Leveraging a Low-Affinity Diazaspiro Orthosteric Fragment to Reduce Dopamine D3 Receptor (D3R) Ligand Promiscuity across Highly Conserved Aminergic G-Protein-Coupled Receptors (GPCRs)

Article abstract of DOI:10.1021/acs.jmedchem.9b00412

Previously, we reported a 3-(2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (1) compound with excellent dopamine D₃ receptor (D₃R) affinity (D₃R K_i = 12.0 nM) and selectivity (D₂R/D₃R ratio = 905). Herein, we present derivatives of 1 with comparable D₃R affinity (32, D₃R K_i = 3.2 nM, D₂R/D₃R ratio = 60) and selectivity (30, D₃R K_i = 21.0 nM, D₂R/D₃R ratio = 934). Fragmentation of 1 revealed orthosteric fragment 5a to express an unusually low D₃R affinity (K_i = 2.7 μM). Compared to piperazine congener 31, which retains a high-affinity orthosteric fragment (5d, D₃R K_i = 23.9 nM), 1 was found to be more selective for the D₃R among D₁- and D₂-like receptors and exhibited negligible off-target interactions at serotoninergic and adrenergic G-protein-coupled receptors (GPCRs), common off-target sites for piperazine-containing D₃R scaffolds. This study provides a unique rationale for implementing weakly potent orthosteric fragments into D₃R ligand systems to minimize drug promiscuity at other aminergic GPCR sites.

Full text of DOI:10.1021/acs.jmedchem.9b00412

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Article
Leveraging a Low Affinity Diazaspiro Orthosteric Fragment to
Reduce Dopamine D3 Receptor (D3R) Ligand Promiscuity Across
Highly Conserved Aminergic G-Protein-Coupled Receptors (GPCRs)
Sean W. Reilly, Aladdin A. Riad, Chia-Ju Hsieh, Kristoffer Sahlholm, Daniel A. Jacome,
Suzy Griffin, Michelle Taylor, Chi-Chang Weng, Kuiying Xu, Nathan Kirschner, Robert
R. Luedtke, Christopher Parry, Shipra Malhotra, John Karanicolas, and Robert H. Mach
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00412 • Publication Date (Web): 25 Apr 2019
Downloaded from http://pubs.acs.org on April 25, 2019
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Leveraging a Low Affinity Diazaspiro Orthosteric
Fragment to Reduce Dopamine D3 Receptor (D3R)
Ligand Promiscuity Across Highly Conserved
Aminergic G-Protein-Coupled Receptors (GPCRs)
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Sean W. Reilly,† Aladdin A. Riad,† Chia-Ju Hsieh,† Kristoffer Sahlholm,† Daniel A. Jacome,‡ Suzy
Griffin,§ Michelle Taylor,§ Chi-Chang Weng,† Kuiying Xu,† Nathan Kirschner,† Robert R. Luedtke,§
Christopher Parry,⊤ Shipra Malhotra,⊤ John Karanicolas,⊤ and Robert H. Mach*,†
†Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States
‡Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, 421
Curie Boulevard, Philadelphia, Pennsylvania 19104, United States
§Department of Pharmacology and Neuroscience, University of North Texas Health Science Center,
3500 Camp Bowie Boulevard, Fort Worth, Texas 76107, United States
⊤Program in Molecular Therapeutics, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia,
Pennsylvania 19111, United States
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ABSTRACT: Previously we reported a 3-(2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-
triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (1) compound with excellent dopamine D3 receptor
(D3R) affinity (D3R Ki = 12.0 nM) and selectivity (D2R/D3R ratio = 905). Herein, we present derivatives
of 1 with comparable D3R affinity (32, D3R Ki = 3.2 nM, D2R/D3R ratio = 60), and selectivity (30, D3R
Ki = 21.0 nM, D2R/D3R ratio = 934). Fragmentation of 1 revealed orthosteric fragment 5a to express an
unusually low D3R affinity (Ki = 2.7 µM). Compared to piperazine congener 31, which retains a high
affinity orthosteric fragment (5d, D3R Ki = 23.9 nM), 1 was found to be more selective for the D3R among
D1- and D2-like receptors, and exhibited negligible off-target interactions at serotoninergic and adrenergic
GPCRs, common off-target sites for piperazine-containing D3R scaffolds. This study provides a unique
rationale for implementing weakly potent orthosteric fragments into D3R ligand systems to minimize drug
promiscuity at other aminergic GPCR sites.
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INTRODUCTION
The dopamine D3 receptor (D3R) is a G protein-coupled receptor (GPCR), viewed as a
pharmacotherapeutic target for numerous neurological and psychiatric disorders as well as drug
addiction.1, 2 Targeting this D2-like receptor is a strategy for schizophrenia drug development due to the
unwanted extrapyramidal side-effects elicited with many typical antipsychotics.3 The appeal of D3R for
drug addiction therapeutics stems from the high expression of this protein in the mesolimbic pathway, a
region of the brain implicated in reward and motivation.4 Human postmortem studies showing enhanced
expression of the D3R in drug exposed brains has further validated the clinical importance of this receptor
for substance abuse disorders (SUD).5 Selective engagement of this receptor is also of considerable
interest for positron-emission tomography (PET) imaging applications to further our understanding and
elucidate the complex molecular mechanisms of drug addiction.6-9
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Development of high affinity D3R selective ligands begins with a fragment-based approach in
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which the binding profiles of amino core synthons are examined at the orthosteric binding site (OBS) of
the receptor. This region contains a highly conserved Asp110 [3.32] residue which forms a salt-bridge
interaction with the protonated nitrogen of the amino core in the ligand scaffold affording receptor
recognintion.10 Due to the high degree of homology between the D3R and the D2R in this domain, the
ligand must then extend into a secondary binding pocket (SBP) containing residues unique to each
receptor in order to confer D3R selectivity.10-12 However, the Asp110 [3.32] that forms the critical
receptor-ligand interaction is also greatly conserved across other aminergic GPCRs.13 Thus, synthesizing
D3R selective compounds, that concomitantly engage the Asp110 [3.32] residue and exhibit minimal off-
target interactions continues to be problematic. As such, there have been no clinically viable D3R selective
therapeutic or PET imaging agent reported to date, preventing us from exploring the therapeutic potential
of this receptor.
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Recently, we reported a new class of D3R selective compounds containing spirodiamine systems
as an alternative to the piperazine amino core (Figure 1).14 In this study, we synthesized and evaluated
analogues and fragments of 1 in order to identify the structural determinants responsible for ligand affinity
and selectivity at the D3R. Compared to the excellent binding profile of aryl piperazine orthosteric
fragment 5d (Ki = 23.9 nM) of ligand 31, we found sython 5a of 1 to be bind with lower affinity (Ki > 2
µM). Computational ligand docking studies were performed in order to elucidate the binding mode of 1,
and other select compounds, within the D3R crystal structure (3PBL). Finally, the pharmacological
behavior of 1 and 31 was screened across all D1 (D1R and D5R)- and D2 (D2R, D3R and D4R)-like
receptors, and select aminergic GPCRs, revealing 1 to be a more selective ligand for the D3R with less
off-target interactions. These results provide rationale for utilizing primary pharmacophores with limited
affinity for the highly conserved orthosteric binding pocket (OBP) of the receptor in order to develop
selective D3R ligands with minimized off-target interactions.
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Figure 1. Lead compounds identified with diazaspiro amino systems.
RESULTS AND DISCUSSION
Chemistry. Synthesis of arylated diazaspiro and piperazine systems was achieved in excellent
yields following our previously reported Pd C–N cross-coupling reports outlined in Scheme 1.15, 16
Unsaturated diazaspiro[5.5]undec-1-ene motif 4a, a presumed cross-coupling β-hydride elimination side-
product, was able to be obtained, albeit in low yields (<10%). Following removal of the BOC protecting
groups from 4b, 4c, and 4e with trifluoroacetic acid (TFA) and basification, the corresponding free-amine
intermediates 4b’, 4c’, and 4e’ were N-alkylated with 1-bromobutane at room temperature in acetone to
afford desired synthons 5a-c. Similarly, S-alkylation of commercially available 4-methyl-5-phenyl-4H-
1,2,4-triazole-3-thiol with the appropriate alkylating reagent yielded 6a-b in good yields. Formation of
1,2,4-triazole fragments 6c-e, and 6g were readily accessed by reacting 6b with the desired amine in the
presence of Cs2CO3 under refluxing conditions. BOC-deprotection with TFA, followed by base
neutralization, afforded the final free-amine fragments 6f and 6h.
