Synthesis and pharmacological investigation of 9-methyl-1,2,3,4, 6,7,12,12b-octahydro-7-oxo-indolo[2,3-a]quinolizine

Article abstract of DOI:10.1016/S0014-827X(99)00056-7

The title ketone, a supposed metabolite of 9-methyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizine (MIQ), was prepared and found different on chromatographic comparison from the isolated metabolite M₄. However, the pharmacological screeni

Full text of DOI:10.1016/S0014-827X(99)00056-7

www.elsevier.nl/locate/farmac
Il Farmaco 54 (1999) 479–485
Synthesis and pharmacological investigation of
9-methyl-1,2,3,4,6,7,12,12b-octahydro-7-oxo-indolo[2,3-a]quinolizine
Teresa Grandi a, Fabio Sparatore a,*, Anna Sparatore b
a Dipartimento di Scienze Farmaceutiche, Uni6ersita` di Geno6a, Viale Benedetto XV 3, 16132 Genoa, Italy
b Istituto di Chimica Farmaceutica e Tossicologica, Uni6ersita` di Milano, Viale Abruzzi 42, 20131 Milan, Italy
Received 27 January 1999; accepted 15 May 1999
Abstract
The title ketone, a supposed metabolite of 9-methyl-1,2,3,4,6,7,12,12b-octahydroindolo[2,3-a]quinolizine (MIQ), was prepared
and found different on chromatographic comparison from the isolated metabolite M4. However, the pharmacological screening
of this compound evidenced, among others, some interesting properties as inhibition of arachidonic acid induced platelet
aggregation and protection from both hypobaric and cyanide induced hypoxia in mice. These activities suggest a possible
cerebroprotective action to be further investigated. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: 7-Oxo-indolo[2,3-a]quinolizidine; Anti-aggregating agents; Cerebroprotective agents
1. Introduction
Therefore, compound 1 has been synthesized through
an unequivocal procedure and its chromatographic be-
haviour was compared with that of metabolite M4,
while its pharmacological profile was investigated thor-
oughly (Scheme 2).
The Fischer indole cyclization applied to several
para-substituted phenylhydrazones of quinolizidin-1-
one afforded 9-substituted-1,2,3,4,6,7,12,12b-octahy-
droindolo[2,3-a]quinolizines, whose pharmacological
properties were reported some years ago [1]. The 9-
methyl derivative (MIQ) at a dose of 5 mg/kg exhibited
a strong inhibition of carrageenan-induced edema in rat
hind paw and anti-hypertensive activity in rats become
hypertensive after DOCA s.c. implantation, as well as a
peculiar biphasic (stimulating and then depressive) ac-
tivity on spontaneous motility in rats.
2. Chemistry
For the synthesis of 1, the sequence followed by
Rosenmund et al. [3–5] to prepare the corresponding
demethylated compound was applied firstly with only
minor modifications.
In an attempt to explain this behaviour, the
metabolic fate of this compound was investigated [2]
and four metabolites (M1–M4) were isolated. The struc-
tures for three of them were characterized by several
mass spectrometry techniques and confirmed by synthe-
sis, while for the fourth (M4) the structure of 9-methyl-
1,2,3,4,6,7,12,12b-octahydro-7-oxo-indolo[2,3-a]quino-
lizine (1) was tentatively suggested on the basis of the
available data (Scheme 1).
Thus, the 5-methyl-2-nitrophenylacetic acid [6] was
converted to the chloride and reacted with N-cyclopen-
tenylmorpholine to obtain 2-[(5-methyl-2-nitrophenyl)-
acetyl]cyclopentanone, whose reduction gave the 2-(5-
methylindol-2-yl)cyclopentanone. Schmidt rearrange-
ment converted the cycloketone into a piperidone
derivative whose reduction gave the 5-methyl-2-(2-pipe-
ridinyl)-1H-indole. Alkylation with ethyl bromoacetate
in the presence of Hu¨nig base, followed by alkaline
hydrolysis furnished the 2-(5-methyl)-2(1H)-indolyl-1-
piperidineacetic acid, which was finally cyclized with
PPA to the required 1.
* Corresponding author. Tel.: +39-010-512 721; fax: +39-010-
353 8358.
E-mail address: sparator@unige.it (F. Sparatore)
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00056-7

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T. Grandi et al. / Il Farmaco 54 (1999) 479–485
Scheme 1.
