1,5-Benzodiazepines XIV. Synthesis of new substituted 9H-bis-[1,2,4] triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepines and relate compounds endowed with in vitro cytotoxic properties

Article abstract of DOI:10.1016/j.farmac.2004.12.005

A series of 11 new 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine derivatives 8e-o was synthesized. Ten of these compounds (8e-m,o), along with four analogues (8a-d) (previously synthesized by us) were tested in vitro in order to evaluate their cytotoxic and anti-HIV-1 properties. In this connection other six original compounds, i.e., five 9-substituted compounds prepared starting from the 6,12-diphenylderivative 8c (compounds 10, 11, 12, 13a,b) and the bis-triazolone derivative 14, were synthesized and tested for the same purpose. While none of the 20 compounds tested exhibited any appreciable anti-HIV-1 activity, some of them exhibited interesting cytotoxic properties, the best results being shown by compounds 8c,d,k and 11 (CC ₅₀ range = 3-12 μM). Therefore, these four compounds were further evaluated for their antiproliferative activity against a panel of human tumor cell lines; actually, compounds 8d, 8k and 11 showed antiproliferative properties against either or both leukemia- and lymphoma-derived cell lines in the low micromolar range.

Full text of DOI:10.1016/j.farmac.2004.12.005

Il Farmaco 60 (2005) 113–125
http://france.elsevier.com/direct/FARMAC/
1,5-Benzodiazepines XIV. Synthesis of new substituted
9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepines and relate
compounds endowed with in vitro cytotoxic properties
Mario Di Braccio a,*, Giancarlo Grossi a, Maurizio Ceruti b, Flavio Rocco b, Roberta Loddo c,
Giuseppina Sanna c, Bernardetta Busonera c, Marta Murreddu c, Maria Elena Marongiu c
a Dipartimento di Scienze Farmaceutiche, Università di Genova, Viale Benedetto XV, 3, I-16132 Genova, Italy
b Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro Giuria 9, I-10125 Torino, Italy
c Dipartimento di Scienze e Tecnologie Biomediche, Sezione di Microbiologia e Virologia Generale & Biotecnologie Microbiche, Cittadella Universitaria,
I-09042 Monserrato (CA), Italy
Received 20 July 2004; received in revised form 10 December 2004; accepted 20 December 2004
Abstract
A series of 11 new 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine derivatives 8e–o was synthesized. Ten of these compounds
(8e–m,o), along with four analogues (8a–d) (previously synthesized by us) were tested in vitro in order to evaluate their cytotoxic and
anti-HIV-1 properties. In this connection other six original compounds, i.e., five 9-substituted compounds prepared starting from the 6,12-
diphenylderivative 8c (compounds 10, 11, 12, 13a,b) and the bis-triazolone derivative 14, were synthesized and tested for the same purpose.
While none of the 20 compounds tested exhibited any appreciable anti-HIV-1 activity, some of them exhibited interesting cytotoxic properties,
the best results being shown by compounds 8c,d,k and 11 (CC50 range = 3–12 µM). Therefore, these four compounds were further evaluated
for their antiproliferative activity against a panel of human tumor cell lines; actually, compounds 8d, 8k and 11 showed antiproliferative
properties against either or both leukemia- and lymphoma-derived cell lines in the low micromolar range.
© 2005 Elsevier SAS. All rights reserved.
Keywords: 1,5-Benzodiazepine fused derivatives; Cytotoxic activity; Antiproliferative activity
1. Introduction
tam group had been replaced by an imidazole or triazole ring
[2], with the aim to improve the anti-HIV-1 activity of the
best 5H-pyrimido[4,5-b] [1,5]benzodiazepine derivative 2d,
we also synthesized the tetracyclic derivatives 5, 6, 7 struc-
turally related to 2d (Fig. 1). Actually, compounds 5, 6 and 7
resulted inactive against HIV-1 and proved to be cytotoxic in
the low micromolar range [1].
We previously described the synthesis and the biological
evaluation of a series of 5H-pyrimido[4,5-b] [1,5]benzodiaz-
epines 2 and pyrazolo[3,4-b] [1,5]benzodiazepines 3, 4
designed as novel analogues of the well known non-nucleo-
side HIV-1 reverse transcriptase inhibitor Nevirapine (1) in
order to test their in vitro anti-HIV-1 activity [1]. Among all
the synthesized compounds, some pyrimidine derivatives 2,
which were more closely related to the Nevirapine structure,
exhibited a slight anti-HIV-1 activity, whereas the pyrazole
derivatives 3, 4 showed mainly cytotoxic properties [1].
Since the literature reported a high anti-HIV-1 activity for
some tetracyclic derivatives of Nevirapine, in which the lac-
This result prompted us to extend our study on other tet-
racyclic 1,5-benzodiazepine derivatives analogues of the com-
pounds 5–7 for their potential cytotoxic properties.
As a first step in this study, we have now synthesized a
number of substituted 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d]
[1,5]benzodiazepines 8. Four examples of these latter (com-
pounds 8a–d) were previously obtained by us as by-products
in the synthesis of the analgesic/anti-inflammatory agents
N,N-disubstituted 4H-[1,2,4]triazolo[4,3-a] [1,5]benzo-
diazepin-5-amines 9 [3] (Fig. 2).
* Corresponding author. Tel.: +39 010 3538352; fax: +39 010 3538358.
E-mail address: dbraccio@unige.it (M. Di Braccio).
0014-827X/$ - see front matter © 2005 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.12.005

