Novel azulene derivatives for the treatment of erectile dysfunction

Article abstract of DOI:10.1016/j.bmcl.2012.09.064

Based on the dopamine D₄ receptor partial agonist FAUC 3019, a series of azulenylmethylpiperazines was synthesized and affinities for the monoaminergic GPCRs including dopamine, serotonin, histamine and α-adrenergic receptor subtypes were deter

Full text of DOI:10.1016/j.bmcl.2012.09.064

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7151–7154
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel azulene derivatives for the treatment of erectile dysfunction
Stefan Löber a, Harald Hübner a, Armin Buschauer b, Fabrizio Sanna c, Antonio Argiolas c,
Maria Rosaria Melis c, Peter Gmeiner a,
⇑
a Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen, Germany
b Institut für Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany
c Department of Biomedical Sciences, Neuroscience and Clinical Pharmacology Section, University of Cagliari, S.S. 554, km 4500, 09042 Monserrato, Cagliari, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the dopamine D4 receptor partial agonist FAUC 3019, a series of azulenylmethylpiperazines was
synthesized and affinities for the monoaminergic GPCRs including dopamine, serotonin, histamine and
Received 18 August 2012
Revised 17 September 2012
Accepted 18 September 2012
Available online 6 October 2012
a-adrenergic receptor subtypes were determined. Ligand efficacies of the most promising test
compounds revealed the N,N-dimethylaminomethyl substituted azulene 11 to be the most potent D4
partial agonist (EC50 = 0.41 nM). This candidate was investigated for its ability to promote penile erection.
Applying an in vivo animal model, test compound 11 turned out to stimulate penile erection in male rats
with superior potency in low concentrations when compared to apomorphine.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Penile erection
Erectile dysfunction
Azulene
Dopamine D4
Partial agonists
The disability to initiate or maintain penile erection sufficient to
permit sexual intercourse is a prevalent ailment among men. It has
been estimated that erectile dysfunction (ED) affects 150 million
individuals worldwide.1 Since the 1970s, substantial progress has
been made to understand the biochemical basis for the physiology
of penile erection and the pathophysiology of erectile dysfunction.2
It is now generally believed that the majority of patients with ED
have an underlying vascular or neurological impairment that
causes insufficient penile erection.3 Starting from the early
1980s, non-selective inhibitors of the phosphodiesterase (PDE) like
papaverine have been used as penile erecting agents,4 but turned
out to cause unpleasant side effects like prolonged erection and
penile fibrosis.5 In the following years, selective PDE5 inhibitors
including sildenafil or tadalafin were launched to the market and
turned out to be highly efficient drugs allowing oral administra-
tion. These agents act through a stimulation of NO release in the
penile smooth muscle inducing its relaxation.6
involved in this response to apomorphine and other dopaminer-
gics.11,12 Interestingly, selective (partial) agonists of the D4 subtype
displayed proerectile activity resulting in a new approach to the
treatment of ED. These findings led to the development of the
novel D4-selective partial agonists ABT-72413,14 and ABT-67015
that have been under investigation in clinical trials for the treat-
ment of erectile dysfunction. Recently, we could demonstrate that
PIP3EA and PD-168,077, two dopamine D4 receptor (partial)
agonists induce penile erection when injected into the paraventric-
ular nucleus of the hypothalamus (PVN) in rats by increasing
O H
N
N
HO
X
N
NH
N
Recent findings indicated that ED therapy needs not necessarily
be based on peripheral modulation of endogenous mediators.7 The
non-selective dopamine receptor agonist apomorphine (Fig. 1)
proved to be able to induce penile erection in rats, rabbits, mon-
keys and men.8–10 Initially, it was hypothesized that the proerectile
effect of apomorphine was mediated by the stimulation of D2
receptors. The discovery of subtypes within the D2 receptor family
(D2, D3 and D4) led to studies aiming to identify the subtype
apomorphine
: X= N
ABT-724
:
X= C-OMe
PIP3EA
N
O
N
X
N
N
H
MeO
:
X= N
: X= C-CN
O
ABT-670
FAUC 3019
PD-168,077
⇑
Corresponding author.
