Investigation of Novel Primary and Secondary Pharmacophores and 3-Substitution in the Linking Chain of a Series of Highly Selective and Bitopic Dopamine D3 Receptor Antagonists and Partial Agonists

Article abstract of DOI:10.1021/acs.jmedchem.9b00607

Dopamine D₃ receptors (D₃R) play a critical role in neuropsychiatric conditions including substance use disorders (SUD). Recently, we reported a series of N-(3-hydroxy-4-(4-phenylpiperazin-1-yl)butyl)-1H-indole-2-carboxamide analogues as high affinity and selective D₃R lead molecules for the treatment of opioid use disorders (OUD). Further optimization led to a series of analogues that replaced the 3-OH with a 3-F in the linker between the primary pharmacophore (PP) and secondary pharmacophore (SP). Among the 3-F-compounds, 9b demonstrated the highest D₃R binding affinity (K_i = 0.756 nM) and was 327-fold selective for D₃R over D₂R. In addition, modification of the PP or SP with a 3,4-(methylenedioxy)phenyl group was also examined. Further, an enantioselective synthesis as well as chiral HPLC methods were developed to give enantiopure R- and S-enantiomers of the four lead compounds. Off-target binding affinities, functional efficacies, and metabolic profiles revealed critical structural components for D₃R selectivity as well as drug-like features required for development as pharmacotherapeutics.

Full text of DOI:10.1021/acs.jmedchem.9b00607

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Article
Investigation of novel primary and secondary pharmacophores,
and 3-substitution in the linking chain of a series of highly selective
and bitopic dopamine D3 receptor antagonists and partial agonists
Anver Basha Shaik, Vivek Kumar, Alessandro Bonifazi, Adrian Guerrero, Sophie Cemaj, Alexandra
Gadiano, Jenny Lam, Zheng-Xiong Xi, Rana Rais, Barbara S. Slusher, and Amy Hauck Newman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00607 • Publication Date (Web): 17 Sep 2019
Downloaded from pubs.acs.org on September 19, 2019
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Investigation of novel primary and secondary pharmacophores, and 3-
substitution in the linking chain of a series of highly selective and
bitopic dopamine D receptor antagonists and partial agonists
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†
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Anver Basha Shaik, Vivek Kumar, Alessandro Bonifazi, Adrian M. Guerrero, Sophie L.
†
†,§
†,§
†
§
§
Cemaj, Alexandra Gadiano, Jenny Lam, Zheng-Xiong Xi, Rana Rais, Barbara S. Slusher,
Amy Hauck Newman†*
†Molecular Targets and Medications Discovery Branch, National Institute on Drug Abuse –
Intramural Research Program, National Institutes of Health, 333 Cassell Drive, Baltimore,
Maryland 21224, United States
§Department of Neurology, Johns Hopkins Drug Discovery, The Johns Hopkins University School
of Medicine, 855 N. Wolfe Street, Baltimore, MD 21205, United States
*
Corresponding Author Phone: (443) 740-2887. Fax: (443) 740-2111. E-mail:
anewman@intra.nida.nih.gov.
KEYWORDS. Dopamine D3 receptor antagonist, partial agonist, chiral resolution,
phenylpiperazine, metabolic stability, pharmacokinetics, VK4-116, VK4-40
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ABSTRACT
Dopamine D receptors (D R) play a critical role in neuropsychiatric conditions including
3
3
substance use disorders (SUD). Recently, we reported a series of N-(3-hydroxy-4-(4-
phenylpiperazin-1-yl)butyl)-1H-indole-2-carboxamide analogues as high affinity and selective
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D R lead molecules for the treatment of opioid use disorders (OUD). Further optimization led to
3
a series of analogues that replaced the 3-OH with a 3-F in the linker between the primary
pharmacophore (PP) and secondary pharmacophore (SP). Among the 3-F-compounds, 9b
demonstrated the highest D R binding affinity (K =0.756 nM) and was 327-fold selective for D R
3
i
3
over D R. In addition, modification of the PP or SP with a 3,4-(methylenedioxy)phenyl group was
2
also examined. Further, an enantioselective synthesis as well as chiral HPLC methods were
developed to give enantiopure R- and S-enantiomers of the four lead compounds. Off-target
binding affinities, functional efficacies, and metabolic profiles revealed critical structural
components for D R selectivity as well as drug-like features required for development as
3
pharmacotherapeutics.
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INTRODUCTION
In the last decade, prescription opioid abuse and misuse has been a major contributor to
mortality in the United States, resulting in a serious national public health crisis.1,2 Nearly 12
million Americans used prescription opioids nonmedically in 2016, with >42,000 people dying
from an opioid overdose.1,3,4 The federal response to the opioid crisis includes research support
toward the development of non-addictive analgesics and additional medications to treat opioid use
disorders (OUD).5
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Medication for addiction treatment in the form of opioid substitution therapies (e.g.,
methadone or buprenorphine) has proven effective for OUD with new formulations adding to the
arsenal of medication options.6-8 Further, the opioid antagonist, naloxone is used to prevent
overdose. However, methadone and buprenorphine continue to be underutilized due to lack of
availability in underserved communities or to undesirable and serious side effects such as
respiratory depression, constipation and abuse liability.9, 10 Recently, the non-opioid 2A
adrenergic receptor agonist, lofexidine, was approved by the U.S. Food and Drug Administration
as the first medication for use in reducing symptoms associated with opioid withdrawal in adults.11
However, lofexidine has no effect on the addictive liability of opioid analgesics.12, 13
Extensive preclinical and clinical studies suggest that all drugs of abuse ultimately activate
the mesolimbic dopaminergic pathway to produce rewarding effects that can lead to addiction.14
Specifically, mediation of dopamine (DA) signaling through dopamine D -like (D R and D R) and
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D -like (D R, D R, D R) G protein-coupled receptors plays a critical role in the pleasurable effects
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of addictive drugs. However, in some people, frequent or chronic stimulation of DA can induce
compulsive and repetitive drug seeking, which can lead to substance use disorders (SUD).15,16 D R
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are highly localized in mesolimbic regions of the brain and are critically involved in motivation to
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drug seeking.17,18 Thus, the discovery of selective D R antagonists and partial agonists has been
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the focus of many research programs interested in the development of medications to treat
neuropsychiatric conditions including SUD.18,19-24
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Over the past two decades, many D R-selective ligands with high affinity have been
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discovered, affording critical tools for understanding mechanisms underlying addiction.21,25-28 In
addition, selective D R antagonists have been extensively investigated preclinically and have
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shown promising results in rodent models of drug self-administration, relapse-like behaviors and
conditioned place preference.23,29, 30 Nevertheless, to date, advancement to human clinical studies
has been limited to only a few selective D R antagonists, including GSK598,809 (1, Figure. 1).31,32
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However, the findings that 1 produced hypertension in dogs when co-administered with cocaine,
diminished prospects for its further development as a cocaine use disorder therapeutic.33
The advancement of bitopic ligands that include a substituted phenyl piperazine as the
primary pharmacophore (PP) and various arylcarboxamides as the secondary pharmacophore (SP)
linked with a variety of substituted alkyl chains have provided excellent tools for investigating
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4-40
D R in animal models of psychostimulant abuse.
