Pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones, a new class of azolotetrazines with potent antitumor activity

Article abstract of DOI:10.1016/S0968-0896(03)00145-7

Pyrrolo[2,1-d][1,2,3,5]tetrazinones 10a-o, compounds that hold the deaza skeleton of the antitumor drug temozolomide, were prepared by reaction of 2-diazopyrroles 9 and isocyanates. Such a synthetic route represents, among those leading to azolotetrazinones reported so far, the only possible one since attempts to cyclize to the title ring system 2-amino-1-carbamoylpyrroles 11 or the mono substituted 2-triazenopyrrole 12 failed. Compounds 10 were screened at the National Cancer Institute (NCI) for their activity against a panel of about 60 human tumor cell lines. Most of them possess remarkable antineoplastic activity having GI₅₀ values in the low micromolar or sub-micromolar range and reaching, in the case of compound 10d, nanomolar concentrations. The most sensitive cell lines were MDA-N and MDA-MB-435 of the breast sub-panel, and SR, K-562, HL60 (TB) and CCRF-CEM of the leukaemia sub-panel. SAR evaluation and COMPARE computations indicate, for compounds 10, a mechanism of action different from that of temozolomide.

Full text of DOI:10.1016/S0968-0896(03)00145-7

Bioorganic & Medicinal Chemistry 11 (2003) 2371–2380
Pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones, a New Class of
Azolotetrazines with Potent Antitumor Activity
Patrizia Diana, Paola Barraja, Antonino Lauria, Alessandra Montalbano,
Anna Maria Almerico, Gaetano Dattolo and Girolamo Cirrincione*
Dipartimento Farmacochimico Tossicologico e Biologico, Universita` degli Studi di Palermo, Via Archirafi 32, 90123 Palermo, Italy
Received 3 January 2003; accepted 28 February 2003
Abstract—Pyrrolo[2,1-d][1,2,3,5]tetrazinones 10a–o, compounds that hold the deaza skeleton of the antitumor drug temozolomide,
were prepared by reaction of 2-diazopyrroles 9 and isocyanates. Such a synthetic route represents, among those leading to azolo-
tetrazinones reported so far, the only possible one since attempts to cyclize to the title ring system 2-amino-1-carbamoylpyrroles 11
or the mono substituted 2-triazenopyrrole 12 failed. Compounds 10 were screened at the National Cancer Institute (NCI) for their
activity against a panel of about 60 human tumor cell lines. Most of them possess remarkable antineoplastic activity having GI50
values in the low micromolar or sub-micromolar range and reaching, in the case of compound 10d, nanomolar concentrations. The
most sensitive cell lines were MDA-N and MDA-MB-435 of the breast sub-panel, and SR, K-562, HL60 (TB) and CCRF-CEM of
the leukaemia sub-panel. SAR evaluation and COMPARE computations indicate, for compounds 10, a mechanism of action dif-
ferent from that of temozolomide.
#
2003 Elsevier Science Ltd. All rights reserved.
2
Introduction
in a wide range of murine and xenograft tumors, but in
the phase II clinical trials, the recommended dose was
too toxic and a deep platelet damage (thrombocyto-
penia) due to cross-linking of the two strands of the DNA,
The outstanding antineoplastic activity exhibited by two
imidazotetrazinone derivatives, mitozolomide 1 and
temozolomide 2, has attracted remarkable attention on
azolotetrazine systems.
3
compromised its clinical use. The 3-methyl congener,
temozolomide, showed to be less potent but less toxic than
mitozolomide and is currently in the market with the trade
1
name temodal and is used against malignant melanoma,
4
mycosis fungoides, and brain tumors.
Mitozolomide and temozolomide are prodrugs which
undergo, in a nucleophilic micro-environment provided
by sequence of guanine residues together with asso-
ciated water molecules, in the major groove of DNA,
ring opening following the nucleophilic attack at C-4 by
an activated molecule of water to afford a monoalkyl-
triazene species, a bioactive alkylating agent. Such
reactive entity likely undergoes an S 2alkylation of
N
nucleophilic residues in the immediate vicinity such as
N-7 and/or O-6 sites of guanine eliminating nitrogen
5
and 5-aminoimidazole-4-carboxamide.
1
Mitozolomide, synthesized in 1980, is the first azolo-
tetrazine derivative to show excellent antitumor effects
Azolotetrazinones have been prepared through the syn-
thetic routes shown in the Scheme 1. The first route,
starting from the diazoazoles 4, obtained by diazotiza-
tion followed by neutralization of the corresponding
*
6
Corresponding author. Tel.: +39-091-6161606; fax: +39-091-
169999; e-mail: gcirrinc@unipa.it
0
968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00145-7

2372
P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
intermediate which undergoes ring closure, since the
reaction is accelerated using polar solvents such as hex-
1
,7
amethylphosphoramide as the reaction medium.
Later on, alternative syntheses of azolotetrazinones
were elaborated. In fact, it was proposed the reaction of
azoloamines 3 with isocyanates or carbamoyl chlorides
or chloroformates/amines to give the carbamoyl azole
derivatives of type 6. The last two reagents allow, in the
preparation of temozolomide, to avoid the use of the
volatile and very dangerous methylisocyanate, but only
the former has preparative interest. Nitrosation of the
intermediates 6 followed by intramolecular coupling led
to the azolotetrazinones 5. This synthetic pathway has
the advantage to avoid the use of diazoazoles, com-
pounds notoriously photo and thermo labile and diffi-
cult to handle. By the intermediacy of the carbamoyl
9
derivatives 6 were synthesised pyrazolotetrazinones 5a
1
0
and imidazotetrazinones 5d. The yields, in pyrazole
series, with some exception, were as good as those
observed in the preceding method; in imidazole series,
instead, this method leads to the final products with
lower yields, sometimes with no preparative interest.
A further synthesis involves the reaction of the diazo-
azoles 4 with primary amines to give the mono sub-
stituted triazenes 7 which undergo carbonylation with
phosgene or phosgene equivalents leading to the bicyclic
azole systems 5. By this route were obtained pyr-
9
,10
azolotetrazinones 5a but not imidazotetrazinones 5d.
The pyrrolo[2,1-d][1,2,3,5]tetrazine ring system 5e, hav-
ing the deaza skeleton of temozolomide, has been pre-
pared by reaction of 2-diazopyrroles 4e with
1
1
isocyanates. The key intermediates for this synthesis
are the 2-diazopyrroles which were only recently
1
2
obtained in preparative yields.
In this paper we report the synthesis of a panel of pyr-
rolo[2,1-d][1,2,3,5]tetrazinones of type 10, by reaction of
2-diazopyrroles with isocyanates which remains the
most convenient method to azolotetrazinones, the
attempts to obtain them by different routes, and their
potent in vitro antitumor activity.
