Synthesis and biological studies of yohimbine derivatives on human α2C-adrenergic receptors

Article abstract of DOI:10.1016/j.bmcl.2005.03.116

A series of yohimbine derivatives was synthesized and evaluated for binding affinity at the human α_2C-adrenergic receptors expressed in Chinese hamster ovary cells. It has been found that compound 5 shows a higher affinity for α_2C-AR than the parent compound yohimbine 1, thereby illustrating that the nature of the linkers affect binding potencies on these receptors.

Full text of DOI:10.1016/j.bmcl.2005.03.116

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2758–2760
Synthesis and biological studies of yohimbine derivatives on
human a2C-adrenergic receptors
Suni M. Mustafa,a Supriya A. Bavadekar,b Guoyi Ma,b Bob M. Moore,a
Dennis R. Fellerb and Duane D. Millera,*
aDepartment of Pharmaceutical Sciences, University of Tennessee, Health Science Center, 847 Monroe Avenue, Memphis,
TN 38163, USA
bDepartment of Pharmacology and National Center for Natural Products Research, School of Pharmacy,
University of Mississippi, Oxford, MS 38677, USA
Received 19 January 2005; revised 29 March 2005; accepted 30 March 2005
Available online 4 May 2005
Abstract—A series of yohimbine derivatives was synthesized and evaluated for binding affinity at the human a2C-adrenergic recep-
tors expressed in Chinese hamster ovary cells. It has been found that compound 5 shows a higher affinity for a2C-AR than the parent
compound yohimbine 1, thereby illustrating that the nature of the linkers affect binding potencies on these receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
Adrenoreceptors (ARs) are membrane proteins belong-
ing to the superfamily of G-protein-coupled receptors.1
With the aid of pharmacological and molecular biolog-
ical techniques, the a-adrenoceptor subtypes a1 and a2
were determined. Detailed studies have shown that these
initial subtypes were further divided into a1A, a1B, and
introduced recently based on the concept that a bivalent
ligand should first undergo univalent binding, followed
by the binding of the second pharmacophore to a recog-
nition site on a neighboring receptor.7 Using this
approach we prepared several yohimbine dimers with
methylene and methylene–diglycine spacer linkages
and it has been found that such compounds with spacers
of n = 3 (2) and n = 24 (3) showed the highest potency
and selectivity for the a2C-AR in receptor binding and
in functional studies measuring cAMP changes using a
cell based luciferase reporter gene assay.8 Since the func-
tional roles for the a2-adrenoceptor subtypes need to be
explored; yohimbine monomeric analogs were synthe-
sized and evaluated for their binding affinity on human
a1D; and a2A, a2B, a2C, and a2D subtypes, depending
on species and tissues.2 This knowledge has led to a
search for selective agonist and antagonists for each sub-
type. Although there are a number of a2-AR antago-
nists,3 only a small set of compounds have shown a
degree of selectivity among the three subtypes of a2-
AR. However, these compounds suffer from either low
subtype selectivity or binding to receptor sites outside
the a2-AR subfamily.3a,4 The importance of a2C-AR
antagonists in treating RaynaudÕs disease was illustrated
by Flavahan et al.5 The most common probes used in
these studies are agonists such as clonidine, and antago-
nists such as yohimbine and yohimbine like compounds
viz rauwolscine, corynanthine.
a
2C-AR with a goal of finding suitable pharmacological
probes and potential therapeutic agents. The signifi-
cance of functional groups in compounds affecting selec-
tivity and affinity in a2-AR system were detailed recently
by Pigini et al.9 The objective of our investigation was to
find whether a second pharmacophore is essential for
binding and how the nature of the linkers affect binding
potencies on these receptors.
Yohimbine 1 is known to be a potent and selective a2-
AR antagonist, and has been used extensively as a phar-
macological probe for studying the a2-AR.6 In order to
improve selectivity, the bivalent ligand approach was
The monomeric analogs of yohimbine were synthesized
by coupling yohimbinic acid with reagents having free
amino groups under standard peptide coupling condi-
tions. 1,3-Dicyclohexylcarbodiimide (DCC) was used
as the coupling agent and N-hydroxybenzotriazole
(HOBT) was used as an additive to catalyze the reaction
*
Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448
3446; e-mail: dmiller@utmem.edu
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.116

