Novel hypotensive agents from Verbesina caracasana. 8. Synthesis and pharmacology of (3,4-dimethoxycinnamoyl)-N1-agmatine and synthetic analogues

Article abstract of DOI:10.1021/jm001017v

The more polar metabolites from the Venezuelan plant Verbesina caracasana, i.e., N³-prenylagmatine, (3,4-dimethoxycinnamoyl)-N¹-agmatine, agmatine, and galegine (prenylguanidine), previously reported (Delle Monache, G.; et al. BioMed

Full text of DOI:10.1021/jm001017v

2950
J . Med. Chem. 2001, 44, 2950-2958
Novel Hyp oten sive Agen ts fr om Ver besin a ca r a ca sa n a . 8. Syn th esis a n d
P h a r m a cology of (3,4-Dim eth oxycin n a m oyl)-N1-a gm a tin e a n d Syn th etic
An a logu es1
Marco Carmignani,*,† Anna Rita Volpe,‡ Bruno Botta,*,§ Romulo Espinal,| Stella C. De Bonnevaux,|
Carlo De Luca, Maurizio Botta,*,∇ Federico Corelli,∇ Andrea Tafi,∇ Rosario Sacco,O 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, Dipartimento Farmaco
Chimico Tecnologico, Universita` di Siena, Siena, Italy, and Istituto di Chirurgia, Universita` di Catanzaro, Catanzaro, Italy
Received J une 26, 2000
The more polar metabolites from the Venezuelan plant Verbesina caracasana, i.e., N3-
prenylagmatine, (3,4-dimethoxycinnamoyl)-N1-agmatine, agmatine, and galegine (prenylguani-
dine), previously reported (Delle Monache, G.; et al. BioMed. Chem. Lett. 1999, 9, 3249-3254),
have been synthesized following a biosynthetic strategy. The pharmacologic profiles of various
synthetic analogues of (3,4-dimethoxycinnamoyl)-N1-agmatine (G5) were also analyzed, to shed
some light on the structure-activity relationship of these compounds. Derivatives with the
(E)-configuration and/or with a p-methoxybenzoyl moiety were found to be responsible for higher
hypotensive effects, which were associated with a slight and, in some cases, not dose-related
increase of cardiac inotropism, with variable and not significant chronotopic responses, and,
only at higher doses, with effects of respiratory depression. Either an increase (to six) or a
decrease (to two) of the number of methylene groups in the alkyl chain of (E)-G5 did not change
blood pressure responses, while slightly increasing the positive inotropic ones. At pharmacologi-
cal doses, all the studied compounds showed hypotensive and slight positive inotropic effects
without relevant chronotropic and respiratory actions.
In tr od u ction
structures of guanidine derivatives may lead to wide
variations of activity.16-18
Many synthetic guanidine derivatives have attracted
pharmacologists in search of new antihypertensive
drugs for their ability to block adrenergic nerve activity
through central and/or peripheral mechanisms.2-4 As
a result, compounds such as guanethidine,5 guanabenz,6
guanfacine,7 and pinacidil8 have been introduced in
antihypertensive drug therapy. In this respect, while
guanethidine induces arterial hypotension only by
peripheral mechanisms including depletion of norad-
renaline in the postganglionic adrenergic endings,9,10 the
long-lasting hypotensive effects of guanabenz and guan-
facine are due to a central mechanism, analogous to that
of clonidine and involving R2-adrenoreceptors in the
central sympathetic pathways.11,12 Conversely pinacidil,
devoid of actions in the central nervous system, is able
to lower blood pressure through arterial vasodilation not
related to changes of the peripheral adrenergic nerve
activity13,14 but depending on its activity as a potassium
channel opener.15 A series of studies on structure-
activity relationships revealed that small changes in the
A crude methanol extract of the Venezuelan plant
Verbesina caracasana Fries (Compositae), intravenously
administered to mice, was found to induce biological
effects such as erection of hair and initial stimulation
and subsequent blockage of breathing. Biologically
controlled purification, culminating in silica gel chro-
matography, yielded a series of active compounds, the
least polar of which was named caracasanamide (G1)
and assigned the structure 1-[(3,4-dimethoxycinnamoy-
l)amino]-4-[(3-methyl-2-butenyl)guanidino]butane (1),
as a mixture of (Z)- and (E)-forms.19 The pharmacologi-
cal profile of the water-soluble (Z)-form and the syn-
thesis of the (E)-form of caracasanamide (1) have been
reported in a previous paper.20 A second metabolite,
named caracasandiamide (G2), was isolated and shown
to be the truxinic-type dimer of 1.21 Similarly to, and
more strongly than, G1, G2 may be considered a
hypotensive and an antihypertensive drug, devoid of the
negative side effects, e.g., the reflex tachycardia and
decreased cardiac inotropism, shown by the majority of
the antihypertensive and vasodilator drugs.22
* To whom correspondence should be addressed. (G.D.M.) Phone:
+39 06 3057612. Fax: +39 06 3053598. E-mail: g.dellemonache@
uniserv.ccr.rm.cnr.it.
Four other hypotensive agents, namely, G3, G5, G6,
and G7, have been isolated and assigned the structures
N3-prenylagmatine (2), (E)-(3,4-dimethoxycinnamoyl)-
N1-agmatine (3), agmatine (4), and galegine (prenylguani-
dine, 5), respectively.23 This paper deals with the
synthesis of the above components of the methanol
extract of V. caracasana. In an effort to shed some light
† Universita` di L’Aquila.
‡ Universita` Cattolica.
§ Universita` La Sapienza.
| Universidad de Carabobo.
Centro Elettrochimica e Chimica Fisica delle Interfasi.
∇ Universita` di Siena.
O Universita` di Catanzaro.
10.1021/jm001017v CCC: $20.00 © 2001 American Chemical Society
Published on Web 08/04/2001

Novel Hypotensive Agents from Verbesina caracasana
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2951
Sch em e 1a
on structure-acivity relationships for these compounds,
the pharmacological profiles of G5 and a number of
synthetic analogues were studied.
Resu lts a n d Discu ssion
Str u ctu r e. Extended chromatography of the more
polar fractions of the extract20 afforded the metabolites
G3, G5, G6, and G7, named according to the elution
order and based on a common guanidino structure.23 On
the basis of the spectral data, the metabolite G3 was
assigned the structure 1-amino-4-[(3-methyl-2-butenyl)-
guanidino]butane (2). The isolation of N-(3-methyl-2-
butenyl)urea (6) in the mixture from alkaline hydrolysis
established the position of the isoprenyl chain in G3.19,20
a
Reaction conditions: (i) R,ω-diaminoalkane, THF/H2O; (ii) 3,4-
dimethoxycinnamic acid, (EtO)2P(O)Cl, Et3N; for 13b-e, Et3N,
CH2Cl2; (iii) CH3SO3H, 1,4-dioxane, reflux; (iv) ion-exchange resin
(OH- form).
