Design and synthesis of bitopic 2-phenylcyclopropylmethylamine (pcpma) derivatives as selective dopamine d3 receptor ligands

Article abstract of DOI:10.1021/acs.jmedchem.9b01835

2-Phenylcyclopropylmethylamine (PCPMA) analogues have been reported as selective serotonin 2C agonists. On the basis of the same scaffold, we designed and synthesized a series of bitopic derivatives as dopamine D3R ligands. A number of these new compounds show a high binding affinity for D3R with excellent selectivity. Compound (1R,2R)-22e and its enantiomer (1S,2S)-22e show a comparable binding affinity for the D3R, but the former is a potent D3R agonist, while the latter acts as an antagonist. Molecular docking studies revealed different binding poses of the PCPMA moiety within the orthosteric binding pocket of the D3R, which might explain the different functional profiles of the enantiomers. Compound (1R,2R)-30q shows a high binding affinity for the D3R (K_i = 2.2 nM) along with good selectivity, as well as good bioavailability and brain penetration properties in mice. These results reveal that the PCPMA scaffold may serve as a privileged scaffold for the design of aminergic GPCR ligands.

Full text of DOI:10.1021/acs.jmedchem.9b01835

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Article
Design and Synthesis of Bitopic 2-Phenylcyclopropylmethylamine
(PCPMA) Derivatives as Selective Dopamine D3 Receptor Ligands
Liang Tan, Qingtong Zhou, Wenzhong Yan, Jian Sun, Alan P.
Kozikowski, Suwen Zhao, Xi-Ping Huang, and Jianjun Cheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01835 • Publication Date (Web): 13 Apr 2020
Downloaded from pubs.acs.org on April 13, 2020
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Design and Synthesis of Bitopic 2-
Phenylcyclopropylmethylamine (PCPMA)
Derivatives as Selective Dopamine D3
Receptor Ligands
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AUTHOR NAMES
Liang Tan,† Qingtong Zhou,†, ‖ Wenzhong Yan, Jian Sun, Alan P. Kozikowski,
, ‖
†
†
§
†
,#
‡,
†,
Suwen Zhao, Xi-Ping Huang, * and Jianjun Cheng *
AUTHOR ADDRESS
†iHuman Institute, ShanghaiTech University, 393 Middle Huaxia Road, Pudong New
District, Shanghai 201210, China
‡Department of Pharmacology, National Institute of Mental Health Psychoactive Drug
Screening Program (NIMH PDSP), University of North Carolina Chapel Hill Medical
School, Chapel Hill, NC 27599, United States
§StarWise Therapeutics LLC, 2020 N Lincoln Park West, Chicago Illinois 60614
#School of Life Science and Technology, ShanghaiTech University, 393 Middle
Huaxia Road, Pudong New District, Shanghai 201210, China
‖These authors contributed equally.
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ABSTRACT
2
-Phenylcyclopropylmethylamine (PCPMA) analogs have been reported as selective
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serotonin 2C agonists. Based on the same scaffold, we designed and synthesized a series
of bitopic derivatives as dopamine D3R ligands. A number of these new compounds
show a high binding affinity for D3R with excellent selectivity. Compound (1R,2R)-
2
2e and its enantiomer (1S,2S)-22e show comparable binding affinity for the D3R, but
the former is a potent D3R agonist while the latter acts as an antagonist. Molecular
docking studies revealed different binding poses of the PCPMA moiety within the
orthosteric binding pocket of the D3R, which might explain the different functional
profiles of the enantiomers. Compound (1R,2R)-30q shows a high binding affinity for
the D3R (K = 2.2 nM) along with good selectivity, as well as good bioavailability and
i
brain penetration properties in mice. These results reveal that the PCPMA scaffold may
serve as a privileged scaffold for the design of aminergic GPCR ligands.
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INTRODUCTION
G protein-coupled receptors (GPCRs) constitute the largest family of membrane
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proteins with more than 800 members. GPCRs are the dominant drug targets of over
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0% of US FDA-approved small molecule drugs. The aminergic subfamily of GPCRs
is particularly rewarding drug targets that have led to the development of many widely
prescribed drugs, and they remain the most actively pursued GPCR drug targets.3
Endogenous ligands of aminergic GPCRs are monoamines (i.e. dopamine, serotonin,
and histamine) which share a chemotype that features a basic nitrogen atom attached to
an aromatic moiety via a linker. Most drugs targeting aminergic GPCRs are small
molecules of a similar chemotype or their structural derivatives. As a result, many drugs
that target aminergic GPCRs are polypharmacological drugs that show limited
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selectivity across receptor subtypes. On the other hand, the shared chemotype among
different aminergic GPCR ligands also offers an opportunity for scaffold repurposing
of a drug targeting a specific GPCR to compounds that show selectivity for another
receptor.
F
F
NH2
NH2
O
O
F
1
2
5-HT2C: EC50 = 3.4 nM
D3R: Ki = 1712 nM
5-HT2C: EC50 = 4.2 nM
D3R: Ki = 811 nM
Figure 1. PCPMA-based serotonin 2C agonists and their binding activity at D3R.
2-Phenylcyclopropylmethylamines (PCPMAs) are a class of compounds that have been
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reported to act as selective serotonin 2C (5-HT ) agonists, which may have potential
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value in treating neurological diseases such as schizophrenia and drug addiction.8, 9
Compounds 1 and 2 (Figure 1), for example, are highly selective 5-HT agonists that
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show in vivo efficacy in schizophrenia-like behavioral models.4, 9 In our previous
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screening data of compounds 1 and 2, we noticed that they showed moderate binding
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affinities for the dopamine D receptor (D3R) (K = 1712 and 811 nM respectively).
