1,5-Benzodiazepines: Part XIII. Substituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines and 4H-imidazo[1,2-a][1,5]benzodiazepin-5-amines as analgesic, anti-inflammatory and/or antipyretic agents with low acute toxicity

Article abstract of DOI:10.1016/S0223-5234(02)01400-9

The reaction of proper N,N-dialkyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines (1) with N-chlorosuccinimide afforded their 4-chloroderivatives 3 which in turn were treated with cyclic amines to give the corresponding 4,5-diaminoderivatives 4. The N,N-dialkyl-4H-imidazo[1,2-a][1,5]benzodiazepin-5-amines (5) were prepared starting from suitable 4-(dialkylamino)-1,3-dihydro-2H-1,5-benzodiazepin-2-ones (8), through multistep synthetic routes. At the 200 mg kg^-1 os dose, some compounds 3 and 4 showed notable analgesic or anti-inflammatory activity but no antipyretic properties, whereas the 5-(dibutylamino) derivatives 5b and 5f proved to be significantly endowed with all these activities. Almost all the compounds 3, 4 and 5 did not show acute toxicity in mice up to 800 mg kg^-1 os dose.

Full text of DOI:10.1016/S0223-5234(02)01400-9

European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
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Original article
1,5-Benzodiazepines
Part XIII. Substituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-
amines and 4H-imidazo[1,2-a][1,5]benzodiazepin-5-amines as
analgesic, anti-inflammatory and/or antipyretic agents with low acute
toxicityꢀ
Giancarlo Grossi a, Mario Di Braccio a, Giorgio Roma a,ꢀ, Vigilio Ballabeni b,
Massimiliano Tognolini b, Francesco Calcina b, Elisabetta Barocelli b
a Dipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV, 3, I-16132 Genova, Italy
b Istituto di Farmacologia e Farmacognosia, Universita` di Parma, Parco Area delle Scienze 27/A, I-43100 Parma, Italy
Received 3 December 2001; received in revised form 26 May 2002; accepted 4 June 2002
Abstract
The reaction of proper N,N-dialkyl-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines (1) with N-chlorosuccinimide afforded
their 4-chloroderivatives 3 which in turn were treated with cyclic amines to give the corresponding 4,5-diaminoderivatives 4. The
N,N-dialkyl-4H-imidazo[1,2-a][1,5]benzodiazepin-5-amines (5) were prepared starting from suitable 4-(dialkylamino)-1,3-dihydro-
2H-1,5-benzodiazepin-2-ones (8), through multistep synthetic routes. At the 200 mg kgꢁ1 os dose, some compounds 3 and 4 showed
notable analgesic or anti-inflammatory activity but no antipyretic properties, whereas the 5-(dibutylamino) derivatives 5b and 5f
proved to be significantly endowed with all these activities. Almost all the compounds 3, 4 and 5 did not show acute toxicity in mice
up to 800 mg kgꢁ1 os dose.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: 1,5-Benzodiazepine tricyclic derivatives; Analgesic; Anti-inflammatory; Antipyretic; Gastrolesivity; Acute toxicity
1. Introduction
substituents endowed compounds 2 with a clear-cut
central nervous system (anticonvulsant) activity, as
expected [3]. Both triazolobenzodiazepines 1 and 2
In previous papers of this series we described the
preparation and pharmacological properties of the
substituted 4H- and 6H-[1,2,4]triazolo[4,3-a][1,5]benzo-
diazepin-5-amines 1 [1,2] and 2 [3], respectively (Fig. 1).
Compounds 1 proved to be completely devoid of
anticonvulsant activity whereas some of them showed
statistically significant analgesic and/or anti-inflamma-
tory properties, depending on the structure [1,2]. On the
other hand, the presence of the 6-phenyl and 8-chloro
usually exhibited rather low acute toxicity [1ꢂ3].
/
No example of [1,2,4]triazolo[4,3-a][1,5]benzodiaze-
pines amino substituted on the diazepine ring had been
previously reported in the literature.
The novelty of the structure and the interesting safety
profile of the analgesic and/or anti-inflammatory agents
1 [1,2] have now prompted us to design and synthesise a
number of their analogues with modified substitution
pattern or different pentatomic heterocycle, in order to
evaluate their analgesic, anti-inflammatory, antipyretic
and toxicological properties. In this paper we just
describe the synthesis and pharmacological properties
of compounds 3, 4 and 5 (Fig. 2).
ꢀ Preliminary results were presented at the Hungarianꢂ
ItalianꢂPolish Joint Meeting on Medicinal Chemistry in Budapest,
Hungary, 2001 (Abstract P-46, p. 74).
/
Germanꢂ
/
/
It must be noted that the pharmacological properties
of some significant examples of compounds 1 have been
ꢀ Correspondence and reprints
E-mail address: roma@unige.it (G. Roma).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 1 4 0 0 - 9

