Bioisosteric phentolamine analogs as potent α-adrenergic antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.07.083

The synthesis and biological evaluation of a new series of bioisosteric phentolamine analogs are described. Replacement of the carbon next to the imidazoline ring of phentolamine with a nitrogen atom provides compounds (2, 3) that are about 1.6 times and 4.1 times more potent functionally than phentolamine on rat α₁-adrenergic receptors, respectively. In receptor binding assays, the affinities of phentolamine and its bioisosteric analogs were determined on the human embryonic kidney (HEK) and Chinese Hamster ovary (CHO) cell lines expressing the human α₁- and α₂-AR subtypes, respectively. Analogs 2 and 3, both, displayed higher binding affinities at the α₂- versus the α₁-ARs, affinities being the least at the α_1B- AR. Binding affinities of the methoxy ether analog 2 were greater than those of the phenolic analog 3 at all six α-AR subtypes. One of the nitrogen atoms in the imidazoline ring of phentolamine was replaced with an oxygen atom to give compounds 4 and 5, resulting in a 2-substituted oxazoline ring. The low functional antagonist activity on rat aorta, and binding potencies of these two compounds on human α_1A- and α_2A-AR subtypes indicate that a basic functional group is important for optimum binding to the α₁- and α_2A-adrenergic receptors.

Full text of DOI:10.1016/j.bmcl.2005.07.083

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4691–4695
Bioisosteric phentolamine analogs as potent
a-adrenergic antagonists
Seoung-Soo Hong,a,* Supriya A. Bavadekar,b Sang-Il Lee,c Popat. N. Patil,d
S. G. Lalchandani,b Dennis R. Fellerb and Duane D. Millere
aDepartment of Manufacturing Pharmacy, College of Pharmacy, Chungbuk National University, Cheongju,
Chungbuk 360-763, Republic of Korea
bDepartment of Pharmacology and National Center for Natural Products Research, School of Pharmacy,
University of Mississippi, University, MS 38677, USA
cDepartment of Food Science, Keimyung College, Taegu 705-037, Republic of Korea
dDivision of Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
eDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis, TN 38163, USA
Received 7 June 2005; revised 28 July 2005; accepted 28 July 2005
Available online 8 September 2005
Abstract—The synthesis and biological evaluation of a new series of bioisosteric phentolamine analogs are described. Replacement
of the carbon next to the imidazoline ring of phentolamine with a nitrogen atom provides compounds (2, 3) that are about 1.6 times
and 4.1 times more potent functionally than phentolamine on rat a1-adrenergic receptors, respectively. In receptor binding assays,
the affinities of phentolamine and its bioisosteric analogs were determined on the human embryonic kidney (HEK) and Chinese
Hamster ovary (CHO) cell lines expressing the human a1- and a2-AR subtypes, respectively. Analogs 2 and 3, both, displayed higher
binding affinities at the a2- versus the a1-ARs, affinities being the least at the a1B-AR. Binding affinities of the methoxy ether analog
2 were greater than those of the phenolic analog 3 at all six a-AR subtypes. One of the nitrogen atoms in the imidazoline ring of
phentolamine was replaced with an oxygen atom to give compounds 4 and 5, resulting in a 2-substituted oxazoline ring. The low
functional antagonist activity on rat aorta, and binding potencies of these two compounds on human a1A- and a2A-AR subtypes
indicate that a basic functional group is important for optimum binding to the a1- and a2A-adrenergic receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
The initial classification of adrenoceptors (AR) into al-
pha (a-AR) and beta (b-AR) was described by Ahlquist
in 1948 on the basis of their pharmacological character-
istics.1 Cloning and expression of a-AR have confirmed
the presence of multiple subtypes of both a1-(a1a, a1b,
a1d) and a2-(a2a, a2b, a2c)AR.2–5 The membrane-span-
ning protein subunits among the a-AR family contain
seven putative transmembrane helices, and a-ARs
are members of the G-protein-coupled superfamily of
receptors.6
for the diagnosis of pheochromocytoma, and (c) for nor-
epinephrine-related dermal necrosis.8,9 Phentolamine is
not useful in the treatment of systemic hypertension or
in patients with myocardial infarctions since it induces
cardiac stimulation. This side-effect is thought to be
caused by the presynaptic a2-AR blockade, which re-
sults in an enhanced neuronal release of norepinephrine.