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The D3R is another potential drug target that is involved in central nervous system
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(
CNS) diseases such as drug addiction. Many highly selective D3R ligands have been
reported in the literature, and BP-897 (3, Figure 2) was the first compound to enter
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clinical trials. Shown in Figure 2 are representative D3R antagonists that share a well-
known scaffold with a primary pharmacophore that binds to the orthosteric binding
pocket (OBP) and a secondary pharmacophore binding to the extended binding pocket
(EBP), both of which are connected with a proper linker. GPCR ligands with such a
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scaffold have been depicted as “bitopic” binders. Interestingly, the 2-fluoroethoxyl
substitution as in compound 1 has been used in the D3R antagonist 4,13 and a
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cyclopropane can be found in compounds such as 5 , 6 and 7 , which share structural
similarity with the PCPMA scaffold of compounds 1 and 2. Also, a cyclopropane was
recently introduced into the linker of a reported D3R bitopic agonist 8.17
Although several highly selective D3R ligands have been reported in the literature, most
compounds suffer from poor bioavailability, low brain penetration or unwanted side
effects in clinical trials.18, 19 Therefore, there is still a need for novel D3R selective
ligands. Based on the co-crystal structures of both the 5-HT and the D3R receptors,20,
2C
21
we envisioned that the small PCPMA scaffold of compounds 1 and 2 binds to the
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orthosteric pockets of both receptors, with the primary amine group interacts with the
conserved Asp3.32 residue through a conserved salt bridge among aminergic GPCRs.3
By attaching a D3R-preferring spacer and an EBP binding motif to the nitrogen atom
of PCPMA, we expect to obtain bitopic PCPMA derivatives as D3R selective ligands
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(Figure 2). Therefore, we started a campaign to design and synthesize derivatives of
compound 1 to discover novel D3R ligands, and these results are reported herein. Our
research shows that in addition to its action at the 5-HT receptor, PCPMA may serve
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as a privileged scaffold that can be repurposed for other aminergic GPCRs.
OBP binding
spacer
EBP binding
S
H
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H
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F3C
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(BP-897)
(GSK598,809)
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H
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O
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H
NH2
N
N
N
N
OH
O
F3C
Cl
H2N
6
7
8
(CJ-1882)
PCPMA
EBP
binding
NH2
N
spacer
Rn
Rn
R'
Bitopic
D3R ligands
5-HT2C agonists
Figure 2. Representative D3R antagonists and the design of PCPMA derivatives.
RESULTS AND DISCUSSION
Chemistry. Based on the structural similarity of the PCPMA with D3R ligands 5 and
6, we first set out to synthesize PCPMA derivatives with the triazole-thiol ether as the
secondary pharmacophore. 4-Methyl-5-phenyl-4H-1,2,4-triazole-3-thiol (9) was
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synthesized according to reported procedures and used as the starting material. A
Michael addition reaction of 9 with methyl acrylate, followed by a saponification
afforded the carboxylic acid 11. For the PCPMA part, Boc-protected intermediates 12a
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and 12b were synthesized according to our published procedures, and both were
alkylated to give 13a-d via Mitsunobu reactions with 2-fluoroethanol or direct
alkylations with iodomethane or iodoethane, as we previously reported.4, 5 After the de-
protection of the Boc, the primary amines 14a-d were coupled with 11 to provide
amides 15a-d, which were subsequently reduced using borane-tetrahydrofuran
complex to provide target compounds 16a-d. These secondary amines could be further
alkylated under reductive amination conditions with simple aldehydes to afford
compounds 17a-l as tertiary amines (for details, see Experimental Section).
Scheme 1. Synthesis of triazole-thiol ether derivatives.a
N
N
N
O
N
N
N
O
N
a
b
HO
S
N
O
S
N
HS
N
N
O
N
9
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X
e
N
S
H
OR1
c
d
X
X
X
15a: X = F, R1 = CH2CH2F
NHBoc
NH2 HCl
.
NHBoc
1
1
1
5b: X = F, R1 = CH2CH3
5c: X = F, R1 = CH3
5d: X = Cl, R1 = CH3
OH
12a: X = F
OR1
OR1
1
1
1
1
3a: X = F, R1 = CH2CH2F
3b: X = F, R1 = CH2CH3
3c: X = F, R1 = CH3
14a: X = F, R1 = CH2CH2F
14b: X = F, R1 = CH2CH3
14c: X = F, R1 = CH3
12b: X = Cl
3d: X = Cl, R1 = CH3
14d: X = Cl, R1 = CH3
N
N
N
N
X
g
f
N
X
N
S
N
S
N
H
OR1
R2
OR1
1
1
1
6a: X = F, R1 = CH2CH2F
6b: X = F, R1 = CH2CH3
6c: X = F, R1 = CH3
2 2 2
17a: X = F, R1 = CH CH F, R = CH3
17b: X = F, R1 = CH CH F, R = CH CH
3
2
2
2
2
17c: X = F, R1 = CH CH , R = CH3
2
3
2
16d: X = Cl, R1 = CH3
2 3 2 2 3
17d: X = F, R1 = CH CH , R = CH CH
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7e: X = F, R1 = CH3, R2 = CH3
7f: X = F, R1 = CH3, R2 = CH2CH3
7g: X = F, R1 = CH3, R2 = CH2CH2CH3
7h: X = F, R1 = CH3, R2 = isopropyl
7i: X = F, R1 = CH3, R2 = cyclopropylmethyl
7j: X = Cl, R1 = CH3, R2 = CH2CH3
17k: X = Cl, R1 = CH3, R2 = CH2CH2CH3
7l: X = Cl, R1 = CH3, R2 = cyclopropylmethyl
1
a
o
Reagents and conditions: (a) methyl acrylate, Cs CO , MeOH, microwave, 100 C, 30
2
3
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min, 69%; (b) LiOH-H O, THF, H O, rt, overnight, 60%; (c) for 13a: 2-fluoroethanol,
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PPh , DEAD, THF, microwave, 60 C, 30 min, 95%; for 13b: EtI, K CO , DMF,
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110 C, 30 min, 96%; (d) 2 M HCl in diethyl ether, rt, overnight, 99%; or 4 M HCl in
dioxane, rt, 4–5 h, 82–94%; (e) HATU, NaHCO , DMF, rt, 3 h, 72–90%; (f) BH -THF
3
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in THF, reflux, 4 h, 58–92%; (g) aldehydes, NaHB(OAc) , THF, rt, 1 h, 24–89%.
3
Compounds with an amide connection between the linker and the secondary
pharmacophore, similar to those in reported D3R ligands 3 and 4, were also designed
and synthesized. As shown in Scheme 2, 4-aminobutan-1-ol (18) was acylated with
either acid chloride or coupled with aromatic acids to give amides 19a-c, and the
primary alcohol group was subsequently oxidized to give aldehydes 20a-c. PCPMAs
1
4c and 14d from Scheme 1 were alkylated under reductive amination conditions to
attach ethyl or propyl substituents thereby affording intermediates 21a-d, which were
then reacted with the former aldehydes under reductive amination conditions to yield
compounds 22a-i (for details, see Experimental Section).
