Low doses of allyphenyline and cyclomethyline, effective against morphine dependence, elicit an antidepressant-like effect

Article abstract of DOI:10.1021/ml300064v

This study demonstrated that cyclomethyline (2) and the corresponding enantiomers (R)-(-)-2 and (S)-(+)-2, displaying α_2C- adrenoreceptor (AR) agonism/α_2A-AR antagonism, similarly to allyphenyline (1) and its enantiomers, significantly decreased the naloxone-precipitated withdrawal symptoms in mice at very low doses. It also highlighted that such positive effects on morphine dependence can even be improved by additional serotoninergic 5-HT_1A receptor (5-HT _1A-R) activation. Indeed, 1 or the single (S)-(+)-1, 2, or both its enantiomers, all behaving as α_2C-AR agonists/α _2A-AR antagonists/5-HT_1A-R agonists, alone and at the same low dose, improved morphine withdrawal syndrome and exerted a potent antidepressant-like effect. Therefore, considering the elevated comorbidity between opiate abuse and depressed mood and the benefit of these multifunctional compounds to both disorders, it is possible that they prove more efficacious and less toxic than a cocktail of drugs in managing opioid addiction.

Full text of DOI:10.1021/ml300064v

Letter
pubs.acs.org/acsmedchemlett
Low Doses of Allyphenyline and Cyclomethyline, Effective against
Morphine Dependence, Elicit an Antidepressant-like Effect
Fabio Del Bello,† Eleonora Diamanti,† Mario Giannella,† Valerio Mammoli,† Carla Marchioro,‡
Laura Mattioli,§ Federica Titomanlio,§ Alessandro Piergentili,† Wilma Quaglia,† Giovanni Benedetti,∥
Maurizio Varrone,∥ and Maria Pigini*,†
†School of Pharmacy, Medicinal Chemistry Unit, University of Camerino, Via S. Agostino 1, 62032 Camerino, Italy
‡Aptuit Verona, via Fleming 4, 37135 Verona, Italy
§School of Pharmacy, Pharmacognosy Unit, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy
∥Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
S
* Supporting Information
ABSTRACT: This study demonstrated that cyclomethyline (2)
and the corresponding enantiomers (R)-(−)-2 and (S)-(+)-2,
displaying α2C-adrenoreceptor (AR) agonism/α2A-AR antagonism,
similarly to allyphenyline (1) and its enantiomers, significantly
decreased the naloxone-precipitated withdrawal symptoms in mice
at very low doses. It also highlighted that such positive effects on
morphine dependence can even be improved by additional
serotoninergic 5-HT1A receptor (5-HT1A-R) activation. Indeed, 1
or the single (S)-(+)-1, 2, or both its enantiomers, all behaving as
α2C-AR agonists/α2A-AR antagonists/5-HT1A-R agonists, alone
and at the same low dose, improved morphine withdrawal syndrome and exerted a potent antidepressant-like effect. Therefore,
considering the elevated comorbidity between opiate abuse and depressed mood and the benefit of these multifunctional
compounds to both disorders, it is possible that they prove more efficacious and less toxic than a cocktail of drugs in managing
opioid addiction.
KEYWORDS: α2-adrenergic ligands, 5-HT1A-R agonists, morphine withdrawal symptoms reduction, antidepressant-like effect
orphine use is the classical approach in the treatment of
chronic or cancer-related pain. Nevertheless, even when
agonist-mediated analgesia.3−5 In detoxification, α2-AR agonists
such as clonidine and lofexidine are often clinically used alone
or in combination with traditional treatments to reduce the
intensity of withdrawal symptoms, thus increasing treatment
duration. However, the α2A-AR activation triggered by these
compounds is responsible for side effects such as sedation and
hypotension.1 It has been reported that in comparison to
clonidine, lofexidine showed decreased incidence and severity
of adverse events.1,6 The partial α2A-AR agonism of lofexidine,
as recently assessed by us,7 might justify its behavior (Table 1).
