Simplified tetrandrine congeners as possible antihypertensive agents with a dual mechanism of action

Article abstract of DOI:10.1021/np030022+

A series of O- and/or N-substituted derivatives of (±)-coclaurine (1a) were synthesized as simplified structural mimics of the antihypertensive alkaloid tetrandrine (2) and assayed for binding to brain cortical sites labeled with the α₁-adrener

Full text of DOI:10.1021/np030022+

954
J . Nat. Prod. 2003, 66, 954-957
Sim p lified Tetr a n d r in e Con gen er s a s P ossible An tih yp er ten sive Agen ts w ith a
Du a l Mech a n ism of Action
Patricio Iturriaga-Va´squez,† Raquel Miquel,‡ M. Dolores Ivorra,‡ M. Pilar D’Ocon,‡ and Bruce K. Cassels*,†
Millennium Institute for Advanced Research in Cell Biology and Biotechnology and Departamento de Qu´ımica, Facultad de
Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile, and Departament de Farmacologia, Facultat de Farma`cia,
Universitat de Valencia, Valencia, Spain
Received J anuary 17, 2003
A series of O- and/or N-substituted derivatives of (()-coclaurine (1a ) were synthesized as simplified
structural mimics of the antihypertensive alkaloid tetrandrine (2) and assayed for binding to brain cortical
sites labeled with the R1-adrenergic radioligand [3H]prazosin or the calcium channel radioligand [3H]-
diltiazem. The introduction of O-benzyl groups on the coclaurine molecule, which exhibits only adrenergic
antagonist activity, led to the appearance of calcium channel blocking activity comparable to that of
tetrandrine while retaining adrenolytic activity in the same concentration range. Contraction of aortal
rings with noradrenaline or KCl was relaxed more potently by some of these coclaurine derivatives than
by tetrandrine, suggesting leads for the development of novel antihypertensive drugs with a dual
mechanism of action.
Bisbenzylisoquinoline (BBIQ) or biscoclaurine alkaloids
make up an extensive family of natural products charac-
terized by the presence of two monomeric 1-benzyl-1,2,3,4-
tetrahydroisoquinoline (BTHIQ) moieties structurally and
biosynthetically related to coclaurine (1a ).1 In the larger,
“tail-to-tail” subfamily, these halves are joined by a biphe-
nyl ether or C-C bond between the benzyl groups and,
usually, one or two extra “head-to-head” ether linkages
between the isoquinoline benzene rings. Many of these
alkaloids have been studied in biological systems, where
they exhibit a wide range of effects including hypotensive,
antiinflammatory, antiarrhythmic, bactericidal, and muscle
relaxant.2
significant.10 These data suggest tetrandrine as a lead for
the development of antihypertensive drugs with a dual
mechanism of action.
Natural BBIQs, though very numerous, are seldom
abundant plant metabolites, and their unsystematic struc-
tural variations weaken any argument regarding their
structure-activity relationships. Their macrocyclic nature,
with very specific bonding between the “eastern” and
“western” regions and a stereogenic center in each, also
makes their synthesis unattractive from a medicinal
chemical viewpoint. On the other hand, it seems reasonable
to predict that simplified, more accessible congeners might
display similar pharmacological activities. In this sense,
the monomeric BTHIQ coclaurine (1a ) may serve as a
template to which lipophilic substituents can be attached
to mimic the structure of either half of the tetrandrine
molecule. This paper describes the preparation and pre-
liminary pharmacological screening of an extensive series
of coclaurine derivatives. In these, one or both phenolic
functions have been methylated or benzylated, the benzyl
groups serving as an approach to the aromatic rings of the
“western” half of the tetrandrine molecule. In most cases,
the basic nitrogen atom has been substituted with methyl,
ethyl, or propyl groups to assess the relevance of N-
alkylation to the adrenergic and calcium channel blocking
components of the smooth muscle relaxant effects of
tetrandrine.