Scheme 2. Synthesis of amide analogs.a
H
H
a
N
Ar
N
Ar
NH2
b
HO
O
HO
O
O
18
1
9a: Ar = A
20a: Ar = A
20b: Ar = B
20c: Ar = C
19b: Ar = B
H
X
N
Ar
19c: Ar = C
d
N
A:
R'
O
O
22a: X = F, R' = ethyl, Ar = A;
22b: X = F, R' = propyl, Ar = A;
22c: X = F, R' = ethyl, Ar = B;
22d: X = F, R' = propyl, Ar = B;
22e: X = F, R' = propyl, Ar = C;
22f: X = Cl, R' = ethyl, Ar = A;
22g: X = Cl, R' = propyl, Ar = A;
HCl
NH2
X
c
X
NH
R'
B:
C:
N
H
O
O
N
1
1
4c: X = F
4d: R = Cl
2
2
1a: X = F, R' = ethyl
1b: X = Cl, R' = ethy
22h: X = Cl, R' = propyl, Ar = B;
21c: X = F, R' = propyl
1d: X = Cl, R' = propyl
22i: X = Cl, R' = propyl, Ar = C;
2
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Reagents and conditions: (a) for 19a: 2-naphthoyl chloride, NEt , CH Cl , rt, 2 h, 87%;
3
2
2
for 19b: 1H-indole-2-carboxylic acid, HATU, NaHCO , DMF, rt, 2 h, 80%; for 19c: 4-
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2-pyridyl)benzoic acid, HATU, NaHCO , DMF, rt, 2 h, 93%; (b) SO -Py, DMSO,
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NEt ; CH Cl ; (c) for 21a and 21b: NaBH , NEt , acetaldehyde, rt, 20 min, 33–34%;
3
2
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3
for 21c and 21d: NaBH , NEt , propionaldehyde, rt, 20 min, 23–31%; (d) NaHB(OAc) ,
4
3
3
THF, rt, overnight, 37–68% for steps b and d.
An alternative route for similar amides is shown in Scheme 3. Briefly, starting from
phenyl aldehydes 23a-f, a sequence of Wittig reaction, ester to Weinreb amide
conversion, Corey-Chaykowsky cyclopropanation and Weinreb amide to aldehyde
reduction provided aldehydes 28a-f.4, 5 Next, the abovementioned aldehydes 20a-c
were converted to propyl amines 29a-c through reductive amination reactions. Further
reductive amination reactions of 28a-f with 29a-c produced compounds 30a-r (for
details, see Experimental Section).
Scheme 3. Synthesis of amide analogs.a
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O
O
O
O
O
a
b
c
N
O
OH
X
X
X
X
2
2
3a: X = 3-F;
3b: X = 3-Cl;
23c: X = 4-F;
3d: X = 4-Cl;
3e: X = 4-CF3;
23f: X = 2,3-diCl
24a: X = 3-F;
24b: X = 3-Cl;
24c: X = 4-F;
24d: X = 4-Cl;
24e: X = 4-CF3;
24f: X = 2,3-diCl
25a: X = 3-F;
25b: X = 3-Cl;
25c: X = 4-F;
25d: X = 4-Cl;
25e: X = 4-CF3;
25f: X = 2,3-diCl
26a: X = 3-F;
26b: X = 3-Cl;
26c: X = 4-F;
26d: X = 4-Cl;
26e: X = 4-CF3;
26f: X = 2,3-diCl
A:
B:
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
H
O
O
N
O
e
d
N
X
X
C:
O
2
2
7a: X = 3-F;
7b: X = 3-Cl;
27c: X = 4-F;
7d: X = 4-Cl;
7e: X = 4-CF3;
28a: X = 3-F;
28b: X = 3-Cl;
28c: X = 4-F;
28d: X = 4-Cl;
28e: X = 4-CF3;
28f: X = 2,3-diCl
H
N
Ar
2
2
N
g
X
27f: X = 2,3-diCl
30a: X = 3-F, Ar = A;
30b: X = 3-F, Ar = B;
30c: X = 3-F, Ar = C;
30j: X = 4-Cl; Ar =A;
30k: X = 4-Cl; Ar =B;
30l: X = 4-Cl; Ar =C;
H
N
H
f
Ar
N
Ar
HN
O
30d: X = 3-Cl, Ar = A; 30m: X = 4-CF ; Ar = A;
O
3
O
3
3
0e: X = 3-Cl, Ar = B;
0f: X = 3-Cl, Ar = C;
30g: X = 4-F, Ar = A;
30h: X = 4-F, Ar = B;
30i: X = 4-F, Ar = C;
30n: X = 4-CF ; Ar = B;
30o: X = 4-CF ; Ar = C;
30p: X = 2,3-diCl, Ar = A;
30q: X = 2,3-diCl, Ar = B;
30r: X = 2,3-diCl, Ar = C;
3
3
2
2
0a: Ar = A
0b: Ar = B
20c: Ar = C
29a: Ar = A
29b: Ar = B
29c: Ar = C
a
Reagents and conditions: (a) Ph P=CHC(=O)OMe, CH Cl , rt, overnight, 67–98%; (b)
3
2
2
LiOH-H O, THF, H O, rt, 2 h, 75–100%; (c) N,O-dimethylhydroxylamine
2
2
+
−
hydrochloride, HATU, NaHCO , DMF, rt, overnight, 81–97%; (d) Me S (O)I , NaH,
3
3
DMSO, rt, overnight, 71–82%; (e) DIBAL-H, THF, −78 °C, 2 h; (f) NaHB(AcO)3,
propylamine, AcOH, rt, 44–64%; (g) NaHB(OAc) , AcOH, THF, rt, overnight, 11–68%
3
for steps e and g.