/H+, OHꢁ; ii RNCO; iii RNCO or
2
Et/RNH or RNHCOCl; iv NaNO /H ; v RNH ; vi COCl or
2 2 2 2 2
Scheme 1. Reagents: i NaNO
ClCO
CDI.
+
azoloamines 3, upon reaction with alkyl or aryl isocyanate
at room temperature in the dark and in a non-hydroxylic
solvent, allowed the isolation of the azolotetrazinones 5 in
yields from good to excellent. This reaction sequence, that
represented the synthetic entry to the azolotetrazinones,
was utilized by Ege to obtain pyrazolotetrazinones 5a,
Chemistry
2-Diazopyrroles 9 were isolated, in preparative yields,
by diazotization of the corresponding 2-aminopyrroles 8
(Scheme 2). The reaction was carried out in 80% acetic
acid with stoichiometric amount of sodium nitrite under
nitrogen atmosphere in the dark followed by neu-
tralisation with sodium carbonate. The strict control of
1
5
,2,3-triazolotetrazinones 5b and indazolotetrazinones
6
c. Using the same procedure Stevens described the
synthesis of many imidazotetrazinones of type 5d further
pyrazolo- and indazolotetrazinone derivatives, and
reported the excellent antineoplastic activity of the imi-
ꢀ
the temperature at 0 C either during diazotization and
12
neutralisation is crucial in obtaining good yields.
1
,7
dazotetrazinones and pyrazolotetrazinones.
Cheng,
still using the same synthetic pathway prepared two
pyrazolotetrazinone derivatives closely related to the
active lead compounds imidazotetrazinones and also
2-Diazopyrroles 9a–d were purified by flash chromato-
graphy. In the case of 3-carboxamide derivative 8c, the
diazotization also led to a small quantity of 5-methyl-6-
phenylpyrrolo[2,3-d][1,2,3]triazine-4-one, due to an
intramolecular coupling reaction of the diazo group with
the carboxamide moiety. It was impossible to obtain
pure diazo compound 9c even after flash chromato-
graphy since it slowly but continuously transformed into
8
reported data on their biological evaluation.
The mechanism of this reaction probably involves a
stepwise ionic pathway going through an initial nucleo-
philic attack at the isocyanate carbon, to give a dipolar

P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
2373
Scheme 2. Reagents: i NaNO /CH
2
3
COOH, Na
2
CO ; ii R
3
3
NCO/DMF; iii NaH/THF, PhNCO; iv NaNO
2
/H+; v aniline/DCM; vi triphosgene or
CDI/THF.
the pyrrolotriazine compound.11 Thus it was used as
crude product obtained from the diazotization.
to the carbonyl group of the tetrazinone ring. The 13
C
NMR spectra showed a pattern compatible with an 1H-
pyrrole structure, completely different from the pattern
of the starting 2H-pyrrole structure exhibited by the
2
-Diazopyrroles were reacted under the same reaction
1
2
conditions that allowed the isolation of the other azo-
lotetrazinones, that is, with stoichiometric amounts of
isocyanates in dichloromethane or ethyl acetate at room
temperature. In this case the formation of pyrrolotetra-
zinones was not observed.
2-diazo derivatives. The signals due to the carbonyl
carbon of the tetrazinone moiety were found at 140–142
ppm and are in agreement with the chemical shifts
1
3
observed in the azole series.
In the attempt to avoid the handling of the rather
unstable 2-diazopyrroles and to prepare a wider panel
of derivatives to explore SAR, we thought to obtain the
pyrrolotetrazinone system by the alternative synthesis
that involves the carbamoylation of the 2-aminopyrroles
and subsequent nitrosation of the corresponding 1-car-
bamoyl-2-amino derivatives. This alternative route might
provide a wider possibility either to functionalize the sys-
tem and to obtain derivatives unsubstituted at position 6
of the pyrrolotetrazinone system, impossible to obtain by
the classical synthesis. In fact, in the preparation of diazo
compounds, upon addition of nitrite to the 2-amino-
pyrroles having the 5 position unsubstituted, the nitrosa-
tion might take place at the nucleus and/or the diazonium
salt, as soon as formed, couples intramolecularly with the
Reaction of 2-diazopyrroles with ten fold excess of iso-
cyanates in dimethylformamide at room temperature in
the dark for 24–72 h afforded the pyrrolotetrazinones in
acceptable yields (45–60%). The reaction was monitored
by IR until the diazo band at ca. 2100 cm disappeared.
A very easy work up, quenching in icy water and flash
chromatography, gave the desired pyrrolotetrazinones
ꢁ
1
1
0a–o. The reduced electrophilicity of the diazo group
bounded to the electron rich heterocycle justifies the use of
a dipolar aprotic solvent and an excess of reactant.
The structure of pyrrolotetrazinones was confirmed by
IR and NMR spectroscopy. In particular, in the IR
ꢁ1
spectra appeared a band at 1719–1741 cm attributable

2374
P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
unsubstituted position of pyrrole. Thus, from the dia-
zotization of 2-amino-3-cyano-4-phenylpyrrole, we
could not isolate the corresponding 2-diazopyrrole.
12 was quantitatively isolated. Also in this case attempts
to cyclise the triazene, at room temperature with tri-
phosgene or carbonyldiimidazole in dry THF in the pre-
sence of triethylamine, failed since the formation of
pyrrolotetrazinones was not observed. In fact, after 72h
the TLC of the reaction mixture showed many spots
likely due to the decomposition of the triazene derivative.
1
4
On the other hand it was interesting to synthesise a 6-
unsubstituted pyrrolotetrazinone derivative since in imi-
dazole series the most active derivatives had no sub-
stituent in that position and the introduction of an alkyl
group decreased the activity proportionally to the bulki-
7
ness of the substituent.
Thus, in conclusion, the only possible synthesis of pyr-
rolotetrazinones involves the reaction of 2-diazopyrroles
with isocyanates.
The carbamoyl derivatives in pyrazole and imidazole
series were prepared reacting the azolo amines with iso-
cyanate in absence of base or in the presence of
Biology
9
,10
triethylamine respectively.
Such conditions were not
suitable for aminopyrroles, in fact in the reaction of these
derivatives with isocyanates was observed the involve-
The pyrrolotetrazinone derivatives 10a–o were sub-
mitted to the National Cancer Insitute (Bethesda, MD)
for testing against a panel of approximately 60 tumor
cell lines that have grouped in disease sub-panels
including leukaemia, non-small-cell lung, colon, central
nervous system, melanoma, ovarian, renal, prostate and
breast tumors cell lines. Details of this in vitro test sys-
tem and the information, encoded by the activity pat-
1
5
ment of the amino group; whereas under strong basic
conditions 2-aminopyrroles were selectively methylated at
1
6
the ring nitrogen. Thus we reacted the amino derivatives
a,b with sodium hydride in anhydrous THF. Upon
addition of phenylisocyanate the carbamoyl derivatives
1a,b were obtained in excellent yields, (88–97%).
