Synthesis and preliminary pharmacological evaluation of some cytisine derivatives

Article abstract of DOI:10.1016/S0014-827X(99)00049-X

Thirty-one N-derivatives of cytisine were prepared in order to modify its pharmacological profile and to obtain compounds of potential therapeutic interest either at a peripheral or central level, particularly as nicotinic ligands with improved ability to cross the blood-brain barrier. Actually, with the introduction of different kinds of substituent on the basic nitrogen of cytisine a variety of activities were observed, both in vivo (analgesic, dopamine antagonism, antihypertensive, inhibition of stress-induced ulcers, antiinflammatory, protection from PAF-induced mortality, hypoglycemic) and in vitro (positive cardio-inotropic, β-adrenergic antagonism, α₁- and α₂-antagonism, inhibition of PAF-induced platelet aggregation). Six randomly selected compounds were tested for the ability to recognize a central nicotinic receptor and four of them exhibited K(i) values in the range 30-163 nM.

Full text of DOI:10.1016/S0014-827X(99)00049-X

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 438–451
Synthesis and preliminary pharmacological evaluation of some
cytisine derivatives
Caterina Canu Boido, Fabio Sparatore *
Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Geno6a, Viale Benedetto XV, 3-16132 Genoa, Italy
Received 26 October 1998; accepted 23 April 1999
Abstract
Thirty-one N-derivatives of cytisine were prepared in order to modify its pharmacological profile and to obtain compounds of
potential therapeutic interest either at a peripheral or central level, particularly as nicotinic ligands with improved ability to cross
the blood–brain barrier. Actually, with the introduction of different kinds of substituent on the basic nitrogen of cytisine a variety
of activities were observed, both in vivo (analgesic, dopamine antagonism, antihypertensive, inhibition of stress-induced ulcers,
antiinflammatory, protection from PAF-induced mortality, hypoglycemic) and in vitro (positive cardio–inotropic, b-adrenergic
antagonism, a1- and a2-antagonism, inhibition of PAF-induced platelet aggregation). Six randomly selected compounds were
tested for the ability to recognize a central nicotinic receptor and four of them exhibited Ki values in the range 30–163 nM.
© 1999 Elsevier Science S.A. All rights reserved.
Keywords: Cytisine derivatives; Quinolizidine derivatives; Nicotine receptors; Cardiovascular agents
1. Introduction
bodies [4]. Cytisine, at a dose of 1 mg e.v., is preferable
to the widely used lobeline [5] by Russian pharma-
cologists.
Recent Japanese patents suggested hypoglycemic and
antiinflammatory activities for cytisine and its N-
methyl derivative [6,7].
Recent in vitro research [8,9] has demonstrated a
very high affinity of cytisine to cerebral nicotinic recep-
tors (KdB1 nM), which is superior to that of any other
tested drug (nicotine, acetylcholine, carbachol as ago-
nists and dihydro-b-erythroidine as antagonists).
Central nicotinic receptors are presently considered
as a possible target for the development of drugs useful
in cognitive and attention disorders, Alzheimer’s dis-
ease and other CNS disfunctions [10], and several natu-
Pursuing our systematic research on the structural
modification of quinolizidine alkaloids, in order to
obtain compounds of pharmacological interest, we
devoted our attention to cytisine present in several
plants of the Leguminosae family (Cytisus, Sophora,
Ulex, Baptisia, Genista and others).
From a pharmacological point of view, cytisine
strictly resembles nicotine (Fig. 1): it acts mainly at
ganglionic level, exhibiting more stimulating than
blocking effects; however, central effects are also
shown. These two alkaloids are almost indistinguish-
able as far as peripheral effect is concerned but they
differ somewhat for the central ones (kind of convul-
sions), which are related to cholinergic stimulation [1,2].
Such a pharmacological profile accounts for the
present lack of therapeutic use of cytisine in western
countries (in the past it was used as diuretic [3]), while
in the former Soviet Union it is used as a respiratory
analeptic for its stimulating activity on respiratory cen-
ters and on chemoreceptors in the aortic and carotid
* Corresponding author. Fax +39-010-353 8358.
E-mail address: sparator@unige.it (F. Sparatore)
Fig. 1.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00049-X