Scheme 2. Reagents: (a) SOCl2/CHCl3; (b) N-(1-cyclopentenyl)morpholine/TEA; (c) SnCl2/ether; (d) NaN3, H2SO4, CHCl3/C6H6; (e) LiAlH4/
THF; (f) BrCH2COOC2H5, N-ethyldicyclohexylamine/CH3OH; (g) KOH, CH3OH; (h) PPA, N2, 75–100°C; (i) PPA, N2, 180°C; (j) H2/PtO2, HCl,
CH3OH.
Since the overall yield of this synthetic sequence was
only 3.5%, an alternative route was sought. Actually,
the 5-methyl-2-(2-piperidinyl)-1H-indole was obtained
more easily by catalytic hydrogenation [7] of 5-methyl-
2-(2-pyridyl)indole that was produced through the
Fischer cyclization of 2-acetylpyridine p-tolylhydra-
zone in analogy with known methods [8,9]. Starting
from the last compound the overall yield of 1 was
doubled.
3. Experimental
3.1. Chemistry
Melting points were determined by the capillary
method on a Bu¨chi apparatus and are uncorrected.
The elemental analyses were performed at the Micro-
analytical Laboratory of the ‘Dipartimento di Scienze
Farmaceutiche’, University of Genoa, and the analyti-

T. Grandi et al. / Il Farmaco 54 (1999) 479–485
481
cal results for the indicated elements were within
90.3% of the calculated values.
2 h. The solution was then poured into ice/water,
extracted with chloroform and the solvent was removed
under reduced pressure. The resulting solid was crystal-
lized from methanol to yield 5, m.p. 235°C (dec.) (0.84
g, 66%). IR (KBr, cm−1): 3264 (NH indole), 3180 (NH
UV spectra were recorded on a Perkin–Elmer 550 S
spectrophotometer; IR spectra were recorded on a
1
Perkin–Elmer spectrometer Paragon FT-100. H NMR
1
spectra were taken on Varian Gemini 200 or Bruker
300 spectrometers, using CDCl3 or DMSO-d6 as sol-
vents, with TMS as internal standard.
piperidinone), 1667 (CO). H NMR (DMSO-d6): 10.95
(br s, 1H, H(1%)); 7.84 (br s, 1H, H(1)); 7.30–7.19 (m,
2H, H(4%)+H(7%)); 6.88 (dd, 1H, J1=7.9 Hz, J2=2.0
Hz, H(6%)); 6.17 (s, 1H, H(3%)); 4.68 (br.t 1H, J about 6
Hz, H(6)); 2.35 (s, 3H, CH3); 2.23 (t, 2H, J=6.2 Hz,
H(3)); 2.15–1.62 (m, 4H, H(4)+H(5)). Anal.
(C14H16N2O) C, H, N.
3.1.1. 2-[(5-Methyl-2-nitrophenyl)acetyl]cyclopentanone
(3)
A mixture of 5-methyl-2-nitrophenylacetic acid [6]
(8.00 g, 0.041 mol), thionyl chloride (5.94 g, 0.050 mol),
DMF (1 ml) and anhydrous chloroform (50 ml) was
heated for 4 h at 40°C under magnetic stirring. The
solvent was removed, the residue was dissolved in dry
chloroform and it was slowly added to a solution of
N-(1-cyclopentenyl)morpholine (6.13 g, 0.040 mol) and
triethylamine (6.07 g, 0.060 mol) in anhydrous chloro-
form (50 ml). The mixture was stirred for 2 h at room
temperature (r.t.), then the solvent was removed at
reduced pressure. The resulting oil was stirred overnight
with 37% HCl (15 ml) and water (15 ml). Finally, water
was added (50 ml) and the solution was extracted with
chloroform. After removal of the solvent the viscous oil
was chromatographed on a silica gel column (CHCl3 as
eluent). The first fractions were pooled and evaporated
to afford 3 as a yellow oil which gave a positive colour
test with FeCl3 and was used without further character-
ization (6.30 g, 59%).
3.1.4. 5-Methyl-2-(2-piperidinyl)-1H-indole (6)
A suspension of 5 (0.84 g, 0.0037 mol) in anhydrous
THF (20 ml) was refluxed with LiAlH4 (1.68 g, 0.044
mol) for 2 h. Water was added slowly (3 ml) and the
product was extracted with ether. The organic phases
were evaporated and the white solid was crystallized
from cyclohexane, m.p. 148–150°C (0.56 g, 71%). IR
(KBr, cm−1): 3283 (NH indole), 3143 (NH piperidine).