1
Since in the H and 13C NMR spectra of G5 (M+ 320 in
the electron impact mass spectrum), compared with
those of G1 (M+ 388), the signals of the 3-methyl-2-
butenyl chain were absent, G5 was assigned the struc-
ture 1-(3,4-dimethoxycinnamoyl)amino-4-guanidinobu-
tane (3). Accordingly, compound 6 was absent in the
hydrolysis mixture. J ust as G1 (1), the natural G5 was
a mixture of (Z)- and (E)-forms (in a ratio of 2:1), and
pharmacological tests were run with the (Z)-form, more
soluble in water.20
The natural product G6 was identical with a com-
mercial sample of agmatine (4), whereas the last
isolated metabolite, G7, was coincident with galegine
(N-(3-methyl-2-butenyl)guanidine, 5), the toxic principle
of Verbesina enceloioides24 and Galega officinalis (goats
rue).25
In summary, the metabolites of V. caracasana appear
to derive from the combination of four elements, which
are all present in G1: the guanidine group, the 1,4-
diaminobutane base, the 3,4-dimethoxycinnamoyl resi-
due, and the prenyl substituent. Biogenetically, G1
seems to be originated from guanidine by three opera-
tions: in detail, the addition of the base, the prenylation,
and the acylation. The first two reactions may happen
at each level, whereas the last one must obviously follow
the addition of the base.
in turn acylated with the mixed anhydride obtained
from (E)-3,4-dimethoxycinnamic acid and diethyl chlo-
rophosphate to afford 9a . Removal of the Boc groups
with excess trifluoroacetic acid at room temperature
gave a complex mixture of products, whereas treatment
of 9a with a catalytic amount of p-toluensulfonic acid
(PTSA) in 1,4-dioxane at reflux, followed by filtration
through anionic exchange resin, afforded (E)-G5 (3) as
the free base in poor yield. However, when 9a was
heated in 1,4-dioxane in the presence of a stoichiometric
quantity of methanesulfonic acid, the mesylate salt of
(E)-G5 (10a ) was obtained in good yield and easily
purified by column chromatography on silica gel. Filtra-
tion through anionic exchange resin gave (E)-G5 (3),
identical with the (E)- component of the natural product.
Phase-transfer-catalyzed (PTC) N-alkylation of 720
with 4-bromo-2-methyl-2-butene at room temperature
(Scheme 2) afforded 11 in 97% yield. The reaction of 11
(Scheme 2) with 1,4-diaminobutane in THF gave the
intermediate 12, which by heating in 1,4-dioxane with
methanesulfonic acid yielded the diacetate salt 13.
Treatment of the last one with anionic exchange resin
yielded G3 (2), identical with the natural sample.
Conversely, the reaction of 11 with 25% NH3 solution
in ethanol provided 14, which was deprotected under
the same conditions as used for 13 to afford 12. The
acetate 12 was again neutralized by ion-exchange resin
to give G7 (5), identical with the natural product.
With the aim at both developing new hypotensive
agents and gaining an insight into the structure-
activity relationships for the series of these guanidino
derivatives, we have synthesized homologues of 10a (the
mesylate salt of G5) bearing a diaminoalkane spacer,
A similar strategy addressed our synthetic routes to
G3, G5, and G7.
Syn th esis. To confirm the assigned stuctures, com-
pounds 2, 3, and 5 were synthesized according to the
pathways summarized in Schemes 1 and 2.
The reaction of N,N′-bis(tert-butoxycarbonyl)-S-me-
thylisothiourea (7)20 with 1,4-diaminobutane in THF
(Scheme 1) gave in 75% yield compound 8a , which was

2952 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Carmignani et al.
Sch em e 2a
a
Reaction conditions: (i) 4-bromo-2-methyl-2-butene, KOH,
Bu4NBr, CH2Cl2/MeCN; (ii) 1,4-diaminobutane, THF/H2O; (iii)
CH3SO3H, 1,4-dioxane, reflux, ion-exchange resin (MeCO2- form);
(iv) ion-exchange resin (OH- form); (v) NH3, EtOH.
F igu r e 1. Changes in systolic blood pressure following iv
administration of (E)-G5, its synthetic analogues 16a and
10b-e, and Natural (Z)-G5 in Anesthetized rats (means ( SE,
n ) 8 for each point).
Sch em e 3a
G120 and G222 being the most potent among them (G2
> G1). In general, these compounds affected breathing
moderately and biphasically (stimulation at low/middle
doses, depression at high doses), without changing
consistently the heart rate (HR), with the exception of
G2, which induced dose-related bradycardia.
In the present study, all the synthetic compounds
were tested as mesylate salts. Either (E)-G5 (10a ) or
its analogue 16a was found, in urethane-anesthetized
rats, to lower BP and to increase cardiac inotropism (as
the maximum rate of rise of the left ventricular isovolu-
metric pressure, dP/dt) and, slightly, HR; an evident
dose-response relationship was observed, for the hy-
potensive effect, in the dose range 1600-12800 µg/kg
(Figures 1-4). The averaged basal values of the mea-
sured cardiovascular parameters were 106 ( 6 mmHg
(systolic BP), 91 ( 5 mmHg (diastolic BP), 313 ( 24
beats/min (HR), and 4910 ( 463 mmHg/s (dP/dt) (means
( SE; n ) 56 for each parameter). Whereas natural (Z)-
G5 slightly increased the respiratory frequency (RF) and
tidal volume (TV) at lower iv doses (200-1600 µg/kg)
and depressed these two parameters not dose-depen-
dently at higher doses (3200-12800 µg/kg),23 synthetic
(E)-G5 and 16a reduced the RF and TV in a dose-related
manner (1600-12800 µg/kg): such effects began a few
seconds after iv administration of both drugs, lasted
a
Reaction conditions: (i) 4-methoxybenzoic acid, (EtO)2P(O)Cl,
Et3N, CH2Cl2; (ii) CH3SO3H, 1,4-dioxane, reflux.
shorter (10b, 10c) or longer (10d , 10e) than that of the
natural compound. Moreover, the analogue compound
16a , modified in the acyl moiety, was prepared from 8a
as outlined in Scheme 3.
