New tricyclic systems of biological interest. Annelated 1,2,3-triazolo[1,5-a]pyrimidines through domino reaction of 3-azidopyrroles and methylene active nitriles

Article abstract of DOI:10.1016/S0040-4020(02)01245-0

Anionic hetero-domino reaction of 3-azidopyrroles and acetonitriles constitutes the synthetic entry to annelated 1,2,3-triazolo[1,5-a]pyrimidines. Upon slight variations of the experimental conditions the method is of general application either with 2 or 4 substituted pyrroles. Thus derivatives of the new ring system pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine, isomers of the previously synthesized pyrrolo[3,4-e][1,2,3]triazolo[1,5-a]pyrimidine, were prepared in high yields. The condensed pyrrolo-triazolo-pyrimidines, although only moderately active, can be used as model for the design of DNA-interactive compounds.

Full text of DOI:10.1016/S0040-4020(02)01245-0

Tetrahedron 58 (2002) 9723–9727
New tricyclic systems of biological interest. Annelated
1,2,3-triazolo[1,5-a]pyrimidines through domino reaction of
3-azidopyrroles and methylene active nitriles
Antonino Lauria, Patrizia Diana, Paola Barraja, Alessandra Montalbano, Girolamo Cirrincione,
*
Gaetano Dattolo and Anna Maria Almerico
`
Dipartimento Farmacochimico, Tossicologico e Biologico, Universita degli Studi, Via Archirafi 32, I-90123 Palermo, Italy
Received 27 June 2002; revised 12 September 2002; accepted 3 October 2002
Abstract—Anionic hetero-domino reaction of 3-azidopyrroles and acetonitriles constitutes the synthetic entry to annelated 1,2,3-
triazolo[1,5-a]pyrimidines. Upon slight variations of the experimental conditions the method is of general application either with 2 or 4
substituted pyrroles. Thus derivatives of the new ring system pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine, isomers of the previously
synthesized pyrrolo[3,4-e][1,2,3]triazolo[1,5-a]pyrimidine, were prepared in high yields. The condensed pyrrolo-triazolo-pyrimidines,
although only moderately active, can be used as model for the design of DNA-interactive compounds. q 2002 Elsevier Science Ltd. All
rights reserved.
1. Introduction
In the course of our researches devoted to the development
of new classes of heteroaromatic systems which incorpor-
ated the pyrrole/indole moiety, we have been interested in
the synthesis and evaluation of the biological activity of
several flat polycondensed nitrogen heterocycles, either
tricyclic and tetracyclic, that can potentially intercalate into
double-stranded DNA. In this context we have prepared and
reported the interesting antiproliferative activity of
indolo[3,2-c]cinnolines,1 indolo [1,2-c][1,2,3]benzotria-
zines,2 and pyrrolo[1,2-f]phenanthridines,3 that can be
Figure 1.
synthetic approach to a new tricyclic angular system,
namely pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine of
type 3, isomer of derivatives of type 1, and report the
results of preliminary antitumor screening tests carried
out on all these classes of annelated pyrrolo-triazolo-
pyrimidines.
related to the well known classes of intercalators with
linear or angular structure such as acridines, anthracyclines
and phenanthridines. These compounds possess such a
property whose principal driving forces are p-stacking and
charge-transfer interactions as well as hydrogen bonding
and electrostatic forces.4 More recently we explored the
synthetic access to angular heterotricycles and we reported
the preparation5 of the new system, pyrrolo[3,4-
e][1,2,3]triazolo[1,5-a]pyrimidine of type 1 and its
rearrangement to pyrrolo[3,4-d][1,2,3]triazolo[1,5-a]pyri-
midine of type 2. The above systems can both be related to
DNA-interactive drugs (Fig. 1).
Routes to ring systems 1 and 3 can be provided by domino
reactions between acetonitriles and azidopyrroles 4 under
basic conditions (Scheme 1). These last through the azido
moiety and can act as a 1,3-dipolar compound in
cycloaddition reactions with dipolarophiles such as the
anions obtained from the methylene active derivatives.