8
1
1
7
tern over all cell lines, have been previously reported.
Unfortunately the nitrosation reaction of carbamoyl
derivatives 11a,b under several reaction conditions
The antitumor activity of a test compound is given by
three parameters for each cell line; pGI50 value (GI50 is
the molar concentration of the compound that inhibits
50% net cell growth), pTGI value (TGI is the molar
concentration of the compound leading to total inhibi-
tion of net cell growth), and pLC50 value (LC50 is the
molar concentration of the compound that induces 50%
net cell death). Moreover, a mean graph midpoint
(MG_MID) is calculated for each of the mentioned
parameters, giving an average activity parameter over
all cell lines. For the calculation of the MG_MID,
insensitive cell lines are included with the highest con-
centration tested. The discovery of compounds with
[
sodium nitrite in acids (acetic, trifluoroacetic, hydro-
chloric, sulfuric, nitric, tartaric); isoamyl nitrite in ace-
tic; trifluoroacetic acid, or isoamyl nitrite in DMF and
DMAP as base] did not evidence the formation, even in
traces, of the pyrrolotetrazinone derivatives 10.
To complete the picture of the possible synthetic routes
to the pyrrolotetrazinones it was undertaken the proce-
dure through the mono-substituted triazene. To this pur-
pose 2-diazopyrrole 9a was reacted with aniline at room
temperature in DCM and in 2h the monophenyltriazene
Table 1. Overview of the results of the in vitro antitumor screening for compounds 10a–oa
b
c
d
Compd
No. of the cell
Lines investigated
No. of cell lines giving positive pGI50 , pTGI , and pLC50
pGI50b
pTGIc
pLC50d
No.
Range
MG_MIDe
No.
1
Range
4.13
MG_MID
4.00
No.
0
Range
MG_MID
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
0g
0h
0i
0j
0k
0l
0m
0n
0o
45
58
58
60
59
59
59
59
59
59
55
55
59
53
58
18
58
57
60
59
59
8
28
50
54
4.76–4.01
7.04–4.45
6.71–4.25
>8.00–5.04
6.52–4.49
5.52–4.59
4.37–4.024.03
4.54–4.01
5.43–4.07
6.17–4.05
4.11
5.7230
5.63
7.16
5.50
5.04
0
4.11
4.37
4.72
36.51–4.24.51
4
6.01–4.36
4.07
28
59
27
54
6.33–4.20
4.54
5.10
4.45
4.50
5
31
8
5.84–4.40
6.11–4.03
5.22–4.04
4.42–4.02
4.09
4.22
4.06
4.15
7.74–4.08
5.63–4.27
5.00–4.10
37
0
0
9
8
0
1
2
4.40–4.03
4.80–4.28
4.01
4.03
4.06
4.08
4.27–4.18
4.00
4.01
424.90–4.05
6
4.2
5
4.17–4.01
0
54
34
25
58
5.72–4.39
6.37–4.01
4.89–4.00
5.88–4.13
4.82
4.27
4.14
5.25
34
7
2
50
5.02–4.02
5.16–4.09
4.11–4.05
5.55–4.10
4.24
4.04
4.00
4.72
12
1
0
43
4.41–4.06
4.11
4.04
4.00
5.27–4.01
4.33
a
Data obtained from the NCI’s in vitro disease-oriented human tumor cells screen.
pGI50 is theꢁlog of the molar concentration that inhibits 50% net cell growth.
b
c
pTGI is theꢁlog of the molar concentration giving total growth inhibition.
d
e
pLC50 is theꢁlog of the molar concentration leading to 50% net cell death.
MG_MID=mean graph midpoint=arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used
concentration interval, the highest tested concentration was used for the calculation.

P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
2375
new selectivity patterns is one of the targets of the
screening program. Selectivity of a compound with
respect to a certain cell line of the screen is characterized
by a high deviation of the particular cell line parameter
compared to the MG_MID value.
respect to CAKI-1 and UO-31 cell lines (pGI =6.37
50
and 6.13, respectively), belonging to the renal cancer
sub-panel.
It is worthy to note that the pyrrolotetrazinones, with
the exception of 10j, which showed selectivity with
respect to LOX IMVI, SK-MEL-5 and UACC-62cell
lines (melanoma sub-panel), did not show any selectivity
with respect to the CNS cancer melanoma sub-panels in
which temozolomide showed curative properties.
An evaluation of the data reported in the Table 1
revealed that compounds belonging to the 8-cyano-7-
methyl-6-phenyl series, 10a–f, are generally more active
than the corresponding compounds belonging to the
8
-cyano-6-methyl-7-phenyl series, 10g–m. Surprisingly
the 3-methyl derivatives 10a and 10g are the least active
in terms either of pGI50 (mean values 4.11 and 4.03,
respectively) and number of sensitive cell lines (18 and 8,
respectively). Also surprising is the low activity of the 8-
carboxamide derivative 10n which is active on 25 cell
lines having a pGI50 mean value 4.14. In the 3-aryl ser-
ies 10b–f,h–l, the effect of the substituents on the phenyl
ring does not play any significant role. In fact in the
In the Table 3 are reported the pTGI and pLC50 of
selected compounds which gave positive values with
respect to a congruous number of cell lines.
At TGI level the most interesting compound was 10d
either in terms of pTGI (MG_MID=5.10) and in terms
of activity against a wide number of cell lines (59) fol-
lowed by 10o, 10c, 10b and 10f (MG_MID in the range
4.72–4.50 and number of cell lines in the range 54–28).
Compound 10d showed good selectivity with respect to
the MDA-MB-435 and MDA-N cell lines of the breast
cancer sub-panel, to the HCC-2998 and HT29 cell lines
of the colon sub-panel, and NCI-H522 of the non-small
cell lung cancer sub-panel pTGI of which differed from
the MG_MID of two magnitude order or more. Com-
pounds 10b and 10c beside the selectivity shown with
respect to the same breast cancer cell lines above men-
tioned, selectively inhibited CCRF-CEM, HL-60(TB)
and K-562of the leukaemia sub-panel.