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Scheme 1. Synthesis of Fragmented Synthonsa
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aReagents and conditions: (i) Pd2(dba)3, RuPhos, aryl halide, diazaspiro reagent, NaOt-Bu, dioxane, 100
°C, 20 min; (ii) TFA, CH2Cl2, rt, 3 h; (iii) 1-bromobutane, K2CO3, acetone, rt, 12 h; (iv) alkylating reagent,
K2CO3, acetone, rt, 12 h; (v) amine, Cs2CO3, ACN, 70 °C, 8h.
Scheme 2 briefly illustrates the general synthesis, disclosed in our previous report14, used to obtain
ligands 1-3, and 7-34. Aryl amide scaffolds 35-37 were prepared by reacting 4b’ with 2-(4-
bromobutyl)isoindoline-1,3-dione, to afford precursor E in a modest yield of 64%. Next, E was treated
with hydrazine in refluxing EtOH to afford free-amine intermediate F in excellent yield (97%). Finally,
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benzamide compounds 35-36 were synthesized by coupling F with the respective benzoic acid in the
presence of 1-hydroxybenzotriazole (HOBt) hydrate and 1-ethyl-3-(3'-
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dimethylaminopropyl)carbodiimide (EDC) in dichloromethane at room temperature. Following our 2011
report17, synthon 4b’ was N-alkylated with intermediate G, to afford the aripiprazole analogue 37 in
modest yield (62%).
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Scheme 2. General Synthesis of Compounds 1-3 and 7-37a
aReagents and conditions: (i) A, TFA, CH2Cl2, rt, 3 h; (ii) B, alkylating reagent, K2CO3, acetone, rt, 12 h;
(iii) C, D, TEA, EtOH, 75 °C, 12 h; (iv) 4b’, 2-(4-bromobutyl)isoindoline-1,3-dione, KI and Cs2CO3,
ACN, 75 °C for 3 h; (v) E, hydrazine, EtOH, 75 °C for 2 h; (vi) F, benzoic acid, HOBt, EDC, CH2Cl2, rt,
2 h; (vii) G, KI, and K2CO3, ACN, 90 °C for 12 h.
Radioligand Binding Profiles of Compound 1 Analogues and Fragments. Structural
modifications were made to 1 in attempts to further enhance D3R binding affinity and probe the structural
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determinants of this ligand template responsible for receptor selectivity. Compounds and synthons of
select ligands were then evaluated in radioligand binding assays using [125I]IABN with HEK 293 cells
stably expressing human D3R and D2L (Tables 1-5). Values of cLogP and topological polar surface area
(tPSA) are also included in Tables 1-4 to provide an estimate of lipophilicity and cell permeability,
respectively, for each full-length ligand.
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We first modified the aryl 1,2,4-triazole ring system in 1, and evaluated several aryl moieties found
to be tolerable in azaspirocyclic D3R ligand architectures previously disclosed by Micheli and co-
workers.18-20 However, we observed a drastic decrease in receptor selectivity after installing the 4-(1,3-
oxazol-2-yl)-phenyl (11) and cyclohexyl (14) substitutions (D2R/D3R ratio = 62 and 221, respectively),
indicating our 1,2,4-triazole ligand architecture follows a different structure-activity relationship (SAR)
within the D3R. When modifying the aryl ring with fluorine substitutions, we found the ortho position to
be more preferred (12, Ki = 25.9 nM, D2R/D3R ratio = 759), as the 4-fluoro analogue resulted in a 2-fold
decrease in receptor selectivity (13, Ki = 39.8 nM, D2R/D3R ratio = 396).
Next, binding profiles of ligands containing structural modifications to the 1,2,4-triazole ring
system were evaluated (15-19). With respect to 1, replacing the –CH3 group for a –CH2CH3 (15) or –NH2
(16) substituent resulted in a 7-fold and 5-fold decrease in receptor selectivity, respectively. Similarly,
deletion of the –CH3 group resulted in drastic loss of receptor selectivity, affording a 35-fold D2R/D3R
ratio for 17, combined with a moderate decrease in D3R affinity (Ki = 32.5). Imidazole systems were also
evaluated as triazole alternatives, however, we found a significant loss in receptor affinity and binding
selectivity for ligands 18 and 19.
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Table 1. D3R/D2R Binding Profiles of 1 Analogues with Modifications to the 1,2,4-Triazole Systema
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2
3
4
5
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7
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Ki ± SEM (nM)b
Compound
R1
R2
Y
D2/D3 Ratioe
cLogPf
tPSAf
D3Rc
D2Rd
1g
7
phenyl
4-N,N-dimethylaniline
2-methoxyphenyl
pyrimidin-3-yl
4-(thiophen-3-yl)phenyl
4-(1,3-oxazol-2-yl)-phenyl
2-fluorophenyl
4-fluorophenyl
cyclohexyl
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH2CH3
NH2
H
N
N
12.0 ± 2.8
138 ± 17.1
63.0 ± 7.5
213 ± 21.4
101 ± 32.3
25.4 ± 2.7
25.9 ± 3.7
39.8 ± 11.9
57.0 ± 13.6
48.7 ± 8.9
75.0 ± 6.3
32.5 ± 2.6
383 ± 53.7
418 ± 49.8
10,895 ± 2,069
12,137 ± 2,713
2,112 ± 269
905
88
4.88
5.21
4.37
3.69
6.41
4.66
5.03
5.03
5.17
5.55
4.90
5.06
6.24
6.26
43.67
46.91
52.90
56.03
43.67
65.26
43.67
43.67
43.67
43.67
69.69
52.46
40.1
8
N
33.5
229
32.2
62.1
759
396
221
116
185
35.7
15.4
85.4
9
N
48,584 ± 10,378
3,250 ± 782
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12
13
14
15
16
17
18
19
N
N
1,574 ± 396
N
19,655 ± 4,441
15,768 ± 1,661
12,584 ± 1,732
5,650 ± 893
N
N
phenyl
N
phenyl
N
13,904 ± 453
1,162 ± 229
phenyl
N
phenyl
H
CH
CH
5,891 ± 267
phenyl
CH3
3,5720 ± 5,804
31.21
aSpiperone assayed under the same conditions as a reference blocker: D2R, 0.06 ± 0.001 nM; D3R, 0.33
± 0.02 nM; D4R, 0.45 ± 0.01 nM. bMean ± SEM; Ki values were determined by at least three experiments.
cKi values for D3 receptors were measured using human D3 expressed in HEK cells with [125I]IABN as
the radioligand. dKi values for D2 receptors were measured using human D2 expressed in HEK cells with
[125I]IABN as the radioligand. e(Ki for D2 receptors)/(Ki for D3 receptors). fCalculated using ChemDraw
Professional 15.1. gCompound reported in our initial work ref. 14.
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We then explored the effect on D3R binding by manipulating the linker length in compounds 20
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2
and 21 (Table 2). However, this modification afforded an ~8-fold reduction in D3R selectivity,
demonstrating the N-propyl linker to be the more optimal spacer. Next, we probed the influence of the 2-
methoxy substituent in compound 1 on D3R ligand binding. When replacing the 2-methoxy with a 2-
ethoxy in 22, receptor selectivity was diminished (D2R/D3R ratio = 114), along with a moderate decrease
in binding affinity (Ki = 45.1 nM). Compared to 20, the 2-fluoroethoxy substituent showed to be more
compatible, affording an increase in overall binding and selectivity (Ki = 33.3 nM, D2R/D3R ratio = 192).
In our initial report, we found moving the aryl methoxy substituent from the 2-position in 1 to the 4-
position in 25 further enhanced D3R selectivity (D2R/D3R ratio = ~1,000-fold), but also diminished ligand
affinity for the receptor (Ki = 97.7 nM).14 Thus, we evaluated 26, a compound containing methoxy
substituents in the ortho and para positions of the aryl ring, to determine if affinity could be enhanced
while maintaining similar selectivity. Instead, we found the receptor affinity for 26 (Ki = 112 nM) to be
comparable to 25, along with a ~2-fold reduction in D3R selectivity (D2R/D3R ratio = 584).