Therefore, selective α2C-AR agonists, devoid of the side effects
associated with α2A-AR stimulation, alone or in combination
with opioids, might afford an improvement over current
therapies with clonidine-like drugs. We reported the interesting
behavior of allyphenyline (1), which displays a significant α2C-
AR agonist/α2A-AR antagonist profile8 ascribed to its preferred
extended molecular conformation.7 In in vivo studies, 1
enhanced morphine analgesia (due to its α2C-agonism), was
devoid of a sedative effect (due to its α2A-antagonism),8,9 and
contrasted and prevented morphine tolerance and dependence
M
legally allowed and closely monitored, long-term opioid
administration alters central pain-related systems, inducing
tolerance and dependence. Opioid addiction, termed a “chronic
relapsing disease”, is associated with a myriad of health and
social problems; hence, its management is an extremely
important area of research.1
α2-Adrenoreceptors (α2-ARs), which have been demonstra-
ted to be extremely sensitive to opioid exposure and to play a
key role in opiate withdrawal symptoms,2 belong to the
superfamily of G-protein-coupled receptors. The three α2A-,
α2B-, and α2C-AR subtypes are widely distributed in the central
nervous system (CNS) and peripheral tissues.3 Studies with
genetically engineered mice have demonstrated that the α2A
subtype mediates hypotension, sedation, and analgesia, as well
as inhibition of monoamine release and metabolism in the
brain. The α2B subtype mediates vasoconstriction, while the α2C
subtype appears to be involved in many CNS processes, such as
the startle reflex, stress responses, and control of locomotion as
well as feedback inhibition of adrenal cathecolamine release.
Moreover, in the brain, α2A- and α2C-ARs, as “heteroreceptors”,
inhibit dopamine and serotonin release. Finally, the α2C subtype
can contribute to adrenergic-opioid synergy and spinal α2-
Received: March 15, 2012
Accepted: June 8, 2012
Published: June 8, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
a
Table 1. Affinity (pKi), Antagonist Potency (pKb), Agonist Potency (pEC50), and Intrinsic Activity (ia) on Human α2-AR
b
c
Subtypes and Affinity (pKi), Agonist Potency (pD2), and Relative Efficacy (%Emax) on Human 5-HT1A-R
α2A
α2B
pEC50 (pKi)
α2C
pEC50 (pKi)
5-HT1A
compd
( )-1
pKb (pKi)
ia
ia
pD2 (pKi)
6.86 0.09
%Emax
d
7.40 0.06
(7.24 0.11)
7.40 0.09
(7.00 0.08)
7.80 0.13
(7.28 0.05)
7.70 0.12
(7.44 0.09)
7.20 0.11
(7.35 0.10)
7.70 0.09
(7.38 0.07)
e
NA
7.30 0.09
(7.07 0.14)
6.73 0.11
(6.75 0.11)
7.60 0.14
(7.15 0.09)
8.70 0.08
(6.56 0.21)
6.50 0.07
(6.63 0.05)
8.70 0.11
(6.56 0.13)
7.24 0.14
0.90
67
(6.47 0.20)
(7.55 0.16)
<5
d
(R)-(−)-1
(S)-(+)-1
( )-2
NA
0.50
0.90
0.80
0.75
0.85
0.60
0.80
(6.25 0.12)
6.00 0.09
(6.40 0.09)
5.48 0.15
(6.39 0.05)
5.40 0.11
(6.47 0.07)
5.80 0.06
(6.39 0.10)
5.95 0.27
(<5)
0.65
0.70
0.65
0.70
0.45
0.80
7.19 0.10
(7.45 0.15)
7.20 0.13
(7.98 0.07)
6.80 0.10
(7.40 0.08)
7.40 0.02
(8.24 0.16)
96
75
68
97
(R)-(−)-2
(S)-(+)-2
b
clonidine
(5.49 0.13)
f
lofexidine
g
6.90 0.14
8.86 0.20
(8.36 0.11)
(7.17 0.18)
(7.16 0.07)
(6.90 0.11)
7.60 0.06
8-OH-DPAT
100
(8.47 0.13)
a
b
c
The data were expressed as means SEMs of 3−6 separate experiments. According to ref 9. pKi values were derived from displacement of [3H]-
8-OH-DPAT binding on human cloned 5-HT1A-R. pD2 values are the negative logarithm of the agonist concentration required to obtain 50% of the
maximal stimulation of [35S]GTPγS binding. Maximal stimulation (%Emax) is expressed as a percentage of the maximal 8-OH-DPAT response.