For the PCPMA moiety of all the new compounds, we obtained only the trans isomers
through using E olefins as the cyclopropanation precursors, the same as our previous
5
2
-HT ligands. Of course, the racemic, trans compound is composed of a pair of (1R,
2C
R)- and (1S, 2S)-enantiomers. In this research, the racemic compounds 22e, 30p, 30q,
and 30r were separated by chiral HPLC to give optically pure (−)- and (+)-enantiomers
for further pharmacological profiling. To determine the absolute configurations of these
enantiomers, we re-synthesized the enantiomers of 22e using a method shown in
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 4 (for details, see Experimental Section). Thus, the Boc-protected intermediate
1
3c from Scheme 1 was separated using chiral HPLC to give both isomers (+)-13c and
(−)-13c, which were de-protected subsequently to give HCl salts (−)-14c and (+)-14c
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
respectively. We had previously determined that the (−)-isomer has the (1R, 2R)
configuration and the (+)-isomer the (1S, 2S) configuration for very similar
compounds.4, 23 Therefore, the configuration of (−)-14c was assigned as (1R, 2R) and
that of (+)-14c as (1S, 2S). Both (−)-14c and (+)-14c were next converted to (−)-21c
and (+)-21c, and then to (−)-22e and (+)-22e. Thus, the configuration of (−)-22e was
assigned as (1R,2R), and (+)-22e as (1S,2S). As will be discussed below, based on
structural similarities and the consistency observed in the 5-HT2C binding data for
compounds 30p, 30q and 30r, namely that the (+)-isomer shows better 5-HT activity,
2
C
which is in agreement with both the results from the enantiomers of 22e and our
previous results, the absolute configurations of the (−)-isomers of 30p, 30q and 30r
were assigned as (1R,2R) and the (+)-isomers as (1S,2S).
Scheme 4. Synthesis of compounds (1R,2R)- 22e and (1S,2S)-22e.
N
O
F
O
O
F
O
H
N
b
NH2 HCl
c
NH
d
N
NHBoc
O
F
F
F
(+)-
13c
(-)-21c
(-)-22e
a
(-)-14c
NHBoc
N
O
O
O
F
O
O
H
b
c
d
N
rac-13c
NHBoc
NH2 HCl
NH
N
O
F
F
F
(
-)-13c
(+)-14c
(+)-21c
(+)-22e
Reagents and conditions: (a) chiral prep-HPLC separation; (b) 4 M HCl in dioxane, rt,
overnight, 98%; (c) propionaldehyde, NaBH , NEt , dioxane, rt, 10 min, 34–63%; (d)
4
3
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20c, NaHB(AcO) , THF, rt, overnight, 32–33%.
3
Structure-Activity Relationships (SARs). Based on the structural similarity between
the PCPMA scaffold and the cyclopropane-containing substructures in compounds 5
and 6, we introduced the 1,2,4-triazolylthiol ether moiety as the EBP binding motif,
connected to the N atom of PCPMA via a three-carbon spacer. As shown in Table 1,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
the binding affinity of compounds 16a and 17a at D3R increased slightly compared to
4
PCPMA compound 1 (K = 1712 nM), but none of them showed significant binding
i
selectivity among five dopamine receptors and the 5-HT2C receptor. The N-ethyl
compound 17b showed more than a 10-fold increase in binding affinity compared to
the PCPMA compound 1 (113.1 nM versus 1712 nM). The 2-fluoroethoxy substituent
on the PCPMA part was reduced to an ethoxy group in compounds 16b, 17c and 17d,
which resulted in slight increases in binding affinities, as can be seen by comparing 17d
versus 17b, 17c versus 17a and 16b versus 16a. When the ethoxy substituent on the
benzene ring was further truncated to a methoxy group, binding affinities were further
increased, as can be seen from compounds 16c, 17e and 17f, with the last showing a Ki
value of 44.0 nM at D3R. The N-ethyl group was changed to a propyl (17g), an
isopropyl (17h) or a cyclopropylmethyl group (17i), but none of these compounds
showed increased affinity for D3R compared to the N-ethyl compound 17f. The fluorine
atom on the left-hand benzene ring was changed to a Cl atom in compounds 17j, 17k
and 17l, with various N substitutions, and these compounds showed comparable
binding affinities for D3R and moderate selectivity against D2R, D4R and 5-HT2C,
while possessing a lower affinity for the D1R and D5R. Taken together, the introduction
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9
1
1
1
1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
of the 1,2,4-triazolylthiol ether significantly enhanced compound affinity for D3R and
reduced their affinity to 5-HT , but compared to compound BP-897, their affinity and
2
C
selectivity still required further improvement. In our assays, BP-897 showed a binding
affinity of 3.1 nM at D3R, and 365-, 80-, 205-, 395-, and 70-fold selectivity against
D1R (1132 nM), D2R (247.0 nM), D4R (634.0 nM), D5R (1223 nM), and 5-HT2C
(218.0 nM) receptors, respectively.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 1. Binding affinities of thiol ethers at D1-5 and 5-HT receptors.
2C
K (nM)
i
Compd
Structureb
D1
D2
D3
D4
D5
5-HT2C
N
N
F
F
F
N
S
N
H
NTc
1
1
6a
7a
368.7
1502.0
524.8
962.3
1079.8
O
O
O
F
N
N
N
S
N
1467.8
584.3
2435.9
844.6
562.3
113.1
977.2
179.2
2154.4
1995.3
NT
NT
F
N
N
N
S
N
1
1
7b
6b
F
N
N
F
F
F
N
S
N
H
295.1
1122.0
1148.2
631.0
660.7
>5000
380.2
253.1
218.8
549.6
55.0
501.2
988.6
100.8
132.8
588.8
>5000
1584.9
926.1
NT
NT
NT
NT
O
O
O
N
N
N
N
S
S
N
1
7c
N
N
N
17d
16c
N
N
F
N
S
N
H
89.8
O
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5
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7
8
9
N
N
N
N
N
F
N
N
N
N
N
S
S
S
S
N
1
7e
794.3
1724.6
1668
323.6
275.4
1161.4
512.9
>5000
238.0
233.0
216.0
247.0
84.5
44.0
87.1
104.7
64.6
31.6
36.7
23.7
3.1
85.1
30.0
1000.0
1847.9
>5000
>5000
1204.0
>5000
>5000
>5000
1223
NT
O
O
O
O
O
N
F
N
1
7f
NT
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
F
N
1
7g
85.8
412.1
495.4
125.9
203.0
535.0
274.0
218.0
N
F
N
1
7h
>5000
1078.0
1509
326.1
291.7
241.0
130.0
163.0
634.0
N
N
F
S
N
1
7i
N
N
Cl
Cl
Cl
N
S
N
1
7j
O
N
N
N
N
S
N
17k
1918
O
O
N
N
S
N
17l
636
H
N
O
N
BP-897
1132
N
O
a
K values were calculated from mean pK values (mean pK ± SEM values from at least
i
i
i
three individual experiments are provided in the Supporting Information, Table S1).
b
c
All compounds were tested as HCl salts. NT, not tested.