C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
439
ral compounds (anabaseine, arecoline, lobeline, anatox-
ine-a, epi-batidine) able to activate them, were added to
nicotine and cytisine.
for nicotinic receptors appears still more outstanding
and quite peculiar.
On the whole, 31 N-substituted derivatives of cytisine
were prepared (Fig. 2).
Many years ago Bovet and his collaborators [11]
demonstrated that suitable doses of nicotine favored
learning and memory in rats. More recently, beneficial
effects of nicotine in both Alzheimer’s and Parkinson’s
disease were asserted [12]. Lobeline also exhibited good
activity in pharmacological models for learning and
memory [13], and for analgesia [14], supporting that
nicotinic receptors play a modulatory role in these
processes.
The foregoing considerations justify the present re-
search aimed at the structural modification of cytisine
in order to obtain compounds of potential therapeutic
interest either at peripheral or central levels with a
particular concern for degenerative pathologies
(Alzheimer’s and related diseases).
Of these, compounds 1–3 were intermediates for the
preparation of other derivatives or could be of interest
as nicotinic ligands. The ketone 3 corresponds to the
alkaloid extracted from Echinosophora koreensis [21].
In the alkyl and arylalkyl derivatives 4–12 the steric
hindrance around the cationic center and the lipophilic-
ity were largely varied so that their influence on the
affinity for nicotinic receptors in vitro and on the
distribution pattern in vivo could be evaluated.
The arylalkyl derivatives could also be interesting as
cardiovascular agents having some structural similarity
with the aryl- and benzyl-sparteins prepared by Boido
et al. [22] and found endowed with positive inotropic
activity.
The structural modifications of cytisine should in-
crease, on the one hand, its passage through the blood–
brain barrier (strongly limited by its relevant
hydrophilic character), as well as reducing its affinity
for ganglionic receptors. However, the selectivity for
central nicotinic receptorial subtypes [10,15] should also
be increased, eventually to the detriment of the
potency.
Structural modification of cytisine was already at-
tempted in the past, but was mainly aimed at improving
its respiratory analeptic property [16,17] or obtaining
local anesthetics [18].
The chemical modifications that can be operated on
cytisine apply, either singularly or in succession, to
secondary amino groups, conjugated double bonds and
carbonyl groups; however, in order to maintain the
central nicotinic activity, the cationic character should
be secured, leaving the typical distance of the basic
nitrogen from the carbonyl dipole unchanged, which
must form a hydrogen bond with a donating group of
the receptor [2,19,20].
In the present work, as a first possibility, the intro-
duction of saturated or unsaturated alkyl or arylalkyl
residues on the amino group was undertaken in order
to increase the overall lipophilicity, but the introduction
of more complex moieties to enable reinforcement or
selection of the activities of cytisine was also
considered.
The presence of a phenylethanolamine or phenyl-
ethanonamine moiety in compounds 13 and 14 could
give rise to some interaction with adrenergic receptors,
influencing the sympathomimetic component of cytisine
activity due to the stimulated release of adrenaline from
adrenal medulla.
Compound 15 is structurally related to the classic
butyrophenone antipsychotics, but still more to the
recent derivatives having as basic head a bridged g-car-
boline moiety characterized by simultaneous affinity for
D2 and 5HT2 receptors [23]. On the other hand, p-
fluorobutyrophenone derivatives could also be of inter-
est as HIV-1 protease inhibitors [24,25].
Compounds 16 and 17 represent examples of a set of
compounds possessing two cytisine units connected
through a polymethylene chain of growing length,
which could generate selective ligands for nicotine re-
ceptors subtypes. It is well known that the elongation
of the carbon atom chain joining two ammonium heads
can shift the selective blockade from ganglionic (hex-
amethonium) to muscular (decamethonium, tubo-
curarine) nicotinic receptors. Moreover, alkanbiguani-
dinium compounds were described recently as being
able to recognize selective nicotinic receptor subtypes
[26].
Compound 16 and its homologs (where n=5, 6 and
10) were already described [27], but, to the best of our
knowledge, their biological activity was not described.
The aryl/heteroaryl–piperazinyl alkyl derivatives 18–
24 and also, to some extent, compound 25 fall within
the multitude of aryl piperazinyl derivatives that, de-
pending on the nature of the aryl residue and of the
moiety attached to the other piperazine nitrogens, can
interact with serotonin and/or a1-adrenergic receptors.
Particularly, they closely resemble the 2-[4-arylpiper-
In order to address the choice of such moieties, the
capability of cytisine to displace specific ligands from
28 different receptors was firstly evaluated.
In this study, cytisine displayed affinity for a number
of receptors (cholecystokinin A, histamine H3, kainate,
muscarinic M1 and M2, N-methyl-D-aspartic acid
NMDA, phencyclidine, serotonin 5HT3, thyrotropin-re-
leasing hormone TRH, vasoactive intestinal polypep-
tide VIP) but the IC50 was always in the range
10−4–10−5 M; thus, the very high affinity of cytisine
azinyl-1-alkyl]-perhydroimidazo[1,5-a]pyridines
[28],
which exhibited high affinity and selectivity for 5-HT1A
-
versus a1-receptor.

440
C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
Fig. 2.
The aroyl derivatives 26–29 supplement the amido
toluenesulphonylurea with the imino group of cytisine
(30) could represent a symbiotic approach to enhance
the hypoglycemic activity of cytisine.
Finally, compound 31 was also the result of a symbi-
otic approach to design an antiarrhythmic agent, taking
into account that lidocaine and N,N%-disubstituted bis-
pidines [29] share such activity.
derivatives formerly studied as respiratory analeptics
[16,17], but the acyl residues now utilized are com-
monly encountered among 5HT3 and 5HT4 agonists
and antagonists, and also among sedative agents (trime-
tozine).
The superimposition of the terminal nitrogen of p-

C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
441
Compound 31 is the 2,6-dimethylanilide of 12-
cytisineacetic acid, an unusual amphoteric alkaloid iso-
lated from Euchresta japonica [30].
CDCl3 or DMSO-d6 as solvent with TMS as internal
standard.
In the NMR spectra, signals of the cytisine moiety
protons are indicated as ‘pyr’ if pertinent to the a-pyri-
done ring and as ‘bisp’ if pertinent to the bispidine
portion of the molecule.
2. Chemistry
For the preparation of compounds 1, 2, 4–12, 14–18
and 31, cytisine was reacted with the suitable halo
derivatives; however, due to steric hindrance around
the imino group, a thorough selection of experimental
conditions is often very important for a successful
alkylation.
N-(3-Chloropropyl)cytisine (2) was obtained without
any particular problem, but all attempts to prepare
N-(2-chloroethyl)cytisine by reacting cytisine with 1-
bromo-2-chloroethane gave always the 1,2-bis-cytisinyl-
ethane (16). Therefore, compound 18 was obtained by
3.1. General method for the alkylation of cytisine (1, 2,
4–12, 14, 31)
Cytisine (0.5–1 g, 2.63–5.26 mmol) was dissolved in
dry acetone (10–20 ml) and the suitable aliphatic or
arylaliphatic halide (1.32–2.63 mmol, ratio 2:1) was
added. For the preparation of compound 2 a cytisine:
1-bromo-3-chloropropane ratio of 1:1 was used. The
mixture was refluxed for a time varying from 1 to 4 h.
In some cases, longer heating times were required (up
to 20 h for 1-bromo-3-chloropropane); however, even
after 36 h, the alkylation of cytisine with ethylene
chlorohydrin was far from completion.
reacting
cytisine
with
1-(2-chloroethyl)-4-(2-
methoxyphenyl)piperazine.
For the synthesis of the piperazine derivatives 19–25,
the suitable aryl- or heteroaryl-piperazines were reacted
with N-(3-chloropropyl)cytisine (2). All but one of the
required piperazines were commercially available; N-
furoylpiperazine was prepared as described in Ref. [31].
Compounds 3 and 13 were obtained by treating
cytisine in dioxane with methylvinylketone and styrene
oxide, respectively.
Finally, for the preparation of amides 26–30, cytisine
was treated with the suitable aroyl chlorides in acetone,
or with p-toluenesulfonyl isocyanate in toluene.
The structures of the prepared compounds were sup-
ported from elemental analyses and spectral data.
The IR spectra of ketones 3, 14, 15 and amides
25–31 exhibited the expected peak for the carbonyl
group, clearly separated from or as partially superim-
posed with that of a-pyridone ring occurring at 1650
cm−1 [2].
The alkylation progression was monitored through
TLC on alumina using dry ether+2% methanol as
eluent.
After cooling, the cytisine hydrohalide was filtered
and acetone was removed under reduced pressure. The
residue, when taken up with ether, may crystallize;
hence, it was partitioned between ether and acidic
water. The acidic solution was basified and extracted
with ether. This method was used to prepare com-
pounds 1, 2, 4–9, 11, 12 and 31. Generally, the crude
products were crystallized from the solvents indicated
in Table 1. In the case of compound 31, purification
was achieved through chromatography on alumina,
eluting with ether+2% methanol. By replacing acetone
with ethylene glycol monomethylether, the method was
also used to prepare compounds 10 and 14.
3.2. N-(2-Hydroxyethyl)cytisine (1)
The NMR spectra did not exhibit any unusual fea-
tures; thus, only the spectra of compounds 3 and 18 are
described as examples (see Section 3).
Since the yield of the cytisine alkylation with ethylene
chlorohydrin was rather poor, the title compound was
also prepared by the method reported by Ing and Patel
[18]. Cytisine dissolved in chloroform was treated with
ethylene oxide and heated for 4 h at 45°C in a closed
tube. After removing the solvent, the residue was taken
up with a little dry ether and formed crystals which
melted at 83–85°C and corresponded to the hydrated
N-(2-hydroxyethyl)cytisine C13H18N2O2·H2O. Ing and
Patel reported a m.p. of 73–74°C. The anhydrous
compound was a glassy solid.
3. Experimental
Melting points were determined by the capillary
method (often sealed under vacuum) on a Bu¨chi ap-
paratus and are uncorrected.
The elemental analyses were performed at the Micro-
analytical Laboratory of the ‘Dipartimento di Scienze
Farmaceutiche’ of Genoa University and the analytical
results for the indicated elements were within 90.3%
of the calculated values.
3.3. N-(3-Oxobutyl)cytisine (3)
IR spectra were recorded with a Perkin–Elmer
Paragon 1000 PC spectrophotometer. H NMR spectra
were taken on a Varian Gemini 200 spectrometer, using
Cytisine (2 g, 10.5 mmol) was dissolved in 7 ml of
warm anhydrous dioxane and treated with 0.95 ml (11.4
mmol) of freshly-distilled methylvinylketone. The mix-
1

