Synthesis of Triazenoazaindoles: A New Class of Triazenes with Antitumor Activity

Article abstract of DOI:10.1002/cmdc.201100027

Despite improvements in the treatment and prevention of cancer, the number of new diagnoses continues to rise; this has fuelled substantial interest in the development of new and effective chemotherapeutic agents. Compounds of the triazene class, such as

Full text of DOI:10.1002/cmdc.201100027

DOI: 10.1002/cmdc.201100027
Synthesis of Triazenoazaindoles: a New Class of Triazenes
with Antitumor Activity
Patrizia Diana,*[a] Antonina Stagno,[a] Paola Barraja,[a] Anna Carbone,[a] Barbara Parrino,[a]
Francesco Dall’Acqua,[b] Daniela Vedaldi,[b] Alessia Salvador,[b] Paola Brun,[c]
Ignazio Castagliuolo,[c] Olaf Georg Issinger,[d] and Girolamo Cirrincione[a]
Despite improvements in the treatment and prevention of
cancer, the number of new diagnoses continues to rise; this
has fuelled substantial interest in the development of new and
effective chemotherapeutic agents. Compounds of the triazene
class, such as dacarbazine, have been used in the clinical man-
agement of many cancer types including brain, leukemia, and
melanoma. A new compound class bearing a triazenoazaindole
scaffold was synthesized with the aim of identifying new anti-
proliferative agents. Compounds 5a–g and 6a–c were
screened against a panel of human tumor cell lines, and two
of them, 5e and 5 f, showed cytotoxicity (GI50 range: 2.2–
8.2 mm) in all cell lines. These two compounds even maintained
their cytotoxicity in some multidrug-resistant cell lines. Flow
cytometry analysis demonstrated their ability to induce cell
death by apoptosis with involvement of lysosomes.
Introduction
Cancer is a growing public problem estimated to have approxi-
mately seven million new incidences per year worldwide. Im-
provements in treatment and prevention have led to a de-
crease in cancer deaths, but the number of new diagnoses
continues to rise. There is substantial and continuing interest
in artificial molecules that bind and interact with DNA. Chemo-
therapeutic agents of the triazene class have been used in the
clinical management of many tumors including brain, leuke-
mia, and melanoma.[1] Dacarbazine, 5-(3,3-dimethyl-1-triazenyl)-
imidazole-4-carboxamide (DTIC), has been one of the most
widely used, as a single drug, to treat malignant melanoma
and Hodgkin tumors resistant to the combination mustargen–
oncovin–procarbazine–prednisone (MOPP) therapy.[2] The
potent antitumor activity shown by dacarbazine led to the de-
velopment of aryl and heteroaryl triazeno derivatives.[3] Among
the derivatives belonging to the latter class of compounds,
azole derivatives were the most important. In fact, triazenotria-
zoles,[4] –pyrazoles,[5] and -imidazoles[6] were active against sev-
eral types of tumors and, within these series, the heterocyclic
moiety seemed to modulate the antitumor activity: the more
electron-rich the heterocyclic ring is, the higher is the potency.
Thus, we synthesized 3-triazenopyrrole of type 1,[7] the most
electron-rich of the azoles, which showed cytotoxic activity
against Friend erythroleukemia cells (FLC) in the range of 1.1–
3.1 mm and a mutagenic effect on Streptomyces coelicolor.[8]
Benzocondensation on this series led to 3-triazenoindoles of
type 2 which were 20–40-fold more active than the pyrrole de-
rivatives against erythroleukemia and multidrug-resistant cells
with IC50 values of 0.053–0.080 mm and 0.10–0.14 mm, respec-
tively. This activity is similar to that of doxorubicin, even if
there is no structural relationship.[9] Moreover, 2-triazenopyr-
roles of type 3 proved to be cytotoxic against leukemia, lym-
phoma, and carcinoma with an IC50 value of 3.9–21 mm[10] and
[a] Prof. P. Diana, Dr. A. Stagno, Prof. P. Barraja, Dr. A. Carbone, Dr. B. Parrino,
Prof. G. Cirrincione
Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari
Sezione di Chimica Farmaceutica e Biologica
Universitꢀ degli Studi di Palermo
Via Archirafi 32, 90123 Palermo (Italy)
Fax: (+39)091-238-60-854
E-mail: diana@unipa.it
[b] Prof. F. Dall’Acqua, Prof. D. Vedaldi, Dr. A. Salvador
Dipartimento di Scienze Farmaceutiche
Universitꢀ degli Studi di Padova
Via Marzolo 5, 35131 Padova (Italy)
[c] Dr. P. Brun, Prof. I. Castagliuolo
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche
Universitꢀ degli Studi di Padova
Via Gabelli 63, 35121 Padova (Italy)
[d] Prof. O. G. Issinger
Institute for Biochemistry and Molecular Biology
University of Southern Denmark
Campusvej 55, 5230 Odense M (Denmark)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201100027.
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P. Diana et al.
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the corresponding benzofused triazenes of type 4 showed ac-
tivity on breast, ovarian, and CNS cancers with IC50 values in
the range of 23–36 mm.[11]
Table 1. 2-(3,3-Dialkyltriaz-1-en-1-yl)-1H-pyrido[2,3-c]pyrroles 5a–g and 2-
(3,3-dialkyltriaz-1-en-1-yl)-1H-pyrido[3,2-b]pyrroles 6a–c.