Although this type of reaction is well described in aromatic
series, only in a few cases has it been applied to pentatomic
heterocycles.6 Moreover in azole series the only two
examples reported so far have demonstrated that the nature
of the substrate and the reaction conditions can widely
influence the nature of the reaction products.7 The
intermediate resulting from the cycloaddition reaction
In connection with these studies in this paper we discuss the
Keywords: heterotricyclic systems; pyrrolo-triazolo-pyrimidine; domino
reaction; antiproliferative activity.
*
Corresponding author. Tel.: þ39-091-6161606; fax: þ39-091-6169999;
e-mail: almerico@unipa.it
0040–4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(02)01245-0

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A. Lauria et al. / Tetrahedron 58 (2002) 9723–9727
Scheme 2. (i) EtONa, EtOH, 708C, 6 h; (ii) NaNO2, AcOH, 08C; NaN3,
08C–rt; (iii) RCH2CN, EtONa, rt.
Scheme 1.
forming efficiency depends on the amount of ethanol present
in the reaction environment in contrast with what was
observed in the case of ethyl 3-azidopyrrole-4-carboxylate.
could be the 3-(triazol-1-yl)pyrrole of type 5 which bears an
amino group susceptible to further reactions. In the presence
of a vicinal carboxylate function, intermediate 5 further
cyclizes to provide the pyrimidine ring.
The new compounds 9a–c, derivatives of the new ring
system 4H-pyrrolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine,
were generally isolated in high yields (70–90%). Their
structure was confirmed by spectroscopic data; in particular
by the presence in the IR spectra of the typical absorption
bands (at 3450–3221 and 1667–1649 cm21) due to the
presence of a cyclic-amide structure. In the 1H NMR
the amide NH signal was found at 11.15–11.17 ppm, and in
the 13C NMR the carbonyl peak was found at 148.6–
158.5 ppm.
This type of anionic hetero-domino reaction has been
successful when the starting compound was ethyl 3-azido-
pyrrole-4-carboxylate, which represented the first
example of such a sequence in pyrrole series.5 However,
since the reactivity of pyrrole derivatives varies greatly
switching the positions of the substituents or moving
them from a b to an a position,8 and is strongly dependent
on the reaction conditions, we thought to explore whether
ethyl 3-azidopyrrole-2-carboxylate compounds behave
similarly.
As already demonstrated in the case of pyrrolo[3,4-
e][1,2,3]triazolo[1,5-a]pyrimidine derivatives of type 1,
which rearranged to pyrrolo[3,4-d][1,2,3]triazolo[1,5-a]
pyrimidine of type 2,5 compound 9a was heated under
reflux in aqueous DMSO to give 7-benzyl-3,5-diphenyl-4,7-
dihydro-8H-pyrrolo[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-
8-one (10) in nearly quantitative yield (Scheme 3). This
conversion reaction can be envisaged as a ring-opening
ring-closure of the pyrimidine and triazole rings in the
presence of water.5 The participation of water in this
rearrangement came from the observation that the reaction
does not occur in dry solvents and is in agreement with a
well acknowledged literature report on the Dimroth
rearrangement in pyrimidine series.10 In our opinion, the
direct rearrangement involving only opening of the triazole
ring has to be ruled out because of the failure of the
transformation under thermal conditions.
2. Results and discussion
The starting material 8 was easily prepared from ethyl
3-amino-1-benzyl-4-phenylpyrrole-2-carboxylate (7),
obtained in turn by cyclization of 6Z under basic conditions
according to literature procedure9 (Scheme 2). The
3-aminopyrrole 7 was diazotized with sodium nitrite in
acetic acid. Addition of sodium azide to the intermediate
diazonium salt led to the 3-azidopyrrole 8 in high yield.