Tetrandrine (2) is the major BBIQ of “han fangji”
(Stephania tetrandra, Menispermaceae) and is the most
characteristic active constituent of this Chinese herbal
remedy, which has been used for centuries in the treatment
of hypertension.3 A number of BBIQs have been compared
with tetrandrine and in most cases have been found to be
less effective as smooth muscle relaxants or L-type calcium
channel blockers.4-7 Within this set of BBIQs, higher
smooth muscle relaxant potencies have been ascribed to
the presence of two biphenyl ether linkages, the (1S,1′S)
configuration, and extensive N- and O-methylation.4,6,7
Although the smooth muscle relaxant and hypotensive
action of tetrandrine is attributed to the selective blockade
of calcium channels,5,8,9 its interaction with R1-adrenergic
receptors, which also modulate calcium channels, may be
Coclaurine (1a ) and its O-benzylated and/or O-methy-
lated derivatives were prepared by the classical Bischler-
Napieralski 3,4-dihydroisoquinoline synthesis and subse-
quent reduction of the intermediates with NaBH4,11-13
followed in most cases by N-alkylation. N-Methylation was
best carried out with aqueous formaldehyde and NaBH4,11,12,14
* To whom correspondence should be addressed. Tel: 562-271-3881.
Fax: 562-271-3888. E-mail: bcassels@uchile.cl.
† Universidad de Chile.
‡ Universitat de Valencia.
10.1021/np030022+ CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/02/2003

Simplified Tetrandrine Congeners
J ournal of Natural Products, 2003, Vol. 66, No. 7 955
Ta ble 1. Coclaurine and Derivatives Discussed in this Papera
Ta ble 2. Displacement of [3H]Prazosin and [3H]Diltiazem from
Rat Brain Cortical Sites by Coclaurine and Derivativesa
compound
R7
R12
RN
compound
[3H]prazosin pKi
[3H]diltiazem pKi
1a
1b
1c
3a
3b
3c
4a
4b
5a
5b
6a )
6b
6c
6d
7a
7b
8a
8b
9a
H
H
H
CH3
CH3
CH3
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
Bn
Bn
Bn
Bn
H
H
CH3
CH2CH3
H
CH3
CH2CH3
H
CH3
H
CH3
H
CH3
CH2CH3
(CH2)2CH3
H
CH3
H
1a
1b
1c
3a
3b
3c
4a
4b
5b
6a
6b
6d
7a
7b
8a
8b
9a
5.97 ( 0.12
5.63 ( 0.07
5.29 ( 0.07
5.23 ( 0.12
5.14 ( 0.03
5.07 ( 0.09
4.92 ( 0.08
5.29 ( 0.12
5.10 ( 0.07
5.02 ( 0.04
5.12 ( 0.09
5.05 ( 0.01
5.49 ( 0.07
5.49 ( 0.05
5.34 ( 0.06
5.73 ( 0.03
5.69 ( 0.16
6.1615
≈30%
4.64 ( 0.08
4.86 ( 0.58
N.D.
5.02 ( 0.05
4.51 ( 0.04
4.49 ( 0.04
4.90 ( 0.08
5.38 ( 0.05
4.61 ( 0.31
5.74 ( 0.11
5.75 ( 0.22
5.60 ( 0.35
5.70 ( 0.25
N.D.
H
CH3
CH3
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
H
H
CH3
CH3
Bn
5.82 ( 0.10
5.57 ( 0.28
6.13 ( 0.22
CH3
H
tetrandrine (2)
a
a
Bn = C6H5CH2-.
pKi ) -log(Ki); Ki ) inhibition constant (µM) of each alkaloid
for the binding of [3H]prazosin or [3H]diltiazem to their specific
sites in rat brain cortical membranes; all binding experiments were
carried out 4-6 times. N.D.: not determined.
and N-ethylation and N-propylation were performed by
treating the secondary amines with ethyl iodide or propyl
bromide in acetonitrile containing NaHCO3, respectively.