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For example, the first enantioselective D R
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4
1
antagonist (R)-PG648 (2, Figure. 1) was a recent lead molecule in this class that showed promise
in rodent models, but not in nonhuman primates, possibly due to metabolic instability.26
Modification of the 3-OH in the linking chain between the PP and the SP with a F- group led to
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BAK2-66 (3, Figure. 1) where the (R)-enantiomer was also the eutomer, as was also reported
for R-2.41
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N
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, GSK598,809
2, (R)-PG648
CF3
N
N
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HN
O
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Cl
Cl
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O
OH
O
Cl
N
H
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, (±)-BAK 2-66
4, (±)-VK4-116
Cl
N
N
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O
OH
N
H
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, (±)-VK4-40
Figure 1. Chemical structures of lead D R antagonists/partial agonists
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The high-resolution crystal structure of D R in complex with the D R/D R antagonist,
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2
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4
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eticlopride, provided a platform for further optimization leading to two D R-selective analogues,
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VK4-116 (4) and VK4-40 (5) in Figure. 1. These structural analogues showed high affinity and
selective binding at D R over D R and excellent metabolic stability in mouse, rat and nonhuman
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primate liver microsomes.30 In addition, lead compound 4 dose-dependently inhibited the
acquisition and maintenance of oxycodone self-administration, lowering the break-point for
oxycodone self-administration under a progressive-ratio schedule of reinforcement and inhibited
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oxycodone extinction response and reinstatement of oxycodone-seeking behaviors. Moreover,
pretreatment with 4 dose-dependently reduced naloxone-precipitated conditioned place aversion
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in rats chronically treated with oxycodone, but had no effect on the antinociceptive actions of
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oxycodone, tested in a hot plate assay. Herein we report further investigation of the PP and SP
in these bitopic molecules and substitution of the linking chain with 3-F. We had previously shown
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that replacing the 3-OH with a 3-F in the linking chain resulted in some of the most selective D R
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antagonists/partial agonists reported 37,38 and we also predicted that this substitution might
increase metabolic stability. Chiral resolution of the lead molecules (Figure. 2) was pursued in
order to further optimize this template and provide new pipeline compounds for development.
Figure 2. Drug design strategy
CHEMISTRY
In Scheme 1, the synthesis of the 3-F-analogues 9a,b and 10a,b began with the secondary
3
9
alcohol intermediates 6a,b synthesized using our previously published strategy. Replacement of
the 3-OH group with F was carried out in the presence of the fluorinating agent diethylaminosulfur
trifluoride (DAST) to generate the 3-F intermediates (7a,b).37,38 Subsequently, removal of the
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phthalimide protecting group yielded primary amine intermediates (8a,b) in quantitative yields,
which underwent amide coupling with indole- or benzofuran-2-carboxylic acids in the presence of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to generate the 3-F-analogues 9a,b and
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0a,b. In Scheme 2, to further extend SAR, we synthesized 13a,b with the 3,4-(methylenedioxy)
moiety as the SP, by using the primary amine intermediates 12a,b (generated in a single step
starting with 11a,b) via a general amidation procedure. The 3,4-(methylenedioxy)phenyl group
was selected as a SP to see how another biologically relevant heterocyclic moiety would affect the
profile of these compounds. This moiety exists in other CNS active agents (e.g., MDMA) and we
wanted to expand the SAR to determine if this bicyclic ring structure would affect D R selectivity.
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As we have noted previously, the PP typically affects the ligand affinity for D R and D R, but the
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25, 40
SP and linker are key to D R selectivity.
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Scheme 1. Synthesis of 3-Fluoro analoguesa
O
O
N
H N
N
R1
2
N
R1
N
R1
N
O
F
N
F
N
O
OH
N
a
b
R2
R2
R2
R3
R3
6
a,b
R3
7a,b
8a,b
c
X
6
a-9a
H
N
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N
R1
R = H, R = OMe, R = Cl
9
a
b
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R = H, R = OMe, R = Cl, X=NH
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2
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O
F
N
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1
R = Cl, R = H, R = H, X=NH
6b-9b
1
2
3
0a R = H, R = OMe, R = Cl, X=O
9a,b
R2
1
2
3
1
2
3
R = Cl, R = H, R = H
10b R = Cl, R = H, R = H, X=O
10a,b
R3
aReagents and conditions: (a) DAST, anhydrous DCM, -78 °C to rt, overnight; (b) hydrazine,
EtOH, reflux, overnight; (c) Indole-2-carboxylic acid for 9a,10a; benzfuran-2-carboxylic acid for
9
b,10b, EDC/HOBt, DIPEA, CHCl /DMF, 0 °C to rt 8h.
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Scheme 2. Synthesis of 3,4-(methylenedioxy) SP analoguesa
O
H N
2
HN
R1
N
R1
H
N
O
R1
N
OH
N
N
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13a,b
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11a, 12a, 13a R = H, R = OMe, R = Cl
1
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11b, 12b, 13b R = Cl, R = H, R = H
aReagents and conditions: (a) (1) 19, 2-PrOH reflux, 3h 2) hydrazine, reflux, overnight; (b)
benzo[d][1,3]dioxole-5-carboxylic acid, EDC/HOBt, DIPEA, CHCl /DMF, 0 °C to rt, 8h.
