1,5-Benzodiazepines. Part XII. Synthesis and biological evaluation of tricyclic and tetracyclic 1,5-benzodiazepine derivatives as nevirapine analogues

Article abstract of DOI:10.1016/S0223-5234(01)01283-1

A number of properly substituted 5H-pyrimido[4,5-b][1,5]benzodiazepines (2) and pyrazolo[3,4-b][1,5]benzodiazepines (3 and 4), as well as compounds 5-7, which are derivatives of new tetracyclic systems, were prepared as nevirapine analogues through multistep synthetic routes. The cytotoxic and anti-HIV-1 properties of compounds 2-7 were evaluated in cell-based assays, together with their inhibitory activity against the HIV-1 recombinant reverse transcriptase (rRT) in enzyme assays. The modifications introduced into nevirapine heterocyclic skeleton proved to have a negative effect for the anti-HIV-1 activity. It is worth noting that some of the new derivatives proved to be cytotoxic in the low micromolar range.

Full text of DOI:10.1016/S0223-5234(01)01283-1

Eur. J. Med. Chem. 36 (2001) 935–949
´
© 2001 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(01)01283-1/SCO
Short communication
1,5-Benzodiazepines. Part XII. Synthesis and biological evaluation of tricyclic
and tetracyclic 1,5-benzodiazepine derivatives as nevirapine analoguesꢀ
Mario Di Braccioa, Giancarlo Grossia, Giorgio Romaa,*, Laura Vargiub, Massimo Murab,
Maria Elena Marongiub
aDipartimento di Scienze Farmaceutiche, Universita` di Genova, Viale Benedetto XV, 3, I-16132 Genoa, Italy
bDipartimento di Biologia Sperimentale, Sezione di Microbiologia, Universita` di Cagliari, Cittadella Universitaria, I-09042
Monserrato, Italy
Received 16 July 2001; revised 14 September 2001; accepted 27 September 2001
Abstract – A number of properly substituted 5H-pyrimido[4,5-b][1,5]benzodiazepines (2) and pyrazolo[3,4-b][1,5]benzodiazepines (3
and 4), as well as compounds 5–7, which are derivatives of new tetracyclic systems, were prepared as nevirapine analogues through
multistep synthetic routes. The cytotoxic and anti-HIV-1 properties of compounds 2–7 were evaluated in cell-based assays, together
with their inhibitory activity against the HIV-1 recombinant reverse transcriptase (rRT) in enzyme assays. The modifications
introduced into nevirapine heterocyclic skeleton proved to have a negative effect for the anti-HIV-1 activity. It is worth noting that
´
some of the new derivatives proved to be cytotoxic in the low micromolar range. © 2001 Editions scientifiques et me´dicales Elsevier
SAS
1,5-benzodiazepine fused derivatives / NNRTIs
1. Introduction
new broad spectrum HIV-1 RT inhibitors, such as
compound 1b and other its 8-[(arylthio)methyl]-2-halo
and 8-[(aryloxy)methyl]-2-halo analogues [2].
Nevirapine (1a) [1] is one of the most active non-
nucleoside HIV-1 reverse transcriptase inhibitors
Therefore, we considered it interesting to investi-
(NNRTIs) and was the first to reach regulatory ap-
gate whether also suitable changes in the nevirapine
proval. It acts as an allosteric inhibitor and is at
heterocyclic skeleton could result in potent anti-HIV-
present used for treatment of HIV-1 infections, in
1 activity and, possibly, inhibition of clinically rele-
combination with nucleoside reverse transcriptase in-
vant NNRTI resistant mutants.
hibitors (NRTIs) and HIV-1 protease inhibitors, in
order to avoid the appearance of the drug resistance
observed in nevirapine monotherapy, resulting from
the enzyme mutation. Recently, proper modifications
in the substitution pattern of nevirapine resulted in
Thus, as a first step in this investigation, we have
synthesised
a
number of 5H-pyrimido[4,5-b]-
[1,5]benzodiazepin-5-ones (2) and pyrazolo[3,4-b]-
[1,5]benzodiazepin-4-ones (3 and 4) as well as tetra-
cyclic derivatives 5, 6, and 7b, as analogues of nevi-
rapine and its very active tetracyclic derivatives [3],
respectively (figure 1).
Here we report the synthesis of the above com-
pounds and their effect against the HIV-1 multiplica-
tion in acutely infected MT-4 cells and the HIV-1 rRT
in enzyme assays.
ꢀ Preliminary results were presented at the Italian–Hungar-
ian–Polish Joint Meeting on Medicinal Chemistry in Giardini
Naxos-Taormina (Catania), Italy, 1999 (Abstract PA 36, p.
122).
* Correspondence and reprints:
E-mail address: roma@unige.it (G. Roma).

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
Compound 2a was treated with Lawesson’s reagent
2. Chemistry
to give thione 10, from which the diethyl derivative
11b was obtained by treatment with KOH and ethyl
iodide.
4-(Dialkylamino)-1,3-dihydro-2H-1,5-benzodia-
zepin-2-ones 8a, c and the derivative 9a, previously
described by us [4, 5], were used as starting com-
pounds to prepare tricyclic derivatives 2a–n, 3a–i and
4a, b.
Thus, treatment of 8a [4] with KOH and methyl
iodide gave the methyl derivative 8b. Reaction of
compounds 8b and 8c [4] with N,N-dimethylfor-
mamide in the presence of PCl5 afforded then the
3-[(dimethylamino)methylene]derivatives 9b and 9c,
respectively. Cyclocondensation of compound 9a [5]
or 9b with proper dinucleophilic reagents (H+) gave
the substituted 6,11-dihydro-5H-pyrimido [4,5-
b][1,5]benzodiazepin-5-ones 2a, b, f, h, k. Treatment
of these compounds with KOH and suitable alkyl
iodides led to the desired derivatives 2c–e, g, i, j, l–n
(see figure 2 and table I).
Analogously, cyclocondensation of phenylhy-
drazine with 9b in refluxing butanol (H+) afforded
only the phenyl derivative 3d, whereas methylhy-
drazine reacted with 9b or 9c under the same condi-
tions to give both compounds 3a or 3g and their
isomers 4a (in very low yield) or 4b, respectively, as
we previously observed in a similar case [5]. 10-
Alkylderivatives 3b, c, e, f, h and i were then easily
obtained from the substituted 5,10-dihydropyra-
zolo[3,4-b][1,5]benzodiazepin-4(1H)-ones 3a, d and g
(KOH and alkyl iodide) (figure 2, table I).
Finally, the reaction of the (ethylthio) derivative
11b with propargylamine or acetohydrazyde, in the
presence of p-toluenesulphonic acid afforded the 9-
ethyl-3-methyl substituted 9H-imidazo[1,2-a]pyri-
mido[5,4-c][1,5]benzodiazepine (5) or 9H-pyrim-
ido[5,4-c][1,2,4]triazolo[4,3-a][1,5]benzodiazepine (6),
respectively. On the other hand, the reaction of
11b with hydroxylamine (NH2OH·HCl+NaHCO3)
yielded the (hydroxyamino)derivative 12b (figure 3).
Two different synthetic pathways were tried for
compound 7b. The first one, based on cyclocondensa-
tion of the corresponding (hydroxyamino) derivative
12b with acetaldehyde dimethylacetal to give com-
pound 7b, was discarded due to the very low yield. A
better result was reached through the procedure
herein described, starting from the (ethylthio) deriva-
tive 11a which was prepared by treating thione 10
with anhydrous K2CO3 and ethyl iodide. By heating a
mixture of 11a, NH2OH·HCl and NaHCO3 in reflux-
ing methanol the (hydroxyamino) derivative 12a was
obtained [6], which was in turn treated with acetalde-
hyde dimethylacetal in the presence of p-toluenesul-
phonic acid to yield 3-methyl-3H,9H-[1,2,4]-
oxadiazolo[4,3 - a]pyrimido[5,4 - c][1,5]benzodiazepine
(7a). Finally, treatment of 7a with KOH and ethyl
iodide afforded the desired 9-ethyl derivative 7b
(figure 3).
Figure 1. Structures of nevirapine (1a), its analogue 1b and compounds 2–7.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
937
Figure 2. Synthetic route to compounds 2a–n, and 3a–i, 4a, b.