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M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Fig. 1. Structures of compounds 1–7.
Fig. 2. Structures of compounds 8a-d previously obtained by us [3] as by-products in the synthesis of the analgesic/anti-inflammatory agents 9.
For the preparation of the novel compounds 8e–o
(Scheme 1) we planned a synthesis aimed at obtaining high
yields of the tetracyclic derivatives 8 as unique reaction prod-
ucts: on the basis of the results of a preliminary screening for
the cytotoxicity, we have developed chiefly the 9-methyl-
substituted compounds (8h–o).
In this connection, we have also prepared other
9-substituted compounds starting from the most active
9-unsubstituted compound 8c: the 9-[(dimethylamino)-
methylene]derivative 10, the 9-chloro- and the 9,9-
dichloroderivatives 11 and 12, respectively, and the 9-(dialkyl-
amino)derivatives 13a,b.
nitroaniline, in the presence of dry pyridine (room tempera-
ture, 1 h) to give the N-(2-nitrophenyl)malonamates 16a–c
which in turn were transformed into the corresponding N-(2-
aminophenyl)malonamates 17a–c by hydrogenation at
5–15 psi in a Parr apparatus in the presence of 5% palladium
on charcoal. The intermediates 17a–c underwent to cycliza-
tion to desired 3-methyl-1H-1,5-benzodiazepine-2,4(3H,5H)-
diones 18b–d by treatment with an ethanolic solution of
sodium ethoxide (30 °C, overnight) followed by acidification
with 2 N aqueous HCl. The diones 18a [5] and 18b–d were
heated with Lawesson’s reagent (Dowtherm A, 150 °C, 2 h)
to give the corresponding dithiones 19a–d from which the
2,4-bis(methylthio)derivatives 20a–d were obtained by reac-
tion with iodomethane (dry acetone at reflux, in the presence
of anhydrous K2CO3, 2 h). Finally, the reaction of com-
pounds 20a–d with suitable hydrazides in excess (Dowtherm
A, 200 °C, 2 h) in the presence of p-toluenesulfonic acid gave
usually good yields of the desired 9-alkyl-6,12-dialkyl(diaryl)-
9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepines
8e–o (Scheme 1).
Moreover, we also introduced a change in the triazole moi-
ety by synthesizing the bis-triazolone derivative 14 (Fig. 3).
2. Chemistry
The
3-methylsubstituted
1H-1,5-benzodiazepine-
2,4(3H,5H)diones 18b–f were prepared by a method analo-
gous to that described by Weber et al. [4] for the preparation
of similar compounds. Thus, the monoethylester of
2-methylmalonic acid (15) was treated with excess PCl5
(CH2Cl2, room temperature, 3 h), then with the suitable 2-
The reaction of 8c [3] with a large excess of N,N-
dimethylformamide dimethyl acetal in pyridine at reflux
(24 h), afforded the 9-[(dimethylamino)methylene]derivative
10.

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
115
Scheme 1
.
Synthesis of substituted 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepines 8e–o.
On the other hand, the reaction of 8c with excess
N-chlorosuccinimide [CCl4–CHCl3 (1:1) at reflux, 24 h]
yielded a mixture of 9-chloroderivative 11 and 9,9-
dichloroderivative 12.
Finally, the bis-triazolone derivative 14 was obtained from
the 2,4-bis(methylthio)derivative 20b through the reaction
with excess ethyl carbazate (Dowtherm A, 200 °C, 2 h)
(Scheme 2).
The reaction of 11 with a large excess of morpholine or
1-methylpiperazine (dimethyl sulfoxide, 120 °C, 1–2 h)
afforded then the 9-(dialkylamino)derivatives 13a,b, respec-
tively.
The structures attributed to the compounds described in
this paper are consistent with the results of elemental analy-
ses, IR and H-NMR spectral data (See Section 4 and
1
Tables 2–4).

116
M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Fig. 3
dione (14).
.
9-Substituted derivatives of 8c (compounds 10, 11, 12, and 13a,b) and 9-methyl-9H-bis[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine-6,12(7H,11H)-
The 1H-NMR spectra were particularly useful in order to
Also the 9-methylsubstituted bis-triazolone derivative 14
appears to be in only one conformation, i.e., the same of
9-methylsubstituted compounds 8h–o, as confirmed by its
1H-NMR spectral data (DMSO-d6 solution, H-9: quartet at d
4.07 ppm; 9-CH3: doublet at d 1.54 ppm). It is reasonable to
think that, also in the case of the other 9-monosubstituted
compounds (11, 13a,b), the only conformer obtained is that
one with the bulky 9-substituent located in the more stable
study the conformational features of compounds 8e–o and
their analogues 11, 12, 13a,b and 14. It is worth noting that
the tetracyclic system of compounds 8 is not planar (as shown
by the structures drawn by MacroModel software, version
8.0 [6]) and its seven membered diazepine ring, in the solid
state and in solution (e.g., in CDCl3) at room temperature, is
blocked in only one boat conformation (Fig. 4), no confor-
mational inversion occurring, very likely due to the restrict-
ing effect of the R′″ substituents of the triazole rings. There-
fore, in the 9-unsubstituted derivatives the two protons at
C-9 are diastereotopic and, in the 1H-NMR spectra, the quasi-
equatorial proton is clearly more deshielded than the quasi-
axial one, because this latter projects into the shielding cone
of the benzene ring; similar remarks have been previously
reported for 1,4-benzodiazepine derivatives [7]. This situa-
tion is indeed confirmed by the 1H-NMR spectra (in CDCl3
solutions) of the 9-unsubstituted compounds 8e–g that show
for the two protons at C-9 two distinct signals [doublets
(J = 16 Hz) as a half of AB quartet] at d ~3.8 ppm (quasi-
axial proton) and at d ~5 ppm (quasi-equatorial proton),
respectively (See Section 4, Table 4). When C-9 is substi-
tuted with an alkyl group, the interconversion barrier of the
seven membered ring is further increased and the R substitu-
ent is very likely located in the thermodynamically more
favoured quasi-equatorial position, according to the litera-
ture suggestions for analogous cases [8,9] (Fig. 4).
1
quasi-equatorial position, even though the H-NMR data
regarding the H-9 chemical shifts of these compounds does
not allow any comparison with those of compounds 8h–o,
due the electronic effect of 9-substituent (chloro or dialky-
lamino group).
The first examples of the 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-
d] [1,5]benzodiazepine system were described by us [3] in
1990; 1 year later the synthesis of a group of compounds
belonging to the general structure 8 (six compounds, among
which two had been already described by us [3]) has been
reported [10]. Even though the last steps of the synthetic route
followed by these authors are very similar to those now used
by us, we can point out that in that paper [10] no 9-substituted
bistriazolobenzodiazepine derivative has been reported,
whereas we now describe mainly 9-substituted compounds
(8h–o, 10, 11, 12, 13a,b, 14). Furthermore, in the above men-
tioned paper no detailed melting point (both for intermedi-
ates and final compounds) was reported but all melting points
were given as > 250 °C, or > 300 °C, or > 350 °C; actually, if
these data are likely for the intermediate benzodiazepine-
diones, -dithiones, and for the bistriazolobenzodiazepine
derivatives, they appear quite unlikely for the 2,4-
bis(methylthio)-3H-1,5-benzodiazepine derivatives. For
instance, the 2,4-bis(methylthio)-3H-1,5-benzodiazepine
(20a, Scheme 1) was there described as a crystalline solid
Actually, we found only one conformer in all the
9-methylsubstituted compounds 8 (8h–o), and in these com-
pounds H-9 appears to be in quasi-axial position (1H-NMR
signal: quartet at d ~4.1 ppm) and, therefore, the CH3 group
is in quasi-equatorial position (1H-NMR signal: doublet at d
~2.2 ppm).