442
C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
Table 1
Molecular formulae, melting points, recrystallization solvents and yields for compounds 1–31
Comp.
Formula
M.p. (°C)
Solvent
Yield (%) a
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
C13H18N2O b
C14H19ClN2O·0.75H2O
C15H20N2O2
C16H24N2O·HCl·0.5H2O
C23H38N2O·HCl·H2O d
C18H20N2O·0.25H2O
C18H19FN2O
C21H26N2O
C19H22N2O·HCl·0.5H2O
C20H24N2O2
C20H24N2O·HCl·0.5H2O
C20H22N2O
C19H22N2O2
83–85
oil
dry ether
75
48
90
76
57.3
77.6
46.4
47.5
54.5
25
80
59
47.2
26
28
68
22
80
53
56
45
26
52
30
58
96
54
50
75
34
26
c
115–118
250–260
275–277
142–143
154–155
115–117
\250
dry ether
isopropanol/ether
isopropanol/ether
dry ether
dry ether (washed)
dry ether
absolute ethanol/dry ether
dry ether
ethanol/ether
ether
ethanol
acetone
dry ether (washed)
acetone
ethyl acetate
100–101
\250
86–87
144–146
146–149
179–183
182–185
138–141
153–154
oil
240
oil
oil
oil
C19H19FN2O2
C21H23FN2O2·HCl·1.5H2O
C24H30N4O2·0.25H2O
C25H32N4O2·0.5H2O
C24H32N4O2·0.25H2O
C24H32N4O
C25H34N4O2·2HCl
C24H31ClN4O·0.5H2O
C25H31F3N4O·H2O
dry ether
chromatography (dry ether+methanol)
dry ether (washed)
chromatography (ether)
chromatography (dry ether+methanol)
chromatography (dry ether+methanol)
chromatography (dry ether+methanol)
dry ether (washed)
ethanol
ethanol
ethanol/dry ether
ethanol
ethanol
C23H31N5O·0.25H2O
C22H30N6O·0.25H2O
C23H30N4O3·2HCl·0.5H2O
oil
\280
C18H16Cl2N2O2
212–213
136–140
185–186
290–292
267
C19H10ClN2O3·0.25H2O
C21H24N2O5
C20H19N3O2
C19H21N3O4S
C21H25N3O2
120–122
chromatography (dry ether+methanol)
a The yields are referred always to the free bases; the eventual conversion to the hydrochlorides gave yields superior to 90%.
b Ref. [18].
c Ref. [21].
d M.p. of free base: 40°C.
ture was stirred in a closed tube for 3 h at room
Dioxane was removed under reduced pressure and
the residue was dissolved in a little warm ethanol. After
cooling, some crystals were collected and the ethanol
solution was added of dry ether that produced a further
precipitation of the compound. The combined product
was crystallized from ethanol to give 0.77 g of pure
compound.
temperature (r.t.) and then for 1 h at 60°C. After
cooling, dry ether was added and the separated crystals
were collected after standing in the refrigerator for
some time.
1H NMR [200 MHz, l ppm, CDCl3]: 7.24 (dd,
J=9.1 Hz, 6.9 Hz, 1H pyr), 6.38 (dd, J=9.1 Hz, 1.4
Hz, 1H pyr), 5.93 (dd, J=6.8 Hz, 1.3 Hz, 1H pyr), 3.96
(d, J=15.4 Hz, 1H bisp), 3.81 (dd, J=15.4 Hz, 6.4 Hz,
1H bisp), 2.9–2.8 (m, 3H bisp), 2.49–2.23 (m, 7H: 3H
bisp+2H NꢀCH2ꢀCH2ꢀ+2H CH2ꢀCH2ꢀCO), 1.91 (s,
3H, CH3ꢀCO), 1.9–1.7 (m, 2H bisp).
3.5. N-{[4-(4-Fluorophenyl)-4-oxo]butyl}cytisine (15)
To a solution of freshly distilled v-chloro-4-fluoro
butyrophenone (1.2 g, 6 mmol) in 10 ml of ethylene
glycol monomethylether, 2.3 g (12.1 mmol) of cytisine
dissolved in 10 ml of the same solvent were added. The
mixture was refluxed for 7 h under nitrogen and then
stirred overnight at r.t.
Cytisine hydrochloride was filtered and the solvent
was removed under reduced pressure. The residue was
partitioned between ether and acidic water. The acid
solution was made alkaline and extracted with ether to
3.4. N-[(2-Hydroxy-2-phenyl)ethyl]cytisine (13)
Cytisine (1 g, 5.26 mmol) was dissolved in 7.5 ml of
anhydrous dioxane; styrene oxide (0.7 ml, 6.14 mmol)
was added and the mixture was refluxed for 6 h and
then heated at 130–150°C for 12 h.