3
Likewise, as for the SP, we extended SAR by replacing the substituted phenyl rings in the
PP of compounds 2-5 with the 6-ethylbenzo[d][1,3]dioxol moiety. We thought this may be an
interesting replacement for the more classic substituted 2,3-diCl-phenyl substitution in previous
series of D R-selective antagonists.2
5,26
In Scheme 3, the synthesis began with the nitration of
3
benzo[d][1,3]dioxole-5-carbaldehyde 14 in fuming nitric acid to give a nitro intermediate 15.
Conversion of 15 to the alkene 16 was accomplished under Wittig reaction conditions and the nitro
group as well as the alkene were reduced via catalytic hydrogenation to yield aniline 17, in one
pot. Compound 17 was reacted with bis(2-chloroethyl)amine to generate the novel piperazine PP,
1
8. The primary amine 20 was prepared via epoxide opening of 2-(2-oxiran-2-yl)ethyl)isoindoline-
1
,3-dione 19 with piperazine 18, followed by deprotection of the phthalimide using hydrazine in
the same step. Coupling of 20 with indole- or benzofuran-2-carboxylic acids gave the 6-
ethylbenzo[d][1,3]dioxol analogues 21 and 22, respectively.
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Scheme 3. Synthesis of 3,4-(methylenedioxy) PP analoguesa
H
H
O
O
O
O
O
O
a
O
b
O
NO2
NO2
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d
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e
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NH2
O
17
18
H N
2
N
NH
H
OH
N
N
f
f
N
O
OH
N
O
20
O
21
O
O
O
H
N
N
O
OH
N
22
O
O
a
Reagents and conditions: (a) Fuming HNO , 0 °C to rt, 2h; (b) Methyltriphenylphosphonium
3
bromide, lithium tert-butoxide, THF -78 to 0 °C to rt, 8h; (c) 10% Pd/C, H , 50 psi, EtOAc, 45
2
.
min; (d) bis(2-chloroethyl)amine HCl, diethyleneglycol monoethylether, 150 °C, 7h; (e) 1) 2-
PrOH, reflux, 3h, 2) hydrazine, reflux, overnight; (f) Indole-2-carboxylic acid for 21; benzfuran-
2
-carboxylic acid for 22, EDC/HOBt, DIPEA, CHCl /DMF, 0 °C to rt 8h.
3
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5
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5
5
5
5
5
6
Synthesis of the (R)- and (S)-enantiomers of lead compounds 4 and 5 are shown in Scheme
4
. Enantioselective synthesis was achieved by applying similar procedures described above.
Briefly, the chiral primary amine intermediates (R)-12a,b and (S)-12a,b were generated by epoxide
0
1
2
3
4
5
6
7
8
9
0
1
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3
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9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
39
38
opening of chiral 2-(2-oxiran-2-yl)ethyl)isoindoline-1,3-dione (R)-19 and (S)-19 respectively
with N-arylpiperazine intermediates 11a,b followed by deprotection of the phthalimide.
Subsequently, (R)-12a,b and (S)-12a,b were coupled with indole 2-carboxylic acid, to afford the
enantiopure (R)-4,5 and (S)-4,5 (HPLC: >98% ee, chromatograms in S.I. Figures. S1 and S2).
Alternatively, (R)-4,5 and (S)-4,5 were also prepared using chiral preparative HPLC from their
corresponding racemates achieving >99% ee. The enantiomers of the 3-F-analogues (R)-9a,b and
(S)-9a,b were synthesized starting from 11a,b which were reacted with enantiopure phthalimide
intermediates (R)-19 and (S)-19 to yield chiral hydroxy intermediates (R)-6a,b and (S)-6a,b
(
Scheme 5). Further replacement of the chiral 3-OH group with F was achieved in the presence of
the fluorinating agent (DAST) to generate the 3-F intermediates (R)-7a,b and (S)-7a,b, retaining
the absolute configuration at the chiral center, as previously observed.38 Subsequently,
deprotection of phthalimide group yielded chiral primary amine intermediates (R)-8a,b and (S)-
8
a,b in quantitative yields, that underwent amide coupling with indole carboxylic acids in the
presence of EDC and hydroxybenzotriazole (HOBt) to generate the enantiomers of 3-F-analogues
R)-9a,b and (S)-9a,b (Scheme 5). Alternatively, (R)-9b and (S)-9b were also prepared using chiral
(
preparative HPLC from corresponding racemate 9b resulting in >99% ee for each enantiomer
(Supplementary Figures. S3 and S4).
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Scheme 4. Synthesis of (R)- and (S)-4 and 5a
HN
R1
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
R2
O
O
R3
N
N
1
1a,b
O
O
O
R)-19
O
a
a
(
(
S)-19
H N
2
H N
2
R1
R1
N
N
OH
N
OH
N
(R)-12a,b
R2
R2
(S)-12a,b
R3
R3
b
b
NH
NH
H
N
H
N
R1
N
R1
N
O
OH
N
O
OH
(S)-4,5
N
R2
(R)-4,5
R2
R3
R3
11a, (R)-12a, (S)-12a, (R)-4, (S)-4
1
2
3
R = H, R = OMe, R = Cl
11b, (R)-12b, (S)-12b, (R)-5, (S)-5
1
2
3
R = Cl, R = H, R = H
aReagents and conditions: (a) (1) 2-PrOH reflux, 3h, 2) hydrazine, reflux, overnight; (b) Indole-2-
carboxylic acid, EDC/HOBt, DIPEA, CHCl /DMF, 0 °C to rt, 8h.
