Novel hypotensive agents from Verbesina caracasana. 6. Synthesis and pharmacology of caracasandiamide

Article abstract of DOI:10.1021/jm991004l

Caracasandiamide, a second hypotensive agent isolated from Verbesina caracasana, is the cyclobutane dimer (truxinic type) of the previously reported 1-[(3,4-dimethoxycinnamoyl)amino]4-[(3-methyl-2- butenyl)guanidino]butane (caracasanamide) (Delle Monache, G.; et al. BioMed. Chem. Lett. 1992, 25, 415-418). The structure was confirmed by synthesis starting from β-truxinic acid obtained by photoaddition of 3,4- dimethoxycinnamic acid. The dimer was coupled with 2 mol of prenylagmatine to give caracasandiamide in satisfactory yield. By contrast, the direct photodimerization of caracasanamide was unsuccessful. Caracasandiamide, assayed by the iv route in anesthetized rats at doses ranging from 50 to 3200 μg/kg of body weight, was found to have no appreciable effect on heart rate. At lower doses, the drug stimulates breathing and increases cardiac inotropism, stroke volume, and cardiac output, thus augmenting blood pressure and aortic flow. At higher doses, caracasandiamide depresses breathing likely through central neurogenic mechanisms (not involved in the cardiovascular effects), continues to stimulate cardiac inotropism, and induces, by reducing peripheral vascular resistance, arterial hypotension with reduction of both aortic flow and stroke volume. These cardiovascular effects appear to involve complex interactions at the level of the peripheral β₁-, β₂-, and α₂- adrenoreceptor-dependent as well as M₂- and M₄-cholinergic receptor- dependent transductional pathways both in cardiovascular myocells and at the level of the postganglionic sympathetic endings (with reserpine- and guanethidine-like mechanisms). The cardiovascular effects of caracasandiamide, different from those of caracasanamide, do not depend on significant actions on the central nervous system and on baroreflex pathways. In a similar manner and more effective than caracasanamide, caracasandiamide may be considered a hypotensive and antihypertensive drug. It is devoid of some of the negative side effects, e.g., reflex tachycardia and decreased cardiac inotropism, which are shown by the majority of the most common antihypertensive and vasodilator drugs.

Full text of DOI:10.1021/jm991004l

3116
J . Med. Chem. 1999, 42, 3116-3125
Novel Hyp oten sive Agen ts fr om Ver besin a ca r a ca sa n a . 6. Syn th esis a n d
P h a r m a cology of Ca r a ca sa n d ia m id e1
Marco Carmignani,*,† Anna R. Volpe,‡ Franco Delle Monache,‡ Bruno Botta,*,§ Romulo Espinal,∇
Stella C. De Bonnevaux,∇ Carlo De Luca,| Maurizio Botta,*, Federico Corelli, Andrea Tafi,
Giuseppe Ripanti,† and Giuliano Delle Monache*,‡
Dipartimento di Biologia di Base e Applicata, Sezione di Farmacologia, Universita` di L’Aquila, 67010 Coppito (AQ), Italy,
Centro Chimica dei Recettori, Universita` Cattolica, Largo F. Vito 1, 00168 Rome, Italy, Dipartimento di Chimica e Tecnologia
delle Molecole Biologicamente Attive, Universita` La Sapienza, Rome, Italy, Departamento de Farmacologia, Universidad de
Carabobo, Carabobo, Venezuela, Centro Elettrochimica e Chimica Fisica delle Interfasi, Rome, Italy, and Dipartimento
Farmaco Chimico Tecnologico, Universita` di Siena, Siena, Italy
Received J anuary 7, 1999
Caracasandiamide, a second hypotensive agent isolated from Verbesina caracasana, is the
cyclobutane dimer (truxinic type) of the previously reported 1-[(3,4-dimethoxycinnamoyl)amino]-
4-[(3-methyl-2-butenyl)guanidino]butane (caracasanamide) (Delle Monache, G.; et al. BioMed.
Chem. Lett. 1992, 25, 415-418). The structure was confirmed by synthesis starting from
â-truxinic acid obtained by photoaddition of 3,4-dimethoxycinnamic acid. The dimer was coupled
with 2 mol of prenylagmatine to give caracasandiamide in satisfactory yield. By contrast, the
direct photodimerization of caracasanamide was unsuccessful. Caracasandiamide, assayed by
the iv route in anesthetized rats at doses ranging from 50 to 3200 µg/kg of body weight, was
found to have no appreciable effect on heart rate. At lower doses, the drug stimulates breathing
and increases cardiac inotropism, stroke volume, and cardiac output, thus augmenting blood
pressure and aortic flow. At higher doses, caracasandiamide depresses breathing likely through
central neurogenic mechanisms (not involved in the cardiovascular effects), continues to
stimulate cardiac inotropism, and induces, by reducing peripheral vascular resistance, arterial
hypotension with reduction of both aortic flow and stroke volume. These cardiovascular effects
appear to involve complex interactions at the level of the peripheral â1-, â2-, and R2-
adrenoreceptor-dependent as well as M2- and M4-cholinergic receptor-dependent transductional
pathways both in cardiovascular myocells and at the level of the postganglionic sympathetic
endings (with reserpine- and guanethidine-like mechanisms). The cardiovascular effects of
caracasandiamide, different from those of caracasanamide, do not depend on significant actions
on the central nervous system and on baroreflex pathways. In a similar manner and more
effective than caracasanamide, caracasandiamide may be considered a hypotensive and
antihypertensive drug. It is devoid of some of the negative side effects, e.g., reflex tachycardia
and decreased cardiac inotropism, which are shown by the majority of the most common
antihypertensive and vasodilator drugs.
In tr od u ction
to have hypotensive effects on mice. The least polar
product was named caracasanamide (G1) and assigned
the structure 1-[(3,4-dimethoxycinnamoyl)amino]-4-[(3-
methyl-2-butenyl)guanidino]butane (1) (Chart 1), as a
mixture of (Z)- and (E)-forms.2
Synthetic derivatives of guanidine (e.g., guanethi-
dine,3 guanabenz,4 and guanfacine5) have been intro-
duced in antihypertensive drug therapy for their ability
to block adrenergic nerve activity through central and/
or peripheral mechanisms.6,7 Also pinacidil,8 an (ary-
lamino)guanidine, is able to lower blood pressure, acting
on arterial vasodilation.
The studies on several synthetic guanidines have
established that small changes in structure may lead
to wide variations of the hypotensive activity.9-11 Re-
cently, we isolated, by utilizing a biologically controlled
purification, a series of active compounds from the crude
methanol extract of the Venezuelan plant Verbesina
caracasana Fries (Compositae), which had been shown
The water-soluble (Z)-form of 1 was shown to be a
hypotensive drug of low-mild potency, devoid of signifi-
cant tachycardic effects. It also provided, via central and
peripheral mechanisms of action, an effect on cardio-
vascular function and revealed stimulating respiratory
effects when administered at nontoxic doses. The phar-
macological profile of the (Z)-form and the synthesis of
the (E)-form of caracasanamide (1) have been reported
in a previous publication.12
1
The H and 13C NMR spectra of a second metabolite,
named caracasandiamide (G2), showed a close similarity
with those of caracasanamide (G1), but the signals of
two aliphatic methine protons and carbons replaced
those of the olefinic unit in the cinnamoyl moiety. Since
the FAB-MS spectrum of caracasandiamide showed a
MH+ peak at m/z 777 and the molecular weight of
† Universita` di L’Aquila.
‡ Universita` Cattolica.
§ Universita` La Sapienza.
∇ Universidad de Carabobo.
| Centro Elettrochimica e Chimica Fisica delle Interfasi.
Universita` di Siena.