C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
443
yield the crude compound 15. Further extraction with
dichloromethane allowed the recovery of unreacted
cytisine.
The crude free base was purified by converting it into
the hydrochloride.
acetone (for 21 and 23), the suitable N-aryl-piperazine
(2–6 mmol) was added and the mixture was generally
refluxed for about 20 h, monitoring the reaction pro-
gression through TLC. For the preparation of com-
pound 23, 7 h of heating were sufficient, while for
compound 19 the reflux was carried out for 32 h.
After cooling, the aryl-piperazine hydrochloride was
filtered and the solvent was removed under reduced
pressure; after taking up the residue with dry ether
some more insoluble material was separated. The ether
solution was concentrated and chromatographed on
alumina (ꢀ20 g) eluting with dry ether or dry ether
plus increasing amounts of methanol (0.5–2%).
3.6. 1,2-bis(N-Cytisinyl)ethane (16)
Cytisine (1 g, 5.26 mmol) was dissolved in dry ace-
tone and added to 0.22 ml (2.6 mmol) of 1-bromo-2-
chloroethane. The mixture was refluxed for 8 h and the
solvent removed. The oily residue was treated with
dilute sodium hydroxide solution and extracted with
dichloromethane. After evaporation of the solvent, a
solid was obtained that crystallized from acetone.
3.10. N-{3-[4-(Pyrimidin-2-yl)piperazin-1-yl]propyl}-
cytisine and N-[3-(4-furoyl piperazin-1-yl)propyl]-
cytisine (24, 25)
3.7. 1,3-bis(N-Cytisinyl)propane (17)
To a solution of N-(3-chloropropyl)cytisine (2) (1–2
mmol) in 10–20 ml of acetonitrile, an equivalent
amount plus a 20–30% excess of the N-(pyrimidin-2-
yl)piperazine or N-furoylpiperazine [31], together with
a large excess (2–6 mmol) of anhydrous sodium car-
bonate, were added. The mixture was refluxed for 48
and 7 h for 24 and 25, respectively. The insoluble
material was filtered and the solvent was removed. The
residue was taken up with water and extracted with
chloroform.
Cytisine (0.57 g, 3 mmol) was dissolved in 10 ml of
diglyme and added to 0.4 g (1.5 mmol) of N-(3-chloro-
propyl)cytisine dissolved in a few milliliters of the same
solvent. The solution was refluxed for 48 h. After
cooling, the precipitate was filtered and the solvent was
removed under reduced pressure. The oily residue was
rinsed several times with dry ether to leave 0.14 (21.2%
yield) of crystals that were further purified from ethyl
acetate.
The solvent was evaporated and the residue was
chromatographed on alumina, eluting with ether+2%
methanol (24) or dichloromethane+1% methanol (25).
The oily compound was analyzed either as such or after
conversion to the dihydrochloride (25).
3.8. N-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl}-
cytisine (18)
To a solution of 1-(2-chloroethyl)-4-(2-methoxy-
phenyl)piperazine (0.45 g, 1.76 mmol) in 15 ml of
acetonitrile, 0.48 g of cytisine and 0.21 g of sodium
hydrogen carbonate (2.52 mmol each) were added. The
mixture was refluxed with stirring for 12 h. The inor-
ganic material was filtered and the solvent removed.
The residue was taken up with water and extracted with
dichloromethane. The oily compound was chro-
matographed on silica gel (30 g) eluting with a mixture
of dichloromethane:methanol (9:1), yielding 0.58 g of
oil that crystallized from dry ether.
3.11. N-Aroylcytisines (26–29)
Cytisine (4–10 mmol) was dissolved in dry acetone
(10–20 ml); an equivalent amount plus 20% excess of
triethylamine was added, followed by an equivalent
amount of the aroyl chloride. For the preparation of
the amide 29, no triethylamine was added, but a mix-
ture of cytisine:aroylchloride (2:1 ratio) was used.
The mixture was refluxed for 1–2 h. After cooling,
the triethylamine hydrochloride or the cytisine hy-
drochloride was filtered and the solvent was removed.
The residue was rinsed with water, diluted sodium
hydroxide solution and again with water and, finally,
crystallized from the suitable solvent (see Table 1).
1H NMR [200 MHz, l ppm, CDCl3]: 7.25 (dd,
J=9.0 Hz, 6.8 Hz, 1H, pyr), 7.1–6.8 (m, 4H, arom),
6.41 (dd, J=9.0 Hz, 1.4 Hz, 1H, pyr), 5.96 (dd, J=6.8
Hz, 1.3 Hz, 1H pyr), 4.05 (d, J=15.4 Hz, 1H bisp),
3.90 (dd, J=15.4 Hz, 6.5 Hz, 1H bisp), 3.86 (s, 3H,
OCH3), 3.05–2.87 (m, 7H: 5H bisp and 2H NꢀCH2),
2.7–2.2
(m,
11H:
1H
bisp
and
10H
ꢀCH2ꢀN(CH2ꢀCH2)2ꢀNꢀ), 1.9–1.6 (m, 2H bisp).
3.12. N-[N-(p-Toluenesulfonyl)carbamoyl]cytisine (30)
3.9. N-[3-(4-Arylpiperazin-1-yl)propyl]cytisine (19–23)
To a solution of cytisine (0.5 g, 2.6 mmol) in 25 ml of
anhydrous toluene was dropped a suspension of 0.57 g
(2.86 mmol) of p-toluenesulfonyl isocyanate in 15 ml of
toluene. The mixture was refluxed for 1 h and then
To a solution of N-(3-chloropropyl)cytisine (2) (1–3
mmol) in 10–20 ml of toluene (for 19, 20 and 22) or