We then turned to a longer spacer group comprised of four carbon atoms, coupled with
an amide connection to the EBP-binding aromatic moiety. The results for these analogs
are summarized in Table 2. For the aromatic moiety, naphthyl, indolyl and 4-
pyridylphenyl were used as they have been reported in numerous D3R selective
compounds. Comparing compounds 22a and 22b, it can be seen that although they
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
show identical binding affinities to D3R, the N-propyl substitution led to a higher
selectivity profile for 22b versus the N-ethyl substitution present in 22a, especially
against D2R and D4R. Also in compound 22d, the N-propyl substituent resulted in a 2-
fold increase in binding affinity compared to that of 22c which has an ethyl substituent.
The introduction of a 4-pyridylphenylgroup led to a further increase in binding affinity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
as in compound 22e, showing a K value of 4.0 nM for D3R. Moreover, compound 22e
i
showed excellent selectivity against all the other dopamine receptors (439-fold against
D1R (1758 nM), 786-fold against D2R (3144 nM), 285-fold against D4R (1142 nM)
and >5,000 nM for D5R). However, it showed only moderate selectivity as the racemic
mixture over 5-HT (53.0 nM versus 4.0 nM, or 13-fold). When the fluorine atom was
2
C
replaced by a chlorine, compound affinity was maintained within the low nM range,
but none of these analogs were more potent, as shown for compounds 22f-i.
a
Table 2. Binding affinities of amide analogs at D1-5 and 5-HT receptors.
2C
K (nM)
i
Structureb
Compd
D1
D2
D3
D4
D5
5-HT2C
H
F
F
N
N
N
2
2a
986
407.4
15.0
384.6
1721
57.5
63.1
49.0
68.0
O
O
O
O
H
N
2
2b
967
1084
871
1083.9
380
14.5
23.4
11.2
844.0
220.0
376.0
2203
>5000
789
H
F
N
N
H
2
2c
N
O
O
H
N
F
N
H
22d
N
507
O
O
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N
H
2
2e
F
N
1758
3144
4.0
1142
>5000
53.0
N
O
O
H
Cl
N
N
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
22f
1981
930
408
1116
1421
23.4
26.9
11.5
553.0
945.0
575
571
1104
4227
49.0
85.0
85.1
O
O
O
O
H
N
Cl
2
2g
H
Cl
N
N
N
H
818
2
2h
O
O
N
H
2
2i
Cl
N
>5000
1308
1105
2105
4823
3494
39.4
26.7
18.8
1833
1298
671
>5000
>5000
>5000
136.0
165
N
O
O
H
F
N
N
3
0a
O
H
F
N
N
N
H
106
3
0b
O
N
H
N
3
0c
F
2906
940
>5000
4012
3494
45.4
15.5
11.1
2222
1318
878
>5000
8670
1339
136
N
O
H
N
Cl
Cl
N
30d
O
H
N
N
N
992
>5000
132
3
0e
H
O
N
H
N
3
0f
Cl
832
992
>5000
5623
26.5
55.8
2326
2171
5580
898
238
N
O
H
N
N
3
0g
>5000
O
F
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H
N
N
N
1457
5934
45.7
1609
10532
219
3
0h
H
O
F
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
H
3
0i
N
3631
509
>5000
4606
3162
106.3
32.6
25.7
2884
1585
510
>5000
6109
9290
2171
179
N
O
F
H
N
N
30j
O
Cl
H
N
N
N
H
649
137
3
0k
O
Cl
N
H
3
0l
N
1308
>5000
17.6
1413
8844
284
N
O
Cl
H
N
N
3
0m
1711
1278
>5000
>5000
43.0
33.1
215
5799
8191
1259
1308
O
F3C
F3C
H
N
N
N
H
2073
3
0n
O
N
H
N
3
0o
1423
891
>5000
2105
1502
23.4
2.0
2171
1339
1131
7703
4018
3500
2362
123
N
O
F3C
Cl
Cl
H
N
N
3
0p
O
Cl
H
Cl
N
N
N
H
891
2.6
105
30q
O
Cl
N
H
Cl
N
3
0r
1647
2362
1.2
2011
>5000
97.7
N
O
a
K values were calculated from mean pK values (mean pK ± SEM values from at least
i
i
i
three individual experiments are provided in the Supporting Information, Table S2).
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bAll compounds were tested as HCl salts.
Next, we removed the 2-methoxy group in the PCPMA moiety. Compounds 30a-c,
which retain only the meta fluorine substitution, showed good affinities for D3R and
excellent selectivity against D2R (Table 2). For example, compound 30c, with a 4-
pyridylphenyl EBP-binding group, showed 45.4 nM binding affinity for D3R,
but >5,000 nM binding affinity for D2R and only micromolar affinities for all other
tested receptors. When a meta chorine was introduced to replace the fluorine atom
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(compounds 30d-f), similar results were observed. Compound 30f showed 26.5 nM
binding affinity for D3R, with excellent selectivity against D2R, D4R and D5R and
good selectivity over D1R (832 nM) and 5-HT (898 nM). We then moved the halogen
2
C
substitution to the para position. As can be seen with compounds 30g-i (4-F) and 30j-l
4-Cl), these compounds showed good binding profiles and in particular, compound 30l
(
represents the best of them, with a K value of 17.6 nM for the D3R and good selectivity
i
against the other four dopamine receptors (1308 nM at D1R, no affinity at D2R, 1413
nM at D4R and 8844 nM at D5R, respectively), and moderate selectivity over 5-HT2C
(284 nM). The substitution of a para-CF group, as present in compounds 5 and 6, was
3
also introduced. As can be seen from an examination of the data obtained for
compounds 30m-o, although their binding affinities were not improved compared to
the para-F or para-Cl substitution, all three compounds fail to bind to D2R while
showing very good selectivity against the other tested receptors. Notably, the presence
of a di-chloro substitution pattern, like that present in the drugs aripiprazole and
cariprazine, provided compounds 30p-r which show significantly enhanced binding
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5
5
6
affinities for D3R (1.2-2.6 nM). Compound 30r, which has a K of 1.2 nM, showed
i
over 1000-fold selectivity against all other dopamine receptors, and an 81-fold
selectivity against 5-HT (K = 97.7 nM). The overall profiles of 30p and 30q were
2
C
i
0
1
2
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4
5
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2
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3
4
5
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9
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2
3
4
5
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9
0
also very encouraging. Based on these results, we selected compounds 22e, 30p, 30q
and 30r for further studies.