The released norepinephrine activates adrenergic recep-
tors in the heart, producing cardiac stimulation.8
Recently, phentolamine mesylate (VasomaxÒ) has been
marketed in Mexico for use in the treatment of male
erectile dysfunction (ED).6,10 The drug induces relaxa-
tion of corpus cavernosum erectile tissue by direct
antagonism of a1- and a2-adrenergic receptors and by
indirect functional antagonism via a non-adrenergic,
endothelium-mediated mechanism, suggesting nitric
oxide synthase activation.11,12 A better understanding
of the structural requirements for the subtypes of
a-adrenergic receptors could result in the design and
Since phentolamine (1), a non-selective a-AR antago-
nist, was discovered by Urech et al.,7 this drug has been
used in a number of clinical situations: (a) for the treat-
ment of pheochromocytoma-related hypertension, (b)
*
Corresponding author. Tel.: +1 9 01 52 39 7 00; fax: +1 9 01 52 39
772; e-mail: shong@gtxinc.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.07.083

4692
S.-S. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4691–4695
H3C
H3C
H3C
N
OR
N
OR
N
OH
HN
HN
N
N
N
HN
O
HN
R = CH3 (4), H (5)
R = CH3 (2), H (3)
Phentolamine (1)
synthesis of more selective drugs for treating erectile
dysfunction and a number of other diseases, including
nasal decongestion, hypertension, hypotension, hyper-
glycemia, depression, liver cell proliferation, hyperag-
gregability of platelets, and benign prostatic
hyperplasia (BPH).
nitrogen atoms in the imidazoline ring of phentolamine
was replaced with an oxygen atom to give the corre-
sponding oxazoline analogs (4, 5). The pharmacological
properties of phentolamine (1) and newly synthesized
bioisosteric analogs (2–5) were compared for their func-
tional activities on rat thoracic aorta and for binding
potencies on human a1- and a2-AR subtypes expressed
in HEK and CHO cells, respectively.
In a previous paper,13 we reported the preparation of
geometrical isomers by replacing the nitrogen atom with
a carbon at the benzylic position of phentolamine; the
ethylene analog having the Z configuration was less po-
tent than phentolamine, but the a1-AR selectivity was
considerably increased. Our next goal was to see if bio-
isosteric analogs (2, 3) of phentolamine, in which the
carbon atom bridging the 2-position of the imidazoline
ring with the diphenylamine nitrogen is replaced by
nitrogen, might bind to a-ARs with an enhanced affini-
ty. As an additional test modification, intended to par-
tially evaluate the importance of basicity, one of the
The synthesis of imidazoline derivatives 2 and 3 is
shown in Scheme 1. The hydrazine 9 was the key
intermediate in the synthesis of all of the target com-
pounds (Schemes 1 and 2). This hydrazine was synthe-
sized with the initial condensation of p-toluidine and
resorcinol in the presence of a catalytic amount of
p-toluidine HCl to give diphenylamine 6 in 74% yield.14
N-Amination of 6 was not successful with hydroxyl-
amine-O-sulfonic acid. Diphenylamine 6 in ethanol
was treated with concentrated HCl, followed by the
H3C
H3C
OH
CH3
NH2
a
b
N NO
OH
NH
OH
+
OH
6
7
H3C
H3C
e
c
d
N NO
OCH3
N NH2
.
HCl
OCH3
9
8
H3C
H3C
N
N
f
N NH
N NH
N
H
N
H
.
(COOH)2
.
OH
3
HCl
OCH3
2
Scheme 1. Reagents and conditions: (a) heat at 160 °C; (b) NaNO2, concd HCl; (c) dimethyl sulfate, K2CO3; (d) 1—LiAlH4/Et2O, 2—HCl;
(e) 1—N-acetyl imidazolinone, POCl3, 2—oxalic acid; (f) 1—satd NaHCO3, 2—BBr3, 3—satd NaHCO3, 4—HCl.

S.-S. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4691–4695
4693
H3C
H3C
a
b
N NO
OCH3
N NH2
OCH3
H3C
8
H3C
O
d
c
N NH
OCH3
N NH
O
NH (CH2)2Cl
N
OCH3
10
4
H3C
N NH
O
N
OH
5
Scheme 2. Reagents and conditions: (a) LiAlH4/Et2O; (b) Cl(CH2)2NCO; (c) CH3OH/H2O, reflux; (d) BBr3.
addition of a cold solution of sodium nitrite in water,
to give nitrosamine 7 in 73% yield,15 and this interme-
diate was then treated with dimethyl sulfate in acetone
in the presence of K2CO3 to provide the desired meth-
oxy compound 8 in 91% yield. In earlier attempts,
treatment of N-nitrosamine 8 with Zn in the presence
of acetic acid gave extensive decomposition (N–N bond
cleavage) and the yield was very poor. The N-nitroso
group was more successfully reduced by LiAlH4 in
diethyl ether; the optimum reaction time was 5 h be-
cause slow N–N bond cleavage became significant
thereafter. The hydrazine free base readily decom-
posed, which could not be purified by flash chromato-
graphy, so it was directly converted to the HCl salt
(70% yield from nitrosamine 8) using excess HCl gas.