444
C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
stirred for 12 h at r.t. After removing the solvent, the
residue was taken up with water adjusting the pH to
around 7 and extracted with dichloromethane. After
evaporation of the solvent, 0.8 g of solid were obtained.
Procedures not previously described are outlined
below.
4.2. h2-Adrenoceptor antagonism (in 6itro) [38,39]
The field stimulated (95% of maximum, 0.2 Hz)
prostatic portion of rat vas deferens, bathed in physio-
logical salt solution at 32°C, was used. If no significant
response was elicited by the test substance (3 mg/ml),
subsequent inhibition of clonidine (2 mg/ml) induced
reduction in twitch responses by more than 50% indi-
cated significant activity.
4. Pharmacology
The prepared compounds were subjected to a wide
pharmacological investigation in order to point out their
central and/or peripheral activities. Due to the growing
interest on the pharmacological properties of cytisine
and other potential nicotine agonists, we deemed it
worthy to describe the results so far achieved, though
concerning only 23 of the 31 prepared compounds.
Initially, some of the compounds (cytisine plus 3, 4, 7,
12, 16, 17) were tested for their ability to recognize
nicotinic receptors, while others (5, 9, 12, 13, 15, 20, 26,
29, 31) were subjected to a general screening concerning
about sixty in vitro and in vivo tests.
On the basis of these results, some other compounds
were selected for investigating single biological activities
such as antihypertensive (18, 19, 22–25), cardio-
inotropic (7, 8, 28), antiinflammatory (22), passive cuta-
neous anaphylaxis (24) and hypoglycemic (30).
Other compounds are still under study and the corre-
sponding results will be the object of a forthcoming
report.
4.3. Central nicotinic acetylcholine receptor binding [9]
Brain cerebral cortices were removed from Wistar rats
and a membrane fraction was prepared by standard
techniques. Membrane preparation (600 mg) was incu-
bated with [3H]cytisine at a concentration of 2 nM for
75 min at 0°C. Non-specific binding was estimated in the
presence of 100 mM nicotine. Membranes were filtered
and washed three times with binding buffer and the
filters were counted to determine the [3H]cytisine bound.
Compounds were screened at 10 mM.
4.4. Cholecystokinin A receptor binding [40]
A membrane preparation was isolated from guinea-
pig pancreas using standard techniques. Membrane
preparation (0.25 mg) was incubated with 0.2 nM [3H]
L-
4.1. Materials and methods
364718 (see Table 2) for 30 min at 37°C. Non-specific
binding was estimated in the presence of 0.3 mM
364718. Membranes were filtered and washed three
times and filters were counted to determine the [3H]
364718 specifically bound. Compounds were screened at
10 mM.
Most of the basic compounds were tested as hy-
drochlorides, while a few (cytisine, 3, 9, 13, 16, 17, 31)
were used as free bases, due to their sufficient solubility
in water.
For in vivo tests, the substances were generally admin-
istered by the oral route using a gastric tube; they were
prepared as aqueous solutions or finely homogenized
suspensions in ‘Tween 80’ (2%). In a few cases the
substances were introduced intraperitoneally (i.p.), dis-
solved in water. Groups of three or five animals (mice or
rats) were used.
For in vitro assays, it was sometimes necessary to
increase the solubility by means of dimethylsulfoxide at
a concentration that would not interfere with the tests
(0.1% for platelet aggregation and 0.5% for all others).
Doses (mg/kg) or concentration (mg/ml or mM) indi-
cated in the methods were the highest utilized routinely
depending on toxicity; when significant activity was
detected, lower doses or concentrations were tested in
order to define the minimal effective ones, and sec-
ondary tests were performed to have some insight for the
possible mechanism of action.
L
-
L
-
4.5. Vasoacti6e intestinal peptide receptor binding [41]
Lungs were obtained from male guinea-pigs and a
membrane fraction was prepared by standard tech-
niques. Membrane preparation (0.2 mg) was incubated
with 0.01 nM [125I]VIP for 30 min at 22°C. Non-specific
binding was estimated in the presence of 13.5 mM VIP.
Membranes were filtered and washed three times and the
filters were counted to determine the [125I]VIP bound.
Compounds were screened at 10 mM.
5. Results and discussion
The most significant results of the pharmacological
investigations are collected in Tables 2–8; some addi-
tional results are mentioned within the following
discussion.
Concerning the sought-after affinity for the nicotinic
acetylcholine receptors, all the citysine derivatives
The procedures used for most assays were already
described [32–37]; the pertinent reference is indicated
near each assay name in Tables, which are relative to the
more significant ones only.

C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
445
Table 2
Cytisine displacement of specific ligands from several receptors
Binding sites a
Preparation source
Ligand b
% inhibition at
10 mM conc.
Cholecystikinin A
Histamine H3
Kainate
Muscarinic M1
Muscarinic M2
guinea-pig pancreas
entire rat brain
entire rat brain except cerebellum
rat brain cortices
rat heart
rat brain cortices
rat brain cortices
rabbit ileum muscularis
rat brain
[3H]
L
-364718
22
17
19
32
19
33
23
18
26
18
[3H]NAMH
[3H]kainate
[3H]pirenzepine
[3H]NMS
N-Methyl-
D
-aspartic acid (NMDA)
[3H]CGS-19755
[3H]TCP
Phencyclidine
Serotonin 5HT3
Thyrotropin-releasing hormone (TRH)
Vasoactive intestinal polypeptide (VIP)
[3H]GR-65630
[3H](Me) TRH
guinea-pig lung
[
125I]VIP
a Cytisine at 10 mM conc. was found inactive for the displacement of the specific ligands from the following binding sites: adenosine A1 and A2,
angiotensine II, bradykinin, cholecystokinin B, galanin, insulin, interleukin 1a, leukotriene B4, neurokinin 1, neuropeptide Y, platelet activating
factor, phorbol ester, serotonin 5HT1A, s, sodium channel, thromboxane A2, and tumor necrosis factor.
b
L-364718=3-(S)-(−)1,3-dihydro-3-(2-indolecarbonylamino)-1-methyl-5-phenyl-2H-1[1,4]benzodiazepin-2-one; NAMH=a-methylhistamine;
NMS=methylscopolamine; CGS 19755=cis-4-phosphonomethyl-2-piperidine carboxylic acid; TCP=thienylcyclidine; GR-65630=1-(1-
methylindol-3-yl)-3-(4-methyl-imidazol-5-yl)-1-oxo-propane; MeTRH=methyl thyrotropin-releasing hormone.
tested so far exhibited an affinity quite lower than that
of cytisine itself (Table 3), with compound 17 being the
most active (Ki=30 nM). However, while the growing
bulkiness of the substituents on the basic nitrogen was
clearly detrimental for the affinity to the receptor, it is
also responsible for a proportional increase in the
lipophilicity which should affect favorably the passage
of the blood–brain barrier.
Of the nine compounds (5, 9, 12, 13, 15, 20, 26, 29
and 31) subjected to a general pharmacological screen-
ing, and therefore tested for toxicity (Table 4), only
compounds 9, 12, 15 and 29 produced mortality in mice
at the dose of 300 mg/kg p.o. or 100 mg/kg i.p. On the
whole, with sublethal doses, the autonomic symptoms
observed during a 72 h period from drug administra-
tion were rather modest.
analgesia, dopamine antagonism and the inhibition of
stress-induced ulcers.
N-Cinnamylcytisine (12) exhibited strong analgesic
activity in the writhing test and against formalin, al-
though only at doses approaching toxic, but the 2-
methoxyphenylpiperazinylpropyl derivative 20 was still
active as an analgesic at 30 mg/kg, while no toxicity was
seen up to a dose of 300 mg/kg p.o. and 100 mg/kg i.p.
Compound 20 was also active in the apomorphine-in-
duced climbing behavior, suggesting dopamine antago-
nistic activity. The observed response was practically
superimposable with that of buspirone.
Taking into account that the arylpiperazinyl moieties,
present in compound 20 and in buspirone, are valid
templates for 5-HT1A receptor affinity, further investi-
gations are warranted to check the coexistence of
affinity for serotonin and dopamine receptors in com-
pound 20 and its analogs 18–24.
Concerning the in vivo assays directed at showing
any specific activity on the CNS (anticonvulsive, 5-
methoxy-N,N-dimethyltryptamine potentiation, oxo-
tremorine antagonism, tetrabenazine hypothermia an-
tagonism), most of them gave negative results; however,
some interesting responses were obtained concerning
On the other hand, the rather weak response of
compound 15 in the climbing assay for dopamine an-
tagonism was somewhat surprising, in spite of it being
a p-fluorobutyrophenone derivative.
All the tested compounds except one (i.e. 31) inhib-
ited to some extent stress-induced ulcers in mice, sug-
gesting that all were able to cross the blood–brain
barrier. Compounds 9 and 12 exhibited significant ac-
tivity, which was unrelated to anticholinergic activity
since tremors, lacrimation and salivation induced by s.c.
oxotremorine injection were not inhibited.
Table 3
Displacement of [3H]cytisine from central nicotinic acetylcholine
binding sites by cytisine and compounds 3, 4, 7, 12, 16 and 17
Comp.
IC50 (nM)
Ki (nM)
Compounds 15 and 20 both inhibited carrageenan-in-
duced rat paw edema without inducing gastric irrita-
tion. Of interest, the complete failure to prevent
arachidonic acid-induced platelet aggregation suggests
that inhibition of the cyclooxygenase is not involved;
this biological profile is rarely observed and may, there-
fore, warrant further investigation.
Cytisine
3
4
5
265
70
1600
6700
156
48
3
163
43
985
4100
96
7
12
16
17
30