Influence of chirality on binding affinity and functional activity. The significance
of chirality is a critical consideration in medicinal chemistry and has also been
1
7
showcased in previous D3R ligand discovery. With respect to the chiral centers on
the PCPMA skeleton, although all compounds were synthesized as the trans isomers,
they were tested as a mixture of (1R,2R) and (1S,2S) isomers in our primary screening.
We thus selected the four best compounds 22e, 30p, 30q and 30r for chiral separations
and further pharmacological profiling. Chiral separations were conducted using chiral
HPLC columns and the absolute configurations of all the isomers were assigned as
discussed above. All four pairs of enantiomers were profiled in the same binding assays
against the dopamine receptors and the 5-HT receptor. As can be seen in Table 3, the
2
C
(1R,2R) enantiomers showed slightly better binding affinities for D3R than the (1S,2S)
for compounds 30p, 30q and 30r (4.4 versus 20.8 nM, 2.2 versus 12.8 nM and 1.5
versus 5.3 nM, respectively). For compound 22e, the enantiomers displayed equal
affinities for D3R (4.1 versus 3.8 nM). Interestingly, the enantiomers of all four
compounds showed comparable affinities for all dopamine receptors within each pair,
but their affinities for 5-HT were different. The (1R,2R) enantiomers showed 3-22×
2C
weaker 5-HT binding affinity than their (1S,2S) isomers, consistent with the results
2
C
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of our previous PCPMA analogs. It would appear that the D3R has little binding
preference for the chiral conformations of the PCPMA unit, while the 5-HT is more
2
C
sensitive.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Enantiomer activity of selected compounds.a
K (nM)
i
Compd
Structureb
D1
D2
D3
D4
D5
5-HT2C
N
H
(
1R,2R)-22e
F
N
4898
1349
4.1
575
851
>5000
1122
N
O
O
N
H
(
1S,2S)-22e
F
N
1071
1230
3.8
>5000
50.1
N
O
O
Cl
Cl
H
Cl
Cl
N
N
N
(
1R,2R)-30p
1288
1047
676
4.4
813
776
>5000
>5000
427
138
O
O
H
N
(
1S,2S)-30p
1148
20.8
Cl
H
Cl
N
N
H
(
1R,2R)-30q
N
1380
1122
1349
2344
537
992
2.2
12.8
1.5
1047
676
676
912
>5000
>5000
>5000
>5000
513
61.7
417
O
Cl
H
N
Cl
(1S,2S)-30q
N
N
H
O
Cl
N
H
N
Cl
(
1R,2R)-30r
550
N
O
Cl
N
H
N
Cl
(
1S,2S)-30r
1023
5.3
44.7
N
O
a
K values were calculated from mean pK values (mean pK ± SEM values from at least
i
i
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4
4
4
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5
5
5
5
5
5
5
5
5
6
three individual experiments are provided in the Supporting Information, Table S3).
bAll compounds were tested as HCl salts.
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These enantiomers were further profiled for their functional activity at the D and 5-
3
HT2C receptors. As shown in Table 4 and Figure 3A, for compounds 30p-r, both
enantiomers showed full or partial agonist activity at D3R with comparable potency
between each pair of enantiomers. Interestingly, (1R,2R)-22e showed very potent
agonist activity (EC = 3.6 nM, E = 77.9%), while (1S,2S)-22e showed no activity
5
0
max
in the agonist mode but behaved as a potent antagonist of D3R (K = 16.7 nM). At 5-
i
HT2C (Figure 3C-D), both 22e enantiomers were weak antagonists with micromolar
activity. For compounds 30p-r, (1S,2S) isomers were partial agonists with low potency
while the (1R,2R) isomers showed very weak antagonist activity at 5-HT receptors.
2
C
These results reveal that the selected compounds show better selectivity for D3R versus
-HT in the functional assays compared to their selectivity as determined from their
5
2
C
binding affinities. Furthermore, these enantiomers were tested at 5-HT and 5-HT
2B
2
A
receptors for their agonist activity to exclude potential hallucinogenic or valvulopathic
side effects related to these receptors respectively. All enantiomers showed very weak
and only partial 5-HT2A agonist activity, and no 5-HT2B agonism was observed (see
Supporting Information, Table S1).
The Tango assay was also performed at D3R to evaluate compound activity for β-
arrestin2 recruitment (Table 4 and Figure 3B). Compared to their G activity, most
i/o
compounds showed very weak β-arrestin2 recruitment at D3R. In particular,
compounds (1S,2S)-22e, (1R,2R)-30p, (1R,2R)-30q and (1R,2R)-30r showed no
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measurable activity in the Tango assay, indicating their preference for the G protein
signaling.24
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
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4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Functional data of selected compounds at D3R and 5-HT .
2C
2+
Compound
D3R Gi
D3R Tango
5-HT2C G (Ca )
q
Antagonist:
b
(
1R,2R)-22e
EC = 3.58 nM (77.9% )
EC = 126.4 nM (50.2%)
5
0
50
IC = 14.5 µM
5
0
No agonism;
Antagonist
(1S,2S)-22e
NTc
Antagonist: K = 16.7 nM
IC = 0.86 µM
i
50
Antagonist:
(
1R,2R)-30p
EC = 177.5 nM (71.7%)
9.2% at 3 µM
44.4% at 3 µM
<5% at 3 µM
5
0
IC = 16.1 µM
5
0
Agonist
EC = 3538 nM (30.3 %)
(1S,2S)-30p
EC = 99.2 nM (83.4%)
5
0
5
0
Antagonist:
(
1R,2R)-30q
EC = 87.0 nM (40.7%)
50
IC > 30 µM
5
0
Agonist
(
1S,2S)-30q
EC = 142.8 nM (63.4%)
EC = 1000.2 nM (27.1%)
5
0
50
EC = 2549 nM (44.2 %)
50
Antagonist
(
1R,2R)-30r
EC = 12.5 nM (68.1 %)
3.1% at 3 µM
5
0
IC = 10.1 µM
5
0
Agonist
EC = 738.3 nM (51.9%)
(
1S,2S)-30r
EC = 29.6 nM (96.2%)
EC = 11086 nM (119.1%)
5
0
50
5
0
Quinpirole
Dopamine
5-HT
EC = 0.42 nM (95.4%)
EC = 35.0 nM (95.2%)
NT
NT
5
0
50
EC = 0.11 nM (100%)
NT
5
0
NT
NT
EC = 0.41 nM (99.0%)
50
a
b
All compounds were tested as HCl salts. For agonist activity, E values are shown
max
c
in brackets. NT, not tested.