Recently, within the framework of a novel tumor targeting
concept, it was demonstrated that the potency of triazenes
could be enhanced by appending them to the classical struc-
ture of inhibitors of the epidermal growth factor receptor
(EGFR) tyrosine kinase. This may ultimately confer new indica-
tions in refractory tumors expressing this oncogene. For exam-
ple, Bcr-Abl is a constitutively activated tyrosine kinase that is
known to be the cause of chronic myelogenous leukemia
(CML), a malignancy of hematopoietic stem cells.[12] Moreover,
it has been shown that Bcr-Abl induces resistance to cytotoxic
drugs by upregulating DNA repair mechanisms. These results
lend support to strategies that seek to combine a DNA damag-
ing agent and a Bcr-Abl inhibitor into a single molecule to
induce enhanced potency in CML cells.[13] It was demonstrated
that some triazene derivatives inhibited the EGF receptor at
the level of ATP binding.[14]
Compd
X
Y
R
Yield [%]
5a
5b
5c
5d
5e
5 f
5g
6a
6b
6c
CH
CH
CH
CH
CH
CH
CH
N
N
N
N
N
N
N
N
CH
CH
CH
Et
53
53
48
46
52
49
59
45
43
43
RÀR: -(CH2)4-
iPr
Me
Bu
Bn
RÀR: -(CH2)5-
Et
RÀR: -(CH2)4-
iPr
N
N
Considering the interest in triazene chemistry, and conse-
quently in their biological properties, and the inhibitory activity
of EGFR by several heterocyclic systems such as pyrroles, in-
doles, and diazaindoles,[15] we extended our research to include
the synthesis of a new triazene class, having an azaindole nu-
cleus. Our aim was to evaluate their anti-neoplastic activity,
both as alkylating agents and potential EGFR kinase inhibitors.
In particular, we focused our attention on the synthesis of 2-
(3,3-dialkyltriaz-1-en-1-yl)-1H-pyrido[2,3-c]pyrroles of type
5
and 2-(3,3-dialkyltriaz-1-en-1-yl)-1H-pyrido[3,2-b]pyrroles of
type 6.
Scheme 1. Synthesis of 2-(3,3-dialkyltriaz-1-en-1-yl)-1H-pyrido[2,3-c]pyrroles 5
and 2-(3,3-dialkyltriaz-1-en-1-yl)-1H-pyrido[3,2-b]pyrroles 6. Reagents and con-
ditions: a) AcOH, NaNO2, 08C, RT, N2 atmosphere, 1 h, then Na2CO3/H2O, 08C,
RT, N2 atmosphere; b) CH2Cl2, RT, N2 atmosphere, 22 h.
Biology
Results and Discussion
Antiproliferative activity
Synthesis of triazenopyridopyrroles 5 and 6
Cytotoxicity was determined by the MTT test after 72 h of
treatment with compounds in five human tumor cell lines:[17]
Jurkat (T-cell leukemia); K-562 (chronic myeloid leukemia); CEM
(T-cell leukemia); LoVo (colon carcinoma), and A-431 (vulvar
squamous cell carcinoma). Table 2 shows the extent of cell sur-
vival expressed as GI50, the concentration which induces 50%
of inhibition of cell growth after 72 h of cell incubation in the
presence of the test compounds. Dacarbazine was used as ref-
erence compound because of its structural similarity with tria-
zenopyridopyrrolo derivatives.
2-Triazenopyridopyrroles of type 5 and 6 were prepared in two
steps starting from the 2-amino-3-ethoxycarbonylpyrrolopyri-
dines 7 or 8. The 2-aminopyridopyrroles 7 and 8 were convert-
ed, in nearly quantitative yields (96–98%), into the correspond-
ing 2-diazo-3-ethoxycarbonylpyridopyrroles 9 and 10, by diazo-
tization in acetic acid with a stoichiometric amount of sodium
nitrite under nitrogen atmosphere in the dark followed by neu-
tralization with sodium carbonate. The strict control of the
temperature at 08C both during diazotization and neutraliza-
tion is crucial to obtain a high yield.[16] The 2-(3,3-dialkyltriaz-1-
en-1-yl)-1H-pyrido[2,3-c]pyrroles 5a–g and 2-(3,3-dialkyltriaz-1-
en-1-yl)-1H-pyrido[3,2-b]pyrroles 6a–c were obtained in accept-
able yields (43–59%; see Table 1) through coupling reaction,
with a large excess (ratio 1:10), of the suitable secondary
amines 11 a–g in anhydrous dichloromethane in the dark at
room temperature under nitrogen atmosphere or with bub-
bling anhydrous dimethylamine (Scheme 1). The reaction was
carried out until the diazo stretch band (~2100 cmÀ1) disap-
peared (22 h).
Compounds 5e and 5 f showed good cytotoxicity in all
tumor cells and they presented GI50 values lower than 10 mm in
all test cell lines. These last two compounds are characterized
by highly hydrophobic substituents at the nitrogen at posi-
tion 3 of the triazene group such as benzyl and butyl groups.
Other compounds showed cytotoxic activity at the concentra-
tions used only toward the EGFR-overexpressing cell line (A-
431),[18] and this fact could be linked to an inhibitory action
toward EGFR. Dacarbazine was not cytotoxic in vitro, as it re-
quires in vivo hepatic enzymatic activation.[1c]
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Antitumor Triazeno Derivatives
results indicate that the compounds inhibited long-term sur-
vival of A-431 cells at micromolar concentrations.
Table 2. Growth inhibitory activity of triazenopyridopyrroles 5 and 6
against a panel of human tumor cell lines.
GI50 [mm]
Jurkat[a]
>20
>20
>20
>20
2.2Æ0.4
2.5Æ0.3
>20
K-562[a]
>20
>20
>20
>20
5.5Æ0.4
8.2Æ0.8
>20
CEM[a]
LoVo[a]
>20
>20
>20
>20
3.3Æ0.6
4.5Æ0.5
>20
A-431[a]
Evaluation of cell death
Compd
5a
5b
5c
5d
5e
5 f
5g
6a
6b
6c
>20
12.6Æ2.7
14.8Æ2.5
11.5Æ1.4
5.1Æ1.4
4.8Æ0.6
4.4Æ0.9
The evaluation of the mode of cellular death was carried out
with the two most cytotoxic compounds: 5e and 5 f. We per-
formed a flow cytometry test that allows us to investigate the
kind of cellular death (necrosis or apoptosis) induced by 2-tria-
zenopyridopyrroles. Apoptosis is characterized by a variety of
morphological changes, for example, in the plasma membrane.
In apoptotic cells, the phospholipid phosphatidylserine (PS) is
early translocated from the inner to the outer leaflet of the
plasma membrane, thereby exposing PS to the external cellular
environment. Annexin V is a Ca2+-dependent phospholipid
binding protein with high affinity for PS and it easily binds to
cells with exposed PS. We performed biparametric cytofluori-
metric analysis using propidium iodide (PI) and Annexin V–FITC
conjugates, which stain DNA and PS residues, respectively.[21]
Experiments were performed in Jurkat cells after 6 and 24 h
of incubation with the chosen compounds. After 6 h of incuba-
tion, no significant changes in Annexin-positive cells and cellu-
lar survival were detected with respect to control, whereas
after 24 h, a concentration-dependent increase of late apoptot-
ic cells was observed (Figure 2).