The hydrazine 9 was treated with N-acetyl-2-imidazoli-
none16 in the presence of POCl3, followed by base
workup, to give imidazoline 2 in 69% yield, obtained
as the oxalic acid salt. The methoxy-protecting group
of 2 in anhydrous methylene chloride was deprotected
by BBr3, and base workup, followed by conversion to
the hydrochloride salt, gave 3 in 69% yield.
yield. The urea 10 was heated in aqueous MeOH solu-
tion to give a cyclized compound, oxazoline 4, in 40%
yield. This methyl ether group was also cleaved with
BBr3, and subsequent base workup gave phenol 5 in
47% yield.
Radioligand binding analyses of phentolamine and its
bioisosteric analogs were performed in CHO and
HEK293 cells stably expressing homogeneous popula-
tions of human a2A-, a2B-, and a2C-ARs, and human
a1A-, a1B-, and a1D-ARs, respectively. Initial studies
were conducted on human a1A- and a2A-AR subtypes,
and the rank order of affinities of these compounds (Ki
values) was phentolamine, 1 (4.1 nM) > 2 (20 nM) > 3
(27 nM) >> 5 (5620 nM) = 4 (5740 nM), and
2
(6.3 nM) > 3 (24 nM) > phentolamine, 1 (56 nM) >> 5
(18,600 nM) > 4 (24,600 nM), respectively (Table 1).
These data revealed that the parent compound, phentol-
amine, had a 14-fold higher binding affinity at the a1A
-
versus the a2A-AR. Bioisosteric analogs of phentolamine
in which the original imidazoline ring was retained (2
and 3), however, exhibited a different binding pattern.
When compared to phentolamine (1), both the analogs
Scheme 2 outlines the synthesis of oxazoline deriva-
tives 4 and 5. N-Nitrosamine 8 in diethyl ether was
treated with LiAlH4 as described above. The resulting
mixture of the hydrazine and N-nitrosamine was
directly treated with 2-chloroethylisocyanate in tetra-
hydrofuran to provide chloroethylurea 10 in 67%
(2, 3) showed decreased binding potencies at the a1A-
AR, whereas the binding potencies at the a2A-AR im-
proved slightly. The a1A- affinity decreases were 5- and
7-fold, while the a2A- affinity increases were 9- and 2-
fold for analogs 2 and 3, respectively. Analog 2 showed
a 3-fold greater binding affinity at the a2A- versus the

4694
S.-S. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4691–4695
Table 1. Binding affinities of phentolamine (1), bioisosteric analogs (2,
3) and 2-substituted oxazoline ring analogs (4, 5) on human a1- and a2-
AR subtypes stably expressed in human embryonic kidney (HEK293)
and Chinese Hamster ovary (CHO) cells, respectivelya,b,c
showed much lower binding affinities at both the a1A
-
as well as the a2A-AR, indicating that replacement of
one of the nitrogen atoms of the imidazoline ring by
oxygen is detrimental for a-AR binding. This likely sug-
gests that the basicity of this ring system is important for
binding to the a-ARs.
Compound
a1A
a1B
a1D
Ki SEMb,c
(nM)
Ki SEMb
(nM)
Ki SEMb
(nM)
With these results, we extended the evaluation of phen-
tolamine 1 and bioisosteric analogs 2, 3 to all six a-AR
subtypes (Table 1). The rank order of binding affinities
exhibited by phentolamine at human a-ARs was
1
2
3
4.12 0.49
19.9 1.6
26.8 1.7
47.4 4.4
102.8 27.3
843 220.1
19.4 4.2
17.5 1.5
49.7 6.8
a2A
a2B
a2C
a
1A > a1D > a1B = a2A = a2B P a2C. Analogs 2 and 3,
1
2
3
55.9 3.6
6.3 0.2
23.8 3.7
48.0 5.8
7.3 0.6
18.0 2.1
80.3 19.0
3.8 0.4
12.8 1.0
both, displayed higher binding affinities at the a2- versus
the a1-ARs, affinities being the least at the a1B-AR.