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
Table I. Structures of compounds 2a–n and 3a–i.
was reacted with N,N-dimethylformamide in the pres-
ence of PCl5 to give unambiguously compound 4e
which is the isomer of 3d. Treatment of compound 4e
with KOH and the proper alkyl iodide gave the
10-alkyl derivatives 4f or 4g (figure 4).
The structures attributed to the compounds de-
scribed in this paper are consistent with the results of
1
elemental analyses, IR and H-NMR spectral data
(see Section 4 and table III).
Compound
R
R%
R¦
1
Concerning in particular the H-NMR spectra, the
multiplicities shown by the 3-CH2 signal of compound
8b, the NꢀCH2 signals of 2d, g, i, l, n, 3b, e, h, 5, 6, 7b,
and by the HC(CH3)2 signals of 2e, j, m and 3c, f, i
depend on non-equivalence of the two geminal hydro-
gens (CH2) or methyl groups [HC(CH3)2], respec-
tively, as we reported for previously described
1,5-benzodiazepine derivatives [7–10].
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
C2H5
CH3
CH3
CH3
CH3
CH3
CH3
C6H5
C6H5
C6H5
CH3
C2H5
i-C3H7
H
C2H5
H
C2H5
i-C3H7
H
C2H5
i-C3H7
C2H5
H
C2H5
i-C3H7
H
C2H5
i-C3H7
H
H
CH3
CH3
C6H5
C6H5
C6H5
SCH3
SCH3
SCH3
H
CH3
CH3
CH3
C6H5
C6H5
C6H5
CH3
CH3
CH3
1
Furthermore, we can point out that the H-NMR
2j
2k
2l
spectra of 9b and 9c clearly confirm the structure
attributed to these compounds, as we previously eluci-
dated for their structural analogues [5, 11]. In this
connection, the singlet corresponding to the N(CH3)2
group of the 3-[(dimethylamino)methylene] sub-
stituent is particularly significant, supporting the free
rotation of this group.
Compounds 5–7 are derivatives of novel hetero-
cyclic systems.
Intermediates 2a [12] and 2b, h [13] were reported
previously in the literature, but obtained through a
different procedure.
2m
2n
3a
3b
3c
3d
3e
3f
3g
3h
3i
C2H5
i-C3H7
The structures attributed to compounds 3a, g and
their corresponding isomers 4a, b have been chemi-
cally confirmed (see figure 4). Actually, in the case of
3a and 4a, taking the previously described isomers 3k
and 4c [5] as reference compounds, treatment of both
3a and 3k with KOH and methyl iodide gave the
same trimethyl derivative 3j, thus confirming the
structure of 3a. Furthermore, the methylation of 4c
afforded the trimethyl derivative 4d, isomer of 3j,
along with the dimethyl derivative 4a, the isomer of
3a.
On the other hand, reaction of compound 8d [4]
with N-methyl formic hydrazide in the presence of
p-toluenesulphonic acid afforded compound 4b, so
confirming both its structure and, consequently, the
one attributed to its isomer 3g.
3. Biological results and conclusions
The title compounds were tested for cytotoxicity
and anti-HIV-1 activity in vitro. Most of them were
also tested in enzyme assays against HIV-1 recombi-
nant reverse transcriptase (rRT).
Table II summarizes the results of biological evalu-
ations, from which the following conclusions can be
drawn.
The skeletal differences between the tricyclic system
of nevirapine and the corresponding ones of com-
pounds 2–7 proved to have a negative effect for the
anti-HIV-1 activity of the latter compounds. In the
case of compounds 2 and 5–7, this effect can be
reasonably attributed to an unfavourable electronic
configuration of their pyrimido[4,5-b][1,5]benzo-
diazepine ring system (compounds 2) or ring system
moiety (compounds 5–7). Nevertheless, it can be
Finally, in order to confirm the structure of com-
pound 3d, the 4-(2-phenylhydrazino) derivative 13

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
939
noted that 2d, 2e and 2n, whose substitution patterns
are closely related to that of nevirapine, are the only
three compounds showing some anti-HIV-1 activity.
On the other hand, the modifications now intro-
duced into the nevirapine heterocyclic skeleton led to
compounds with increased cytotoxic activity. Some of
them, in particular 3c, 3f and 7b, proved cytotoxic in
the low micromolar range (CC50 =4–7 mM).
When tested in vitro against the rRT, the title
compounds resulted inactive with the sole exceptions
of 2d, 2n and, in particular, 3e which showed some
inhibitory effect.
ter, using (CH3)4Si as an internal reference (l=0), and
chemical shifts (l) are reported in ppm. Analyses of all
new compounds, indicated by the symbols of the ele-
ments, were within 0.4% of the theoretical values and
were performed by the Laboratorio di Microanalisi,
Dipartimento di Scienze Farmaceutiche, 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
Carlo Erba silica gel (0.05–0.20 mm) or Carlo Erba
neutral aluminium oxide (Brockmann activity I).
4.1.1. 4-(Dimethylamino)-1,3-dihydro-1-methyl-2H-1,5-
benzodiazepin-2-one (8b)
4. Experimental protocols
A mixture of 20.0 mmol (4.06 g) of compound 8a [4],
80.0 mmol (4.49 g) of finely powdered KOH and 80 mL
of dry acetone was refluxed for 10 min, then the solution
of 30.0 mmol (4.26 g) of iodomethane in 15 mL of dry
acetone was added and the mixture was further refluxed
for 30 min, with stirring. The solvent was then removed
in vacuo, the residue was partitioned between water and
dichloromethane, and the aqueous phase was extracted
4.1. Chemistry
Melting points were determined using a Fisher–Johns
(electrothermal when above 300 °C) apparatus and are
uncorrected. IR spectra were recorded in a Perkin–
Elmer 398 spectrophotometer. 1H-NMR spectra were
recorded in a Varian Gemini 200 (200 MHz) spectrome-
Figure 3. Synthetic route to compounds 5, 6, 7a, b.

