8 Hz, 1H), 6.80 (dt, J ¼ 8.7, 1.9 Hz, 1H),
6.37e6.30 (m, 2H), 3.84 (d, J ¼ 1.4 Hz, 3H), 3.70 (s, 2H), 3.11 (t,
J ¼ 5.7 Hz, 2H), 2.84 (d, J ¼ 11.6 Hz, 2H), 2.71e2.64 (m, 4H), 2.24 (td,
J ¼ 13.5, 4.2 Hz, 2H), 1.80 (d, J ¼ 13.8 Hz, 2H). 13C NMR (101 MHz,
12.8 Hz), 148.79 (dd, JC-F ¼ 119.9, 12.8 Hz), 140.86, 130.54, 127.77,
127.62 (d, JC-F ¼ 11.4 Hz), 126.18 (t, JC-F ¼ 3.5 Hz), 123.68 (dd, JC-
¼ 6.6, 4.8 Hz), 116.10, 115.93, 111.47, 108.62, 100.55, 56.06, 55.41,
F
50.52, 48.74, 39.10, 36.43, 23.21. HRMS (ESI) m/z for C23H26F2N3O
[MþH]. calcd 398.2044, found 398.2037.
CDCl3) d 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,
6′-Methoxy-1-(2,4,5-trifluorobenzyl)-2′,3′,4′,9′-tetrahy-
35.91, 23.33. HRMS (ESI) m/z for
352.2025, found 352.2024.
C
21H26N3O2 [MþH]. calcd
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(2i).
Yield ¼ 193 mg (93%) 1H NMR (400 MHz, CDCl3)
d
8.06 (s, 1H), 7.30
6′-Methoxy-1-(pyridin-2-ylmethyl)-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(ddd, J ¼ 10.7, 8.9, 6.6 Hz, 1H), 7.19 (d, J ¼ 8.6 Hz, 1H), 6.98 (d,
J ¼ 2.4 Hz, 1H), 6.95e6.86 (m, 1H), 6.84 (dd, J ¼ 8.7, 2.5 Hz, 1H), 3.89
(s, 3H), 3.59 (d, J ¼ 1.3 Hz, 2H), 3.16 (t, J ¼ 5.7 Hz, 2H), 2.81e2.66 (m,
4H), 2.60 (dd, J ¼ 23.9, 2.7 Hz, 2H), 2.10 (td, J ¼ 13.7, 4.6 Hz, 2H), 1.80
(3f).
Yield ¼ 118 mg (65%).1H NMR (400 MHz, CDCl3)
8.59 (d, J ¼ 4.9 Hz,
d
1H), 8.34 (s, 1H), 7.63 (t, J ¼ 7.7 Hz, 1H), 7.41 (d, J ¼ 7.8 Hz, 1H), 7.17
(d, J ¼ 8.3 Hz, 2H), 6.93 (s, 1H), 6.78 (dd, J ¼ 8.7, 2.6 Hz, 1H), 3.84 (s,
3H), 3.76 (s, 2H), 3.12 (t, J ¼ 5.7 Hz, 2H), 2.77 (d, J ¼ 11.4 Hz, 2H),
2.71e2.58 (m, 4H), 2.15 (td, J ¼ 13.4, 12.8, 4.3 Hz, 2H), 1.75 (d,
(dd, J ¼ 14.1, 2.7 Hz, 2H).13C NMR (101 MHz, CDCl3)
d 156.20 (ddd, JC-
¼ 247.5, 10.1, 2.2 Hz), 154.03, 149.1 (ddd, JC-F ¼ 251.7, 14.9, 14.0),
F
146.8 (ddd, JC-F ¼ 245.3, 12.5, 4.0), 140.79, 130.64, 127.76, 121.72 (dt,
JC-F ¼ 16.9, 4.6 Hz), 118.72 (dd, JC-F ¼ 18.9, 5.7 Hz), 111.52, 111.47,
108.60, 105.31 (dd, JC-F ¼ 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 [MþH].
calcd 416.1950, found 416.1940.
J ¼ 13.6 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 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 [MþH]. calcd 363.2185, found 363.2188.