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G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
/
diazepinone 8b and the 2-(2-benzimidazolyl)-N,N-dibu-
tylacetamide (9). Compound 8b was then treated with
Lawesson’s reagent in refluxing anhydrous toluene to
give the corresponding 1,5-benzodiazepine-2-thione 10,
which in turn was reacted with methyl iodide, in the
presence of anhydrous K2CO3 (acetone at reflux),
affording 11b (Fig. 4).
Fig. 1. Structures of the substituted 4H- and 6H-[1,2,4]triazolo[4,3-
a][1,5]benzodiazepin-5-amines 1 and 2.
The raw thick oil obtained from the reaction of
(methylthio)derivative 11a or 11b with aminoacetalde-
hyde dimethyl acetal (ethylene glycol, 160 8C) was then
dissolved and heated in refluxing 99% formic acid to
obtain the cyclocondensation of the intermediate ami-
noderivative to the tricyclic compound 5a or 5b,
respectively.
When compound 11a or 11b was treated with
propargylamine in the presence of p-toluenesulphonic
acid (Dowtherm A, 180 8C) the N,N-dialkyl-1-methyl-
4H-imidazo[1,2-a][1,5]benzodiazepin-5-amine 5e or 5f
was obtained, respectively.
Fig. 2. Structures of compounds 3 and 4 (4-substituted derivatives of
compounds 1) and of compounds 5 (isosteric analogues of compounds
1).
recently further investigated. In summary, they showed
no affinity for the central and peripheral 1,4- and 1,5-
benzodiazepine receptors and proved to affect the
inflammatory response in mice by inhibiting interleu-
kin-6 and prostaglandin E2 production [4].
On the other hand, the reaction of compound 8a with
aminoacetaldehyde dimethyl acetal, using p-toluenesul-
phonic acid as the catalyst (Dowtherm A, 180 8C),
directly afforded a small amount of the tricyclic
compound 12 along with its intermediate aminoderiva-
tive 8c, which in turn gave 12 by treatment with 99%
formic acid at reflux. The treatment of 4H-imidazo[1,2-
a][1,5]benzodiazepin-5(6H)-one (12) with an excess of
piperidine or morpholine, in the presence of titanium
tetrachloride and anisole (refluxing anhydrous toluene),
yielded the desired compound 5c or 5d, respectively (Fig.
4).
2. Chemistry
The reaction of the substituted 4H-[1,2,4]triazolo[4,3-
a][1,5]benzodiazepin-5-amines 1aꢂ
cinimide in refluxing carbon tetrachloride afforded the
corresponding 4-chloroderivatives 3aꢂd (Fig. 3, Table
/d with N-chlorosuc-
/
Compounds 5aꢂf are the first examples of imi-
/
1). In the case of compound 3c also a small amount of
4,4-dichloroderivative 7 was obtained.
dazo[1,2-a][1,5]benzodiazepines amino substituted on
the diazepine ring.
The starting compounds 1a,c [1] and 1b [2] were
previously described by us, whereas 1d was prepared by
the reaction of compound 6 [2] with excess morpholine
(refluxing anhydrous toluene), in the presence of tita-
nium tetrachloride and anisole (Fig. 3).
The structures attributed to the compounds described
in this paper are supported by the results of elemental
analyses and IR and 1H-NMR spectral data (see Section
5 and Table 2).
As we reported for previously described compounds 1
The substituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzo-
[1,2], also for the tricyclic compounds 1d (CDCl3), 5aꢂf
/
diazepin-4,5-diamines 4aꢂ
treatment of 4-chloroderivatives 3aꢂ
excess amines (ethylene glycol, 160 8C for 4a,b; di-
methyl sulphoxide, 120 8C for 4cꢂg) (Fig. 3, Table 1).
The substituted 4H-imidazo[1,2-a][1,5]benzodiaze-
pin-5-amines 5aꢂf were synthesised through literature
procedures [5] properly modified, starting from the
substituted 4-(methylthio)-3H-1,5-benzodiazepin-2-
/
g were obtained from the
(CDCl3) and 12 (DMSO-d6) the 4-CH2 1H-NMR signal
appears as an AB quartet (1d, 5e, 5f), or as a sharp (12)
/
c with proper
or broad (5aꢂd) singlet, depending on the presence or
/
/
lack, respectively, of 1-substituent and its steric hin-
drance. Actually, the AB quartet signal for 4-CH2 is
evidently due to the geminal coupling of the two non-
equivalent protons, as a result of the presence of only
one of the two boat conformations of the diazepine ring,
no interconversion occurring in the solution at the
registration temperature (21 8C). On the contrary,
when a high or slow rate interconversion occurs, the 4-
CH2 signal appears as a sharp or broad singlet,
respectively [8].
/
amines 11a [6], 11b, or the 4-(dimethylamino)-1,3-
dihydro-2H-1,5-benzodiazepin-2-one (8a) [7] (Fig. 4,
Table 1).
The starting compound 11b was prepared through the
following three-step synthetic route. The reaction of o-
phenylenediamine with ethyl N,N-dibutylmalonamate
in the presence of phosphorus oxychloride (110 8C)
afforded a mixture of the desired (dibutylamino)benzo-
Concerning then the 1H-NMR spectra of the 4,5-
disubstituted tricyclic derivatives 3 and 4, it can be
observed that the signal (a sharp singlet) corresponding

G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ
/944
935
Fig. 3. Synthesis of compounds 3aꢂ
/
d and 4aꢂg.
/
to the unsymmetrically substituted 4-CH is in accor-
dance with the presence (previously reported in the
literature for similar compounds [8]) of only one
conformation (the most stable) of the diazepine ring in
the solution at the registration temperature (CDCl3,
which exhibited a statistically significant activity at this
dose were further tested at doses decreasing by a factor
of two, until statistically significant activity was ob-
served. Only for compounds 5b and 5f, in the case of
LPS induced fever test in rats, the highest dose used was
100 mg kgꢁ1. Gastric tolerability and acute toxicity
were evaluated in mice after oral administration of
compounds at the 400 and 800 mg kgꢁ1 doses,
respectively (see Section 5).
21 8C). Besides, the multiplicities of the H2CÃ
/
NÃ
/
CH2
signals of compounds 3b and 4f,g seem to be due only to
the chirality of the molecules, whereas the more complex
multiplicities of the H2CÃ
/
NÃ
/
CH2 signals of the 4,5-
e are most likely
bis(dialkylamino) derivatives 4aꢂ
/
The pharmacological results obtained for the above
compounds are reported in Table 1, as well as those of
attributable to both the molecule chirality and the
reciprocally hindered free rotation of the two bulky
(diakylamino) substituents.
the ‘lead compounds’ 1aꢂc [1,2] and the unpublished
/
data concerning the antipyretic activity and gastrolesiv-
ity of compound 1b, in order to compare it with its
isosteric analogue 5b.
Among the novel compounds tested, a statistically
significant activity at the 200 mg kgꢁ1 dose was shown
3. Pharmacological results and discussion
by 3d, 4c, 5b and 5f (inhibition range 61ꢂ
acetic acid writhing test in mice (analgesic activity), by
3c, 4b, 4f, 5b, 5f (inhibition range 46ꢂ80%) in the
carrageenan induced rat paw edema test (anti-inflam-
/
90%) in the
Compounds 3aꢂ
/
d, 4aꢂ
/
g and 5aꢂf were tested in vivo
/
for their antinociceptive, anti-inflammatory and anti-
pyretic activities. They were administered orally and
assayed at the initial dose of 200 mg kgꢁ1. Compounds
/

936
G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
/
Fig. 4. Synthetic routes to compounds 5aꢂf.
/
matory activity), and by the 4H-imidazo[1,2-a][1,5]ben-
zodiazepine derivatives 5b,f (100 mg kgꢁ1) and 5d (200
On the whole, the highest activities were afforded by
5f as analgesic agent (90% protection at the 200 mg
kgꢁ1 dose) and 5b as anti-inflammatory (80% edema
inhibition at the 200 mg kgꢁ1 dose) and antipyretic
agent (92% protection at the 100 mg kgꢁ1 dose).
However, compounds 4c and 5f still exhibited at the
100 mg kgꢁ1 dose a statistically significant analgesic
(45% protection) or anti-inflammatory (35% edema
inhibition) activity, respectively, whereas 5b afforded
mg kgꢁ1) (inhibition range 80ꢂ
92%) in the LPS induced
/
fever test in rats (antipyretic activity). In this connec-
tion, it is interesting to point out that all the 4,5-
disubstituted 4H-[1,2,4]triazolo[4,3-a][1,5]benzodiaze-
pine derivatives 3 and 4 reported herein proved to be
completely devoid of antipyretic activity, as well as the
4-unsubstituted precursor 1b [2].