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
117
Scheme 2. Synthetic routes to the compounds 10, 11, 12, 13a,b and 14.
melting over 300 °C [10], whereas we found that this com-
pound is a low-melting solid (53.5–55 °C, Table 3).
HIV-1 activity and the results of biological evaluation are
reported in Table 1.
None of the 20 compounds tested exhibited any anti-HIV-
1 activity; on the other hand, six compounds showed a clear
cytotoxic activity (CC50 < 50 µM), while further four com-
pounds afforded only a moderate cytotoxic effect (CC50 rang-
ing from 50 to 137 µM).
3. Biological results and conclusions
Compounds 8a–d [3] and the novel 8e–m,o, 10, 11, 12,
13a,b and 14 were tested in vitro for cytotoxicity and anti-

118
M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Fig. 4
.
Conformations of 6,9,12-trimethyl-9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine 8h (chosen as an example) drawn with MacroModel ver-
sion 8.0 [6]: the conformer with 9-methyl group in quasi-equatorial position (I) is more likely than that one with the 9-methyl group in the quasi-axial position
(II).
As regards the compounds 8, phenyl groups as R′″ sub-
stituents afforded the highest cytotoxicity (compounds 8c and
8d). Compounds devoid of cytotoxicity (CC50 > 200 µM)
were obtained when R′″ were alkyl groups (compounds
8a,b,e,h,i) or 4-pyridyl groups (8g,m), while when R′″ were
benzyl groups (compounds 8f,j), in only one case a slight
cytotoxic effect was observed (8f, CC50 = 58 µM).
As the 9-methylsubstituted derivative 8d was clearly more
cytotoxic (CC50 = 4 µM) than the 9-unsubstituted analogue
8c (CC50 = 12 µM), further seven 9-methylderivatives (8 h–
m,o) were prepared and tested. However, the biological results
were quite disappointing, with the sole exception of 8k which
displayed fairly good cytotoxic properties (CC50 = 12 µM).
Furthermore, the 9-methyl substitution was negative in the
case of 6,12-dibenzylderivatives, since the 9-unsubstituted
compound 8f was more cytotoxic than the 9-methyl ana-
logue 8j (CC50 = 58 and > 200 µM, respectively).
In general, test compounds specifically inhibited the pro-
liferation of cells derived from haematological tumours
(Table 2) at concentrations (IC50 range = 1.9–25 µM, Table 2)
significantly lower than those inhibitory for solid tumors
(Table 3). Exceptions were compound 8k, which was fairly
active also against skin melanoma, breast adenocarcinoma
and lung squamous carcinoma (IC50 range = 8.4–8.6 µM,
Table 3), and compound 11, which was also active against
breast adenocarcinoma (IC50 = 10 µM, Table 3). It is worth
noting that the test compounds did not show significant anti-
proliferative properties against the normal tissue-derived cells
[IC50 = 100 or >100 µM, except for 8k against CRL7065 cells
(25 µM), Table 3].
In conclusion, among the 20 9H-bis-[1,2,4]triazolo[4,3-
a:3′,4′-d] [1,5]benzodiazepine derivatives tested, only com-
pounds 8d, 8k and 11, showed antiproliferative activity against
either or both leukaemia- and lymphoma-derived cell lines in
the low micromolar range. Therefore, it can be concluded that,
in this molecular class, the substitution with phenyl or
p-chlorophenyl groups at C-6 and C-12, and with a methyl or
chloro at C-9, are the main structural features favouring the
antiproliferative activity.
Compounds bearing substituents on the benzene ring (8o)
or on the phenyl R′″ groups (8k,l) showed a decreased cyto-
toxicity compared to unsubstituted compounds. In particular,
the substitution with 3-methoxyphenyl as R′″ groups resulted
very unfavourable (compound 8l).
Further experiments aimed at defining target and mecha-
nism of action of title compounds are in progress.
Among the compounds derived from the cytotoxic com-
pound 8c, a very good result was afforded by the 9-chloro-
derivative 11 (CC50 = 3 µM), while the 9,9-dichloroderivative
12 displayed lower cytotoxicity (CC50 = 30 µM) similar to
that of the 9-morpholino derivative 13a (CC50 = 32 µM). More
modest results were given by the 9-(4-methyl-1-pipera-
zinyl)derivative 13b and by the 9-[(dimethylamino)-
methylene]derivative 10. On the other hand, the bis-triazolone
derivative 14 did not result cytotoxic (CC50 >200 µM).
In an effort to more completely define the biological pro-
file of title compounds, those resulted most cytotoxic against
MT-4 cells (Table 1), i.e., 8c, 8d, 8k and 11, were evaluated
for antiproliferative activity against a panel of cell lines
derived from either haematological (CCRF-CEM, WIL-
2NS, and CCRF-SB) or solid (SKMEL28, MCF7, SKMES-1,
HepG2, and DU145) human tumors. They were also evalu-
ated against cell lines derived from normal human tissues
(MRC-5, CRL7065).
4. Experimental
4.1. Chemistry
Melting points were determined using a Fisher–Johns appa-
ratus (Electrothermal when above 300 °C) and are uncor-
rected. IR spectra were recorded on a Perkin-Elmer “Spec-
trum One” spectrophotometer (abbreviations relative to IR
bands: br = broad, s = strong, w = weak, sh = shoulder).
1H-NMR spectra were recorded on a Varian Gemini 200
(200 MHz) spectrometer and chemical shifts (d) are reported
in ppm using (CH3)4Si as an internal reference (d = 0). Spin
multiplicities are given as follows: s (singlet), d (doublet), dd
(double doublet), t (triplet), q (quartet), m (multiplet). Analy-

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
119
Table 1
In vitro biological activities of compounds 8a–m,o, 10, 11, 12, 13a,b and 14