6′-Methoxy-1-(pyridin-3-ylmethyl)-2′,3′,4′,9′-tetrahy-
6′-Methoxy-2′,3′,4′,9′-tetrahydrospiro[piperidine-4,1′-pyr-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole] (3g). Yield ¼ 61 mg
ido[3,4-b]indole] (3a). 1H NMR (400 MHz, MeOD)
d
7.18 (d,
(34%).1H NMR (400 MHz, MeOD)
d
8.53 (s, 1H), 8.44 (d, J ¼ 4.9 Hz,
J ¼ 8.6 Hz, 1H), 6.90 (d, J ¼ 2.4 Hz, 1H), 6.72 (dd, J ¼ 8.7, 2.5 Hz, 1H),
3.80 (s, 3H), 3.11e2.94 (m, 4H), 2.84 (dt, J ¼ 12.7, 3.9 Hz, 2H), 2.66 (t,
J ¼ 5.7 Hz, 2H),1.96 (td, J ¼ 13.5, 4.7 Hz, 2H),1.72 (d, J ¼ 12.8 Hz, 2H).
1H), 7.80 (dd, J ¼ 7.8, 2.2 Hz,1H), 7.39 (dd, J ¼ 7.9, 5.0 Hz,1H), 7.15 (d,
J ¼ 8.9 Hz, 1H), 6.88 (d, J ¼ 2.3 Hz, 1H), 6.70 (dt, J ¼ 8.8, 1.8 Hz, 1H),
3.78 (s, 3H), 3.58 (s, 2H), 3.02 (t, J ¼ 5.7 Hz, 2H), 2.64 (t, J ¼ 5.6 Hz,
4H), 2.46 (t, J ¼ 12.1 Hz, 2H), 2.06 (tt, J ¼ 14.1, 7.0 Hz, 2H), 1.69 (d,
13C NMR (101 MHz, MeOD)
d 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 [MþH]. 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 ¼ 44 mg (36%)1H
J ¼ 13.7 Hz, 2H). 13C NMR (101 MHz, MeOD)
d 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 [MþH]. calcd 363.2185, found 363.2180.
NMR (800 MHz, MeOD)
d
7.45e7.42 (m, 2H), 7.39 (t, J ¼ 7.7 Hz, 2H),
1-(4-(tert-Butyl)benzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
7.34e7.30 (m, 1H), 7.21 (d, J ¼ 8.8 Hz, 1H), 6.92 (d, J ¼ 2.4 Hz, 1H),
6.76 (dd, J ¼ 8.7, 2.4 Hz,1H), 3.81 (s, 3H), 3.21 (td, J ¼ 6.0, 2.8 Hz, 2H),
3.18e3.15 (m, 1H), 2.82 (s, 1H), 2.80e2.76 (m, 2H), 2.65 (s, 1H), 2.56
(s, 1H), 2.32 (ddd, J ¼ 14.4, 12.8, 4.5 Hz, 1H), 2.21 (ddd, J ¼ 14.3, 12.9,
4.4 Hz, 1H), 2.05 (s, 1H), 1.99e1.94 (m, 1H), 1.85 (dd, J ¼ 14.5, 2.9 Hz,
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(3h).
8.52 (br, 1H), 7.32
Yield ¼ 111 mg (53%). 1H NMR (400 MHz, CDCl3)
d
(s, 4H), 7.22 (d, J ¼ 8.8 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.82 (dd,
J ¼ 8.8, 2.6 Hz, 1H), 3.88 (s, 3H), 3.63 (s, 2H), 3.15 (t, J ¼ 5.7 Hz, 2H),
2.82 (d, J ¼ 11.6 Hz, 2H), 2.71 (t, J ¼ 5.7 Hz, 2H), 2.57 (td, J ¼ 12.1,
2.4 Hz, 2H), 2.20 (td, J ¼ 13.4, 4.4 Hz, 2H), 1.79 (s, 2H), 1.34 (s, 9H).
1H), 1.53 (d, J ¼ 6.8 Hz, 3H). 13C NMR (201 MHz, MeOD)
d 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 [MþH]. calcd 376.2389, found
376.2389.
13C NMR (101 MHz, CDCl3)
d 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
C
27H36N3O [MþH]. calcd 418.2858, found 418.2849.