E-mail address: peter.gmeiner@medchem.uni-erlangen.de (P. Gmeiner).
Figure 1. Dopamine D4 (partial) agonists showing proerectile effects.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.064

7152
S. Löber et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7151–7154
central oxytocinergic neurotransmission.16 This effect is associated
by an increased NO production in the PVN. The findings boosted
the search for new D4 ligands, which has been an emerging field
of drug discovery within the last two decades.17
fluoride to obtain the unprotected alkyne 4. We also planned to
introduce functional groups with H-bond donor or acceptor proper-
ties. Starting from the readily available azulenedicarbaldehyde 7,26
reductive amination yielded the N-phenylpiperazine derivative 8.
Subsequent reduction of the formyl group resulted in formation of
the alcohol 9. As a further structural variation, the carbaldehyde
group of 8 was converted into a nitrile function upon reaction with
NH3 in the presence of iodine. The attachment of a third tertiary
amine moiety was performed by reductive amination of the
aldehyde 8 with secondary amines to give the respective bis-
aminomethyl azulenes 11–16. Alternatively, the dimethylamino-
methyl derivative 11 could be synthesized by treatment of FAUC
3019 with Eschenmoser’s salt.
On the course of our investigations on subtype selective dopa-
mine receptor agonists and antagonists,18–20 we reported on the
piperazinylmethyl substituted azulene FAUC 3019 showing subn-
anomolar affinity and partial agonism at the D4 subtype.21 FAUC
3019 revealed a strong pro-erectile effect in rats superior to the
in vivo efficacy of apomorphine. The effect could be prevented in
a dose-dependent manner by L-745,870,22 a highly selective D4
receptor antagonist. The rationale for the incorporation of the quite
uncommon azulene moiety has been based on its non-uniform
charge distribution with a significant negative molecular electro-
static field (MEP) below and above the five-membered ring and a
positive MEP map at the seven-membered ring. The large dipole
moment of azulenes accounts not only for an intense charge-
transfer absorption in the visible region and the tendency to
undergo electrophilic aromatic substitution at the five-membered
ring, but also leads to receptor recognition properties similar to
that of the pyrazolo[1,5-a]pyridine nucleus which has proved to
be a potent recognition element in dopaminergic D4 ligands.23–25
To further validate scope and limitations of the azulene scaffold
for the design of potent dopamine D4 partial agonists, we herein
present the synthesis and SAR investigations on a series of FAUC
3019 derivatives bearing additional substituents in position 3 of
the azulene moiety.
Receptor binding experiments were established to evaluate
the binding properties of the azulene derivatives 4–6 and 9–16 to
the most relevant monoaminergic GPCRs. D1 receptor affinities
were determined utilizing porcine striatal membranes and the D1
selective radioligand [3H]SCH23390.27 D2long, D2short, D3, and D4
receptor affinities were investigated employing the cloned human
dopamine receptor subtypes D2long, D2short,
28 D3,29 and D4.430 stably
expressed in Chinese hamster ovary cells (CHO) and the radioligand
[3H]spiperone.27 Affinities to further aminergic GPCRs were deter-
mined to check the selectivities over connatural receptor families.
Therefore, binding affinities to the porcine serotonin receptors
5-HT1A, 5-HT2 and the adrenoceptors a1, a2 as well as to the human
histamine receptor subtypes H1, H2 H3, H4 were determined
utilizing the radioligands [3H]WAY100,635, [3H]ketanserin, [3H]
a
Our initial chemical investigations were directed to the introduc-
tion of a bromo or iodo substituent into the electron rich position 3 of
the azulene ring system. Thus, FAUC 3019 was converted to the
halogenated derivatives 1 and 2 in 52% and 49% yield, respectively
(Scheme 1). The iodo azulene 2 was employed as a synthetic
intermediatefor an introduction ofethynyl substituents. Thus, Sono-
gashira cross-coupling with TMS-acetylene, phenylacetylene and 1-
pentyne led to the test compounds 3, 5 and 6. The trimethylsilyl
group of 3 could be easily removed with tetrabutylammonium
prazosin, [3H]RX821002, [3H]mepyramine, [9H]UR-DE257, [3H]N -
methylhistamine and [3H]UR-PI294, respectively. The binding data
was analyzed according to a sigmoid model by nonlinear regression.