1H NMR (DMSO-d6): 0.79 (br s, 1H, H(1)); 7.27–7.14
(m, 2H, H(4)+H(7)); 6.84 (dd, 1H, J1=8.0 Hz, J2=
2.0 Hz, H(6)); 6.13 (s, 1H, H(3)); 3.75 (dd, 1H, J1=
10.0 Hz, J2=3.0 Hz, H(2%)); 3.03 (br d, 1H, J=12.8
Hz); 2.79–2.60 (m, 2H); 2.38 (s, 3H, CH3); 2.00–1.87
(m, 1H+NH); 1.79–1.32 (m, 4H). Anal. (C14H18N2) C,
H, N.
3.1.5. 2-(5-Methyl-2(1H)-indolyl)-1-piperidineacetic
acid (7)
3.1.2. 2-(5-Methyl-1H-indol-2-yl)cyclopentanone (4)
Dry hydrogen chloride was bubbled for 30 min into
a solution of 3 (3.40 g, 0.013 mol) and anhydrous
stannous chloride (7.40 g, 0.039 mol) in dry ether (100
ml). The mixture was stirred at r.t. for 1 h, then the
solvent was removed under reduced pressure to give a
yellow residue which, in turn, was treated at 0°C with
15% NaOH solution (70 ml). After 15 min the solution
was extracted with CHCl3, the extracts were dried
(Na2SO4), the solvent was removed and the residue was
purified by chromatography on a silica gel column
(eluent CHCl3). The first fractions gave a solid which
was crystallized from ethanol to give 4, m.p. 147–
148°C (1.20 g, 43%). IR (KBr, cm−1): 3330 (NH), 1726
(CO). 1H NMR (CDCl3): 9.93 (br s, 1H, H(1%)); 7.34 (d,
1H, J=2.0 Hz, H(4%)); 7.25 (d, 1H, J=8.5 Hz, H(7%));
6.97 (dd, 1H, J1=8.5 Hz, J2=2.0 Hz, H(6%)); 6.22 (m,
1H, H(3%)); 3.48 (m, 1H, H(2)); 2.70–1.92 (m, 9H,
H(CH3)+aliphatic protons). Anal. (C14H15NO) C, H,
N.
A solution of 6 (1.00 g, 0.0047 mol), ethyl bromo-
acetate (7.0 ml), N-ethyldicyclohexylamine (7.0 ml) in
methanol (40 ml) was stirred at r.t. for 48 h. The
solution was evaporated to dryness and the residue
treated with benzene/pentane (1:1) to allow the precipi-
tation of the N-ethyldicyclohexylamine hydrobromide.
After filtration, the organic phase was extracted with 1
N HCl and the acid extracts made alkaline with
NH4OH 2 N up to pH 10. Finally, the aqueous phase
was extracted with benzene, the organic phase was
dried (Na2SO4) and evaporated to give an oil which was
dissolved in 7 ml of 20% KOH methanolic solution.
The mixture was stirred at r.t. for 20 h and added with
water (20 ml). The pH value was adjusted to 5.5, the
white solid which precipitated was filtered, washed with
water and finally crystallized from methanol to yield 7,
m.p. 217–219°C (dec.) (0.67 g, 52%). IR (KBr, cm−1):
3600–2600 (very broad, NH indole+ꢀNH+), 1616
1
and 1368 (COO−). H NMR (DMSO-d6, 300 MHz):
10.87 (br s, 1H, H(1%)); 7.23–7.18 (m, 2H, H(4%)+
H(7%)); 6.84 (dd, 1H, J1=8.0 Hz, J2=2.0 Hz, H(6%));
6.18 (d, 1H, J=2.0 Hz, H(3%)); 3.78 (dd, 1H, J1=11.0
Hz, J2=3.0 Hz, H(2)); 3.11–3.04 (m, 1H); 3.10 (d, 1H,
J=16.0 Hz, H(a)); 2.80 (d, 1H, J=16.0 Hz, H(a));
2.65–2.54 (m, 1H); 2.33 (s, 3H, CH3); 1.86–1.30 (m,
3.1.3. 6-(5-Methyl-1H-indol-2-yl)piperidin-2-one (5)
A mixture of 4 (1.2 g, 0.0056 mol), sodium azide
(0.75 g, 0.011 mol) and conc. H2SO4 (10 ml) in chloro-
form (60 ml) and benzene (60 ml) was stirred at 0°C for

482
T. Grandi et al. / Il Farmaco 54 (1999) 479–485
1
1
water protons allows no H NMR assignment for the
(1.80 g, 43%). IR (KBr, cm−1): 3350 (NH). H NMR
NH+. Anal. (C16H20N2O2) C, H, N.