6′-Methoxy-1-phenethyl-2′,3′,4′,9′-tetrahydrospiro[piperi-
1-(2,4-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
dine-4,1′-pyrido[3,4-b]indole] (3c). Yield ¼ 50 mg (40%)1H NMR
drospiro[azepane-4,1′-pyrido[3,4-b]indole] (4a). Yield ¼ 56 mg
(800 MHz, MeOD)
d
7.32 (t, J ¼ 7.5 Hz, 2H), 7.28 (d, J ¼ 7.4 Hz, 2H),
(76%)1H NMR (400 MHz, MeOD)
d
7.48e7.31 (m, 1H), 7.23 (d,
7.24e7.11 (m, 2H), 6.92 (d, J ¼ 2.5 Hz, 1H), 6.76 (dd, J ¼ 8.7, 2.5 Hz,
1H), 3.81 (s, 3H), 3.17 (t, J ¼ 5.8 Hz, 2H), 3.00 (d, J ¼ 11.9 Hz, 2H), 2.92
(dd, J ¼ 10.7, 6.0 Hz, 2H), 2.85 (dd, J ¼ 10.8, 6.0 Hz, 2H), 2.80e2.61
J ¼ 8.7 Hz, 1H), 7.09e6.92 (m, 2H), 6.91 (t, J ¼ 1.8 Hz, 1H), 6.75 (dt,
J ¼ 8.9, 1.9 Hz, 1H), 3.79 (s, 3H), 3.67 (s, 2H), 3.15 (dp, J ¼ 24.7, 6.3,
5.8 Hz, 2H), 2.75 (h, J ¼ 6.8 Hz, 6H), 2.09 (dtd, J ¼ 44.9, 14.8, 7.3 Hz,
3H), 1.86 (dd, J ¼ 13.8, 6.4 Hz, 1H), 1.72 (p, J ¼ 5.9 Hz, 2H). 13C NMR
(m, 4H), 2.20 (td, J ¼ 13.8, 4.4 Hz, 2H), 1.89 (d, J ¼ 14.1 Hz, 2H). 13
C
NMR (201 MHz, MeOD)
d
153.67, 139.20, 138.53, 131.46, 128.34,
(101 MHz, MeOD)
d
163.37 (dd, JC-F ¼ 93.9, 12.1 Hz), 160.91 (dd, JC-
128.25, 127.17, 126.05, 111.18, 110.89, 107.14, 99.77, 59.60, 54.89,
¼ 94.0, 12.2 Hz), 153.70, 138.27, 132.81 (dd, JC-F ¼ 9.7, 6.1 Hz),
F
13

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
131.21, 127.05, 121.39 (dd, JC-F ¼ 14.6, 3.7 Hz), 111.36, 110.90 (dd, JC-
124.23, 120.79, 119.76 (dd, JC-F ¼ 14.9, 3.9 Hz), 112.38, 112.31, 111.19
(dd, JC-F ¼ 21.1, 3.9 Hz),108.39,103.80 (t, JC-F ¼ 25.9 Hz), 55.12, 50.39,
48.46, 38.99, 35.76, 22.99. HRMS (ESI) m/z for C22H23BrF2N3 [MþH].
calcd 446.1043, found 446.1036.
¼ 21.2, 3.7 Hz), 110.89, 105.10, 103.44 (t, JC-F ¼ 26.0 Hz), 99.84,
F
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 [MþH]. calcd 412.2200, found
412.2193.
6′-Chloro-1-(2,4-difluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro
1’-(2,4-Difluorobenzyl)-6-methoxy-2,3,4,9-tetrahydrospiro
[pyrido[3,4-b]indole-1,3′-pyrrolidine] (4b). Yield ¼ 83 mg (43%)
[piperidine-4,1′-pyrido[3,4-b]indole] (5c). Yield
¼
134 mg
(67%).1H NMR (400 MHz, CDCl3)
d
8.86e8.49 (m, 1H), 7.43 (d,
1H NMR (800 MHz, CDCl3)
d
8.87 (s, 1H), 7.33 (td, J ¼ 8.6, 6.6 Hz,1H),
J ¼ 9.3 Hz, 2H), 7.21 (d, J ¼ 8.7 Hz, 1H), 7.08 (d, J ¼ 8.5 Hz, 1H), 6.82
(q, J ¼ 9.0 Hz, 2H), 3.69 (s, 2H), 3.11 (d, J ¼ 5.8 Hz, 2H), 2.81 (d,
J ¼ 11.3 Hz, 2H), 2.67 (q, J ¼ 8.5, 8.0 Hz, 4H), 2.18 (q, J ¼ 8.0, 5.3 Hz,
7.24 (d, J ¼ 8.7 Hz, 1H), 6.94 (d, J ¼ 2.6 Hz, 1H), 6.89e6.84 (m, 2H),
6.83 (dd, J ¼ 8.7, 2.6 Hz, 1H), 3.87 (s, 3H), 3.79e3.73 (m, 2H), 3.26
(dt, J ¼ 13.0, 4.6 Hz, 1H), 3.18e3.07 (m, 3H), 2.78e2.68 (m, 2H), 2.64
(q, J ¼ 9.0 Hz, 1H), 2.53 (d, J ¼ 8.9 Hz, 1H), 2.34 (dt, J ¼ 13.3, 8.5 Hz,
1H), 2.14 (ddd, J ¼ 13.0, 9.2, 3.1 Hz, 1H). 13C NMR (201 MHz, CDCl3)
2H), 1.76 (d, J ¼ 13.6 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 163.25
(dd, JC-F ¼ 100.2, 12.4 Hz), 160.77 (dd, JC-F ¼ 100.6, 12.3 Hz), 141.11,
133.91, 132.90 (dd, JC-F ¼ 9.8, 5.8 Hz), 128.44, 124.90, 121.74, 119.48
(d, JC-F ¼ 15.4 Hz), 117.69, 111.85, 111.29 (d, JC-F ¼ 20.7 Hz), 108.51,
103.82 (t, JC-F ¼ 25.7 Hz), 54.96, 50.36, 48.43, 39.00, 35.72, 22.99.