d
e
f
g
Compounds exhibiting ia of <0.3 were considered not active (NA). pEC50 = 8.08 0.12; ia = 0.90. Ref 7. pEC50 = 8.20 0.10; ia = 0.60.
at a very low dose (0.05 mg/kg).9 In contrast, clonidine
attenuated the expression of morphine withdrawal symptoms at
a higher dose (5 mg/kg) and was ineffective in preventing
them.10 Because α2-AR antagonists (i.e., yohimbine) also
attenuated the intensity of withdrawal symptoms,11 we
attributed the unique profile of 1 to the synergistic combination
of its effective dual α2C-AR agonism/α2A-AR antagonism.9 The
positive effects of 1 on dependence were also displayed by (S)-
(+)-1 (potent α2C-AR full agonist) and (R)-(−)-1 (α2C-AR
partial agonist); they both were endowed with similar α2A-AR
antagonism.9 To confirm the aforementioned results and
discover novel tools for the management of opioid withdrawal
symptoms that have a major role in relapse to drug-taking
behavior after detoxification, we extended the study to the
recently reported allyphenyline analogue 2,7 now named
cyclomethyline, and its enantiomers, prepared in the present
investigation. Compound 2 showed interesting α2C-AR
agonism/α2A-AR antagonism (Table 1), and according to
what reported for 1,7 this functional profile might be associated
with a preferred extended molecular conformation. Therefore,
the corresponding enantiomers were evaluated by binding and
functional assays on Chinese hamster ovary (CHO) cells
expressing recombinant human α2-AR subtypes according to
previous procedures.9 Moreover, the effects of the racemate 2 at
the dose of 0.05 mg/kg and its enantiomers at the dose of 0.025
mg/kg (ip) on the expression and acquisition of morphine
dependence were determined. To learn more about the
pharmacological properties of this novel class of compounds,
expecially in light of the strong comorbidity between opioid
addiction and depressive disorders seen in several clinical
studies,12 1 and 2 and their enantiomers were evaluated in a
behavioral model of depression in mice. It is known that the
serotoninergic [5-hydroxytryptamine (5-HT)] system is
involved in the neural regulation of mood, and abnormalities
in 5-HT neurotransmission have been observed in the
pathophysiology of depression. Moreover, 5-HT dysfunction
is one of the main mechanisms contributing to mood disorders
in opiate abstinence.13 It has also been suggested that α1A-AR
agonists might represent a novel treatment for depression.14
Therefore, the biological profiles of the aforementioned
compounds at 5-HT1A receptor (5-HT1A-R), particularly
relevant to antidepressant responses in human beings,15 and
at α1A-AR were assessed according to procedures previously
reported.16 Finally, an assessment of the in vitro absorption,
distribution, metabolism, excretion (ADME) properties9 of 2
and of the activity of 1 and 2 on the human ether-a-go-go-
̀
related gene (hERG) channel17 was performed.
The enantiomers of 2 were prepared according to Scheme 1.