The azide 8 was added at room temperature to the sodium
salts of acetonitriles in ethanol, under the identical
experimental conditions successfully used in the case of
the isomeric ethyl 3-azidopyrrole-4-carboxylate. In this case
after 24 h at room temperature the TLC analysis showed
mainly unreacted starting compounds. However, since
ethanol is eliminated in the final step of this domino
sequence, we decided to drive the reaction to completeness
by removing the solvent under reduced pressure. In fact
upon reduction of the volume of the reaction solution, the
appearance of the new compounds was observed (TLC
monitoring). Therefore it seems that in this case the bond-
Scheme 3.

A. Lauria et al. / Tetrahedron 58 (2002) 9723–9727
9725
3. Conclusions
In conclusion, domino reactions between 3-azidopyrroles
and acetonitriles constitute a versatile entry to annelated
1,2,3-triazolo[1,5-a]pyrimidines. Although in the presence
of the same type of complexity (two rings formation in
sequence) for this type of domino reaction the bond-forming
economy greatly depends on the nature of the starting
material. However, minor modification of the experimental
procedure allows the reaction to become suitable for a
general application in pyrrole series.
Figure 2.
Some of the variously condensed pyrrolo-triazolo-pyrimi-
dine ring systems showed only a moderate antiproliferative
activity. However, because of their structural features they
can constitute a model suitable for the design of new
potential DNA-interactive molecules worthy of further
developments.
All the derivatives of the annelated 1,2,3-triazolo[1,5-a]
pyrimidine ring systems, obtained by using this type of
domino reaction, compounds 9a–c, 10, and 11a–c5 (Fig. 2)
were investigated for their biological activity.
Prior to the evaluation of the potential ability of the new
annelated triazolo-pyrimidine ring systems to interact with
DNA, we calculated11 the LUMO and HOMO energies,
considering that these variables are of importance when two
molecules with p electron systems form charge-transfer
complexes, and also the values of some molecular
descriptors [MR (molar refractivity), ASA (accessible
surface area)].
4. Experimental
4.1. General
Melting points (uncorrected) were taken on a Buchi-Tottoli
capillary apparatus; IR spectra were determined in bromo-
form with a Jasco FT/IR 5300 spectrophotometer; H and
1
13C NMR spectra were measured at 200 and 50.3 MHz,
respectively, in (CD3)2SO solution, unless otherwise
specified, using a Bruker AC-E series 200 MHz spec-
trometer (TMS as internal reference). Column chromato-
graphy was performed with a Biotage FLASH40i
chromatography module (prepacked cartridge system).
An analysis of the data reported in Table 1 shows that these
values are comparable with those of well known DNA-
intercalators of the acridine or anthracycline classes such as
Amsacrine (AMSA) and Doxorubicin (DOXO). Therefore,
these pyrrolo-triazolo-pyrimidine derivatives appear to be
good candidate as antitumor drugs.
Compounds 9a–c, 10, and 11a–c were screened at the NCI
(Bethesda) in the Developmental Therapeutics Program. In
the primary anticancer screening assay,12 when tested
against a 3-cell line panel consisting of the MCF7 (Breast),
NCI-H460 (Lung), and SF-268 (CNS) the pyrrolo-triazolo-
pyrimidine resulted inactive up to 1024 M concentrations.
All the derivatives were also tested against the human
intestinal adenocarcinoma (LoVo), however, only com-
pounds 11a–c exhibited a moderate antiproliferative
activity with GI50¼24.0–37.5 mM.13
4.1.1. Ethyl 3-amino-1-benzyl-4-phenyl-1H-pyrrole-2-
carboxylate (7). This aminopyrrole was prepared according
to the literature procedure.9 Thus a solution of 3-oxo-2-
phenylpropanenitrile14 (1.0 g, 6.9 mmol) and N-benzyl-
glicine ethyl ester (1.6 g, 8.3 mmol) in benzene (25 mL)
was heated to reflux under a water separator (Dean-Stark
trap) for 10 h and then evaporated under reduced pressure.