3
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2
2
2
2
2
2
3
3
3
3
3
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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 5. Synthesis of (R)- and (S)-3-Fluoro analoguesa
HN
R1
N
O
O
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
R2
N
N
R3
O
O
a
O
O
a
1
1a,b
(
R)-19
(
S)-19
O
O
N
N
R1
R1
N
N
O
O
OH
N
OH
N
R2
(S)-6a,b
R2
(
R)-6a,b
R3
R3
b
b
O
O
N
N
R1
N
N
R1
O
F
N
O
F
N
R2
(
R)-7a,b
R2
(S)-7a,b
R3
R1
R3
c
c
H N
2
H N
2
N
R1
N
F
N
F
N
(
R)-8a,b
R2
(S)-8a,b
R2
R3
R3
d
d
NH
H
NH
N
H
N
R1
N
R1
N
O
F
N
O
F
S)-9a,b
(R)-6a, (R)-7a, (R)-8a, (R)-9a, (S)-6a, (S)-7a, (S)-8a, (S)-9a
N
R2
(
R)-9a,b
R2
R3
(
R3
1
2
3
R = H, R = OMe, R = Cl
(R)-6b, (R)-7b, (R)-8b, (R)-9b, (S)-6b, (S)-7b, (S)-8b, (S)-9b
1
2
3
R = Cl, R = H, R = H
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aReagents and conditions: (a) 2-PrOH, reflux, 3h; (b) DAST, anhydrous DCM, -78 °C to rt,
overnight; (c) hydrazine, EtOH, reflux, overnight; (d) Indole-2-carboxylic acid, EDC/HOBt,
DIPEA, CHCl /DMF, 0 °C to rt 8h;
3
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
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5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
2
3
4
5
6
7
8
9
0
PHARMACOLOGICAL RESULTS AND DISCUSSION
Dopamine D R and D R binding analysis
2
3
3
The radioligand [ H]N-methylspiperone was employed in all the competition binding
experiments using membrane preparations from stably transfected HEK293 cells expressing
human D and D dopamine receptors to determine binding affinities and D R/D R selectivities
2L
3
2
3
(Table 1). All new analogues showed moderate to high D R binding affinities in the range of
3
K =0.290-99.9 nM. D R binding affinities were generally lower and thus D R/D R selectivities
i
2
2
3
ranged from 15-1735-fold.
As previously discovered, replacing the 3-OH group in the linker with a 3-F atom, and
retaining the PP of 4, resulted in compounds 9a and 10a, where the SPs were either the indole-2-
carboxamide or the benzofuran-2-carboxamide, respectively, with moderately high affinity and
selectivity for D R. Of note, 9a, the analogue with the indole carboxamide as the SP, was 746-
3
fold D R selective over D R. In contrast, the analogues of 5, 9b and 10b had relatively higher D R
3
2
3
affinities compared to 4, but D R affinities were also improved leading to reduced selectivity, as
2
previously seen with 5. For example, the indole analogue 9b had one of the highest affinities in
the series (K = 0.756 nM) but was less selective (D R/D R=327) than its analogue, 9a (D R/D R
i
2
3
2
3
=
746).
Replacing the SP with a benzo[d][1,3]dioxole-5-carboxamide scaffold, resulted in lower
D R binding affinities for 13a and 13b compared to their indole analogues, 4 and 5, respectively.
3
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2
2
2
2
2
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3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Moreover, D R affinity increased for 13a (D R K = 364 nM) compared to 4 resulting in lower
2
2
i
D R-selectivity, further exemplifying the indole as a privileged SP structure.
3
Replacing the PP with 6-ethylbenzo[d][1,3]dioxol-piperazine resulted in compounds 21
and 22 that were relatively well tolerated at D R, but again D R affinities were improved leading
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
3
2
to less D R-selective compounds. Likewise, the indole analogue 21 showed higher D R affinity
3
3
than the benzofuran analogue, 22, with D R K = 4.31 nM and 23.6 nM, respectively. Interestingly,
3
i
4
0
we have previously shown that the PP plays a more prominent role in affinity, but in this case
the PP is identical and the SPs are affecting affinity. These data demonstrate that the entire bitopic
molecule must be taken into account when assessing affinities and selectivities. Further the
chemical composition of each PP, SP and linker plays a critical and sometimes differing role in
the pharmacological profiles of these unique molecules. Although these heterocyclic modifications
to the SP and PP resulted in D R-selective analogues, none were superior to the lead molecules 4
3
and 5.
As the lead molecules 4 and 5 are the racemates, it was important to resolve the chiral
center in the linking chain in order to identify which enantiomer would be taken on for further
development. The binding affinities for the racemates of 4 and 5 were comparable to those
previously reported (Table 1). (R)-4 exhibited very similar D R binding affinity to the racemate
3
with a K = 7.40 nM and was similarly selective over D R (1459-fold). Whereas, (S)-4 was the
i
2
distomer with a K = 37.9 nM, (~5-fold enantioselectivity). As noted with (R)-2, D R
i
3
enantioselectivity was modest; D R affinity showed essentially no enantioselectivity. As
2
previously described, (±)-5, showed sub-nanomolar binding affinity at D R (K = 0.433 nM) and
3
i
(
R)-5 also exhibited similarly high D R binding affinity (K = 0.290 nM). (S)-5 (K = 0.886 nM)
3 i i
was also the distomer, although its binding affinity was only reduced by ~3-fold compared to (R)-5.
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Further, both the R and S enantiomers of 5 were less selective at D R over D R when compared to
3
2
similar enantiomers of 4, although still in the 300-fold range.
In comparison, the 3-F analogues with an indole-3-carboxamide as the SP demonstrated
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
high D R affinity and thus it was of interest to resolve the racemates 9a and 9b as potential pipeline
3
lead molecules. As previously shown, both the R-enantiomers (R)-9a,b were the eutomers at D R.
3
(
R)-9a exhibited ~2-fold higher binding affinity at D R (K = 13.0 nM) compared to (±)-9a and
3 i
~
1650-fold selectivity over D R (2-fold higher than (±)-9a). Whereas, (S)-9a showed reduced D R
2
3
binding affinity and selectivity (K = 35.3 nM, 535-fold over D R). Moreover, (R)-9b exhibited
i
2
sub-nanomolar affinity at D R (K = 0.373 nM). Interestingly, (S)-9b also displayed sub-nanomolar
3
i
binding affinity at D R with K = 0.840 nM and was >1000-fold selective over D R. Thus, the
3
i
2
selectivity profile of (S)-9b at D R over D R was dramatically increased and ~5-fold higher than
3
2
either (±)-9b (327-fold) or (R)-9b (215-fold).
In summary, (R)-9a and (R)-9b with the 3-F in the linking chain showed similar binding
affinities and selectivity profiles to their corresponding 3-OH analogues, (R)-4 and (R)-5. Whereas,
(S)-9a and (S)-9b showed similar binding affinities to (S)-4 and (S)-5 respectively; however, (S)-
9
b exclusively showed higher selectivity over D R and was approximately 4-fold more selective
2
than (S)-5, demonstrating the critical role of substitution and stereochemistry of the linking chain
in the binding profile of this series of molecules. Of note, cLogP and PSA values were obtained
for all final compounds and are listed in Table 1. In general, these values suggest that most of these
compounds are highly lipophilic, which is typical of bitopic compounds selective for D R.