The competition experiments clearly showed that the introduc-
tion of an alkyne side chain to the five-membered ring of the
azulene core results in a decrease of dopamine D4 receptor binding
when compared to FAUC 3019 (Table 1). Thus, Ki values of approx-
imately 10–20 nM were determined for the Sonogashira products 4,
5 and 6, whereas the affinity for D2long and D2short was more or less
N
N
N
N
b
a
FAUC 3019
MeO
MeO
: R= TMS
: R= H
1: X= Br
2: X= I
3
4
5
6
X
c
: R= Ph
: R= C H
R
3
7
CHO
N
N
N
N
d
e
or f
MeO
MeO
: R= CH OH
: R= CN2
9
8
CHO
CHO
R
10
7
g
g
N
N
N
N
h
FAUC 3019
MeO
MeO
R
X
N
N
: X= N(CH )
11
15: R= 2-OMe
16: X= 4-Cl
: X= piperi3din-1-yl
2
12
13
14
: X= morpholin-4-yl
: X= 4-methylpiperazin-1-yl
Scheme 1. Reagents and conditions: (a) NBS or NIS, benzene, 5 °C, 3 h; (b) R-CCH, CuI, Pd(PPh3)2Cl2, THF, rt, 16 h; (c) TBAF, THF, 0 °C, 1 h; (d) 4-(2-methoxyphenyl)piperazine,
Na(OAc)3BH, CH2Cl2, rt, 1 h; (e) NaBH4, THF, rt, 1.5 h; (f) NH4OH, I2, H2O, THF, rt, 16 h; (g) secondary amine, Na(OAc)3BH, CH2Cl2, rt, 16 h; (h) CH2@N(CH3)2I, CH2Cl2, rt, 4 h.

S. Löber et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7151–7154
7153
Table 1
Receptor binding data of compounds 4–6, 9–16 and FAUC 3019 at the porcine dopamine D1, the human dopamine D2long, D2short, D3 and D4.4, the porcine 5-HT1A and 5-HT2, the
procine adrenergic a1 and a2 and the human histamine H1, H2, H3 and H4 receptors
Compound
Ki Valuesa [nM]
b
b
b
b
pD1
hD2long
hD2short
hD3
hD4.4
p5-HT1A
p5-HT2
p
a1
p
a2
hH1
hH2
hH3
hH4
4
5
6
9
10
11
12
13
6400
1300
1400
860
95
65
76
150
8.3
210
51
130
60
76
67
60
68
39
140
37
92
57
34
160
24
370
210
370
580
530
390
160
350
460
33
9.5
16
19
680
770
410
120
110
110
65
99
91
100
760
54
2100
1000
1300
990
230
370
210
57
31
33
32
57
66
8.3
27
n.d.
n.d.
n.d.
44
n.d.
23
140
220
170
120
1200
39
n.d.
n.d.
n.d.
1400
n.d.
2600
5500
6500
>10,000
1000
490
n.d.
n.d.
n.d.
>10,000
n.d.
3200
4200
6100
5500
530
n.d.
n.d.
n.d.
>10,000
n.d.
>10,000
>10,000
>10,000
5500
>10,000
>10,000
n.d.
n.d.
n.d.
n.d.
>10,000
n.d.
>10,000
>10,000
>10,000
>10,000
>10,000
>1000
n.d.
0.71
2.4
1.5
1.9
4.6
3.8
0.79
6.0
0.40
410
380
4500
4100
5000
6400
740
1800
1300
1000
2100
560
14
15
16
17
95
33
1100
500
85
83
1600
92
460
n.d.
FAUC 3019
20
n.d.
n.d. = not determined.
a
Ki Values in nM are based on the means of 2–11 experiments each done in triplicate.