P h a r m a cology. All the natural compounds,23 when
administered by an intravenous (iv) route in anesthe-
tized rats, were able to reduce blood pressure (BP) and
to increase cardiac inotropism in a dose-related manner,

Novel Hypotensive Agents from Verbesina caracasana
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2953
F igu r e 3. Changes in heart rate following iv administration
of (E)-G5, its synthetic analogues 16a and 10b-e, and natural
(Z)-G5 in anesthetized rats (means ( SE, n ) 8 for each point).
F igu r e 2. Changes in diastolic blood pressure following iv
administration of (E)-G5, its synthetic analogues 16a and
10b-e, and natural (Z)-G5 in anesthetized rats (means ( SE,
n ) 8 for each point).
continued to induce biphasic chronotropic responses
(with prevalent bradycardia), and as a consequence of
the primary effect of respiratory depression, also the
increased chronotropic response was followed by a phase
of reduced inotropism. For instance, the initial increase
of HR and dP/dt, which was observed with either 16a
or (E)-G5 at a dose of 12800 µg/kg, was soon followed
by an about 80% reduction of both HR and dP/dt (with
respect to the basal values); HR, dP/dt, BP, RF, and TV
all required about 7 min to return to the levels preceding
administration of this dose of (E)-G5.
The synthetic homologues of (E)-G5, with two (10b),
three (10c), five (10d ), or six (10e) methylene groups
in the alkyl chain, were all able to decrease systolic and
diastolic BPs, the latter giving in all cases the highest
response; the four compounds showed positive inotropic
effects at all the tested doses, but only for 10c and 10d
was it possible to establish a dose-response relationship
(Figures 1-4). On the other hand, the effects of these
drugs on HR were quite different: 10c and 10d were
provided with slight bradycardic and tachycardic ac-
tions, respectively, without a dose-response relation-
ship. 10b did not show effects on HR until a dose of 400
µg/kg, increasing it until a dose of 3200 mg/kg and
decreasing it at doses of 6400-12800 µg/kg (which
induced lower positive inotropic responses). 10e in-
creased HR until a dose of 1600 µg/kg and then induced
a lower tachycardic response which, at the highest dose
from 16 to 110 s, and were concomitant to reduction of
HR and dP/dt (which followed the early increase, lasting
from 11 to 23 s, of these cardiac indices). On the whole,
16a was slightly more active than 10a in determining
tachycardia and arterial hypotension (at higher doses)
as well as in depressing breathing; moreover, these
effects of 16a were slightly longer than those of (E)-G5.
As compared with natural (Z)-G5, which decreased HR
(6400-12800 µg/kg), 16a and (E)-G5 were more potent
in lowering BP, induced slight tachycardia instead of
bradycardia, and depressed breathing at all doses, while
(Z)-G5 showed biphasic respiratory effects (see above);
on the other hand, there were no substantial differences
in the positive inotropic effects of the three compounds
(Figures 1-4).
The analysis of the cardiovascular effects of 16a and
(E)-G5 showed that a slight increase of HR and dP/dt,
induced by the lowest doses (50 and 100 µg/kg), was
concomitant to the maximal hypotensive effect, whereas
a slight bradycardia was observed when BP was return-
ing to its basal levels. Such a bradycardia was not
present at a dose of 200 µg/kg, which induced sporadic
periods of ventricular extrasystolic firing lasting about
3 min. Tachycardia, followed by bradycardia and extra-
systolia, reappeared in the dose range 400-1600 µg/kg.
On the other hand, higher doses of 16a and (E)-G5

2954 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Carmignani et al.
resembled either (E)-G5 or 16a in determining slight
tachycardia, while 10b and, mostly, 10c (at doses higher
than 3200 µg/kg) resembled (Z)-G5 in inducing an
appreciable bradycardia.
When considering structure-activity relationships,
the (E)-configuration [10a vs natural (Z)-G5] and/or a
p-methoxybenzoyl substituent [16a vs (Z)-G5] appeared
to be responsible for a higher hypotensive action (mostly
diastolic), a reversal of the chronotropic effect (tachy-
cardic instead of bradycardic), and a dose-related de-
pression of breathing. On the other hand, the decrease
to two (10b) or three (10c) and the increase to five (10d )
or six (10e) methylene groups in the alkyl chain did not
allow, in the dose range 50-3200 µg/kg (without con-
sidering the highest doses depressing breathing), for
effects on BP significantly different from those of (E)-
G5 (15a , with four dCH2 groups). Nevertheless, in this
dose range, 10b-e appeared to be slightly more active
than 10a in increasing cardiac inotropism. Moreover,
when comparing chemical structures of the studied
compounds with that of guanethidine, it appears quite
likely that these compounds are provided with higher
lipophility because of their higher hydrocarburic com-
ponent (thus being able to cross the blood-brain barrier
much more than guanethidine).9,10
Either natural23 or synthetic guanidine com-
pounds,20-22 including those of the present study, have
been characterized for their respiratory and, mostly,
cardiovascular effects on the basis of a preliminary
extensive investigation, carried out on mice (see above)
and dogs, showing the crude methanol extract of V.
caracasana to have the respiratory and cardiovascular
systems as selective targets.20,22 In particular, this
extract (0.5-4.0 mg/kg of body mass by the iv route)
was able to reduce the mean BP in a dose-related
manner, in anesthetized dogs, with the maximum effect
at a dose of 2.0 mg/kg (-58% ( 4% with respect to the
basal BP values, mean ( SE; n ) 6) and not differing
under barbiturate or chloralose anesthesia.19,20,22 Syn-
thesis of the water-soluble (Z)-forms of a series of
guanidine compounds (G1-G3, G5-G7), previously
obtained as a mixture of the (E)- and (Z)-forms from V.
caracasana, allowed in anesthetized rats (1) their
prevalent cardiovascular effects to be confirmed, (2) such
compounds to be characterized as hypotensive drugs
through a vasodilating action (depending on central and/
or peripheral mechanisms), (3) their pharmacological
potencies to be determined (in the dose range 50-3200
µg/kg, by the iv route), (4) their pharmacological activi-
ties to be compared with those of standard (hypotensive
and/or antihypertensive) drugs, ranging from clonidine
to papaverine, and (5) chronotropic and/or inotropic
components to be excluded (in the above dose range) in
the hypotensive effect.19-23 Therefore, these compounds
have been proposed, in such a dose range, as hypoten-
sive drugs of high (G2),22,23 mild (G1, G7),20,23 or low
potency (G3, G5, G7)23 not presenting significant chro-
notropic effects. In this regard, only G1, G2, and G7 (G2
> G1 > G7) showed appreciable positive inotropic effects
together with stimulatory (G7 > G1) or inhibitory effects
(G2) of variable degree on breathing.20-23 Thus, G5
being one of the studied guanidine derivatives provided
with hypotensive activity but devoid of consistent ino-
tropic and respiratory actions,23 the aim of this study
F igu r e 4. Changes in the maximum rate of rise of the left
ventricular isovolumetric pressure (dP/dt) following iv admin-
istration of (E)-G5, its synthetic analogues 16a and 10b-e,
and natural (Z)-G5 in anesthetized rats (means ( SE, n ) 8
for each point).