10.1021/jm991004l CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/23/1999

Novel Hypotensive Agents from V. caracasana
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3117
Ta ble 1. 1H and 13C NMR Data of Caracasandiamide and Its
Ch a r t 1
Hydrolysis Productsa
2
3
4
5
H2-1
H2-2
H2-3
H2-4
H-R
H-â
H-2′
H-5′
H-6′
3.35 m
1.70 m
1.61 m
3.22 m
4.32 d (6)
3.95 d
6.60 d (2)
6.71 d (8)
6.65 dd
3.22
1.68
1.52 m
3.18 m
4.30
3.52
1.96
1.96
3.19
4.24
3.91
6.59
6.72
6.68
3.61
3.68
3.86
6.58, 6.56
6.70, 6.68
6.66, 6.64
3.59, 3.60
3.67, 3.66
3.63
5.31
1.72
1.69
3′-OMe 3.62 s
4′-OMe 3.68 s
H2-1′′
H-2′′
H3-4′′
H3-5′′
NH
3.94 m
5.32 br t (6.5)
1.73 br s
1.71 br s
8.10, 7.85, 7.55 br s
4.10
5.52
1.61
1.58
C-1
C-2
C-3
C-4
39.44
27.35
27.03
42.11
38.94
27.76
26.18
42.22
40.81
27.68
25.88
41.22
C-R
C-â
44.18
45.54
44.34
45.96
44.18
45.54
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
OMe
OMe
CdO
133.82
113.36
148.32
149.53
112.03
120.92
56.04
136.23, 135.81
114.71, 114.58
148.70, 148.55
150.12, 150.03
113.18, 113.13
121.42, 121.37
56.35 (×2)
56.51, 56.49
179.05, 173.81
158.23
132.57
113.19
148.81
151.44
112.08
120.82
55.91
55.95
173.06
55.89
173.65
CdNH 157.06
158.47
42.64
121.38
137.01
26.55
C-1′′
C-2′′
C-3′′
C-4′′
C-5′′
40.43
120.07
137.32
25.83
18.33
40.31
121.00
136.43
25.69
caracasanamide was 388 Da, we concluded that G2 was
a dimer of G1, that is, a diamide connected to a
cyclobutane skeleton.13
18.17
18.98
A series of hydrolytic experiments allowed the relation
of G2 to methyl â-3,4-dimethoxytruxinate. The latter
was shown to be identical with a synthetic specimen,
obtained by photodimerization, in the solid phase of
ethyl (E)-3,4-dimethoxycinnamate, followed by hydroly-
sis and methylation.13 Therefore, caracasandiamide was
assigned the structure bis(3′,4′-dimethoxy)-â-truxinbis-
[[N-(3-methylbut-3-enyl)guanidobutyl]amide] (2).
This paper deals with the synthesis and the pharma-
cological profile of caracasandiamide.
a
300 and 75 MHz, TMS as internal standard. Solvents:
acetone-d6-DMSO-d6, 4:1, 2, 3 (60 °C); C5D5N, 4; acetone, 5. The
signals in the 1H NMR spectra showed the appropriate integrate
intensities. Multiplicities are indicated in the first row. Coupling
constants are given in parentheses (in Hz). Proton and protonated
carbon signals were correlated by a HETCOR experiment. The
signals for aromatic and truxinic rings in the two moieties of 3
are not equivalent. For compound 5 only the major signal of the
syn-anti pair is reported.
1
signal of the 3′-methoxy groups in the H NMR spec-
trum, requires the two aromatic rings to be face to face.
A strongly acidic hydrolysis (2 N H2SO4 in MeOH) of 3
gave again prenylurea, prenylagmatine (4), and the
methyl ester 5 (Chart 1). The chemical shift (δ 3.68) of
the equivalent carbomethoxy groups in the 1H NMR
spectrum indicated that they do not face the aryl rings.14
Accordingly, DIF NOE experiments revealeded that the
H-â protons and the aryl rings of 5 are on the same face.
Since identical DIF NOE results were obtained for 2
and 3, the same stereochemistry as for â-truxinic acid
was established for the three compounds. The mass
spectra of 2, 3, and 5 are characterized by fragments
typical of a truxinic derivative.15 For instance, the
fragmentation of 3 can be rationalized as shown in
Scheme 1, the diagnostic peak at m/z 300 also being
present in the mass spectra of 2 and 5.
Resu lts a n d Discu ssion
Str u ctu r e. The spectral characteristics of caracasan-
diamide and its hydrolysis products are summarized in
Table 1. The alkaline hydrolysis of 2 gave the product
3 and 1-amino-4-(3-methylbut-2-enylguanidino)butane
(prenylagmatine, 4) (Chart 1). The isolation of preny-
lurea from the reaction mixture confirmed the position
of the prenyl group in 4. The quasi molecular peak (MH+
at 597 in the FAB-MS spectrum) suggested that com-
pound 3 was an amido acid, as supported by the
presence of two distinct carbonyl resonances (at 173.7
and 179.0 ppm) and double signals for the aromatic and
cyclobutane carbons in the 13C NMR spectrum. More-
over, the resistance of compound 3 to the hydrolysis of
the second prenylagmatine unit, as well as to the
methylation of the carboxylic group, was attributed to
the presence of a hydrogen bond between the acid and
amide groups. As a consequence, the two carbonyl
groups must lie on the same side of the cyclobutane ring.
A similar cisoid disposition, revealed by the high-field
In conclusion, caracasandiamide and its hydrolysis
products were assigned the structures summarized in
Chart 1.
Syn th esis. According to the hypotheses put forward
on the biogenesis of caracasandiamide,13 we initially

3118 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Carmignani et al.
Sch em e 1
Sch em e 3a
Sch em e 2a
a
Reagents: (i) hν, rt; (ii) KOH, EtOH (5b); H2, Pd/C (5c); (iii)
a
pentylamine, CDI.
Reagents: (i) CMC metho-p-toluenesulfonate, CH2Cl2; (ii)
CF3COOH; (iii) CH3SO3H, CH2Cl2.
focused on the possibility to synthesize 2 by direct
photoaddition of caracasanamide (Scheme 2). The pho-
tochemical reaction was carried out on the N-Boc-
protected caracasanamide 1a ,12 a more soluble and
stable substrate than 1. Irradiation of 1a in the solid
state16 with a 125-W mercury lamp for 20 h gave a
mixture of photoadducts, which were separated by
preparative TLC. However, none of the isolated products
corresponded to the N-Boc derivative of 2.
Therefore, we followed a different approach, based on
the formation of amide bonds between prenylagmatine
and the suitably substituted â-truxinic acid, which in
turn can be obtained by photodimerization of the
corresponding cinnammate.17 Ethyl 3,4-dimethoxycin-
namate (6a ) and benzyl 3,4-dimethoxycinnamate (6b)
were thus subjected to solid-state photodimerization
(Scheme 2) affording the â-truxinic esters 5a (40%) and
5b (38%), respectively, along with small amounts of
other stereoisomers and starting material (ca. 40%). The
ethyl ester 5a by hydrolysis with ethanolic KOH gave
the â-truxinic acid 7, which by methylation with diaz-
omethane provided the dimethyl ester 5, identical with
the hydrolysis product (H2SO4, MeOH) of the natural
compound 2. Moreover, treatment of 7 with catalytic H2-
SO4/EtOH afforded again the diester 5a , proving that
no epimerization had occurred during the alkaline
hydrolysis. The acid 7 was also obtained by the hydro-
genolysis of the benzyl ester 5b. Finally, the acid 7 was
converted into the bicyclic imide 8, by treatment with
n-pentylamine in the presence of N,N′-carbodiimidazole
(CDI) in excess, thus chemically confirming a cis rela-
tionship between the â-substituents.
