Vascular effects of diphenylmethoxypiperidine-derived dopamine uptake inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.04.040

Vascular effects of 4-aryl methoxypiperidinol compounds previously shown to share with cocaine the ability to inhibit the dopamine transporter are described. All the compounds tested inhibit KCl-induced and noradrenaline- dependent contractions in mesente

Full text of DOI:10.1016/j.bmcl.2014.04.040

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2429–2432
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Vascular effects of diphenylmethoxypiperidine-derived dopamine
uptake inhibitors
a,b,e
Victor M. Pulgar a,b,c,d, , Jill Keith Harp
⇑
a Biomedical Research Infrastructure Center, Winston-Salem State University, Winston Salem, NC, United States
b Department of Life Sciences, Winston-Salem State University, Winston Salem, NC, United States
c Department of Obstetrics & Gynecology, Wake Forest University School of Medicine, Winston Salem, NC, United States
d Hypertension Research & Vascular Center, Wake Forest University School of Medicine, Winston Salem, NC, United States
e Physiology & Pharmacology, Wake Forest University School of Medicine, Winston Salem, NC, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Vascular effects of 4-aryl methoxypiperidinol compounds previously shown to share with cocaine the
ability to inhibit the dopamine transporter are described. All the compounds tested inhibit KCl-induced
and noradrenaline-dependent contractions in mesenteric arteries ex vivo. Thus, diphenylpyraline and its
analogs may have a role as therapeutic options for the treatment of some of the cardiotoxic effects of
cocaine intoxications.
Received 29 January 2014
Revised 8 April 2014
Accepted 9 April 2014
Available online 18 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Piperidinol-4
Arterial contraction
Dopamine transporter
As a powerful sympathomimetic agent, cocaine exerts its
rewarding activity by blocking the dopamine transporter (DAT)
with the subsequent increase in dopamine (DA) concentrations in
the synapse. The search for cocaine antagonists has been the focus
of researchers and several compounds with DAT inhibiting activity
have been described.1 For example, benzotropine (BZT) analogs
possess a diphenylmethoxy moiety and a piperidyl ring; these
compounds were previously studied as potential pharmacothera-
pies for cocaine addiction.2 Similar to cocaine, the well-known
histamine H1 receptor antagonist diphenylpyraline (DPP) increases
brain dopamine, possesses psychostimulant properties in mice3
and also shares the same BZT structural features. The fact that
DPP functions as a potent dopamine reuptake inhibitor without
producing significant rewarding effects suggests that DPP and its
analogs merit further study as potential candidates for pharmaco-
therapies for cocaine addiction. These observations prompted the
synthesis of a series of DPP analogs and it was shown that they
share with DPP the ability to inhibit the DAT.4 In these DPP analogs,
symmetrical para substituents of the benzene ring were found to
be important for high potency in binding to the DAT.4 In addition,
several piperidine derivatives have been shown to block an array of
dopaminergic, serotoninergic and adrenergic monoamine trans-
porters,5 reinforcing the physiological relevance of the interactions
between the piperidine structure and monoamine transporters.
Besides the well-established alterations in behavior, cocaine
exerts powerful effects on the cardiovascular system with chest
pain being one of the most common complaints with acute cocaine
use.6 Several studies have shown that cocaine increases contrac-
tion in isolated arteries and hearts,7–9 supporting the notion that
this increased contraction of coronary arteries caused by cocaine
may be related to the myocardial infarction associated with acute
cocaine intoxication.10,11
Since DPP and its analogs share with cocaine the inhibition of
the DAT, we investigated whether these compounds share vascular
properties with cocaine. In this study, the effects of equimolar
doses of cocaine, DPP and its analogs were tested on KCl- and
noradrenaline (NA)-induced contractions of the rat mesenteric
resistance artery (MRA).
The DPP analogs were synthesized as previously described4
utilizing methods used for the synthesis of the tropane series of
compounds12–14 (see Fig. 1). Final mixtures were purified by flash
chromatography with mass and 1H NMR spectra used for further
analyses.15
Vascular contraction was tested in isolated mesenteric resis-
⇑
Corresponding author at: Biomedical Research Infrastructure Center, Winston-
Salem State University, 115 S. Chestnut Street, Winston Salem, NC 27157, United
States. Tel.: +1 (336) 761 6716; fax: +1 (336) 761 2239.
tance arteries mounted in
a wire myograph as previously
described.16 Arterial segments were normalized to 0.9Á L100, with
L100 being the internal circumference the vessels would have if
E-mail address: vpulgar@wakehealth.edu (V.M. Pulgar).