8
-cyano-6-methyl-7-phenyl series 10b–f, the most active
compound was the 4-chlorophenyl derivative 10d
MG_MID=7.16), followed by the unsubstituted
(
phenyl (10b), 4-methoxyphenyl (10c), and 3-chloro-
phenyl (10e) derivatives which showed similar
MG_MID values (5.72, 5.63 and 5.50, respectively). The
least active was the 4-nitrophenyl derivative 10f
(MG_MID=5.04). In the 8-cyano-7-methyl-6-phenyl
series 10h–l, instead, the 4-nitrophenyl derivative 10l
was the most active compound, closely followed by the
4
-chlorophenyl substituted derivative 10j (MG_MID
values 4.82and 4.7,2 respectively). Derivatives 10i
4-methoxyphenyl), 10k (3-chlorophenyl) and 10h
(
(
(
At LC50 level, the best responses were observed in the
case of the MDA-MB-435 and MDA-N breast cancer
cell lines obtained by 10b and 10c and in the case of
HCC-2998 colon cancer cell line cell line by 10d.
unsubstituted phenyl) showed decreasing activity
MG_MID values in the range 4.37–4.11).
In the Table 2 are reported the pGI50 values of the most
active pyrrolotetrazinones and the anticancer agents 1
and 2. Pyrrolotetrazinones were particularly efficacious
against the leukaemia sub-panel. In fact the calculated
pGI50 MG_MID values of leukaemia sub-panel, related
to the active compounds, are always higher than the
over all cell lines MG_MID values (ÁMG_MID 0.15–
Two pyrrolotetrazinone derivatives, 10b and 10o, were
evaluated as anticancer agents in an in vivo animal
model in which polyvinylidene fluoride (PVDF) hollow
fibres containing various human cancer cell cultures
were implanted intraperitoneally (ip) and sub-
cutaneously (sc) into mice and compounds were admi-
1
8
0.62). The most sensitive leukaemia cell lines are SR,
K-562, HL60 (TB) and CCRF-CEM cell lines.
nistrated by the ip route.
The effects of the
compounds on reduction of viable cancer cell mass
compared to those of controls were determined. Both
compounds were tested in the hollow fiber assay against
a panel of 12human cell lines (Non-Small Cell Lung
Cancer: NCI-H23, NCI-H522; Colon Cancer: SW-620,
COLO-205; CNS Cancer: U251, SF-295; Melanoma:
LOX IMVI, UACC-62; Ovarian Cancer: OVCAR-3,
OVCAR-5; Breast cancer: MDA-MB-231, MDA-MB-
Excellent response was obtained in the breast cancer sub-
panel in which 3-aryl substituted pyrrolotetrazinones
belonging to the 6-phenyl-7-methyl series 10b–f,o had
GI50 generally in the low micromolar range and in the
case of 10d up to nanomolar concentrations. The most
sensitive breast cancer cell lines are MDA-N, MDA-MB-
1
9
4
35, and, at lower level, NCI/ADR-RES cell lines.
435) as described previously. The compounds were
solubilized in 10% DMSO in saline/Tween-80R and
administrated intraperitoneally once daily for a total of
four doses at each of two dose levels (150 and 100 mg/
kg). The day after the last compound dose, the fibers
were collected and assessed for viable cell mass. Unfor-
tunately both compounds were inactive under the con-
ditions of test.
Against the COLO 205, HCT-116, HT29 and KM12 cell
lines of the colon sub-panel as well as the OVCAR-3 of the
ovarian sub-panel pGI50 was generally above the average.
All the compounds tested, with the exception of 10h,
showed a moderate selectivity with respect to the prostate
cancer sub-panel and particularly to the PC-3 cell line.
The data above described suggest that the mode of
action of the pyrrolotetrazinones 10 differs from that of
temozolomide. In fact considering that the crucial step
The 3-cyclohexyl derivative 10m had a low pGI50 mean
value (4.27) but showed a remarkable selectivity with

2376
P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
a
a
a
Table 2. Inhibition of in vitro cancer cell lines by pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones 10 and compounds 1 and 2
Cell line
pGI50b
1
0b
10c
10d
10e
10f
10i
10j
10k
10l
10m10o
1
2
Leukaemia
CCRF-CEM
HL-60 (TB)
K-5626.63
RPMI-8226
MOLT-4
SR
6.05
6.61
6.27
6.27
7.42
7.50
4.30
7.26
7.20
5.44
5.48
5.22
4.83
4.69
5.08
5.43
4.05
4.74
5.35
4.1
4.63
nd
4.55
<4.00
4.1
4.37
nd
5.07
5.72
<4.00
<4.00
5.69
c
4.24.1
4.5
nd
4.1
76.48 5.47 7.55 4.746.2
6.17
5.47
5.88
5.80
6.33
5.63
5.58
6.16
6.20
nd
6.525.33
5.50
nd
4.58
nd
4.68
nd
4.32
nd
nd
5.60
4.0
4.4
4.24.0
4.0
4.1
>8.00
5.13
5.07
4.90
5.14
4.61
5.88