Following the procedures successfully employed for
the synthesis of caracasanamide,12 the diacid was
activated as the corresponding acyl dichloride or mixed
anhydride with diethyl chlorophosphate and reacted
with the suitably protected prenylagmatine 4a (Scheme
3).12 However, the yield of the acylation reaction never
exceeded 13%. This failure may be attributed to the
considerable steric hindrance around the carboxy groups.
In addition, the preferential formation of the cyclic
anhydride of 2, resulting in compouds such as 3, may
be a factor. The reaction between 7 and 4a in the
presence of 1-cyclohexyl-3-(2-morpholinoethyl)carbodi-
imide (CMC) metho-p-toluenesulfonate gave the com-
pound 2a , in fair yield, when the steric congestion of
the molecule is considered.
Although the removal of N-Boc protecting groups can
be accomplished by trifluoroacetic acid, ensuing from
the electrophilic addition of the acid to the prenyl double
bond, deprotection of 2a was better performed with a
catalytic amount of methanesulfonic acid at reflux in
1,4-dioxane. Synthetic 2 (61% yield) was identical by
comparison of spectral and chromatographic data with
an authentic sample of caracasandiamide.

Novel Hypotensive Agents from V. caracasana
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3119
Ta ble 2. Changesa in Systolic and Diastolic Blood Pressure, Heart Rate, Maximum Rate of Rise of the Left Ventricular Isovolumetric
Pressure (dP/dt), Mean Aortic Flow, and Stroke Volume following iv Administration of Caracasandiamide (3) in Anesthetized Rats
blood pressure
systolic
diastolic
mmHg
+16 ( 2 +10 ( 2 +13 ( 3
heart rate
dP/dt
mmHg/s
mean aortic flow
mL/min
stroke volume
µL %
3 (µg/kg) mmHg
%
%
beats/min
%
%
%
50
+17 ( 2
+12 ( 2
-14 ( 1
-20 ( 2
-30 ( 4
-41 ( 5
-9 ( 3
-3 ( 2 +1070 ( 90 +17 ( 2 +19 ( 3 +26 ( 3 +73 ( 5 +36 ( 4
-3 ( 1 +1129 ( 82 +18 ( 2 +11 ( 2 +15 ( 2 +20 ( 3 +10 ( 2
100
200
400
800
1600
3200
+12 ( 2 +10 ( 1 +13 ( 2 -10 ( 2
-13 ( 2 -13 ( 2 -17 ( 3 -13 ( 1.5 -4 ( 1 +1369 ( 170 +22 ( 3 -14 ( 4 -19 ( 2 +23 ( 2 +11 ( 1
-19 ( 2 -17 ( 1 -22 ( 2 -16 ( 2
-29 ( 4 -31 ( 3 -40 ( 4 -16 ( 3
-39 ( 4 -43 ( 7 -55 ( 6 -30 ( 2
-4 ( 1 +2615 ( 181 +43 ( 5 -14 ( 3 -19 ( 3 -46 ( 4 -23 ( 3
-4 ( 2 +3282 ( 269 +54 ( 4 -25 ( 3 -34 ( 4 -86 ( 6 -43 ( 4
-8 ( 1 +3676 ( 264 +60 ( 3 -36 ( 2 -49 ( 3 -97 ( 6 -48 ( 3
-11 ( 3 +4708 ( 430 +77 ( 6 -41 ( 3 -56 ( 4 -117 ( 7 -58 ( 5
-48 ( 6.5 -46 ( 5 -53 ( 4 -68 ( 4 -41 ( 5
baseline 104 ( 4
78 ( 5
365 ( 16
+6131 ( 348
73 ( 4
201 ( 15
a
Values are means( SE (n ) 12 for each dose with the exception of 3200 µg/kg, for which n ) 7).
Ta ble 3. Changesa in Respiratory Frequency and Tidal
Volume following iv Administration of Caracasandiamide (3) in
Anesthetized Rats
well as stimulatory effects on RF and TV until the dose
of 1600 µg/kg (higher doses caused great respiratory
depression and 6400 µg/kg was also able to induce
irreversible respiratory blockade, bradycardia, and ven-
tricular arrhythmias with final cardiac arrest); (Z)-
caracasanamide was already found to be more potent
than guanethidine in lowering BP; the same drug was
as potent as reserpine and papaverine and less potent
than clonidine, hexamethonium, and histamine.12 Like
(Z)-caracasanamide, G2 did not decrease (at nontoxic
doses) cardiac inotropism in contrast with all the tested
hypotensive drugs. Furthermore, the hypotensive effect
of G2 was longer than that of (Z)-caracasanamide, which
was already found to last more than those of drugs (like
guanethidine, papaverine, and histamine) acting by
peripheral vasodilatatory mechanisms.12 The analysis
of the effects of G2 indicated that, if the lower doses of
the drug did not significantly change HR while increas-
ing BP, dP/dt, AF, and SV, doses higher than 200 µg/kg
continued to increase only dP/dt while reducing BP, AF,
and SV. Therefore, this reduction of BP, AF, and SV
was due to a G2-induced decrease of total peripheral
resistance (overcoming the increase of cardiac inotro-
pism) and, only in the case of the doses of 1600 and 3200
µg/kg, also to the slight negative chronotropic effect of
the drug. On the other hand, the increase of BP, AF,
and SV induced by 50 and 100 µg/kg G2 was explained
by an increase of cardiac output due to higher cardiac
inotropism. Since BP and AF were reduced by G2 (200
µg/kg) when RF and TV continued to be increased, it
was quite unlikely that the depression of respiratory
function, induced by the highest doses of the drug, was
involved in the arterial vasodilation responsible of both
blood hypotension and reduction of AF and SV. In this
respect, 5 of the 12 rats treated with the dose of 3200
µg/kg died because of respiratory blockade, which
developed within a period of 15-19 s starting from
administration of the drug. Also at the dose of 1600 µg/
kg, G2 caused, in some cases, short periods of respira-
tory blockade, ranging from 23 to 35 s, that disappeared
spontaneously and were not considered in evaluating
the drug-induced depression of respiratory function.
Even when G2 (400-3200 µg/kg) depressed RF and TV,
there was an initial increase of both RF and TV; this
increase started at 4-6 s, reached the maximum within
8-12 s, and disappeared after 17-23 s following iv
administration of the drug. G2 (800-1600 µg/kg) re-
duced RF and TV beginning from 36 ( 4 to 48 ( 5 s
and reached the maximum between 68 ( 4 and 93 ( 5
s; the basal respiratory values were restored within a
respiratory frequency
tidal volume
µL
3 (µg/kg)
beaths/min
%
%
50
+5 ( 1
+6 ( 2
+5 ( 1
+6 ( 2
+743 ( 55
+1106 ( 61
+1442 ( 108
-530 ( 48
-3191 ( 275
-3654 ( 290
-4229 ( 334
+15 ( 2
+22 ( 2
+29 ( 4
-11 ( 2
-64 ( 7
-74 ( 5
-85 ( 5
100
200
400
800
1600
3200
+9 ( 3
+9 ( 2
-3 ( 1
-3 ( 1
-15 ( 2
-61 ( 5
-67 ( 3
-16 ( 2
-64 ( 4
-70 ( 3
baseline
96 ( 7
4970 ( 371
a
Values are means ( SE (n ) 12 for each dose with the
exception of 3200 µg/kg, for which n ) 7).