http://dx.doi.org/10.1016/j.bmcl.2014.04.040
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

2430
V. M. Pulgar, J. Keith Harp / Bioorg. Med. Chem. Lett. 24 (2014) 2429–2432
R3
HO
R3
OH
O
R2
R1
R1
N
N
b
1a R1 =H, R2 =F, R3 =F
1 R1 H
2a R1 =-CH3, R2 =H, R3 =Cl
2b R1 =-CH3, R2 =F, R3 =F
3a R1 =-iPr, R2 =F, R3 =F
4a R1 =-Bu, R2 =Cl, R3 =Cl
2 R1 -CH3
3 R1 -iPr
4 R1 -Bu
R2
a
Figure 1. Synthesis of substituted diphenylmetoxypiperidines. Reagents and conditions: (a) Hal-R, K2CO3, DMF, 40 °C; (b) p-TSA, benzene/DMF (25:1), reflux. For details see
Refs. 3,4.
they were exposed to a transmural pressure of 100 mmHg.17
Optimal diameters (OD) were calculated as OD = 0.9 L100
/p
.
A
Arteries with an OD of 278 10
l
m were used.18
The contraction to 75 mM KCl was used to evaluate receptor-
independent responses, whereas receptor-dependent responses
were evaluated as the contraction to a dose–response curve to
NA. Maximal contraction to KCl was unaffected by pre-incubation
with cocaine (3 Â 10À6 M), however DPP (3 Â 10À6 M) and all the
diphenylmethoxypiperidines (each one at
a concentration of
3 Â 10À6 M) tested blocked KCl contraction (Fig. 2, p <0.05). The
diphenylmethoxypiperidines studied blocked KCl-induced con-
traction with varying efficacy. Compounds 3a and 4a were the
most effective whereas 1a, 2a and 2b share with DPP approxi-
mately a 70% of inhibition of KCl-induced contraction.
Dose response curves to noradrenaline (NA) in the presence of
cocaine, DPP and diphenylmethoxypiperidine compounds are
shown in Figure 3. Maximal responses to NA were not affected
by pre-incubation with cocaine (Fig. 3A, Table 1), whereas 4a
was the only diphenylmethoxypiperidine compound that
decreased NA maximal contraction (Fig. 3B, Table 1, p <0.05).
Sensitivity to NA was increased by cocaine and decreased by DPP
and all diphenylmethoxypiperidines tested. The greatest inhibitory
effect was observed with compounds 3a and 4a (Fig. 4, Table 1,
p <0.05). In the presence of cocaine the inhibitory effect of
the diphenylmethoxypiperidine compounds on NA contraction
remained unaffected (data not shown).
B
Parameters of the ‘Rule of 5’19 were calculated for the
compounds tested using the online software Molinspiration.20
Figure 3. Effects of cocaine, DPP and diphenylmetoxypiperidines on NA-induced
contraction. MRA were exposed to increasing concentrations of NA in the presence
of cocaine (Coc, n = 6, 3 Â 10À6 M) or DPP (n = 6, A, 3 Â 10À6 M) and DPP analogs 1a,
2a, 2b, 3a and 4a, (n = 5, B, all at 3 Â 10À6 M) as indicated.
The relationship between the calculated octanol/water partition
coefficient (LogP) and the observed effects on NA contraction were
fitted to a linear regression. Compounds 3a and 4a with the great-
est inhibitory effect on NA contraction display the largest LogP
values (Table 1). Figure 5 shows the linear relationship between
LogP and
D
pD2
(D
pD2 = À0.3499 Á LogP + 0.3383) suggesting that
more effective inhibitors should possess increased lipophilicity.
Our results show for the first time that DPP and the DPP analogs
tested inhibit receptor-dependent as well as receptor-independent
contractions in MRA. Compared to cocaine, DPP and its analogs dis-
played opposite effects on vascular contraction; whereas cocaine
treatment increases sensitivity to NA, all diphenylmethoxypiperi-
dines compounds tested diminished sensitivity to NA.
Figure 2. Effects of cocaine, DPP and diphenylmetoxypiperidines on KCl-induced
contraction. MRA (n = 6) were exposed to 75 mM KCl during 5 min in the presence
of cocaine (3 Â 10À6 M), DPP (3 Â 10À6 M) and DPP analogs (all at 3 Â 10À6 M) as
indicated. ⁄p <0.05 versus control arteries.