Trituration of the residue with ethyl ether afforded a white
crystalline solid (900 mg) identified as 6Z. After filtration,
the ethereal mother liquid was chromatographed using
dichloro-methane/methanol 30:1 as eluant. The first product
to be eluted was 6Z (380 mg). Further elution gave a syrup
identified as 6E (230 mg). A solution of 6Z (770 mg,
2.4 mmol) in 0.28 M sodium ethoxide in ethanol (10 mL)
was heated at 708C for 6 h. The reaction mixture was cooled
to room temperature and evaporated to dryness under
reduced pressure. The residue was partitioned between
water and ethyl acetate. After separation of the organic
phase, the water layer was neutralized with acetic acid
and extracted twice with ethyl acetate. The organic layer
and washings were combined, dried over sodium sulfate and
evaporated to dryness. The residue was purified by column
chromatography using dichloromethane/methanol 30:1 as
eluant to give derivative 7 as a colorless oil which solidified
on standing (690 mg, yield 90%), mp 658C (lit.,9 mp 678C).
Table 1. Molecular descriptors for annelated 1,2,3-triazolo[1,5-a]pyrimi-
dines
2
ASA (A )
˚
Compound
LUMO (eV)
HOMO (eV)
MRa
9a
9b
9c
10
11a
11b
11c
AMSA
DOXO
20.7437
20.8271
21.0409
20.7452
20.5561
20.6535
20.8288
21.2638
21.3378
28.7851
29.2677
29.4766
28.4161
28.5358
29.0393
29.2694
28.1826
28.8961
124.70
108.15
105.76
124.78
125.11
108.55
106.16
107.77
134.02
388.52
346.72
333.26
379.05
364.84
334.79
323.32
353.31
467.29
These calculations were performed with the software TSAR (V 3.2),
running on an Indigo II Silicon Graphics work station.
Predicted values were calculated as a sum of the atomic values
determined according to Viswanadhan et al.15
4.1.2. Ethyl 3-azido-1-benzyl-4-phenyl-1H-pyrrole-2-
carboxylate (8). To a solution of 7 (3.2 g, 10 mmol) in
acetic acid (30 mL) and water (4 mL), sodium nitrite
a

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A. Lauria et al. / Tetrahedron 58 (2002) 9723–9727
(0.83 g, 12 mmol) in water (4 mL) was added at 08C, under
vigorous stirring. After 50 min sodium azide (3.25 g,
50 mmol) was added in portions and the reactants were
stirred for a further 3 h at room temperature. The solid was
filtered off and air dried to give 8 as a yellow precipitate:
3.1 g, yield 90%, mp 1108C; IR: 2120 (N3), 1693 (CO) cm1;
1H NMR d: 1.23 (3H, t, J¼6.4 Hz, CH2CH3), 4.22 (2H, q,
J¼6.4 Hz, CH2CH3), 5.54 (2H, s, CH2), 7.12–7.71 (11H, m,
2£C6H5, H-5); 13C NMR d: 14.0 (q), 52.5 (t), 60.1 (t), 108.7
(s), 112.9 (s), 124.2 (d), 126.4 (d), 126.5 (d), 127.0 (d),
127.2 (d), 127.3 (s), 128.5 (d), 129.1 (d), 132.1 (s), 138.2 (s),
159.3 (s). Anal. Calcd for C20H18N4O2: C, 69.35; H, 5.24;
N, 16.17. Found: C, 69.25; H, 5.29; N, 16.25.
127.5 (d), 128.3 (2d), 128.6 (d), 128.9 (d), 129.1 (d), 133.4
(s), 138.5 (s), 142.0 (s), 142.8 (s), 154.2 (s), 158.5 (s). Anal.
Calcd for C21H14N6O: C, 68.84; H, 3.85; N, 22.94. Found:
C, 68.87; H, 3.80; N, 22.79.