Ki Values in nM are based on one orientating experiment done in triplicate.
b
unchanged. Substitution of a hydroxymethyl group in position 3 of
the azulene moiety led to the ligand 9 displaying D4 ligand affinity
that was similar to the lead compound FAUC 3019. Significant
binding to the adrenoreceptors a1 and a2 (Ki: 57 nM and 44 nM,
Table 2
Intrinsic activities of the test compounds 9, 11, 15, the reference compounds FAUC
3019 and quinpirole obtained from the stimulating effect on mitogenesis of D4
receptor expressing cells
Compound
[3H]thymidine incorporation (mitogenesis)
respectively) could be observed while selectivity over 5-HT1A
,
5-HT2 and the histamine receptors H1–H4 was very high. The cyano
derivative 10 showed Ki values in the single digit nanomolar range
for D4 and D2short (Ki: 2.4 nM and 8.3 nM, respectively). Attachment
of an aminomethyl substituent appeared to be an effective strategy
to obtain suitable D4 ligands with pronounced selectivity over
related GPCR subtypes. Thus, the tertiary amines 11–14 showed
Ki between 1.5 and 4.6 nM for D4, when the dimethylaminomethyl
derivative 11 displayed the best selectivity over D1, D2 and D3.
Moreover, the selectivity of 11 over the hallucinogenic anti-target
5-HT2 (1200-fold) was superior when compared to the reference
compound FAUC 3019 (230-fold). Interestingly, the symmetric
bis-(2-methoxyphenylpiperazinyl methyl) substituted azulene 15
exhibited subnanomolar D4 affinity (0.79 nM) as well as strong
a1 (8.3 nM) affinity and moderate H2 binding (550 nM). The
respective 2-chlorophenypiperazine 16 showed significantly
weaker affinities to all tested aminergic GPCRs, but displayed the
highest affinities to H1 (490 nM) and H2 (460 nM) within the
tested group. Due to compound instability, no reliable receptor
binding data could be determined for the halogenated derivatives
1 and 2.
Agonist effecta
EC50 (nM)
b
9
11
15
41%
45%
37%
39%
100%
0.94
0.41
0.98
1.2
FAUC 3019
Quinpirole
1.4
Rate of incorporation of [3H]thymidine as evidence for mitogenesis activity
relative to the maximal effect of the full agonist quinpirole (=100%) used as a
reference.
a
b
EC50 values derived from a mean curve of 4–8 independent experiments each
done in hexaplicates.
To evaluate the efficacy of our D4 ligands, intrinsic activities of
the most promising target compounds were determined in vitro in
a mitogenesis assay by measuring the rate of [3H]thymidine incor-
poration into growing CHO cells stably expressing the human D4.2
receptor (Table 2). In comparison with the reference agonist quin-
pirole (efficacy = 100%), the test compounds 9, 11 and 15 showed
partial agonist activity with ligand efficacies of 41%, 45% and
37%, respectively, which is similar to FAUC 3019 (39%). However,
the dimethylamine 11 displayed D4 potency (EC50 = 0.41 nM).
Thus, the D4 partial agonist 11 was chosen to be investigated for
its ability to promote penile erection in an animal model.
As depicted in Figure 2, the test compound 11 proved to induce
penile erection in male rats after both systemic (subcutaneous, sc)
administration and injection into the paraventricular nucleus
(PVN). The determined minimal effective dose has been 5 lg/kg
sc and 5 ng when injected into the PVN, respectively. When
Figure 2. (A) Effect of 11 injected sc (left) or into the PVN (right) on penile erections
in male rats: dose-response curves in comparison with apomorphine. Values are
means SEM of 8 and 6 rats per group for sc and PVN treatment, respectively. (B)
Effect of the D4 antagonist L-745,870 given sc (1 mg/kg) (left) or into the PVN (1 lg)
compared to the reference agent apomorphine, the azulene 11
showed superior potency in concentrations up to 25 lg/kg (sc) or
25 ng/rat (into PVN), respectively. This is a substantial improve-
ment when compared to FAUC 3019, which required a higher dose
than apomorphine to reach the same efficacy.21 The penile erection
(right) on penile erections induced by 11 given sc (0.025 mg/kg) (left) or into the
PVN (10 ng) (right). Values are means SEM of 10 and 8 rats per group for systemic
and PVN treatments, respectively. ⁄P <0.01 with respect to vehicle-treated rats; #P
<0.01 with respect to the corresponding group of vehicle + 11-treated rats (One-
Way ANOVA followed by Tukey’s multicomparison test).