HRMS (ESI) m/z for C22H23ClF2N3 [MþH]. calcd 402.1549, found
402.1539.
d
162.58 (dd, JC-F ¼ 208.3, 11.7 Hz), 161.34 (dd, JC-F ¼ 208.7, 12.1 Hz),
153.96, 140.73, 132.11e132.03 (m), 130.58, 127.26, 120.86, 111.63,
111.30, 111.21 (dd, JC-F ¼ 21.3, 3.7 Hz), 106.03, 104.09 (t, JC-
¼ 25.7 Hz), 100.49, 65.57, 59.95, 56.05, 52.59, 52.48, 41.80, 39.15,
F
22.30. HRMS (ESI) m/z for C22H24F2N3O [MþH]. calcd 384.1887,
1-(2,4-Difluorobenzyl)-7′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-4,1′-pyrido[3,4-b]indole]
found 384.1894.
(5d).
1-(2,4-Difluorobenzyl)-6′-methoxy-2′,3′,4′,9′-tetrahy-
drospiro[piperidine-3,1′-pyrido[3,4-b]indole] (4c). Yield ¼ 85 mg
Yield ¼ 90 mg (45%).1H NMR (400 MHz, CDCl3)
d
8.24 (s, 1H),
7.48e7.32 (m, 2H), 6.94e6.68 (m, 4H), 3.82 (s, 3H), 3.67 (s, 2H), 3.13
(t, J ¼ 5.7 Hz, 2H), 2.83e2.76 (m, 2H), 2.72e2.54 (m, 4H), 2.17 (td,
J ¼ 13.5, 13.1, 4.4 Hz, 2H), 1.77 (d, J ¼ 13.7 Hz, 2H). 13C NMR
(43%) 1H NMR (400 MHz, CDCl3)
d 9.65 (s, 1H), 7.31e7.27 (m, 1H),
7.22 (d, J ¼ 6.7 Hz, 1H), 6.96 (d, J ¼ 2.2 Hz, 1H), 6.90e6.76 (m, 3H),
3.88 (s, 3H), 3.60 (q, J ¼ 12.9 Hz, 2H), 3.47e3.17 (m, 2H), 3.00 (s, 1H),
2.92e2.61 (m, 3H), 2.43e2.05 (m, 3H), 1.96e1.67 (m, 2H), 1.58 (s,
(101 MHz, CDCl3)
d
163.17 (dd, JC-F ¼ 91.6, 11.9 Hz), 160.70 (dd, JC-
¼ 92.1, 12.1 Hz), 156.28, 138.34, 136.25, 132.75 (dd, JC-F ¼ 9.5,
F
1H). 13C NMR (101 MHz, CDCl3)
d
163.37 (dd, JC-F ¼ 84.4, 12.1 Hz),
5.9 Hz), 121.77, 119.96 (dd, JC-F ¼ 14.8, 3.8 Hz), 118.65, 111.20 (dd, JC-
160.90 (dd, JC-F ¼ 84.5, 12.3 Hz), 153.79, 139.99, 132.55 (dd, JC-
¼ 20.8, 3.8 Hz), 108.93, 108.49, 103.74 (t, JC-F ¼ 25.7 Hz), 94.99,
F
¼ 9.5, 6.2 Hz), 130.43, 127.18, 120.51 (dd, JC-F ¼ 14.3, 3.7 Hz), 111.74,
55.78, 54.99, 50.38, 48.59, 39.15, 35.97, 23.14. HRMS (ESI) m/z for
F
111.31, 111.13 (dd, JC-F ¼ 20.9, 3.7 Hz), 106.14, 104.29 (t, JC-
C
23H26F2N3O [MþH]. calcd 398.2044, found 398.2033.