12-O-Benzylcoclaurine (9a ) was prepared conveniently by
selective debenzylation of 7,12-O,O′-dibenzylcoclaurine (6a )
with SnCl4.15
The affinities of all the coclaurine derivatives for R1-
adrenergic receptors were assessed in binding studies in
rat brain cortical homogenates using the subtype nonselec-
tive radioligand [3H]prazosin. A subset of these compounds
was assayed for affinity for the benzothiazepine modulatory
site of the L-type Ca2+ channel displacing [3H]diltiazem,
and eight of them were selected for functional testing as
R1-adrenergic antagonists or Ca2+ channel blockers in rat
thoracic aorta, contracted with noradrenaline or by depo-
larization with KCl, respectively.10 The results were com-
pared with available data for coclaurine itself and for
tetrandrine.
formation (with the 1-benzyl moiety pointing away from
the isoquinoline benzene ring) predominates. On the other
hand, substitution at the nitrogen atom with methyl, ethyl,
or propyl groups led to progressively increased shielding
of H-8, of the C-7 methoxy protons, or of the C-7 benzyloxy
methylene protons as in compounds 6b-d , indicating that
the N-substituted molecules reside most of the time in a
“folded” conformation. Interestingly, in these molecules the
C-7 benzyloxy methylene protons appeared as two distinct
doublets, showing that this group is conformationally fixed
on the NMR time scale. The N-substituted compounds
studied here are therefore not only structural but also
conformational mimics of the constrained “eastern” half of
the tetrandrine molecule. As will be discussed later, the
conformational preferences of these coclaurine derivatives
may be related to their affinities at the prazosin binding
site of the R1-adrenoceptor and at the benzothiazepine site
of L-type calcium channels.
Resu lts a n d Discu ssion
The specific binding of the R1-adrenergic ligand [3H]-
prazosin at a concentration of 0.2 nM and [3H]diltiazem at
a concentration of 3 nM represented approximately 90%
and 70% of the total binding, respectively. All the com-
pounds competed for both radioligands with the inhibition
constants summarized in Table 2. The interactions of some
representative coclaurine analogues with [3H]prazosin- and
[3H]diltiazem-labeled rat brain cortical membranes are
shown in Figures 1A and 1B. The low micromolar affinity
of coclaurine (1a ) for prazosin binding sites, which is not
significantly different from that of tetrandrine, is slightly
better than those of most of its analogues tested here.
On going from coclaurine to its N-methyl and N-ethyl
derivatives (1b and 1c), affinity for prazosin binding sites
was halved with each additional carbon atom. This loss of
affinity might be related to steric hindrance of a key
interaction between the nitrogen atom, which should be
largely protonated at physiological pH, and an anionic site
common to all monoamine G-protein-coupled receptors. An
alternative explanation could be that since an “extended”
conformation is clearly less accessible in the N-substituted
molecules, this less populated form may approximate the
pharmacophoric conformation of BTHIQs at R1-adrenocep-
tors. However, since flexible molecules may be biologically
active without necessarily preferring a pharmacophoric
conformation, but merely being able to adopt it when
The BTHIQ derivatives prepared in this study are listed
in Table 1. For the sake of clarity, compound numbers
followed by a letter “a” always correspond to the secondary
amines, and by “b”, “c”, or “d” to the N-methylated,
1
-ethylated, or -propylated analogues. Their H NMR spectra
were recorded and analyzed to establish their conforma-
tional preferences. Earlier 1H NMR studies on BTHIQs
suggested that the preferred conformation of the N-
unsubstituted secondary amines has the pendent benzyl
group pointing away from the tetrahydroisoquinoline ben-
zene ring, while N-methylation leads to a preferentially
folded conformation in which the benzyl group shields H-8
and the protons of the methoxyl group bonded to C-7, when
such a group is present.16-18 The “western” half of head-
to-head, tail-to-tail BBIQs is held in an extended conforma-
tion, while the “eastern” half adopts a folded conformation,
as is clearly seen in molecular models and corroborated by
1H and 13C NMR results.19 Analysis of the 1H NMR spectra
of our extensive series of N-unsubstituted and N-substi-
tuted BTHIQs indicated that the preference observed in
simple analogues for extended or folded conformations,
respectively, is quite general, even when C-7 bears a bulky
benzyloxy group.
The 1H NMR data of all the compounds lacking an
N-substituent (1a , 3a , 4a , 5a , 6a , 7a , 8a , 9a ) showed that,
as in simpler BTHIQ derivatives,16,18 an “extended” con-

956 J ournal of Natural Products, 2003, Vol. 66, No. 7
Iturriaga-Va´squez et al.