120
M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Table 2
poured into 800 ml of cold water and the mixture was vigor-
ously stirred at room temperature for 1 h The organic layer
was collected and the aqueous one was thoroughly extracted
with dichloromethane. The combined extracts were washed
with 5% aqueous NaHCO3, then with water, dried (anhy-
drous sodium sulphate) and finally evaporated to dryness in
vacuo. By treating the oily residue with a little petroleum ether
and standing compounds 16a–c separated out as pale yellow
solids, which were then crystallized from the suitable sol-
vents. Data of compounds 16a–c are reported in Table 4.
In vitro antiproliferative activity of compounds 8c,d,k and 11 against human
leukemia/lymphoma-derived cell lines
Compound IC50 (µM) a
MT-4 b
CCRF-CEM c WIL-2NS d
13 25.5 0.9
6.6 0.2
CCRF-SB e
15
10 0.1
7.5
1.9 0.5
8c
8d
8k
11
12 0.2
1
1
4
0.3
1
15
10
2
2
12
3
7
2
2
0.1
4.2 1.5
3.6 0.4
a Compound concentration required to reduce cell proliferation by 50%,
as determined by the MTT method, under conditions allowing untreated
controls to undergo at least three consecutive rounds of multiplication. Data
represent mean values ( SD) for three independent determinations.
b CD4+ human T-cells containing an integrated HTLV-1 genome.
c CD4+ human acute T-lymphoblastic leukemia.
4.1.2. General procedure for the preparation of ethyl
N-(2-aminophenyl)-2-methylmalonamates 17a–c
To a solution of 25.00 mmol of the proper compound 16
(6.66 g of 16a, or 7.52 g of 16b, or 8.36 g of 16c) in 150 ml of
ethanol, 5% palladium on charcoal (0.50 g) was added, and
the mixture was subjected to hydrogenation at 15 psi (5 psi,
in the case of chloro derivative 16b) in a Parr apparatus at
room temperature. When the hydrogen absorption ceased, the
mixture was filtered and the resulting colourless solution was
evaporated to dryness in vacuo to give solid or oily residues
from which, after treatment with little ethyl ether, com-
pounds 17a–c separated out as whitish solids, which were
then crystallized from the proper solvent. Data of com-
pounds 17a–c are reported in Table 4.
d Human splenic B-lymphoblastoid cells.
e Human acute B-lymphoblastic leukemia.
ses of all new compounds, indicated by the symbols of the
elements, were within 0.4% of the theoretical values and
were performed by the Laboratorio di Microanalisi, Diparti-
mento di Scienze Farmaceutiche, University of Genoa.
Thin-layer chromatograms were run on Merck silica gel
60F254 precoated plastic sheets (layer thickness 0.2 mm). Col-
umn chromatography was performed using Carlo Erba silica
gel (0.05–0.20 mm) or Carlo Erba neutral aluminium oxide
(Brockmann activity I).
4.1.3. General procedure for the preparation of 3-methyl-
1H-1,5-benzodiazepine-2,4(3H,5H)-diones 18b–d
4.1.1. General procedure for the preparation of ethyl
2-methyl-N-(2-nitrophenyl)malonamates 16a–c
Sodium (30.0 mmol, 0.69 g) was dissolved in 150 ml of
anhydrous ethanol: to this solution 20.0 mmol of the proper
compound 17 (4.73 g of 17a, or 5.41 g of 17b, or 6.09 g of
17c) were added and the mixture was stirred at 30 °C for 8 h.
The solvent was then removed in vacuo and the residue was
treated with aqueous 2 N HCl: the crude compound 18 that
separated out as a whitish solid was collected by filtration,
washed with water, dried, and crystallized from the suitable
solvent. Data of compounds 18b–d are reported in Table 5.
In a two-neck flask, protected by the moisture with a cal-
cium chloride tube, phosphorus pentachloride (11.44 g,
55.0 mmol) was carefully added, in little amounts, to the solu-
tion of 50.0 mmol (7.30 g) of 2-methylmalonic acid monoet-
hyl ester 15 in 150 ml of dichloromethane. The resulting mix-
ture was then stirred at room temperature for 3 h, then the
solution of the 35.0 mmol of the proper aromatic amine [4.83 g
of 2-nitroaniline, or 6.04 g of 4-chloro-2-nitroaniline, or 7.21 g
of 4-(trifluoromethyl)-2-nitroaniline] in 50 ml of dichlo-
romethane and 15 ml of dry pyridine was slowly added by a
dropping funnel (an exothermic reaction with emission of
white fumes occurred during the addition). The resulting warm
mixture was stirred at room temperature for 30 min, then
4.1.4. General procedure for the preparation of 1H-1,5-
benzodiazepine-2,4(3H,5H)-dithiones 19a–d
A mixture of 5.0 mmol of the proper compound 18 (0.88 g
of 18a [5]; 0.95 g of 18b; 1.12 g of 18c; 1.21 g of 18d),
Table 3
In vitro antiproliferative activity of compounds 8c,d,k and 11 against human solid tumor-derived cell lines and normal tissue-derived cell lines
Compound
IC50 (µM) a
SK-MEL-28 b
74 14
MCF7 c
44 0.2
67 0.4
8.5 0.5
SK-MES-1 d
HepG2 e
DU145 f
>100
MRC-5 g
>100
CRL7065 h
>100
8c
8d
8k
11
89
1
75
8
>100
>100
>100
35
>100
>100
>100
8.4 0.4
8.6 0.4
8
3
20
2
100
25
2
35
2
10
1
64 13
36
>100
>100
100
a Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method, under conditions allowing untreated controls to
undergo at least three consecutive rounds of multiplication. Data represent mean values ( SD) for three independent determinations.
b Human skin melanoma.
c Human breast adenocarcinoma.
d Human lung squamous carcinoma.
e Human hepatocellular carcinoma.
f Human prostate carcinoma.
g Human lung fibroblasts.
h Human foreskin fibroblasts.