4.2.4. 7-Benzyl-3,5-diphenyl-4,7-dihydro-8H-pyrrolo-
[3,2-d][1,2,3]triazolo[1,5-a]pyrimidin-8-one (10).
solution of 6-benzyl-3,8-diphenyl-4H-pyrrolo[2,3-
A
e][1,2,3]triazolo[1,5-a]pyrimidin-5(6H)-one (9a) (0.21 g,
0.5 mmol) was heated under reflux in dimethylsulfoxide
(99.8%, 10 mL) for 1 h. The cooled reaction mixture was
then poured onto crushed ice and the solid was filtered off,
air dried to give derivative 10 (206 mg) as a light yellow
solid. Yield 98%, mp 2108C; IR: 3295 (NH), 1685 (CO)
1
4.2. General method for the preparation of 3-substituted
6-benzyl-5-oxo-8-phenyl-5,6-dihydro-4H-pyrrolo[2,3-e]-
[1,2,3]triazolo[1,5-a]pyrimidines (9a–c)
cm21; H NMR d: 5.78 (s, 2H, CH2), 7.50–7.33 (m, 11H,
2£C6H5, H-6), 7.79–7.97 (m, 5H, C6H5), 12.30 (s, 1H,
H-4); 13C NMR d: 50.7 (t), 111.9 (s), 113.3 (s), 117.5 (s),
124.9 (d), 126.5 (d), 127.1 (d), 127.3 (d), 127.4 (d), 127.7
(d), 128.1 (d), 128.5 (d), 128.6 (d), 129.2 (d), 129.8 (s),
130.2 (s), 131.4 (s), 131.5 (s), 137.9 (s), 153.9 (s). Anal.
Calcd for C26H19N5O: C, 74.80; H, 4.59; N, 16.78. Found:
C, 74.70; H, 4.55; N, 16.95.
To a solution of 1 mM sodium ethoxide in ethanol (3.9 mL)
substituted acetonitriles (3.9 mmol) in absolute ethanol
(10 mL) were added at room temperature. After being
stirred for 15 min a solution of azido-pyrrole 8 (1.2 g,
3.6 mmol) in absolute ethanol (10 mL) was added and the
mixture was stirred for a further 24 h at room temperature.
Evaporation of the solvent under reduced pressure gave a
solid which was purified by column chromatography using
dichloromethane/ethyl acetate 95:5 as eluant.
Acknowledgements
This work was financially supported in part by Ministero
`
dell’Istruzione, dell’Universita e della Ricerca.
4.2.1. 6-Benzyl-3,8-diphenyl-4H-pyrrolo[2,3-e][1,2,3]tri-
azolo[1,5-a]pyrimidin-5(6H)-one (9a). From
2-phenylacetamide, white solid (1.15 g, yield 80%)
8
and
1
mp.3008C; IR: 3350 (NH), 1649 (CO) cm21; H NMR d:
References
5.71 (2H, s, CH2), 7.13–7.42 (11H, m, 2£H-30, H-40, 2£H-
300, H-400, 2£H-3000, H-4000, 020 £H-20), 8.09 (1H, s, H-7), 8.34
(2H, d, J¼7.4 Hz, 2£H-2 ), 8.52 (2H, d, J¼7.3 Hz, 2£H-
2000), 11.15 (1H, s, H-4); 13C NMR d: 50.9 (t), 110.6 (s),
111.9 (s), 123.2 (d), 124.1 (d), 124.4 (d), 125.1 (d), 126.6
(s), 127.2 (d), 127.3 (d), 128.2 (2d), 128.4 (d), 130.7 (d),
134.4 (s), 135.2 (s), 139.6 (s), 143.2 (s), 147.5 (s), 148.6 (s).
Anal. Calcd for C26H19N5O: C, 74.80; H, 4.59; N, 16.78.
Found: C, 74.76; H, 4.65; N, 16.80.
1. Barraja, P.; Diana, P.; Lauria, A.; Passannanti, A.; Almerico,
A. M.; Minnei, C.; Longu, S.; Congiu, D.; Musiu, C.; La Colla,
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Lauria, A.; Passannanti, A.; Musiu, C.; Pani, A.; Murtas, P.;
Minnei, C.; Marongiu, M. E.; La Colla, P. J. Med. Chem. 1999,
42, 2561–2568.