Binding affinities of the methoxy ether analog 2 were
greater than those of the phenolic analog 3 at all six
a-AR subtypes.
a [3H]Prazosin and [3H]rauwolscine were used as the radioligands in the
equilibrium competition radioligand binding assays for the a1- and
a2-ARs, respectively, and the nonspecific binding was measured in
the presence of 10 lM of phentolamine or yohimbine.
The parent compound, phentolamine (1), and its bio-
isosteric analogs (2–5) were also tested for functional
activities on rat aorta. Responses in the rat aorta have
b Ki was calculated according to the Cheng–Prusoff equation17 and the
data for all analogs are means SEM of N = 4–6.
c The oxazoline ring analogs were evaluated for binding only in human
been known to be mediated predominantly by the a1D
-
a
1A- and a2A-AR subtypes and the mean pKi SEM (Ki, nM) values
AR.20 All test compounds blocked the phenylephrine-
induced contractions of rat aorta and produced parallel
shifts in the concentration-response curve of the a-AR
agonist, phenylephrine. Table 2 shows the potency of
the test compounds expressed as their KB values. As
compared to phentolamine (KB, 11.8 nM), analog 3
was 4.1 times more potent blocker of the vascular
for analogs 4 and 5 on human a1A- and a2A-ARs are 5.24 0.05
(5740 nM) and 4.61 0.17 (24,600 nM), and 5.25 0.04 (5620 nM)
and 4.73 0.04 (18,600 nM), respectively.
Table 2. Functional activities of phentolamine (1) and bioisosteric
analogs (2–5) as assessed by the antagonism of phenylephrine-induced
contractions of rat aorta
response (KB, 2.9 nM). Methoxy analog
2 (KB,
Compound
Rat aorta (a1D-AR18–20
)
7.4 nM) was also more potent than phentolamine. As
observed in our binding studies, oxazoline analogs 4
(KB, >30,000 nM) and 5 (KB, 13,000 nM) were much
less potent than phentolamine or analogs 2 and 3;
and these findings suggest that the basicity of this ring
system is important for binding to a1-adrenoceptors.
However, the binding and functional data for analogs
2 and 3 at cloned human a1D and rat aorta a1D are
not in complete agreement. Our binding studies suggest
that analog 2 binds with a 2.8-fold higher affinity than
analog 3 at cloned human a1D-AR (Table 1), whereas
the functional potency of analog 3 is 2.6-fold higher
than that of analog 2 (Table 2). A possible explanation
for this discrepancy is that the organization of cloned
receptors in host cell lines such as the one used for
determining binding affinities in the present case may
be different from those of native receptors in integrated
tissue systems, such as the rat aorta, used for our func-
tional assays. However, there may exist other possibil-
ities leading to this disparity (see Table 3).
KB (nM) 95% CLa
Potency ratiob
1
2
3
4
5
11.8 0.8
7.4 0.6
2.9 0.7
1.0
1.6
4.1
—
>30,000
13,000 0.8
—
a The KB value for each compound, as determined by rightward shifts
in the agonist (phenylephrine) concentration–response curves, was
calculated according to the Schild equation:21 KB = antagonist con-
centration/dose ratio-1 where dose ratio is the ratio of the EC50 for
phenylephrine in the presence and absence of the antagonist. Data
were analyzed using GraphPad Prism software [GraphPad Prism,
San Diego, CA] to obtain EC50 values and are expressed as
means 95% confidence limits (CL) of N = 4–6 experiments.
b Potency ratio = KB(phentolamine, 1)/KB(bioisosteric analog).
a
1A-AR, whereas analog 3 was equipotent in binding to
the a1A- and a2A-AR. Bioisosteric analogs of phentol-
amine containing an oxazoline ring, analogs 4 and 5,
Table 3. Elemental analyses
Compound
Calculated (%)
H
Formula
Found (%)
H
C
N
C
N
7
68.41
63.51
57.71
60.28
61.17
64.74
62.84
5.30
6.47
5.86
6.01
6.04
6.71
6.43
12.27
10.58
14.17
17.57
12.59
13.32
13.73
C
13H12N2O2
C14H16N2OÆHCl
17H20N4OÆ(COOH)2Æ0.5H2O
C16H18N4OÆHCl
17H20N3O2Cl
C17H19N3 O2Æ1.0H2O
16H17N3 O2Æ1.25H2O
68.32
63.31
58.03
60.17
61.15
64.35
62.85
5.30
6.45
5.70
6.04
6.04
6.71
6.38
12.24
10.51
14.01
17.31
12.51
13.25
13.68
9
2
C
3
10
4
C
5
C

S.-S. Hong et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4691–4695
4695
7. Urech, E.; Marxer, A.; Miescher, K. Helv. Chim. Acta
1950, 33, 1386.