V. M. Pulgar, J. Keith Harp / Bioorg. Med. Chem. Lett. 24 (2014) 2429–2432
2431
Table 1
Effects of cocaine, DPP and DPP analogs on noradrenaline-mediated contractions in MRA
R1
R2
R3
Molecular Formula
pD2
NAMAX (% KMAX
)
LogP
Noradrenaline
5.95 0.03
6.52 0.09*
5.38 0.04*
5.17 0.07*
5.08 0.08*
5.10 0.12*
4.48 0.15*
4.26 0.07*
122
130
129
116
113
121
121
2
7
4
4
8
6
1
Cocaine, 3 Â 10À6
M
DPP
1a
2a
2b
3a
4a
CH3
H
CH3
CH3
i-Pr
Bu
H
F
H
F
F
Cl
F
F
Cl
F
F
C19H23NO
C18H19F2NO
C19H22ClNO
C19H21F2NO
3.161
2.869
3.815
3.465
4.137
5.931
C
C
21H27F2NO
22H27Cl2NO
Cl
99 6*
Sensitivity to NA is expressed as pD2 and maximal contraction as % KMAX. Calculated values of LogP are indicated for DPP and the DPP analogs studied.
*
p <0.05 versus control arteries.
Our linear regression analysis showed that more active com-
pounds should also be more lipophilic. The N-alkyl substituents
appear to be more important than halogen substituents on the
aromatic rings in terms of the activity described here. As the size
of the N-alkyl substituent increases in compounds 1a (–H), 2b
(–CH3) and 3a (–iPr), all with para-F substituents in the aromatic
rings, the inhibitory effect on NA contraction increases. The most
effective compound 4a has a N-butyl group, its activity may be
due to the fact that the potential targets are integral membrane
proteins such as ion channels and/or receptors, and agrees with
the previously described importance of properly positioned
lipophilic groups.19
Since DPP and its analogs are able to inhibit receptor-dependent
and receptor-independent contractions, they may be acting upon
global mechanisms of vascular control. The inhibitory effect is
maintained in the presence of cocaine, suggesting that DPP and
its analogs may act through different vascular mechanisms.
Vascular contraction, both receptor-dependent and receptor-
independent, is mediated by rises in [Ca+2]i. As an inhibitor of the
H1-histamine receptor, DPP and its analogs may interact with
the complex vascular actions of histamine.25,26 It was reported
earlier that H1-antihistaminic compounds relax norepinephrine-
contracted tissues;27 the fact that this effect is also observed in
KCl-contracted tissues27 suggests that the inhibitory effects on
adrenergic contraction are not mediated by direct interactions
Figure 4. Effects of cocaine, DPP and diphenylmetoxypiperidines on sensitivity to
NA-induced contraction. MRA were exposed to increasing concentrations of NA in
the presence of cocaine (Coc, n = 6, 3 Â 10À6 M), DPP (n = 6, 3 Â 10À6 M) and DPP
analogs (n = 5, all at 3 Â 10À6 M) as indicated. Effects on NA sensitivity are
expressed as
DpD2, letters indicate statistically significant differences.
with
a-adrenergic receptors. H1-antihistaminics have also been
shown to modulate [Ca+2]i. The H1-antihistaminic astemizole
lowers [Ca+2]i by inhibiting Ca+2 influx in mast cells through the
inhibition of store operated Ca+2 channels (SOC).28 SOC channels
are important in maintaining tonic contractions and are involved
in adrenergic activation of the vasculature.29 It is conceivable that
DPP and its analogs modulate [Ca+2]i by inhibiting SOC in MRA.
However, the inhibitory effects of DPP and its analogs on NA
contraction is not altered by cocaine suggesting an effect down-
stream direct modulation of [Ca+2]i.
Figure 5. Correlation between lipophilicity and inhibition of NA contraction. Linear
regression analysis between effects on NA sensitivity (DpD2) and Log P for DPP and
DPP analogs tested. R2 = 0.766, p <0.02.
Compounds with H1-antihistaminic activity have also been
shown to block the K+ channel human ether-a-go-go related gene
HERG1 encoding the main subunit of a cardiac K+ channel.30
HERG1 activity is responsible for the rapid component of the
ventricular repolarizing current IKr and the interaction of
H1-antihistaminics with HERG1 has been suggested as responsible
for the cardiotoxic effects displayed by some of these drugs.31 Since
HERG channels are expressed in smooth muscle cells32, our results
also emphasize the importance of examining the effects of DPP and
its analogs on HERG channels in future studies. Due to the high
structural homology between K+ channels, it is possible that
H1-antihistaminic compounds may interact with additional
members of this important family of ion channels and modulate
their activities. Thus, the effects of DPP and its analogs on Ca+2
and K+ channels may be operating in the vasculature to inhibit
KCl- and NA-dependent contractions.