(12800 µg/kg), was reversed with the appearance of a
bradycardic effect; also in this case, doses of 6400-
12800 µg/kg were characterized by lower positive ino-
tropic responses (Figures 1-4). In conclusion 10a , 16a ,
and 10b-e appeared to be, within a wide dose range,
drugs lowering BP according to a small-slope dose-
response curve. The hypotensive effect coexisted with
variable and negligible chronotropic ones not exceeding,
at the highest dose, a 10% or a 20% change (positive or
negative, respectively) with regard to the basal values.
The same compounds showed stimulatory actions on
cardiac inotropism not related to the doses (10b, 10e),
not according to clear dose-response relationships (10a ,
16a , 10c, 10d ), or accompanied by negative chronotropic
responses associated with respiratory depression (10a ,
16a ). In this regard, the depressive effects on breathing
of all the studied compounds were slight and similar
(in the dose range 50-1600 µg/kg). Only at the highest
(subtoxic) doses, 10b and 10e induced lower increases
of dP/dt, in the presence of reversal of the tachycardic
response and potentiation of the hypotensive effect.
Therefore, as compared with 10a in the dose range 50-
3200 µg/kg, its analogues 10b-e were found to have
similar effects on BP and to be slightly more active in
increasing cardiac inotropism. As far as the chronotropic
effects were concerned, 10d and 10e (50-6400 µg/kg)

Novel Hypotensive Agents from Verbesina caracasana
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2955
Ta ble 1. Changesa in Systolic and Diastolic Blood Pressures
Following iv Administration of Several Guanidine Derivatives
and Vasodilating or Antyhypertensive Drugs in Anesthetized
Rats
RF and TV, and G2 in reducing HR. Differences in
pharmacological potency were found for these natural
compounds, G2 being the most active substance in
lowering BP and increasing cardiac inotropism (only G2
depressed breathing also at nontoxic doses)22 and G120
and G723 the most active substances in stimulating
breathing. It seems quite likely that also (E)-G5, 16a ,
and 10b-e act by central and/or peripheral mechanisms
already found for the natural guanidine derivatives
obtained from V. caracasana. In this respect, pharma-
codynamic differences among the synthesized drugs and
between the synthetic mesylate salts with an (E)-
configuration and the natural products with a (Z)-
configuration are likely to depend on their ability to
cross the blood-brain barrier, on the lower or higher
lipophilicity, and on the pharmacokinetic proper-
ties.20,22,23
blood pressure
drug
systolic
diastolic
(Z)-G5 (4.12 µmol/kg)
(E)-G5 (10a) (4.12 µmol/kg)
16a (4.12 µmol/kg)
-12 ( 2
-21 ( 2
-28 ( 4
-29 ( 3
-24 ( 3
-17 ( 2
-14 ( 3
-32 ( 4
-21 ( 2
-46 ( 6
-34 ( 5
-21 ( 3
-28 ( 4
-10 ( 3
-30 ( 3
-31 ( 2
-33 ( 4
-28 ( 4
-27 ( 2
-36 ( 3
-23 ( 4
-15 ( 2
-30 ( 3
-27 ( 4
-16 ( 2
-23 ( 2
10b (4.12 µmol/kg)
10c (4.12 µmol/kg)
10d (4.12 µmol/kg)
10e (4.12 µmol/kg)
guanethidine (25 µmol/kg)
clonidine (0.108 µmol/kg)
hexamethonium (12 µmol/kg)
reserpine (8 µmol/kg)
papaverine (5 µmol/kg)
histamine (0.044 µmol/kg)
a
Values are means ( SE (n ) 4 in each group).
All the synthetic guanidine compounds presented in
this study (as well as their natural homologue and
analogues from V. caracasana) appear to be drugs
provided with selective dose-related hypotensive effects
within a wide range (50-3200 µg/kg by the iv route).
These effects are generally accompanied, mostly in the
cases of 10a and 16a , by a slight increase of cardiac
inotropism but not by significant respiratory or chro-
notropic changes. If a hypotensive response (mostly
when dependent on arterial vasodilation) should be
expected to be followed by a reverse effect (associated
with tachycardia), due to baroreceptor-dependent sym-
pathetic activation, the absence of such an effect for the
compounds of this study may be explained as the result
of interactions among the above central and peripheral
mechanisms previously described for the natural com-
pounds.19-23 Accordingly, while the hypotensive re-
sponses observed in this study are constantly ac-
companied by increased dP/dt, HR does not change
consistently [(E)-G5] or appears to decrease [(Z)-G5,
10c], to increase (16a , 10d ), or to change biphasically
(10b, 10e) as, in general, the respiratory responses.
Therefore, the increased inotropism by the studied
compounds does not seem to depend on a reflex increase
of sympathetic activity by itself, their hypotensive
effects being related to arterial vasodilation and not to
reduction of HR. In this respect 10a , 16a , and 10b-e
seem to be, in a clinical perspective, more suitable
hypotensive (antihypertensive) drugs because of the
lack, as compared with some natural analogues, of
consistent positive inotropic (as G1 and G2),20,22 respira-
tory, and chronotropic side effects (as G2 and G7).22,23
Although this study has not been directed to investigate
the action mechanisms of the tested guanidine com-
pounds, it is likely, considering the results obtained with
their natural analogues,20-23 that they too are provided
with multiple action mechanisms (as also found for
other guanidine derivatives such as guanethidine5 and
guanabenz6). Moreover, although a guanidine moiety is
also shown by complex with nitric oxide (NO) synthase
to have inhibitory activity [like L-NAME (nitroarginine
methyl ester) and aminoguanidine], a similar mecha-
nism is quite unlikely for our guanidine compounds,
inducing, on the contrary, arterial vasodilation and
slight positive inotropic effects (NO is known to induce
vascular relaxation, to depress cardiac activity, and to
be an antiarrhythmic agent).26-28
was also to assess the pharmacological profile of its (E)-
homologue 10a and analogues 16a and 10b-e to obtain
compounds provided with a higher hypotensive activity
and potentially more useful as antihypertensive agents.