F igu r e 2. Semifolded (A) and extended (B) conformers of benzyltet-
rahydroisoquinoline, conformations optimized at the ab initio RHF-
631g [d,p] level.
5b) were able to displace the radioligand completely,
although their affinities for the diltiazem binding site are
only in the 10-5 M range. On the other hand, the O-
benzylated derivatives tested had uniformly higher affini-
ties than any of the other compounds, suggesting that large
lipophilic substituents at C-7 and/or C-12 of the BTHIQ
skeleton favor binding to the diltiazem site. Significantly,
these are the positions occupied by the benzene rings of
one of the halves of tetrandrine. When a direct comparison
can be made between N-unsubstituted and N-substituted
analogues, no definite conclusion can be drawn regarding
the conformational preferences of these compounds at the
diltiazem site, since their affinities do not show any
consistent variation. In this regard, a recent molecular
modeling study suggests that BTHIQs may be superim-
posed upon the diltiazem structure in an extended confor-
mation and that this also appears to be the situation for
the “western” half of tetrandrine.21 All four mono-O-
benzylated derivatives tested by us were statistically
indistinguishable from tetrandrine as [3H]diltiazem dis-
placers, and as they are racemic compounds, the affinities
of their eutomers might be expected to be greater by a
factor of 2.
F igu r e 1. Displacement of the specific binding of [3H]prazosin (A) or
[3H]diltiazem (B) (concentration-response curves) to rat cerebral
cortical membranes by coclaurine, tetrandrine, and some derivatives.
Each point is the mean of the results from three or five experiments
performed in duplicate.
interacting with their macromolecular target, these results
should be interpreted with caution. A similar, though less
pronounced trend was seen in the more weakly binding
7-O-methylated norarmepavine series (3a -c) but not for
12-O-methylcoclaurine (4a ) and its N-methyl analogue
(4b), suggesting that a free hydroxyl group at C-12 might
be a key feature of BTHIQ binding to R1-adrenoceptors.
This idea seems to be borne out by the low affinities of the
7,12-di-O-benzylated series (6a -d ). The 7-O-benzyl com-
pounds tested with a free 12-hydroxyl group (7a , 7b) have
higher affinities than coclaurine. 7-O-Benzyl-12-O-meth-
ylcoclaurine also (8a ) bound somewhat better, but this gain
was lost upon N-methylation. Finally, the affinity of 12-
O-benzylcoclaurine (9a ) was indistinguishable from that
of the parent compound, possibly reflecting a different
binding mode.
The IC50 values obtained for the different coclaurine
analogues from functional studies in rat thoracic aortal
rings are shown in Table 3. These results extend an earlier
study showing that both coclaurine (1a ) and norarmepa-
vine (3a ) relax this tissue, suggesting that these alkaloids
are calcium channel blockers.22 In our hands, cumulative
increments of the concentrations of these compounds
(10-9-10-4 M) relaxed both noradrenaline (100 µM)- and
KCl (80 mM)-induced contractions in the presence of Ca2+
,
confirming their antagonism at adrenoceptors and calcium
channels.
In these studies it is noteworthy that, in addition to being
more potent adrenolytics, the 7-O-benzyl derivatives 7a and
7b are more potent than tetrandrine as blockers of KCl-
elicited aortal contraction, and all four O-benzylated
derivatives tested are more effective than coclaurine or
tetrandrine as relaxants of vascular smooth muscle con-
tracted with noradrenaline. Although tetrandrine has
slightly higher affinities than these four compounds for the
Micromolar coclaurine (1a ) was unable to displace [3H]-
diltiazem from its brain cortical binding sites by more than
30%, while tetrandrine was a fairly potent displacer. In
the present study we found that the N-substituted and
O-methylated derivatives of coclaurine (1b,c, 3b,c, 4a ,b,

Simplified Tetrandrine Congeners
J ournal of Natural Products, 2003, Vol. 66, No. 7 957
Ta ble 3. Inhibitory Potencies (pIC50) of Coclaurine, Some
Coclaurine Derivatives, and Tetrandrine on Contractions
Induced in Rat Thoracic Aorta by Noradrenaline (1 µM) or KCl
(80 mM)a
gether, these results support our hypothesis that large,
lipophilic substituents attached to the phenol groups of the
coclaurine molecule are able to mimic the binding and
functional interactions of tetrandrine with the prazosin and
benzothiazepine sites in R1-adrenoceptors and L-type cal-
cium channels. Thus, extension of the coclaurine structure
(which acts predominantly via antagonism at R1-adreno-
ceptors) with O-benzyl substituents leads to enhanced
smooth muscle relaxant potency involving both R1-adren-
ergic antagonism and calcium channel blockage in the same
narrow concentration range, suggesting the possibility of
developing useful hypotensive drugs with a dual mecha-
nism of action based on the BTHIQ scaffold.