¼ 25.9 Hz), 100.22, 63.38, 56.48, 56.07, 53.34, 52.17, 38.90, 36.31,
1-(2,4-Difluorobenzyl)-6′-methoxy-9′-methyl-2′,3′,4′,9′-tet-
F
22.64, 22.09. HRMS (ESI) m/z for C23H26F2N3O [MþH]. calcd
rahydrospiro[piperidine-4,1′-pyrido[3,4-b]indole]
(5e).
398.2044, found 398.2040.
Yield ¼ 47 mg (23%).1H NMR (400 MHz, CDCl3)
d
7.45 (q, J ¼ 8.1 Hz,
1-Benzyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)piperidin-4-
1H), 7.20 (d, J ¼ 8.7 Hz, 1H), 6.97 (d, J ¼ 2.4 Hz, 1H), 6.87 (dtd,
J ¼ 28.7, 9.4, 2.5 Hz, 3H), 3.89 (s, 6H), 3.65 (s, 2H), 3.10 (t, J ¼ 5.7 Hz,
2H), 2.72 (tt, J ¼ 18.0, 9.9 Hz, 6H), 2.52e2.22 (m, 2H), 1.72 (d,
amine (4d). Yield ¼ 105 mg (53%)1H NMR (400 MHz, CDCl3)
d 8.37
(s, 1H), 7.34 (q, J ¼ 7.8 Hz, 1H), 7.30e7.18 (m, 1H), 7.01 (s, 1H), 6.83
(dt, J ¼ 32.9, 9.1 Hz, 3H), 3.88 (s, 3H), 3.53 (s, 2H), 2.98 (dq, J ¼ 11.1,
6.8 Hz, 4H), 2.84 (d, J ¼ 11.2 Hz, 2H), 2.50 (tt, J ¼ 10.0, 4.3 Hz, 1H),
2.07 (t, J ¼ 11.5 Hz, 2H), 1.86 (d, J ¼ 12.4 Hz, 2H), 1.54e1.24 (m, 2H).
J ¼ 13.7 Hz, 2H). 13C NMR (101 MHz, CDCl3)
d 162.63 (d, JC-
¼ 11.9 Hz), 160.15 (d, JC-F ¼ 12.1 Hz), 153.93, 140.67, 132.74, 132.27
F
(br), 126.69, 120.93 (br), 111.39, 111.02 (d, JC-F ¼ 21.6 Hz), 109.59,
109.17, 103.62 (t, JC-F ¼ 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 [MþH].
calcd 412.2200, found 412.2191.
13C NMR (101 MHz, CDCl3)
d
162.87 (dd, JC-F ¼ 77.0, 12.3 Hz), 160.41
(dd, JC-F ¼ 78.0,11.8 Hz),153.88,132.19 (dd, JC-F ¼ 9.5, 6.2 Hz),131.61,
127.85, 122.84, 120.95 (dd, JC-F ¼ 15.0, 3.7 Hz), 113.56, 112.16, 111.91,
110.96 (dd, JC-F ¼ 20.9, 3.7 Hz), 103.48 (t, JC-F ¼ 25.7 Hz), 100.75,
55.97, 54.82, 54.65, 52.15, 46.76, 32.66, 26.02. HRMS (ESI) m/z for
(þ/¡)-(2R,11aS)-6′-methoxy-1,2′,3,3′,4,4′,6,9′,11,11a-decahy-
drospiro[pyrido[1,2-b]isoquinoline-2,1′-pyrido[3,4-b]indole]
C
23H28F2N3O [MþH]. calcd 400.2200, found 400.2188.