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Next, we examined the binding profiles of fluorinated derivatives 28-30. When moving the
fluorine atom from the 4-position (27) to the 2-position in 28, a loss in receptor affinity (Ki = 208 nM)
and selectivity (D2R/D3R ratio = 126) is observed. Upon installing a –CF3 functional group in the para
position of the aryl ring in 29, we observed a further decrease in both D3R affinity (Ki = 370 nM) and
selectivity (D2R/D3R ratio = 57). Due to the excellent D3R binding properties observed with compounds
1 and 27, we evaluated the 4-fluoro-2-methoxyphenyl ring system in ligand 30. Compared to 1, compound
30 was found to have a comparable D3R binding affinity (Ki = 21.0 nM) to 1, along with a modest
improvement in receptor selectivity (D2R/D3R ratio = 934).
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Table 2. D3R/D2R Binding Profiles of 1 Analogues with Modifications to the Linker and Aryl Ringa
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7
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9
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11
12
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17
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20
21
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25
26
27
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29
30
31
32
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Ki ± SEM (nM)b
Compound
n
R
D2/D3 Ratioe
cLogPf
tPSAf
D3Rc
D2Rd
20
21
22
23
24
25g
26
27g
28
29
30
0
2
1
1
1
1
1
1
1
1
1
2-methoxy
2-methoxy
89.7 ± 22.5
59.2 ± 5.1
45.1 ± 9.7
396 ± 17.3
33.3 ± 1.7
97.7 ± 17.4
112 ± 24.5
25.6 ± 5.6
208 ± 9.6
370 ± 17.8
21.0 ± 1.4
9,004 ± 1,133
9,248 ± 1,745
5,165 ± 525
100
156
114
44.4
192
1,073
584
383
126
57.2
934
4.54
5.06
5.41
4.33
5.27
4.88
4.89
5.32
5.32
6.38
5.17
43.67
43.67
43.67
54.67
43.67
43.67
52.90
34.44
34.44
34.44
43.67
2-ethoxy
2-hydroxy
17,595 ± 4,185
6,379 ± 964
2-fluorethoxy
4-methoxy
104,847 ± 29,076
65,229 ± 37,252
9,792 ± 1,790
26,253 ± 6,866
21,172 ± 1,499
19,634 ± 3,691
2,4-dimethoxy
4-fluoro
2-fluoro
2-fluoro-4-trifluoromethyl
2-methoxy-4-fluoro
aSpiperone assayed under the same conditions as a reference blocker: D2R, 0.06 ± 0.001 nM; D3R, 0.33
± 0.02 nM; D4R, 0.45 ± 0.01 nM. bMean ± SEM; Ki values were determined by at least three experiments.
cKi values for D3 receptors were measured using human D3 expressed in HEK cells with [125I]IABN as
the radioligand. dKi values for D2 receptors were measured using human D2 expressed in HEK cells with
[125I]IABN as the radioligand. e(Ki for D2 receptors)/(Ki for D3 receptors). fCalculated using ChemDraw
Professional 15.1. gCompound reported in our initial work ref. 14.
Next, we evaluated two additional analogues containing modified diazaspiro cores (Table 3). In
contrast to 1 and 31, unsaturated ligand 32, containing a novel diazaspiro[5.5]undec-1-ene amino core,
was found bind with higher affinity at the D3R (Ki = 3.2 nM), although receptor selectivity was not
maintained (D2R/D3R ratio = 60). Interestingly, when reversing the diazaspiro[4.5]decane moiety found
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in 33, a two-fold increase in receptor selectivity was observed for ligand 34 (D2R/D3R ratio = 758), along
with a similar D3R binding affinity (Ki = 27.8 nM).
1
2
3
4
5
6
7
Table 3. D3R/D2R Binding Profiles of 1 Analogues with Modifications to the Amino Corea
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Ki ± SEM (nM)b
Compound
31g
Amino Core
D2/D3 Ratioe
cLogPf
tPSAf
D3Rc
D2Rd
6.5 ± 0.88
260 ± 44.2
40.2
4.09
43.67
32
33g
34
3.2 ± 0.71
197 ± 24.4
60.7
315
758
5.38
4.32
4.32
43.67
43.67
43.67
19.6 ± 4.7
6,168 ± 939
27.8 ± 4.4
21,098 ± 3,069
aSpiperone assayed under the same conditions as a reference blocker: D2R, 0.06 ± 0.001 nM; D3R, 0.33
± 0.02 nM; D4R, 0.45 ± 0.01 nM. bMean ± SEM; Ki values were determined by at least three experiments.
cKi values for D3 receptors were measured using human D3 expressed in HEK cells with [125I]IABN as
the radioligand. dKi values for D2 receptors were measured using human D2 expressed in HEK cells with
[125I]IABN as the radioligand. e(Ki for D2 receptors)/(Ki for D3 receptors). fCalculated using ChemDraw
Professional 15.1. gCompound reported in our initial work ref. 14.
Finally, we evaluated the diazaspiro[5.5]undecane amino core in two aryl amide templates that
were previously identified as excellent D3R-prefering scaffolds when conjugated to the 1-(2-
methoxyphenyl)piperazine system (Table 4).21, 22 While maintaining the 2-methoxyphenyl aryl system,
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we investigated 4-(dimethylamino)- and 4-(thiophen-3-yl)-benzamides with a four carbon spacer linked
to the diazaspiro[5.5]undecane moiety. However, using this ligand framework, compounds 35 and 36 both
exhibited poor D3R receptor affinity and selectivity. This diazaspiro core was then evaluated in a D2R-
prefering aripiprazole ligand architecture disclosed in our previous work.17 Although 37 appears to be
slightly D2R-selective, receptor affinity was low.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. D3R/D2R Binding Profiles of 1 Analogues with Modifications to the Aryl Head Groupa
Ki ± SEM (nM)b
Compound
R
D2R/D3R Ratioe
cLogPf
tPSAf
D3Rc
D2Rd
35
92.9 ± 20.5
2,766 ± 582
29.8
5.64
44.81
36
37
61.5 ± 15.0
3,762 ± 776
2,630 ± 400
61.2
0.1
4.43
4.50
48.05
54.04
14,587 ± 1,772
aSpiperone assayed under the same conditions as a reference blocker: D2R, 0.06 ± 0.001 nM; D3R, 0.33
± 0.02 nM; D4R, 0.45 ± 0.01 nM. bMean ± SEM; Ki values were determined by at least three experiments.
cKi values for D3 receptors were measured using human D3 expressed in HEK cells with [125I]IABN as
the radioligand. dKi values for D2 receptors were measured using human D2 expressed in HEK cells with
[125I]IABN as the radioligand. e(Ki for D2 receptors)/(Ki for D3 receptors). fCalculated using ChemDraw
Professional 15.1.
Lead compound 1 was fragmented in order to identify synthons acting as primary (PP) or
secondary pharmacophores (SP). Unexpectedly, fragments 4b’ and 5a were found to have micromolar
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affinity for the receptor, with 5a exhibiting nominal D3R selectivity (Table 5). Low receptor binding was
also observed with synthons 5b and 5c, despite the Ki values of 19.6 nM and 24.2 nM obtained from the
parent compounds 2 and 3, respectively. The binding affinities obtained with 5a-c are in contrast to those
reported for alkylated arylpiperazine synthons (i.e., 5d11 and 5e21, 23) of potent D3R ligand scaffolds. These
PP typically show Ki values of ~ 10-20 nM at the receptor by engaging the orthosteric binding pocket
(OBP) of the protein. This allows the ionizable nitrogen of these PP to form an ionic interaction with the
highly conserved Asp110 [3.32] residue within the OBP, affording the favorable receptor affinity of these
orthosteric fragments.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Binding studies continued with evaluation of synthons from the right-half of lead compound 1 to
determine if receptor affinity was being engaged by an unexpected 1,2,4-triazole pharmacophore. Similar
to the displacement observed with synthon 5a, low receptor binding was observed with fragments 6a, 6c,
6d, and 6f. Although minimal potency was observed with these 1,2,4-trizole synthons, a trend of
increasing affinity was observed when elongating fragment 6a with piperdine (6c), and 4,4-
dimethylpiperidine (6d). In addition, we found 6f to be ~197-fold selective for the D3R over the D2R,
while piperazine analogue 6h illustrated no receptor selectivity. We hypothesized the 1,2,4-triazole
system may be key in directing, and subsequently, stabilizing the bulky diazaspiro PPs of 1-3, within the
OBP of the receptor. Once stabilized, these diazaspiro systems may be able to interact with residues within
the OBP due to the large structural space these systems inhabit which are not accessible to the piperazine
PP of 31.