G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ
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937
Table 1
Structures and pharmacological data of compounds 3aꢂ
/
d, 4aꢂ
/
g, 5aꢂ
/
f and 1aꢂc
/

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G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
/
an interesting statistically significant antipyretic effect
(73% protection) at the 50 mg kgꢁ1 dose.
Actually, on the whole, the 4H-imidazo[1,2-a][1,5]-
benzodiazepine derivatives 5b and 5f appear to be the
most interesting of all compounds synthesised and tested
in this study, combining remarkable analgesic, anti-
inflammatory and antipyretic activities at doses devoid
of acute adverse effects.
Besides, the data for acute gastrolesivity and toxicity
in mice clearly provided preliminary satisfactory indica-
tions about the safety profile of both the tricyclic 1,2,4-
triazole derivatives 3, 4 and imidazole derivatives 5,
being only compound 5d endowed with toxicity at the
800 mg kgꢁ1 dose. In this connection, indomethacin
was previously reported to produce gastric lesions in
mice at anti-inflammatory doses (10 mg kgꢁ1 os) [9].
Finally, we must remark that the steric hindrance of
the 5-(dialkylamino) substituent of starting compounds
1a,b and 1c (i.e. the most effective compounds 1 as
analgesic or anti-inflammatory agents, respectively [1,2])
only allowed the introduction of low hindering cyclic
amino groups as 4-substituents of compounds 4 (com-
5. Experimental
5.1. Chemistry
M.p.s were determined using a Fisherꢂ
tus and are uncorrected. IR spectra were recorded on a
Perkinꢂ
Elmer 398 spectrophotometer. 1H-NMR spectra
/Johns appara-
/
were recorded on a Varian Gemini 200 (200 MHz)
spectrometer, using (CH3)4Si as an internal reference
pounds 4aꢂg).
/
(dꢃ
Analyses of all new compounds, indicated by the
symbols of the elements, were within 90.4% of the
/0), and chemical shifts (d) are reported in ppm.
/
theoretical values and were performed by the Labor-
atorio di Microanalisi, Dipartimento di Scienze Farm-
aceutiche, Universita` di Genova.
Thin layer chromatograms were run on Merck silica
gel 60 F254 precoated plastic sheets (layer thickness 0.2
mm). Column chromatography was performed using
4. Conclusions
Concerning the structureꢂ
(SAR), pharmacological data listed in Table 1 seem to
/
activity relationships
suggest the following conclusions:
Carloꢂ
/
Erba silica gel (0.05ꢂ
/
0.20 mm) or CarloꢂErba
/
a) The 4-chloro substituent of compounds 3 can
produce (compounds 3a and 3b) the loss of the
analgesic activity shown by the corresponding
starting compounds 1 (1a [1] and 1b [2], respec-
tively).
neutral aluminium oxide (Brockmann activity I).
5.1.1. 5-Morpholino-1-phenyl-4H-[1,2,4]triazolo[4,3-
a][1,5]benzodiazepine (1d)
b) The introduction of a cyclic 4-amino substituent
into analgesic (1a,b) or anti-inflammatory (1c)
compounds 1 generally lowers the activity of these
compounds (partial exceptions are here the 4-amino
substituted compounds 4c and 4f).
c) In the cases of both 4-chloroderivatives 3 and 4-
aminoderivatives 4, the presence of a morpholino
substituent in proper position can yield notable
activity modifications with respect to the reference
compounds devoid of this group: compare the high
anti-inflammatory and low analgesic activities of 5-
(dimethylamino)derivative 3c with the high analge-
sic and null anti-inflammatory activities of 5-mor-
pholinoderivative 3d, as well as the remarkable anti-
inflammatory properties of 4-morpholinoderivative
4b with the analgesic ones of the corresponding 4-
unsubstituted compound 1a [1].
To an ice bath cooled solution of 10.9 mmol (1.2 mL)
of TiCl4 and 2 mL of anisole in 40 mL of dry C6H5CH3,
a solution of 5 mL of morpholine in 5 mL of dry
C6H5CH3 was added, while stirring. 1-Phenyl-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5(6H)-one (6)
(10.0 mmol, 2.76 g) [2] and the solution of 3 mL of
morpholine in 10 mL of dry C6H5CH3 were then added
and the resulting mixture was refluxed with stirring for 6
h.
After cooling, 3 mL of 2-propanol, 2 g of diatomac-
eous earth and 3 mL of 28% aq. NH3 were added to the
mixture. After stirring, the resulting suspension was
filtered, the solid collected was thoroughly washed with
CH2Cl2, and the filtrate and washings were dried
(anhydrous Na2SO4), then evaporated to dryness under
reduced pressure. By adding a little EtOAc to the oily
residue, the nearly pure whitish compound 1d separated
d) The replacement of the 1,2,4-triazole of compounds
1 with the imidazole fused ring affords compounds
5, some of which exhibited not only appreciable
analgesic and anti-inflammatory, but also, depend-
ing on the substitution pattern, notable antipyretic
properties which were not shown by the 1,2,4-
triazole derivatives 1b, 3 and 4.
out (2.75 g, 80%); white crystals, m.p. 182ꢂ183.5 8C,
/
after crystallisation from the same solvent with charcoal.
IR (CHCl3, cmꢁ1): 1610, 1586 strong, 1530 weak, 1476.
1H-NMR (CDCl3): d 3.16 and 4.42 (AB system, Jꢃ
/
14
3.90 (m, 8H, morpholine
6.94 and 7.23ꢂ7.63 (2m, 2Hꢄ7H, H-
phenyl H’s). Anal. C20H19N5O (C, H, N).
Hz, 1Hꢄ
CH2’s), 6.81ꢂ
7,8,9,10ꢄ
/
1H, 4-CH2), 3.58ꢂ
/
/
/
/
/