>20
>20
>20
4.9Æ0.8
5.1Æ0.7
>20
>20
>20
>20
12.6Æ1.1 >20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
7.6Æ0.9
>20
10.9Æ1.5
>20
DITC[b] >20
[a] Concentration causing 50% growth inhibition of tumor cells after 72 h
incubation; values are the mean ÆSEM of at least three independent ex-
periments. [b] DITC=dacarbazine.
Cytotoxicity in multidrug-resistant cell lines
One of the main problems of cancer chemotherapy is the
onset of tumor cell resistance, which can derive from different
mechanisms. Among these mechanisms, one is the overexpres-
sion of P-glycoprotein, which increases cell efflux of antitumor
drugs. The cytotoxicity of the two most cytotoxic triazenopyri-
dopyrroles was also evaluated in two resistant cell lines, be-
cause of the importance of resistance phenomena in the effica-
cy of cancer therapy: CEMVbl100, a T-cell leukemia line selected
under continuous treatment with vinblastine which shows a
classical multidrug-resistance phenotype and overexpresses
the mdr1 gene,[19] and LoVoDoxo, a colon adenocarcinoma cell
line resistant to doxorubicin and to a number of intercalating
agents such as various anthracyclines, mitoxantrone, and ame-
tantrone.[20] As shown in Table 3, the examined compounds are
almost equally potent toward cells resistant to vinblastine and
doxorubicin relative to the parent cell lines.
Evaluation of cell cycle
Cells were fixed and labeled with PI. This analysis was per-
formed after 24 h of treatment of Jurkat cells with 5e and 5 f.
The results are presented in Table 4 and are expressed as per-
centage of cells in each cell-cycle phase for every sample, ex-
amples of cell-cycle analysis are shown in Figure 3. The most
important change in the cell-cycle profile following incubation
with triazenopyridopyrroles is the onset of a novel peak with
lower DNA content than the G1 peak. This new peak, often re-
ferred as sub-G1, is found in cells
undergoing apoptosis. Moreover,
Table 3. Effect of triazenopyridopyrroles against various drug-resistant cell lines.
GI50 [mm]
the percentages of the sub-G1
peak in treated cells are consis-
tent with the amount of dead
cells found in the previous ex-
periment. Thus, triazeno deriva-
tives were able to induce cell
death by apoptosis.
Compd
CEM
CEMVbl100
LoVo
LoVoDoxo
5e
5 f
Vbl[c]
Doxo[e]
4.9Æ0.8[a]
5.1Æ0.7[a]
0.004Æ0.0002[a]
ND[d]
8.6Æ0.9[a] (1.7)[b]
9.5Æ1.5[a] (1.9)[b]
0.21Æ0.03[a] (525)[b]
ND[d]
3.3Æ0.6[a]
4.5Æ0.5[a]
ND[d]
0.12Æ0.03[a]
6.1Æ0.9[a] (1.8)[b]
7.1Æ1.4[a] (1.6)[b]
ND[d]
13.5Æ0.12[a] (112.5)[b]
[a] Concentration causing 50% growth inhibition of tumor cells after 72 h of incubation; values are the mean
ÆSEM of at least three independent experiments. [b] Values in brackets are fold resistance, indicating the de-
crease potency of the compound in the resistant cell lines. [c] Vbl=vinblastine. [d] ND=not determined.
[e] Doxo=doxorubicin.
Involvement of organelles in
cellular death
Some cytofluorimetric experi-
ments were performed to check
Colony formation assay
the involvement of some organelles such as mitochondria and
lysosomes in cellular death induced by triazenopyridopyrroles.
One typical feature of mitochondrial dysfunction during cell
death is the loss of mitochondrial membrane potential. This
loss was checked by 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylben-
zimidazole carbocyanine (JC-1) cell staining after 24 h of Jurkat
Treating cells with the most active compounds for 24 h de-
creased the growth of colonies (7–14 days) in the clonogenic
survival assay in a concentration-dependent manner. Exposure
of cells to compounds 5e and 5 f at 50 and 20 mm almost com-
pletely eliminated the formation of colonies (Figure 1). These
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Figure 2. Exposure of phosphatidylserine and analysis of PI staining after
a) 6 and b) 24 h of Jurkat cell treatment with 5e and 5 f at 5 or 10 mm. Cells
were stained with Annexin V–FITC (FL1) and of PI (FL3) and analyzed by flow
cytometry; the percentages are expressed as the mean of three independ-
ent experiments. A+/PIÀ: fluorescence in FL-1, no fluorescence in FL-3;
A+/PI+: fluorescence in FL-1 and FL-3.
uptake method described by Zhao et al.[23] Although mitochon-
dria seemed to be only marginally involved in apoptosis prop-
agation, lysosomes played a major role in inducing cell death
as can be observed by the concentration-dependent increase
of lysosomal rupture in Figure 4.
Figure 1. Colony formation assays were performed as described in the Ex-
perimental Section. a) Results are represented as percent colony formation
relative to untreated cells (0 mm=100%; 5e: white bars, 5 f: hashed bars);
error bars represent the mean ÆSEM of three independent experiments.
The images below show representative wells of colonies stained with crystal
violet after treatment with the indicated concentrations of b) 5e or c) 5 f.