3
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1
1
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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 1. Chemical structures and human D -Like Receptor Binding affinity data of newly
2
developed analoguesa
Linker
H
N
N
N
Ar1
Ar2
X
O
PP
SP
Compound
Ar
1
X
Ar
2
cLogP tPSA
D
2
R
D
3
R
2 3
D /D
K
(nM)
i
± SEM
K
i
± SEM
(nM)
5.40
56.84 20800 ± 7060 27.9 ± 5.64
(n = 6) (n = 6)
746
F
(9a)
O
N
H
Cl
5
5
.40
.40
56.84 21400 ± 1500 13.0 ± 2.94
n = 3) (n = 3)
1646
535
(
R)-9a
(
F
F
F
F
F
F
F
O
O
N
H
Cl
Cl
56.84 18900 ± 2720 35.3 ± 5.15
n = 3) (n = 3)
(
S)-9a
(
N
H
Cl
Cl
Cl
5.63
47.61 247 ± 57.4
n = 7)
0.756 ± 0.185 327
(n = 9)
(
(9b)
N
H
5
5
5
.63
.63
.37
47.61 80.2 ± 13.8
n = 4)
0.373 ± 0.061 215
(n = 3)
(R)-9b
(
N
H
47.61 891 ± 305
(n = 4)
0.840 ± 0.156 1060
(n = 3)
(
S)-9b
N
H
54.04 10800 ± 2290 99.9 ± 13.4
108
134
1
0a
(n = 3)
(n = 3)
O
O
O
Cl
Cl
5.60
4.53
44.81 328 ± 33.5
(n = 4)
2.45 ± 0.510
(n = 7)
1
0b
O
O
83.5
364 ± 101
n = 3)
24.5 ± 4.44
(n = 4)
15
1
3a
(
OH
O
Cl
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1
3b
O
O
Cl
4.76
4.27
74.27 424 ± 79.9
n = 3)
3.02 ± 0.742
(n = 5)
140
125
(
OH
OH
21
86.3
537 ± 41.3
n = 3)
4.31 ± 1.07
(n = 3)
(
N
H
O
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
O
O
2
2
4.24
83.5
1440 ± 48.8
n = 3)
23.6 ± 7.35
(n = 3)
61
(
OH
O
O
4
.93
77.07 13200 ± 3770 7.61 ± 1.17
n = 4) (n = 4)
1735
1459
(
OH
OH
4b
O
N
H
Cl
4
.93 77.07
10800 ± 2590 7.40 ± 1.09
n = 4) (n = 4)
(
(R)-4
O
N
H
Cl
4.93
5.16
77.07 13500 ± 1230 37.9 ± 9.70
n = 3) (n = 4)
356
(
OH
OH
(S)-4
O
N
H
Cl
Cl
67.84 132 ± 12.7
n = 4)
0.433 ± 0.116 305
(n = 6)
(
5b
N
H
Cl
Cl
5.16
5.16
67.84 75.8 ± 13.7
n = 4)
0.290 ± 0.085 261
(n = 6)
(
(R)-5
(S)-5
OH
OH
N
H
67.84 219 ± 66.2
n = 4)
0.886 ± 0.296 247
(n = 6)
(
N
H
aThe values represent the arithmetic mean ± SEM of triplicate determinations from at least three
independent experiments. IC values for each compound were determined from dose-response
5
0
4
3
curves and K values were calculated by the Cheng-Prusoff equation using GraphPad Prism
i
version 6.00 for Macintosh. n = number of independent experiments, each performed in triplicate.
bCompound reported in reference 39
Functional and Off-Target Data
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5
5
5
5
5
6
The in vitro functional efficacies of a subset of 3-F-analogues 9a,b and 10a,b and R- and
S-enantiomers of both 4 and 5 were examined for stimulation or inhibition of quinpirole-stimulated
mitogenesis in CHO cells (Table 2). These data revealed that all the 3-F analogues were D R
3
0
1
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5
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9
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5
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7
8
9
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1
2
3
4
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9
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1
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5
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7
8
9
0
antagonists, as were (R)- and (S)-4 and (R)-5. However, (S)-5 was a relatively low efficacy partial
3
9
agonist, like (±)-5. This difference in efficacies between enantiomers was also observed with
previous lead compound 2, where (R)-2 was an antagonist and (S)-2 was a weak partial agonist at
4
1
D R.
3
In addition to binding data for D R and D R, the same set of compounds was tested for
2
3
binding affinities at serotonin 5-HT , 5-HT and 5-HT receptors. As depicted in Table 3, none
1A
2A
2C
of these analogues showed high affinity binding to any of the 5-HT receptor subtypes tested
compared to their D R affinities. Compound 10b showed the highest affinities of the 3-F
3
analogues, across the 5-HT receptor subtypes, and was a low potency partial agonist at 5HT1A
(EC = 351 nM). (R)- and (S)-5 showed higher affinities across the 5-HT receptor subtypes than
5
0
(
R)- and (S)-4, however selectivities for D R remained high ((R)-4, 5-HT /D R = 469, 5-
3 1A 3
HT /D R = 324, 5-HT /D R = 211, and (S)-4, 5-HT /D R = 149, 5-HT /D R = 228, 5-
2
A
3
2c
3
1A
3
2A
3
HT /D R = 170).
2c
3
Table 2. Functional data for selected compounds using stimulation or inhibition of
a
quinpirole-stimulated mitogenesis in CHO cells with human dopamine D R
3
Compound
Agonist
%
Antagonist
EC ± S.E.M., nM
Stimulation
IC ± S.E.M., nM
5
0
50
9
a
>5400
>6800
2.7
5.4
510 ± 130
95 ± 31
9
b
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1
0a
0b
(R)-4
S)-4
R)-5
S)-5
>4000
>7500
>3200
>6800
>2200
46 ± 17
5.1
490 ± 150
450 ±160
350 ± 110
1020 ± 200
18.7 ± 6.6
660 ± 260
1
15.5
17.7
17.0
20.2
25.9
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
(
(
(
aData were obtained through the NIDA Addiction Treatment Discovery Program contract
ADA12013) with Oregon Health & Science University.