S. M. Mustafa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2758–2760
2759
1
and to suppress the epimerization at C-16. Accordingly
the yohimbine monomeric analogs were prepared as
shown in Scheme 1. The yohimbine monomeric analogs
10 and 11 were prepared by the deprotection of 6 using
HCl in ether and by the catalytic hydrogenation of 5
using 10% Pd/C in ethyl acetate, respectively. Attempts
to prepare compound 11 by ester hydrolysis was success-
ful although isolation was not and this prompted us to
prepare its benzyl derivative followed by reduction.
The structures of all the yohimbine monomeric analogs
synthesized were characterized by H NMR, MS, and
CHN analyses. The mono-N-protected-1,3-diaminopro-
panes 16 and 17 were prepared as illustrated in Scheme
210 whereas 21 was prepared by the deprotection of the
coupled product 20 of glycine methyl ester hydrochlo-
ride 18 and N-t-Boc glycine 19 with TFA at 0 °C in
methylene chloride medium.
The effects produced by the introduction of functional
groups in the side chain at C-16 of yohimbine were
Scheme 1. Reagents and conditions: (a) DCC, HOBT, THF, Et3N, rt; (b) NH2(CH2)3COOBz; (c) NH2(CH2)3NHBoc 16; (d) NH2(CH2)3NHCbz 17;
(e) NH2CH2COOMe 18; (f) NH2CH2CONHCH2COOMe 21; (g) HCl in ether, 30 min; (h) H2, 10% Pd/C, EtOAc. [Compound 13, the diazidopropane
is a very dangerous explosion hazard and it should be handled carefully.]
Scheme 2. Reagents and conditions: (a) NaN3, DMF/H2O, 80 °C, 20 h; (b) Ph3P, Et2O/EtOAc–5% HCl, rt, 24 h; (c) (Boc)2O/CbzCl, NaOH, THF–
H2O, rt; (d) Ph3P, THF, rt, 24 h; PG = Boc, Cbz; (e) EDC, HOBT, Et3N, DCM; (f) TFA, THF, 0 °C.