DPP and its analogs share with cocaine the ability to inhibit the
DAT and our results point to a potential additional role of these
compounds as therapeutic options for the treatment of the cardio-
toxic effects of cocaine intoxications. The cocaine-induced
increased sensitivity to NA in rat MRA has been previously
described21 and is consistent with its role as an inhibitor of
catecholamines’ reuptake by terminal nerves. This effect is
ascribed to the blockade of the noradrenaline transporter and is
potentially responsible for the increased coronary contraction as
a contributor for the cardiotoxic effects observed in cocaine
intoxications.7,10,22,23 Moreover, cocaine in concentrations seen in
drug users has also been shown to increase intracellular Ca+2
concentrations ([Ca+2]i) in cultured vascular smooth muscle cells
from cerebral vessels.24

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V. M. Pulgar, J. Keith Harp / Bioorg. Med. Chem. Lett. 24 (2014) 2429–2432
benzophenones were reduced to the benzhydrols using sodium borohydride in
We have shown that substituted diphenylmethoxypiperidines
isopropanol in nearly quantitative yield. The ethers were synthesized by
condensation of benzhydrols with 25% molar excess of N-substituted
are able to inhibit both receptor-dependent and receptor-
independent contractions in isolated MRA. The role of
piperidin-4-ols or N-methylpiperidin-4-ol in benzene with
a Dean–Stark
diphenylmethoxypiperidines on the regulation of [Ca+2
vascular smooth muscle cells warrants further investigation.
] in
i
trap. For details see Ref. 4.
15. For all compounds the mass spectra (Agilent 1100 HPLC and Micromass
Quattro Ultima spectrometer; Waters, Milford, MA) contained weak (MÀH)+
ions characteristic of piperidines and the typical isotope patterns for one or
more halogen atoms in the structures. Characteristic ions that occurred were
m/z 99 (methylpiperidine fragment) as the base peak and m/z 114
(methylpiperidinol fragment) at approximately 50% R.A. in each mass
spectrum. Significant diarylmethane fragments also occurred for all
compounds. For compounds with very weak (MÀH)+ ions in the EI mass
spectrum, electrospray ionization mass spectra from a methanol solution were
taken to confirm the molecular weight as the (M+H)+ ion. The relative response
of all compounds was from 95.8% up to 99.1%. The 1H NMR (300 MHz, CDCl3,
Varian EM-360 spectrometer) spectra of N-methylpiperidines showed typical
signals: 1.30–1.35 (m, 2H, H-2,6 ax), 1.85–1.90 (m, 2H, H-2,6 equiv), 2.00–2.05
(m, 2H, H-3,5 ax), 2.40–2.45 (s, 3H, N-CH3), 2.90–3.00 (m, 2H, H-3,5 equiv),
3.50–3.55 (m, 1H, H-4 ax), 5.35–5.45 (s, 1H, Ar-CH-Ar), 7.20–7.60
(m, aromatic C-H).
Source of funding
Funded by NIH MD00232.
Acknowledgments
This work was supported by the National Center for Minority
Health and Health Disparities Grant MD00232. The authors want
to thank to Gennady B. Lapa, Antoinette Helm and Shelesa Perry
for their help in the synthesis of the DPP analogs used in this study.
The authors want to thank to Drs. Jeff Overholt and Manjunatha B.
Bhat for critical reading of the manuscript and Dr. Azeez Aileru for
logistical support in conducting these studies.
16. Pulgar, V. M.; Jeffers, A. B.; Rashad, H. M.; Diz, D. I.; Aileru, A. A. J. Cardiovasc.
Pharmacol. 2013, 62, 174.
17. Mulvany, M. J.; Halpern, W. Nature 1977, 260, 619.
18. For each arterial segment, the maximal response to KCl was calculated, and the
response to NA was expressed as percent of the corresponding maximal
response to KCl (%KMAX). Concentration-response curves to NA were analyzed
Supplementary data
by fitting individual experimental data to
a logistic curve to determine
maximal response and sensitivity. The curve was of the form
Y = bottom + (top À bottom)/(1 + 10(LogEC50 À X) * Hill Slope)) where X is the
logarithm of the concentration and Y is the response; the sensitivity values
reported are derived from these fits. Sensitivity was expressed as pD2
(pD2 = Àlog[EC50]) with EC50 being the concentration of agonist producing
50% of the maximal response. Effects on NA sensitivity were calculated for each
experiment as the difference on sensitivity between NA and NA in the presence
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.
04.040. These data include MOL files and InChiKeys of the most
important compounds described in this article.
of the different compounds (DpD2). One segment per animal was used to test
each compound; n (between 5 and 6) refers to the number of animals used.
Data are expressed as mean SEM. One-way analysis of variance (ANOVA)
with Bonferroni’s multiple comparisons was used to determine significant
differences. A p <0.05 was accepted as an indication of statistical significance.
For details see Ref. 16.
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