G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ
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939
5.1.2. General procedure for N,N-dialkyl-4-chloro-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amines 3aꢂ
The thick oil deriving from reaction of 3a (2.32 g) with
piperidine was subjected to column chromatography
(silica gel) eluting with EtOAc: by adding a little Et2O to
the residue obtained from the eluate, 1.58 g of 4a
separated out.
/
d
A mixture of 5.0 mmol of 1a (1.28 g) [1], 1b (1.56 g)
[2], 1c (1.52 g), or 1d (1.73 g), 5.0 mmol (0.67 g) of N-
chlorosuccinimide [6 mmol (0.80 g) in the case of
reaction with 1c] and 50 mL of CCl4 was refluxed for
30 min (1a,c), 1 h (1b), or 3 h (1d), while stirring. After
cooling, 100 mL of CH2Cl2 and 100 mL of aq. 0.5 N
NaOH were added to the mixture which was then
vigorously stirred at room temperature (r.t.) for 30
min. The organic phase was collected and the aqueous
one was extracted several more times with CH2Cl2. The
combined extracts were washed with water, then dried
(anhydrous Na2SO4) and evaporated to dryness in
vacuo to give an oily residue.
5.1.3.2. N,N-Diethyl-4-morpholino-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4b).
The oil obtained from reaction of 3a (2.32 g) with
morpholine was chromatographed on a silica gel column
eluting with CH2Cl2ꢂ
/
petroleum etherꢂEt3N (5:5:1). The
/
eluate collected, after removal of solvents and treatment
of the residue with a little isopropyl ether, afforded 1.77
g of 4b.
Compounds 3a,b,d: By adding a proper solvent
(isopropyl ether for 3a,b and Et2O for 3d) to the oils
obtained starting from 1a, 1b or 1d, compounds 3a, 3b
or 3d, respectively, separated out as white solids, which
were crystallised from the suitable solvent.
5.1.3.3. N,N-Diethyl-4-(4-methyl-1-piperazinyl)-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4c).
The oily residue resulting from reaction of 3a (2.32 g)
with 4-methylpiperazine was chromatographed on a
silica gel column eluting first with EtOAcꢂ
/
Me2CO
Compounds 3c and 7: The oily residue obtained
starting from 1c was subjected to column chromatogra-
(1:1) to remove some impurities, then with CH2Cl2ꢂ
/
Et3N (9:1) to recover 4c. The thick oil finally obtained
from this eluate was treated with a little petroleum ether
to give 1.79 g of 4c.
phy (silica gel), eluting first with CH2Cl2ꢂEtOAc (1:1)
/
to recover dichloroderivative 7, then with EtOAc to
collect the desired 4-chloroderivative 3c. Each com-
pound separated out as a white solid by adding a little
isopropyl ether to the oil recovered from the respective
eluate.
5.1.3.4. N,N-Dibutyl-4-(1-piperidinyl)-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4d).
The reaction of 2.77 g of 3b with piperidine gave an oil
which was chromatographed on a silica gel column
Data for compounds 3aꢂd are reported in Table 2.
/
eluting with petroleum etherꢂEtOAc (1:1); after dis-
/
5.1.2.1. 4,4-Dichloro-N,N-dimethyl-1-phenyl-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (7).
carding the first fractions containing a little amount of
starting compound 3b, the eluate subsequently collected,
after a treatment identical to that above described for
the recovery of 4b, yielded 1.59 g of 4d.
White crystals (7.5%); m.p. 179ꢂ
ether). IR (CHCl3, cmꢁ1): 1616, 1596, 1495 weak, 1478.
1H-NMR (CDCl3): d 3.10 [s, 6H, N(CH3)2], 6.75ꢂ
7.10
and 7.28ꢂ7.75 (2m, 2Hꢄ7H, H-7,8,9,10ꢄphenyl H’s).
Anal. C18H15Cl2N5 (C, H, N, Cl).
/
181 8C (from isopropyl
/
/
/
/
5.1.3.5. N,N-Dibutyl-4-morpholino-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4e).
The residue resulting from the reaction of 2.77 g of 3b
with morpholine was subjected to column chromato-
5.1.3. General procedure for N,N,N?,N?-tetralkyl-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-4,5-diamines
graphy [Al2O3, CH2Cl2ꢂEtOAc (1:1)]. The eluate col-
/
4aꢂ
/
g
lected afforded a thick oil from which, after the same
treatment described above for compounds 4b,d, 1.71 g
of 4e separated out.
A mixture of chloroderivative 3a, 3b, or 3c (8.0
mmol), 7 mL of the suitable dialkylamine, and 7 mL
of DMSO (compounds 4cꢂ
pounds 4a,b) was heated at 120 8C for 1 h (4dꢂ
/
g) or ethylene glycol (com-
g) or 2 h
/
5.1.3.6. N,N-Dimethyl-1-phenyl-4-(1-pyrrolidinyl)-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4f).
The oil obtained from reaction of 2.70 g of 3c with
pyrrolidine was chromatographed on a silica gel col-
(4c), or at 160 8C for 1 h (4a,b), while stirring. After
cooling, the mixture was poured into ice-cooled water
(200 mL) and the resulting emulsion or suspension was
exhaustively extracted with CH2Cl2. From combined
extracts (washed with water and dried, then evaporated
to dryness in vacuo) an oily residue was obtained from
which compound 4 was recovered (white solid) as
described below for each case.
umn, eluting first with CH2Cl2ꢂEtOAc (1:1) to recover
/
0.40 g of starting compound 3c, then with EtOAc to
collect 4f. This eluate, after removal of solvent, afforded
sticky oil from which, after treatment with a little Et2O
and standing, 1.21 g of 4f separated out.
5.1.3.1. N,N-Diethyl-4-(1-piperidinyl)-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4a).
5.1.3.7. N,N-Dimethyl-1-phenyl-4-(1-piperidinyl)-4H-
[1,2,4]triazolo[4,3-a][1,5]benzodiazepin-5-amine (4g).