compound
NA pIC50
KCl pIC50
1a
1b
3b
4b
5b
7a
7b
8b
9a
4.76 ( 0.09
4.61 ( 0.14
3.68 ( 0.13
4.60 ( 0.22
4.18 ( 0.13
4.88 ( 0.10
5.06 ( 0.21
5.37 ( 0.12
4.79 ( 0.07
4.53
(17.2%)
4.17 ( 0.20
3.94 ( 0.07
4.14 ( 0.06
4.83 ( 0.07
5.12 ( 0.07
5.24 ( 0.16
4.94 ( 0.15
4.86 ( 0.07
4.87
tetrandrine (2)15
Exp er im en ta l Section
a
Data are expressed as mean ( SEM; all binding experiments
were repeated 4-6 times. Number in parentheses indicates %
relaxation of contraction at 100 µM drug concentration.
Some of the compounds reported here are known inter-
mediates in the synthesis of natural BTHIQs. Most of them,
however, have not been described previously, and their
physical, spectroscopic, and analytical data are given as
Supporting Information. Descriptions of the binding and
functional studies are also included in the Supporting
Information.
[3H]prazosin and [3H]diltiazem binding sites, its lower
functional potency, particularly at R1-adrenoceptors, might
result from different receptor subtype and ion channel
populations in brain cortex and aorta or might be a
consequence of its excessive molecular weight (623), limit-
ing its access to its sites of action.
Regarding the effect of N-substitution, the R1-adreno-
ceptor affinity of coclaurine (1a ) and its O-methylated
analogues (3a and 4a ) tends to fall off with increasing size
of the N-alkyl groups in the N-alkylated compounds 1c and
3c. Two alternative but not exclusive interpretations may
be put forth: either bulky substituents interfere with the
presumably ionic interaction of the protonated nitrogen
atom with an anionic residue, or they destabilize an
extended conformation necessary for optimal binding to the
receptor.
Ack n ow led gm en t . This work was supported by
a
CONICYT scholarship (P.I.), by the Presidential Chair in
Sciences (B.K.C.), ICM Grant No. P99-031-F, and by a research
grant from the Generalitat Valenciana (GV01-292). The
authors thank Dr. Gerald Zapata-Torres for the optimization
of the benzyltetrahydroisoquinoline conformers depicted in
Figure 2.
Su p p or tin g In for m a tion Ava ila ble: Experimental Section and
descriptions of the binding and functional studies. This material is
available free of charge via the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
The affinity of 1a and its O-methylated analogues (3a
and 4a ) for R1 receptors decreases with increasing methy-
lation, suggesting that at least one of the hydroxyl groups
may contribute to binding to the prazosin site. On the other
hand, a benzyl ether moiety on the coclaurine structure
allows affinity for this site to be preserved, even if the
remaining hydroxyl group is methylated, suggesting that
the additional benzyl group may establish hydrophobic
interactions with relatively distant residues, compensating
for the loss of the putative hydrogen bond donating
hydroxyl groups. It must be kept in mind that different
drugs competing for a common binding site may bind in
different orientations. This goes to say that different
pharmacophoric conformations of R1-adrenolytic BTHIQs
are likely to exist, in line with the wide structural varia-
tions among R1-adrenergic antagonists.
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The results obtained in the [3H]diltiazem displacement
experiments were quite different and practically indepen-
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of experiments, a single O-benzyl substituent, either at C-7
or C-12 (as in 7b, 8a ,b, and 9a ), or two O-benzyl groups
(6b and 6d ) seem to favor binding to the benzothiazepine
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NP030022+