(S)-(+)- and (R)-(−)-2-(2-cyclopropylmethyl)propionic acid
methyl ester [(S)-(+)-3 and (R)-(−)-3, respectively] were
obtained by Mitsunobu reaction from 2-(cyclopropylmethyl)-
phenol18 and methyl (R)-(−)- or (S)-(+)-lactate, respectively,
with inversion of configuration.19 The reaction of (S)-(+)- or
(R)-(−)-3 with ethylenediamine in the presence of (CH3)3Al
yielded the corresponding imidazolines (S)-(+)- and (R)-
1
(−)-2, whose e.e. determined by H NMR spectroscopy was
about 85%, due to partial racemization occurring under the
conditions used in the reactions. Indeed, their spectra, as well as
that of the racemic ( )-2 on addition of the chiral shift reagent
(S)-(+)-2,2,2-trifluoro-1-(9-anthryl)-ethanol, showed a double
doublet at δ 1.38 ppm for the methyl protons. The resolution
of the racemic imidazoline ( )-2 was performed by HPLC
using Chiralcel OD-H (25 cm × 0.46 cm, 5 μm particle size) as
the chiral stationary phase and n-hexane/2-propanol 90/10 v/v
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dx.doi.org/10.1021/ml300064v | ACS Med. Chem. Lett. 2012, 3, 535−539

ACS Medicinal Chemistry Letters
Letter
a
Scheme 1. Synthesis of (S)-(+)-2 and (R)-(−)-2
Figure 1. Effects of acute (A, B) or repeated (C, D) ip administration
of ( )-2 (0.05 mg/kg), (R)-(−)-2, and (S)-(+)-2 (0.025 mg/kg) on
expression and acquisition of morphine dependence, respectively.
Naloxone-precipitated withdrawal symptoms, in both control and
morphine-treated mice, are given as a measure of the frequency of
jumping (A, C) and somatic signs (B, D) expressed as a summary of
rearing, forepaw tremors, and teeth chatter. Significant differences: *p
< 0.05, **p < 0.01, as compared to vehicle; ++p < 0.01, as compared to
the morphine group.
a
Reagents and conditions: (a) DIAD, Ph3P, THF, rt. (b) Ethylenedi-
amine, Al(CH3)3, toluene, 0−65 °C, 7 h.
as the mobile phase. Detection was monitored at a wavelength
of 220 nm, and the retention times were 5.9 and 10.3 min for
(+)-2 and (−)-2, respectively; (+)-2: [α]D = +19.28, e.e.
20
20
1
100%; (−)-2: [α]D = −19.59, e.e. 100%. In this case, the H
NMR spectra, similarly performed, showed for the methyl
group of (+)-2 and (−)-2 only one doublet at δ 1.36 and δ 1.40
ppm, respectively. The absolute configuration of the stereo-
center in the bridge of these enantiomers was determined by
comparing the sign of their optical rotations with those of the
enantiomers obtained by stereoselective synthesis. Conse-
quently, the (S) absolute configuration was assigned to the
dextrorotatory enantiomer (+)-2 and (R) to the levorotatory
(−)-2.
The affinity values (pKi), antagonist properties (pKb), agonist
potencies, and intrinsic activities (pEC50 and ia, respectively) of
the enantiomers of 2 on α2-AR subtypes are reported in Table
1. The biological profiles of 2, 1, and its enantiomers were also
included for useful comparison. This study indicates that (i)
both enantiomers of 2 displayed binding affinities for the three
α2-AR subtypes comparable to those of the racemic compound;
(ii) (S)-(+)-2 was endowed with the highest α2C-AR agonist
activity; (iii) (R)-(−)-2 was characterized by lower but still
significant α2C-AR agonism; and (iv) (R)-(−)-2 and (S)-(+)-2
showed weak α2B-AR activation but effective α2A-AR antagonist
potencies. Interestingly, 2, (R)-(−)-2 and (S)-(+)-2 affected
expression and acquisition of morphine dependence (Figure 1)
as observed previously for 1 and its enantiomers.9 The
frequencies of naloxone-precipitated jumping and other somatic
signs (rearing, forepaw tremors, and teeth chatter) decreased
significantly. To study possible antidepressant-like activity, the
mouse forced swimming test (FST), a behavioral model with
good reliability and predictive validity for screening anti-
depressants,20 was used. Figure 2A shows that 1 induced
significant reduction of immobility time. This effect was
maximal at the dose of 0.05 mg/kg and comparable to that
obtained with the dose of 20 mg/kg of the selective 5-HT
reuptake inhibitor fluoxetine, included as reference com-
pound.21 Significant antidepressant activity was also observed
Figure 2. Immobility time in the FST in mice following ip
administration of ( )-1 at 0.012, 0.025, 0.05, 0.2, and 0.5 mg/kg
(A), ( )-2 at 0.025, 0.05, and 0.2 mg/kg (B), and fluoxetine (Fluox;
20 mg/kg) as a reference drug. Data represent means ( SEMs) of 8−
9 animals. Differences from vehicle-treated: *p < 0.05, **p < 0.01;
where not indicated, the differences are not statistically significant.