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
121
Table 4
Data of compounds 16a–c and 17a–c
Compound
RIV
H
Yield
(%)
92
m.p. (°C)
Molecular formulab
IRc (cm–1
)
1H-NMRd (d, ppm)
(solvent) a
59.5–60.5 (A)
16a
C
C
C
12H14N2O5
3330 (NH), 1740 s, br
(ester CO), 1702 s, br
(amide CO), 1609, 1588. 7.23 (near t, 1H, phenyl H-4), 7.70 (near t, 1H,
phenyl H-5), 8.27 (near d, 1H, phenyl H-3),
8.76 (near d, 1H, phenyl H-6), 10.95 e (broad
s, 1H, NHCO).
1.30 (t, 3H, CH2CH3), 1.55 (d, 3H, CHCH3),
3.50 (q, 1H, CHCH3), 4.29 (q, 2H, CH2CH3),
16b
16c
Cl
92
65
67.5–68.5 (B)
53–54 (B)
12H13ClN2O5
3330 (NH), 1740 s, br
(ester CO), 1708 s, br
(amide CO), 1610, 1580. 7.67 (dd, J5,6 = 9 Hz, J5,3 = 3 Hz, 1H, phenyl
H-5), 8.27 (d, J3,5 = 3 Hz, 1H, phenyl H-3),
8.82 (d, J6,5 = 9 Hz,1H, phenyl H-6), 10.84 e
(broad s, 1H, NHCO).
1.34 (t, 3H, CH2CH3), 1.55 (d, 3H, CHCH3),
3.60 (q, 1H, CHCH3), 4.35 (q, 2H, CH2CH3),
CF3
12H13F3N2O5
3345 (NH), 1748 s, br
(ester CO), 1719 s, br
1.34 (t, 3H, CH2CH3), 1.58 (d, 3H, CHCH3),
3.62 (q, 1H, CHCH3), 4.31 (q, 2H, CH2CH3),
(amide CO), 1633, 1588, 7.90 (near d, 1H, phenyl H-5), 8.53 (near s,
1523.
1H, phenyl H-3), 8.98 (d, J6,5 = 9 Hz, 1H, phe-
nyl H-6), 11.21 e (broad s, 1H, NHCO).
17a
17b
H
94
79
97–98 (C)
C
C
12H16N2O3
3416 and 3340 sh (NH2), 1.29 (t, 3H, CH2CH3), 1.50 (d, 3H, CHCH3),
3251 (NH), 1749 s (ester 3.49 (q, 1H, CHCH3), 3.80 e (s, 2H, NH2),
CO), 1642 s (amide CO), 4.25 (q, 2H, CH2CH3), 6.60–7.40 (m, 4H, phe-
1604 w, 1592 w, 1543.
3446 and 3387 (NH2),
nyl H-3,4,5,6), 8.44 e (broad s, 1H, NHCO).
1.26 (t, 3H, CH2CH3), 1.50 (d, 3H, CHCH3),
Cl
140–141 (D)
12H15ClN2O3
3294 (NH), 1729 s (ester 3.45 (q, 1H, CHCH3), 3.86e (broad s, 2H,
CO), 1667 s (amide CO), NH2), 4.23 (q, 2H, CH2CH3), 6.50–7.37 (m,
1629, 1600 w, 1546.
3H, phenyl H-3,5,6), 8.26 e (broad s, 1H,
NHCO).
17c
CF3
71
172–172.5 (D)
C
13H15F3N2O3
3444 and 3370 (NH2),
1.30 (t, 3H, CH2CH3), 1.57 (d, 3H, CHCH3),
3272 (NH), 1740 s (ester 3.51 (q, 1H, CHCH3), 4.00 e (broad s, 2H,
CO), 1654 s (amide CO), NH2), 4.30 (q, 2H, CH2CH3), 6.90–7.70 (m,
1605 w, 1537.
3H, phenyl H-3,5,6), 8.60 e (broad s, 1H,
NHCO).
a Crystallization solvent: A = ethanol/petroleum ether, B = petroleum ether, C = ethyl acetate/isopropyl ether, D = ethanol.
b Anal. C,H,N. (C, H, N, Cl for 16b, 17b).
c In CHCl3 solutions (compounds 16a–c); in Kbr pellets (compounds 17a–c). Abbreviations: s = strong, br = broad, w = weak, sh = shoulder.
d In CDCl3 solutions. Abbreviations: s = singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet.
e Disappeared with D2O.
7.5 mmol (3.03 g) of the Lawesson’s reagent and 10 ml of
DowthermA was heated at 150 °C for 2 h with stirring. In the
case of the reactions carried out with compounds 18a–c, the
suspension obtained was diluted with a little acetone and the
whitish insoluble solid was collected by filtration, washed
with the same solvent and dried to yield the nearly pure com-
pounds 19a–c. Due the very low solubility of these com-
pounds in most solvents, only a little amount of them was
crystallized from the proper solvent in order to prepare the
analytical sample; the remaining amount was used rough in
the subsequent synthetic step. On the contrary, compound 19d
was very soluble in many solvents: therefore the final mix-
ture of the reaction carried out with 18d was chromato-
graphed on a aluminium oxide column eluting first with
dichloromethane until Dowtherm A was removed, then with
tetrahydrofuran in order to recover 19d. The eluate collected,
after removal of solvent afforded a whitish solid, which was
taken up in a little petroleum ether, filtered and crystallized
from the suitable solvent. Data of compounds 19a–d are
reported in Table 5.
4.1.5. General procedure for the preparation of 2,4-bis(m-
ethylthio)-3H-1,5-benzodiazepines 20a–d
A mixture of 5.0 mmol of the proper dithione 19 (1.04 g of
19a; 1.11 g of 19b; 1.28 g of 19c; 1.45 g of 19d), 1.40 g of
anhydrous potassium carbonate, 1.60 ml (~25 mmol) of
iodomethane and 50 ml of dry acetone was refluxed for 2 h.
The solvent was then removed in vacuo and the residue par-
titioned between water and dichloromethane. The aqueous
phase was extracted several more times with dichloromethane.