Table 4
Maximum tolerated dose in mice for compounds 5, 9, 12, 13, 15, 20, 26, 29 and 31 (n.t.=not tested)
Administration
route
Dose
(mg/kg)
No. of animals dead (and effects) after treatment with compounds
5
9
12
13
15
20
26
29
31
Oral
300
100
0/3
2/3
3/3
0/3 irritation;
slight increase in
touch sensitivity
n.t.
1/3
1/3
0/3 no effect
0/3 no effect
0/3 no effect
n.t.
1/3
0/3 no effect
0/3 no effect
0/3 slight
n.t.
n.t.
n.t.
decrease in
abdominal tone
and limb tone
n.t.
30
n.t.
0/3 no effect
3/3
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
Intraperitoneal
100
0/3 slight
decrease in
limb tone
and grip
3/3
0/3 slight
decrease in limb
tone and grip
3/3
0/3 slight
0/3 slight
0/3 no effect
0/3 slight
decrease in
abdominal
tone and limb
tone
tremor and
behavioral
depression;
decrease in
spontaneous
activity; slight
/
slight muscular muscular
relaxation and
ataxia; slight
respiratory
relaxation and
ataxia
5
(
fastening;
vasodilation
438
30
no effect
0/3 slight
muscular
0/3 slight
decrease in
limb tone
no effect
0/3 slight
decrease in limb relaxation;
tone and grip
slight muscular slight decrease
n.t.
n.t.
no effect
in abdominal
tone; slight
decrease in limb
tone
451
relaxation; slight
decrease in limb
tone
slight decrease in
limb tone
slight
respiratory
fastening
10
3
n.t.
n.t.
no effect
n.t.
n.t.
n.t.
no effect
n.t.
slight decrease
in limb tone
no effect
no effect
n.t.
n.t.
no effect
n.t.

Table 5
Some in vivo activities of compounds 5, 9, 12, 15 and 20 a,b
c
Test
Route; dose
Effect
Response to compound d
Reference drug
Route; dose
Effect
(mg/kg)
(mg/kg)
5
9
12
15
20
67
e
f
Dopamine antagonist [34] (mice)
Phenyl quinone writhing [33] (mice)
Formalin analgesia [37] (mice)
i.p.; 30
10
p.o.; 100
30
p.o.; 100
30
10
p.o.; 100
30
p.o.; 100
30
p.o.; 30
10
p.o.; 100
30
p.o.; 100
30
p.o.; 100
30
0
17
0
17
buspirone
trifluoroperazine
aspirin
i.p.; 25
i.p.; 1
p.o.; 50
67
50
68
n.t.
40
n.t.
n.t.
n.t.
n.t.
0
n.t.
0
n.t.
n.t.
36
n.t.
n.t.
n.t.
n.t.
0
n.t.
80
20
100
38
n.t.
n.t. 34
0
n.t.
0
50
13
n.t. 25
13
25
3
31
19
n.t.
43
n.t.
n.t.
n.t.
n.t.
17
100
47
96
64
0
40
0
67
0
g
aspirin
p.o.; 50
60
h
i
Antiinflammatory [32] (rats)
aspirin
phenylbutazone
promethazine
p.o.; 150
p.o.; 50
p.o.; 10
40
33
73
8
0
n.t.
25
Passive cutaneous anaphylaxis [34] (rats)
Antiulcer [32] (stress induced in rats)
Antiulcer [33] (ethanol induced in rats)
Hypoglycemic [32] (mice)
n.t.
0
n.t.
j
13
n.t.
38
n.t.
0
n.t.
n.t.
n.t.
50
13
n.t.
38
n.t.
0
n.t.
n.t.
13
chlorpromazine
n.t. omeprazole
p.o.; 20
p.o.; 10
p.o.; 300
71
63
65
/
n.t.
j
0
n.t.
26
3
0
carbenoxolone
n.t.
0
n.t.
n.t.
n.t.
k
l
phenformin
p.o.; 100
p.o.; 3
35
22
5
Hypoglycemic [32] (fasted mice)
glibenclamide
)
n.t.
a Only assays for which at least one compound gave a significant or boderline response are reported.
438
b Compounds 13, 26, 29 and 31 were also tested for all the indicated assays and found inactive; moreover, compounds 22, 24 and 30 were tested for antiinflammatory, passive cutaneous
anaphylactic and hypoglycemic activity, respectively, and were found to be inactive or very weakly active.
c References for the methods employed.
451
d n.t., not tested.
e % Reduction of apomorphine-induced climbing behavior.
f % Inhibition of number of writhes.
g % Reduction of induced paw licking time recorded during the following 20–30 min period after formalin injection (tested only compounds active in the preceding assay).
h % Inhibition of rat paw edema 3 h after carrageenan administration.
i % Inhibition of PCA blue-colored wheals.
j % Reduction of scores attributed to gastric ulcerative lesions.
k % Reduction of tolerance curve after a glucose load (1 g/kg s.c.).
l % Reduction of glycemia in fasted mice 1 h after drug administration (only compounds active in the preceding assay were tested).