(
Table 3. In vitro binding and functional data for selected compounds at 5-HT , 5-HT
1
A
2A
a
and 5-HT receptors
2
C
5
-HT1A
5-HT2A
5-HT2C
5-HT1A
[35S]GTPS
binding
[3H]-8-OH-
DPAT
[ I]DOI
125
[ I]DOI
125
Compound
K ± SEM,
K ± SEM,
i
i
K ± SEM, nM
nM
nM
i
Agonist
EC ±
%
Stimula
tion
5
0
SEM, nM
9
a
9500 ± 3000
910 ± 120
2660 ± 570
117 ± 14
6600 ± 1400
620 ± 120
2990 ± 530
336 ± 19
>8900
NDb
-
9b
580 ± 160
1780 ± 390
464 ± 36
NDb
1
0a
NDb
-
10b
351 ± 84
43
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1
1
1
1
1
1
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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
(R)-4
2800 ± 340
1933 ± 20
7600 ± 1800
21.3 ± 6.6
940 ± 130
1260 ± 320
NDb
NDb
-
(S)-4
4980 ± 270
248 ± 18
131 ± 12
5670 ± 930
50 ± 13
-
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
(R)-5
1050±260
2060±580
74
69
(S)-5
1110 ± 360
aData were obtained through the NIDA Addiction Treatment Discovery Program contract
b
(
ADA12013) with Oregon Health & Science University. ND = Not determined; Functionalassays
for each receptor was not conducted if the K value for the binding assay was >250 nM for 5-HT
i
receptors.
Rat Liver Microsome Metabolic Stability
Test compounds including 3-F analogues (9a, 10a, 9b, 10b) and 4, 5 as well as the R-, S-
enantiomers of 9a, 9b, 4 and 5 were screened for phase I metabolic stability in rat liver microsomes
following our previously described procedure.39 As seen in Figure 3A, of all the four 3-F
analogues, 9a was the most stable with ~75% intact remaining, whereas 9b, 10b, 10a showed
lower stability with 38%, 34% and 12% parent remaining, respectively. Individual R- and S-
enantiomers of 9a and 9b (Figure 3B) showed higher stability with 57-70% intact remaining.
Lastly, compounds 4 and 5 as well as their R- and S-enantiomers were found to be metabolically
stable with 57-86% remaining at 60 min.
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2
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4
4
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4
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0
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Phase I metabolic stability of 3-F analogues (9a, 9b, 10a, 10b), 4, 5, and R-, S-
enantiomers of 9a, 9b, 4 and 5 in rat liver microsomes fortified with NADPH. The R- and S-
enantiomers of 9a, 9b, 4 and 5 were found to be stable in rat liver microsomes with >50% intact
remaining at 60 min.
Pharmacokinetics of R- and S-enantiomers in rats.
Owing to the promising in vitro metabolic stability and potency of the (R)- and (S)-
enantiomers of 4 and 5, pharmacokinetic evaluation was conducted in rats following oral (PO)
administration at a 10 mg/kg dose. As shown in Figure 4, all compounds were orally available,
with (R)- and (S)-4 achieving maximum plasma concentration (Cmax) of 454.4 ± 58.8 pmol/mL and
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1
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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
2
26.4 ± 28.9 pmol/mL, respectively, at 4h (Tmax) post administration. In contrast, brain
concentrations were higher with Cmax values of 1796.6 ± 228 pmol/g and 959.9 ± 53.3 pmol/g
for (R)-4 and (S)-4 enantiomers, respectively. Similarly, the overall exposure calculated from area
under the curve (AUC ) was higher for both the enantiomers in brain versus plasma, with a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
-t
corresponding brain to plasma (b/p) ratio of 3.8 and 4.1 for (R)-4 and (S)-4, respectively.
Likewise, (R)-5 and (S)-5 achieved a plasma Cmax of 305.5 ± 112.8 pmol/mL and 600.6 ±
1
08.1 pmol/mL and a brain Cmax of 3850 ± 513.7 pmol/g and 3666.7 ± 312.7 pmol/g respectively.
The corresponding plasma exposures (AUC ) for (R)-5 and (S)-5 were 1985 ± 278.6 h*pmol/mL
0
-t
and 2678 ± 207.5 h*pmol/mL and brain exposures were 21608 ± 1411 h*pmol/g and 16665 ± 1205
h*pmol/g, respectively. Significant brain/plasma ratios for (R)-5 (~11) and (S)-5 (~6), suggests
excellent brain penetrability.
Notably, both (R)- and (S)-4 and showed 12- and 88- fold higher brain levels, respectively,
versus their D R K values (Table 1) at 8h post drug administration. Moreover (R)- and (S)-5
3
i
showed nearly 1000- and 7000- fold higher levels at 8h versus their respective D R K values
3
i
(Table 1). Given this, 10 mg/kg oral dose may serve as an effective starting dose in rodent self-
administration studies, although this will need further confirmation.
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0
1
2
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5
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1
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3
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Pharmacokinetic evaluation of (R)- and (S)- enantiomers of 4 and 5 in rats
following oral administration (10 mg/kg). Plasma and brain concentration vs time profile of (R)-
and (S)-enantiomers of 4 and 5; concentration data is expressed as mean ± SD, n=3, per time-point.
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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
Pharmacokinetic parameters are expressed as mean ± SD. Brain to plasma ratios are calculated
from mean AUCbrain to AUCplasma. Data for (R)-5 is adapted from previously reported data.44
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
CONCLUSION
Novel 3-F and 3,4-(methylenedioxy) analogues of lead molecules 4 and 5 were synthesized
and evaluated for binding affinities at D R and D R; all modifications reported herein resulted in
2
3
compounds with high D R affinities and selectivities. Although modification of the PP or SP with
3
a 3,4-(methylenedioxy) group was generally well tolerated, none of these analogues proved
superior to 4 and 5, in terms of D R binding affinities or selectivities. The R- and S-enantiomers
3
of both 4 and 5 were resolved by two methods (1) enantioselective synthesis using resolved chiral
intermediates and (2) chiral preparative HPLC. The R-enantiomers of both the compounds were
the eutomers and displayed D R and D R affinities comparable to the racemates, with the S-
3
2
enantiomers having lower affinities at D R and thus lower selectivities. R-4, (S)-4 and (R)-5 were
3
D R antagonists, whereas, (S)-5 was a low efficacy partial agonist, a change in efficacy previously
3
observed with (R)- and (S)-2. All four enantiomers displayed significant metabolic stability in rat
liver microsomes, and very high brain to plasma ratios when administered orally, in rats. Finally,
inconsequential off target binding affinities further support the development of lead candidates
(R)-4 and (R)-5 as potential pharmacotherapeutics to treat OUD.