Table 2
Physical and chemical data of compounds 3aꢂ
/
d and 4aꢂg
/
c
d
Compound Yield
(%)
M.p.(8C) (sol- Molecular for-
IR (cmꢁ1
)
1H-NMR (d, ppm)
a
b
vent)
128ꢂ
120ꢂ
205ꢂ
224ꢂ
193ꢂ
184ꢂ
mula
3a
3b
3c
3d
4a
4b
90
82
71
72
59
65
/
129 (A)
122 (A)
206 (B)
C14H16ClN5
C18H24ClN5
C18H16ClN5
1612, 1588s, 1512
br, w.
1.30 [t, 6H, N(CH2CH3)2], 3.56 [q, 4H, N(CH2CH3)2], 6.40 (s, 1H, H-4), 7.09ꢂ7.50 (m, 4H, H-7,8,9,10), 8.64 (s, 1H,
/
H-1).
/
1610, 1585s, 1510
br, w.
0.97 [t, 6H, N(CH2CH2CH2CH3)2], 1.38 [m, 4H, N(CH2CH2CH2CH3)2], 1.50ꢂ
3.35ꢂ3.64 [m, 4H, N(CH2CH2CH2CH3)2], 6.39 (s, 1H, H-4), 7.08ꢂ7.50 (m, 4H, H-7,8,9,10), 8.63 (s, 1H, H-1).
3.23 [s, 6H, N(CH3)2], 6.48 (s, 1H, H-4), 6.70ꢂ6.88 and 7.20ꢂ7.60 (2m, 2Hꢄ7H, H-7,8,9,10ꢄphenyl H’s).
/
1.90 [m, 4H, N(CH2CH2CH2CH3)2],
/
/
/
1613, 1588s,
1510w, 1481.
/
/
/
224.5 (C) C20H18ClN5O
1615, 1590, 1512w, 3.50ꢂ
/
3.94 (m, 8H, morpholine CH2’s), 6.35 (s, 1H, H-4), 6.80ꢂ
/
6.98 and 7.23ꢂ7.63 (2m, 2Hꢄ7H, H-7,8,9,10ꢄphenyl
/
1480.
H’s).
0.90ꢂ
a-CH2’s), 3.19ꢂ
1.04ꢂ1.48 [m, 6H, N(CH2CH3)2], 2.00ꢂ
morpholine OCH2’s), 3.25ꢂ3.75 [m, 4H, N(CH2CH3)2], 5.11 (s, 1H, H-4), 6.95ꢂ
H-1).
1612, 1582s, 1525, 1.07ꢂ
/
194 (D)
185 (A)
C19H26N6
1608, 1578s,
1521w, 1500w.
1610, 1581s,
1523w, 1496w.
/
1.50 [m, 12H, N(CH2CH3)2ꢄpiperidine b-CH2’sꢄg-CH2], 1.83ꢂ
3.73 [m, 4H, N(CH2CH3)2], 5.04 (s, 1H, H-4), 6.83ꢂ7.33 (m, 4H, H-7,8,9,10), 8.55 (s, 1H, H-1).
2.19 and 2.32ꢂ2.51 (2m, 2Hꢄ2H, morpholine NCH2’s), 3.16 (near t, 4H,
7.40 (m, 4H, H-7,8,9,10), 8.57 (s, 1H,
/
2.09 and 2.17ꢂ
/
2.40 (2m, 2Hꢄ2H, piperidine
/
/
/
C18H24N6O
/
/
/
/
/
4c
4d
4e
63
51
54
129ꢂ
/
130 (E)
C19H27N7
/
1.42 [m, 6H, N(CH2CH3)2], 1.70ꢂ
NCH3), 2.32ꢂ2.52 (m, 2H, 2H of piperazine NCH2’s), 3.27ꢂ
(m, 4H, H-7,8,9,10), 8.56 (s, 1H, H-1).
0.80ꢂ1.88 [m, 20H, N(CH2CH2CH2CH3)2ꢄpiperidine b-CH2’sꢄg-CH2], 1.90ꢂ
piperidine a-CH2’s), 3.10ꢂ3.68 [m, 4H, N(CH2CH2CH2CH3)2], 5.03 (s, 1H, H-4), 6.85ꢂ
(s, 1H, H-1).
0.74ꢂ1.11 [m, 6H, N(CH2CH2CH2CH3)2], 1.16ꢂ
2Hꢄ2H, morpholine NCH2’s), 2.95ꢂ3.70 [m, 4H, N(CH2CH2CH2CH3)2], 3.17 (m, 4H, morpholine OCH2’s), 5.09 (s,
1H, H-4), 6.93ꢂ7.42 (m, 4H, H-7,8,9,10), 8.57 (s, 1H, H-1).
1.32ꢂ1.51 (m, 4H, pyrrolidine b-CH2’s), 2.08ꢂ2.17 and 2.33ꢂ
N(CH3)2], 5.23 (s, 1H, H-4), 6.62ꢂ6.76 and 7.12ꢂ7.64 (2m, 2Hꢄ7H, H-7,8,9,10ꢄphenyl H’s).
0.89ꢂ1.28 (m, 6H, piperidine b-CH2’sꢄg-CH2), 2.00ꢂ2.17 and 2.23ꢂ2.40 (2m, 2Hꢄ2H, piperidine a-CH2’s), 3.20 [s,
6H, N(CH3)2], 5.14 (s, 1H, H-4), 6.65ꢂ6.76 and 7.12ꢂ7.63 (2m, 2Hꢄ7H, H-7,8,9,10ꢄphenyl H’s).
/
2.19 [m, 6H, piperazine CH3N(CH2)2ꢄ2H of piperazine NCH2’s], 2.05 (s, 3H,
1498w.
/
/
3.70 [m, 4H, N(CH2CH3)2], 5.09 (s, 1H, H-4), 6.94ꢂ7.35
/
113.5ꢂ
/
114 (F) C23H34N6
1610, 1582s,
1522w, 1495w.
/
/
2.12 and 2.22ꢂ
/
2.43 (2m, 2Hꢄ2H,
/
/7.33 (m, 4H, H-7,8,9,10), 8,54
142ꢂ
/
143 (A)
188 (A)
C22H32N6O
1610, 1583s,
1523w, 1498w.
/
/
1.92 [m, 8H, N(CH2CH2CH2CH3)2], 1.97ꢂ
/
2.20 and 2.30ꢂ2.52 (2m,
/
/
/
4f
41
40
187ꢂ
/
C22H24N6
C23H26N6
1611, 1587s,
1521br,w, 1480.
1614, 1590s,
/
/
/
2.52 (2m, 2Hꢄ2H, pyrrolidine a-CH2’s), 3.21 [s, 6H,
/
/
4g
192.5ꢂ
/
193.5
/
/
/
(A)
1520br,w, 1480.
/
/
a
b
c
Crystallization solvent: Aꢃisopropyl ether, Bꢃethyl acetate/petroleum ether, Cꢃethyl acetate, Dꢃethyl acetate/isopropyl ether, Eꢃethyl ether/petroleum ether, Fꢃpetroleum ether.
Anal. C, H, N (C, H, N, Cl for 3aꢂd).
In CHCl3 solutions. Abbreviations: sꢃstrong, brꢃbroad, wꢃweak.
In CDCl3 solutions.
/
d