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
Figure 4. Structure confirmation for compounds 3a, d, g, 4a, b and synthesis of compounds 4e–g.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
941
Table II. In vitro biological activities of compounds 2c–e, g, i,
j, l–n, 3b, c, e, f, h, i, 4f, g and 5, 6, 7b.
mamide, with stirring. The resulting solution was stirred
at room temperature for 20 h, then poured onto ice-wa-
ter. After addition of 6 N aqueous NaOH (until alka-
line) and 150 mL of dichloromethane, the mixture was
vigorously stirred at room temperature for 30 min. The
organic solution was collected and the aqueous phase
was further extracted several more times with
dichloromethane. The combined extracts, after drying
and removal of solvent, afforded a thick oil from which,
by addition of a little ethyl ether, the nearly pure
compounds 9b or 9c, respectively, separated out as white
solids which were recrystallised from the proper solvent.
a
b
d
Compound CC50
MT-4
EC50
SI c
IC50
HIV-1
HIV-1 rRT
2c
2d
2e
2g
2i
\200
\200
\200
\200
40.4
\200
139
61
\200
91
4.6
43
5
50
34.4
]200
\200
14.5
17.6
6.9
\200
51
117
–
\4
\1.7
–
–
–
–
–
\30
\30 (26%)
\30
\200
\40.4
\200
\139
\61
76
\91
\4.6
\43
\5
\50
\34.4
\200
\200
\14.5
\17.6
\6.9
0.2
\30
\30
2j
\30
2l
\30
2m
2n
3b
3c
3e
3f
3h
3i
\30
\2.6
]30 (39%)
–
–
–
–
–
–
–
–
–
–
–
\30
\30
4.1.2.1. Compound 9b
=30 (45%)
Yield: 2.87 g (88%) from 2.61 g of 8b; m.p. 151–
152 °C, after crystallisation from ethyl acetate–ethyl
ether. IR (CHCl3, cm−1): 1640 (CO), 1605 shoulder,
\30
\30
\30
1590, 1560. 1H-NMR (CDCl3):
l
2.80 [s, 6H,
4f
4g
5
6
7b
–
–
–
–
–
ꢁCHꢀN(CH3)2], 3.00 and 3.14 [2s, 3H+3H, 4-N(CH3)2],
3.27 (s, 3H, 1-CH3), 6.82 [s, 1H, ꢁCHꢀN(CH3)2], 6.95–
7.40 (m, 4H, H-6,7,8,9). Anal. C15H20N4O (C, H, N).
Nevirapine \200
\1000 0.6
4.1.2.2. Compound 9c
a Compound concentration (mM) required to reduce the viabil-
ity of mock-infected MT-4 cells by 50%, as determined by the
MTT method.
Yield: 4.00 g (92%) from 3.69 g of 8c [4]; m.p.
158–159 °C (from ethyl acetate–petroleum ether). IR
1
(CHCl3, cm−1): 1658 (CO), 1616, 1600, 1560. H-NMR
b Compound concentration (mM) required to achieve 50%
protection of MT-4 cells from the HIV-1 induced cytopatho-
genicity, as determined by the MTT method.
(CDCl3): l 1.10 and 1.29 (2t, 3H+3H, CH2CH3), 2.82 [s,
6H, ꢁCHꢀN(CH3)2], 3.13–3.53 (m, 3H, 3H of CH2CH3),
3.93 (m, 1H, 1H of CH2CH3), 6.63–6.85 and 6.97–7.44
(2m, 2H+7H, H-6,7,8,9+phenyl Hs), 6.73 [s, 1H,
ꢁCHꢀN(CH3)2]. Anal. C22H26N4O (C, H, N).
c Selectivity index, CC50/EC50 ratio.
d Compound concentration (mM) required to inhibit the HIV-1
rRT activity by 50%. Data in parentheses represent the per-
centage of inhibition of enzyme activity at 30 mM.
4.1.3. General procedure for compounds 2a, b, f, h and
k
several more times with the same solvent. The combined
extracts were dried (anhydrous Na2SO4), then evapo-
rated to dryness under reduced pressure to give a thick
oil from which, after addition of some ethyl ether and
standing, 3.74 g (86%) of pure 8b separated out; white
crystals, m.p. 122–123 °C, after crystallisation from
ethyl acetate–petroleum ether. IR (CHCl3, cm−1): 1660
A mixture of 6.0 mmol of 9a [5] (1.55 g) or 9b (1.63
g), an excess of the proper amidine [12.0 mmol of
formamidine acetate (1.25 g) or acetamidine acetate
(1.42 g), 9.0 mmol (1.41 g) of benzamidine hydrochlo-
ride plus 1.5 mL of triethylamine, or 6.0 mmol (1.67 g)
of 2-methylisothiourea sulphate plus 2.5 mL of tri-
ethylamine], and 20 mL of the suitable solvent was
stirred at the temperature and for the time reported
below for each case.
1
(CO), 1610, 1581. H-NMR (CDCl3): l 2.89 and 3.71
(AB system, J=12 Hz, 2H, 3-CH2), 3.18 [s, 6H,
N(CH3)2], 3.36 (s, 3H, 1-CH3), 6.98–7.22 (m, 4H, H-
6,7,8,9). Anal. C12H15N3O (C, H, N).
After cooling, compound 2 was recovered as de-
scribed below for each case.
4.1.2. General procedure for 1-substituted 4-(dialkyl-
amino)-3-[(dimethylamino)methylene]-1,3-dihydro-
2H-1,5-benzodiazepin-2-ones (9b, c)
4.1.3.1. 6,11-Dihydro-5H-pyrimido[4,5-b][1,5]benzo-
diazepin-5-one (2a)
The suspension finally obtained from the reaction of
PCl5 (3.60 g) was slowly added to the solution of 12.0
mmol of 8b or 8c [4] in 42 mL of N,N-dimethylfor-
9a [5] with formamidine acetate (Dowtherm A, 160 °C,

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M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
1 h) was diluted with a little acetone and filtered to
recover 0.61 g of pure compound 2a as a white solid.
matographed on a silica gel column eluting with
dichloromethane–ethyl acetate (4:1) until pure com-
pound 2k was completely recovered (0.26 g, white solid).
Data for compounds 2a, b, f, h, k are reported in table
III.
4.1.3.2. 6,11-Dihydro-6-methyl-5H-pyrimido[4,5-b][1,5]-
benzodiazepin-5-one (2b)
The mixture obtained from the reaction of 9b with
formamidine acetate (Dowtherm A, 160 °C, 1 h) was
treated with 10% aqueous Na2CO3, then exhaustively
extracted with dichloromethane. The combined extracts
were dried, then evaporated to give an oil which was
subjected to column chromatography (silica gel), eluting
first with dichlorometane to remove Dowtherm A and
some impurities, then with ethyl acetate to collect, after
removal of solvent, 0.54 g of pure compound 2b as a
white solid.
4.1.4. General procedure for 6,11-dialkyl-6,11-dihydro-
5H-pyrimido[4,5-b][1,5]benzodiazepin-5-ones (2c–e, g, i,
j, l–n)
A mixture of 1.5 mmol of 2b (0.34 g), 2f (0.36 g), 2h
(0.45 g), or 2k (0.41 g), 9.0 mmol (0.50 g) of finely
powdered KOH and 50 mL of dry acetone was refluxed
for 10 min, with stirring. The solution of 9.0 mmol of
iodoethane (1.40 g) or 2-iodopropane (1.53 g) in 15 mL
of dry acetone was then added and the mixture was
further refluxed for 1 h (compounds 2d, g, i, l) or 2 h
(compounds 2e, j, m), while stirring.
In the case of reactions carried out with 2a (1.5 mmol,
0.32 g), 18.0 mmol (1.0 g) of KOH and 18.0 mmol of
iodomethane (2.55 g) (preparation of 2c) or iodoethane
(2.81 g) (preparation of 2n) were used and the reaction
mixture was refluxed for 1 h.
The solvent was then removed in vacuo and the
residue was partitioned between water and
dichloromethane. The aqueous phase was extracted sev-
eral more times with dichloromethane. The combined
organic phases, after drying and removal of solvent,
afforded an oil from which the pure dialkylated com-
pound 2 was recovered as a white solid after a suitable
column chromatography [aluminium oxide, ethyl acetate
as eluent, in the case of compounds 2c, g, i, j, l–n; silica
gel, dichloromethane–petroleum ether–triethylamine
(10:10:1) as eluent, in the case of compounds 2d, e].
Data for compounds 2c–e, g, i, j, l–n are reported in
table III.
4.1.3.3. 6,11-Dihydro-2,6-dimethyl-5H-pyrimido[4,5-b]-
[1,5]benzodiazepin-5-one (2f)
After the reaction of 9b with acetamidine acetate
(Dowtherm A, 160 °C, 2 h) was carried out, the result-
ing mixture was subjected to the same procedure de-
scribed above for the preparation of compound 2b. The
oil ultimately obtained was chromatographed on a sil-
ica gel column, eluting first with dichloromethane
until Dowtherm A was completely removed, then
with dichloromethane–petroleum ether–triethylamine
(10:10:1), discarding the first fractions containing impu-
rities. Further exhaustive elution with the same mixture
afforded the nearly pure compound 2f (0.25 g), as a
white solid.
4.1.3.4. 6,11-Dihydro-6-methyl-2-phenyl-5H-pyrimido-
[4,5-b][1,5]benzodiazepin-5-one (2h)
The mixture finally obtained from the reaction of 9b
with benzamidine (refluxing butanol, 24 h), was evapo-
rated to dryness under reduced pressure to give an oily
residue that was treated, then vigorously stirred (room
temperature, 30 min) with 0.5 N aqueous NaOH (100
mL) and (1:1) ethyl ether–petroleum ether (100 mL).
This way the nearly pure compound 2h (0.36 g) sepa-
rated out as a yellowish solid.
4.1.5. General procedure for compounds 3a, 4a, 3g, 4b
and 3d
A mixture of 6.0 mmol of 9b (1.63 g) or 9c (2.17 g),
9.0 mmol of methylhydrazine (0.41 g) or phenylhy-
drazine (0.97 g), 3.0 mL of glacial acetic acid and 30 mL
of n-butanol was refluxed for 4 h, while stirring. The
solvent was then removed in vacuo and the residue was
partitioned between 10% aqueous Na2CO3 and
dichloromethane. The organic layer was collected and
the aqueous phase was extracted several more times with
dichloromethane. The combined extracts were dried,
then evaporated to dryness under reduced pressure to
give a residue from which compounds 3a, 4a, or 3g, 4b,
or 3d were recovered as described below.
4.1.3.5. 6,11-Dihydro-6-methyl-2-(methylthio)-5H-
pyrimido[4,5-b][1,5]benzodiazepin-5-one (2k)
The solution resulting from the reaction of compound
9b with 2-methylisothiourea (refluxing dry pyridine, 24
h) was poured into cold water and the mixture was
exhaustively extracted with dichloromethane. The com-
bined extracts were dried and the solvents removed in
vacuo to give an oily residue which was chro-