3. Aiello, E.; Dattolo, G.; Cirrincione, G.; Almerico, A. M.;
Diana, P.; Grimaudo, S.; Mingoia, F.; Barraja, P. Il Farmaco
1995, 50, 365–368.
4.2.2. 6-Benzyl-5-oxo-8-phenyl-5,6-dihydro-4H-pyrrolo
[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine-3-carboxamide
(9b). From 8 and 2-cyanoacetamide, white solid (1.24 g,
yield 90%) mp.3258C; IR: 3450–3250 (NH2 and NH),
1662 (CO) cm21; 1H NMR d: 5.72 (2H, s, CH2), 7.16–7.40
(9H, m, H-7, NH2, 2£H-30, H-40, 2£H-300, H-400), 8.08–8.11
(4H, m, 2£H-20, 2£H-200), 11.17 (1H, bs, H-4); 13C NMR d:
51.0 (t), 110.8 (s), 112.0 (s), 123.2 (s), 124.9 (d), 125.2 (d),
127.3 (2d), 128.3 (d), 128.5 (d), 131.1 (d), 134.1 (s), 139.3
(s), 145.0 (s), 146.5 (s), 148.6 (s), 163.1 (s). Anal. Calcd for
C21H16N6O2: C, 65.62; H, 4.20; N, 21.86. Found: C, 65.59;
H, 4.22; N, 21.83.
4. Wakelin, L. P. G.; Waring, M. J. Comprehensive Medicinal
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4.2.3. 6-Benzyl-5-oxo-8-phenyl-5,6-dihydro-4H-pyr-
rolo[2,3-e][1,2,3]triazolo[1,5-a]pyrimidine-3-carbo-
nitrile (9c). From 8 and malononitrile, white solid (922 mg,
yield 70%) mp.3258C; IR: 3221 (NH), 2236 (CN), 1667
(CO) cm21. 1H NMR d: 5.10 (2H, s, CH2), 6.63–6.79 (8H,
m, 2£H-20, 2£H-30, H-40, 2£H-300, H-400), 7.58 (1H, s, H-7),
7.67 (2H, d, J¼7.8 Hz, 2£H-200), 11.17 (1H, s, H-4); 13C
NMR d: 51.0 (t), 115.7 (s), 116.5 (s), 125.8 (s), 127.3 (d),
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A. Lauria et al. / Tetrahedron 58 (2002) 9723–9727
9727
11. Structure optimization in vacuo and in (CH3)2SO was carried
out, by using the software PIMMS (V 1.43) and Vamp (V 6.1),
supplied by Oxford Molecular, to obtain the interatomic
distances and molecular orbital energies.
13. Test compounds, dissolved in (CH3)2SO at an initial
concentration of 200 mM and serially diluted in culture
medium, were incubated in the presence of LoVo cells for 72 h
at 378C. The number of viable cells was determined by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) method Denizot, F.; Lang, R. J. Immunol.
Meth. 1986, 89, 271–277, Tumor cell growth at each drug
concentration was expressed as percentage of untreated
controls and the concentration resulting in 50% (GI50) growth
inhibition was determined by linear regression analysis.
14. Anderson, E. L.; Casey, J. E.; Greene, L. C.; Lafferty, J. J.;
Reiff, H. E. J. Med. Chem. 1964, 7, 259–268.
12. In the protocol, each cell line is inoculated and preincubated
on a microtiter plate. Test agents are then added at a single
concentration and the culture incubated for 48 h. End-point
determinations are made with alamar blue Gray, G. D.;
Wickstrom, E. Biotechniques 1996, 21, 780–782, Results for
each test agent are reported as the percent of growth of the
treated cells when compared to the untreated control cells.
Compounds which reduce the growth of any one of the cell
lines to approximately 32% or less (negative numbers indicate
cell kill) are passed on for evaluation in the full panel of 60 cell
lines over a 5-log dose range.
15. Viswanadhan, V. N.; Ghose, A. K.; Revankar, G. R.; Robin,
R. K. J. Chem. Inf. Comput. Sci. 1989, 29, 163–172.