Non-small cell lung cancer
A549/ATCC
EKVX
6.21
5.93
6.98
5.71
5.75
6.05
<4.00
5.80
5.49
5.35
5.54
7.13
7.13
5.35
5.24
4.45
4.59
5.65
5.06
5.41
5.50
4.99
5.05
5.15
5.09
4.90
5.41
4.73
5.23
4.96
<4.00
4.21
4.43
4.62
4.07
4.09
4.02
4.29
4.67
4.29
4.87
<4.00
4.18
4.43
nd
4.48
4.83
4.35
nd
5.20
5.49
4.78
4.88
4.67
4.64
4.65
4.08
4.43
4.0
4.624.13
4.45
5.00
5.24
4.0
4.0
4.0
4.0
HOP-625.39
HOP-924.81
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
N C2 I-H52
Colon cancer
COLO-205
HCC-2998
HCT-116
HCT-15
5.39
5.14
4.3
7.05
7.34
7.09
7.28
7.39
4.0
4.0
5.53
4.83
5.47
6.06
4.31
5.10
4.87
4.80
4.51
5.16
4.0
4.1
4.0
4.3
4.0
4.0
4.0
4.0
4.0
4.0
<4.00
4.11
<4.00
<4.00
<4.00
5.32
<4.00
<4.00
<4.00
nd
nd
7.64
5.71
5.424.64
4.324.99
6.31
5.60
6.21
6.14
nd
5.53
5.50
6.30
5.35
nd
7.72
7.64
7.49
7.31
7.55
7.42
7.32
6.07
5.46
5.69
4.98
5.13
5.18
4.47
4.14
4.36
4.95
4.97
4.89
4.35
4.10
4.15
5.15
4.58
4.61
4.05
<4.00
4.28
<4.00
4.94
5.00
4.71
5.83
5.48
5.76
5.58
5.70
4.0
4.0
4.0
4.0
4.0
4.0
4.3
4.0
4.0
4.0
4.0
4.0
4.0
4.0
5. 41 25.2 4.50
5.00
4.39
4.68
HT29
KM12
SW-620
5.89
6.05
5.37
5.07
5.25
4.99
4.36
4.31
4.57
4.80
4.62
4.60
4.51
4.25
4.33
5.05
4.90
5.11
6.40
6.21
5.63
6.24
<4.00
<4.00
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-25.38
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
5.26
6.15
6.09
6.01
5.59
5.78
5.49
5.69
5.88
5.91
5.62nd
5.60
7.12
7.49
7.58
7.21
5.75
7.26
5.17
5.84
5.80
4.99
4.70
5.63
4.83
5.45
4.84
4.94
4.08
4.26
4.50
4.10
4.49
5.15
4.68
<4.00
4.09
4.90
4.73
5.00
nd
<4.00
4.53
4.325.46
4.18
4. 74 2. 6
4.88
5.20
5.0
4.2
4.0
4.24.0
4.0
4.0
4.40
4.48
nd
4.5
5.40
4.1
5.79
4.57
4.51
4.51
4.84
4.84
4.55
5.37
4.99
4.94
4.71
4.48
4.86
4.16
5.00
4.27
4.0
4.0
5.39
4.58
6.34
5.48
5.40
6.23
5 4. 9. 50 5
6.02
5.55
4.65
5.42
7.26
7.33
7.61
5.0 24 .57
5.25
7.49
6.05
7.13
5.12
4.47
<4.00
4.09
5.05
<4.00
4.83
6.15
4.09
5.29
4.06
4.06
nd
4.76
4.62
nd
4.0
4.49
<4.00
<4.00
4.06
5.49
4.71
5.40
4.7
4.0
4.0
4.1
4.0
4.0
5.024.83
5.38
4.56
5.51
7.05
4.50
<4.00
5.94
4.0
<4.00
4.15
<4.00
<4.00
5.44
6.33
4.51
5.40
4.49
<4.00
5.45
4.0
4.0
4.0
4.3
4.0
5.424.90
4.58
5.35
4.48
4.14
4.84
4.0
4.77
5.21
4.07
4.27
4.45
6.06
<4.00
4.43
<4.00
<4.00
4.81
5.10
4.0
4.0
5.83
6.40
4.76
6.18
5.18
6.18
5.65
6.17
4.25
5.63
5.40
5.88
7.36
7.50
7.04
6.73
7.16
7.05
5.60
5.78
5.06
5.43
5.27
5.51
4.82
4.62
4.49
4.26
4.99
4.22
<4.00
4.39
4.03
4.36
5.42
5.47
4.94
5.19
5.68
4.0
4.0
4.2
4.0
4.0
4.0
4.0
4.0
4.0
4.0
5.524.55
5.09
4.77
4.97
4.61
4.79
4.37
4.82
4.54
<4.00
4.56
4.59
<4.00
4.60
4.12
<4.00
4.27
4.98
4.56
4.96
<4.00
4.05
4.424.63 4 2. 95 4.2 4.3
4.2
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
5.78
5.30
4.86
6.01
5.70
5.72
4.45
5.525.54
5.71
5.24
5.13
5.48
5.95
5.50
4.73
7.24
7.56
6.05
7.07
7.425.73
7.24
6.59
5.53
5.63
5.19
5.18
4.78
5.42
5.00
4.91
5.25
5.31
4.924.34
4.72
4.42
4.525.37
4.88
4.74
4.16
7
4.54
4.58
4.2 4.65
<4.00
4.51
5.29
4.70
4.30
4.61
4.18
4.1
5. 64 2. 0
4.70 1 5.2 4.0
5.72
4.97
4.0
4.0
4.3
nd
4.0
nd
5.37
4.526.37
4.55
4.55
4.76
4.0
4.0
4.0
4.94
4.70
4.20
4.07
4.65
4.45
4.11
4.14
<4.00
nd
4.65
4.55
6.13
4.01
<4.00
5.62
4.89
4.1
4.0
4.0
TK-10
UO-31
7
5. 25 .05
4.95
4.15
nd
5.68
4.0
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
5.76
5.76
5.89
5.79
7.39
7.43
6.06
5.46
5.37
5.03
4.54
4.13
4.65
4.41
4.58
4.16
5.46
5.27
4.69
<4.00
5.39
5.30
4.0
4.0
4.0
4.0
5.85
5.94
4.67
5.35
6.75
7.04
4.525.37
5.38
5.53
5.67
5.40
5.34
6.55
6.71
6.38
7.56
7.03
7.40
>8.00
>8.00
4.74
5.40
5.51
5.80
5.18
6.25
6.37
5.21
5.31
4.76
4.59
4.89
4.99
4.40
4.18
4.60
<4.00
<4.00
<4.00
<4.00
4.30
5.12
4.75
4.44
4.15
4.51
4.62
4.25
5.21
4.36
<4.00
4.60
<4.00
<4.00
<4.00
4.05
4.79
4.80
5.09
4.85
4.61
4.39
4.57
4.66
<4.00
<4.00
4.67
5.63
5.46
5.07
5.08
5.00
5.35
4.98
5.69
4.24.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.0
4.01
<4.00
<4.00
<4.00
<4.00
6.56
4.84
5.37
T-47D
5.75
5.04
5.46
4.22
MG_MIDd
5.725.63
7.16
5.50
5.04
4.37
64 .724.2 74 . 58 24. 25 . 24 .1
4.0
a
Data obtained from NCI’s in vitro disease-oriented tumor cells screen.
pGI50 is the ꢁlog of the molar concentration causing 50% growth inhibition of tumor cells.
b
c
Not determined.
MG_MID=mean graph midpoint=arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concen-
tration interval, the highest tested concentration was used for the calculation.