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Figure 3. Functional characterization of lead compounds at D dopamine receptors (A,
3
B) and 5-HT receptors (C, D). (A) Inhibition of cAMP production at D (G -agonist
2
C
3
i
activity), (B) β-arrestin2 recruitment (Tango agonist activity), and calcium mobilization
at 5-HT (G agonist activity in C and antagonist activity in D) represented means ±
2
C
q
SEM from a minimum of 3 independent assays, each in triplicate or quadruplicate.
Reference agonist (5-HT) was used at 1 nM final concentration in antagonist assays
(D), in which mesulergine served as a reference antagonist.
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Molecular Docking Studies. To understand the molecular basis of the binding profiles
of these new compounds, especially compounds (1R,2R)- and (1S,2S)-22e, we carried
out molecular docking studies on them using Schrödinger’s Maestro program. (1R,2R)-
and (1S,2S)-22e, as well as eticlopride, were docked to the antagonist-bound structure
1
1
1
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2
1
of D3R (PDB code: 3PBL). The salt bridge between the positive charged tertiary
amine and the carboxylate of D1103.32 was observed for all three ligands (Figure 4A),
3
which has been reported as highly conserved for most aminergic receptors. Consistent
with previous research21, 25, 26 and our expectations, the PCPMA moiety of both
(1R,2R)- and (1S,2S)-22e occupies the OBP of D3R, in good alignment with eticlopride,
while the attachment to the PCPMA extends into the EBP. Notably, although both
(1R,2R)- and (1S,2S)-22e fit nicely in the OBP with the substituted benzene rings
forming π-π stacking and hydrophobic interactions, the orientations of the substituted
phenyl ring of (1R,2R)- and (1S,2S)-22e are quite different. For (1R,2R)-22e, the 2-
methoxy substituent on the benzene ring is deeply buried within the OBP, forming
hydrophobic contacts with C1143.36, S1965.46 and F3466.52; while the 2-methoxy group
of the antagonist (1S,2S)-22e flips up towards the extracellular side. As a result, for
antagonist (1S,2S)-22e, the cyclopropane linker between the benzene ring and the
protonated N overlays perfectly with amide linker of eticlopride, while for the agonist
(1R,2R)-22e the cyclopropane points to another direction due to the flip of the benzene
ring. Since a structure of the active state of D3R is currently unavailable, the docking
of agonist (1R,2R)-22e to the active state of D3R has not been conducted. This flip of
the benzene ring in the inactive conformation of D3R might explain the different
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functional results observed for these enantiomers.
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9
0
Figure 4. Comparison of binding poses of D3R antagonist eticlopride, (1R,2R)-22e and
(1S,2S)-22e. The receptor structure employed for the molecular docking study was
extracted from the crystal structure of the eticlopride-D3R complex (PDB code: 3PBL).
Carbon atoms of eticlopride, (1R,2R)-22e and (1S,2S)-22e are colored in green, orange
and cyan, respectively.
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Figure 5. Comparison of binding poses of 5HT2C representative agonists (A) and
antagonists (B). The receptor structures used in the molecular docking studies were
extracted from the crystal structures of the ergotamine-5HT2C complex (PDB code:
6
BQG) and the ritanserin-5HT2C complex (PDB code: 6BQH). Carbon atoms of
ergotamine, compound 2, ritanserin, and (1S,2S)-22e are colored in orange, pink, green
and cyan, respectively.
A further observation from the functional data is that while the PCPMA compound 2 is
a potent agonist of 5-HT , the bitopic derivative (1S,2S)-22e turned out to be a
2
C
moderate antagonist (IC = 858.5 nM). For the di-Cl substituted compounds 30p, 30q
50
and 30r, although the (1S,2S) eutomers showed partial agonist activity, their efficacy
was very low (Emax = 30.3%, 44.2% and 51.9% respectively). To explore the molecular
basis for this conversion from PCPMA-based 5-HT2C agonists to bitopic
antagonists/weak partial agonists, we also performed molecular docking of compounds
2
and (1S,2S)-22e to the crystal structures of 5-HT . Thus, compound 2 was docked to
2C
the ergotamine-bound active structure of 5-HT (PDB code: 6BQG), while compound
2
C
(1S,2S)-22e was docked to the ritanserin-bound inactive structure of 5-HT (PDB code:
2
C
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6BQH). As shown in Figure 5, all of these ligands form salt bridges between the
protonated nitrogen atoms of the ligands and D1343.32, a key interaction for aminergic
GPCR ligands. In the co-crystal structures, the deep binding pose of ritanserin, with the
phenyl ring inserted more deeply than the ergoline scaffold of ergotamine, was
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proposed as a possible explanation for its antagonistic activity. In our docking poses,
in comparison to the PCPMA compound 2, the bitopic derivative (1S,2S)-22e inserts
approximately one helical turn deeper into the TM bundle, which likely explains its
antagonistic action at the 5-HT receptor. The fluorophenyl groups form halogen-
2
C
aromatic interactions with F2235.47and F3206.44, aromatic edge-to-face π-π stacking
with F3286.52 and W3246.48, and hydrophobic interactions with T1393.37, S1383.36 and
I1423.40. Similar to ritanserin, the deep binding pose of (1S,2S)-22e likely prevents the
conformational changes of the toggle switch in TM6 (W3246.48) and rotamer switches
20
in the conserved P-I-F motif, thus stabilizing the inactive state of 5-HT . It may thus
2
C
be postulated that the attachment of the four carbon linker and the EBP-binding motifs
to the PCPMA scaffold results in a deeper binding mode for compound (1S,2S)-22e
thus leading to its antagonism of the receptor.
Pharmacokinetic and brain penetration properties. Based on their good binding
affinity and selectivity for D3R, we selected compounds (1R,2R)-22e, (1R,2R)-30p,
(
1R,2R)-30q and (1R,2R)-30r for further PK profiling in male ICR mice. As shown in
Table 5, intravenous (iv) administration (5 mg/kg) of these compounds showed
moderate exposure in plasma, with high clearance. Oral administration (10 mg/kg)of
compounds (1R,2R)-22e, (1R,2R)-30p and (1R,2R)-30r gave very low bioavailability
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(3.8%, 6.6% and 13.0% respectively), but compound (1R,2R)-30q showed a reasonably
good oral bioavailability of 34.1%. Furthermore, this compound showed an oral half-
life of 4.1 h and an MRT of 6 h.