Table III. Data of compounds 2a–n, 3a–i and 4a, b.
Compound Yield M.p. (°C)
Molecular
formula b
IR c (cm-1)
1H-NMR d (l, ppm)
(%)
(solvent) a
2a
48
340 dec. e (A) C11H8N4O
3260 (NH), 3190 (NHCO),
6.78–7.32 (m, 4H, H-7,8,9,10), 8.67 and 8.82 (2s, 1H+1H,
1680s (CO), 1620, 1595, 1570w, H-2, 4), 9.72 f (broad s, 1H, NH), 10.00 f (broad s, 1H,
1530w, 1510
3370 (NH), 1640s (CO), 1606w, 3.40 (s, 3H, 6-CH3), 7.08–7.48 (m, 4H, H-7,8,9,10), 8.73
1580, 1565sh, 1495br
and 8.85 (2s, 1H+1H, H-2,4), 9.74 f (broad s, 1H, NH)
1640s (CO), 1595, 1571, 1540w, 3.49 and 3.51 (2s, 3H+3H, 6-CH3+11-CH3), 7.15–7.31 (m,
1502 4H, H-7,8,9,10), 8.81 and 8.94 (2s, 1H+1H, H-2,4)
1640s (CO), 1596, 1570, 1540w, 1.28 (t, 3H, CH2CH3), 3.52 (s, 3H, 6-CH3), 3.54–3.84 (m,
NH)
2b
2c
2d
40
58
58
236–237 e (B)
145–146 (C)
150–151 (C)
C12H10N4O
C13H12N4O
C14H14N4O
1501
1H, 1H of CH2CH3), 4.20–4.56 (m, 1H, 1H of CH2CH3),
7.02–7.37 (m, 4H, H-7,8,9,10), 8.79 and 8.89 (2s, 1H+1H,
H-2,4)
2e
58
142–143 (C)
C15H16N4O
1640s (CO), 1596, 1570, 1533w, 1.49 and 1.56 [2d, 3H+3H, CH(CH3)2], 3.52 (s, 3H,
1500
6-CH3), 4.52 [m, 1H, CH(CH3)2], 7.15–7.37 (m, 4H,
H-7,8,9,10), 8.80 and 8.88 (2s, 1H+1H, H-2,4)
2f
17
47
223–224 (D)
157–158 (C)
C13H12N4O
C15H16N4O
3240 (NH), 1650s (CO), 1613w, 2.50 (s, 3H, 2-CH3), 3.38 (s, 3H, 6-CH3), 7.00–7.47 (m, 4H,
1591, 1563, 1514br
H-7,8,9,10), 8.78 (s, 1H, H-4), 9.70 f (broad s, 1H, NH)
1640s (CO), 1595, 1575, 1540w, 1.28 (t, 3H, CH2CH3), 2.62 (s, 3H, 2-CH3), 3.51 (s, 3H,
2g
1501
6-CH3), 3.54–3.84 (m, 1H, 1H of CH2CH3), 4.25–4.57 (m,
1H, 1H of CH2CH3), 7.12–7.25 (m, 4H, H-7,8,9,10), 8.83 (s,
1H, H-4)
2h
2i
20
65
233–234 e (D) C18H14N4O
3375 (NH), 1640s (CO), 1608w
1584s, 1548, 1500br
3.52 (s, 3H, 6-CH3), 6.71–7.70 (m, 8H, H-7,8,9,10+phenyl
H-3%,4%,5%+NH; 7H after treatment with D2O), 8.28–8.64
(m, 2H, phenyl H-2%,6%), 9.17 (s, 1H, H-4)
128–129 (C)
C20H18N4O
1638s (CO), 1592w, 1566s,
1530w, 1500
1.38 (t, 3H, CH2CH3), 3.54 (s, 3H, 6-CH3), 3.67–4.08 (m,
1H, 1H of CH2CH3), 4.40–4.70 (m, 1H, 1H of CH2CH3),
7.16–7.33 (m, 4H, H-7,8,9,10), 7.45–7.58 (m, 3H, phenyl
H-3%,4%,5%), 8.40–8.53 (m, 2H, phenyl H-2%,6%), 9.02(s, 1H,
H-4)
2j
85
162–163 (C)
C21H20N4O
1640s (CO), 1593w, 1566s,
1528w, 1501
1.61 and 1.65 [2d, 3H+3H, CH(CH3)2], 3.52 (s, 3H,
6-CH3), 4.63 [m, 1H, CH(CH3)2], 7.12–7.41(m, 4H,
H-7,8,9,10), 7.46–7.55 (m, 3H, phenyl H-3%,4%,5%), 8.40–8.52
(m, 2H, phenyl H-2%,6%), 8.98 (s, 1H, H-4)
2k
2l
16
87
206–206.5 (B) C13H12N4OS 3270 (NH), 1640 (CO), 1600br, 2.52 (s, 3H, SCH3), 3.32 (s, 3H, 6-CH3), 7.09–7.35 (m, 4H,
1582, 1540, 1510
C15H16N4OS 1638s (CO), 1594, 1565s, 1525,
1500
H-7,8,9,10), 8.65 (s, 1H, H-4), 9.74 f (s, 1H, NH)
1.29 (t, 3H, CH2CH3), 2.57 (s, 3H, SCH3), 3.50 (s, 3H,
6-CH3), 3.57–3.87 (m, 1H, 1H of CH2CH3), 4.22–4.52 (m,
1H, 1H of CH2CH3), 7.10–7.25 (m, 4H, H-7,8,9,10), 8.73 (s,
1H, H-4)
135–136 (E)