As previously shown,20-23 some of the tested natural
compounds were more potent, on a molar basis, than
guanethidine, papaverine, reserpine, or hexamethonium
in reducing BP, evidenced more prolonged hypotensive
actions, and did not reveal the negative inotropic and
chronotropic properties of such vasodilating or antihy-
pertensive drugs (with the only exception of G2). On the
whole, as discussed above in comparison with natural
(Z)-G5, its (E)-homologue 10a and analogue 16a showed
a higher hypotensive activity and confirmed properties
of consistent respiratory depression only at high (sub-
toxic) doses. On the other hand, 10b-e did not differ
from 10a and 16a in the mean hypotensive effect, when
considering the dose range 50-3200 µg/kg (excluding
the highest doses depressing breathing), despite a
slightly higher positive inotropic activity. In this regard,
Table 1 compares, on a molar basis, the hypotensive
effects of (Z)-G5, 10a , 16a , 10b-e, and several vasodi-
lating or antihypertensive drugs.
From a pharmacodynamic point of view, it was found
that (E)-G5, 16a , and 10b-e decreased BP (lasting a
few seconds after their iv administration) before affect-
ing HR, dP/dt, RF, and TV, thus suggesting a vascular
level of action in determining systemic arterial vasodi-
lation, in a way similar to that shown by the natural
products G2,22 G3, G5, G6, and G7,23 but not G1.20 This
last compound showed a peripheral (cardiac) â1-adreno-
receptor-like action (causing an increase of HR and dP/
dt) and reduced both the central sympathetic tone and
baroreceptor reflex activity (causing a reduction of BP).
On the other hand, G2 showed only peripheral mecha-
nisms including reserpine- and guanethidine-like ac-
tions, â1- and â2-adrenoreceptor-like and M2-4-cholinergic
receptor-like components, and R2-adrenoreceptor an-
tagonistic properties. Moreover, the cardiovascular ef-
fects of G120 and G222 were not related, in the dose range
50-3200 µg/kg by the iv route, to the respiratory ones
(consisting of an increase of RF and TV at lower doses
and a decrease of RF and TV at higher doses). Similar
effects were shown by G3 and G5-G7,23 which re-
sembled G1 and G2 in lowering BP, G1 in increasing

2956 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Carmignani et al.
OMe), 41.76 (t, C-1), 40.03 (q, Me), 38.95 (t, C-4), 27.15, 26.22
(t each, 2 × CH2); FABMS (TDEG/Gly) m/z 321 (M+ + 1). Anal.
(C17H28N4O6S) C, H, N.
(E)-(3,4-Dim eth oxycin n a m oyl)-N1-a gm a tin e (5). Treat-
ment of 10a (126 mg) with anion-exchange resin gave (E)-G5
(5; 92 mg), identical (1H, 13C NMR, FABMS) with the natural
(E)-component.
A further study will deal with structure-activity
relationships among the compounds of this study, their
natural analogues from V. caracasana, and other syn-
thetic analogues to select some of them for a clinical
evaluation in the treatment of arterial hypotension of
various genesis.
N2,N3-Bis(ter t-bu toxyca r bon yl)-N-(γ,γ-d im eth yla llyl)-
S-m eth ylisoth iou r ea (11). A solution of 720 (3.5 g, 12 mmol)
in CH2Cl2/CH3CN (60 mL; 19:1, v/v) was added dropwise to a
stirred mixture of KOH (1.9 g, 34.3 mmol) and Bu4NBr (720
mg, 2.27 mmol) in the same solvent (60 mL). After the
resulting solution was stirred for 15 min, a solution of 4-bromo-
2-methyl-2-butene (4.3 g, 28.8 mmol) in CH2Cl2/CH3CN (100
mL, 19:1) was slowly added over 1 h. After being stirred for 2
h further, the reaction mixture was diluted with water, and
the organic layer was washed with brine, dried (Na2SO4), and
evaporated. The residue was filtered through a short pad of
silica gel eluting with hexanes/EtOAc (9:1) to yield 11 (5.6 g,
97%) as a clear oil. Anal. (C17H30N2O4S) C, H, N.
1-Am in o-4-[N2,N3-bis(ter t-bu toxycar bon yl)-N3-(γ,γ-dim -
eth yla llyl)gu a n id in o]bu ta n e (12). A solution of 11 (3.58 g,
10 mmol) in THF (25 mL) was added dropwise to a stirred
solution of 1,4-diaminobutane (2.29 g, 26 mmol) in THF/H2O
(40 mL; 20:1, v/v). After being stirred for 1 h at 50 °C, the
reaction mixture was concentrated in vacuo, and the residue
was partitioned between CHCl3 and 10% aqueous NaHCO3.
The organic layer was dried (Na2SO4) and evaporated. The
residue by chromatography on a silica gel column (CHCl3/Et3N,
19:1) gave 12 (2.79 g, 70%) as a clear oil. Anal. (C20H38N4O4)
C, H, N.
N-(4-Am in obu tyl)-N′-(γ,γ-d im eth yla llyl)gu a n id in e Di-
a ceta te (13). A solution of 12 (518 mg, 1.3 mmol) and
methanesulfonic acid (0.17 mL, 2.6 mmol) in dry 1,4-dioxane
(15 mL) was held at reflux overnight, then and concentrated
in vacuo. The residue was applied to a column packed with
Dowex 1x2-200 ion-exchange resin (CH3COO- form). Elution
with MeOH afforded crude 13, which was further purified by
a column of Sephadex LH-20 preswelled in MeOH, to give pure
13 (125 mg, 30%) as an oil. Anal. (C14H30N4O4) C, H, N.
N3-P r en yla gm a tin e (2). Treatment of 13 (125 mg) with
anion-exchange resin gave G3 (2; 59 mg), identical (1H, 13C
NMR, FABMS) with the natural sample.
N,N′-Bis(t er t -b u t oxyca r b on yl)-N-(γ,γ-d im et h yla llyl)-
gu a n id in e (14). To a solution of 11 (1.0 g, 2.8 mmol) in EtOH
(30 mL) was added a 25% NH4OH solution (14 mL) with
stirring at room temperature over 7 h in small portions. The
reaction mixture was evaporated, and the residue was parti-
tioned between CH2Cl2 and water. The organic layer was
washed with water, dried (Na2SO4), and evaporated to give
14 (560 mg, 61%) as an oil. Anal. (C16H29N3O4) C, H, N.
N-(γ,γ-Dim eth yla llyl)gu a n id in e Aceta te (15). To a solu-
tion of 14 (262 mg, 0.8 mmol) in dry 1,4-dioxane (20 mL) was
added methanesulfonic acid (52 mL, 0.8 mmol) at 0 °C with
stirring. The reaction mixture was held at reflux for 20 h and
evaporated. The residue by chromatography on silica gel (CH2-
Cl2/MeOH, 7:3) gave 15 (168 mg, 90% yield) as an oil. Anal.