G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ
/944
941
By proceeding exactly as above described for the
preparation of 4f, from the reaction of 3c (2.70 g) with
piperidine, 0.70 g of starting compound 3c and 1.24 g of
the desired 4g were finally recovered.
N(CH2CH2CH2CH3)2], 1.40ꢂ
/
1.66 [m, 4H, N(CH2CH2-
CH2CH3)2], 3.35 [m, 4H, N(CH2CH2CH2CH3)2], 4.05
(s, 2H, CH2CO), 7.15ꢂ7.26 and 7.48ꢂ7.62 (2m, 4H, H-
4,5,6,7). Anal. C17H25N3O (C, H, N).
/
/
Data for compounds 4aꢂg are reported in Table 2.
/
5.1.5. 4-(Dibutylamino)-1,3-dihydro-2H-1,5-
benzodiazepine-2-thione (10)
5.1.4. 4-(Dibutylamino)-1,3-dihydro-2H-1,5-
benzodiazepin-2-one (8b) and 2-(2-benzimidazolyl)-
N,N-dibutylacetamide (9)
A mixture of 10.0 mmol (2.87 g) of 8b, 5.5 mmol (2.22
g) of Lawesson’s reagent and 20 mL of dry C6H5CH3
was stirred at reflux for 30 min. The solvent was then
removed in vacuo and the residue taken up in CH2Cl2
and subjected to column chromatography [Al2O3,
Phosphorus oxychloride (0.10 mol, 15.33 g) was
added dropwise with stirring to an ice-cooled suspension
of 0.10 mol (10.81 g) of 1,2-phenylenediamine in 0.20
mol (48.67 g) of ethyl N,N-dibutylmalonamate [10], and
the resulting mixture was then heated at 110 8C for 4 h,
while stirring. Water (400 mL) was then added to the
dark viscous slurry obtained and the mixture was
vigorously stirred at 100 8C until a fluid emulsion was
CH2Cl2ꢂEtOAc (1:1)]. The eluate collected afforded a
/
thick oil from which, after a treatment with a little
petroleum ether, pure compound 10 separated out (2.79
g, 92%); ivoryꢂ
/
white crystals, m.p. 114.5ꢂ115 8C, after
/
crystallisation from the same solvent. IR (CHCl3,
1
obtained. After cooling, 200 mL of Et2Oꢂ/EtOAc (1:1)
cmꢁ1): 3360 (NH), 1609, 1570 strong, 1510 weak. H-
NMR (CDCl3): d 0.95 [t, 6H, N(CH2CH2CH2CH3)2],
were added and the mixture was further stirred at r.t. for
30 min. After discarding some insoluble tars, the acidic
aqueous layer was collected and the organic phase was
exhaustively extracted with 2 N aq. HCl. The combined
aqueous phases were decolourised with charcoal, then
made alkaline with 28% aq. NH3, and the resulting
emulsion was thoroughly extracted with CH2Cl2. The
combined extracts were dried, then evaporated to
dryness in vacuo to afford a thick oil, which was
dissolved in a little 2-propanol and treated with a
saturated HCl Et2O solution. This way a whitish solid
separated out which was filtered, washed with anhy-
drous Et2O, then treated with 100 mL of 10% aq.
Na2CO3. The resulting emulsion was extracted several
times with CH2Cl2, then the solvent was removed from
the combined extracts (dried over anhydrous Na2SO4)
to give an oil (mixture of 8b and 9), which was subjected
to column chromatography (silica gel), eluting first with
1.36 [m, 4H, N(CH2CH2CH2CH3)2], 1.50ꢂ
N(CH2CH2CH2CH3)2], 3.57 [m, 6H, N(CH2CH2CH2-
CH3)2ꢄ3-CH2], 6.90ꢂ7.22 (m, 4H, H-6,7,8,9), 10.06 (s,
1H, NH; disappeared with D2O). Anal. C17H25N3S (C,
H, N, S).
/
1.75 [m, 4H,
/
/
5.1.6. N,N-Dibutyl-4-(methylthio)-3H-1,5-
benzodiazepin-2-amine (11b)
A mixture of 5.0 mmol (1.51 g) of 10, 0.70 g of
anhydrous K2CO3, 1.0 mL of MeI and 40 mL of dry
Me2CO was refluxed for 2 h, while stirring. The solvent
was then removed, the residue partitioned between
water and CH2Cl2, then the aqueous phase extracted
several more times with the same solvent. The oil
obtained from the combined organic phases (after
drying and removal of solvent) was chromatographed
on a silica gel column eluting with CH2Cl2. The eluate
collected was evaporated to dryness under reduced
pressure to give a thick oil from which, after addition
of a little MeOH and standing at 4 8C, pure compound
C6H5CH3ꢂMe2CO (6:1).
/
The eluate, after removal of solvents in vacuo, gave a
thick oil which was treated with a little petroleum ether
to yield 4.55 g (16%) of pure 8b; white crystals, m.p.
11b crystallised as a white solid (1.45 g, 91%); m.p. 65ꢂ
/
109.5ꢂ
/
110 8C after crystallisation from the same sol-
67 8C after recrystallisation from the same solvent. IR
vent. IR (CHCl3, cmꢁ1): 3395 (NH), 1674 (CO), 1609,
1573. 1H-NMR (CDCl3): d 0.95 [t, 6H, N(CH2CH2-
CH2CH3)2], 1.35 [m, 4H, N(CH2CH2CH2CH3)2], 1.62
[m, 4H, N(CH2CH2CH2CH3)2], 3.21 (s, 2H, 3-CH2),
(CHCl3, cmꢁ1): 1590, 1560. H-NMR (CDCl3): d 0.94
[t, 6H, N(CH2CH2CH2CH3)2], 1.34 [m, 4H, N(CH2-
1
CH2CH2CH3)2], 1.50ꢂ
CH2CH3)2], 2.50 (s, 3H, SCH3), 3.09 (s, 2H, 3-CH2),
3.46 [t, 4H, N(CH2CH2CH2CH3)2], 6.92ꢂ7.34 (m, 4H,
/1.75 [m, 4H, N(CH2CH2-
3.47 [t, 4H, N(CH2CH2CH2CH3)2], 6.90ꢂ
/7.21 (m, 4H,
/
H-6,7,8,9), 8.30 (s, 1H, NH; disappeared with D2O).
Anal. C17H25N3O (C, H, N).
H-6,7,8,9). Anal. C18H27N3S (C, H, N, S).
From the eluate subsequently collected (EtOAc), a
thick oil was finally obtained from which, after addition
of a little isopropyl ether and standing, 2.75 g (10%) of
pure compound 9 separated out; white crystals, m.p.
5.1.7. N,N-Dialkyl-4H-imidazo[1,2-
a][1,5]benzodiazepin-5-amines 5a,b
A mixture of 8.0 mmol of 11a (2.09 g) [6] or 11b (2.54
g), 24.0 mmol (2.52 g) of aminoacetaldheyde dimethyl
acetal and 10 mL of ethylene glycol was stirred at
160 8C for 1 h. After cooling, the resulting solution was
poured into water (300 mL) and the mixture was
exhaustively extracted with CH2Cl2. The combined
84ꢂ85 8C after crystallisation from the same solvent.
/
IR (CHCl3, cmꢁ1): 3380 broad (NH), 1630 broad,
1
strong (CO), 1524. H-NMR (CDCl3): d 0.90 and 0.93
[2t, 6H, N(CH2CH2CH2CH3)2], 1.31 [m, 4H,