at 0.025 and 0.2 mg/kg doses, whereas no activity was observed
at lower and higher doses. Similarly, the maximal effect of 2 was
displayed at the dose of 0.05 mg/kg (Figure 2B). Nevertheless,
the antidepressant response of 1 was only associated with the
(S)-(+)-1 enantiomer, whereas the effect of 2 was similarly
induced by both its enantiomers (Figure 3). In Table 1,
affinities and functional values of all compounds at 5-HT1A-R
are also reported. The 5-HT1A-R agonist 8-hydroxy-2-(di-n-
propylamino)tetralin (8-OH-DPAT) was included for useful
comparison.16 From the data, it emerged that 1 displayed good
5-HT1A-R affinity and behaved as an agonist. These properties
were maintained and even enhanced only by the (S)-(+)-1
enantiomer, whereas (R)-(−)-1 showed negligible affinity (pKi
< 5).
In contrast, 2, (R)-(−)-2 and (S)-(+)-2 displayed similar and
significant 5-HT1A-R affinities and agonist potencies. None of
the compounds studied showed significant α1A-AR agonism (ia
< 0.35). Altogether, the data indicated that dual α2C-AR/5-
HT1A-R activation was requested for the antidepressant-like
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ACS Medicinal Chemistry Letters
Letter
associated with 5-HT1A activation, as verified for the examined
compounds, is equally compatible with an antidepressant-like
effect. The advantage of this peculiar biological profile was also
supported by the observation that yohimbine, a 5-HT1A-R
agonist27 and antagonist at all three α2-AR subtypes,25 did not
display antidepressant activity. The physicochemical and in
vitro ADME9 profile of 2 proved to be similar to that of 1.
Indeed, 2 was highly soluble in water at physiological pH. The
permeation rate in a parallel artificial membrane permeability
assay (PAMPA) assay and the percentage remaining after
incubation for 1 h with recombinant hCYP3A4 were very high.
From an automatic patch clamp assay on HEK293 cells
overexpressing the hERG channel,17 compounds 1 and 2
showed to be weak inhibitors, with IC50 values of 16.9 and 8.9
μM, respectively, suggesting a low cardiotoxicity risk associated
with their potential therapeutic use.
In summary, the present study demonstrated that cyclo-
methyline (2) and its enantiomers, characterized by α2C-AR
agonist/α2A-AR antagonist profile, significantly decreased the
naloxone-precipitated withdrawal symptoms in mice at very low
doses. These properties are in agreement with those shown by
the lead 1 and its enantiomers and confirm that dual α2C-AR
agonism/α2A-AR antagonism represents a favorable condition
for inducing positive effects on morphine dependence.9
Interestingly, such effects can even be improved by additional
5-HT1A-R activation. Indeed, unlike clonidine, 1 or the single
(S)-(+)-1 enantiomer, 2, or both its enantiomers, all behaving
as α2C-AR agonists/α2A-AR antagonists/5-HT1A-R agonists,
alone and at the same low dose, improved morphine
withdrawal syndrome and exerted a potent antidepressant-like
effect. It has been suggested that opiate abuse may induce
depressed mood, or conversely, depression can drive to drug
abuse in a self-medication hypothesis. Therefore, such multi-
functional compounds might be beneficial to both disorders.13
In addition, their therapeutic potential is strongly enhanced by
the lack of sedative side effects (due to their α2A-antagonism)
and alteration of motor activity (data not shown), as well as by
their favorable in vitro ADME profiles and limited activity on
the hERG channel.