(6b). 1H NMR (599 MHz, cdcl3)
d
8.34 (d, J ¼ 20.8 Hz, 1H), 7.14 (dq,
1-(2,4-Difluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro[piperi-
J ¼ 13.2, 6.7, 6.2 Hz, 2H), 7.11e6.99 (m, 3H), 6.94 (d, J ¼ 2.6 Hz, 1H),
6.78 (dt, J ¼ 8.7, 2.3 Hz, 1H), 3.86 (s, 4H), 3.52 (d, J ¼ 15.3 Hz, 1H),
3.22e3.08 (m, 2H), 2.94e2.88 (m, 1H), 2.84 (tq, J ¼ 8.2, 3.1, 2.6 Hz,
1H), 2.77e2.72 (m, 3H), 2.69 (t, J ¼ 5.7 Hz, 2H), 2.29e2.19 (m, 1H),
2.00 (dt, J ¼ 13.9, 3.1 Hz, 1H), 1.92e1.82 (m, 2H). 13C NMR (101 MHz,
dine-4,1′-pyrido[3,4-b]indole] (5a). Yield ¼ 103 mg (56%).1H NMR
(400 MHz, CDCl3)
d
8.94 (s, 1H), 7.51 (d, J ¼ 7.5 Hz, 1H), 7.43e7.25
(m, 2H), 7.13 (hept, J ¼ 8.4, 7.5 Hz, 2H), 6.84e6.61 (m, 2H), 3.66 (s,
2H), 3.13 (t, J ¼ 5.7 Hz, 2H), 2.86e2.69 (m, 4H), 2.69e2.52 (m, 2H),
2.19 (dt, J ¼ 13.0, 7.5 Hz, 2H), 1.73 (d, J ¼ 13.7 Hz, 2H). 13C NMR
CDCl3)
d 153.98, 140.49, 133.80, 133.42, 130.74, 128.10, 127.67,
(101 MHz, CDCl3)
d
163.22 (dd, JC-F ¼ 91.1, 11.9 Hz), 160.74 (dd, JC-
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 [MþH]. calcd 374.2232, found 374.2222.
¼ 91.6, 11.9 Hz), 139.72, 135.68, 132.93 (dd, JC-F ¼ 9.5, 5.9 Hz),
F
127.30, 121.56, 119.77 (dd, JC-F ¼ 15.0, 3.7 Hz), 119.18, 118.12, 111.21
(dd, JC-F ¼ 21.3, 3.7 Hz), 110.99, 108.50, 103.78 (t, JC-F ¼ 25.7 Hz),
55.07, 50.44, 48.50, 39.14, 35.77, 23.15. HRMS (ESI) m/z for
C
22H24F2N3 [MþH]. calcd 368.1938, found 368.1935.
6′-Bromo-1-(2,4-difluorobenzyl)-2′,3′,4′,9′-tetrahydrospiro
5. Associated CONTENT
[piperidine-4,1′-pyrido[3,4-b]indole] (5b). Yield ¼ 51 mg (54%) 1H
NMR (400 MHz, CDCl3)
d
8.83 (s, 1H), 7.60 (d, J ¼ 2.1 Hz, 1H), 7.36
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 ana-
lyses 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 at. Final site for Associated content also needs to be
provided.
(td, J ¼ 8.7, 6.6 Hz, 1H), 7.19 (dd, J ¼ 8.6, 2.0 Hz, 1H), 7.12 (d,
J ¼ 8.5 Hz, 1H), 6.83e6.68 (m, 2H), 3.65 (s, 2H), 3.11 (t, J ¼ 5.7 Hz,
2H), 2.77 (dd, J ¼ 8.4, 2.9 Hz, 2H), 2.70e2.57 (m, 4H), 2.14 (td,
J ¼ 13.3, 4.6 Hz, 2H), 1.72 (dd, J ¼ 14.1, 2.4 Hz, 2H). 13C NMR
(101 MHz, CDCl3)
d
163.19 (dd, JC-F ¼ 93.7, 11.9 Hz), 160.72 (dd, JC-
¼ 94.1,11.9 Hz),141.14,134.23,132.81 (dd, JC-F ¼ 9.7, 5.7 Hz),129.14,
F
14

J.-H. Son, P.-W. Phuan, J.S. Zhu et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
of a defined subset of minimal function CFTR mutations, Sci. Rep. 9 (2019)
17640.
Author CONTRIBUTIONS
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The manuscript was written by J.-H.S., P.-W.P., J.S.Z., K.Y.T., D.J.T.,
A.S.V., P.M.H. and M.J.K. Synthetic work was performed by J.-H.S.,
J.S.Z., E.L., and A.C. Computational studies were performed by
K.Y.T. Biological data collection was performed by P.-W.P. All au-
thors have given approval to the final version of the manuscript.
Declaration of competing interest
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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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 Foun-
dation 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.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112888.
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