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Table 5. D3R/D2R Binding Profiles of Synthonsa
1
2
3
4
5
Ki ± SEM (nM)b
Ki ± SEM (nM)b
Compo
und
D2R/D3R
Ratioe
Compou
nd
D2R/D3R
Ratioe
Synthon
Synthon
D3Rc
D2Rd
D3Rc
D2Rd
6
7
8
9
>39,319 ±
99,379 ±
11,752
>50,316
± NA
180,375
± 70,719
>2.5
>3.6
89.6
NA
4b’
6a
6c
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2,773 ±
384
26,739 ±
NA
1,098 ±
53.8
98,462 ±
18,965
>9.6
3.9
5a
6,647 ±
4,559
25,823 ±
14,830
631 ±
43.7
59,994 ±
22,704
95.1
5b
5c
6d
6f
23,256 ±
9,615
26,050 ±
NA
1,145 ±
97.1
>225,74
8 ± NA
>1.1
0.86
1.5
>197
23.9 ± 5.8
20.6 ± 4.8
5df
72,753
±
13,438
74,272 ±
17,815
1
6h
14.7 ± 1.2
22.3 ± 4.9
5e
aSpiperone assayed under the same conditions as a reference blocker: D2R, 0.06 ± 0.001 nM; D3R, 0.33
± 0.02 nM; D4R, 0.45 ± 0.01 nM. bMean ± SEM; Ki values were determined by at least three experiments.
cKi values for D3 receptors were measured using human D3 expressed in HEK cells with [125I]IABN as
the radioligand. dKi values for D2 receptors were measured using human D expressed in HEK cells with
[125I]IABN as the radioligand. (Ki for D2 receptors)/(Ki for D3 receptors). Compound and receptor
e
f
binding data from ref. 11.
Molecular Docking Studies. To probe these potential protein-ligand interactions, select
compounds were modeled within the D3R (3PBL) to identify residue contacts within orthosteric and SBP
of the receptor (Figure 2, A). A top docking pose of 1 was found to engage in multiple polar and non-
polar contacts between the arylated diazaspiro[5.5]undecane moiety, including π–π stacking interactions
between the aryl ring and the Phe3466.52 residue within the OBP of the receptor (Figure 2, B). Similarly,
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ligand docking also predicted the diazaspiro[5.5]undecane moiety of ligand 17, and diazaspiro[5.5]undec-
1-ene system of high-affinity compound 32, to also engage in hydrophobic interactions with residues
Phe346 [6.52] and Phe345 [6.51] (Figure 2, C and F). When evaluating the potential salt bridge formation
between residue Asp110 [3.32] and the ionizable nitrogens in the diazaspiro cores of scaffolds 1, 17, and
32, we identified distances of 3.4 Å, 3.2 Å and 3.1 Å, respectively. Interestingly, these were all found to
be greater than the predicted 2.7 Å distance observed with binding pose of compound 31 (Figure 2, E).
These results could explain the contrasting receptor binding affinity observed between diazaspiro PP 5a
and piperazine PP 5d. Thus, we surmise the 1,2,4-triazole side of the molecule appropriately positions the
bulky diazaspiro[5.5]undecane moiety within the hydrophobic cavity of the D3R OBP, allowing the amino
core to engage in non-polar interactions with residues Phe346 [6.52] and Phe345 [6.51]. These contacts
then assist in stabilizing this “weaker” ionic interaction with Asp110 [3.32], resulting in the observed
receptor affinity for 1, 17, and 32. This analysis is also consistent with the poor binding affinity for
compound 19, as the docking pose predicted minimal hydrophobic interactions with residues Phe345
[6.52] and Phe346 [6.51], and a distance of 4.2 Å between the protonated nitrogen of the ligand and the
conserved Asp110 [3.32] side chain (Figure 2, D).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Next, we evaluated interactions between the non-conserved residues within the SBP of the D3R,
responsible for ligand selectivity,11 and the predicted binding modes of 1 and its derivatives. In addition
to the significant polar and non-polar interactions between Val86 [2.61] and the 1,2,4-triazole system of
lead compound 1, we also identified a potential weak hydrogen bond interaction between Glu90 [2.65]
and the methyl substituent of the N-heterocycle (Figure 2, B). Disruption of this C–H···O interaction may
explain the reduction in receptor selectivity for compound 17 (Figure 2, C), in which the only structural
difference between this ligand and 1, is the removal of the –CH3 group. When substituting the –CH3 with
–CH2CH3 (15)or–NH2 (16), receptor selectivity is again diminished, while binding affinity is moderately
maintained. Although the N–H···O hydrogen bond interactions of 17 and Glu90 [2.65] would appear to
be more favorable than the C–H···O interactions of 1, previous studies have shown the latter to result in
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more favorable binding profiles due to the potential penalty associated with –NH2 desolvation upon ligand
1
2
3
binding.24, 25
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. (A) Predicted binding modes of compound 1 derivatives in complex with the D3R (3PBL).
Predicted polar (yellow, 3.0 Å cut-off), weak hydrogen bonding (magenta, 3.6 Å cut-off), π–π stacking
(purple, 5.5 Å cut-off), and hydrophobic (black, 4.0 Å cut-off) residue interactions with compounds 1 (B),
17 (C), 19 (D), 31 (E), and 32 (F) within the binding pocket of the receptor. Interaction between the
Asp110 [3.32] residue and the ionizable nitrogen of the ligand scaffold is shown in red.
Previous reports have discussed the critical role EL1 Gly residues can play in determining
D2R/D3R selectivity of ligands.26, 27 Glycine can act as an acceptor in weak hydrogen bond interactions in
many protein-ligand complexes.24 In our study, the predicted binding pose of 1 places the aryl ring off of
the 1,2,4-triazole moiety in close proximity to the Gly93 [EL1] residue, affording potential weak C–H···O
interactions. However, this interaction is found to be absent in the binding poses of less selective ligands
31 and 32. We hypothesize the lack of receptor selectivity observed for compounds 10-11 and 13 may be
a result of steric clashing between the para substituents of these ligands and the Gly93 [EL1] residue in
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the SBS. This steric clash could also explain the lack of receptor selectivity for compounds 21 and 35-36,
which all contain a butyl spacer group, compared the shorter propyl linker of 1. We observed this proposed
SAR trend after installing a C–F bond in the para position of 1, resulting in a loss of receptor selectivity
for 13 (D2R/D3R = 905 vs. 396, respectively). Receptor selectivity was able to be slightly restored,
however, after moving the C–F substitution to ortho site of the aryl ring in 12, potentially alleviating steric
interactions afforded with Gly93 [EL1] and compound 13.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 1 Competitively Inhibits DA at the OBP. To confirm the binding mode of 1 does
indeed require occupancy within the OBP, we evaluated 1 (Figure 3, A), 31 (Figure 3, B), and known
allosteric modulator SB26965228, 29 (Figure 3, C) in a β-arrestin assay with endogenous ligand dopamine
(DA) as the orthosteric ligand. Both 1 and 31 caused a limitless rightward shift in the dopamine-dose
response curves, suggesting a competitive mode of action at the OBP.30 This is in contrast to the limited
dextral shift that is observed with SB269652, resulting from the allosteric pharmacology of the compound
at the receptor. Furthermore, when plotting the concentration-response (CR) curves for 1 and SB269,652
into a Schild plot, a linear regression slope near unity is obtained for 1, indicative of competitive
antagonism,31 while a non-linear regression slope is observed for SB269,652, indicating an allosteric
mode of antagonism (See SI).32-34 Overall, these data clearly indicate 1 behaves in a competitive manner
at the OBP with the orthosteric endogenous ligand DA, ruling out an allosteric binding mechanism for
the diazaspiro ligand.