122
M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Table 5
Data of compounds 18b–d, 19a–d and 20a–d
Compound
R
RIV Yield m.p. (°C)
Molecular
formulab
IRc (cm–1
)
1H-NMRd (d, ppm)
(%)
63
(solvent) a
>360 (A)
18b
CH3
H
C
C
C
10H10N2O2
3170 and 3062 (NH), 1703 s (CO),
1650 s, 1604 w, 1503.
1.11 (d, 3H, 3-CH3), 3.11 (q, 1H, H-3),
7.03–7.30 (m, 4H, H-6,7,8,9), 10.41 e (s,
2H, NHCO).
18c
18d
CH3 Cl
40
359–360 (A)
361–363 (A)
10H9ClN2O2 3193 and 3065 (NH), 1716 s (CO),
1658 s, 1598 w, 1497.
1.12 (d, 3H, 3-CH3), 3.20 (q, 1H, H-3),
7.09–7.30 (m, 3H, H-6,8,9), 10.51 e (s, 2H,
NHCO).
CH3 CF3 54
11H9F3N2O2 3190 and 3065 (NH), 1720 s (CO),
1657 s, 1619, 1500 w.
1.23 (d, 3H, 3-CH3), 3.37 (q, 1H, H-3),
7.30–7.80 (m, 3H, H-6,8,9), 10.50 e (broad
s, 2H, NHCO).
19a
19b
H
H
H
95
98
315–317 (B) C9H8N2S2
3102 (NH), 1662, 1603, 1532 s, 1487, 4.06 (s, 2H, CH2), 7.30 (s, 4H, H-6,7,8,9),
1170 s (CS).
12.47 e (s, 2H, NHCS).
CH3
319–320 (C)
303–304 (C)
280–281 (D)
C10H10N2S2
C10H9ClN2S2
C11H9F3N2S2
3159 and 3110 (NH), 1661, 1605,
1537 s, 1491 , 1168 s (CS).
1.43 (d, 3H, 3-CH3), 3.67 (q, 1H, H-3),
7.14–7.62 (m, 4H, H-6,7,8,9), 12.51 e (s,
2H, NHCS).
19c
19d
CH3 Cl
87
3153 and 3105 (NH), 1664, 1599,
1537 s, 1485, 1167 s (CS).
1.48 (d, 3H, 3-CH3), 3.42 (q, 1H, H-3),
6.70–7.60 (m, 3H, H-6,8,9), 10.90 e (s, 2H,
NHCS).
CH3 CF3 98
3162 and 3115 (NH), 1660 w, 1621,
1601 w, 1533 s, 1505 , 1167 s (CS).
1.60 (d, 3H, 3-CH3), 3.50 (q, 1H, H-3),
7.38–7.78 (m, 3H, H-6,8,9), 12.48 e (s, 2H,
NHCS).
20a
20b
H
H
H
83
62
53.5–55 (E)
150–151 (F)
C
C
11H12N2S2
12H14N2S2
1597 s, 1570, 1545 sh, 1460.
1602, 1583 s, 1545 sh, 1460.
2.50 (s, 6H, SCH3), 3.09 (s, 2H, CH2),
7.09–7.49 (m, 4H, H-6,7,8,9).
1.52-1.83 (m, 3H, 3-CH3), 2.47 (s, 6H,
SCH3), 2.68–2.93 (m, 1H, H-3), 7.13–7.42
(m, 4H, H-6,7,8,9).
CH3
20c
20d
CH3 Cl
76
54–55 (G)
C
C
12H13ClN2S2 1607, 1587 s, 1550 w, 1460.
1.20–1.86 (m, 3H, 3-CH3), 2.45 (s, 6H,
SCH3), 2.60–2.95 (m, 1H, H-3), 7.08–7.50
(m, 3H, H-6,8,9).
CH3 CF3 64
99–100 (G)
13H13F3N2S2 1605, 1582 s, 1550 w, 1450 w, 1410.
1.48–1.80 (m, 3H, 3-CH3), 2.47 (s, 6H,
SCH3), 2.65–2.86 (m, 1H, H-3), 7.33–7.51
(m, 2H, H-8,9), 7.65 (near s, 1H, H-6).
a Crystallization solvent: A = ethanol, B = methanol/dichloromethane, C = ethanol/tetrahydrofuran, D = ethyl acetate/isopropyl ether, E = petroleum ether,
F = isopropyl ether, G = methanol.
b Anal. C,H,N (compounds 18b,d); C,H,N,Cl (compound 18c); C,H,N,S (compounds 19a,b,d, 20 a,b,d); C,H,N,S,Cl (compounds 19c, 20 c).
c In Kbr pellets (compounds 18b–d, 19a–d); in CHCl3 solutions (compounds 20a–d). Abbreviations: s = strong, w = weak, sh = shoulder.
d In DMSO-d6 solutions (18b–d, 19a–d); in CDCl3 solutions (compounds 20a–d). Abbreviations: s = singlet, d = doublet, q = quartet, m = multiplet.
e Disappeared with D2O.
The combined organic extracts were dried (anhydrous sodium
sulphate), then evaporated to dryness to give an oily residue,
which was purified by chromatography on a silica gel col-
umn, eluting with the mixture dichloromethane–petroleum
ether (1:1). The fraction collected was evaporated to dryness
in vacuo and the resulting white oil, after treatment with a
little petroleum ether and standing at 4 °C, afforded com-
pounds 20a–d as white solids, which were then recrystal-
lized from the suitable solvents. The low-melting com-
pounds 20a,c, due their very high solubility in nearly all
solvents, were used as oils in the subsequent synthetic step,
only a small amount of them being recrystallized from the
proper solvent in order to prepare the analytical sample.
Data for compounds 20a–d are reported in Table 5.
4.1.6. General procedure for the preparation
of the substituted 9H-bis-[1,2,4]triazolo[4,3-a:3′,4′-d]
[1,5]benzodiazepines 8e–o
A mixture of 2.5 mmol of the proper 2,4-bis(methyl-
thio)derivative 20 (0.59 g of 20a; 0.63 g of 20b; 0.71 g of
20c; 0.80 g of 20d), 7.5 mmol of the suitable hydrazide (0.56 g
of acetylhydrazine; 0.66 g of propionylhydrazine; 1.08 g of
(phenylacetyl)hydrazine; 1.02 g of benzoylhydrazine; 1.28 g
of (4-chlorobenzoyl)hydrazine; 1.13 g of (3-methoxy-
benzoyl)hydrazine; 1.03 g of isonicotinoylhydrazine), 0.15 g
of p-toluenesulfonic acid and 10 ml of Dowtherm A was