P h a r m a cology. The cardiovascular and respiratory
effects following intravenous (iv) injection of caracasan-
diamide (G2) in anesthetized rats are reported in Tables
2 and 3, respectively. The drug slightly reduced heart
rate (HR) while increasing maximum rate of rise of the
left ventricular isovolumetric pressure (dP/dt), an index
of cardiac inotropism, in a dose-related manner. The
negative chronotropic effect determined, at the highest
tested dose (3200 µg/kg), only an 11% reduction of basal
HR (i.e., preceding the administration of G2), whereas
cardiac inotropism was markedly augmented by the
drug, reaching a 77% maximum increase. On the other
hand, G2 showed biphasic effects on systolic and dias-
tolic blood pressure (BP), aortic flow (AF), stroke volume
(SV), respiratory frequency (RF), and tidal volume (TV).
In this regard, lower doses of the drug slightly increased
systolic and diastolic BP and AF (50 and 100 µg/kg) as
well as SV, RF, and TV (50-200 µg/kg), whereas higher
doses reduced all these indices in a dose-related manner.
On a molar basis and by the iv route, G2 induced
higher hypotensive effects than (Z)-caracasanamide,
guanethidine, hexamethonium, reserpine, and papav-
erine, being, however, less potent than clonidine and
histamine (Table 4). Furthermore, G2 and (Z)-cara-
casanamide increased cardiac inotropism in contrast to
the other tested drugs, and only (Z)-caracasanamide was
able to increase HR (Table 4). The G2-induced hypoten-
sion lasted much more than that following administra-
tion of guanethidine, histamine, papaverine, hexame-
thonium, and (Z)-caracasanamide. Notably, the dimer
G2 was more potent, as a hypotensive and positive
inotropic agent, than the monomer (Z)-caracasanamide.
However, the latter (50-6400 µg/kg by the iv route in
the rat) did not show biphasic effects on BP, while
having slight and no dose-related tachycardic action as

3120 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Carmignani et al.
Ta ble 4. Cardiovascular Responsesa to Caracasandiamide (3) and Several Antihypertensive or Vasodilating Drugs Administered by
iv Route in Anesthetized Rats
blood pressure
drug
systolic
diastolic
heart rate (beats/min)
dP/dt (mmHg/s)
caracasandiamide (4.12 µM/kg)
(Z)-caracasanamide (4.12 µM/kg)
guanethidine (25 µM/kg)
clonidine (0.108 µM/kg)
hexamethonium (12 µM/kg)
reserpine (8 µM/kg)
-43 ( 2
-24 ( 3
-29 ( 4
-18 ( 2
-48 ( 4
-35 ( 5
-20 ( 3
-28 ( 2
-47 ( 3
-20 ( 2
-22 ( 1
-16 ( 1
-40 ( 4
-29 ( 2
-15 ( 2
-24 ( 3
-35 ( 3
+25 ( 2
-24 ( 3
-53 ( 7
-46 ( 5
-51 ( 6
-18 ( 3
-12 ( 2
+3991 ( 242
+3107 ( 190
-1090 ( 84
-1345 ( 126
-4421 ( 284
-1438 ( 146
-1204 ( 49
-1002 ( 68
papaverine (5 µM/kg)
histamine (0.044 µM/kg)
baseline
113 ( 7
92 ( 6
348 ( 14
6420 ( 374
a
Values are means ( SE (n ) 4 in each group).
period of 348 ( 41 and 408 ( 23 s (n ) 7). The
hypotensive effect of G2 (800-1600 µg/kg) began con-
comitantly to those on dP/dt, AF, and SV (4-5 s),
starting from its iv administration, and increased
progressively, reaching a maximum at the maximal
increase of dP/dt. Both drug-induced hypotension and
positive inotropic effect lasted, in the dose interval 800-
1600 µg/kg, from 378 ( 26 to 439 ( 31 s (n ) 12) and
returned to the values preceding drug administration
within the same times of restoration of AF and SV. The
slight bradycardic response to G2 (800-1600 µg/kg)
developed during the course of the other cardiovascular
effects; however, it began later (24 ( 4 to 33 ( 5 s; n )
12) and disappeared concomitantly to the restoration
of the respiratory parameters. Interestingly, the G2
(800-3200 µg/kg)-induced decrease of BP, AF, and SV
and increase of dP/dt soon reached simultaneously their
maximum value, which was followed by a transient
rebound of BP, AF, and SV almost to the basal values
and by a contemporary marked decrease of dP/dt; during
this rebound phase, HR continued to be slightly reduced,
while RF and TV continued to climb up toward their
basal values; the same rebound phase lasted from 22 (
5 to 41 ( 6 s (n ) 7), and afterward, BP, AF, and SV as
well as dP/dt reached again their maximal negative and
positive increments, respectively. At the end of the
rebound phase, G2 (1600-3200 µg/kg) caused the ap-
pearance of sporadic extrasystolia, which rapidly evolved
to extrasystolic firing lasting from 8 to 36 s and, in some
rats surviving to the respiratory blockade induced by
the dose of 3200 µg/kg, also to periods of ventricular
fibrillation. At last, in some cases, the G2 (800-3200
µg/kg)-induced hypotensive response was followed by a
period (128 - 206 s) of blood hypertension, which was
accompanied by increased TV and HR and by reduced
dP/dt, AF, and SV. It was concluded that G2 is a drug
that increases markedly cardiac inotropism when ad-
ministered by iv route in the rat at doses ranging from
50 to 3200 µg/kg. The positive inotropic effect coexists,
at lower doses, with a moderate increase of BP, AF, SV,
and minute volume and, at higher doses (400-3200 µg/
kg), with increasing reduction of BP, AF, and SV and
with respiratory depression. Only at doses of 1600 µg/
kg or higher, administered by iv injection, G2 may
induce reversible or irreversible respiratory blockade,
respectively, with occurrence of ventricular arrhythmias
including fibrillation.
the actions on HR, RF, and TV. Moreover, the few
seconds required for inducing cardiovascular effects
indicated that the drug acts at the cardiac and vascular
levels in determining increase of the inotropism and
increase (at lower doses) and reduction (at higher doses)
of the other cardiovascular indices. According to these
observations, there were no central neurogenic compo-
nents in the cardiovascular effects of G2: either bilat-
eral vagotomy or spinalization (under vagotomy) did not
change cardiovascular responses to the drug (200-1600
µg/kg, ratio ) 2.0, iv route) which, by itself, failed to
change the positive chronotropic and inotropic as well
as pressor responses to bilateral carotid occlusion (BCO).
On the other hand, G2 (400-1600 µg/kg, ratio ) 2.0, iv
route) antagonized cardiovascular responses to iv no-
radrenaline and adrenaline (each at the dose of 1 µg/
kg), while increasing those of iv acetylcholine (2.50 µg/
kg) and, mostly, iv isoprenaline (0.625 µg/kg); these
effects were related to the doses of G2 and remained
for at least over 60 min following its administration
(Table 5). Moreover, phenylephrine (10 µg/kg/min, a
selective R1-adrenoreceptor agonist) did not influence
cardiovascular effects of G2 (400-1600 µg/kg, ratio )
2.0, iv route), while papaverine, lowering BP by relaxing
arterial myocells (through a phosphodiesterase-depend-
ent reduced availability of calcium ions for contractile
mechanisms), reduced significantly only the diastolic
hypotensive effect of G2 (Table 6). Similarly, reserpine,
depleting the catecholamine content in both central and
peripheral adrenergic synapses, strongly reduced car-
diovascular effects of G2, while, on the contrary, the
ganglion blockade by the anticholinergic drug hexam-
ethonium potentiated the same effects of G2 (Table 6).