(C8H17N3O2) C, H, N.
Exp er im en ta l Section
Gen er a l P r oced u r es. Melting points were determined
with a Kofler apparatus and are uncorrected. 1H and 13C NMR
spectra were determined with a Varian Gemini instrument
(300 and 75 MHz, respectively), using tetramethylsilane as
an internal standard in the reported solvents. IR spectra were
determined with
a Perkin-Elmer 237 spectrophotometer.
Electron impact (EI) and fast atom bombardment (FAB) mass
spectra were determined on a VG 7070EQ spectrometer.
1H NMR, IR, and FABMS spectra of the synthetic interme-
diates are reported in the Supporting Information.
P la n t Ma ter ia l. Leaves of V. caracasana were collected in
Valencia, Venezuela. A voucher specimen is deposited at the
herbarium of the Departamento de Physiologia vegetal of the
Universidad Central de Venezuela, Maracaibo, Venezuela.
Extr a ction a n d F r a ction a tion . Extraction and the first
fractionation on silica gel with CHCl3/MeOH mixtures have
been described in our previous paper.20 Extended chromatog-
raphy on silica gel with CHCl3/MeOH mixtures of fractions
containing products more polar than G1 G2 gave the new
23
compounds G3-G7, G4 having not been identified.
Syn th esis. 1-Am in o-4-[N2,N3-bis(ter t-bu toxyca r bon yl)-
gu a n id in o]bu ta n e (8a ). A solution of N2,N3-bis(tert-butoxy-
carbonyl)-S-methylisothiourea20 (7; 2.9 g, 10 mmol) in THF (25
mL) was added dropwise to a stirred solution of 1,4-diami-
nobutane (2.29 g, 26 mmol) in THF/H2O (40 mL; 20:1, v/v).
After being stirred for 1 h at 50 °C, the reaction mixture was
concentrated in vacuo, and the residue was partitioned be-
tween CHCl3 and 10% aqueous NaHCO3. The organic layer
was dried (Na2SO4) and evaporated. The residue by chroma-
tography on a Si gel column (CHCl3/Et3N, 19:1) gave 8a (2.48
g, 75%) as an oil. Anal. (C15H30N4O4) C, H, N.
(E)-4-[N2,N3-Bis(ter t-bu toxycar bon yl)gu an idin o]-1-[(3,4-
d im eth oxycin n a m oyl)a m in o]bu ta n e (9a ). To a solution of
(E)-3,4-dimethoxycinnamic acid (825 mg, 4.2 mmol) and tri-
ethylamine (1.3 mL, 9.3 mmol) in dry THF (30 mL) was added
slowly a solution of diethyl chlorophosphate (870 mg, 5 mmol)
in THF (30 mL). After the resulting solution was stirred for 2
h at room temperature, the precipitate was filtered off and
the filtrate was added dropwise to a solution of 8a (2.0 g, 6
mmol) and triethylamine (1.3 mL, 9.3 mmol) in dry CH2Cl2
(30 mL). The reaction mixture was stirred for 2 h at room
temperature, concentrated to a small volume, and diluted with
CH2Cl2 and 10% aqueous Na2CO3. The organic layer was
washed with water, dried (Na2SO4), and evaporated. The
residue was purified on a silica gel column with AcOEt to
afford 9a (1.88 g, 60%) as a viscous oil. Trituration with Et2O
gave a colorless powder, mp 136-138 °C. Anal. (C26H40N4O7)
C, H, N.
(E)-1-[(3,4-Dim eth oxycin n a m oyl)a m in o]-4-gu a n id in ob-
u ta n e Mesyla te (10a ). To a solution of 9a (416 mg, 0.8 mmol)
in dry 1,4-dioxane (20 mL) was added methanesulfonic acid
(52 mL, 0.8 mmol) at 0 °C with stirring. The reaction mixture
was held at reflux for 20 h and concentrated in vacuo. The
residue was chromatographed on silica gel (CH2Cl2/MeOH, 7:3)
to give 10a (263 mg, 79%) as a pale yellow glass: IR (Nujol)
Ga legin e (P r en yla gm a tin e, 7). Treatment of 15 (168 mg)
with anion-exchange resin gave G7 (7; 108 mg), identical (1H,
13C NMR, FABMS) with the natural sample.
Com p ou n d s 8b-e. The compounds were obtained as oils
following the same procedure reported for 8a and using the
appropriate R,ω-diaminoalkane. Yields:
1-a m in o-4-[N2,N3-bis(ter t-bu t oxyca r bon yl)gu a n idin o]-
ethane (8b), 48%; 1-amino-4-[N2,N3-bis(tert-butoxycarbonyl)-
guanidino]propane (8c), 76%; 1-amino-4-[N2,N3-bis(tert-bu-
toxycarbonyl)guanidino]pentane (8d ), 67%; 1-amino-4-[N2,N3-
bis(tert-butoxycarbonyl)guanidino]hexane (8e), 57%.
Com p ou n d s 9b-e. The compounds were obtained as
viscous oils starting from 8b-e and following the procedure
described for 9a . Yields:
1
νmax 3310, 1620 cm-1; H NMR (C5D5N) δ 8.90 (1H, br t, J )
6 Hz, NH), 8.24 (1H, t, J ) 5 Hz, NH), 8.06 (1H, d, J ) 15.5
Hz, H-R), 7.25 (1H, d, J ) 2 Hz, H-2′), 7.22 (1H, dd, J ) 8 and
2 Hz, H-6′), 7.18 (1H, d, J ) 15.5 Hz, H-â), 6.87 (1H, d, J ) 8
Hz, H-5′), 3.59 (2H, q, J ) 6 Hz, 1-CH2N), 3.82, 3.72 (3H each,
s, 2 × OMe), 3.41 (2H, br q, J ) 5.5 Hz, 4-CH2N), 3.01 (3H, s,
Me), 1.76 (4H, m, 2 × CH2); 13C NMR δ 167.18 (s, CO),
158.56 (s, CdNH), 150.57, 149.15 (s each, C-3′, C-4′), 140.05
(d, C-R), 129.04 (s, C-1′), 122.27, 120.87 (d each, C-6′, C-â),
112.21 (d, C-5′), 110.87 (d, C-3′), 55.90, 55.82 (q each, 2 ×
(E)-4-[N2,N3-bis(tert-butoxycarbonyl)guanidino]-1-[(3,4-
dimethoxycinnamoyl)amino]ethane (9b), 98%;

Novel Hypotensive Agents from Verbesina caracasana
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2957
(E)-4-[N2,N3-bis(tert-butoxycarbonyl)guanidino]-1-[(3,4-
dimethoxycinnamoyl)amino]propane (9c), 87%;
3.25 (2H each, br t, J ) 6 Hz, 2 CH2N), 2.72 (3H, s, Me), 1.64
(4H, m, 2 × CH2); 13C NMR δ 168.93 (s, CO), 162.08 (s, C-4′),
158.78 (s, CdNH), 125.91 (s, C-1′), 133.04 (d each, C-2′, C-6′),
115.27 (d, C-3′, C-5′), 56.48 (q, OMe), 42.25 (t, C-1), 39.63 (q,
Me), 38.89 (t, C-4) 27.90, 27.78 (t each, C-2, C-3); FABMS
(TDEG/Gly) m/z 265 (M+ + 1). Anal. (C14H24N4O5S) C, H, N.