EGFR inhibition
Table 4. Cell-cycle analysis after 24 h of treatment with triazenopyrido-
pyrroles.[a]
The human vulvar cancer cell line A-431, which overexpresses
EGFR, was used to investigate whether these compounds
could block EGF receptor signaling as many triazenopyridopyr-
roles were cytotoxic only toward this kind of cell line. After
treating these cells with compounds for three hours, the block-
age in EGF receptor signaling was detected by Western blot-
ting using phospho-specific antibodies against the EGF recep-
tor and its downstream signaling molecule ERK1/2. As a posi-
tive control, some cell samples were treated with a known
EGFR inhibitor (AG1478). In Figure 5, a clear decrease in pEGFR
and pERK1/2 signals was observed after treatment with
AG1478, as a consequence of the inhibition of the activity of
EGFR. In contrast, no band intensity decrease was detected in
Compd
G1 [%]
S [%]
G2/M [%]
sub-G1 [%]
Control
63.0
34.5
43.7
44.0
49.1
14.5
9.8
11.4
12.0
12.5
20.9
20.6
15.1
14.0
15.4
1.6
35.1
29.8
30.0
23.0
5e (10mm)
5e (5mm)
5 f (10mm)
5 f (5mm)
[a] Data indicate the percentage of cells in each phase.
cell treatment with compounds as described by Salvioli et al.[22]
Lysosome involvement in cell death was also examined by
staining Jurkat cells with acridine orange (AO), following the
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Antitumor Triazeno Derivatives
Figure 3. Representative cell-cycle profile of Jurkat cells incubated for 24 h
with 5e and 5 f at 5 and 10 mm. Cells were fixed and labeled with PI and an-
alyzed by flow cytometry as described in the Experimental Section. a) con-
trol, b) 10 mm 5e, c) 5 mm 5e, d) 10 mm 5 f, e) 5 mm 5 f.
Figure 4. Involvement of organelles in cell death induced by triazenopyrido-
pyrroles. a) Percent of cells with loss of mitochondrial membrane potential
as measured by JC-1 staining after 24 h treatment of Jurkat cells with 5e or
5 f at 5 and 10 mm. b) Percent of cells with lysosomal rupture as measured
by flow cytometry AO fluorescence after 24 h treatment of Jurkat cells with
5e or 5 f. Percentages are expressed as the mean ÆSEM of at least three ex-
periments.
triazeno derivatives samples relative to the EGF-stimulated
control. Thus, the hypothesis is that EGFR is not the target of
triazenopyridopyrrole compounds.
Evaluation of alkylating activity
As a dacarbazine mechanism is linked to covalent binding to
DNA, the alkylation activity of some triazeno derivatives (5d–
5f) was evaluated using a colorimetric assay, in which 4-(4’-ni-
trobenzyl)pyridine (NBP) was used as an alkylation substrate.[24]
Alkylating compounds react with the pyridine nucleophilic ni-
trogen in a basic environment and the reaction can be ob-
served by an absorbance increase. Triazenopyridopyrroles were
not able to induce an absorbance increase at 545 nm; more-
over, spectrophotometric measurements to check the stability
in aqueous medium did not reveal any chemical decomposi-
tion of the compound (see, for example, the behavior of 5e in
Figure 6).
Figure 5. Cells were starved overnight and treated for 10 min with EGF
(100 ngmLÀ1) after 3 h of compound incubation. Cell lysates (100 mg) were
separated by SDS-PAGE and the membrane probed with phospho-specific
antibodies against the EGF receptor (P-Tyr1173) or ERK1/2 (P-Thr202/P-
Tyr204). In addition, the membranes were probed with antibodies against
total EGFR. a) 1: control without EGF, 2: control+EGF, 3: 5 mm AG1478+EGF,
4: 20 mm 5a+EGF, 5: 20 mm 5b+EGF, 6: 20 mm 5c+EGF; b) 1: control with-
out EGF, 2: control+EGF, 3: 20 mm 5e+EGF, 4: 5 mm AG1478+EGF, 5:
20 mm 5 f+EGF, 6: 20 mm 6a+EGF, 7: 20 mm 6c+EGF.
Conclusions
Herein we describe the synthesis and the antiproliferative
properties of new derivatives having a triazenopyridopyrrole
structure. Their antiproliferative activity was evaluated through
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they are cytotoxic in vitro without activation by liver micro-
somes.
As many triazeno derivatives were cytotoxic only toward the
EGFR-overexpressing cell line and some triazene derivatives
were found to be ATP competitive EGFR inhibitors, we also
checked their possible EGFR inhibition. However, EGFR is not
the target of triazeno derivatives. Further investigations are
needed to identify the molecular target and are in progress.
Experimental Section
Chemistry
General: All melting points were taken on a Bꢁchi-Tottoli capillary
apparatus and are uncorrected; IR spectra were determined in bro-
moform with a Jasco FT/IR 5300 spectrophotometer; 1H and
13C NMR spectra were measured at 200 and 50.3 MHz, in CDCl3 so-
lution, using a Bruker Avance II series 200 MHz spectrometer
[(CH3)4Si as internal reference]. Column chromatography was per-
formed with Merck silica gel 230–400 mesh ASTM or with Bꢁchi Se-
pacore chromatography module (pre-packed cartridge system). Ele-
mental analyses (C, H, N) were within Æ0.4% of the theoretical
values.
Figure 6. Representative example of compound stability in aqueous solu-
tion. The aqueous stability of 5e (5 mm in PBS) at 378C at various time
points is shown. The stability was verified by UV/Vis spectroscopy.
the classical MTT test in different human tumor cell lines and
two of them, 5e and 5 f, were cytotoxic in all cell lines investi-
gated (GI50 range: 2.2–8.2 mm). These two compounds are char-
acterized by the presence of two identical hydrophobic sub-
stituents with great steric hindrance at position 3 of the tria-
zene group. As the MTT test only measures the cellular meta-
bolic state, the antiproliferative effect of the two most active
compounds was also checked using the colony formation
assay, confirming their cytotoxicity.
2-Diazo-3-ethoxycarbonylpyrrolo[2,3-c]pyridine (9) and 2-diazo-
3-ethoxycarbonylpyrrolo[3,2-b]pyridine (10) were prepared as
previously described[17] by reacting a solution of 2-amino-3-ethoxy-
carbonylpyrrolopyridine 7 and 8 (0.62 g, 3 mmol) in glacial acetic
acid (6 mL) with a solution of sodium nitrite (0.21 g, 3 mmol) in a
small amount of water (1 mL). The reaction was carried out at 08C
under a nitrogen atmosphere. The mixture was neutralized at 08C
with saturated Na2CO3 and the yellow solid precipitate was filtered
off and dried under vacuum in the dark. The crude products, quick-
ly shaken in cyclohexane and filtered off, gave 2-diazopyrrolopyri-
dine 9 and 10.