Replacement of the 3-OH in the linking chain between the PP and SP resulted in a series
of interesting 3-F-analogues. Compounds 9a,b, direct analogues of 4 and 5 proved to have the
highest D R binding and selectivity profiles among the 3-F-analogues and separation of
3
enantiomers was achieved through enantioselective synthesis or chiral preparative
chromatography in >99% ee. The D R over D R >1000 selectivity profile of (S)-9b was notable
3
2
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and further emphasized the importance of the linker in the binding profiles of these compounds.
Interestingly, D R affinity was decreased by >10-fold between the (R)- and (S)-enantiomers, which
2
has not been observed previously. The introduction of 3-F into the linker also resulted in metabolic
stability in rat liver microsomes, suggesting these analogues will serve as future in vivo tools for
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
further investigation into the role of D R in SUD and as potential medication candidates to treat
3
opioid use disorders.
EXPERIMENTAL METHODS
The reaction conditions and yields were not optimized. Anhydrous solvents and many reagents
were purchased from Sigma-Aldrich Co. and were used without further purification. All other
chemicals and reagents were purchased from LLC, AK Scientific, TCI America, Acros Organics,
Maybridge, and Alfa Aesar. All amine final products with a 3-F linker were converted into the
oxalate salts and the other compounds were converted into HCl salts. Spectroscopic data and yields
refer to the free base form of all compounds. Teledyne ISCO CombiFlash Rf or flash column
1
chromatography was performed using silica gel (EMD Chemicals, Inc.; 230-400 mesh, 60 Å). H,
1
3
19
1
C and F NMR spectra were acquired using a Varian Mercury Plus 400 spectrometer. H
chemical shifts are reported as parts per million (δ ppm) relative to tetramethylsilane (0.00 ppm)
1
3
as an internal standard. Coupling constants are measured in Hz. Chemical shifts for C NMR
1
9
spectra are reported as parts per million (δ ppm) relative to deuterated solvents. F spectra
chemical shifts are reported relative to CDCl /CFCl as internal standard. Chemical shifts,
3
3
multiplicities and coupling constants (J) have been reported and calculated using Vnmrj Agilent-
NMR 400MR or MNova 9.0. Gas chromatography-mass spectrometry (GC/MS) data were
acquired (where obtainable) using an Agilent Technologies (Santa Clara, CA) 6890N GC equipped
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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
with an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. × 0.25 μm film
thickness) and a 5973 mass-selective ion detector in electron-impact mode. Ultrapure grade helium
was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port and transfer line
temperatures were 250 and 280 °C, respectively, and the oven temperature gradient used was as
follows: the initial temperature (100 °C) was held for 3 min and then increased to 295 °C at 15
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
°
C/min over 13 min, and finally maintained at 295 °C for 10 min. Combustion analysis was
performed by Atlantic Microlab, Inc. (Norcross, GA) and the results agree within ±0.4% of
calculated values. HRMS (mass error within 5 ppm) and MS/MS fragmentation analysis were
performed on a LTQ-Orbitrap Velos (Thermo-Scientific, San Jose, CA) coupled with an ESI
source in positive ion mode. HPLC analysis was performed using an Agilent system coupled with
DAD (Diode Array Detector). Separation of the analyte, purity and enantiomeric excess
determinations was achieved employing one of the following different methods. HPLC Method
A: CHIRALCEL OZ-H (Daicel Corporation CPI Company) column (4.6 x 250 mm, 5 μm). The
mobile phase used (1 mL/min flow rate) was composed of 0.2% diethylamine in 2-propanol and
hexanes with gradient elution, starting with 10% of 2-propanol linearly increasing to 30%, total
run time was 30 min. DAD detector wavelength was set at 254 nm, and the column temperature
was maintained at 40 °C. A Sample of 20 μl with concentration of 0.5 mg/mL, was injected for
analytical method. HPLC Method B: CHIRALPAK AD-H (Daicel Corporation CPI Company)
column (4.6 x 250 mm, 5 μm). The mobile phase used (1 mL/min flow rate) was composed of
10% 2-propanol in hexanes with isocratic elution. The total run time was 80 min, DAD detector
wavelength was set at 254 nm and the column temperature was maintained at 40 °C. A Sample of
2
0 μl with concentration of 0.5 mg/mL, was injected for analytical method. Preparative HPLC
Method C: CHIRALCEL OZ-H (Daicel Corporation CPI Company) semi preparative column (20
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x 250 mm, 5 μm) performed on an Agilent Technologies HP series 1200 HPLC. The mobile phase
used (10 mL/min flow rate) was composed of 10% 2-propanol in hexanes with isocratic resolution.
The total run time was 50 min and DAD detector wavelength was set at 254 nm. A sample of 0.6
mL with a concentration of 10 mg/mL, was injected for preparative separation. Preparative
HPLC Method D: CHIRALPAK AD-H (Daicel Corporation CPI Company) semi preparative
column (21 x 250 mm, 5 μm). The mobile phase used (18 mL/min flow rate) was composed of
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
1
0% 2-propanol in hexanes with isocratic resolution. The total run time was 60 min and DAD
detector wavelengths were set at 254 nm and 280 nm. A Sample of 5.0 ml with concentration of
0 mg/mL, was injected for preparative separation. cLogP and polar surface area (PSA) values
1
were calculated using ChemDraw Professional Ultra 15.0. Melting point determination was
conducted using an OptiMelt automated melting point system and are uncorrected. Optical
rotations were determined using a Jasco DIP-370 polarimeter. Based on NMR, HPLC and
combustion analysis data, all final compounds are ≥95% pure, unless otherwise stated.
General Amidation Procedure. The appropriate aryl carboxylic acid (1.2 mmol) was dissolved
in mixture of CHCl and dimethylformamide (8:2) and cooled to 0 °C. To this solution was added
3
EDCI (1-Ethyl-3-(3- dimethylaminopropyl)carbodiimide) HCl (1.3 mmol) and HOBt
4
5
(
Hydroxybenzotriazole) (1.2 mmol mmol). After 30 min, the aryl amine (1.0 mmol) was added,
at 0 °C. To this reaction mixture was added DIPEA (N,N-Diisopropylethylamine) (1.5 mmol) and
the mixture continued to stir at room temperature for 8h. The reaction progress was monitored by
TLC (thin layer chromatography). After completion of the reaction, the mixture was basified to
pH 9 with saturated NaHCO water solution. The product was extracted with CHCl (50 ml x 4).