8. Hoffman, B. B.; Lefkowitz, R. J. N. Eng. J. Med. 1980,
302, 1390.
9. Hoffman, B.. In Katzung, B. G., Ed., 3rd ed.; Basic and
Clinical Pharmacology; Appleton Lange: Norwalk, 1987,
pp 95–104.
10. Zorgnioti, A. W. Int. J. Impot. Res. 1994, 6, 37.
11. Traish, A.; Gupta, S.; Gallant, C.; Huang, Y-H.; Gold-
stein, I. Int. J. Impot. Res. 1998, 10, 215.
12. Silva, L. F.; Nascimento, N. R.; Fonteles, M. C.; Moraes,
M. E.; Vasconcelos, P. R.; Moraes, M. O. Int. J. Impot.
Res. 2005, 17, 27.
Overall, these results indicate that replacement of the
carbon bridging the 2-position of the imidazoline ring
with the diphenylamine nitrogen by a nitrogen atom as
in compounds (2 and 3) maintains or modestly increases
the activity on selected a-AR subtypes. Replacing one of
the nitrogen atoms of the imidazoline ring of phentol-
amine with an oxygen atom drastically reduces activity.
Compounds 2 and 3 serve as promising lead compounds
for further investigations.
Acknowledgments
13. Hong, S-S.; Lee, H.; Miller, D. D. Med. Chem. Res. 1997,
7, 53.
This research was financially supported in part by
RESEARCH FUND offered by Chungbuk National
University Development Foundation 2000. The authors
also wish to thank the National Center for Natural
Products Research (USDA ARS Agreement No. 58-
6408-2-009) at the University of Mississippi, School of
Pharmacy, for the partial support of this research.
14. Hatsohek, A.; Zega, A. J. Prakt. Chem. 1886, 209.
15. Vogelꢀs Textbook of Practical Organic Chemistry; Furnies,
B. S., Hannaford, A. J., Smith, P. W. G., Tatchell, A. R.,
Eds., 5th ed.; Longman Scientific & Technical: London,
1989; pp 962–963.
16. Hall, H. K., Jr.; Schneider, A. K. J. Am. Chem. Soc. 1958,
80, 6409.
17. Cheng, Y.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22,
3099.
References and notes
18. Piascik, MT.; Guarino, R. D.; Smith, M. S.; Soltis, E. E.;
Saussy, D. L.; Perez, D. M. J. Pharmacol. Exp. Ther. 1995,
275.
19. Lamba-Kanwal, V. K.; Hamada, A.; Adejare, A.; Clark,
M. T.; Miller, D. D.; Patil, P. N. J. Pharmacol. Exp. Ther.
1988, 245, 793.
1. Ahlquist, R. P. Am. J. Physiol. 1948, 153, 586.
2. Michel, M. C.; Kenney, B.; Schwinn, D. A. Naunyn-
Schmiedebergs Arch. Pharmacol. 1995, 352, 1.
3. Ford, A. P. D. W.; Williams, T. J.; Blue, D. R.; Clarke,
D. E. Trends Pharmacol. Sci. 1994, 15, 167.
20. Venkataraman, B. V.; Shams, G.; Hamada, A.; Amem-
iya, Y.; Tantishaiyakul, V.; Hsu, F.; Fashempour, J.;
Romstedt, K. J.; Miller, D. D.; Feller, D. R.; Patil, P.
N. Naunyn-Schmiedebergs Arch. Pharmacol. 1991, 344,
454.
4. Bylund, D. B. Trends Pharmacol. Sci. 1988, 9, 356.
5. Buscher, R.; Herrmann, V.; Insel, P. A. Trends Pharmacol.
Sci. 1999, 20, 94.
6. Hrometz, S. A.; Edelmann, S. E.; McCune, D. F.;
Olges, J. R.; Hadley, D. M.; Perez, D. M.; Piascik, M.
T. J. Pharmacol. Exp. Ther. 1995, 290, 452.
21. Schild, H. O. Br. J. Pharmacol. 1947, 2, 189.