One of the main problems of tumor treatment with chemo-
therapeutic drugs, together with the severe side effects, is the
initiation of resistance, which is often mediated by the overex-
pression of the P-glycoprotein drug efflux pump. The most
active compounds 5e and 5 f also exhibited cytotoxic activity
against drug-resistant cell lines (CEMVbl100 and LovoDoxo), which
overexpress that drug efflux pump. These results are promising
as they suggest that these compounds might be useful in the
treatment of drug refractory tumors.
General method for the synthesis of 2-triazen-1-yl-1H-pyrido-
[2,3-c]pyrrolo-3-carboxylates (5a–g) and 2-triazen-1-yl-1H-
pyrido[3,2-b]pyrrolo-3-carboxylates (6a–c). A solution of secon-
dary amine (30 mmol) in anhydrous CH2Cl2 (20 mL) or bubbling an-
hydrous dimethylamine was added to a stirred solution of 2-diazo-
pyrrolopyridine 9 or 10 (0.65 g, 3 mmol) in anhydrous CH2Cl2
(30 mL). The reaction mixture was kept in the dark at RT and under
nitrogen atmosphere until the diazo stretch band at ~2100 cmÀ1
disappeared (22 h). Removal of the solvent and the excess of
amine, under reduced pressure, gave the 2-triazenopyrrolopyri-
dines 5 or 6, respectively. The crude products were purified by
column chromatography on silica gel using CH2Cl2/EtOAc (95:5) as
eluent.
The treatment of Jurkat cells with these derivatives caused
cell death by apoptosis, as determined by the loss of mem-
brane asymmetry (Annexin V positivity). Moreover, the cell-
cycle effects of triazenopyridopyrroles were investigated to
confirm the previous test results and to gain insight into their
potential mechanism of action. Although the onset of the sub-
G1 peak was detected as a consequence of apoptotic machi-
nery activation, there were no other clear changes in cell-cycle
profiles of treated samples. Lysosomes seemed to be involved
in apoptosis induction, whereas mitochondria had only a mar-
ginal role.
Ethyl
2-(3,3-diethyl-1-triazenyl)-1H-pyrido[2,3-c]pyrrolo-3-car-
boxylate (5a): yellow powder (0.46 g, 53%): Rf =0.30 (CH2Cl2/
MeOH 9:1); mp: 173–1748C; 1H NMR (200 MHz, CDCl3): d=1.32–
1.43 (m, 9H, 3ꢂCH3), 3.89–3.94 (m, 4H, 2ꢂCH2), 4.36–4.43 (m, 2H,
CH2), 8.02 (d, J=5.5 Hz, 1H, H-4), 8.25 (d, J=5.5 Hz, 1H, H-5), 8.73
(s, 1H, H-7), 9.50 ppm (s, 1H, NH); 13C NMR (50 MHz, CDCl3): d=
10.9 (q), 14.2 (q), 14.6 (q), 43.0 (t), 50.3 (t), 59.8 (t), 97.3 (s), 115.8
(d), 130.7 (s), 131.4 (d), 134.5 (s), 138.4 (d), 153.9 (s), 164.7 ppm (s);
IR (KBr): n˜ =1681 cmÀ1 (CO); Anal. calcd (%) for C14H19N5O2: C 58.12,
H 6.62, N, 24.21%; found: C 58.41, H 6.80, N 24.08.
A possible alkylating activity was evaluated through a colori-
metric method but triazenopyridopyrroles were found to be
devoid of alkylating properties in vitro. Thus, the target of tria-
zeno derivatives was not DNA, and this result seemed to be
confirmed by the absence of accumulation of cells in the G2
phase in the cell-cycle analysis. Moreover, 5e and 5 f certainly
have a different mechanism of action than dacarbazine, as
Ethyl
2-(3,3-tetramethylelene-1-triazenyl)-1H-pyrido[2,3-c]pyr-
rolo-3-carboxylate (5b): yellow powder (0.46 g, 53%): Rf =0.26
1
(CH2Cl2/MeOH 9:1); mp: 191–1928C; H NMR (200 MHz, CDCl3): d=
1296
www.chemmedchem.org
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2011, 6, 1291 – 1299

Antitumor Triazeno Derivatives
1.46 (t, J=7.1 Hz, 3H, CH3), 2.05 (t, J=3.3 Hz, 4H, 2ꢂCH2), 3.82 (t,
J=3.3 Hz, 2H, CH2), 3.92 (t, J=3.3 Hz, 2H, CH2), 4.43 (q, J=7.1 Hz,
2H, CH2), 7.98 (d, J=5.4 Hz, 1H, H-4), 8.31 (d, J=5.4 Hz, 1H, H-5),
8.68 (s, 1H, H-7), 9.48 ppm (s, 1H, NH); 13C NMR (50 MHz, CDCl3):
d=14.6 (q), 23.3 (t), 23.9 (t), 47.9 (t), 52.0 (t), 59.8 (t), 97.0 (s), 115.9
(d), 130.5 (s), 132.5 (d), 133.5 (s), 140.2 (d), 153.4 (s), 164.8 ppm (s);
IR (KBr): n˜ =1980 (NH), 1671 cmÀ1 (CO); Anal. calcd (%) for
C14H17N5O2: C 58.52, H 5.96, N 24.37%; found: C 58.34, H 5.59, N
25.54.
1H, NH); 13C NMR (50 MHz, CDCl3): d=14.6 (q), 23.9 (t), 24.6 (t),
26.4 (t), 44.6 (t), 53.9 (t), 59.8 (t), 97.2 (s), 115.8 (d), 130.6 (s), 132.9
(d), 133.3 (s), 140.5 (d), 152.9 (s), 164.7 ppm (s); IR (KBr): n˜ =2937
(NH), 1675 cmÀ1 (CO); Anal. calcd (%) for C15H19N5O2: C 59.79, H
6.36, N 23.24; found: C 60.15, H 6.57, N 22.93.