3
3
The combined organic fractions were evaporated to afford the crude amide product. All final
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1
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1
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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
products were purified using flash column chromatography eluting with CHCl and acetone
3
solvent systems, as described.
(R)-N-(4-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-
indole-2-carboxamide (R-4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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5
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1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
The compound was prepared from indole-2-carboxylic acid (0.22 g, 1.33 mmol) and (R)-12a (0.38
g, 1.11 mmol) according to the general amidation procedure. The crude product was purified by
flash chromatography using 30% acetone/CHCl as eluent to give the desired product as a colorless
3
1
solid (0.274 g, 51% yield). H NMR (400 MHz, CDCl ) δ 9.33 (s, 1H), 7.64 (d, J = 7.9 Hz, 1H),
3
7
.43 (t, J = 8.1 Hz, 1H), 7.40 (s, 1H), 7.32 – 7.22 (m, 1H), 7.13 (t, J = 7.6 Hz, 1H), 6.85 (d, J = 8.4
Hz, 2H), 6.62 (s, 1H), 3.91 (m, 2H), 3.84 (s, 3H), 3.58 – 3.41 (m, 1H), 3.16 (br s, 4H), 2.85 (s,
H), 2.56 (dd, J = 15.1, 7.6 Hz, 4H), 2.45 (d, J = 6.4 Hz, 2H), 1.82 (m, 1H), 1.64 (m, 1H), 1.21 (t,
2
1
3
J = 7.6 Hz, 3H); C NMR (101 MHz, CDCl ) δ 161.52, 146.26, 145.95, 140.77, 136.06, 131.15,
3
128.30, 127.72, 124.25, 122.33, 121.73, 120.52, 116.67, 111.87, 101.88, 66.43, 63.75, 59.09,
23
D
5
3.69, 50.39, 38.05, 33.26, 28.47, 15.40; Mp 194-195 °C Anal (C H ClN O ) C, H, N. [푎] -
2
6
33
4
3
27.89 (CHCl , c 0.23), (R-4) was analyzed by HPLC Method A, retention time = 11.004 min and
3
enantiomeric excess was >99%. (SI. Figure S1, panel B)
(S)-N-(4-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-
2-carboxamide (S-4).
The compound was prepared from indole-2-carboxylic acid (0.127 g, 0.78 mmol) and (S)-12a
(0.225 g, 0.658 mmol) according to the general amidation procedure. The crude product was
purified by flash chromatography using 30% acetone/CHCl as eluent to give the desired product
3
1
as a colorless solid (0.176 g, 55% yield). H NMR (400 MHz, CDCl ) δ 9.42 (s, 1H), 7.64 (d, J =
3
8
.0 Hz, 1H), 7.43-7.40 (m, 2H), 7.31 – 7.22 (m, 1H), 7.13 (t, J = 7.5 Hz, 1H), 6.89 – 6.81 (d, J =
28
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Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
4
.1 Hz, 2H), 6.62 (d, J = 1.5 Hz, 1H), 4.02 – 3.86 (m, 2H), 3.85 (s, 3H), 3.50 (m, 1H), 3.15 (br s,
H), 2.84 (m, 2H), 2.55 (m, 4H), 2.48 – 2.34 (d, J = 2.1 Hz, 2H), 1.91 – 1.77 (m, 1H), 1.64 (m,
1
3
1H), 1.21 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, CDCl ) δ 161.47, 146.41, 145.76, 140.76,
3
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
1
6
36.16, 131.14, 128.16, 127.61, 124.18, 122.44, 121.84, 120.38, 116.67, 111.77, 101.69, 66.48,
3.74, 58.98, 53.68, 50.39, 37.96, 33.10, 28.46, 28.21, 15.42. Mp 191-192 °C Anal
2
3
(
C H ClN O ) C, H, N. [푎] +34.54 (CHCl , c 0.16), (S-4) was analyzed by HPLC Method A,
26 33 4 3 3
D
retention time = 18.356 min and enantiomeric excess was >99%. (SI. Figure S1, panel C)
R)-N-(4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-
(
carboxamide ((R)-5).
The compound was prepared from indole-2-carboxylic acid (0.64 g, 3.95 mmol) and (R)-12b (1.12
g, 3.59 mmol) according to the general amidation procedure. The crude product was purified by
flash chromatography using 30% acetone/CHCl as eluent to give the desired product as a pale-
3
1
pink solid (0.98 g, 60% yield). H NMR (400 MHz, CDCl ) δ 9.44 (s, 1H), 7.64 (d, J = 7.7 Hz,
3
1H), 7.44 (d, J = 7.9 Hz, 2H), 7.33 – 7.21 (m, 1H), 7.15 (m, 2H), 6.95 (dd, J = 17.8, 7.6 Hz, 2H),
6
7
3
1
2
.85 (s, 1H), 4.01 – 3.82 (m, 2H), 3.51 (m, 1H), 3.07 (br s, 4H), 2.89 (m, 2H), 2.78 (dd, J = 14.8,
.3 Hz, 2H), 2.63 (m, 2H), 2.52 – 2.40 (m, 2H), 1.83 (m, 1H), 1.64 (m, 1H), 1.23 (t, J = 7.4 Hz,
1
3
H). C NMR (101 MHz, CDCl ) δ 161.54, 149.26, 143.23, 136.18, 131.14, 128.72, 127.64,
3
26.78, 124.22, 124.16, 121.70, 120.49, 117.97, 111.85, 101.83, 66.45, 63.70, 51.63, 38.09, 33.26,
7.46, 14.02. The HCl salt was precipitated from acetone; Mp 233-234 °C Anal
2
3
(C H ClN O •HCl) C, H, N. [푎] -45.29 (CHCl , c 0.23), (R-5) was analyzed by HPLC Method
2
5
31
4
2
3
D
A, retention time = 14.698 min and enantiomeric excess was >98%. (SI. Figure S2, panel B)
(S)-N-(4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-hydroxybutyl)-1H-indole-2-
carboxamide (S-5).
29
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