Figure 3. Immobility time in the FST in mice following ip
administration of (R)-(−)-1, (S)-(+)-1, (R)-(−)-2, and (S)-(+)-2
(0.025 mg/kg). Data represent means ( SEMs) of eight animals.
Differences from vehicle-treated: **p < 0.01; where not indicated, the
differences are not statistically significant.
effect induced by low doses of 1 and the single (S)-(+)-1
enantiomer, 2, and both its enantiomers. This consideration
does not contrast with the results of previous studies.22,23
Indeed, they indicated that low doses of clonidine (0.01−0.06
mg/kg) increased 5-HT neurotransmission by attenuating the
release of endogenous noradrenaline via activation of α2-AR
autoreceptors on adrenergic neurones22 and, hence, induced
anti-immobility effects with subactive doses of 5-HT1A-R
agonists (i.e., 8-OH-DPAT) in mouse FST.23 On the contrary,
higher doses (0.1−0.4 mg/kg) decreased 5-HT neuro-
transmission through the direct activation of the α2-AR
heteroreceptors on the 5-HT terminals.22 The peculiar
relationship between α2-AR activation and 5-HT function,
highlighted by such studies, might justify the dose-dependent
U-shaped trend exhibited by the anti-immobility effect of our
α2C-AR/5-HT1A-R agonists. To support our observations,
experiments in the presence of the 5-HT1A-R antagonist
WAY 10013524 and the α2-AR antagonist yohimbine25 were
carried out. Both antagonists, ineffective when given alone,
significantly and similarly contrasted the actions evoked by
0.025 mg/kg dose of (S)-(+)-1, (R)-(−)-2, and (S)-(+)-2
(Figure 4). Therefore, these results confirmed that dual α2C-
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedure, hERG activity assay, detail of biological
assay, and elemental analysis of the final compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 4. Influence of the 5-HT1A-R antagonist WAY 100635 (0.1
mg/kg, sc) (A) and the α2-AR antagonist yohimbine (1.250 mg/kg,
ip) (B) on the antidepressant-like effect of the enantiomers (S)-(+)-1,
(R)-(−)-2, and (S)-(+)-2 (0.025 mg/kg, ip) on the FST. Data
represent means ( SEMs) of 7−8 animals. Significant differences: **p
< 0.01, as compared with vehicle group; ++p < 0.01, as compared with
enantiomers related-treated mice; and °p < 0.05, as compared with
antagonist-treated mice; where not indicated, the differences are not
statistically significant.
AUTHOR INFORMATION
Corresponding Author
*Tel: +39-0737-402257. Fax: +39-0737-637345. E-mail: maria.
pigini@unicam.it.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We thank the MIUR (Rome), the University of Camerino, and
the Fondazione Monte dei Paschi (Siena) for financial support.
■
AR/5-HT1A-R activation, favorably triggered by the same
molecule, took part in the anti-immobility effect. It has been
reported that selective α2C-AR antagonists, allowing α2A-AR
stimulation, produced important antidepressant effects in the
FST.26 Because α2A- and α2C-AR subtypes complement each
other to integrate CNS function and behavior,5 it is possible
that also the α2C-AR agonism/α2A-AR antagonism combination,
ABBREVIATIONS
■
AR, adrenoreceptor; CNS, central nervous system; CHO,
Chinese hamster ovary; 5-HT, 5-hydroxytryptamine; 5-HT1A-R,
5-HT1A receptor; ee, enantiomeric excess; ip, intraperitoneally;
538
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ACS Medicinal Chemistry Letters
Letter
antagonists, 5-HT1A full agonists, and cytotoxic agents. J. Med. Chem.
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sc, subcutaneously; ADME, absorption, distribution, metabo-
lism, excretion; PAMPA, parallel artificial membrane perme-
ability assay; DIAD, diisopropyl azodicarboxylate; CYP,
̀
cytochrome P450; hERG, human ether-a-go-go-related gene
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