(CDCl3): 9.61 (br s, 1H, H(1)); 8.57 (m, 1H, H(6%)); 7.81
(m, 1H, H(3%)); 7.72 (td, 1H, J1=7.5 Hz, J2=1.6 Hz,
H(4%)); 7.44 (s, 1H, H(4)); 7.30 (d, 1H, J=8.0 Hz,
H(7)); 7.16 (m, 1H, H(5%)), 7.05 (dd, 1H, J1=8.0 Hz,
J2=2.0 Hz, H(6)); 6.95 (s, 1H, H(3)); 2.46 (s, 3H,
CH3). Anal. (C14H12N2) C, H, N.
3.1.6. 9-Methyl-1,2,3,4,6,7,12,12b-octahydro-7-
oxoindolo[2,3-a]quinolizine (1)
A mixture of 7 (0.3 g, 0.0011 mol) and polyphospho-
ric acid (25.57 g) was heated under nitrogen atmosphere
for 0.5 h at 75°C, for 0.5 h at 85°C and for 3 h at
90–100°C. The reaction mixture was poured into ice/
water, alkalinized to pH 10 with concentrated ammonia
and extracted with chloroform. The organic phase was
dried (Na2SO4) and the solvent removed to afford a
solid which was purified by a chromatography on a
silica gel column (ethyl acetate as eluent). After a
fore-run which was discarded, the fractions were col-
lected and evaporated to give 1 as a white solid which
was crystallized from methanol, m.p. 240°C (dec.) (0.16
g, 57%). UV (CH3OH) (umax, nm (log m)): 295 (4.00),
265 (4.06), 245 (4.25). IR (KBr, cm−1): 3191 (NH),
Compound 9 was converted into the hydrochloride
as described for 1; m.p. 209–212°C (dec.).
3.1.9. 5-Methyl-2-(2-piperidinyl)-1H-indole (6)
A solution of 9 as hydrochloride (1.80 g, 0.0073 mol)
in methanol (60 ml) was reduced in a Parr apparatus
using PtO2 (0.21 g) as catalyst. After removal of the
catalyst, the solvent was evaporated under reduced
pressure, the residue dissolved in dichloromethane and
was shaken with a 10% solution of Na2CO3. Evapora-
tion of the organic phase yielded 6, which was crystal-
lized from cyclohexane. M.p. 148–150°C (0.88 g, 58%).
1
1620 (CO). H NMR (DMSO-d6, 300 MHz): 11.80 (br
s, 1H, H(12)); 7.84 (s, 1H, H(8)); 7.31 (d, 1H, J=8.4
Hz, H(11)); 7.03 (dd, 1H, J1=8.4 Hz, J2=2.0 Hz,
H(10)); 3.55–3.48 (m, 1H); 3.31 (d, 1H, J=16.5 Hz,
H(6)); 3.10 (dd, 1H, J1=16.5 Hz, J2=2.5 Hz, H(6));
2.98–2.89 (m, 1H); 2.50–2.36 (m, 2H); 2.42 (s, 3H,
CH3); 1.95–1.53 (m, 5H). Anal. (C16H18N2O) C, H, N.
Compound 1 was converted into the hydrochloride
by means of the stoichiometric quantity of 1 N ethano-
lic HCl; the solution was evaporated to dryness and the
residue washed several times with dry ether. M.p.
\260°C (dec.).
3.2. Chromatography
For mono- and bidimensional thin layer chromato-
graphy, precoated silica gel 60 F254 TLC plates (5×20
and 20×20 cm, thickness 0.25 mm), from Merck
(Bracco, Milan, Italy) were used. Compound 1 was laid
down on plates as methanolic solution. Mixtures of
iso-propanol, ethyl acetate, benzene, 25% ammonia
(30:30:30:9) or toluene, ethyl formate, formic acid
(50:40:10) were used as eluents. Spots were visualized
under UV light (254 nm) and by spraying the plates
with Dragendorff’s reagent.