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Figure 3. Curve shift analysis of 1 (A), 31 (B), and SB269,625 (C) using a dopamine (DA) mediated β-
arrestin 2 recruitment assay. The receptor was stimulated with the indicated concentration of DA along
with and without concentrations of the test compound. Data points represent the mean ± SEM of
dopamine-induced luminescence response obtained from seven to sixteen replicates.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PDSP Pharmacological Evaluation. Compounds 1 and 31 were then submitted to the
Psychoactive Drug Screening Program (PDSP)35, 36 to evaluate the binding (Table 6) and functional
behavior of the ligands at D1- and D2-like receptors (Figure 4). Compared to our initial report, the PDSP
obtained Ki values that differed slightly from our initial report14 for both compounds at the D3R and D2R.
However, this is most likely due to the use of [3H]N-methylspiperone by the PDSP in their binding assay,
compared to our use of [125I]IABN, a high-affinity radioligand with negligible non-specific binding37.
Compound 1 showed no interaction at the D4R or D1-like receptors, while 31 exhibited Ki values of 721
nM and ~ 3 µM at the D4R and D5R, respectively (Table 6). Lead compound 1 was found to behave as a
preferential D3R antagonist (IC50 = 41.4 nM) with negligible functional potency at the D2R (IC50 > 1.5
µM) (Figure 4, D). While antagonist affinities are known to vary depending on the competing agonist
used38, we are currently evaluating 1 in other cell-signaling cascade models to further assess the 39-fold
D2R/D3R selectivity ratio observed in the PDSP Tango functional assays. Ligand 31 however, was found
to be a weak agonist/antagonist at both the D2R and D3R (Figure 4, C-F), a common nuance that has been
disclosed with previously reported D3R selective piperazine-based scaffolds.11, 39 This can be attributed
to the lack of receptor selectivity that is engaged with the aryl piperazine orthosteric PP of the ligand
scaffold. Finally, compound 31 was also identified as a weak partial agonist at the D4R (IC50 = 427 nM)
(Figure 4, G).
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
Figure 4. Agonist and antagonist selectivity profiling with G-protein-independent β-arrestin recruitment
Tango assays at human D1- and D2-like receptors. (A) Concentration-response to varying doses of D1R
agonists and control SKF81297. (B) Response to varying doses of D1R antagonists and control
SCH23390, added 30 min before addition of a final EC80 concentration (3 µM) of reference agonist
SKF81297. (C) Concentration-response to varying doses of D2R agonists and control quinpirole. (D)
Concentration-response to varying doses of D2R antagonists and control haloperidol, added 30 min before
addition of a final EC80 concentration (3 nM) of reference agonist quinpirole. (E) Concentration-response
to varying doses of D3R agonists and control quinpirole. (F) Concentration-response to varying doses of
D3R antagonists and control haloperidol, added 30 min before addition of a final EC80 concentration (3
nM) of reference agonist quinpirole. (G) Concentration-response to varying doses of D4R agonists and
control lisuride. (H) Concentration-response to varying doses of D4R antagonists and control
nemonapride, added 30 min before addition of a final EC80 concentration (100 nM) of reference agonist
lisuride. Data are shown as mean ± SEM of 3 independent experiments. N/A, not active. N/C, data not
converged.
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PDSP also evaluated compounds 1 and 31 for off-target interactions with other GPCRs (Table 6).
Similar to the findings in our initial publication14, ligand 31 was found to bind with high affinity at the 5-
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HT1A (Ki = 0.9 nM) and 5-HT2B (Ki = 42 nM) receptors, and was also found to exhibit marginal inhibitions
at the 5-HT2C (Ki = 159 nM) and 5-HT7A (Ki = 128 nM). Compound 1, however, exhibited negligible
affinities (Ki > 1 µM) for all the serotonin subclass receptors in the study. These findings show the
potential of 1 as a D3R PET agent, as serotoninergic interactions are known to hinder many piperazine
1
2
3
4
5
6
7
8
9
containing scaffolds40-42, 23, 43
.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Among the subclass of histamine GPCRs screened, both compounds bind at the H1 histamine
receptor, with high affinity (1, Ki = 8 nM; 31, Ki = 40 nM). No appreciable opioid receptor inhibition was
identified during the primary screen for 1 and 31 at 10 µM drug concentrations. Lead compound 1 was
found to weakly interact at the muscarinic M1 (54% inhibition at 10 µM of compound) and M3 (Ki = 514
nM) receptors as well.
Off-target interactions were discovered with compounds 1 and 31 at the subfamilies of the α1- and
α2-adrenergic receptors, which are GPCRs known to play a role in vasoconstriction.44 While 1 showed no
appreciable interactions at the examined adrenergic sites, compound 31 was found to be active at the α1A
(Ki = 49 nM) and the α1B (Ki = 94 nM) receptors, with moderate inhibitive action at the α2 subclass group.
Adrenergic interactions have been observed with many piperazine-containing D3R ligand scaffolds45,
including BP 897.46 This is consistent with a recent study indicating α2-adrenoceptors are targets for
known D2-like receptor ligands including 7-OH-PIPAT, RO-105824, and dopamine.47 Unsurprisingly,
the liability of D3R antagonists to induce hypertension, observed with GSK598809 in dog models48, has
hindered these class of compounds from progressing in clinical trials as potential addiction therapeutics49,
making the selective nature of 1 over the adrenergic receptors particularly encouraging.
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Table 6. Binding Affinities of Compounds 1 and 31 at Select GPCRsa
1
2
3
Ki (nM)a or % Inhibition at 10 µMb
Ki (nM)a or % Inhibition at 10 µMb
4
5
6
7
8
9
GPCR
GPCR
1
31
N/A
577
6
1
31
N/A
N/A
94
54%
52
N/A
N/A
NT
D1
D2
M1
M2
Dopamine
D3
Muscarinic
M3
514
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N/A
N/A
1,297
N/A
N/A
N/A
N/A
2,144
N/A
N/A
N/A
N/A
N/A
8
721
3,016
0.9
590
N/A
N/A
49
D4
M4
N/A
2,178
2,173
N/A
N/A
1,557
N/A
N/A
N/A
8,523
N/A
N/A
N/A
N/A
N/A
N/A
931
D5
M5
5-HT1A
5-HT1B
5-HT1D
5-HT1E
5-HT2A
α1A
α1B
α2A
α2B
α2C
α2β2
α2β4
α3β2
α3β4
α4β2
α4β4
β1
2,032
>50%
N/A
1,415
42
94
221
241
433
N/A
N/A
N/A
N/A
N/A
N/A
N/A
60%
N/A
N/A
N/A
N/A
N/A
Serotonin 5-HT2B
159
N/A
N/A
N/A
128
40
5-HT2C
5-HT3
5-HT5A
5-HT6
5-HT7A
H1
Adrenergic
NT
NT
H2
Histamine
H3
β2
73%
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
β3
H4
µ
Dopamine Active Transporter (DAT)
Norepinephrine Transporter (NET)
Serotonin Transporter (SERT)
GABAA
Opioid
κ
δ
295
N/A
N/A, not active (<50% inhibition obtained in primary assay at 10 µM of compound). NT, not tested. aKi
values determined by at least three experiments. b% of receptor inhibition at 10 µM of compound, values
determined by at least three experiments.
CONCLUSIONS
In our previous report, we identified 1 as a new lead D3R selective compound containing a novel
diazaspiro[5.5]undecane amino core.14 In this study, we imposed structural modification to 1, affording
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analogues with slightly enhanced D3R selectivity (30, D3R Ki = 21.0 nM, D2R/D3R ratio = 934) and
affinity (32, D3R Ki = 3.2 nM, D2R/D3R ratio = 60). Fragmentation of 1 revealed diazaspiro[5.5]undecane
synthon 5a to be a low-affinity PP for the receptor (5a, D3R Ki > 2.7 µM), despite the excellent potency
observed with the full length compound (1, D3R Ki = 12.0 nM). Molecular docking studies predicted the
1,2,4-triazole half of 1 to favorably position the bulky diazaspiro within the OBP of the receptor, allowing
the amino core to engage in hydrophobic interactions with the Phe345 [6.52] and Phe346 [6.51] residues,
ultimately stabilizing the “weak” ionic interaction between the ionizable nitrogen of the PP and the highly
conserved Asp110 [3.32]. To verify 1 was not interacting at another receptor site, (i.e., allosteric behavior)
we utilized a dopamine-mediated β-arrestin 2 recruitment assay to show the competitive manner in which
1 behaves with the orthosteric ligand, revealing the binding mode of 1 does indeed require OBP
occupancy. Upon examining the functional behavior of compounds 1 and 31 at D1- and D2-like receptors,
we found 1 to be a more D3R selective ligand for both receptor binding and cell signaling. These results,
combined with the minimal off-target interactions observed with 1 at other GPCRs, suggests the unique
binding contacts between the PP of 1 and the highly conserved residues within OBP of the D3R receptor
play a pivotal role in diminishing non-specific ligand binding with other closely related serotoninergic
and adrenergic sites. As such, installing low affinity amino fragments, such as 5a, into D3R ligand systems
may be beneficial in overcoming drug promiscuity, commonly encountered with piperazine-based ligand
scaffolds. This unorthodox approach could be exploited for the development of clinically viable D3R
selective compounds for addiction therapeutic and PET imaging applications.