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
123
stirred at 200 °C for 2 h.After cooling, compounds 8e–o were
recovered from the final mixture as follows.
Compounds 8e,g,i,l,m: the white solid that separated out
was taken up in a little ethanol and filtered to give the nearly
pure compound 8.
Compounds 8f,j: the solid that separated out (0.30 g) was
recovered by filtration and washed with a little ethyl ether. It
was shown to be (elemental analysis, IR, and 1H-NMR spec-
tra) 1,2-diphenylacetylhydrazine, white needles, m.p.: 231–
233 °C, after crystallization from ethanol (Ref. [11]: m.p.:
231 °C). The filtrate and washings, after removal of solvent
gave an oil which was chromatographed on a silica gel col-
umn, eluting first with dichloromethane until Dowtherm A
was removed, then with ethyl acetate in order to remove some
impurities and finally with the mixture dichloromethane/
methanol (9:1). This last eluate, after removal of solvents gave
the nearly pure compound 8.
Compounds 8 h,k: the reaction mixture was directly chro-
matographed on a silica gel column; the pure compound 8
was recovered proceeding exactly as above described for the
chromatography of compounds 8f,j.
Compounds 8n,o: the solid that separated out (0.25 g) was
recovered by filtration and washed with a little acetone. It
was shown (elemental analysis, IR, and 1H-NMR spectra) to
be 1,2-dibenzoylhydrazine, white needles, m.p.: 239–240 °C,
after crystallization from ethanol (Ref. [12]: m.p.: 238 °C).
The filtrate and washings, after removing of acetone afforded
an oily residue which was subjected to a chromatography on
a silica gel column carried out as in the two last cases, to
yield the pure compound 8.
mixture was shaken in a separatory funnel: the organic layer
was collected and the aqueous one was further extracted twice
with chloroform. The combined organic phase was washed
with water, then dried (anhydrous sodium sulphate), concen-
trated in vacuo to a little volume and finally subjected to chro-
matography on a silica gel column, first eluting with the mix-
ture chloroform–ethyl acetate (1:1). The first eluate collected
gave, after removal of solvents the pure dichloro derivative
12 as a white solid (0.14 g, 16%), which decomposes without
melting after 330 °C after crystallization from acetone/ethyl
acetate. IR (KBr): 1602 w, 1579 w, 1530 w, 1508, 1470, 1447,
1416, 1390 cm–1. 1H-NMR (CDCl3): d 6.98–7.13 and 7.24–
7.38 (2 m, 2H + 2H, H-1,2,3,4), 7.42–7.65 (m, 10H, phenyl
H’s). Anal. C23H14Cl2N6 (C, H, N, Cl).
The elution was completed with ethyl acetate: the fraction
collected gave, after removal of solvent, the pure compound
11 as a white solid (0.57 g, 69%) which decomposes without
melting after 320 °C after crystallization from acetone/ethyl
acetate. IR (KBr): 2947 (9-CHCl), 1610 w, 1600 sh, 1581 w,
1
1530 sh, 1505, 1472, 1449, 1422, 1410 cm–1. H-NMR
(CDCl3): d 6.96 (s, 1H, H-9), 7.03–7.17 and 7.24–7.38 (2 m,
2H + 2H, H-1,2,3,4), 7.43–7.66 (m, 10H, phenyl H’s). Anal.
C23H15ClN6 (C, H, N, Cl).
4.1.9. General procedure for the preparation
of 9-(dialkylamino)derivatives 13a,b
A mixture of 1.5 mmol (0.62 g) of chloro derivative 11,
5 ml of the suitable dialkylamine, and 7 ml of dimethyl sul-
phoxide was heated at 120 °C for 1 h (compound 13b) or for
2 h (compound 13a), while stirring. After cooling, the mix-
ture was poured into ice-cooled water (200 ml) and the result-
ing emulsion was exhaustively extracted with dichlo-
romethane.
Compounds 8e–o were then crystallized from the suitable
solvents. Their data are reported in Table 6.
From combined extracts (washed with water and dried over
anhydrous Na2SO4 then evaporated to dryness in vacuo), an
oily residue was obtained from which compound 13 was
recovered as below described for each case.
4.1.7. 9-[(Dimethylamino)methylene]-6,12-diphenyl-9H-bis
[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine (10)
A mixture of 2 mmol (0.75 g) of 8c [3], 7.0 ml of N,N-
dimethylformamide dimethyl acetal, and 5.0 ml of anhy-
drous pyridine was refluxed (120 °C), with stirring, for 24 h.
The mixture was then evaporated to dryness in vacuo and the
solid residue was taken up in a little acetone and filtered to
give the nearly pure compound 10 · 0.5 H2O (0.55 g, 62%),
pale orange crystals, m.p. 355–357 °C after crystallization
from ethanol. IR (KBr): 3440 br (H2O), 1630 s, 1505, 1472,
4.1.9.1.9-Morpholino-6,12-diphenyl-9H-bis[1,2,4]triazolo-
[4,3-a:3′,4′-d] [1,5]benzodiazepine (13a). The oil obtained
from the reaction carried out with morpholine was subjected
to chromatography on a silica gel column first eluting with
the mixture dichloromethane–petroleum ether–triethylamine
(5:5:1) in order to remove some impurities, then with the mix-
ture dichloromethane–triethylamine (9:1): the eluate col-
lected, after removal of solvents afforded the nearly pure com-
pound 13a · H2O (0.47 g; 65%) as a white solid, m.p. 332–
334°C, after crystallization from ethanol. IR (KBr): 3420 br
(H2O), 1596 w, 1578 w, 1522, 1502, 1473, 1448, 1421,
1
1440, 1419 cm–1. H-NMR (DMSO-d6): d 3.10 [s, 6H,
N(CH3)2], 6.85–6.98 and 7.08–7.22 (2 m, 2H + 2H,
H-1,2,3,4), 7.37–7.62 (m, 11H, = CH- + phenyl H’s). Anal.
C26H21N7 · 0.5 H2O (C, H, N).
1
1402 cm–1. H-NMR (CDCl3): d 2.36 (near t, 4H, morpho-
4.1.8. 9-Chloro-6,12-diphenyl-9H-bis
line N–CH2s), 3.18 (near t, 4H, morpholine O–CH2s), 5.53
(s, 1H, H-9), 6.93–7.06 and 7.21–7.35 (2 m, 2H + 2H,
H-1,2,3,4), 7.38–7.65 (m, 10H, phenyl H’s). Anal. C27H23N7O
· H2O (C, H, N).
[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine (11)
and 9,9-dichloro-6,12-diphenyl-9H-bis [1,2,4]triazolo[4,3-
a:3′,4′-d] [1,5]benzodiazepine (12)
A mixture of 2.0 mmol (0.75 g) of 8c [3], 10.0 mmol
(1.33 g) of N-chlorosuccinimide and 100 ml of chloroform–
carbon tetrachloride (1:1) was refluxed with stirring for 24 h.
After cooling 2 N aqueous NaOH (50 ml) was added and the
4.1.9.2. 9-(4-Methyl-1-piperazinyl)-6,12-diphenyl-9H-bis
[1,2,4]triazolo[4,3-a:3′,4′-d] [1,5]benzodiazepine (13b). The
oil resulting from the reaction carried out with 1-methyl-