d

P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
2377
a
Table 3. Inhibition of in vitro cancer cell Lines by pyrrolo[2,1-d][1,2,3,5]tetrazine-4(3H)-ones 10
Cell line
pTGIb
pLC50c
10b
10c
10d
10e
10f
10l
10o
10b
10c
10d
10e
10f
10l
10o
Leukaemia
CCRF-CEM
HL-60 (TB)
K-5626.10
RPMI-8226
MOLT-4
SR
5.37
5.825.45
5.48
<4.00
<4.00
<4.00
<4.00
4.22
4.24
4.86
<4.00
<4.00
5.03
<4.00
5.15
d
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
5.16
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.52
5.11
nd
<4.00
<4.00
<4.00
<4.00
nd
<4.00
4.08
<4.00
<4.00
nd
<4.00
nd
4.01
nd
<4.00
<4.00
4.48
4.51
5.03
nd
nd
5.89
4.91
4.65
4.524.47
5.39
4.88
nd
<4.00
5.58
nd
5.57
<4.00
nd
4.33
4.37
4.46
<4.00
4.49
nd
4.11
<4.00
<4.00
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon Cancer
COLO-205
HCC-2998
HCT-116
4.68
4.925.16
4.824.79
4.71
<4.00
<4.00
5.11
<4.00
4.90
<4.00
<4.00
<4.00
5.07
<4.00
4.49
4.70
4.50
4.57
4.73
4.07
4.58
4.86
nd
4.624.69
4.35
4.66
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
nd
4.17
<4.00
4.33
<4.00
4.47
4.09
<4.00
4.53
4.42
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.34
<4.00
4.24
4.09
<4.00
<4.00
4.39
<4.00
4 .81 24.2
4.33
<4.00
<4.00
4.87
4.74
6.19
4.85
4.13
5.89
7.09
4.93
<4.00
<4.00
4.06
<4.00
<4.00
<4.00
<4.00
4.43
<4.00
4.13
<4.00
4.27
<4.00
<4.00
<4.00
<4.00
4.23
<4.00
<4.00
<4.00
<4.00
4.81
4.34
4.47
4.06
4.05
<4.00
4.48
<4.00
4.59
4.40
4.53
<4.00
4.10
<4.00
<4.00
nd
nd
5.47
4.81
4.97
4.96
6.47
7.18
4.93
4.92
6.78
5.00
4.61
5.63
4.74
4.424.72
<4.00
5.04
4.83
4.64
4.70
4.39
<4.00
<4.00
<4.00
4.12
4.66
4.45
5.55
4.47
<4.00
4.36
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
nd
5.11
6.11
4.39
4.28
4.924.09
4.16
5.22
<4.00
<4.00
<4.00
2
4.04
4.30
4.35
4.35
4.12
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.32
4.20
5.27
<4.00
5.12
4.54
4.88
<4.00
nd
5.58
<4.00
nd
HCT-15
HT29
KM124.67
SW-620
4.61
4.63
4.69
4.29
<4.00
5.44
5.09
4.2
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.21
<4.00
<4.00
<4.00
5.37
<4.00
<4.00
5.05
CNS Cancer
SF-268
SF-295
<4.00
4.94
5.44
nd
5.06
<4.00
5.12
5.40
5.33
5.07
4.76
4.87
5.66
6.36
4.79
4.43
4.91
<4.00
5.30
4.98
<4.00
5.11
4.95
4.49
4.85
4.55
4.44
4.40
4.84
4.34
4.13
4.57
nd
4.36
4.67
<4.00
4.74
5.27
4.82
4.44
5.49
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.40
<4.00
<4.00
<4.00
4.19
4.38
4.71
4.06
<4.00
4.31
<4.00
<4.00
<4.00
<4.00
<4.00
4.37
4.14
4.37
4.27
<4.00
4.10
<4.00
<4.00
4.14
nd
<4.00
<4.00
4.31
5.09
4.41
4.17
SF-539
SNB-19
SNB-75
U251
<4.00
4.424.33
5.19
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-24.2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian Cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
<4.00
<4.00
4.90
<4.00
<4.00
5.15
<4.00
<4.00
4.77
4.89
4.46
4.89
4.88
4.57
5.924.724.60
4.68
4.80
<4.00
<4.00
<4.00
5.05
4.66
4.51
4.60
4.66
4.10
4.37
<4.00
nd
<4.00
4.32
4.91
4.55
4.89
4.56
4.48
4.67
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.44
<4.00
4.40
4.20
4.12
5.25
4.26
4.32
<4.00
<4.00
<4.00
<4.00
<4.00
4.16
4.33
4.19
4.26
4.31
<4.00
4.30
<4.00
4.35
<4.00
<4.00
nd
<4.00
<4.00
4.40
4.27
4.44
4.28
4.15
4.31
7
4.54
4.06
<4.00
4.78
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.44
4.32
4.71
<4.00
<4.00
<4.00
5.29
<4.00
<4.00
<4.00
5.06
<4.00
nd
<4.00
4.48
4.30
4.87
5.83
4.45
4.49
4.86
4.52
<4.00
5.25
<4.00
<4.00
<4.00
<4.00
4.47
5.00
4.56
4.40
4.59
4.10
4.60
4.40
4.424.61
4.024.73
4.63
<4.00
4.92
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.06
4.21
<4.00
<4.00
4.03
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.13
4.35
4.08
4.03
4.21
<4.00
4.20
<4.00
<4.00
<4.00
4.29
<4.00
4.45
4.28
4.36
<4.00
4.29
5.25
4.62
5.30
<4.00
<4.00
<4.00
Renal Cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
4.525.08
7
5.2
<4.00
4.88
<4.00
<4.00
5.23
4.21
<4.00
<4.00
4.58
4.53
4.79
4.28
4.51
4.60
<4.00
4.59
4.74
4.31
<4.00
<4.00
4.32
4.16
4.05
nd
5.48
4.61
4.82
5.18
4.69
5.20
4.58
5.33
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.22
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.24
<4.00
4.35
<4.00
4.23
4.27
4.25
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
nd
5.24
4.24
4.41
4.63
4.30
4.65
4.27
4.94
<4.00
<4.00
<4.00
5.23
<4.00
<4.00
<4.00
5.34
6.00
4.08
4.56
4.61
4.88
4.46
4.58
4.49
<4.00
<4.00
4.39
<4.00
<4.00
<4.00
4.07
<4.00
4.24
UO-31
Prostate Cancer
PC-3
DU-145
<4.00
5.07
<4.00
5.10
4.84
4.74
5.00
4.39
4.83
4.58
5.024.82
4.63
<4.00
<4.00
<4.00
<4.00
4.23
<4.00
<4.00
<4.00
4.424.41
4.15
4.41
4.08
4.74
4.36
Breast Cancer
MCF7
4.35
4.58
<4.00
<4.00
6.17
6.51
<4.00
4.06
<4.00
5.04
4.20
<4.00
5.95
6.33
4.63
4.82
5.80
4.50
7.44
7.74
4.73
4.48
4.45
<4.00
5.21
<4.00
nd
nd
4.71
4.18
4.46
<4.00
4.37
4.50
4.50
4.84
4.16
4.19
4.57
<4.00
<4.00
<4.00
4.16
4.80
<4.00
4.63
<4.00
4.65
4.77
<4.00
<4.00
<4.00
<4.00
5.20
6.01
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
5.05
5.84
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.49
nd
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
<4.00
4.31
<4.00
4.16
<4.00
<4.00
4.02
<4.00
<4.00
4.08
<4.00
<4.00
<4.00
<4.00
<4.00
4.18
<4.00
4.24
<4.00
4.31
4.35
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
T-47D
<4.00
<4.00
<4.00
4.27
4.61
5.14
4.16
<4.00
4.23
4.16
<4.00
MG_MIDe
4.51
4.54
5.10
4.45
4.50
4.24
4.72
4.07
4.09
4.22
4.06
4.15
4.04
4.33
a
Data obtained from NCI’s in vitro disease-oriented tumor cells screen.
pTGI is theꢁlog of the molar concentration giving total growth inhibition.
b
c
pLC50 is theꢁlog of the molar concentration leading to 50% net cell death.
d
e
Not determined.