1
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5
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EXPERIMENTAL SECTION
Chemistry: Arylated amine synthons 4 and A-B were synthesized following our previously disclosed
Pd-catalyzed C–N cross-coupling reports.15, 16 Compounds C, G, 6b, 1-3, 25, 27, 31, and 33 were obtained
following the synthetic procedures outlined in our initial publication.14 Finally, 1,2,4-triazole compounds
D, and all other commercial reagents were purchased and used as without further purification. However,
D analogues were also able to be obtained in good yield following the protocol described by Micheli and
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co-workers.18 NMR spectra were taken on a Bruker DMX 500 MHz. Compound structures and identity
1
2
3
4
5
6
7
8
1
13
were confirmed by H and C NMR, and mass spectrometry. Compound purity greater than 95% was
determined by LCMS analysis using a 2695 Alliance LCMS. Purification of organic compounds were
carried out on a Biotage Isolera One with a dual wavelength UV−Vis. detector. Chemical shifts (δ) in the
9
13
NMR spectra (1H and C) were referenced by assigning the residual solvent peaks. Compounds were
10
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taken up with CH2Cl2 followed by dropwise addition of a 2.0 M HCl solution in diethyl ether. After stirring
at room temperature for 1 h, the solvent was removed under reduced pressure to afford the desired
compound as a hydrochloride salt for in vitro studies.
General Method A: Synthesis of Fragmented Synthons 4-5. The appropriate arylated diazaspiro and
piperazine precursors (4), obtained following our previous reports,15, 16 were dissolved in CH2Cl2 (2 mL),
followed by dropwise addition of CF3COOH (2 mL), and stirred at room temperature for 3 h. Volatiles
were then removed under reduced pressure and the crude product was neutralized with a saturated
NaHCo3(aq) solution (10 mL). The reaction mixture was extracted with CH2Cl2 (3 x 20 mL), and the
organic layers were combined, dried, and concentrated to afford the free-amine intermediates (4’) which
were used without further purification. A mixture of 4’ (1.0 mmol), 1-bromobutane (1.1 mmol), and
K2CO3 (1.5 mmol) was stirred in acetone (5 mL) at room temperature for 5 h. The crude reaction mixture
was then filtered, and solvent was removed under reduced pressure. The residue was purified by flash
chromatography on silica gel eluting with MeOH solution/CH2Cl2 (1:10) affording target compounds (5).
General Method B: Synthesis of Diazaspiro Analogues 1-3 and 7-34. Target compounds can be
obtained following the synthetic procedures outlined in our initial.14 Briefly, the appropriate arylated
diazaspiro and piperazine precursors (A), obtained following our previous reports,15, 16 were dissolved in
CH2Cl2 (2 mL), followed by dropwise addition of CF3COOH (2 mL), and stirred at room temperature for
3 h. Volatiles were then removed under reduced pressure and the crude product was neutralized with a
saturated NaHCo3(aq) solution (10 mL). The reaction mixture was extracted with CH2Cl2 (3 x 20 mL), and
the organic layers were combined, dried, and concentrated to afford the free-amine intermediates (B)
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which were used without further purification. A mixture of B (1 mmol), alkylating reagent (2 mmol), and
K2CO3 (1.5 mmol) was stirred in acetone (5 mL) at room temperature for 12 h. The crude reaction mixture
was then filtered and solvent was removed under reduced pressure. The residue was loaded onto a Biotage
SNAP flash purification cartridge and eluded with 5% MeOH in CH2Cl2 affording intermediates C.
Finally, a mixture of D (1 mmol), TEA (1.5 mmol), and ethanol (10 mL) was stirred at 75 °C for 15 min.
The appropriate intermediate C (1 mmol) was then added, and the solution was stirred at 75 °C for 12 h.
Solvent from the crude reaction mixture was then removed under reduced pressure. The residue was
loaded onto a Biotage SNAP flash purification cartridge and eluded with 10% 7N NH3 in MeOH
solution/CH2Cl2 to give the target compounds 1-3 and 7-34. The residue was loaded onto a Biotage SNAP
flash purification cartridge and eluded with 10% 7N NH3 in MeOH solution/CH2Cl2 to give the target
compounds 1-3 and 7-34.
1
2
3
4
5
6
7
8
9
10
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13
14
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2-(4-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)butyl)isoindoline-1,3-dione (E). 4b’ (1
mmol), 2-(4-bromobutyl)isoindoline-1,3-dione (1.2 mmol), KI (1.0 mmol) and Cs2CO3 (2.5 mmol) were
dissolved in acetonitrile (5 mL), and the reaction mixture was stirred at 75 °C for 3 h. The reaction mixture
was then filtered and solvent was removed under reduced pressure. Crude residue was purified by silica
gel column chromatography using ethyl acetate/hexane (1:1) as the mobile phase to afford E as a light-
orange solid (Yield 64%). 1H NMR (500 MHz, CDCl3) δ 7.75-7.74 (m, 2H), 7.62-7.61 (m, 2H), 6.90-6.87
(m, 2H), 6.84-6.82 (m, 1H), 6.77 (d, J = 7.9 Hz, 1H), 3.77 (s, 3H), 3.63 (t, J = 6.9 Hz, 2H), 2.91-2.89 (m,
13
4H), 2.33-2.28 (m, 6H), 1.62-1.59 (m, 6H), 1.52-1.45 (m, 6H); C NMR (125 MHz, CDCl3) δ 168.1,
152.0, 142.0, 133.7, 131.9, 122.9, 122.4, 120.7, 118.0, 110.8, 58.3, 55.1, 49.1, 46.5, 37.6, 35.6, 29.0, 26.5,
24.2; LC-MS (ESI) m/z: 462.64 [M+H]
4-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)butan-1-amine (F). E (1 mmol) and
hydrazine hydrate (50-60%) were dissolved in ethanol (5 mL), and the reaction mixture was stirred at 75
°C for 2 h. The reaction mixture was then filtered and solvent was removed under reduced pressure. Crude
residue was purified by silica gel column chromatography 10% 7 N NH3 in MeOH solution/CH2Cl2
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(1.5:1) to afford F as a clear oil (Yield 97%). 1H NMR (500 MHz, CDCl3) δ 6.89-6.86 (m, 2H), 6.82-6.78
(m, 1H), 6.75-6.73 (m, 2H), 3.75 (s, 3H), 2.89-2.87 (m, 4H), 2.62-2.59 (t, J = 6.7 Hz, 2H), 2.31 (bs, 4H),
2.26-2.23 (t, J = 7.0 Hz, 2H), 1.59-1.1.57 (m, 4H), 1.50-1.49 (m, 4H), 1.46-1.41 (m, 2H), 1.38-1.34 (m,
2H), 1.21 (bs, 2H); 13C NMR (125 MHz, CDCl3) δ 152.0, 142.0, 122.3, 120.6, 117.9, 110.8, 58.8, 55.1,
49.1, 46.5, 41.9, 35.6, 31.7, 28.9, 24.3; LC-MS (ESI) m/z: 332.65 [M+H]
1
2
3
4
5
6
7
8
9
10
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13
14
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Tert-butyl 9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undec-7-ene-3-carboxylate (4a). Compound 4a
can be synthesized following using the reaction conditions disclosed in Pd C–N cross-coupling report.15