3.1.7. 2-Acetylpyridine-p-tolylhydrazone (8)
A mixture of 2-acetylpyridine (4.83 g, 0.040 mol) and
p-tolylhydrazine (4.88 g, 0.040 mol) in ethanol (3 ml)
was heated for 1 h at 90–100°C under magnetic stir-
ring. On cooling, a pale yellow crystalline solid precipi-
tated and was crystallized from n-hexane, to give
yellow needles, m.p. 95–97°C (8.70 g, 97%). IR (KBr,
3.3. Pharmacology
The pharmacological profile of compound 1 was
explored through a preliminary broad screening per-
formed by Panlabs Inc. (Bothell, WA, USA). This
assay package, named PharmaScreen®, is used in the
determination of the maximum tolerated dose (MTD,
p.o. and i.p.), with simultaneous behavioural examina-
tion (Irvin test), and in 34 primary in vivo tests (using
a suitable MTD fraction, depending on the test type)
and in 26 in vitro tests concerning CNS, cardiovascular
and gastrointestinal apparatuses, intermediary meta-
bolism, allergy and inflammation.
1
cm−1): 3337 (NH). H NMR (CDCl3): 8.55 (m, 1H,
H(6)); 8.19 (m, 1H, H(3)); 7.67 (td, 1H, J1=7.50 Hz,
J2=1.60 Hz, H(4)); 7.48 (br s, 1H, NH); 7.21–7.08 (m,
5H, H(5)+phenylene protons); 2.46 (s, 3H, acetyl
CH3); 2.38 (s, 3H, CH3 on the phenyl ring). Anal.
(C14H15N3) C, H, N.
3.1.8. 5-Methyl-2-(2-pyridyl)indole (9)
Compound 1 was used as hydrochloride. For in vivo
tests 1 was generally administered by oral route using a
gastric tube, as aqueous solution or finely homogenized
suspension in Tween 80 (2%) when the highest dose
(300 mg/kg) was used. In a few cases it was introduced
intraperitoneally as aqueous solution (10 ml/kg).
Groups of three or five animals (rats or mice) were
used. For in vitro assays, the dissolution of test com-
pound in buffer or saline solution was speeded up by
4H). A broad water signal between 3.65 and 3.40 hides
A mixture of 8 (4.50 g, 0.020 mol) and polyphospho-
ric acid (13.5 g) was heated slowly from 25 to 180°C
under nitrogen. After 5 min at 180°C, the mixture was
cooled to r.t., added to water (20 ml), 6 N NaOH (20
ml) and extracted with ether. The ethereal extracts were
dried (Na2SO4), evaporated at reduced pressure and the
residue was submitted to column chromatography on
alumina (ratio 1:20) using ether as eluent to give 9,
which was crystallized from methanol, m.p. 125–126°C

T. Grandi et al. / Il Farmaco 54 (1999) 479–485
483
Table 1
Some in vivo activities of compound 1 and reference drugs
a
Test
Dose (mg/kg)
Effect
Response
Ref. Drug
Dose (mg/kg)
Response
route
route
b
c
Phenylquinone writhing (mice) [11]
Tetrabenazine hypothermia (mice)
100 p.o.
30 i.p.
18
15
ibuprofen
25 p.o.
3 p.o.
3 p.o.
30 i.p.
65
50
50
imipramine
tranylcypromine
diazepam
d
e
f
Hypobaric hypoxia (mice)
100 i.p.
30
100 p.o.
30
128; 113
0
60
20
−6
−8
−10
−8
88; 85
42
50; 50
38
120
Hypoxia cyanide induced (mice) [13]
Blood pressure (SH rats) [10,11]
Heart rate (SH rats) [10,11]
Bleeding time (mice) [13]
flunarizine
nifedipine
nifedipine
aspirin
30 p.o.
5 p.o.
60
(1 h)
(4 h)
(1 h)
(4 h)
100 p.o.
−18
−14
–7
−10
55
f
100 p.o.
5 p.o.
g
h
100 p.o.
30
100 p.o.
30
30 p.o.
30 p.o.
10
30 p.o.
10
30 p.o.
300 p.o.