942
G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
/
extracts were dried and solvent was removed to give an
oil which was dissolved in 10 mL of 99% HCOOH: this
solution was then refluxed (100 8C) for 1.5 h, while
stirring. The final reaction mixture was poured onto ice-
water and the resulting solution was carefully treated
with Na2CO3 until alkaline and extracted several times
with CH2Cl2. From the combined extracts, after drying
and removal of solvent, an oily residue was obtained
which was chromatographed on an Al2O3 column
was collected and the organic one was further extracted
twice with 2 N aq. HCl. The combined aqueous phases
were treated with 28% aq. NH3 until alkaline and the
mixture was exhaustively extracted with CH2Cl2. The
combined extracts were dried and evaporated to dryness
under reduced pressure to afford an oily residue, which
was chromatographed on Al2O3 column, eluting with
CH2Cl2ꢂEtOAc (9:1). The eluate collected, after re-
/
moval of solvents, gave a thick oil from which com-
pound 5e or 5f was recovered as reported below.
[CH2Cl2ꢂEtOAc (9:1)]. The eluate collected was evapo-
/
rated to dryness under vacuo to give a thick oil from
which compound 5a or 5b was recovered as reported
below.
5.1.8.1. N,N-Diethyl substituted derivative 5e. By add-
ing a little isopropyl ether to the oil obtained from 11a,
pure compound 5e (1.14 g, 53%) separated out as a
5.1.7.1. N,N-Diethyl substituted derivative 5a. By add-
ing a little petroleum ether to the oil obtained from 11a,
pure compound 5a (1.04 g, 51%) separated out as a
white solid, m.p. 106 8C after crystallisation from
isopropyl ether. IR (CHCl3, cmꢁ1): 1608, 1573 strong,
1525 weak, 1500. 1H-NMR (CDCl3): d 1.25 [t, 6H,
white solid, m.p. 129ꢂ130 8C after crystallisation from
/
the same solvent. IR (CHCl3, cmꢁ1): 1608, 1574 strong,
1
1514. H-NMR (CDCl3): d 1.24 [t, 6H, N(CH2CH3)2],
2.32 (s, 3H, 1-CH3), 2.95 and 4.11 (AB system, Jꢃ
Hz, 1Hꢄ1H, 4-CH2), 3.53 [near q, 4H, N(CH2CH3)2],
6.84 (s, 1H, H-2), 6.98ꢂ7.10 and 7.22ꢂ7.30 (2m, 1Hꢄ
3H, H-7,8,9,10). Anal. C16H20N4 (C, H, N).
/
14
/
/
/
/
N(CH2CH3)2],
N(CH2CH3)2ꢄ
2Hꢄ4H, H-1,2,7,8,9,10). Anal. C15H18N4 (C, H, N).
3.34ꢂ/3.83
[qꢄ
/
broad
s,
6H,
/
4-CH2], 6.97ꢂ
/
7.13 and 7.21ꢂ/7.43 (2m,
/
5.1.8.2. N,N-Dibutyl substituted derivative 5f. By using
exactly the same procedure that was employed to obtain
5b dimaleate and the free base 5b (see above), 5f
5.1.7.2. N,N-Dibutyl substituted derivative 5b. The oil
deriving from the reaction carried out with 11b was
treated with a solution of 16.0 mmol (1.86 g) of maleic
acid in 10 mL of anhydrous EtOH. The resulting
solution was stirred for few minutes at r.t., then diluted
with anhydrous Et2O and petroleum ether until it
dimaleate (5f×/2H4C4O4) (2.47 g, 56%) and the free
base 5f were obtained from the oil afforded by the
reaction of 11b.
5f Dimaleate: White solid, m.p. 124 8C after crystal-
lisation
C28H36N4O8 (C, H, N).
from
anhydrous
EtOH/Et2O.
Anal.
became cloudy. After standing, the 5b dimaleate (5b×
/
2H4C4O4) separated out as a white solid which was
recovered by filtration and dried (2.23 g, 51%), then
recristallysed from anhydrous EtOH/Et2O to give white
Compound 5f: Colourless thick oil. IR (CHCl3,
1
cmꢁ1): 1609, 1572 strong, 1515. H-NMR (CDCl3): d
0.94 [t, 6H, N(CH2CH2CH2CH3)2], 1.18ꢂ
N(CH2CH2CH2CH3)2], 2.31 (s, 3H, 1-CH3), 2.94 and
4.12 (AB system, Jꢃ14 Hz, 1Hꢄ1H, 4-CH2), 3.18ꢂ3.69
[m, 4H, N(CH2CH2CH2CH3)2], 6.84 (s, 1H, H-2), 6.98ꢂ
7.12 and 7.20ꢂ7.35 (2m, 1Hꢄ3H, H-7,8,9,10).
/
1.82 [m, 8H,
crystals, m.p. 114ꢂ
/
115 8C. Anal. C27H34N4O8 (C, H,
N).
/
/
/
An analytical sample of this salt was treated with 10%
aq. Na2CO3, the mixture was extracted with CH2Cl2 and
the extract was dried then evaporated in vacuo. A thick
colourless oil was obtained which was thoroughly dried
in vacuo to afford the pure free base 5b. IR (CHCl3,
/
/
/
5.1.9. 4-[(2,2-Dimethoxyethyl)amino]-1,3-dihydro-2H-
1,5-benzodiazepin-2-one (8c) and 4H-imidazo[1,2-
a][1,5]benzodiazepin-5(6H)-one (12)
1
cmꢁ1): 1610, 1575 strong, 1525 weak, 1501. H-NMR
(CDCl3): d 0.94 [t, 6H, N(CH2CH2CH2CH3)2], 1.14ꢂ
1.48 [m, 4H, N(CH2CH2CH2CH3)2], 1.50ꢂ1.78 [m,
4H, N(CH2CH2CH2CH3)2], 3.30ꢂ3.76 [tꢄbroad s, 6H,
N(CH2 CH2CH2CH3)2ꢄ4-CH2], 6.98ꢂ7.15 and 7.20ꢂ
7.44 (2m, 2Hꢄ4H, H-1,2,7,8,9,10).
/
A mixture of 10.0 mmol (2.03 g) of 8a [7], 30.0 mmol
(3.15 g) of aminoacetaldheyde dimethyl acetal, 0.30 g of
monohydrate p-toluenesulphonic acid and 10 mL of
Dowtherm A was heated at 180 8C for 3 h, while
stirring. After cooling, the reaction mixture was dis-
solved in CH2Cl2 (200 mL) and this solution was washed
with 5% aq. NaHCO3, then with water and dried over
anhydrous Na2SO4. After removal of solvent in vacuo,
the liquid residue obtained was chromatographed on a
silica gel column, eluting first with CH2Cl2 to remove
Dowtherm A, then with EtOAc to recover 8c. The eluate
collected afforded a solid residue, which was taken up in
a little Et2O and filtered. There was so obtained pure 8c
/
/
/
/
/
/
/
5.1.8. N,N-Dialkyl-1-methyl-4H-imidazo[1,2-
a][1,5]benzodiazepin-5-amines 5e,f
A mixture of 8.0 mmol of 11a (2.09 g) [6] or 11b (2.54
g), 16.0 mmol (0.88 g) of propargylamine, 0.15 g of
monohydrate p-toluenesulphonic acid and 10 mL of
Dowtherm A was stirred at 180 8C for 1 h. After
cooling, Et2O (100 mL) and 2 N aq. HCl (100 mL) were
added to the mixture. After stirring, the aqueous phase
(1.07 g, 41%); white crystals, m.p. 199.5ꢂ200 8C after
/