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
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5
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5
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6
0
1
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9
0
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9
0
Table 5. PK and brain penetration properties of selected compounds in ICR mice.a
(
1R,2R)-22e
(1R,2R)-30p
(1R,2R)-30q
(1R,2R)-30r
iv po
Parameters
iv
-
po
43.2
0.25
2.64
49.5
-
iv
-
po
iv
-
po
Cmax (ng/mL)
tmax (h)
37.3
0.25
2.08
81.1
-
63.9
1.00
4.11
428
-
-
118
0.50
2.12
254
-
-
-
-
-
t1/2 (h)
0.437
660
126
0.415
3.13
-
1.51
616
134
2.58
20.8
-
3.12
629
117
3.40
23.8
-
0.576
976
84.8
0.808
4.11
-
AUC (h·ng/mL)
0-t
CL (mL/min/kg)
MRT(h)
3.72
-
2.59
-
6.00
-
2.77
-
VdSS (L/kg)
F
3.8%
6.6%
34.1%
13.0%
brain concentration
at 0.5 h (ng/mL)
plasma concentration
at 0.5 h (ng/mL)
brain/plasma ratio
at 0.5 h
530±37
8.21±2 3424±286 23.2±4.0 833±187 51.3±21 980±164 26.7±9.0
4
46±15 16.8±5.0
270±22
12.7
28.4±15
0.82
215±13
3.88
35.5±7.0 511±67
1.45 1.92
118±7.0
0.23
1
.19
0.49
6.57
brain concentration
at 2.0 h (ng/mL)
plasma concentration
at 2.0 h (ng/mL)
brain/plasma ratio
at 2.0 h
19.4±11
913±97
23.2±4.0
543±99
160±45 227±17.0 9.1±5.3
1
2.7±3.1 8.4±2.4
1.53 0.79
82.6±9.7 16.4±3.5 94.8±19.3 67.7±20 118±16.5 44.1±7.6
11.1 1.61 5.72 2.36 1.93 0.207
aFor all four compounds, iv dose is 5 mg/kg and po dose 10 mg/kg; “-”, no applicable.
In our previous studies, PCPMA analogs showed very high brain penetration as 5-HT2C
agonists.4, 5 Accordingly, these four selected compounds were further tested for their
brain penetration properties in ICR mice. Brain and plasma drug concentrations after
both iv and oral dosing were measured at 0.5 and 2.0 h time points, and brain/plasma
ratios were calculated. As shown in Table 5, all four compounds showed good brain
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penetration, although significant differences were observed for their brain
concentrations following oral dosing, which can be attributed to their different overall
PK profiles. Thus, the brain exposures of compounds (1R,2R)-22e, 30p and 30r were
very low following oral dosing, despite good brain/plasma ratios. As for compound
0
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(1R,2R)-30q, high brain/plasma ratios were observed for both iv and oral dosing, at
both time points. Calculated concentrations of 108.6 (51.3 ng/mL) and 338.6 nM (160
ng/mL) were observed at 0.5 and 2.0 h, respectively. Moderate but long-lasting brain
exposure could be expected for compound (1R,2R)-30q, given the relatively long half-
life and MRT observed in PK studies. The overall PK and brain penetration profiles of
(1R,2R)-30q support it as a good candidate for further in vivo studies in disease models.
Polypharmacological profiling at other aminergic GPCRs. As mentioned above,
polypharmacology is a common feature of many aminergic GPCR ligands. To further
profile the binding selectivity of our bitopic D3R ligands, we selected compounds
(
1R,2R)-22e and (1R,2R)-30q and screened them against 29 other aminergic GPCRs,
including serotonin, adrenaline, histamine, and muscarinic receptors. Compounds
1R,2R)-22e and (1R,2R)-30q show weak binding affinity for most receptors (pK < 6),
(
i
and modest binding for 5-HT , 5-HT , α , α , and H1R (see Supporting Information,
1
A
2B 2A 2C
Table S4). These data show that compounds (1R,2R)-22e and (1R,2R)-30q have good
selectivity for the D3R against most other aminergic GPCRs.
Conclusions
In the past decade, co-crystal structures of aminergic GPCRs have greatly facilitated
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our understanding of ligand-receptor interactions at these receptors as well as the
structure-based drug design of novel GPCR ligands. Starting from the PCPMA scaffold,
we have designed and synthesized a series of bitopic derivatives that possess excellent
binding affinities for the D3R with good selectivity against other dopamine receptors.
Among the optimized compounds, (1R,2R)-22e showed a binding affinity of 4.1 nM
for D3R, with 1195-, 329- and 140-fold selectivity over D1R (4898 nM), D2R (1349
nM) and D4R (575 nM), while possessing weak 5-HT2C affinity (1122 nM) and no
affinity for the D5R. Its enantiomer, (1S,2S)-22e, displayed a comparable affinity for
1
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D3R (K = 3.8 nM), with 282-, 324- and 224-fold selectivity against D1R (1071 nM),
i
D2R (1230 nM) and D4R (851 nM), no affinity for D5R, and moderate selectivity over
5-HT2C (K = 50.1 nM). Notably, although the enantiomers showed almost identical
i
binding affinity for D3R, (1R,2R)-22e behaved as a potent agonist (EC = 3.6 nM),
5
0
while (1S,2S)-22e showed potent antagonist activity (K = 16.7 nM) in the G -cAMP
i
i
assays. Molecular docking results revealed that these enantiomers adopt different
binding poses in the D3R OBP, which emphasizes the importance of compound
chirality in medicinal chemistry.
Due to high lipophilicity and large molecular weight, most bitopic ligands suffer from
poor PK properties and low brain penetration. In this study, we determined that
compound (1R,2R)-30q, which shows a high binding affinity for the D3R (K = 2.2 nM)
i
with excellent selectivity against all other dopamine receptors (627-, 244- and 476-fold
against D1R, D2R and D4R, no binding affinity for D5R) and most other aminergic
GPCRs, is a bioavailable (F = 34.1%) and brain penetrant D3R partial agonist (EC =
5
0
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