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S. M. Mustafa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2758–2760
Table 1. Binding data (Ki) of compounds 1–11 on human a2C-AR
References and notes
Compound
Ki SEM (nM)
1. (a) Hoffman, B. B.; Lefkowitz, R. J. Annu. Rev. Pharma-
col. Toxicol 1980, 20, 581; (b) Gerhardt, M. A.; Neubig, R.
R. Mol. Pharmacol. 1991, 40, 707; (c) Eason, M. G.;
Kurose, H.; Holt, B. D.; Raymond, J. R.; Liggett, S. B. J.
Biol. Chem. 1992, 267, 15795.
1
0.88 0.03
8.50 1.00
19.00 2.30
0.65 0.03
2.80 0.63
1.1 0.24
2
4
5
6
2. (a) Bylund, D. B. FASEB J. 1992, 6, 832; (b) Bylund, D.
B.; Eikenberg, D. C.; Hieble, J. P.; Langer, S. Z.;
Lefkowitz, R. J.; Minneman, K. P.; Molinoff, P. B.;
Ruffolo, R. R., Jr.; Trendelenburg, U. Pharmacol. Rev.
1994, 46, 121; (c) Hieble, J. P.; Bondinell, W. E.; Ruffolo,
R. R., Jr. J. Med. Chem. 1995, 38, 3415; (d) Hieble, J. P.;
Ruffolo, R. R., Jr. Prog. Drug Res. 1996, 47, 81; (e) Hieble,
J. P.; Ruffolo, R. R., Jr.; Sulpizio, A. C.; Naselsky, D. P.;
Conway, T. M.; Ellis, C.; Swift, A. M.; Ganguly, S.;
Bergsma, D. J. Pharmacol. Commun. 1995, 6, 91.
3. (a) Ruffolo, R. R., Jr.; Bondinell, W. E.; Hieble, J. P. J.
Med. Chem. 1995, 38, 3415; (b) Clark, R. D.; Michel, A.
D.; Whiting, R. L. Prog. Med. Chem. 1986, 23, 1.
4. (a) Yound, P.; Berge, J.; Chapman, H.; Cawthorne, M. A.
Eur. J. Pharmacol. 1989, 168, 381; (b) Okumura, K.;
Koike, K.; Asai, H.; Takayanagi, I. Gen. Pharmacol. 1988,
19, 463; (c) Beeley, L. J.; Berge, J. M.; Chapman, H.;
Hieble, J. P.; Kelly, J.; Naselsky, D. P.; Rockell, C. M.;
Young, P. W. Bioorg. Med. Chem. 1995, 3, 1693; (d)
Michel, A. D.; Loury, D. N.; Whiting, R. L. Br. J.
Pharmacol. 1990, 99, 560; (e) Blaxall, H. S.; Murphy, T. J.;
Baker, J. C.; Ray, C.; Bylund, D. B. J. Pharmacol. Exp.
Ther. 1991, 259, 323; (f) Bylund, D. B.; Blaxall, H. S.;
Iverson, L. J.; Caron, M. G.; Lefkowitz, R. J.; Lomasney,
J. W. Mol. Pharmacol. 1992, 42, 1.
7
8
8.00 0.70
5.20 0.70
32.00 3.40
3.00 0.70
9
10
11
The radioligand binding studies of yohimbine monomeric analogs were
carried out using CHO cells expressing homogeneous population of
a
2C-ARs. The competition binding assays were performed with
[3H]rauwolscine (0.1 lCi, 0.7 nM) and nonspecific binding was deter-
mined in the presence of 10 lM phentolamine. Ki values were calcu-
lated from the equation of Cheng and Prusoff11 and the data represent
the mean of four to nine experiments. The IC50 values were determined
by nonlinear regression analysis of competition data using the
GRAPHPAD PRISM computer program.
noticeable as seen in Table 1. In general, a comparison
of Ki values clearly points out that yohimbine dimer 2
has weak affinity for human a2C-ARs as compared to
monomeric analogs. Results show that among the
monomeric derivatives, compound 5 exceeds the binding
affinity of the parent compound yohimbine 1 toward
a
2C-ARs. Compounds 6, 7, and 11 possessed compara-
ble affinities to the parent compound while 8 and 9 dis-
played relatively lower binding potency than yohimbine.
The introduction of a carboxyl group at the side chain
led to derivative 11 a structure similar to yohimbinic
acid 4 on the other hand surpasses the affinity of 4 for
the human a2C-ARs whereas the amino analog 10 exhib-
ited weaker affinity.
5. Flavahan, N.; Flavahan, S.; Chotani, M.; Mitra, S.; Su, B.
U.S. Patent. 6,444,681, 2002; Chem. Abstr. 2002, 137,
103.
6. Goldberg, M. R.; Robertson, D. Pharmacol. Rev. 1983,
35, 143.
7. Portoghese, P. S. J. Med. Chem. 2001, 44, 2259.
8. (a) Zheng, W.; Lei, L.; Lalchandani, S.; Sun, G.; Feller, D.
R.; Miller, D. D. Bioorg. Med. Chem. Lett. 2000, 10, 627;
(b) Lalchandani, S. G.; Lei, L.; Zheng, W.; Suni, M. M.;
Moore, B. M.; Liggett, S. B.; Miller, D. D.; Feller, D. R. J.
Pharmacol. Exp. Ther. 2002, 303, 979; (c) Miller, D. D.;
Zheng, W.; Moore, B. M.; Mustafa, S. U.S. Patent
6,638,943 B2, 2003; Chem. Abstr. 2002, 137, 794.
9. (a) Pigini, M.; Bousquet, P.; Carotti, A.; Dontenwill, M.;
Giannella, M.; Moriconi, R.; Piergentili, A.; Quaglia, W.;
Tayebati, S. K.; Brasili, L. Bioorg. Med. Chem. 1997, 5,
833; (b) Pigini, M.; Quaglia, W.; Gentili, F.; Marucci, G.;
Cantalamessa, F.; Franchini, S.; Sorbi, C.; Brasili, L.
Bioorg. Med. Chem. 2000, 8, 883; (c) Gentili, F.;
Bousquet, P.; Brasili, L.; Caretto, M.; Carrieri, A.;
Dontenwill, M.; Giannella, M.; Marucci, G.; Perfumi,
M.; Piergentili, A.; Quaglia, W.; Rascente, C.; Pigini, M.
J. Med. Chem. 2002, 45, 32; (d) Gentili, F.; Bousquet, P.;
Brasili, L.; Dontenwill, M.; Feldman, J.; Ghelfi, F.;
Giannella, M.; Piergentili, A.; Quaglia, W.; Pigini, M. J.
Med. Chem. 2003, 46, 2169; (e) Gentili, F.; Ghelfi, F.;
Giannella, M.; Piergentili, A.; Pigini, M.; Quaglia, W.;
Vesprini, C.; Crassous, P. A.; Paris, H.; Carrieri, A. J.
Med. Chem. 2004, 47, 6160.
The order of binding affinities exhibited by yohimbine 1
on human a2-AR subtypes was a2C (0.88 nM) P a2A
(1.4 nM) > a2B (7.1 nM), while the order of the most
potent compound 5 was found to be a2C (0.65 nM) >
a2A (29 nM) > a2B (1300 nM).
In conclusion we describe herein the synthesis and radio-
ligand binding studies of a series of yohimbine deriva-
tives which led to the conclusion that a second
pharmacophore is not essential and, binding affinity de-
pends on the nature of the substituent in the side chain
found in the bivalent ligand approach.12 Our future
efforts will be oriented in optimizing the potent
compound 5 in order to understand the mechanism of
binding on these receptors.
Acknowledgements
10. Lee, J. W.; Jun, S. I.; Kim, K. Tetrahedron Lett. 2001, 42,
2709.
11. Cheng, Y. C.; Prusoff, W. H. Biochem. Pharacol. 1973, 22,
3099.
12. Mustafa, S. M.; Bavadekar, S. A.; Moore, B. M.; Liggett,
S. B.; Feller, D. R.; Miller, D. D. Abstract of papers,
228th National Meeting of the American Chemical Soci-
ety: Philadelphia, PA, 2004, Abstract 60.
Funding of this research was from Van Vleet founda-
tion, Ole Miss and in part by the USDA, ARS Specific
Cooperative Agreement No.# 58-6408-2-0009 at the
University of Mississippi. The authors are grateful to
Dr. Stephen B. Liggett (College of Medicine, University
of Cincinnati, OH) for the provision of CHO cells
expressing human a2C-AR used in this study.