Ethyl
2-(3,3-diethyl-1-triazenyl)-1H-pyrido[3,2-b]pyrrolo-3-car-
boxylate (6a): yellow powder (0.37 g, 45%): Rf =0.20 (CH2Cl2/
MeOH 9:1); mp: 167–1688C; 1H NMR (200 MHz, CDCl3): d=1.28–
1.42 (m, 9H, 3ꢂCH3), 3.76–3.97 (m, 4H, 2ꢂCH2), 4.43 (q, J=7.1 Hz,
2H, CH2), 7.04 (dd, J=4.8, 8.1 Hz, 1H, H-6), 7.52 (dd, J=1.4, 8.1 Hz,
1H, H-7), 8.48 (dd, J=1.4, 4.8 Hz, 1H, H-5), 9.45 ppm (s, 1H, NH);
13C NMR (50 MHz, CDCl3): d=11.0 (q), 14.3 (q), 14.7 (q), 42.8 (t),
49.9 (t), 59.8 (t), 98.3 (s), 117.2 (d), 117.4 (d), 127.0 (s), 144.1 (d),
145.5 (s), 152.0 (s), 164.2 ppm (s); IR (KBr): n˜ =2973 (NH), 1683 cmÀ1
(CO); Anal. calcd (%) for C14H19N5O2: C 58.12, H 6.62, N 24.21;
found: C 58.37, H 6.48, N 24.40.
Ethyl
2-(3,3-diisopropyl-1-triazenyl)-1H-pyrido[2,3-c]pyrrolo-3-
carboxylate (5c): yellow powder (0.46 g, 48%): Rf =0.23 (CH2Cl2/
MeOH 9:1); mp: 254–2558C; 1H NMR (200 MHz, CDCl3): d=1.35–
1.45 (m, 15H, 5ꢂCH3), 4.17 (sept, J=6.5 Hz, 1H, CH), 4.41 (q, J=
7.1 Hz, 2H, CH2), 5.38 (sept, J=6.7 Hz, 1H, CH), 7.97 (d, J=5.4 Hz,
1H, H-4), 8.30 (d, J=5.4 Hz, 1H, H-5), 8.63 (s, 1H, H-7), 9.39 ppm (s,
1H, NH); 13C NMR (50 MHz, CDCl3): d=14.6 (q), 19.0 (qꢂ2), 23.5
(qꢂ2), 49.0 (d), 51.2 (d), 59.6 (t), 96.8 (s), 115.7 (d), 130.4 (s), 132.8
(d), 133.5 (s), 140.7 (d), 152.9 (s), 164.8 ppm (s); IR (KBr): n˜ =2975
(NH), 1708 cmÀ1 (CO); Anal. calcd (%) for C16H23N5O2: C 60.55, H
7.30, N 22.07; found: C 60.84, H 7.08, N 22.21.
Ethyl 2-(3,3-tetramethylelene 1-triazenyl)-1H-pyrido[3,2-b]pyr-
rolo-3-carboxylate (6b): yellow powder (0.37 g, 43%): Rf =0.18
1
(CH2Cl2/MeOH 9:1); mp: 164–1658C; H NMR (200 MHz, CDCl3): d=
1.41 (t, J=7.1 Hz, 3H, CH3), 2.05–2.10 (m, 4H, 2ꢂCH2), 3.84 (t, J=
6.4 Hz, 2H, CH2), 3.94 (t, J=6.2 Hz, 2H, CH2), 4.45 (q, J=7.1 Hz, 2H,
CH2), 7.06 (dd, J=4.8, 8.1 Hz, 1H, H-6), 7.53 (dd, J=1.3, 8.1 Hz, 1H,
H-7), 8.49 (dd, J=1.3, 4.8 Hz, 1H, H-5), 9.49 ppm (s, 1H, NH);
13C NMR (50 MHz, CDCl3): d=14.7 (q), 23.4 (t), 23.9 (t), 47.8 (t), 51.8
(t), 59.9 (t), 98.2 (s), 117.3 (d), 117.5 (d), 127.0 (s), 144.0 (d), 145.3 (s),
152.5 (s), 164.3 ppm (s); IR (KBr): n˜ =1685 cmÀ1 (CO); Anal. calcd
(%) for C14H17N5O2: C 58.52, H 5.96, N 24.37; found: C 58.69, H 5.72,
N 24.21.
Ethyl 2-(3,3-dimethyl-1-triazenyl)-1H-pyrido[2,3-c]pyrrolo-3-car-
boxylate (5d): yellow powder (0.36 g, 46%): Rf =0.69 (CH2Cl2/
MeOH 9:1); mp: 186–1878C; H NMR (200 MHz, CDCl3): d=1.45 (t,
J=7.1 Hz, 3H, CH3), 3.39 (s, 3H, CH3), 3.58 (s, 3H, CH3), 4.42 (q, J=
7.1 Hz, 2H, CH2), 7.98 (d, J=5.5 Hz, 1H, H-4), 8.31 (d, J=5.5 Hz, 1H,
H-5), 8.67 (s, 1H, H-7), 9.56 ppm (s, 1H, NH); 13C NMR (50 MHz,
CDCl3): d=14.6 (q), 37.1 (q), 43.9 (q), 59.8 (t), 97.4 (s), 115.9 (d),
130.4 (s), 132.9 (d), 133.2 (s), 140.5 (d), 152.4 (s), 164.7 ppm (s); IR
(KBr): n˜ =3444 (NH), 1685 cmÀ1 (CO); Anal. calcd (%) for C12H15N5O2:
C 55.16, H 5.79, N 26.80; found: C 55.48, H 5.44, N 26.96.