G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ
/944
943
crystallisation from EtOAc with charcoal. IR (KBr,
cmꢁ1): 3350 (NH), 3120 broad (NHCO), 1676 (CO),
1
1615, 1585, 1562, 1534. H-NMR (CDCl3): d 3.08 (s,
2H, 3-CH2), 3.42 (s, 6H, OCH3’s), 3.58 (t, 2H, NHCH2;
5.1.11.1. 5-(1-Piperidinyl)-4H-imidazo[1,2-
a][1,5]benzodiazepine (5c). The oily residue obtained
from the reaction carried out with piperidine was treated
with a little isopropyl ether to give pure compound 5c
d
after treatment with D2O), 4.57 [t, 1H,
CH2CH(OCH3)2], 5.26 (near t, 1H, NHCH2; disap-
peared with D2O), 6.94ꢂ7.29 (m, 4H, H-6,7,8,9), 8.25 (s,
(1.64 g, 62%), white crystalline solid, m.p. 131ꢂ132 8C
/
after recrystallisation from the same solvent. IR (CHCl3,
cmꢁ1): 1605, 1568 strong, 1530 shoulder, 1500 weak.
/
1H, 1-NH; disappeared with D2O). Anal. C13H17N3O3
(C, H, N).
The column was further eluted with Me2CO and the
fraction collected, after removal of solvent, afforded
compound 12 as a pale brown solid (0.15 g, 7.5%);
1H-NMR (CDCl3): d 1.62 (m, 6H, piperidine b-CH2’sꢄ
/
g-CH2), 3.55ꢂ
/
3.80 (mꢄ
/
broad s, 6H, piperidine a-
CH2’sꢄ4-CH2), 7.02ꢂ
/
/
7.14 and 7.23ꢂ
/
7.44 (2m, 2Hꢄ
/
4H, H-1,2,7,8,9,10). Anal. C16H18N4 (C, H, N).
whitish crystals, m.p. 282ꢂ284 8C (dec.), after crystal-
/
5.1.11.2. 5-Morpholino-4H-imidazo[1,2-
lisation from EtOAc with charcoal. IR (KBr, cmꢁ1):
3190 broad (NH), 1685 strong (CO), 1600 weak, 1532
weak, 1510. 1H-NMR (DMSO-d6): d 3.63 (s, 2H, 4-
a][1,5]benzodiazepine (5d). The nearly solid residue
deriving from the reaction carried out with morpholine
was taken up in a little Et2O and filtered. There was so
obtained 1.74 g (65%) of pure 5d; white crystals, m.p.
CH2), 7.09 (d, Jꢃ
/
1.4 Hz, 1H, H-2), 7.28ꢂ
/
7.50 and
1.4
7.62ꢂ7.69 (2m, 3Hꢄ
/
/
1H, H-7,8,9,10), 7.71 (d, Jꢃ
/
154ꢂ
charcoal. IR (CHCl3, cmꢁ1): 1608, 1580 strong, 1525
weak, 1498. 1H-NMR (CDCl3): d 3.40ꢂ
4.05 (mꢄbroad
s, 10H, morpholine CH2’sꢄ4-CH2), 7.04ꢂ7.17 and
7.21ꢂ7.48 (2m, 2Hꢄ4H, H-1,2,7,8,9,10). Anal.
/
155 8C after crystallisation from EtOAc with
Hz, 1H, H-1), 10.36 (s, 1H, NH; disappeared with D2O).
Anal. C11H9N3O (C, H, N).
/
/
/
/
/
/
5.1.10. Cyclocondensation of 8c to 12
C15H16N4O (C, H, N).
HCOOH (99%, 10 mL) was added to 4.0 mmol (1.05
g) of 8c and the resulting solution was refluxed for 1.5 h,
while stirring. The reaction mixture was then poured
onto ice-water and the solution obtained was carefully
treated with Na2CO3 until alkaline. The resulting
suspension was stirred for 30 min at r.t., and then the
solid was recovered by filtration, washed with water and
dried. There was so obtained 0.54 g of nearly pure
compound 12. By further extraction with CH2Cl2 of the
aqueous filtrate and washings, an additional crop (0.11
g) of 12 was recovered (total yield 0.65 g, 82%).
5.2. Pharmacology
Male Wistar rats (250ꢂ/300 g) and male Swiss mice
(25ꢂ35 g) were used. The test compounds were sus-
/
pended in 0.5% methylcellulose and administered by
oral gavage in groups of eight animals fasted overnight.
Indomethacin (10 mg kgꢁ1 os) was used as reference
drug in all the experimental tests while control animals
received an equivalent volume of vehicle alone.
The ethical guidelines for investigation of experimen-
tal pain in conscious animals were followed and all of
the tests were carried out according to the EEC ethical
regulation (EEC Council 86/609; D.L.27/01/1992, No.
116).
5.1.11. N,N-Dialkyl-4H-imidazo[1,2-
a][1,5]benzodiazepin-5-amines 5c,d
A solution of 5 mL of the proper amine in 5 mL of dry
C6H5CH3 was added to an ice bath cooled solution of
1.2 mL (10.9 mmol) of TiCl4 and 2 mL of anisole in 40
mL of dry C6H5CH3, while stirring, then 10.0 mmol
(1.99 g) of compound 12 and the solution of 3 mL of the
amine in 10 mL of dry C6H5CH3 were further added and
the resulting mixture was stirred at reflux for 6 h.
After cooling, 3 mL of 2-propanol, 2 g of diatomac-
eous earth and 3 mL of 28% aq. NH3 were added to the
mixture. After stirring, the resulting suspension was
filtered, the solid collected was thoroughly washed with
CH2Cl2, and the filtrate and washings were dried
(anhydrous Na2SO4), then evaporated to dryness under
reduced pressure. The oily residue was chromato-
graphed on a silica gel column eluting with EtOAc
5.2.1. Anti-inflammatory activity
Anti-inflammatory activity was studied by inducing
paw edema according to Winter’s method [11]. Carra-
geenan 1% (0.1 mL) was injected into the plantar surface
of the rat hind paw simultaneously with the oral
administration of the test compounds. Paw volume
was determined immediately after the injection of
phlogogen agent and again 3 h later by means of a
plethysmometer.
5.2.2. Analgesic activity
Antinociceptive activity was studied by writhing test
[12] through the intraperitoneal injection of 0.2 mL/
mouse of AcOH solution (0.6%) 1 h after oral treatment
with the test drugs. Complete extension of either hind
limb was regarded as a writhing response. The number
(compound 5c) or EtOAcꢂMe2CO (1:1) (compound 5d);
/
the eluate collected, after removal of solvents, gave a
residue from which compound 5c or 5d was recovered as
reported below.

944
G. Grossi et al. / European Journal of Medicinal Chemistry 37 (2002) 933ꢂ944
/
of writhings was recorded for 5 min between 10 and 15
min after the injection of the noxious agent.
calculated from the aforementioned differences in the
response between the treated and the control groups.
5.2.3. Antipyretic activity
Acknowledgements
Antipyretic activity was determined in rats in which
fever was induced by intraperitoneal injection of 100 mg
kgꢁ1 Escherichia coli lipopolysaccharides (LPS) (Ser-
otype 0111:B4 Sigma, Milan, Italy) according to Roma-
novsky et al. [13]. The test compounds were
administered orally 1 h before the injection and rectal
temperature was recorded in an air-conditioned room
(23 8C) immediately before and 5 h after pyretogen
injection.
The authors wish to thank Dr G. Domenichini for
skillful technical assistance in pharmacological experi-
ments, A. Panaro for elemental analyses, F. Tuberoni
for IR spectra and Dr R. Raggio for H-NMR spectra.
The financial support from University of Genoa and
University of Parma is gratefully acknowledged.
1
References
5.2.4. Ulcerogenicity
The acute gastric mucosal damage of the test com-
pounds was evaluated by examining the stomachs
excised 5 h after oral administration of the drugs in
mice. The stomachs, fixed in 2% formalin, were opened
and examined with a stereomicroscope by an observer
unaware of the treatment the mice received. The number
and the length of the gastric lesions are measured by
means of an image analyser.
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/
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223.
5.2.6. Data analysis
/
672.
Results were calculated as mean9
between treated and control groups were determined by
Student’s t-test. ꢀP B0.05 was reported when such
differences were statistically significant and ꢀꢀP B0.01
was reported when the differences were highly signifi-
cant. Then, the pharmacological activities of the com-
pounds were expressed as the percentages of inhibition
/
S.E.M. Differences
/
/
/
/
/
421.
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