448
C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
Table 6
tempt was made to improve this action by joining the
cytisine moiety with a p-toluenesulfonylamidocarbonyl
residue. The obtained compound 30 was completely
inactive at a dose of 30 mg/kg p.o., while killing the
mice at 100 mg/kg p.o., thus resulting in the most toxic
of the cytisine derivatives tested so far.
Although cytisine and N-methylcytisine seem en-
dowed with hypoglycemic activity [6], once more the
observed activity of 12 and 20 must rely on their entire
peculiar structures, since all the remaining tested com-
pounds were completely lacking activity.
Among the previously cited nine compounds sub-
jected to a general screening, compound 20 exhibited a
very strong and long-lasting antihypertensive activity in
rats, while compounds 9, 12 and 13 showed only mod-
est activity. Therefore, six more compounds structurally
related to 20 (18, 19 and 22–25) were tested, and two of
them (19 and 24) were found to exert a good dose-re-
lated and long-lasting activity. Table 6 indicates that
compound 19 was the most potent, but 20 was the most
active. Compound 20 exerted a moderate bradycardic
effect (−8% heart rate) at the highest dose employed
(100 mg/kg), but this was not seen at a lower dose (30
mg/kg), still resulting in a strong antihypertensive
response.
The antihypertensive activity was related neither to
ganglionic blockade (pupil dilation test in mice [35]) nor
to a1- or a2-adrenoceptor antagonism (inhibition of
nor-epinephrine-induced mydriasis in mice [35] and re-
versal of clonidine-induced bradycardia in rats [37]),
although a1- and a2-adrenoceptor antagonisms were
observed in vitro (Table 7). Further b-adrenergic antag-
onism and negative chronotropic activity were shown in
vitro on isolated atria, but calcium antagonism, potas-
sium channel activation, or angiotensin inhibition were
not observed on atria or ileum; thus, the mechanism of
the antihypertensive action remains somewhat
intriguing.
Still concerning the cardiovascular system, an inter-
esting inotropic activity was observed in vitro on iso-
lated guinea-pig left atria for compounds 9 and 15
(Table 7). This activity was not blocked by propranolol;
moreover, a lack of reduction of spontaneous tone in
guinea-pig trachea was observed (Table 8). Therefore,
neither adrenergic stimulation nor phosphodiesterase
inhibition seem to be involved in the inotropic activity.
Inotropic activity is not commonly found in p-
fluorobutyrophenone derived drugs, but it was ob-
served by Platou et al. [42] in melperone.
Both compounds 9 and 15, but particularly the latter,
exhibited negative chronotropic activity in sponta-
neously beating guinea-pig right atria. It is worth not-
ing that compound 13, differing from 9 by the presence
of an hydroxyl group on the phenylethyl moiety, was
completely devoid of inotropic and chronotropic activi-
ties on isolated atria.
Antihypertensive activity in spontaneously hypertensive rats [32,33] of
compounds 9, 12, 13, 18–20 and 24 (n.d.=not determined)
a
Comp.
Dose p.o.
(mg/kg)
% Variation of blood pressure b
after
1 h
2 h
4 h
9
12
13
18
19
30 c
30 c
100
100
100
30
10
3
100
30
10
−5
−3
−3
4
−5
−8
−4
2
−5
−2
−8
−6
−27
−21
−20
2
−20
−20
−18
0
−18
−19
−15
−4
20
24
−36
−18
−5
−38
−12
−3
−34
−12
−2
100
30
10
−19
−8
−17
−6
−12
−16
n.d.
n.d. −4
Reserpine
Clonidine
25
0.1
−20
−22
−24
−22
−26
−20
a In addition to the above, compounds 5, 15, 22, 23, 25, 26, 29 and
31 were also tested for antihypertensive activity and found to produce
B5% variation of blood pressure in each of the three measures. A
variation ]10% is considered highly significant.
b The variation of blood pressure was associated with only a
negligible (B5%) variation of heart rate; compound 20 at 100 mg/kg
produced an 8% reduction of heart rate. A variation ]20% is
considered highly significant.
c Higher doses were not used because of toxicity (see Table 4).
According to a Japanese patent [7], cytisine and its
N-methyl derivative are endowed with antiinflamma-
tory activity; nevertheless, the observed activity of com-
pounds 15 and 20 can be hardly ascribed to the mere
presence of the cytisine moiety in their structure, since
the remaining tested derivatives (including compound
22, which is almost identical to 20) were completely
devoid of antiedema action. Thus, the entire structures
of compounds 15 and 20 are responsible for their
action.
It is worth noting that compound 20 (but not 15)
exhibited antiallergic activity in the passive cutaneous
anaphylaxis test [34] in rats as well as moderate antihis-
tamine activity, inhibiting by 40% the histamine-in-
duced wheal in rats. These activities may relate to the
observed in vitro antihistamine and antiserotonin activ-
ities (Table 8), which also might be, at least in part,
responsible for the antiedema effect.
A significant reduction of glycemia was produced by
compounds 12 and 20 in mice after a glucose load and
also by compound 12 in fasting mice. Both compounds
were inactive in mice previously treated with streptozo-
tocin, indicating that they favor the release of insulin,
as do the well-known sulfonylureas. Therefore, an at-