Conversely, these effects of G2 were not changed by
pretreatment with the R1,2-adrenoreceptor-blocking drug
phentolamine (2 mg/kg, iv route). All the above tested
drugs were ineffective in changing significantly the
respiratory effects of G2. On the whole, these results
showed that G2 does not affect the central sympathetic
and parasympathetic tone (spinalization, vagotomy) as
well as the baroreflex activity (BCO) and does not
interact with the cardiovascular R1- and R2-adrenore-
ceptors (phenylephrine, phentolamine). On the other
hand, considering that reserpine (depleting the content
of catecholamines at both postganglionic adrenergic
endings and central adrenergic presynaptic terminals
by blocking their accumulation into storage vesicles)
strongly opposed the G2-induced reduction of BP, AF,
and SV, it was deduced that this guanidine compound
acts presynaptically at the peripheral (cardiovascular)
As far as the mechanisms of action of G2 were
concerned, it was noted that the effects of this drug on
BP, dP/dt, AF, and SV preceded and lasted more than

Novel Hypotensive Agents from V. caracasana
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3121
Ta ble 5. Cardiovascular Responsesa to iv Doses of Physiological Agonists Before (Control) and After Administration of
Caracasandiamide (3) in Anesthetized Ratsb
blood pressure (mmHg)
agonist
3 (µg/kg)
systolic
diastolic
heart rate (beats/min)
dP/dt (mmHg/s)
noradrenaline (1 µg/kg)
-
400
1600
-
400
1600
-
400
1600
-
400
1600
+43 ( 3
+31 ( 2*
+24 ( 2*
+25 ( 3
+21 ( 2
+14 ( 1*
-20 ( 2
-36 ( 3*
-43 ( 4*
-25 ( 1
-27 ( 2
-38 ( 4*
+25 ( 2
+15 ( 1*
+10 ( 1*
+16 ( 3
+5 ( 1*
+3 ( 1*
-31 ( 3
-49 ( 3*
-66 ( 5*
-31 ( 2
-45 ( 3*
-54 ( 3*
+28 ( 3
+23 ( 2
+13 ( 2*
+41 ( 3
+24 ( 3*
+7 ( 2*
+46 ( 4
+69 ( 5*
+79 ( 5*
-27 ( 4
-30 ( 5
-34 ( 5
+5564 ( 420
+5136 ( 364
+2225 ( 174*
+8416 ( 147
+6848 ( 240*
+2739 ( 168*
+9844 ( 704
+13696 ( 975*
+14552 ( 1021*
-4451 ( 172
-5820 ( 304*
-6821 ( 491*
adrenaline (1 µg/kg)
isoprenaline (0.625 µg/kg)
acetylcholine (2.50 µg/kg)
a
b
Values are means(SE (n ) 8 in each group). *p < 0.05 (compared with the control mean). Cardiovascular responses to the agonists
were repeated beginning 30 min after administration of each dose of caracasandiamide.
Ta ble 6. Cardiovascular Responsesa to iv Doses of Caracasandiamide (3) Before (Control), During, and After Administration of
Papaverine, Reserpine, and Hexamethonium in Anesthetized Rats
blood pressure (mmHg)
treatment
3 (µg/kg)
systolic
diastolic
heart rate (beats/min)
dP/dt (mmHg/s)
control
200
400
800
1600
800
1600
800
1600
-
-11 ( 2
-16 ( 2
-26 ( 3
-36 ( 3.5
-22 ( 4
-33 ( 4
-11 ( 2*
-16 ( 5*
-25 ( 3*
-41 ( 5*
-50 ( 5*
-13 ( 1
-16.5 ( 3
-27.5 ( 2
-40 ( 3
-16 ( 3*
-29 ( 2*
-12 ( 3*
-15 ( 3*
-27 ( 2*
-45 ( 6*
-53 ( 3*
-15 ( 3
-17 ( 3
-22 ( 2
-36 ( 5
-17 ( 3
-31 ( 4
-9 ( 1*
-14 ( 4*
-34 ( 4*
-46 ( 4*
-57 ( 5*
+1129 ( 152
+2436 ( 205
+3154 ( 271
+3597 ( 299
+2967 ( 147
+3321 ( 168
+720 ( 57*
+1238 ( 81*
+3424 ( 212*
+3938 ( 181*
+4965 ( 322*
papaverine (1 mg/kg/min, iv route)
reserpine (5 mg/kg/day, for 2 days, ip route)
hexamethonium (5 mg/kg, iv route)
400
800
a
Values are means ( SE (n ) 6 in each group). *p < 0.05 (compared with the control mean).
sympathetic synapses through a reserpine-like mecha-
nism.18 In this regard, a central neurogenic “reserpine-
like” mechanism of G2 was excluded since the inter-
ruption of the central sympathetic pathways by spinali-
zation failed to change the effects of this drug. An
explanation of such conclusion may be that G2, differ-
ently from (Z)-caracasanamide and similarly to guanethi-
dine and guanadrel, does not cross easily the blood-
brain barrier.19 Moreover, it is interesting to note that
the guanidine derivative guanethidine (not crossing this
barrier) is concentrated within the neurosecretory vesicles
of the postganglionic sympathetic endings, where it
replaces noradrenaline; then, this drug depletes the
normal transmitter with a transient increase of its
release which is responsible, for example, for transient
blood hypertension; at large acute doses, guanethidine
also appears to block excitation-secretion coupling (“brety-
lium-like” mechanism).20 These mechanisms of action
of guanethidine seem to well explain the biphasic effects
of G2 on cardiovascular function: i.e., lower doses may
cause a transient increase of the release of noradrena-
line, whereas higher doses may first oppose this release
by a “bretylium-like” effect and then deplete the normal
transmitter (“reserpine-like” effect). As compared to (Z)-
caracasanamide, only partially lipophilic in the cis
form,13,21 G2 has a double molecular weight, a higher
steric hindrance, and a lower molecular flexibility.
On the basis of the above considerations, the antago-
nistic effect of G2 on the cardiovascular responses to
noradrenaline and adrenaline (the latter having been
used at a dose activating prevalently the R1- and R2-
adrenoreceptors) may be explained only through inter-
actions of G2 with the intracellular biochemical path-
ways transducing, in the cardiac and vascular myocells,
the R-adrenoreceptor activation toward the related
functional effects. The same interpretation may be given
for explaining the potentiating effects of G2 on the
cardiovascular responses to acetylcholine (activating the
M-cholinergic receptors) and isoprenaline (activating the
cardiac â1- and vascular â2-adrenoreceptors). In this
respect, (Z)-caracasanamide was found to increase HR
and dP/dt also by interacting at the cardiac â1-adreno-
receptors.12 Moreover, the ability of papaverine to
reduce the diastolic hypotensive response to G2 seems
to confirm a “transductional” level of action of this drug
in the vascular myocells, i.e., on the adenosine cyclic
3′,5′-monophosphate (cAMP)-dependent availability of
calcium ions for contractile mechanisms (reduced by
papaverine).22 With regard to this, activation of the â2-
adrenoreceptors leads to increased levels of cAMP by
stimulation of adenylyl cyclase, thus causing reduced
availability of the free calcium in vascular myocells.
Conversely, in the heart, stimulation of the â1-adreno-
receptors increases cAMP, which enhances phosphory-
lation of contractile proteins and activates voltage-
sensitive calcium channels in the plasma membrane,
thus leading to positive inotropic and chronotropic
responses.23 On the other hand, stimulation of the
cardiac and vascular M(M2,M4)-cholinergic receptors
results in activation of receptor-operated potassium
channels, increased levels of guanosine cyclic 3′,5′-
monophosphate (cGMP, reducing the availability of free
calcium) with consequent lower or inhibited activity of
adenylyl cyclase, and, possibly, suppression of the

3122 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Carmignani et al.
activity of receptor-operated calcium channels, thus
causing negative inotropic and chronotropic responses,
and vasodilation.24,25 Finally, activation of the R(R1,R2)-
adrenoreceptors in the cardiac and vascular myocells
ultimately causes contraction as a result of increased
concentrations of intracellular calcium (mainly depend-
ing on inhibition of adenylyl cyclase, stimulation of
phospholipases, increased polyphosphoinositide hydrol-
ysis, and activation of protein kinase C).23,26 Since
phenylephrine (R1-adrenoreceptor agonist) did not alter
the effects of G2, it may be thought that this drug
interacts, in opposing the responses to noradrenaline
and adrenaline, only with transductional pathways
coupled to activation of the cardiac and vascular R2-
adrenoreceptors.
ceptors, while potentiating the â1-adrenoreceptor-de-
pendent cardiac reactivity.12 (Z)-Caracasanamide re-
duces central sympathetic tone and baroreflex reactivity
and relaxes vascular myocells by a direct effect not
involving a â2-adrenoreceptor component.12 G2 is not
provided with central neurogenic effects, while also
having the above vascular â2-adrenoreceptor-like and
cardiac M-cholinoreceptor-like components.