1
Ethyl 2-(3,3-diisopropyl-1-triazenyl)-1H-pyrido[3,2-b]pyrrolo-3-
carboxylate (6c): yellow powder (0.41 g, 43%): Rf =0.15 (CH2Cl2/
1
MeOH 9:1); mp: 154–156.08C; H NMR (200 MHz, CDCl3): d=1.32–
Ethyl 2-(3,3-dibuthyl-1-triazenyl)-1H-pyrido[2,3-c]pyrrolo-3-car-
boxylate (5e): yellow powder (0.55 g, 52%): Rf =0.45 (CH2Cl2/
MeOH 9:1); mp:147–1488C; 1H NMR (200 MHz, CDCl3): d=0.88–
1.00 (m, 7H), 1.34–1.47 (m, 7H), 1.65–1.78 (m, 4H), 2.84–2.92 (m,
1H), 3.72–3.91 (m, 3H), 4.41 (q, J=7.1 Hz, 2H, CH2), 8.01 (d, J=
5.5 Hz, 1H, H-4), 8.26 (d, J=5.5 Hz, 1H, H-5), 8.69 (s, 1H, H-7),
9.60 ppm (s, 1H, NH); 13C NMR (50 MHz, CDCl3): d=13.7 (q), 13.8
(q), 14.6 (q), 19.9 (t), 20.6 (t), 27.7 (t), 30.9 (t), 48.2 (t), 55.5 (t), 59.7
(t), 97.3 (s), 115.8 (d), 130.2 (s), 132.7 (d), 133.6 (s), 140.8 (d), 152.2
(s), 164.7 ppm (s); IR (KBr): n˜ =3438 (NH), 1675 cmÀ1 (CO); Anal.
calcd (%) for C18H27N5O2: C 62.58, H 7.88, N 20.27; found: C 62.47,
H 8.12, N, 19.97.
1.40 (m, 15H, 5ꢂCH3), 4.12 (sept, J=6.6 Hz, 1H, CH), 4.41 (q, J=
7.1 Hz, 2H, CH2), 5.39 (sept, J=6.8 Hz, 1H, CH), 7.03 (dd, J=4.8,
8.0 Hz, 1H, H-6), 7.55 (dd, J=1.3, 8.0 Hz, 1H, H-7), 8.46 (dd, J=1.3,
4.8 Hz, 1H, H-5), 9.53 ppm (s, 1H, NH); 13C NMR (50 MHz, CDCl3):
d=14.7 (q), 19.1 (qꢂ2), 23.5 (qꢂ2), 48.8 (d), 50.9 (d), 59.8 (t), 97.7
(s), 117.1 (d), 117.6 (d), 127.1 (s), 143.7 (d), 145.3 (s), 152.6 (s),
164.3 ppm (s), IR (KBr): n˜ =3315 (NH), 1691 cmÀ1 (CO); Anal. calcd
(%) for C16H23N5O2: C 60.55, H 7.30, N 22.07; found: C 60.85, H 7.15,
N 22.00.
Biology
Ethyl
2-(3,3-dibenzyl1-triazenyl)-1H-pyrido[2,3-c]pyrrolo-3-car-
Chemicals: If not otherwise indicated, all the chemicals were pur-
chased from Sigma–Aldrich (Milan, Italy).
boxylate (5 f): yellow powder (0.61 g, 49%): Rf =0.34 (CH2Cl2/
MeOH 9:1); mp: 133–1358C; H NMR (200 MHz, CDCl3): d=1.37 (t,
1
J=7.1 Hz, 3H, CH3), 4.39 (q, J=7.1 Hz, 2H, CH2), 4.88 (s, 2H, CH2),
5.04 (s, 2H, CH2), 7.15–7.34 (m, 10H, 10ꢂCH), 8.00 (dd, J=0.9,
5.5 Hz, 1H, H-4), 8.30 (d, J=5.5 Hz, 1H, H-5), 8.68 (d, J=0.9 Hz, 1H,
H-7), 9.46 ppm (s, 1H, NH); 13C NMR (50 MHz, CDCl3): d=14.6 (q),
49.6 (t), 57.8 (t), 59.8 (t), 98.26 (s), 116.0 (d), 127.9 (d), 128.0 (dꢂ2),
128.4 (d), 128.7 (dꢂ2), 129.0 (dꢂ2), 129.3 (dꢂ2), 130.8 (s), 132.9
(d), 133.5 (s), 134.4 (s), 135.0 (s), 140.3 (d), 151.9 (s), 164.6 ppm (s);
IR (KBr): n˜ =3438 (NH), 1683 cmÀ1 (CO); Anal. calcd (%) for
C24H23N5O2: C 69.72, H 5.61, N 16.94; found: C 69.93, H 5.33, N
17.04.
Cell lines: Jurkat cells (human T-cell leukemia), K-562 cells (human
chronic myeloid leukemia), and CEM cells (human T-cell leukemia)
were grown in RPMI-1640 medium; LoVo cells (human intestinal
adenocarcinoma) were grown in Ham’s F12 medium, and A-431
cells (vulvar squamous cell carcinoma EGFR-overexpressing) were
grown in Dulbecco’s modified Eagle’s medium (DMEM). All media
were supplemented with 115 UmLÀ1 penicillin G, 115 mgmLÀ1
streptomycin, and 10% fetal bovine serum (Invitrogen, Milan, Italy).
CEMVbl–100 is a multidrug-resistant line selected against vinblastine
and were grown in complete RPMI-1640 medium supplemented
with vinblastine (0.1 mgmLÀ1).[19] LoVoDoxo is a doxorubicin resistant
subclone of LoVo cells and the cells were grown in complete
Ethyl 2-(3,3-pentamethylelene-1-triazenyl)-1H-pyrido[2,3-c] pyr-
rolo-3-carboxylate (5g): yellow powder (0.53 g, 59%): Rf =0.15
Ham’s
F12
medium
supplemented
with
doxorubicin
1
(CH2Cl2/MeOH 9:1); mp: 198–1998C; H NMR (200 MHz, CDCl3): d=
(0.1 mgmLÀ1).[20]
1.45 (t, J=7.1 Hz, 3H, CH3), 1.72 (s, 6H, 3ꢂCH2), 3.80 (s, 2H, CH2),
4.07 (s, 2H, CH2), 4.42 (q, J=7.1 Hz, 2H, CH2), 7.97 (d, J=5.5 Hz,
1H, H-4), 8.29 (d, J=5.5 Hz, 1H, H-5), 8.68 (s, 1H, H-7), 9.58 ppm (s,
Cellular toxicity: Individual wells of a 96-well tissue culture microtit-
er plate (Falcon, Becton-Dickinson, Italy) were inoculated with
ChemMedChem 2011, 6, 1291 – 1299
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
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P. Diana et al.