Table III. (Continued)
Compound Yield M.p. (°C)
Molecular
formula b
IR c (cm-1)
1H-NMR d (l, ppm)
(%)
(solvent) a
2m
2n
68
142–142.5 (E) C16H18N4OS 1640s (CO), 1592w, 1560s,
1.48 and 1.57 [2d, 3H+3H, CH(CH3)2], 2.57 (s, 3H, SCH3),
3.47 (s, 3H, 6-CH3), 4.50 [m, 1H, CH(CH3)2], 7.12–7.37 (m,
4H, H-7,8,9,10), 8.69 (s, 1H, H-4)
1520w, 1500
38
83–84 (F)
C15H16N4O
1640s (CO), 1597, 1571, 1542w, 1.23 and 1.27 (2t, 3H+3H, 6-CH2CH3+11-CH2CH3),
1501
3.60–3.89 (m, 2H, 1H of 6-CH2CH3+1H of 11-CH2CH3),
4.25–4.63(m, 2H, 1H of 6-CH2CH3+1H of 11-CH2CH3),
7.17–7.33 (m, 4H, H-7,8,9,10), 8.79 and 8.88 (2s, 1H+1H,
H-2,4)
3a
3b
61
70
218–220 (G)
129–130 (C)
C12H12N4O
C14H16N4O
3280 (NH), 1620s, br (CO),
1566br, 1502
1630s, br, (CO), 1600w, 1550w, 1.13 (t, 3H, CH2CH3), 3.46 (q, 2H, CH2CH3), 3.47 (s, 3H,
3.23 (s, 3H, 5-CH3), 3.74 (s, 3H, 1-CH3), 7.05–7.28 (m, 4H,
H-6,7,8,9), 7.54 (s, 1H, H-3), 8.66 f (s, 1H, NH)
1510, 1495
5-CH3), 3.81 (s, 3H, 1-CH3), 7.11–7.30 (m, 4H, H-6,7,8,9),
7.82 (s, 1H, H-3)
3c
74
142.5–143 (C) C15H18N4O
1630s, br, (CO), 1598w, 1546w, 1.06 and 1.12 [2d, 3H+3H, CH(CH3)2], 3.47 (s, 3H,
1490br
5-CH3), 3.60 [m, 1H, CH(CH3)2], 3.81 (s, 3H, 1-CH3),
7.11–7.30 (m, 4H, H-6,7,8,9), 7.83 (s, 1H, H-3)
3.28 (s, 3H, 5-CH3), 7.06–7.33 (m, 4H, H-6,7,8,9), 7.46–7.69
(m, 5H, phenyl Hs), 7.80 (s, 1H, H-3), 8.51 f (s, 1H, NH)
0.97 (t, 3H, CH2CH3), 3.03–3.40 (m, 2H, CH2CH3), 3.48 (s,
3H, 5-CH3), 7.15–7.30 (m, 4H, H-6,7,8,9), 7.38–7.66 (m,
5H, phenyl Hs), 7.97 (s, 1H, H-3)
3d
3e
82
75
199–200 (G)
143–144 (C)
C17H14N4O
C19H18N4O
3385 (NH), 1632s, br (CO),
1598, 1568w, 1543, 1498
1630s, br, (CO), 1597, 1546 w,
1495br
3f
3g
3h
3i
84
68
59
78
182–183 (B)
C20H20N4O
1635s, br, (CO), 1598, 1554w,
1496s, br
0.88 and 0.91 [2d, 3H+3H, CH(CH3)2], 3.43 [m, 1H,
CH(CH3)2], 3.50 (s, 3H, 5-CH3), 7.19–7.79 (m, 9H,
H-6,7,8,9+phenyl Hs), 8.01 (s, 1H, H-3)
267–268.5 (D) C17H14N4O
3280–3000 br (NH), 1641s (CO), 3.79 (s, 3H, 1-CH3), 6.68, 6.91, 7.08 and 7.22–7.48 (4m,
1607, 1590w, 1566, 1492
1H+1H+1H+6H, H-6,7,8,9+phenyl Hs), 7.61 (s, 1H,
H-3), 8.85 f (s, 1H, NH)
155–156 (C)
C19H18N4O
1645s, br, (CO), 1595w, 1555w, 1.31 (t, 3H, CH2CH3), 3.62–3.77 (m, 2H, CH2CH3), 3.85 (s,
1508, 1490
3H, 1-CH3), 6.81, 6.96–7.17 and 7.22–7.49 (3m,
1H+2H+6H, H-6,7,8,9+phenyl Hs), 7.87 (s, 1H, H-3)
124.5–125 (C) C20H20N4O
1653s, br, (CO), 1598w, 1552w, 1.17 and 1.26 [2d, 3H+3H, CH(CH3)2], 3.86 (s, 3H,
1507, 1487
1-CH3), 3.90 [m, 1H, CH(CH3)2], 6.75–6.86, 7.02–7.14 and
7.19–7.50 (3m, 1H+2H+6H, H-6,7,8,9+phenyl Hs), 7.91
(s, 1H, H-3)
4a
4b
3
185–187 (B)
240–241 (G)
C12H12N4O
C17H14N4O
3380 (NH), 1630 s, br (CO),
1608, 1578s, 1500
3280–3000 br (NH), 1650s (CO), 3.72 (s, 3H, 2-CH3), 6.63, 6.71, 7.01, 7.17 and 7.23–7.49
3.23 (s, 3H, 5-CH3), 3.68 (s, 3H, 2-CH3), 6.89–7.27 (m, 4H,
H-6,7,8,9), 7.97 (s, 1H, H-3), 8.44 f (s, 1H, NH)
16
1610, 1592w, 1574, 1492
(5m, 1H+1H+1H+1H+5H, H-6,7,8,9+phenyl Hs), 8.06
(s, 1H, H-3), 8.64 f (s, 1H, NH)
a Crystallisation solvent: A=ethanol–chloroform, B=ethyl acetate–isopropyl ether, C=isopropyl ether, D=ethanol, E=isopropyl ether–petroleum
ether, F=petroleum ether, G=ethyl acetate.
b Anal. C, H, N.
c In CHCl3 solutions, except for 2a, f, k, 3a, g, 4b for which a KBr pellet was used. Abbreviations: s=strong, w=weak, sh=shoulder, br=broad.
d In CDCl3 solutions, except for 2a, b, f, k, 3a, d, g, 4a, b for which DMSO-d6 was used.
e Lit. [12]: 2a, m.p. \300 °C. Lit. [13]: 2b, m.p. 236–237 °C; 2h, m.p. 227 °C.
f Disappeared with D2O.