MG_MID=mean graph midpoint=arithmetical mean value for all tested cancer cell lines. If the indicated effect was not attainable within the used concen-
tration interval, the highest tested concentration was used for the calculation.

2378
P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
for the activation of the temozolomide is the ortogonal
nucleophilic addition by water at C-4 of the tetrazine
nucleus, the bulky phenyl should hinder the attack of
the molecule of water at the tetrazinone carbonyl con-
sequently the methyl derivative should be more active
than phenyl derivative. Also the phenyl on the position
the literature.24 Derivatives 8c,d were prepared from
derivative 8a by acetylation (acetic anhydride) and sub-
sequent reaction with bubbling dry HCl in anhydrous
ꢀ
ꢀ
EtOH for 10 h at 0 C to obtain 8c or at 60 C to obtain
1
1
8d, as described previously.
6
should hinder the approach of the nucleophile to the
General procedure for the synthesis of 2-diazopyrroles
9a–d. 2-diazopyrroles 9a–d were prepared by diazotiza-
tion at 0 C of the corresponding amines with sodium
carbonyl, but compounds 10b–f are at least one magni-
tude order more active than compounds 10h–m bearing
the smaller methyl group in the same position. In imi-
dazole series, as already mentioned in this paper, the
presence of an hydrogen or a small group bounded to
the position 6 is crucial to maintain good activity.
ꢀ
nitrite in acetic acid followed by neutralization with
1
1
aqueous Na CO as described previously. In the
2
3
case of the diazotization of 8c it was impossible to
obtain pure 9c which was reacted without further
1
1
purification.
Moreover, at variance with the imidazotetrazinone ser-
ies in which compounds bearing a cyano group at the
General procedure for the synthesis of pyrrolo[2,1-
d][1,2,3,5]tetrazine (10a–o). To a solution of 9a–d (2
mmol) in dry dimethylformamide (10 mL), the suitable
isocyanate (20 mmol) in dry dimethylformamide (10
mL) was added dropwise at rt in the dark. The reaction
2
0
position 8 are unactive, pyrrolotetrazinones with a
cyano bounded at the same position are more active
than the corresponding derivatives bearing, a carbamoyl
or an ethoxycarbonyl moieties in spite of the fact that
the cyano group is not capable of the intramolecular
hydrogen bonding which, in the monomethyl triazene
intermediate of the imidazole series, brings about the
elimination of the alkylating species.
ꢁ
1
mixture was stirred until the diazo band at ꢃ2110 cm
disappeared (24–72 h) then poured onto crushed ice.
The solid precipitate was filtered off, air dried and pur-
ified by flash chromatography with dichloromethane as
1
1
eluant to give 10a–o.
This evaluation is confirmed by the computerised ana-
2
1
ꢀ
1741 (CO) 1525 and 1346 (NO ), 853 and 690 (Ar CH)
lysis COMPARE performed for derivative 10b. In fact
the compound showed against temozolomide a Pearson
Correlation Coefficient (PCC) very poor, 0.149, at GI50
level and even lower, 0.045, at TGI level. In fact there is
practically no correlation strongly suggesting a different
mechanism of action. COMPARE computations per-
formed for 10b and 10d against the NCI ‘Standard
Agents’ Database were negative (PCC<0.6) suggesting
that the antiproliferative activity of pyrrolo[2,1-d]
10f. Yield 46%; mp 132–134 C (dec.); IR 2233 (CN),
2
ꢁ
1 1
cm ; H NMR d 2.29 (3H, s, CH ), 7.45–7.53 (5H, m,
3
Ph), 7.92( 2H , d, J=9.1 Hz, C H ), 8.44 (2H, d, J=9.1
1
6
4
3
Hz, C H ); C NMR d 10.4 (q, CH ), 93.3 (s, C-3),
6
4
3
112.6 (s, CN), 124.3 (d, Ar) 127.4 (d, Ar), 127.6 (d, Ar),
127.8 (s, C-4), 128.3 (s, Ar), 128.4 (s, C-5), 128.8 (d, Ar),
130.8 (d, Ar), 138.8 (s, C-2), 139.6 (s, CO), 142.6 (s, Ar),
147.1 (s, Ar). Anal. calcd for C H N O (MW 372.34):
1
9
12
6
3
[
1,2,3,5]tetrazines is mechanistically unrelated to that of
C, 61.29; H, 3.25; N, 22.57%; found: C, 61.46; H, 3.18;
N, 22.69%.
any known drug and make this class of compounds
worthy of great attention. It is our intention to under-
take studies directed to elucidate the biochemical
mechanism of action.
ꢀ
10g. Yield 52%; mp 186–187 C; IR 2231 (CN), 1734
1 1
ꢁ
(CO) 738 and 688 (Ar CH) cm ; H NMR d 2.84 (3H,
1
3
s, CH ), 3.97 (3H, s, CH ), 7.43–7.52(5H, m, Ph);
C
3
3
NMR d 12.9 (q, CH ), 36.9 (q, CH ), 92.9 (s, C-3), 112.3
3
3
(
s, CN), 126.0 (s, C-4), 128.9 (d, Ph), 129.0 (d, Ph), 129.4
Experimental
(d, Ph), 129.8 (s, Ph), 131.6 (s, C-5), 139.3 (s, C-2), 141.6
All melting points were taken on a Buchi-Tottoli capil-
lary apparatus and are uncorrected; IR spectra were
determined in bromoform with a Jasco FT/IR 5300
spectrophotometer; H and C NMR spectra were
measured at 200 and 50.3 MHz, respectively in DMSO-
d6 solution, using a Bruker AC series 200 MHz
spectrometer (TMS as internal reference). Column
chromatography was performed with Merck silica gel
(s, CO). Anal. calcd for C H N O (MW 265.27): C,
14 11 5
63.39; H, 4.18; N, 26.40%; found: C, 63.12; H, 4,07; N,
26.33%.
1
13
ꢀ
(CO), 1253 and 1027 (C–O), 826, 736 and 691 (Ar CH)
10i. Yield 60%; mp 170–171 C; IR 2234 (CN), 1719
ꢁ
1 1
cm ; H NMR d 2.74 (3H, s, CH ), 3.85 (3H, s, CH ),
3
3
3
1
7.47 (2H, d, J=8.6 Hz, Ar), 7.53–7.57 (7H, m, Ar);
C
2
30–400 Mesh ASTM or with Biotage FLASH40i chro-
NMR d 12.7 (q, CH ), 55.4 (q, CH ), 90.2(s, C-3), 113.0
3
3
matography module (prepacked cartridge system). Ele-
mental analysis were within ꢂ0.4% of the theoretical
values.