(E)-4-[N2,N3-bis(tert-butoxycarbonyl)guanidino]-1[(3,4-
dimethoxycinnamoyl)amino]pentane (9d ), 68%;
(E)-4-[N2,N3-bis(tert-butoxycarbonyl)guanidino]-1-[(3,4-
dimethoxycinnamoyl)amino]hexane (9e), 80%.
P h a r m a cology. An im a ls. Adult male Wistar rats (average
mass 280 ( 5 g, mean ( SE; n ) 56) were provided by the
Catholic University farm, Rome, Italy. Maintenance, feeding,
and general treatment of the animals were as previously
described.20,22
Com p ou n d s 10b-e. Following the same procedure de-
scribed for 10a , the title compounds were prepared.
(E)-1-[(3,4-Dim eth oxycin n a m oyl)a m in o]-2-gu a n id in o-
1
eta n e Mesyla te (10b): yield 74%; mp 148-151 °C; H NMR
(C5D5N) δ 9.35 (1H, t, J ) 6 Hz, NHCO), 9.30 (1H, br t, J ) 5
Hz, NH), 8.04 (1H, d, J ) 15.5 Hz, H-R), 7.30 (1H, d, J ) 2
Hz, H-2′), 7.23 (1H, dd, J ) 8 and 2 Hz, H-6′), 7.12 (1H, d, J
) 15.5 Hz, H-â), 6.86 (1H, d, J ) 8 Hz, H-5′), 3.84 (2H, q, J )
6 Hz, 1-CH2N), 3.76, 3.72 (3H each, s, 2 × OMe), 3.72 (2H, q,
J ) 5.5 Hz, 2-CH2N), 3.12 (3H, s, Me); 13C NMR δ 167.68 (s,
CO), 159.03 (s, CdNH), 150.09, 149.19 (s each, C-3′, C-4′),
140.50 (d, C-R), 128.69 (s, C-1′), 122.20, 120.28 (d each, C-6′,
C-â), 112.09 (d, C-5′), 110.83 (d, C-3′), 55.75 (q, OMe), 42.14
(t, C-1), 40.29 (q, Me), 39.06 (t, C-2); FABMS (TDEG/Gly) m/z
293 (M+ + 1). Anal. (C15H24N4O6S) C, H, N.
Car diovascu lar Deter m in ation s an d Respir ator y Mon i-
tor in g. Animals were anesthetized with 10% (w/v) ethylure-
thane (1 mL/100 g of body mass), which was dissolved in 0.9%
NaCl solution (saline) and administered with a single intra-
peritoneal (ip) injection. The trachea was cannulated to allow
spontaneous breathing. Polyethylene catheters, containing
sodium heparin (100 US pharmacopeia U/mL), were inserted
into the left femoral artery for recording aortic BP and into
the right femoral vein for drug administration. Cardiac inot-
ropism was evaluated as the maximum rate of rise of the left
ventricular isovolumetric pressure (dP/dt), which was obtained
by means of a calibrated 3F catheter-tip pressure transducer
(Millar Instruments, Houston, TX) inserted into the left
common carotid artery and advanced into the left ventricle.29,30
A P23Db pressure transducer (Statham Medical Instruments,
Los Angeles, CA) was used for measuring systolic and diastolic
BPs, which were averaged electronically.31 HR was obtained
by a 9875B Beckman cardiotachometer coupler (Beckman
Instruments, Inc., Shiller Park, IL), which was triggered by
the R-peak of the lead II electrocardiogram.32 The pulsatile
BP registered in the left ventricle was differentiated through
a BL 622 derivative computer (Biotronex Laboratories, Inc.,
Kensington, MA) for determining positive and negative dP/dt
(measured in mmHg/s), as previously described.26,30 All car-
diovascular parameters were continuously monitored by means
of a polygraphic recorder (Mangoni Elettronica, Pisa, Italy),
which was interfaced to a computer system giving the cardio-
vascular measures in real time.26 A light-heated table was
employed to mantain a constant 37 °C body temperature of
the animals. They were heparinized, as previously de-
scribed,27,29 and respiration was also monitored by using a
pneumotachograph adapted to a Biotronex BL 620 integrator
to yield the full respiratory wave. In this regard, the tracheal
cannula was connected to the pneumotachograph to assess RF
and TV under spontaneous breathing.28,30 Also RF and TV were
continuously monitored polygraphically. After completion of
the surgical procedures, the rats received no treatment for 60
min to allow for the stabilization of the cardiovascular and
respiratory parameters.
(E)-1-[(3,4-Dim eth oxycin n a m oyl)a m in o]-3-gu a n id in o-
p r op a n e Mesyla te (10c): yield 47%; mp 170-173 °C; 1H
NMR (C5D5N) δ 9.02 (1H, br t, J ) 5 Hz, NH), 9.00 (1H, t, J
) 6.5 Hz, NHCO), 8.04 (1H, d, J ) 15.5 Hz, H-R), 7.31 (1H, d,
J ) 2 Hz, H-2′), 7.22 (1H, dd, J ) 8 and 2 Hz, H-6′), 7.14 (1H,
(1H, d, J ) 15.5 Hz, H-â), 6.86 (1H, d, J ) 8 Hz, H-5′), 3.79,
3.73 (3H each, s, 2 × OMe), 3.72 (2H, q, J ) 6 Hz, 1-CH2N),
3.58 (2H, q, J ) 6 Hz, 4-CH2N), 3.12 (3H, s, Me), 2.05 (2H, m,
J ) 6 Hz, CH2); 13C NMR δ 167.17 (s, CO), 158.61 (s, CdNH),
150.08, 149.18 (s each, C-3′, C-4′), 140.07 (d, C-R), 128.91 (s,
C-1′), 122.23, 120.75 (d each, C-6′, C-â), 112.12 (d, C-5′), 110.75
(d, C-3′), 55.79 (q, OMe), 40.37 (q, Me), 40.05 (t, C-1), 37.24 (t,
C-3), 29.49 (t, C-2); FABMS (TDEG/Gly) m/z 307 (M+ + 1).