Anti-ulcer ethanol induced (rats)
carbenoxolone
50
h
i
Anti-ulcer stress induced (rats) [10]
Urine volume change (rats) [10,11]
25
chlorpromazine
hydroflumethiazide
20 p.o.
3 p.o.
71
+70
+80
+20
+200
+10
+40
j
j
Saluresis (rats) [10,11]
Kaluresis (rats) [10,11]
hydroflumethiazide
hydroflumethiazide
3 p.o.
+200
+200
30 p.o.
10 p.o.
a References for the methods employed (when no reference is cited, the method is described in the present paper).
b % Inhibition of number of writhes.
c % Reduction of tetrabenazine-induced hypothermic response.
d % Prolongation of survival time of mice placed in hypobaric (200 mmHg) chamber.
e % Surviving mice 60 min after i.v. administration of submaximal lethal dose of KCN.
f % Variation of blood pressure and heart rate in spontaneously hypertensive rats; variation higher than 10 and 20%, respectively, are considered
highly significant.
g % Prolongation of bleeding time in mice after transection of the tip (0.5 mm) of each tail.
h % Reduction of scores attributed to ulcerative lesions, 1 h after the administration of 1 ml of absolute ethanol or after 4 h of forced partial
immersion in water.
i % Variation of urine volume in test versus control animals 6 h from administration.
j % Variation of Na+ or K+ excretion expressed as meq/100 g body weight 6 h from administration.
the H(5%) signals; moreover, the rapid exchange with
means of DMSO; the final concentration of DMSO,
not interfering with tests, was 0.1% for platelet aggrega-
tion and 0.5% for all others.
3.3.1. Tetrabenazine hypothermia [17]
Test sample is administered p.o. (30 mg/kg) to a
group of three mice 30 min before injection of tetra-
benazine methan sulfonate (50 mg/kg, i.p.) and body
temperature is recorded 60 min later. Reduction of
tetrabenazine-induced hypothermic response by more
than 50% is considered significant and may indicate
anti-depressant activity.
The procedures for most of these assays were already
described [10–16]; the pertinent reference is indicated
near each assay name in the Tables 1 and 2, which are
relative only to the more significant ones. Procedures
not previously described are given in the following
section. A pre-established level of response which is
high enough to suggest a significant activity is indicated
for each assay. Doses (mg/kg) or concentrations (mg/ml
or mM) indicated in the methods were the highest
utilized routinely, depending on toxicity; when signifi-
cant activity was detected, lower doses or concentra-
tions were tested in order to define the minimal effective
ones, and secondary tests were performed to provide
some insight for the possible mechanism of action.
3.3.2. Hypobaric hypoxia [18]
The test substance is administered i.p. (100 mg/kg) to
a group of three mice 30 min before being placed in a
chamber at a hypobaric pressure of 200 mmHg. Prolon-
gation of survival time relative to a vehicle-treated
control group of animals by more than 100% in the
absence of CNS depressant effect may indicate cerebro-
protective activity.

484
T. Grandi et al. / Il Farmaco 54 (1999) 479–485
Table 2
Some in vitro activities of compound 1 and reference drugs
a
Test
Conc. (mM)
Effect
Response Ref. drug
Conc. (mM)
Response
70
b
c
Adrenergic a1 antagonism [15] (rat vas deferens)
Cardiac inotropy (guinea pig left atria) [11]
Cardiac chronotropy (guinea pig right atria) [11]
Rat portal vein spontaneously activated [11]
Tracheal contraction (guinea pig) [11]
30
30
30
30
30
15
−20
−5
20
prazosin
0.08
c
clonidine
cromakalim
epinephrine
3
0.3
0.3
−18
81
79
d
e
27
Inhibition of platelet aggregation induced by:
Arachidonic acid [11]
f
30
10
30
30
30
30
30
30
100
0
0
4
8
12
18
25
aspirin
14
0.3
3
0.3
10
0.03
0.03
1
100
100
74
68
60
60
70
74
indomethacin
2-chloroadenosine
apafant
sulotroban
nifedipine
f
ADP [11]
PAF [11]
Tromboxane A2 [12]
Bradykinin B2 antagonism [11]
Cholecystokynin CCKA antagonism [11]
Leukotriene LTD4 antagonism [12]
f
f
g
g
g
devazepide
LY-171883
a References for the methods employed.
b % Reduction of the phenylephrine-induced contractile response.
c % Variation in contractile force or rate, variations higher than 40 and 10%, respectively, are considered highly significant.
d % Inhibition of spontaneous movements.
e % Inhibition of tracheal tone relative to relaxation induced by 0.3 mg/ml epinephrine.
f % Inhibition of maximum non reversible rabbit platelet aggregation induced by sodium arachidonate (50 mg/ml), ADP (0.8 mg/ml), PAF (10–20
ng/ml) and U-46619 (5 mg/ml).
g % Reduction of guinea pig ileum contractile response to the various agents.