Separation of 4a and the saturated major product 4b was achieved by flash chromatography on silica gel
eluting with a 40% EtOAc/hexane gradient to afford a tan oil. (Yield 9%). 1H NMR (500 MHz, CDCl3) δ
7.02-6.98 (m, 1H), 6.95-6.93 (m, 1H), 6.91-6.86 (m, 2H), 6.28 (d, J = 8.1 Hz, 1H), 4.64 (d, J = 8.2 Hz,
1H), 3.83 (s, 3H), 3.53-3.50 (m, 2H), 3.46-3.43 (m, 2H), 3.41-3.35 (m, 2H), 1.74-1.72 (m, 2H), 1.54 (bs,
2H), 1.46 (s, 9H), 1.44-1.42 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 155.1, 152.4, 137.6, 132.0, 123.7,
122.2, 121.1, 112.1, 105.5, 79.3, 55.7, 44.2, 39.9, 38.5, 34.5, 30.3, 28.6; LC-MS (ESI) m/z: 303.47 [M-
C(CH3)3+H]
3-Butyl-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (5a). Following general method A, 5a was
obtained as a white semi-solid. (Yield 38%). 1H NMR (500 MHz, CDCl3) δ 6.97-6.93 (m, 2H), 6.90-6.88
(m, 1H), 6.83-6.82 (m, 1H), 3.83 (s, 3H), 2.97-2.95 (m, 4H), 2.45 (bs, 4H), 2.38-2.35 (m, 2H), 1.68-1.65
(m, 4H), 1.61-1.59 (m, 4H), 1.52-1.46 (m, 2H), 1.34-1.28 (m, 2H) 0.90 (t, J = 7.3 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 152.3, 142.2, 122.6, 120.9, 118.2, 111.0, 58.8, 55.3, 49.3, 46.7, 35.6, 29.1, 29.0, 20.9,
14.1; LC-MS (ESI) m/z: 317.68 [M+H]
8-Butyl-2-(2-methoxyphenyl)-2,8-diazaspiro[4.5]decane (5b). Following general method A,
compound 5b was obtained as an off-white semi-solid. (Yield 59%). 1H NMR (500 MHz, CDCl3) δ 6.88-
6.82 (m, 3H), 6.70 (d, J = 7.0 Hz, 1H), 3.80 (s, 3H), 3.37 (t, J = 6.9 Hz, 2H), 3.21 (s, 2H), 2.98 (bs, 2H),
2.81-2.78 (m, 2H), 1.99 (bs, 4H), 1.82 (t, J = 6.8 Hz, 2H), 1.76-1.73 (m, 2H), 1.38-1.34 (m, 2H), 0.94 (t,
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J = 7.3 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 150.3, 139.2, 121.3, 120.0, 115.3, 112.0, 55.7, 50.5, 48.9,
1
2
3
38.9, 33.3, 26.5, 20.4, 13.7; LC-MS (ESI) m/z: 303.52 [M+H]
4
5
6
2-Butyl-7-(2-methoxyphenyl)-2,7-diazaspiro[4.4]nonane (5c). Following general method A, 5c was
7
8
9
1
obtained as an oil. (Yield 38%). H NMR (500 MHz, CDCl3) δ 6.87-6.80 (m, 3H), 6.68-6.67 (m, 1H),
10
11
12
13
14
15
16
17
18
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3.80 (s, 3H), 3.45-3.35 (m, 3H), 3.29 (d, J = 9.4 Hz, 1H), 3.12 (bs, 2H), 3.00 (bs, 2H), 2.78-2.75 (m, 2H),
2.07-1.91 (m, 4H), 1.65-1.58 (m, 2H), 1.38-1.33 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 150.0, 139.1, 121.2, 119.6, 114.8, 112.0, 63.9, 61.8, 56.3, 55.6, 49.6, 47.6, 37.3, 35.7, 29.9,
20.4, 13.8; LC-MS (ESI) m/z: 289.93 [M+H]
1-Butyl-4-(2-(2-fluoroethoxy)phenyl)piperazine (5e). Following general method A, 5e50 was obtained
as a colorless oil. (Yield 41%). 1H NMR (360 MHz, CDCl3) δ 6.97-6.95 (m, 3H), 6.85 (m, 1H), 4.77 (dt,
J = 47.5, 4.0 Hz, 2H), 4.25 (dt, J = 29.0, 4.0 Hz, 2H), 3.16 (s, 4H), 2.68 (s, 4H), 2.43 (t, J = 7.8 Hz, 3H),
1.54 (m, 2H), 1.35 (m, 2H) 0.93 (t, J = 7.3 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 150.9, 141.9, 122.6,
122.1, 118.4, 113.7, 82.5, 81.2, 67.6, 67.4, 58.5, 53.4, 50.3, 28.6, 20.7, 11.9; LC-MS (ESI) m/z: 281.20
[M+H]
4-Methyl-3-phenyl-5-(propylthio)-4H-1,2,4-triazole (6a). A mixture of 4-methyl-5-phenyl-4H-1,2,4-
triazole-3-thiol (1.0 mmol), 1-bromobutane (1.1 mmol), and K2CO3 (1.5 mmol) was stirred in acetone (5
mL) at room temperature for 12 h. The crude reaction mixture was then filtered, and solvent was removed
under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10%
1
7 N NH3 in MeOH solution/CH2Cl2 (1:10) affording 6a as a white solid. (Yield 81%). H NMR (500
MHz, CDCl3) δ 7.58-7.56 (m, 2H), 7.44-7.41 (m, 3H), 3.53 (s, 3H), 3.19 (t, J = 7.2 Hz, 2H), 1.78-1.74
13
(quint, J = 7.2 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H); C NMR (125 MHz, CDCl3) δ 155.8, 129.9, 128.8,
128.5, 127.1, 35.4, 31.6, 22.9, 13.2; LC-MS (ESI) m/z: 234.17 [M+H]
1-(3-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)piperidine (6c). A mixture of 6b14 (1.0
mmol), piperdine (1.1 mmol), and Cs2CO3 (1.5 mmol) was stirred in acetonitrile (3 mL) at 70 °C for 12
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h. The crude reaction mixture was then filtered, and solvent was removed under reduced pressure. The
residue was purified by flash chromatography on silica gel eluting with 10% 7 N NH3 in MeOH
solution/CH2Cl2 (1:10) affording 6c as a white solid. (Yield 20%). 1H NMR (500 MHz, CDCl3) δ 7.60-
7.58 (m, 2H), 7.47-7.45 (m, 3H), 3.56 (s, 3H), 3.26 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 7.3 Hz, 2H), 2.44 (bs,
4H), 2.04-1.98 (m, 2H), 1.61-1.57 (quint, J = 5.6 Hz, 4H), 1.41 (bs, 2H); 13C NMR (125 MHz, CDCl3) δ
155.9, 151.8, 130.0, 128.9, 128.6, 127.1, 57.5, 54.5, 31.6, 31.5, 26.5, 25.5, 24.1; LC-MS (ESI) m/z: 317.15
[M+H]
1
2
3
4
5
6
7
8
9
10
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4,4-Dimethyl-1-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)piperidine (6d). A mixture
of 6b14 (1.0 mmol), 4,4-dimethylpiperdine (1.1 mmol), and Cs2CO3 (1.5 mmol) was stirred in acetonitrile
(3 mL) at 70 °C for 8 h. The crude reaction mixture was then filtered, and solvent was removed under
reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N
NH3 in MeOH solution/CH2Cl2 (1:10) affording 6d as a white solid. (Yield 26%).1H NMR (500 MHz,
CDCl3) δ 7.59-7.58 (m, 2H), 7.46-7.44 (m, 3H), 3.57 (s, 3H), 3.25 (t, J = 6.7 Hz, 2H), 2.92-2.89 (m, 2H),
2.80 (bs, 4H), 2.24-2.20 (m, 2H), 1.60 (bs, 4H), 0.93 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 156.1, 151.3,
130.1, 128.9, 128.6, 126.9, 56.1, 49.6, 36.4, 31.7, 30.6, 28.0, 25.2; LC-MS (ESI) m/z: 345.18 [M+H]
Tert-butyl
9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-
diazaspiro[5.5]undecane-3-carboxylate (6e). A mixture of 6b14 (1.0 mmol), tert-butyl 3,9-
diazaspiro[5.5]undecane-3-carboxylate (1.1 mmol), and Cs2CO3 (1.5 mmol) was stirred in acetonitrile (5
mL) at 70 °C for 12 h. The crude reaction mixture was then filtered, and solvent was removed under
reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N
1
NH3 in MeOH solution/CH2Cl2 (1:10) affording 6e as an off-white solid (Yield 47%). H NMR (500
MHz, CDCl3) δ 7.55-7.54 (m, 2H), 7.42-7.40 (m, 3H), 3.52 (s, 3H), 3.28-3.26 (m, 4H), 3.21 (t, J = 7.0
Hz, 2H), 2.51 (t, J = 7.1 Hz, 2H), 2.42 (bs, 4H), 1.99-1.93 (quint, J = 7.1 Hz, 2H), 1.48 (t, J = 5.6 Hz,
4H), 1.36 (s, 9H), 1.34 (bs, 4H); 13C NMR (125 MHz, CDCl3) δ 155.7, 154.8, 151.7, 129.9, 128.8, 128.4,
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126.9, 79.1, 65.7, 56.9, 48.9, 39.6, 38.8, 34.8, 31.5, 30.9, 29.4, 28.3, 26.4, 15.2; LC-MS (ESI) m/z: 486.17
[M+H]
1
2
3
4
5
6
3-(2-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane
(6f).