As observed with (Z)-caracasanamide, the biphasism
of the respiratory effects of G2 is not related to the
cardiovascular ones. In fact, G2 increases dP/dt and
decreases HR in the presence of either stimulation or
depression of RF and TV. Moreover, the cardiovascular
effects of G2 precede and last more than those on RF
and TV. Only at subtoxic or toxic doses (like 1600 and
3200 µg/kg, respectively), greatly depressing breathing,
G2 reverses for a short period the reduction of BP, AF,
and SV. The relatively long latency time for observing
the effects of G2 on breathing is likely to depend on the
difficulty to cross the blood-brain barrier and to reach
significant concentrations in the central nervous system.
As for (Z)-caracasanamide, the obtained data do not
allow us to hypothesize on the mechanisms by which
G2 stimulates, and then depresses, the central respira-
tory pathways, in the latter case with consequent
cardiac arrhythmias and possible cardiac arrest.
G2 is the second compound of a series of six guanidine
derivatives, isolated from Verbesina caracasana, for
which a pharmacological profile has been defined. A
structure-activity relationship among all these com-
pounds will be established when the pharmacological
studies of the remaining four drugs have been com-
pleted. The present study shows that, at low dosage
(50-100 µg/kg, by iv route), G2 is a positive inotropic
drug increasing cardiac output without significant chro-
notropic effects. At dosages ranging from 200 to 800 µg/
kg, G2 becomes a hypotensive drug of mild potency not
affecting significantly the chronotropism and mantain-
ing a high positive inotropic action. At doses of 1600
µg/kg or higher, G2 is a hypotensive compound of high
potency that, despite the high positive inotropic effect
and the poor chronotropic action, may associate effects
of excessive respiratory depression leading to severe
cardiac arrhythmias. As a cardiovascular agent, G2
appears to act preponderantly by peripheral mecha-
nisms of action.
Having excluded central neurogenic components (sym-
pathetic, parasympathetic, baroreflex) in the cardiac
effects of G2, these effects then appear to be related,
more than to direct actions of this drug on receptors, to
modulations on peripheral transductional pathways
physiologically activated by â1- and â2-adrenoreceptors
as well as R2-adrenoreceptor and M(2,4)-cholinoreceptor
agonists (adrenaline, noradrenaline, and acetylcholine,
respectively).23-27 The peripheral reserpine-like compo-
nent of G2 may only be involved in the duration of the
effects of G2 concerning the reduction of BP, HR, AF,
and SV, but not in the induction of the same effects
because reserpine requires some time for depleting the
catecholamine content in the postganglionic sympathetic
endings.18 In this way, “lower” and “higher” doses of G2
are likely to modulate the above transductional path-
ways in a quantitatively different manner. So, a “â1-
adrenoreceptor-like component” may explain, through
an increased cardiac inotropism, the lower dose-induced
increase of BP, dP/dt, AF, and SV; this component may
also explain the increase of dP/dt induced by higher
doses of G2, as was found earlier for (Z)-caracasan-
amide.12
Analogously,
“â2-adrenoreceptor-
and
M-cholinoreceptor-like components” as well as an “R2-
antagonistic-like” component are likely to cooperate in
contributing (by systemic arterial vasodilation) to de-
crease total peripheral resistance (to levels overcoming
the increase of cardiac inotropism) and, only the cho-
linergic component, to reduce HR. Moreover, the in-
creased effects of G2 during the hexamethonium-
induced ganglionic blockade (reducing or impeding the
release of catecholamines from adrenals into blood and
from sympathetic endings in both heart and vessels as
well as from parasympathetic endings in the heart) may
be referred to the above R2-, â1-, and â2-adrenoreceptor
antagonistic (R2) and -like (â1,â2) components (involved
in either vasodilation or the positive inotropic effect) as
well as to the above M-cholinoreceptor-like component
(involved in the negative chronotropic response) through
a mechanism of receptor hypersensibility (up-regula-
tion) due to the reduced levels of both adrenergic and
cholinergic neurotransmitters at the autonomous neu-
roeffector synapses.23,28-30
Exp er im en ta l Section
Gen er a l. Melting points were determined with a Gellen-
kamp apparatus and are uncorrected. The NMR spectra were
determined with a Varian Gemini 300 spectrometer (300 MHz)
using tetramethylsilane as internal standard. IR spectra
were recorded on a Perkin Elmer 298 instrument. Electron
impact (EIMS) spectra were recorded with a Kratos MS 80
instrument. High-resolution FABMS were determined on a
VG7070EQ spectrometer. Microanalyses were performed by
Dipartimento Farmaco Chimico Tecnologico, Siena, Italy.
P la n t Ma ter ia l. Leaves of V. caracasana were collected in
Valencia (Venezuela). A voucher specimen was deposited at
the herbarium of the Departamento de Physiologia Vegetal,
Universidad Central de Venezuela, Maracaibo (Venezuela).
Extr a ction a n d F r a ction a tion . The crude extract (26 g)
from leaves (5 kg), obtained as described in our previous
paper,12 was fractionated on silica gel with chloroform-
methanol mixtures. The second fraction, eluted with CHCl3-
MeOH, 8:2, gave caracasandiamide (2; 1.2 g) after extensive
chromatographic separation.
Between (Z)-caracasanamide and G2 there are phar-
macodynamic analogies and differences, besides differ-
ences in potency and duration of action (as hypotensive
and positive inotropic drugs) and in the quality of some
effects (on HR and respiratory function). Both drugs do
not interact with the peripheral R1- and R2-adrenore-

Novel Hypotensive Agents from V. caracasana
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16 3123
3â,4â-Bis(3′,4′-d im eth oxyp h en yl)-1r,2r-bis[[N-(3-m eth -
y lb u t -2-e n y l)g u a n i d i n o ]b u t y l]c y c lo b u t a n e c a r b o x -
a m id e (ca r a ca sa n d ia m id e, 2): foam; IR νmax 3320, 3220,
(20 mL), and the mixture was held at reflux for 6 h, concen-
trated to a small volume, and diluted with H2O. The solution
was washed with EtOAc, acidified with concd HCl to pH 2,
and extracted with EtOAc. The organic layer was washed with
water, dried (Na2SO4), and evaporated to give the product 5
(383 mg, 97%; homogeneous on TLC with 1% AcOH in
EtOAc): glassy solid, softens at 99-125 °C; IR (CHCl3) νmax
1655, 1638, 1515, 1260, 1205, 1138, 1020 cm-1 1H and 13C
;
NMR in Table 1; FABMS m/z (rel int) 777 [MH]+ (100), 747
[M - OCH2]+ (6), 709 [MH - C5H8]+ (11), 650 [M - pre-
nylguanidine]+ (3), 596 [M - prenylguanidinobutane]+ (3), 579
[MH - prenylagmatine]+ (25), 389 [a]+ (15), 300 [b]+ (8), 191
[ArCHdCHCO]+ (57).