MED
100 mL of complete medium containing 5ꢂ103 of the above cited
cells. Plates were incubated at 37 C8 in a humidified 5% incubator
for 24 h prior to the experiments. After medium removal, 100 mL
of the drug solution, dissolved in DMSO and diluted with the suita-
ble complete medium, were added to each well and incubated at
378C for 72 h. DMSO concentration was always lower than 1%.
Cell viability was assayed by the MTT [(3-(4,5-dimethylthiazol-2-yl)-
2,5 diphenyl tetrazolium bromide)] test as described previously.[17]
Analogous experiments were performed with drug-resistant cell
lines.
blocked with 0.2% casein, 0.1% Tween-20 in PBS (blocking buffer)
for 1 h and incubated with polyclonal anti-phospho-EGFR (Tyr1173)
(Santa Cruz), anti-phospho-ERK1/2 (P-p42/p44) (Thr202/Tyr204)
(Cell Signaling) overnight or with anti-EGFR (Santa Cruz) for 2 h.
After washing in blocking buffer, the membranes were incubated
with the proper secondary antibody coupled to alkaline phospha-
tase (Jackson Immunoresearch Laboratories) for 1 h. Visualization
was performed with CDP-star (Tropix) according to the manufac-
turer’s instructions.[25]
Alkylation of 4-(4’-nitrobenzyl)pyridine (NBP): 5 mL of NBP in DMSO
(80 mgmLÀ1) and 5 mL of compounds in DMSO (final concentra-
tions: 10–100 mm) were added to 500 mL of 100 mm phosphate
buffer (pH 7.4). Mixtures were incubated at 378C for 30 min; then,
1 mL of 1-octanol and 0.1 mL 10m NaOH were added. Solutions
were shaken and centrifuged at 10000 g for 2 min. The extend of
alkylation was checked spectrophotometrically by recording the
absorbance of the upper layer at 545 nm.[24]
Colony formation assay: A-431 cells were plated at 1000 per well in
six-well plates to provide an optimal counting density. Cells were
treated with compounds at different concentrations for 24 h. After
24 h, medium was replaced with fresh medium and cells were cul-
tured for one to two weeks until well-defined colonies had formed
(replacing culture medium every two to three days). Cells were
briefly permeabilized with 0.9% saline solution and stained with
0.5% crystal violet in 20% methanol. Colonies of ꢀ50 cells were
then counted visually. Data points represent the average of three
values and error bars represent the SD.
Evaluation of aqueous stability: Solutions of compounds were pre-
pared in PBS and were incubated at 378C. Their stability was
checked by looking for changes in UV/Vis spectra on a PerkinElmer
Lambda 15 spectrophotometer after precise incubation times.
Externalization of phosphatidylserine: Surface exposure of phospha-
tidylserine (PS) by apoptotic cells was measured by flow cytometry
by adding Annexin V–FITC to cells according to the manufacturer’s
instructions (Annexin V Fluos, Roche Diagnostic). Simultaneously,
cells were stained with propidium iodide (PI). Samples were incu-
bated in the dark for 15 min and then analyzed using a BD FACS
Calibur (Becton Dickinson, New York, USA) flow cytometer. The
fluorescence of FITC and PI were collected in FL1 and FL3, respec-
tively.[21] At least 10000 events for each sample were acquired.
Acknowledgements
This work was supported financially by the Ministero dell’Istru-
zione dell’Universitꢀ e della Ricerca (MIUR). This work was also
supported by the Danish Cancer Society (grant 252-1109-210 to
O.G.I.). We thank Tine D. Rasmussen (Institute for Biochemistry
and Molecular Biology, University of Southern Denmark) for ex-
cellent technical assistance.
Cell-cycle analysis: Jurkat cells were incubated for 24 h with or
without the test compounds, then fixed with ice-cooled ethanol
(70%), treated overnight with RNAse (100 mgmLÀ1) in phosphate
saline buffer and finally stained with PI (10 mgmLÀ1). Samples were
analyzed on a BD FACS Calibur flow cytometer. Results of cell-cycle
analysis were examined using WinMDI 2.9 (Windows Multiple
Document Interface for Flow Cytometry).
Keywords: antiproliferative activity · antitumor agents · EGF
receptors · triazeno derivatives
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Mitochondrial dysfunction: Jurkat cells were incubated with or with-
out different concentrations of test compounds for 24 h. Cells were
collected by centrifugation (5 min, RT, 300 g) and resuspended in
Hank’s Balanced Salt Solution (HBSS) containing JC-1 at a concen-
tration of 1 mm. The cytofluorimetric analysis (BD FACS Calibur flow
cytometer) was performed collecting green (FL1) and orange (FL2)
fluorescence in at least 10000 events for each sample.[22]
Lysosome dysfunction: After 24 h of treatment with different con-
centrations of test compounds, Jurkat cells were stained with AO
at a concentration of 1 mm in RPMI-1640 at 378C for 15 min. The
fluorescence was directly recorded with a flow cytometer (BD FACS
Calibur) using 488 nm wavelength as excitation and emission in
the FL3 channel.[23]
Protein extraction and Western blotting: A-431 cells (1ꢂ106) were
seeded in 6 cm Petri dishes; after 24 h, they were washed with PBS
and starved overnight. Then, cells were incubated with or without
20 mm of compounds for 3 h and stimulated with EGF 100 ngmLÀ1
for 10 min. Cell extracts were prepared after scraping in lysis buffer
[50 mm Tris-HCl pH 8.5, 150 mm NaCl, 1% Triton X-100, 10% glyc-
erol, 1 mm DTT, 30 mm NaPPi, 10 mm NaF, 1 mm Na3VO4, 100 nm
okadaic acid, complete protease inhibitors (Roche)], incubating on
ice for 15 min and clearing by centrifugation (20 min, 48C,
16000 g); 100 mg of protein was loaded in 12.5% SDS-PAGE and
then blotted to a PVDF membrane (Bio-Rad). Membranes were
[7] G. Cirrincione, A. M. Almerico, E. Aiello, G. Dattolo, S. Grimaudo, P.
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ChemMedChem 2011, 6, 1291 – 1299

Antitumor Triazeno Derivatives
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Received: January 28, 2011
Revised: March 14, 2011
Published online on April 26, 2011
ChemMedChem 2011, 6, 1291 – 1299
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1299