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
945
4.1.5.1. 5,10-Dihydro-1,5-dimethylpyrazolo[3,4-b][1,5]-
benzodiazepin-4(1H)-one (3a) and 5,10-dihydro-2,5-
dimethylpyrazolo[3,4-b][1,5]benzodiazepin-4(2H)-one
(4a)
g), or 3g (1.5 mmol, 0.43 g) were 10-alkylated with 9.0
mmol of iodoethane (1.40 g) or 2-iodopropane (1.53 g)
under the same conditions above described for the
preparation of compounds 2c–e, g, i, j, l–n (the reaction
time was always 1 h). From the final reaction mixture a
thick oily residue was then recovered by a procedure
identical to that used for compounds 2c–e, g, i, j, l–n.
From this oil the pure 10-alkylated compound 3 was
obtained as a white solid after addition of a little ethyl
ether (compounds 3e, f), or through a suitable column
chromatography [silica gel, ethyl acetate as eluent, in the
case of compounds 3b, c; silica gel, dichloromethane–
petroleum ether–triethylamine (10:10:1) as eluent for
compound 3h; aluminium oxide, ethyl acetate as eluent
for compound 3i].
The oily residue obtained from the reaction carried
out with 9b and methylhydrazine was chromatographed
on a silica gel column, eluting first with the mix-
ture dichloromethane–petroleum ether–triethylamine
(9:3:1). The eluate collected afforded a solid residue
which was crystallised from ethyl acetate to give a little
amount (0.10 g) of 1-methyl-1H-1,5-benzodiazepine-
2,4(3H,5H)-dione (clearly deriving from decomposition
of 9b) which was identified by spectral data and com-
parison with an authentic sample [14]. After standing,
from the mother liquor a white solid separated out
which was recrystallised from ethyl acetate–isopropyl
ether (1:1) to give a low yield of compound 4a.
By further eluting the column with acetone, then
evaporating the eluate, a white solid residue was recov-
ered, which was treated with a little ethyl ether to give a
good yield of 3a.
Data for compounds 3b, c, e, f, h, i are reported in
table III.
4.1.7. 6,11-Dihydro-5H-pyrimido[4,5-b][1,5]benzo-
diazepine-5-thione (10)
A mixture of 4.0 mmol (0.85 g) of 2a, 4.0 mmol (1.62
g) of Lawesson’s reagent and 6 mL of Dowtherm A was
stirred at 150 °C for 30 min. After cooling, the resulting
suspension was diluted with a little ethyl acetate and
filtered. There was so obtained the nearly pure com-
pound 10 (0.90 g, 99%); orange–yellow solid, m.p.
330–332 °C (dec.) (from methanol). IR (KBr, cm−1):
3260–2880 broad (NH), 1629 weak, 1586 strong, broad,
1504. 1H-NMR (DMSO-d6): l 6.97–7.17 (m, 4H, H-
7,8,9,10), 8.60 and 9.04 (2s, 1H+1H, H-2,4), 9.45 (s, 1H,
NH; disappeared with D2O), 12.02 (near s, 1H, NH;
disappeared with D2O). Anal. C11H8N4S (C, H, N, S).
4.1.5.2. 5,10-Dihydro-1-methyl-5-phenylpyrazolo-
[3,4-b][1,5]benzodiazepin-4(1H)-one (3g) and
5,10-dihydro-2-methyl-5-phenylpyrazolo[3,4-b][1,5]-
benzodiazepin-4(2H)-one (4b)
From the reaction of 9c with methylhydrazine a
nearly solid residue was obtained which was treated with
a little ethyl acetate to afford a good yield of pure
compound 3g, as a white solid. The oil obtained from
evaporation of the mother liquor was chromatographed
on a silica gel column, eluting first with dichloro-
methane–petroleum ether–triethylamine (6:3:1) to re-
cover, after removal of solvents, white compound 4b.
Further elution of the column with ethyl acetate–ace-
tone (1:1) afforded an additional crop of 3g.
4.1.8. Compounds 11a and 11b
For the preparation of 11a, a mixture of 4.0 mmol
(0.91 g) of 10, 1.0 g of anhydrous K2CO3, 80.0 mmol
(12.48 g) of iodoethane and 100 mL of dry acetone was
refluxed for 5 h, with stirring.
4.1.5.3. 5,10-Dihydro-5-methyl-1-phenylpyrazolo[3,4-b]-
[1,5]benzodiazepin-4(1H)-one (3d)
The thick oily residue obtained from the reaction of
9b with phenylhydrazine was treated with a little ethyl
acetate and allowed to stand until white crystals of pure
compound 3d separated out.
In the case of 11b, the mixture of 4.5 mmol (1.03 g) of
10, 27.0 mmol (1.51 g) of finely powdered KOH and 100
mL of dry acetone was refluxed for 15 min, then
iodoethane (27.0 mmol, 4.21 g) was added and the
mixture was further stirred at reflux for 1.5 h.
In both cases the solvent was then removed in vacuo
and the residue was partitioned between water and
dichloromethane. The organic layer was collected and
the aqueous phase was exhaustively extracted with
dichloromethane. The combined extracts were dried and
solvent removed to give a thick oil which was chro-
matographed on a silica gel column.
Data for compounds 3a, d, g and 4a, b are reported in
table III.
4.1.6. General procedure for the 1,5,10-trisubstituted
5,10-dihydropyrazolo[3,4-b][1,5]benzodiazepin-4(1H)-
ones (3b, c, e, f, h, i)
Compound 3a (1.5 mmol, 0.34 g), 3d (1.5 mmol, 0.43

946
M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
4.1.8.1. 5-(Ethylthio)-11H-pyrimido[4,5-b][1,5]benzo-
diazepine (11a)
4.32 (2m, 1H+1H, 9-CH2CH3), 7.14 (s, 1H, H-2), 7.20–
7.46 (m, 4H, H-5,6,7,8), 8.83 and 8.98 (2s, 1H+1H,
H-11,13). Anal. C16H15N5 (C, H, N).
After eluting with dichloromethane–ethyl acetate
(1:1) and evaporating the eluate, the nearly solid residue
was treated with a little isopropyl ether to afford orange
crystals of pure compound 11a (0.65 g, 63%); m.p.
192–193 °C, after crystallisation from the same solvent;
IR (CHCl3, cm−1): 3375 (NH), 1619, 1596, 1574, 1560
4.1.10. 9-Ethyl-3-methyl-9H-pyrimido[5,4-c][1,2,4]-
triazolo[4,3-a][1,5]benzodiazepine (6)
A mixture of 2.0 mmol (0.57 g) of 11b, 6.0 mmol (0.44
g) of acetohydrazide, 0.10 g of monohydrate p-toluene-
sulphonic acid and 5 mL of Dowtherm A was stirred at
200 °C for 1 h. Following then the same procedure
described above for isolation of compound 5, pure
compound 6 (0.26 g, 47%) was obtained; white crystals,
m.p. 254–254.5 °C (from ethyl acetate). IR (CHCl3,
cm−1): 1600 shoulder, 1586, 1557, 1501. 1H-NMR
(CDCl3): l 1.21 (t, 3H, 9-CH2CH3), 2.71 (s, 3H, 3-CH3),
3.72 and 4.35 (2m, 1H+1H, 9-CH2CH3), 7.23–7.51 (m,
4H, H-5,6,7,8), 8.94 and 9.11 (2s, 1H+1H, H-11,13).
Anal. C15H14N6 (C, H, N).
1
shoulder. H-NMR (CDCl3): l 1.40 (t, 3H, CH2CH3),
3.15 (q, 2H, CH2CH3), 6.11 (s, 1H, NH; disappeared
with D2O), 6.68 and 6.93–7.17 (2m, 1H+3H, H-
7,8,9,10), 8.54 and 8.62 (2s, 1H+1H, H-2,4). Anal.
C13H12N4S (C, H, N, S).
4.1.8.2. 11-Ethyl-5-(ethylthio)-11H-pyrimido[4,5-b]-
[1,5]benzodiazepine (11b)
The column was eluted with toluene–triethylamine
(9:1) and the eluate evaporated to dryness to afford
nearly pure compound 11b (0.96 g, 75%), from which an
analytically pure sample (yellow–orange thick oil) was
obtained by repeating the same procedure. IR (CHCl3,
4.1.11. Preparation of compounds 12a and 12b
The mixture of 3.0 mmol of 11a (0.77 g) or 11b (0.85
g), 3.9 mmol (0.27 g) of hydroxylamine hydrochloride,
3.9 mmol (0.33 g) of NaHCO3, and 25 mL of methanol
was refluxed for 2 h, with stirring. Compound 12a or
12b, respectively, was isolated from the final reaction
mixture as described below.
1
cm−1): 1611, 1591, 1560, 1540 weak. H-NMR (CDCl3):
l 1.28 (t, 3H, SCH2CH3), 1.43 (t, 3H, NCH2CH3), 3.20
(q, 2H, SCH2CH3), 3.90 (q, 2H, NCH2CH3), 6.93 and
7.05–7.19 (2m, 1H+3H, H-7,8,9,10), 8.65 and 8.77 (2s,
1H+1H, H-2,4). Anal. C15H16N4S (C, H, N, S).
4.1.9. 9-Ethyl-3-methyl-9H-imidazo[1,2-a]pyrimido-
[5,4-c][1,5]benzodiazepine (5)
4.1.11.1. 5-(Hydroxyamino)-11H-pyrimido[4,5-b][1,5]-
benzodiazepine (12a)
A mixture of 2.0 mmol (0.57 g) of 11b, 12.0 mmol
(0.66 g) of propargylamine, 0.10 g of monohydrate
p-toluenesulphonic acid and 5 mL of Dowtherm A was
heated at 180 °C for 2 h, with stirring. After cooling,
the mixture was stirred with 10% aqueous Na2CO3 and
dichloromethane, then allowed to stand until two homo-
geneous layers were obtained. The organic phase was
collected and the aqueous one further extracted several
times with dichloromethane. The combined organic
phases were dried, then evaporated to afford an oil
which was chromatographed on a silica gel column,
eluting first with dichloromethane until Dowtherm A
was completely removed, then with toluene–triethyl-
amine (9:1). After elimination of some impurities, this
eluate was collected and evaporated to dryness to give a
thick oily residue from which, by adding a little ethyl
ether and petroleum ether, compound 5 separated out as
a white solid (0.16 g, 29%); m.p. 187–188 °C (from
isopropyl ether). IR (CHCl3, cm−1): 1603 weak, 1580
After cooling, the suspension obtained was filtered
and the recovered whitish solid was washed with water,
then dried to give 0.48 g (70%) of 12a; white crystals,
m.p. 254–255 °C (from ethanol). IR (KBr, cm−1): 3385,
3300–2700 broad, 1650, 1592, 1572, 1538, 1515. 1H-
NMR (DMSO-d6): l 6.82–6.98 and 7.15–7.27 (2m,
2H+2H, H-7,8,9,10), 8.26 (s, 1H, NH; disappeared with
D2O), 8.59 and 8.80 (2s, 1H+1H, H-2,4), 9.54 (s, 1H,
NH; disappeared with D2O), 10.97 (s, 1H, OH; disap-
peared with D2O). Anal. C11H9N5O (C, H, N).
4.1.11.2. 11-Ethyl-5-(hydroxyamino)-11H-pyrimido-
[4,5-b][1,5]benzodiazepine (12b)
The residue obtained after removal of solvent was
partitioned between water and dichloromethane, then
the aqueous phase was exhaustively extracted with
dichloromethane. The combined organic phases were
dried and evaporated to dryness to give a thick oil from
which, after addition of a little ethyl ether and standing,
0.30 g (39%) of nearly pure 12b separated out as a
whitish solid; m.p. 184–184.5 °C (from ethyl acetate–
1
shoulder, 1568, 1551, 1500 broad. H-NMR (CDCl3): l
1.21 (t, 3H, 9-CH2CH3), 2.42 (s, 3H, 3-CH3), 3.73 and