7154
S. Löber et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7151–7154
7. Andersson, K. E. Pharmacol. Rev. 2001, 53, 417.
promoting effect of 11 reached 60–65% of the maximal response of
apomorphine. In order to prove that this effect is attributed to the
dopamine D4 receptor agonism of 11, the highly selective D4 antag-
onist L-745,870 was used to block the observed response. Accord-
ingly, administration of L-745,870 sc (1 mg/kg) or into the PVN
8. Bitran, D.; Hull, E. Neurosci. Biobehav. Rev. 1987, 11, 365.
9. Barros, H.; Braz, S.; Carlini, E. Pharmacology 1989, 38, 335.
10. Heaton, J. P.; Morales, A.; Adams, M. A.; Johnston, B.; el-Rashidy, R. Urology
1995, 45, 200.
11. Hsieh, G.; Hollingsworth, P.; Martino, B.; Chang, R.; Terranova, M.; O’Neill, A.;
Lynch, J.; Moreland, R.; Donnelly-Roberts, D.; Kolasa, T.; Mikusa, J. P.; McVey, J.
M.; Marsh, K. C.; Sullivan, J. P.; Brioni, J. D. J. Pharmacol. Exp. Ther. 2004, 308,
330.
(1 lg) antagonized almost completely the effect of 11 indicating
a D4 receptor mediated penile erection promoting effect.
In summary, we could demonstrate that the introduction of an
additional substituent into the azulene moiety of our D4 partial
agonist FAUC 3019 allows an effective tuning of affinity, subtype
selectivity and in vivo potency. The N,N-dimethylaminomethyl
substituted azulene 11 combines excellent D4 affinity and good
selectivity over a collection of relevant monaminergic GPCRs with
high ligand efficacy. Applying an in vivo model, 11 turned out to
stimulate penile erection in male rats with superior potency in
low concentrations when compared to apomorphine, whereas
the penile erection promoting effect of 11 reached 60–65% of the
maximal response of apomorphine. Compared to FAUC 3019,
which required a higher dose than apomorphine to reach the same
efficacy, the test compound 11 displayed higher potency than
apomorphine.
12. Sanna, F.; Succu, S.; Hubner, H.; Gmeiner, P.; Argiolas, A.; Melis, M. R. Behav.
Brain Res. 2011, 225, 169.
13. Brioni, J. D.; Moreland, R. B.; Cowart, M.; Hsieh, G. C.; Stewart, A. O.; Hedlund,
P.; Donnelly-Roberts, D. L.; Nakane, M.; Lynch, J. J., III; Kolasa, T.; Polakowski, J.
S.; Osinski, M. A.; Marsh, K.; Andersson, K.-E.; Sullivan, J. P. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 6758.
14. Cowart, M.; Latshaw, S. P.; Bhatia, P.; Daanen, J. F.; Rohde, J.; Nelson, S. L.; Patel,
M.; Kolasa, T.; Nakane, M.; Uchic, M. E.; Miller, L. N.; Terranova, M. A.; Chang,
R.; Donnelly-Roberts, D. L.; Namovic, M. T.; Hollingsworth, P. R.; Martino, B. R.;
Lynch, J. J., III; Hsieh, G. C.; Moreland, R. B.; Brioni, J. D.; Stewart, A. O. J. Med.
Chem. 2004, 47, 3853.