Anal. (C16H26N4O6S) C, H, N.
(E)-1-[(3,4-Dim eth oxycin n a m oyl)a m in o]-5-gu a n id in o-
p en ta n e Mesyla te (10d ): yield 57%; mp 193-196 °C; 1H
NMR (C5D5N) δ 8.95 (1H, br t, J ) 6 Hz, NH), 8.79 (1H, t, J
) 7 Hz, NHCO), 8.12 (1H, d, J ) 15.5 Hz, H-R), 7.50 (1H, d,
J ) 2 Hz, H-2′), 7.25 (1H, dd, J ) 8 and 2 Hz, H-6′), 7.39 (1H,
(1H, d, J ) 15.5 Hz, H-â), 6.86 (1H, d, J ) 8 Hz, H-5′), 3.84,
3.72 (3H each, s, 2 × OMe), 3.62 (2H, q, J ) 6 Hz, 1-CH2N),
3.34 (2H, q, J ) 6 Hz, 5-CH2N), 3.11 (3H, s, Me), 1.64 (4H, m,
J ) 6 Hz, 2- CH2,4-CH2), 1.58 (2H, br m, 3-CH2); 13C NMR δ
166.81 (s, CO), 158.61 (s, CdNH), 150.08, 149.18 (s each, C-3′,
C-4′), 139.75 (d, C-R), 129.16 (s, C-1′), 122.32, 121.36 (d each,
C-6′, C-â), 112.02 (d, C-5′), 110.57 (d, C-3′), 55.84, 55.73 (q each,
OMe), 41.49 (t, C-1), 40.41 (q, Me), 39.00 (t, C-5), 29.20, 28.13
(t each, C-2, C-4), 24.09 (t, C-3); FABMS (TDEG/Gly) m/z 335
(M+ + 1). Anal. (C18H30N4O6S) C, H, N.
P r otocol. Eight rats were used to determine the dose-
response relationship for each of the tested compounds [(E)-
G5, (Z)-G5, 16a , 10b-e]. Saline solutions of these compounds
were prepared daily and injected in a volume of 50 µL; the
doses ranged from 50 to 12800 µg/kg of body mass (ratio 2.0)
and were expressed in terms of free bases. The control
administration of the solvent alone did not change the
cardiovascular and respiratory parameters. Peak effects were
considered for each assay. Each of the consecutive tests was
not performed until the parameters had returned to the values
preceding the first administration and had stabilized.
Fifty-two rats were used to compare, on a molar basis, the
mean BP responses to some antihypertensive or vasodilating
drugs with those to (E)-G5, (Z)-G5 (10a ), 16a , and 10b-e (n
) 4 for each drug). These rats received, by iv injection under
the above experimental conditions, a selected dose (4.12 µM/
kg) of each of the guanidine compounds or a dose of guanethi-
dine (25 µM/kg), clonidine (0.108 µM/kg), hexamethonium (12
µM/kg), reserpine (8 µM/kg), papaverine (5 µM/kg), or hista-
mine (0.044 µM/kg). All drugs were dissolved in saline solution,
and all doses were expressed in terms of free bases. General
experimental conditions were as above.
(E)-1-[(3,4-Dim eth oxycin n a m oyl)a m in o]-6-gu a n id in o-
h exa n e Mesyla te (10e): yield 72%; mp 150-153 °C; 1H NMR
(C5D5N) δ 9.11 (1H, br t, J ) 6 Hz, NH), 8.75 (1H, t, J ) 7 Hz,
NHCO), 8.12 (1H, d, J ) 15.5 Hz, H-R), 7.51 (1H, d, J ) 2 Hz,
H-2′), 7.38 (1H, (1H, d, J ) 15.5 Hz, H-â), 7.23 (1H, dd, J ) 8
and 2 Hz, H-6′), 6.86 (1H, d, J ) 8 Hz, H-5′), 3.83, 3.71 (3H
each, s, 2 × OMe), 3.58 (2H, br q, J ) 6 Hz, 1-CH2N), 3.32
(2H, br q, J ) 6 Hz, 5-CH2N), 3.10 (3H, s, Me), 1.62 (4H, m,
2-CH2,5-CH2), 1.55 (4H, br m, 3- CH2, 4-CH2); 13C NMR δ
166.76 (s, CO), 158.62 (s, CdNH), 150.68, 149.16 (s each, C-3′,
C-4′), 139.68 (d, C-R), 129.18 (s, C-1′), 122.35, 121.48 (d each,
C-6′, C-â), 111.98 (d, C-5′), 110.66 (d, C-3′), 55.82, 55.76 (q each,
OMe), 41.42 (t, C-1), 40.28 (q, Me), 39.16 (t, C-6), 29.20, 28.13
(t each, C-2, C-5), 24.02, 23.89 (t each, C-3, C-4); FABMS
(TDEG/Gly) m/z 349 (M+ + 1). Anal. (C19H32N4O6S) C, H, N.
4-[N2,N3-Bis(ter t-bu toxycar bon yl)gu an idin o]-1-[(4-m eth -
oxyben zoyl)a m in o]bu ta n e (16). Compound 16 was prepared
as a white amorphous solid from 4-methoxybenzoic acid
following the same procedure described for 9a ; yield 54%.
1-[(4-Meth oxyben zoyl)a m in o]-4-gu a n id in obu ta n e Me-
syla te (16a ). Compound 16a was prepared as described for
10a in 59% yield: pale yellow glass; IR (CHCl3) νmax 3240, 1730
cm-1; 1H NMR (CD3OD) δ 7.87 (2H, d, J ) 8.5 Hz, H-2′, H-6′),
6.88 (2H, d, J ) 8.5 Hz, H-3′, H-5′), 3.79 (3H, s, OMe), 3.42,
Sta tistics. Data were expressed as means ( SE. Dose-
response relationships of the guanidine compounds were

2958 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Carmignani et al.
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Ack n ow led gm en t. This study was supported by
grants from the Italian Ministry for University and
Scientific and Technological Research (>40%; 1995,
1996) and Research National Council (Progetto Strate-
gico Ambiente e Territorio, 1996-1998; Progetto singolo,
1998, 1999) to M.C. M.B., F.C., and A.F. thank the
University of Siena (60% funds) for financial support.
Su p p or tin g In for m a tion Ava ila ble: 1H NMR, IR, and
FABMS spectral data of the synthetic intermediates. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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