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
947
isopropyl ether). IR (KBr, cm−1): 3390, 3200–2600
broad, 1630, 1600, 1580, 1543 weak, 1510. 1H-NMR
(DMSO-d6): l 1.17 (t, 3H, CH2CH3), 4.01 (q, 2H,
CH2CH3), 6.94–7.34 (m, 4H, H-7,8,9,10), 8.68 (s, 1H,
NH; disappeared with D2O), 8.70 and 8.82 (2s, 1H+1H,
H-2,4), 10.75 (s, 1H, OH; disappeared with D2O). Anal.
C13H13N5O (C, H, N).
CH2CH3), 6.44 (q, J=5 Hz, 1H, H-3), 6.92–7.29 (m,
4H, H-5,6,7,8), 8.85 and 8.88 (2s, 1H+1H, H-11,13).
Anal. C15H15N5O (C, H, N).
4.1.14. 5,10-Dihydro-1,5,10-trimethylpyrazolo[3,4-b]-
[1,5]benzodiazepin-4(1H)-one (3j)
4.1.14.1. Method A ( from compound 3k)
4.1.12. 3-Methyl-3H,9H-[1,2,4]oxadiazolo[4,3-a]-
A mixture of 1.5 mmol (0.32 g) of 3k [5], 18.0 mmol
(1.00 g) of finely powdered KOH and 50 mL of dry
acetone was refluxed for 10 min, with stirring. The
solution of 18.0 mmol (2.55 g) of iodomethane in 15 mL
of dry acetone was then added and the mixture was
further refluxed for 1 h. After cooling, the solvent was
removed in vacuo, the residue was partitioned between
water and dichloromethane, then the aqueous phase was
further extracted several times with the same solvent.
The combined organic phases were dried and solvent
was removed affording an oily residue which was
treated with ethyl ether to give 0.26 g (72%) of pure 3j;
white solid, m.p. 155–155.5 °C (from ethyl acetate). IR
(CHCl3, cm−1): 1635 strong, broad (CO), 1600 weak,
pyrimido[5,4-c][1,5]benzodiazepine (7a)
A mixture of 2.0 mmol (0.45 g) of 12a, 4 mL of
acetaldehyde dimethylacetal, 0.10 g of monohydrate p-
toluenesulphonic acid and 10 mL of dimethyl sulphox-
ide was stirred at 140 °C for 1 h, then cooled and
poured into water (200 mL). The resulting mixture was
made alkaline with Na2CO3 then exhaustively extracted
with ethyl ether–ethyl acetate (1:1). The combined ex-
tracts were dried and the solvents removed in vacuo to
give a thick oil, which was chromatographed on a silica
gel column, eluting with toluene–acetone (4:1). The
eluate collected gave a solid residue which was taken up
in a little ethyl ether and filtered, yielding 0.18 g (36%)
of 7a; white crystals, m.p. 198–199 °C (from ethyl
acetate–isopropyl ether). IR (CHCl3, cm−1): 3380 (NH),
1
1545 shoulder, 1515 broad, 1498. H-NMR (CDCl3): l
3.32 (s, 3H, 10-CH3), 3.45 (s, 3H, 5-CH3), 3.84 (s, 3H,
1-CH3), 7.09–7.30 (m, 4H, H-6,7,8,9), 7.78 (s, 1H, H-3).
Anal. C13H14N4O (C, H, N).
1
1600, 1586, 1512 shoulder, 1500. H-NMR (CDCl3): l
1.63 (d, J=5Hz, 3H, 3-CH3), 6.52 (q, J=5Hz, 1H,
H-3), 6.85–7.16 (m, 5H, H-5,6,7,8+NH; 4H after treat-
ment with D2O), 8.69 and 9.03 (2s, 1H+1H, H-11,13).
Anal. C13H11N5O (C, H, N).
4.1.14.2. Method B ( from compound 3a)
Starting, as a whole, from a mixture of 1.5 mmol
(0.34 g) of 3a, 9.0 mmol (0.50 g) of KOH, 9.0 mmol
(1.28 g) of iodomethane and 65 mL of dry acetone, and
following the same procedure described above for the
preparation of 3j from 3k, a white solid was finally
obtained (0.28 g, 77%) which proved to be compound 3j
(m.p., TLC, IR).
4.1.13. 9-Ethyl-3-methyl-3H,9H-[1,2,4]oxadiazolo-
[4,3-a]pyrimido[5,4-c][1,5]benzodiazepine (7b)
A mixture of 1.5 mmol (0.38 g) of 7a, 4.5 mmol (0.50
g) of finely powdered KOH and 80 mL of dry acetone
was refluxed for 15 min, then iodoethane (9.0 mmol,
1.40 g) was added and the resulting mixture was refluxed
for 30 min, with stirring. The solvent was then removed
in vacuo, the residue partitioned between water and
dichloromethane, and the aqueous phase exhaustively
extracted with dichloromethane. The combined organic
phases were dried and solvent was removed to afford a
thick oil which was chromatographed on a silica gel
column, eluting with toluene–triethylamine (9:1). The
eluate, after elimination of solvents, afforded a thick oil
which was treated with petroleum ether to give com-
pound 7b as a white solid (0.11 g, 26%); m.p. 113–
113.5 °C (from isopropyl ether–petroleum ether). IR
4.1.15. Preparation of 5,10-dihydro-2,5-dimethyl-
pyrazolo[3,4-b][1,5]benzodiazepin-4(2H)-one (4a) and
5,10-dihydro-2,5,10-trimethylpyrazolo[3,4-b][1,5]-
benzodiazepin-4(2H)-one (4d) from compound 4c
Following exactly the same procedure described
above for the preparation of 3j from 3k (except for a
reaction time of 24 h), from the reaction of 1.5 mmol
(0.32 g) of 4c [5] with 18.0 mmol (2.55 g) of
iodomethane in the presence of 18.0 mmol (1.00 g) of
finely powdered KOH, after the subsequent treatment of
the reaction mixture, a final oily residue was obtained
and chromatographed on a silica gel column, eluting
first with dichloromethane–petroleum ether–triethyl-
(KBr, cm−1): 1595 strong, 1573, 1528 weak, 1502. H-
NMR (CDCl3): l 1.23 (t, 3H, 9-CH2CH3), 1.70 (d, J=5
Hz, 3H, 3-CH3), 3.75 and 4.44 (2m, 1H+1H, 9-
1