15. Patel, M. V.; Kolasa, T.; Mortell, K.; Matulenko, M. A.; Hakeem, A. A.; Rohde, J. J.;
Nelson, S. L.; Cowart, M. D.; Nakane, M.; Miller, L. N.; Uchic, M. E.; Terranova,
M. A.; el-Kouhen, O. F.; Donnelly-Roberts, D. L.; Namovic, M. T.; Hollingsworth,
P. R.; Chang, R.; Martino, B. R.; Wetter, J. M.; Marsh, K. C.; Martin, R.;
Darbyshire, J. F.; Gintant, G.; Hsieh, G. C.; Moreland, R. B.; Sullivan, J. P.; Brioni,
J. D.; Stewart, A. O. J. Med. Chem. 2006, 49, 7450.
16. Melis, M. R.; Succu, S.; Sanna, F.; Melis, T.; Mascia, M. S.; Enguehard-Gueiffier,
C.; Hübner, H.; Gmeiner, P.; Gueiffier, A.; Argiolas, A. Eur. J. Neurosci. 2006, 24,
2021.
17. Löber, S.; Hübner, H.; Tschammer, N.; Gmeiner, P. Trends Pharmacol. Sci. 2011,
32, 148.
Acknowledgment
18. Haubmann, C.; Huebner, H.; Gmeiner, P. Bioorg. Med. Chem. Lett. 1999, 9, 3143.
19. Haubmann, C.; Huebner, H.; Gmeiner, P. Bioorg. Med. Chem. Lett. 1999, 9, 1969.
20. Ehrlich, K.; Gotz, A.; Bollinger, S.; Tschammer, N.; Bettinetti, L.; Haerterich, S.;
Huebner, H.; Lanig, H.; Gmeiner, P. J. Med. Chem. 2009, 52, 4923.
21. Löber, S.; Tschammer, N.; Huebner, H.; Melis, M. R.; Argiolas, A.; Gmeiner, P.
ChemMedChem 2009, 4, 325.
This research was funded by
Forschungsgemeinschaft (DFG).
a Grant of the Deutsche
Supplementary data
22. Kulagowski, J. J.; Broughton, H. B.; Curtis, N. R.; Mawer, I. M.; Ridgill, M. P.;
Baker, R.; Emms, F.; Freedman, S. B.; Marwood, R. M.; Patel, S.; Patel, S.; Ragan,
C. I.; Leeson, P. D. J. Med. Chem. 1996, 39, 1941.
23. Löber, S.; Hübner, H.; Gmeiner, P. Bioorg. Med. Chem. Lett. 1999, 9, 97.
24. Löber, S.; Hübner, H.; Utz, W.; Gmeiner, P. J. Med. Chem. 2001, 44, 2691.
25. Löber, S.; Aboul-Fadl, T.; Hübner, H.; Gmeiner, P. Bioorg. Med. Chem. Lett. 2002,
12, 633.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.09.
064.
References and notes
26. Hafner, B. Justus Liebigs Ann. Chem. 1959, 92, 108.
27. Hübner, H.; Haubmann, C.; Utz, W.; Gmeiner, P. J. Med. Chem. 2000, 43, 756.
28. Hayes, G.; Biden, T. J.; Selbie, L. A.; Shine, J. Mol. Endocrinol. 1992, 6, 920.
29. Sokoloff, P.; Andrieux, M.; Besancon, R.; Pilon, C.; Martres, M.-P.; Giros, B.;
Schwartz, J.-C. Eur. J. Pharmacol. 1992, 225, 331.
30. Asghari, V.; Sanyal, S.; Buchwaldt, S.; Paterson, A.; Jovanovic, V.; Van Tol, H. H.
M. J. Neurochem. 1995, 65, 1157.
1. Dean, R. C.; Lue, T. F. Urol. Clin. North Am. 2005, 32, 379.
2. Aytac, I. A.; McKinlay, J. B.; Krane, R. J. Br. J. Urol. 1999, 84, 50.
3. Lue, T. F. New Engl. J. Med. 1802, 2000, 342.
4. Virag, R. Lancet 1982, 2, 938.
5. Zentgraf, M.; Ludwig, G.; Ziegler, M. Eur. Urol. 1989, 16, 165.
6. Gresser, U.; Gleiter, C. H. Eur. J. Med. Res. 2002, 7, 435.