1720 cm-1 1H NMR (CDCl3) δ 9.84 (2H, br s, COOH), 6.66
;
(2H, d, J ) 8.3 Hz, H-5′), 6.56 (2H, dd, J ) 8.3 and 2 Hz, H-6′),
6.32 (1H, d, J ) 2 Hz), 4.33 (2H, d, J ) 6.3 Hz, H-a), 3.84 (2H,
d, J ) 6.3 Hz), 3.77 (6H, s, 4′-OMe), 3.61 (6H, s, 3′-OMe); EIMS
m/z (rel int) 416 [M]+ (2), 300 [b]+ (100), 208 [a]+ (100), 191 [a
- OH]+ (62). Anal. (C22H24O8) C, H.
Meth yl a n d Eth yl Der iva tives of 7. The diacid 7, when
treated with CH2N2, gave the dimethyl ester 5, identical with
an authentic sample of the hydrolysis product, whereas with
catalytic H2SO4/EtOH it afforded again the diethyl ester 5a .
Hyd r olysis of Ca r a ca sa n d ia m id e. A solution of cara-
casandiamide (300 mg) and 2 N NaOH in MeOH (15 mL) was
left under stirring for 4 days. The reaction mixture was poured
into water, and MeOH was evaporated.The aqueous solution
was acidified with concd H2SO4 and extracted with EtOAc
(×3). The residue of the pooled organic extracts, after purifica-
tion on silica gel with CHCl3-MeOH, 99:1, gave the monoa-
mide 3 (158 mg). The H2O lyophilysate by chromatography
on LH-20 with MeOH gave 1-amino-4-[(3-methyl-2-butenyl)-
guanidino]butane acetate (4) (53 mg) and prenylurea (12 mg).
3â,4â-Bis(3′,4′-d im eth oxyp h en yl)-1r-ca r boxy-2r-[[N-(3-
m eth ylbu t-2-en yl)gu a n id in o]bu tyl]cyclobu ta n eca r box-
a m id e (3): mp 179-180 °C; IR νmax 3320, 3220, 1655, 1638,
1515, 1260, 1205, 1138, 1020 cm-1; 1H and 13C NMR in Table
1; FABMS m/z 597 MH+ (71), 389 [a]+ (40), 300 [b]+ (20), 297
[b′]+ (10), 207 [a′]+ (46), 191 [ArCHdCHCO]+ (100); HRMS
found 279.1594, C16H23O4 requires 279.1596.
N-(n -P en t yl)-3â,4â-b is(3′,4′-d im et h oxyp h en yl)-1r,2r-
cyclobu ta n ed ica r boxim id e (8). n-Pentylamine (0.02 mL, 0.2
mmol) was added to a refluxing solution of 7 (62 mg, 0.15
mmol) and CDI (71 mg, 0.44 mmol) in dry 1,4-dioxane. After
heating for 8 h, the reaction mixture was cooled to room
temperature; further CDI (35 mg, 0.22 mmol) was added and
refluxed overnight. After removal of the solvent the residue
was dissolved in EtOAc. The solution was washed with 1 N
HCl, 10% NaHCO3, and water, then concentrated to give an
oil, which was purified by column chromatography on silica
gel with CH2Cl2-EtOAc, 1:1, to give 8 (56 mg, 80%): mp 93-
1-Am in o-4-[(3-m eth ylbu t-2-en yl)gu a n id in o]bu ta n e a c-
eta te (p r en yla gm a tin e, 4): oil; IR (KBr) νmax 3450, 3360,
95 °C (Et2O); IR (Nujol) νmax 1700 cm-1 1H NMR (CDCl3) δ
;
1665, 1310, 1150, 838 cm-1 1H and 13C NMR in Table 1;
;
FABMS m/z 199 MH+.
6.69 (2H, d, J ) 8.4 Hz, H-5′), 6.60 (2H, dd, J ) 8.3 and 1.5
Hz, H-6′), 6.32 (1H, d, J ) 1.5 Hz), 4.03 (2H, d, J ) 3.8 Hz,
H-R), 3.79 (6H, s, 4′-OMe), 3.62 (4H, m, H-â, N-CH2), 3.60 (6H,
s, 3′-OMe) 1.67 (2H, m, CH2), 1.36 (4H, m, 2xCH2), 0.92 (3H,
t, J ) 7 Hz, Me); EIMS m/z (rel int) 467 [M]+ (22), 300 [b]+
(46), 167 [b]+ (100). Anal. (C27H33NO6) C, H, N.
Acid ic Hyd r olysis. The monoamide 3 (100 mg) was re-
fluxed in 2 N H2SO4 (MeOH, 1 mL) for 3 h. After evaporation
of MeOH, the reaction mixture was extracted with EtOAc. The
residue of the organic layer chromatographed on silica gel with
CHCl3 gave methyl ester 5a (42 mg). The aqueous layer was
lyophilized and chromatographed on Sephadex LH-20 to give
prenylagmatine (4) (21 mg) and prenylurea (6 mg).
Tetr a k is(Boc-ca r a ca sa n d ia m id e) (2a ). CMC metho-p-
toluenesulfonate (93 mg, 0.22 mmol) was added to a solution
of 5 (45 mg, 0.11 mmol) and protected prenylagmatine 4a (90
mg, 0.22 mmol) in dry CH2Cl2 (8 mL). The mixture was stirred
at room temperature for 2 h, then washed with water, dried
(Na2SO4), and concentrated under reduced pressure. The
residue, by column chromatography on silica gel with EtOAc,
afforded 2a (39 mg, 30%; homogeneous on TLC with EtOAc):
Meth yl 3â,4â-bis(3′,4′-d im eth oxyp h en yl)-1r,2r-cyclo-
1
bu ta n ed ica r boxyla te (5): oil; H and 13C NMR in Table 1;
EIMS m/z (rel int) 444 [M]+ (9), 413 [M - OMe]+ (49), 300
[b′]+ (100), 285 [b′ - Me]+ (32), 222 [a]+ (100), 207 [a - Me]+
(100), 191 [a - OMe]+ (100); m* 351.5 (413 f 381), 270.8 (300
f 285), 193.0 (222 f 207), 164.3 (222 f 191).
1
glassy solid; IR (Nujol) νmax 3250, 1720, 1640 cm-1; H NMR
Syn t h esis. E t h yl 3â,4â-Bis(3′,4′-d im et h oxyp h en yl)-
1r,2r-cyclobu ta n ed ica r boxyla te (5a ). Powdered ethyl (E)-
3,4-dimethoxycinnamate (6a ) was dissolved in the minimum
volume of CH2Cl2 and spread between pairs of glass plates
(20 × 20 cm). After complete evaporation of the solvent, the
plates were irradiated for 6 h with a 125-W mercury lamp
(Hanovia) at room temperature. The oily reaction mixture by
silica gel column and preparative TLC chromatographies
(hexane-EtOAc, 3:2) afforded 5a (100 mg, 40%): oil; IR
(CDCl3) δ 7.12 (2H, t, J ) 8 Hz, exchg D2O, NHCO), 6.68 (2H,
br s, H-2′), 6.65 (1H, d, J ) 8 Hz, H-5′), 6.55 (2H, br d, J )
8Hz, H-6′), 5.45 (2H, t, J ) 7.5 Hz, exchg D2O, NH), 5.15 (2H,
dd, J ) 10 and 6 Hz, H-2′′), 4.22 (4H, m, H-R, H-â), 3.76 (6H,
s, 4′-OMe), 3.60 (6H, s, 3′-OMe), 3.23 (8H, br t, J ) 7 Hz, H2-
1, H2-4), 3.04 (4H, m, H2-1′′), 1.70 (12H, br s, 2 x Me), 1.69-
1.56 (8H, m, CH2), 1.49, 1.45 (18H each, s, Me3); FABMS
(TDEG/Gly) m/z 1178 [MH]+. Anal. (C62H96N8O14) C, H, N.