948
M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
amine (10:10:1). The eluate collected, after removal of
solvents in vacuo, gave the trimethylderivative 4d as a
white solid (0.12 g, 33%); m.p. 173–174 °C (from iso-
propyl ether). IR (CHCl3, cm−1): 1630 strong, broad
4.1.18. 5,10-Dihydro-5-methyl-2-phenylpyrazolo-
[3,4-b][1,5]benzodiazepin-4(2H)-one (4e)
PCl5 (2.40 g) was slowly added to the solution of 8.0
mmol (2.24 g) of 13 in 28 mL of N,N-dimethylfor-
mamide, while stirring. The resulting solution was
stirred at room temperature for 60 h, then slowly poured
onto ice-water, made alkaline with 6 N aqueous NaOH,
then vigorously stirred at room temperature for 30 min.
A solid separated out that was recovered by filtration,
washed with water and dried, then crystallised from
ethyl acetate–dichloromethane (1:1) to yield 0.60 g
(26%) of pure 4e, whitish solid, m.p. 277–278 °C. IR
(KBr, cm−1): 3318 and 3115 (NH), 1618 broad (CO),
1
(CO), 1594 weak, 1566 strong, 1491. H-NMR (CDCl3):
l 3.31 (s, 3H, 10-CH3), 3.40 (s, 3H, 5-CH3), 3.75 (s, 3H,
2-CH3), 7.04–7.23 (m, 4H, H-6,7,8,9), 7.69 (s, 1H, H-3).
Anal. C13H14N4O (C, H, N).
By subsequent elution with dichloromethane–tri-
ethylamine (9:1) a white solid was recovered (0.11 g,
32%) which proved to be (m.p., TLC, IR) compound 4a
(see Section 4.1.5.1 and table III).
1
1598 shoulder, 1573, 1498. H-NMR (DMSO-d6): l 3.29
4.1.16. Preparation of compound 4b from 8d
(s, 3H, 5-CH3), 7.04–7.38, 7.50 and 7.83 (3m, 5H+2H+
2H, H-6,7,8,9+phenyl Hs), 8.85 (s, 2H, H-3+NH; 1H
after treatment with D2O). Anal. C17H14N4O (C, H, N).
A mixture of 5.0 mmol (1.40 g) of 8d [4], 20.0 mmol
(1.48 g) of N-methyl formic hydrazide [5], 0.10 g of
monohydrate p-toluenesulphonic acid and 10 mL of
dimethyl sulphoxide was stirred at 130 °C for 22 h, then
cooled, poured onto ice-water and made alkaline with
Na2CO3: a solid separated out that was recovered by
filtration, washed with water and dried, then chro-
matographed on an aluminium oxide column. After
discarding some impurities eluted with dichloromethane,
the elution with dichloromethane–ethyl acetate (1:1)
afforded a white solid (0.12 g, 8.3%) which proved to be
(m.p., TLC, IR) compound 4b (see Section 4.1.5.2 and
table III).
4.1.19. Preparation of compounds 4f, g
A mixture of 1.5 mmol (0.43 g) of 4e, 50 mL of dry
2-butanone and 18.0 mmol (1.00 g) of finely powdered
KOH was refluxed for 10 min, with stirring. The solu-
tion of 18.0 mmol of iodoethane (2.81 g) (compound 4f)
or 2-iodopropane (3.06 g) (compound 4g) in 15 mL of
2-butanone was then added and the resulting mixture
was further refluxed for 16 h. After removing the solvent
in vacuo, the residue obtained was partitioned between
water and dichloromethane and the aqueous phase was
extracted several more times with the same solvent. The
combined organic phases were dried, then evaporated
to dryness to give an oily residue, which was
4.1.17. 1,3-Dihydro-1-methyl-4-(2-phenylhydrazino)-
2H-1,5-benzodiazepin-2-one (13)
chromatographed on
a silica gel column, eluting
with petroleum ether–dichloromethane–triethylamine
(8:2:1). The eluate collected, after removal of solvents,
afforded compound 4f or 4g as a white solid, which was
taken up in a little ethyl ether and recovered by
filtration.
A mixture of 10.0 mmol (2.17 g) of 8b, 30.0 mmol
(3.24 g) of phenylhydrazine, 0.10 g of monohydrate
p-toluenesulphonic acid and 20 mL of dimethyl
sulphoxide was stirred at 130 °C for 16 h, then cooled
and poured onto ice-water. The suspension resulting was
made alkaline with Na2CO3, then ethyl ether (50 mL)
was added and the mixture was stirred at room temper-
ature for 30 min. The solid that separated out was
recovered by filtration, washed with water and ethyl
ether and dried to give 1.95 g (70%) of compound 13;
whitish crystals, m.p. 242–244 °C (from ethanol). IR
(KBr, cm−1): 3310 strong (NH), 1665, 1631 strong,
4.1.19.1. 10-Ethyl-5,10-dihydro-5-methyl-2-phenyl-
pyrazolo[3,4-b][1,5]benzodiazepin-4(2H)-one (4f)
Yield: 0.39 g (82%); white crystals, m.p. 194–195 °C
(from ethyl acetate). IR (CHCl3, cm−1): 1629 (CO), 1596
1
weak, 1567 strong, 1490. H-NMR (CDCl3): l 1.39 (t,
3H, CH2CH3), 3.45 (s, 3H, 5-CH3), 3.94 (q, 2H,
CH2CH3), 7.07–7.35, 7.43 and 7.62 (3m, 5H+2H+2H,
H-6,7,8,9+phenyl Hs), 8.25 (s, 1H, H-3). Anal.
C19H18N4O (C, H, N).
1
1600, 1531 weak, 1498. H-NMR (DMSO-d6): l 3.03
and 3.28 (AB system, J=12 Hz, 2H, 3-CH2), 3.26 (s,
3H, 1-CH3), 6.68, 6.98, 7.07–7.32 and 7.43 (4m, 1H+
2H+5H+1H, H-6,7,8,9+phenyl Hs), 8.22 (s, 1H, NH;
disappeared with D2O), 8.75 (s, 1H, NH; disappeared
with D2O). Anal. C16H16N4O (C, H, N).
4.1.19.2. 5,10-Dihydro-10-isopropyl-5-methyl-2-phenyl-
pyrazolo[3,4-b][1,5]benzodiazepin-4(2H)-one (4g)
Yield: 0.41 g (82%); white crystals, m.p. 193–194 °C

M. Di Braccio et al. / European Journal of Medicinal Chemistry 36 (2001) 935–949
949
(from ethyl acetate). IR (CHCl3, cm−1): 1628 (CO), 1596
weak, 1565 strong, 1488. 1H-NMR (CDCl3): l 1.40–1.80
[m, 6H, CH(CH3)2], 3.45 (s,3H, 5-CH3), 4.20 [m, 1H,
CH(CH3)2], 7.07–7.49 and 7.63 (2m, 7H+2H, H-
6,7,8,9+phenyl Hs), 8.25 (s, 1H, H-3). Anal. C20H20N4O
(C, H, N).
37 °C, samples were spotted on glass fibre filters (What-
man GF/A), and the acid-insoluble radioactivity was
determined.
Acknowledgements
The authors thank the Italian MURST and CNR
(Rome), as well as the Italian Ministero della Sanita`-Isti-
tuto Superiore di Sanita` (Rome)-Progetto AIDS 2000
(grant no. 40 C.47), for financial support. The authors
wish to thank Dr Daniela Ghiani for editing this
manuscript, Mr Antonio Panaro for elemental analyses,
Mr Franco Tuberoni for IR spectra, Dr. Carlo Rossi and
4.2. Biological evaluation
4.2.1. Compounds
Test compounds were solubilized in DMSO at 200
mM and then diluted into culture medium.
4.2.2. Cells
1
Dr Riccardo Raggio for H-NMR spectra.
MT-4 cells were grown at 37 °C in a 5% CO2 atmo-
sphere in RPMI 1640 medium, supplemented with 10%
foetal calf serum (FCS), 100 UI mL−1 penicillin G and
100 mg mL−1 streptomycin. Cell cultures were checked
periodically for the absence of mycoplasma contamina-
tion with the MycoTect Kit (Gibco).
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mM DTT, 0.1 mg mL−1 BSA, 0.5 OD260 unit mL−1
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