7
8
9
Compound was dissolved in CH2Cl2 (2 mL), followed by dropwise addition of CF3COOH (2 mL), and
stirred at room temperature for 3 h. Volatiles were then removed under reduced pressure, and the crude
product was neutralized with a saturated NaHCO3 (aq) solution (10 mL). The reaction mixture was
extracted with CH2Cl2 (3 × 20 mL), and the organic layers were combined, dried, and concentrated to
afford 6f as an off-white solid (Yield 97%). (HCl salt) 1H NMR (500 MHz, (CD3)2SO) δ 10.97 (bs, 1H),
7.78-7.76 (m, 2H), 7.62-7.59 (m, 3H), 3.65 (s, 3H), 3.35 (t, J = 6.9 Hz, 2H), 3.29-3.26 (m, 2H), 3.21-3.16
(m, 2H), 2.99 (bs, 6H), 2.22-2.17 (m, 2H), 1.83-1.77 (m, 6H), 1.54-1.52 (m, 2H); 13C NMR (125 MHz,
(CD3)2SO) δ 154.7, 151.2, 130.8, 128.9, 128.8, 125.0, 53.7, 47.0, 38.7, 38.6, 34.6, 32.2, 31.1, 30.1, 27.9,
26.7, 23.4; LC-MS (ESI) m/z: 386.68 [M+H]
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1-(3-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)piperazine (6h). Following the procedure
for 6f, compound 6h was obtained as a light tan oil. (Yield 12%). 1H NMR (500 MHz, CDCl3) δ 7.56-
7.54 (m, 2H), 7.43-7.41 (m, 3H), 3.52 (s, 3H), 3.25-3.22 (m, 2H), 3.21 (s, 1H), 2.85-2.83 (t, J = 4.8 Hz,
4H), 2.42-2.38 (m, 6H), 1.95-1.89 (quint, J = 7.1 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 155.7, 151.9,
130.0, 128.8, 128.5, 127.0, 57.2, 53.8, 45.6, 31.5, 31.0, 26.5; LC-MS (ESI) m/z: 318.66 [M+H]
4-(5-((3-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)thio)-4-methyl-4H-1,2,4-
triazol-3-yl)-N,N-dimethylaniline (7). Following general method B, 7 was obtained as a tan oil. (Yield
1
48%). H NMR (500 MHz, CDCl3) δ 7.46 (d, J = 8.8 Hz, 2H), 6.92-6.89 (m, 2H), 6.86-6.83 (m, 1H),
6.80-6.78 (m, 1H), 6.72 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.54 (s, 3H), 3.20 (t, J = 7.1 Hz, 2H), 2.96 (s,
3H), 2.93-2.91 (m, 4H), 2.65 (t, J = 7.1 Hz, 2H), 2.57 (bs, 4H), 2.08-2.02 (quint, J = 7.1 Hz, 2H), 1.63
13
(bs, 8H); C NMR (125 MHz, CDCl3) δ 156.4, 152.1, 151.1, 150.5, 141.9, 129.3, 122.6, 120.7, 118.1,
113.9, 111.7, 110.9, 56.7, 55.2, 48.9, 46.5, 45.8, 40.1, 35.8, 34.7, 31.6, 31.0, 28.9, 26.1; LC-MS (ESI)
m/z: 535.32 [M+H]
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Page 29 of 48
Journal of Medicinal Chemistry
3-(2-Methoxyphenyl)-9-(3-((5-(2-methoxyphenyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-
diazaspiro[5.5]undecane (8). Following general method B, 8 was obtained as a clear oil. (Yield 39%).
1H NMR (500 MHz, CDCl3) δ 7.46-7.43 (m, 2H), 7.05-7.02 (m, 1H), 6.97-6.95 (m, 1H), 6.93-6.92 (m,
2H), 6.88-6.86 (m, 1H), 6.81 (d, J = 7.8 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.35 (s, 3H), 3.28 (t, J = 6.9
Hz, 2H), 2.94 (t, J = 4.6 Hz, 4H), 2.54 (t, J = 7.1 Hz, 2H), 2.47 (bs, 4H), 2.03-2.00 (m, 2H), 1.66-164 (m,
4H), 1.60-1.58 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 157.2, 154.3, 152.2, 151.1, 142.1, 132.2, 132.0,
122.6, 121.0, 120.8, 118.2, 116.3, 111.0 (2xCH), 57.2, 55.5, 55.3, 49.2, 46.6, 36.0, 35.5, 31.1, 30.9, 29.1,
26.7; LC-MS (ESI) m/z: 522.23 [M+H]
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3-(2-Methoxyphenyl)-9-(3-((4-methyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-
diazaspiro[5.5]undecane (9). Following general method B, 9 was obtained as a light tan oil. (Yield 53%).
1H NMR (500 MHz, CDCl3) δ 8.85 (s, 1H), 8.69-8.68 (m, 1H), 7.98-7.96 (m, 1H), 7.43-7.40 (m, 1H),
6.94-6.90 (m, 2H), 6.86-6.83 (m, 1H), 6.80 (d, J = 7.7 Hz, 1H), 3.80 (s, 3H), 3.59 (s, 3H), 3.29 (t, J = 6.9
Hz, 2H), 2.94-2.92 (m, 4H), 2.56 (t, J = 6.3 Hz, 2H), 2.49 (bs, 4H), 2.06-2.00 (quint, J = 6.9 Hz, 2H),
1.64-1.62 (m, 4H), 1.60-1.58 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 153.1, 152.7, 152.2, 151.0, 148.8,
142.0, 136.0, 123.7, 123.5, 122.6, 120.8, 118.1, 110.9, 57.0, 55.3, 49.2, 46.6, 36.0, 35.3, 31.6, 31.1, 29.0,
26.5, 22.6; LC-MS (ESI) m/z: 493.24 [M+H]
3-(2-Methoxyphenyl)-9-(3-((4-methyl-5-(4-(thiophen-3-yl)phenyl)-4H-1,2,4-triazol-3-
yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (10). Following general method B, 10 was obtained as a
white solid. (Yield 15%) 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H),
7.51-7.50 (m, 1H), 7.40-7.39 (m, 2H), 6.96-6.93 (m, 2H), 6.89-6.87 (m, 1H), 6.82 (d, J = 8.0 Hz, 1H),
3.82 (s, 3H), 3.59 (s, 3H), 3.29 (t, J = 7.2 Hz, 2H), 2.96-2.94 (m, 4H), 2.49 (t, J = 7.5 Hz, 2H), 2.42-2.40
(m, 4H), 2.02-1.96 (quint, J = 7.1 Hz, 2H), 1.66-1.64 (m, 4H), 1.57-1.55 (m, 4H); 13C NMR (125 MHz,
CDCl3) δ 155.5, 152.2, 152.0, 142.1, 141.1, 137.2, 128.9, 126.7, 126.6, 126.0, 125.6, 122.5, 121.3, 120.8,
118.1, 110.9, 57.3, 55.3, 49.2, 46.6, 36.1, 35.8, 31.6, 31.3, 29.1, 26.9; LC-MS (ESI) m/z: 574.19 [M+H]
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