1
(CHCl3) νmax 1720 cm-1; H NMR (CDCl3) δ 6.65 (2H,d, J )
Ca r a ca sa n d ia m id e (2). A mixture of 11 (20 mg, 0.017
mmol) and methanesulfonic acid (0.2 µL) in dry 1,4-dioxane
(1 mL) was refluxed under N2 for 4 h. The residue from
evaporation, by preparative TLC (CHCl3-MeOH, 8:2), gave
caracasandiamide (8 mg, 61%), identical with an authentic
sample of the natural product. Anal. (C42H64N8O6) C, H, N.
P h a r m a cology. An im a ls. Adult male Wistar rats, weigh-
ing 299 + 3 g (mean + SE; n ) 118), were housed in stainless
steel cages and fed a standard laboratory diet. They received
“ad libitum” deionized drinking water and were kept undis-
turbed for 2 weeks in controlled conditions of dampness, light,
temperature, and noise.
Ca r d iova scu la r a n d Resp ir a tor y Deter m in a tion s. Rats
were anesthetized with 10% (w/v) ethylurethane (1 mL/kg of
body weight), which was dissolved in 0.9% NaCl solution
(saline) and administered with a single ip injection. The
trachea was cannulated to allow spontaneous breathing.
Polyethylene catheters (PE 20 tubing) were placed in the left
femoral artery for recording aortic blood pressure (BP) and
into the femoral veins for drug administration. A calibrated
3F catheter-tip pressure transducer (Millar Instruments,
Houston, TX), inserted in the right common carotid artery and
8.3 Hz, H-5′), 6.58 (2H, dd, J ) 8.3 and 2 Hz, H-6′), 6.30 (1H,
d, J ) 2 Hz), 4.25 (2H, m, H-R), 4.14 (4H, q, J ) 7 Hz, CH2),
3.73 (6H, s, 4′-OMe), 3.72 (2H, m, H-â), 3.58 (6H, s, 3′-OMe),
1.24 (6H, t, J ) 7 Hz, Me); EIMS m/z (rel int) 472 [M]+ (2),
427 [M - OEt]+ (5), 300 [b]+ (100), 236 [a]+ (100), 221 [a -
Me]+ (6), 191 [a - OEt]+ (100); m* 340.0 (427 - 381), 207.0
(236 - 221), 154.5 (236 - 191). Anal. (C26H32O8) C, H.
Ben zyl 3â,4â-Bis(3′,4′-d im eth oxyp h en yl)-1r,2r-cyclo-
bu ta n ed ica r boxyla te (5b). Benzyl (E)-3,4-dimethoxycin-
namate (6b) by the same procedure as above (but with 20-h
irradiation time) gave 5b (38%): oil; IR (CHCl3) νmax 1720 cm-1
;
1H NMR (CDCl3) δ 7.33 (10H, br s, Ph), 6.65 (2H, d, J ) 8.3
Hz, H-5′), 6.54 (2H, dd, J ) 8.3 and 2 Hz, H-6′), 6.33 (1H, d,
J ) 2 Hz), 5.05 (4H, m, OCH2), 4.34 (2H, m, H-R), 3.82 (6H, s,
4′-OMe), 3.79 (2H, m, H-â), 3.59 (6H, s, 3′-OMe); EIMS m/z
(rel int) 596 [M]+ (2), 505 [M - OBn]+ (5), 300 [b]+ (100), 298
[a]+ (100), 283 [a - Me]+ (6), 191 [a - OBn]+ (100). Anal.
(C36H36O8) C, H.
3â,4â-Bis(3′,4′-d im eth oxyp h en yl)-1r,2r-cyclobu ta n ed i-
ca r boxylic Acid (7). Ethyl ester 5a (447 mg, 0.95 mmol) was
added to a solution of KOH (533 mg, 9.5 mmol) in 95% EtOH

3124 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 16
Carmignani et al.
advanced in the left ventricle, was used for determining the
maximum rate of rise of the left ventricular isovolumetric
pressure (dP/dt), an index of cardiac inotropism.31,32 Systolic
BP and diastolic BP were measured by a P23Db Statham
pressure transducer (Statham Medical Instruments, Los An-
geles, CA) and averaged electronically. Heart rate (HR) was
obtained by a 9875B Beckman cardiotachometer coupler
(Beckman Instruments, Inc., Schiller Park, IL), which was
erine (1 mg/kg/min), and after treatment with reserpine (5 mg/
kg/day for 2 days, by ip route) or hexamethonium (5 mg, iv
route).
Thirty-two rats were randomly divided into eight groups in
order to compare on a molar basis the cardiovascular effects
of some antihypertensive or vasodilating drugs with those of
G2. These rats received, by iv injection under the above
experimental conditions, (Z)-caracasanamide (4.12 µM/kg of
body weight), G2 (4.12 µM/kg), guanethidine (25 mM/kg),
clonidine (0.108 mM/kg), hexamethonium (12 mM/kg), reser-
pine (8 mM/kg), papaverine (5 mM/kg), or histamine (0.044
mM/kg). All drugs were dissolved in saline solution, and all
doses were expressed in terms of free bases. The control
administration of solvent alone caused insignificant changes
in both cardiovascular and respiratory parameters. Peak
effects were considered for each assay. Each of the consecutive
tests was not made until the parameters had returned to the
values preceding the previous administration of (Z)-cara-
casanamide and had stabilized.
triggered by the R-peak of the lead II electrocardiogram.33
A
Biotronex derivative computer (model BL622; Biotronex Labo-
ratories, Inc., Kensington, MA) was used for determining dP/
dt, by differentiating the pulsatile BP registered in the left
ventricle. The computer was adjusted to minimize the expres-
sion of preload and afterload, as previously described.34 The
unit of measurement of dP/dt was mmHg/s.
Systolic BP, diastolic BP, or mean aortic flow (AF) was
measured electromagnetically by inserting, after having per-
formed a laparatomy of about 3 cm, a probe (1-mm i.d.) around
the abdominal aorta (about 1 cm above iliac bifurcation), using
a Statham K2002 flowmeter. To obtain an index of stroke
volume (SV), the pulsatile AF was integrated by using a BL620
Biotronex apparatus.35 BP, HR, dP/dt, AF, and SV were
continuously monitored on a Beckman RM dynograph re-
corder.36 The body temperature of the animals was kept
constant at 37 °C by using an electrically heated table. Each
rat received by iv route 1 mL of 0.9% saline solution containing
100 USP of sodium heparin.
Sta tistics. Data were expressed as means ( SE and
compared by analysis of variance.38 Only a P value less than
0.05 was considered to be significant.
Ack n ow led gm en t. Three of us (M.B., F.C., and
A.T.) wish to thank the University of Siena for financial
support (60% funds). M.B. also thanks the Merck
Research Laboratories (Academic Development Program
in Chemistry) for a grant. M.C. and A.R.V. were
supported by grants from the Italian Ministry for
University and Scientific and Technological Research
(40% funds, 1995, 1996) and Italian CNR (1995, 1996).
Respiration was monitored by means of a pneumot-
achograph adapted to a Biotronex BL 620 integrator to yield
the full respiratory wave.37 Respiratory frequency (RF) and
tidal volume (TV) were assessed under spontaneous breathing
by connecting the tracheal cannula to the pneumotachograph.
RF and TV were monitored polygraphycally along with the
cardiovascular parameters. After completion of the surgical
procedure, the rats were kept undisturbed for 60 min to allow
for the stabilization of all cardiovascular and respiratory
parameters.
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