124
M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
Table 6
Data of compounds 8e–o
Compound
R
H
R′″
RIV
H
Yield m.p. (°C)
Molecular
formulab
IRc (cm–1
)
1H-NMRd (d, ppm)
(%)
62
(solvent) a
8e
C2H5
345–347 (A)
C
C
C
15H16N6
25H20N6
21H14N8 ·
1545, 1523,
1500, 1460,
1427, 1410.
1.34 (t, 6H, CH2CH3), 2.69–2.92 and
2.96–3.18 (2 m, 2H + 2H, CH2CH3),
3.74 and 4.90 (AB q, J = 16 Hz, 1H + 1H,
9-CH2), 7.52–7.76 (m, 4H, H-1,2,3,4).
3.70 and 4.92 (AB q, J = 16 Hz, 1H + 1H,
9-CH2), 3.97 and 4.26 (AB q, J = 16 Hz,
2H + 2H, CH2C6H5), 6.94–7.56 (m, 14H,
H-1,2,3,4 + phenyl H’s).
8f
H
H
H
H
H
H
30
81
48
73
281–281.5 (B)
333–334 (A)
358–359 (A)
336–338 (A)
1595w, 1580w,
1532, 1499,
1468, 1452,
1430w, 1412.
3395br (H2O),
1603, 1542,
1501, 1465,
1418br.
8g
8h
8i
3.94 and 5.14 (AB q, J = 16 Hz, 1H + 1H,
9-CH2), 7.10–7.20 and 7.42–7.51 (2 m,
2H + 6H, H-1,2,3,4 + pyridyl H-3′,5′),
8.79 (near d, 4H, pyridyl H-2′,6′).
0.33 H2O
CH3 CH3
C
C
14H14N6
1595w, 1527s,br 2.16 (d, 3H, 9-CH3), 2.59 and 2.61 (over-
1509s,br, 1460 , lapped singlets, 6H, 6,12-CH3), 3.93 (q,
1420, 1402.
1H, H-9), 7.47–7.60 and 7.63–7.75 (2 m,
2H + 2H, H-1,2,3,4).
CH3 C2H5
16H18N6
1530, 1501,
1468, 1430,
1405.
1.17 (t, 6H, CH2CH3), 1.86 (d, 3H,
9-CH3), 2.65–2.86 and 2.94–3.17 (2 m,
2H + 2H, CH2CH3), 4.20 (q, 1H, H-9)
7.65–7.78 and 7.85–7.96 (m, 2H + 2H,
H-1,2,3,4).
8j
8k
8l
CH3
CH3
CH3
H
H
H
20
72
75
284–286 (B)
367–369 (C)
255.5–257 (A)
C
C
C
26H22N6
1602w, 1590w,
1528, 1502,
1468, 1458,
1431, 1412.
1600, 1570w,
1530, 1518sh,
1501, 1468,
1430, 1417.
1615sh, 1602,
1583, 1522,
1501, 1488,
1460br, 1438,
1420.
2.17 (d, 3H, 9-CH3), 3.87 (q, 1H, H-9),
3.93 and 4.27 (AB q, J = 16 Hz,2H + 2H,
CH2C6H5), 7.02–7.53 (m, 14H,
H-1,2,3,4 + phenyl H’s).
24H16Cl2N6
2.26 (d, 3H, 9-CH3), 4.09 (q, 1H, H-9),
7.04–7.15 and 7.32–7.52 (2 m, 2H+10H,
H-1,2,3,4+p-chlorophenyl H’s).
26H22N6O2
2.27 (d, 3H, 9-CH3), 3.79 (s, 6H, OCH3),
4.11 (q, 1H, H-9), 6.97–7.16 and 7.24–
7.40 (2 m, 8H + 4H, H-1,2,3,4 + m-
methoxyphenyl H’s).
8m
CH3
H
82
354–355.5 (A)
C
22H16N8 ·
3405br (H2O),
1608, 1560,
1530, 1501,
1470, 1455 sh,
1427.
2.28 (d, 3H, 9-CH3), 4.11 (q, 1H, H-9),
7.09–7.20 and 7.37–7.57 (2 m, 2H + 6H,
H-1,2,3,4 + pyridyl H-3′,5′), 8.77 (near d,
4H, pyridyl H-2′,6′).
1.75 H2O
8n
8o
CH3
CH3
Cl
15
47
315–317 (D)
331–333 (A)
C
C
24H17ClN6
1602w, 1585w,
1525, 1501,
1473, 1445,
1420.
2.27 (d, 3H, 9-CH3), 4.10 (q, 1H, H-9),
6.98–7.06 and 7.22–7.32 (2 m, 1H + 2H,
H-1,3,4), 7.44–7.60 (m, 10H, phenyl H’s).
CF3
25H17F3N6
1628w, 1600w,
1582w, 1528,
1481, 1450,
1423.
2.27 (d, 3H, 9-CH3), 4.12 (q, 1H, H-9),
7.13–7.31 (m, 3H, H-1,3,4), 7.40–7.64
(m, 10H, phenyl H’s).
a Crystallization solvent: A = ethanol, B = methanol, C = ethanol/dichloromethane, D = ethyl acetate/isopropyl ether.
b Anal. C,H,N. (C, H, N, Cl for 8k,n).
c In KBr pellets. Abbreviations: w = weak, br = broad, s = strong, sh = shoulder.
d In CDCl3 solutions, except for 8i (DMSO-d6). Abbreviations: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.

M. Di Braccio et al. / Il Farmaco 60 (2005) 113–125
125
piperazine was chromatographed on a silica gel column, first
eluting with ethyl acetate in order to remove a little amount
(0.04 g) of the starting compound 11, then with the mixture
dichloromethane–triethylamine (9:1): this eluate afforded
0.24 g (42%) of compound 8c (deriving from a dehalogena-
tion of 11) and which was identified by comparison (m.p.,
TLC and IR) with an authentical sample [3]. Finally, the frac-
tion eluted with the mixture chloroform–methanol (9:1) gave,
after removal of solvents, the pure compound 13b · H2O
(0.17 g, 23%) as white crystals melting at 320–322 °C, after
crystallization from ethanol. IR (KBr): 3420 br (H2O), 1600 w,
1580 w, 1522, 1501, 1470, 1448, 1420, 1403 cm–1. 1H-NMR
(CDCl3): d 1.60–2.15 and 2.30–2.47 (2 m, 4H + 4H, pipera-
zine CH2s), 2.04 (s, 3H, NCH3), 5.53 (s, 1H, H-9), 6.92–
7.05 and 7.21–7.33 (2 m, 2H + 2H, H-1,2,3,4), 7.40–7.67 (m,
10H, phenyl H’s). Anal. C28H26N8 · H2O (C, H, N).
cells. The HIV-1 stock solution had a titre of 1.0 × 107 50%
cell culture infectious dose (CCID50)/ml.
4.2.4. Antiviral assays
Activity of compounds against HIV-1 was based on inhi-
bition of virus-induced cytopathogenicity in MT-4 cells
acutely infected at a multiplicity of infection of 0.01. Cyto-
toxicity of test compounds was evaluated in parallel with their
antiviral activity and was based on the viability of mock-
infected cells, as monitored by the MTT method [13].
Acknowledgements
Authors wish to thank Prof. Paola Fossa (Dipartimento di
Scienze Farmaceutiche, Università di Genova) for her valu-
able help in conformational studies; financial support from
Italian MIUR is gratefully acknowledged.
4.1.10. 9-Methyl-9H-bis [1,2,4]triazolo[4,3-a:3′,4′-d]
[1,5]benzodiazepine-6,12(7H,11H)-dione (14)
A mixture of 4 mmol (1.00 g) of 20d, 16.0 mmol (1.67 g)
of ethyl carbazate and 10 ml of Dowtherm A was stirred at
200 °C for 2 h. After cooling a white solid separated out: it
was taken up in a little ethyl acetate and recovered by filtra-
tion, then subjected to chromatography on a silica gel col-
umn, eluting first with the mixture dichloromethane–ethyl
acetate (1:1) in order to remove some impurities. The subse-
quent elution with the mixture acetone–ethyl acetate (2:1)
afforded the pure compound 14 · 0.75 H2O (0.50 g, 44%);
white crystals (from ethanol) which decompose without melt-
ing after 380 °C. IR (KBr): 3420 br (H2O), 3277 and 3069
(NH), 1695 s, br (CO), 1604, 1586, 1513, 1465, 1429 cm–1.
1H-NMR (DMSO-d6): d 1.54 (d, 3H, 9-CH3), 4.07 (q, 1H,
H-9), 7.42–7.67 and 7.77–8.02 (2 m, 2H + 2H, H-1,2,3,4),
12.10 (broad s, 2H, NHCO; disappeared with D2O). Anal.
C12H10N6 O2 · 0.75 H2O (C, H, N).
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4.2.2. Cells
Cell lines were purchased from American Type Culture
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4.2.3. Virus
Human immunodeficiency virus type 1 (HIV-1) was
obtained from supernatants of persistently infected H9/IIIB