3.3.3. Anti-ulcer (ethanol-induced) acti6ity [19]
The test substance is administered p.o. (100 mg/kg)
to overnight-fasted rats 15 min before gavage with 1 ml
On the other hand, compound 1 exhibited the follow-
ing significant activities: saluretic and moderate
kaluretic activities associated with increased urine out-
of absolute ethanol. The animals are sacrificed 1 h later
put; protection from ethanol-induced gastric ulcers in
and gastric ulceration is scored subjectively concerning
rats, unrelated to in vivo anti-cholinergic activity; inhi-
degree of haemorrhage and severity of ulcerative le-
bition of arachidonic acid-induced platelet aggregation
sions. Inhibition of gastric ulcers by more than 50%
in vitro, with strong prolongation of bleeding time in
suggests cytoprotective activity.
mice (although similar concentration failed to inhibit
ADP and tromboxane A2-induced aggregation).
Still more interesting was the capacity of compound
1 at 100 mg/kg i.p. or p.o. to prolong the survival time
of mice placed in a chamber at a hypobaric pressure of
200 torr and to reduce the mortality of mice receiving a
sub-maximal lethal dose (LD95=2.4 mg/kg i.v.) of
potassium cyanide. No toxic or other untoward central
or autonomic signs were seen at similar or higher dose
levels; thus, both assays suggest a possible cerebropro-
tective activity for 1 deserving further investigations.
Although at only moderate level some other activities
were seen both in vivo and in vitro: inhibition of
phenylquinone writings, antagonism to tetrabenazine-
induced hypothermia (indicating possible anti-depres-
sive activity), inhibitions of stress-induced gastric
ulcers; adrenergic a1-antagonism on rat vas deferens,
potassium channel activation (reduction of spontaneous
myogenic movements of strips of rat portal-mesenteric
veins), leukotriene LTD4, bradykinin B2 and cholecys-
tokinin CCKA antagonism on guinea pig ileum and
relaxation of guinea pig tracheal strips.
4. Results and discussion
The chromatographic behaviour of 1 on two different
TLC systems (TCL1, Rf=0.74; TLC2, Rf=0.06) indi-
cated that it is different from the MIQ metabolite M4
previously isolated [2] (TCL1, Rf=0.04; TLC2, Rf=
0.25), whose structure, therefore, remains undefined.
In the general pharmacological screening, compound
1 resulted well tolerated up to the dose of 100 mg/kg
i.p. or 300 mg/kg p.o. in mice, without any sign of
central or autonomic effect during the 72 h of observa-
tion after administration.
Compound 1 actually exhibited an activity profile
quite different from that of MIQ. On the one hand, the
strong anti-inflammatory and anti-hypertensive activi-
ties observed in rats with MIQ at 5 mg/kg were,
respectively, completely abolished or strongly reduced
even when 1 was administered in rats at doses up to 100
mg/kg p.o. Moreover, the biphasic effect of MIQ on
spontaneous motility in rats was not shown by 1 neither
in rats (at 30 mg/kg) nor in mice at 300 mg/kg p.o. or
100 mg/kg i.p.
Concluding, it is worth noting that the pharmacolog-
ical profile of compound 1, bearing a ketonic function

T. Grandi et al. / Il Farmaco 54 (1999) 479–485
485
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7-oxo-indolo[2,3-a]quinolizine (1) was found to be dif-
ferent from the MIQ metabolite M4 whose structure
remains unclear.
Nevertheless, compound 1 exhibited several interest-
ing properties among which are the inhibition of
arachidonic acid-induced platelet aggregation with in-
crease of bleeding time and the capacity to protect mice
from both hypobaric and cyanide-induced hypoxia. The
simultaneous presence of these activities in the same
molecule indicate compound 1 as an interesting new
lead for the search of cerebroprotective agents useful in
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