C. Canu Boido, F. Sparatore / Il Farmaco 54 (1999) 438–451
449
Table 7
In vitro tests related to cardiocirculatory activity for compounds 9, 12, 15 and 20
a
Test
Conc.
(mg/ml)
Effect
Effects produced by compound b
Reference drug
trequinsin
Conc.
(mg/ml)
Effect
9
12
−7
15
20
c
c
Inotropic effect [33]
(guinea-pig left atria)
10
3
+48
+27
+44
−27
10
+48
n.t. +27
n.t.
Chronotropic effect [33]
(guinea-pig right atria)
10
3
1
−21
−15
−7
−16
−6
−11
−3
−12
−7
quinidine
diltiazem
10
10
10
−20
−18
−14
n.t.
n.t.
n.t. mecamylamine
d
e
Ca2+ antagonism [33]
(guinea-pig ileum)
3
26
10
23
5
cinnarizine
practolol
1
82
72
b-Adrenergic antagonism
[35] (guinea-pig left
atria)
10
3
1
n.t.
12
n.t.
8
n.t.
n.t.
20
n.t.
n.t.
83
56
0
0.5
f
a1-Antagonism [37] (rat vas
deferens prostatic
portion)
3
1
0.3
18
n.t.
n.t.
0
n.t.
n.t.
0
n.t.
n.t.
71
57
25
prazosin
0.01
0.2
58
62
65
g
h
a2-Antagonism (rat vas
deferens prostatic
portion)
3
1
0
n.t.
0
n.t.
0
n.t.
58
21
yohimbine
Angiotension I inhibition
[33] (guinea-pig ileum)
3
0
14
0
20
[sar1,thr8]ang. II
0.05
a References for the methods employed (when no reference is cited, the method is as described in this report).
b Compounds 5, 13, 26, 29 and 31 were also assayed using the indicated tests, but did not produce any significant response (compounds 7, 8
and 28 were also tested for inotropic effects but only exhibited weak negative effects). n.t., not tested.
c % Variation in contractile force or rate.
d % Inhibition of Ca2+-induced contractions.
e % Reduction of isoproterenol-induced positive inotropic effect.
f % Reduction of phenylephrine-induced contractile response.
g % Inhibition of clonidine-induced reduction in twitch response.
h % Reduction of contractile response.
Compound 15 exhibited a good inhibition of PAF-in-
duced platelet aggregation, while compounds 9 and 13
had only a moderate activity; the remaining compounds
were inactive. Moreover, compound 15 (at 30 mg/kg)
protected 60% of mice from PAF-induced mortality.
This action is quite unusual, and its presence beside
antiinflammatory activity not related to cyclooxygenase
inhibition makes compound 15 worthy of further inves-
tigations, particularly those aimed at identification of
histoprotective antiiflammatory agents.
Finally, compound 9 increased the electrically stimu-
lated guinea-pig ileum contractions at concentrations
]3 mg/ml (Table 8), but inhibited them at much higher
concentrations (30 mg/ml). Such behavior is suggestive
of a gastroprokinetic activity which will be checked in
vivo.
Acknowledgements
Financial supports from the CNR and from the
Ministero dell’Universita` e della Ricerca Scientifica e
Tecnologica are gratefully acknowledged.
References
[1] D. Bovet, F. Bovet Nitti, Structure et Activite´ Pharmaco–
dynamique des Me´dicaments du Syste`me Nerveux Ve´ge´tatif,
Karger, Basel, 1948, pp. 605–606.
[2] R.B. Barlow, L.J. Mc Leod, Some studies on cytisine and its
methylated derivatives, Br. J. Pharmacol. 35 (1969) 161–174.
[3] P. Lebeau, M.M. Janot, Traite´ de Pharmacie Chimique, tome
IV, Masson, Paris, 1955, p. 2859.
[4] M.J. Dallemagne, C.H. Heymans, Respiratory stimulants, in:
R.H.F. Manske (Ed.), The Alkaloids, vol. 5, Academic Press,
New York, 1955, pp. 109–139.
[5] V.V. Zakusov, Farmacologiia, Medgiz, Moscow, 1960, p. 203.
[6] I. Murakoshi, Y. Fuji, S. Takedo, J. Arai, Lupine alkaloids as
antidiabetics, Jpn. Kokai Tokkyo Koho, Japanese Patent 04-
295480 (20-10-1992); Chem. Abstr. 118 (1993) 45733.
In conclusion, the introduction of different kinds
of substituents on the basic nitrogen of cytisine
gave rise to several new interesting activities, while
an appreciable degree of affinity for central nicotinic
receptors was sometimes retained.

Table 8
Some in vitro activities of compounds 5, 9, 12, 13, 15 and 20 (n.t.=not tested)
b
Test
Conc. (mg/ml)
Effect
Effects produced by compound
Reference drug
Conc. (mg/ml)
Effect
5
9
12
13
15
20
Platelet aggregation induced by:
PAF [33]
c
10
3
100
30
3
3
3
3
30
10
3
1
10
3
8
24
n.t.
8
31
n.t.
67 l
23
26
12
0
13
0
0
15
RP-48740 m
theophylline
5
54
60
n.t.
n.t.
n.t.
c
e
f
d
d
d
d
d
ADP [33]
Tracheal relaxation [33]
LTD4 antagonism [34]
Bradykinin antagonism [33]
Substance P antagonism [33]
Cholecystokinin antagonism [33]
Ileum electric stimulation increase [33]
7
25
0
0
9
25
23
10
26
n.t.
n.t.
−
25
0
0
20
0
n.t.
n.t.
0
11
10
3
30
30
25
15
31
n.t.
n.t.
f
f
15
12
−
+
+
−
f
0
g
h
n.t.
n.t.
−
n.t. metoclopramide
n.t.
−
n.t.
90
68
31
80
55
10
0.3
+
−
n.t.
−
n.t.
/
n.t.
n.t.
i
j
j
j
j
j
j
Antihistamine [34]
Antiserotonin [34]
terfenadine
25
73
56
1
k
j
j
j
j
100
30
10
promethazine
0.1
4
(
a Compounds 26, 29 and 31 were also tested in the indicated assays but were found inactive or very weakly active.
b References for the methods employed.
438
451
c % Inhibition of rabbit platelets aggregation induced by the indicated agent.
d Only compounds active in the preceding assay were tested.
e % Inhibition of guinea-pig tracheal tone relative to relaxation induced by 0.3 mg/ml epinephrine.
f % Inhibition of contractile response of guinea-pig ileum to various agents.
g % Increase of contractions by \15% is recorded as significant (+).
h At 30 mg/ml a significant (\50%) decrease of contractions was observed.
i % Inhibition of histamine-induced guinea-pig ileum contraction.
j Tested only compounds active in PCA assay (see Table 7).
k % Inhibition of serotonin-induced rat aortic strip contractions.
l Compound 15 at 30 mg/kg p.o. reduced PAF-induced mortality in mice by 60%.
m 3-(3-Pyridyl)-1H,3H-pyrrolo[1,2-c]thiazole-7-carboxamide.

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