(s, CN), 114.1 (d, Ar), 126.0 (s, C-4), 128.2 (d, Ar), 128.8
(d, Ar), 129.0 (d, Ar), 129.2 (d, Ar), 129.5 (s, C-5), 129.8
(s, Ar), 130.1 (s, Ar), 139.5 (s, C-2), 141.2 (s, CO), 159.7
(s, Ar). Anal. calcd for C H N O (MW 357.37): C,
20 15 5 2
6
1
7.22; H, 4.23; N, 19.60%; found: C, 66.98; H, 4,37; N,
9.46%.
Synthesis of substituted 2-aminopyrroles 8a–d. Deriva-
tives 8a,b were prepared from 1-acetamido-1-phenyl-
ꢀ
(CO), 1521 and 1346 (NO ), 847, 771 and 698 (ArCH)
10l. Yield 51%; mp 153–155 C; IR 2234 (CN), 1737
propan-2-one or 2-acetamido-1-phenylpropan-1-one23
2
2
2
ꢁ
1 1
and malonitrile according to the procedure described in
cm ; H NMR d 2.78 (3H, s, CH ), 7.50–7.66 (5H, m,
3

P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
2379
Ph), 7.96 (2H, d, J=8.6 Hz, C H ), 8.48 (2H, d, J=8.6
6
nitrite (140 mg, 2mmol) in water (2mL) was added.
4
1
3
Hz, C H ); C NMR d 12.9 (q, CH ), 91.4 (s, C-3),
TLC monitoring of the reaction revealed that no traces
of pyrrolotetrazinoes 10b,h were formed after 4 days at
rt. The same result was obtained when it was used: (a)
sodium nitrite in acetic acid and acetic acid/sodium
acetate; (b) sodium nitrite in trifluoroacetic acid; (c)
sodium nitrite in hydrochloric acid; (d) sodium nitrite in
sulphuric acid; (e) sodium nitrite in nitric acid; (f)
sodium nitrite in tartaric acid; (g) isoamyl nitrite in
acetic acid; (h) isoamylnitrite in trifluoroacetic acid; (i)
isoamyl nitrite in DMF and DMAP as base.
6
4
3
1
1
1
1
12.8 (s, CN), 124.5 (d, Ar), 127.0 (s, C-4), 127.7 (d, Ar),
29.0 (d, Ar), 129.2 (d, Ar), 129.3 (d, Ar), 129.7 (s, Ar),
29.8 (s, C-5), 139.2 (s, C-2), 141.0 (s, CO), 142.5 (s, Ar),
47.3 (s, Ar). Anal. calcd for C H N O (MW 372.34):
1
9
12
6
3
C, 61.29; H, 3.25; N, 22.57%; Found: C, 61.38; H, 3.41;
N, 22.33%.
ꢀ
1
(
0m. Yield 45%; mp 180–182 C; IR 2233 (CN), 1723
1
ꢁ
1
CO), 1452(CH 2), 739 and 698 (Ar CH) cm
; H
NMR d 1.27–2.03 (11H, m, cyclohexyl), 2.84 (3H, s,
1
3
CH ), 7.44–7.57 (5H, m, Ph); C NMR d 13.0 (q, CH3),
3-Cyano-4-methyl-5-phenyl-2-triazenylpyrrole (12). To a
solution of 3-cyano-2-diazo-4-methyl-5-phenylpyrrole
9a (0.63 g, 3 mmol), in dry dichloromethane (30 mL), a
solution of aniline (2.73 mL, 30 mmol) in dry dichloro-
methane (50 mL) was added. The reaction mixture was
kept in the dark, at rt and under nitrogen atmosphere,
3
2
9
1
1
5.0 (t, CH ), 25.7 (t, CH ), 31.7 (t, CH ), 58.1 (d, CH),
2 2 2
2.2 (s, C-3), 112.5 (s, CN), 125.8 (s, C-4), 128.7 (d, Ph),
28.9 (d, Ph), 129.4 (d, Ph), 130.0 (s, Ph), 131.5 (s, C-5),
38.9 (s, C-2), 141.2 (s, CO). Anal. calcd for C H N O
1
9
19
5
(
6
MW 333.39): C, 68.45; H, 5.74; N, 21.01%; found: C,
8.51; H, 5,91; N, 20.83%.
ꢁ
1
until the diazo stretching band at 2110 cm
appeared (2h). Removal of the solvent, under reduced
dis-
Preparation of 2-amino-1-carbamoylpyrrole (11a,b). To
an ice cooled solution of aminopyrroles 8a,b (5 mmol)
in dry THF (50 mL), sodium hydride (50% mineral oil
dispersion, 264 mg, 5.5 mmol) is slowly added. After
bubbling has ceased, phenylisocyanate (0.54 mL, 5
mmol) was added. The mixture was stirred at rt for 15
min and then heated under reflux for 1 h. The mixture
was poured onto crushed ice (500 g). The solid pre-
cipitate was filtered air dried and purified by flash
chromatography using n-hexane/ethyl acetate 6:4 as
eluent.
pressure, gave the 2-triazenylpyrrole 12 in quantitative
ꢀ
yields; mp 134–135 C; IR 3437 e 3246 (NH), 2212
ꢁ
1 1
(CN), 766 and 697 (Ar CH) cm ; H NMR d 2.25 (3H,
s, CH ), 7.05 (1H, t, J=7.4 Hz, Ph) 7.32–7.57 (9H, m,
1
3
3
Ph), 12.08 (1H, s, NH), 12.67 (1H, s, NH); C NMR d
10.8 (q, CH ), 85.3 (s, C-3), 114.5 (d, Ph), 116.6 (s, CN),
3
118.3 (s, C-4), 122.8 (d, Ph), 127.0 (d, Ph), 127.4 (d, Ph),
127.5 (s, C-5), 128.5 (d, Ph), 129.2 (d, Ph), 131.2 (s, C-2),
141.1 (s, Ph), 146.2(s, Ph). Anal. calcd for C 18H N
15
5
(MW 301.35): C, 71.74; H, 5.02; N, 23.24%; found: C,
71.54; H, 5,23; N, 23.55%.
2
nylpyrrole (11a). Yield 97%; mp 227–229 C; IR 3351
-Amino-3-cyano-4-methyl-1-(N-phenylcarbamoyl)-5-phe-
ꢀ
Attempted cyclization of 3-cyano-4-methyl-5-phenyl-2-
triazenylpyrrole (12). To a solution of the triazenyl-
pyrrole 12 (0.3 g, 1 mmol) in dry THF (30 mL) and
(
(
(
(
br, NH and NH ), 2206 (CN), 1706 (CO), 762 and 690
2
ꢁ
1
1
0
Ar CH) cm ; H NMR d 2.23 (3H, s, CH ), 7.01
TEA (0.1 mL, 1 mmol) trifosgene or 1,1 -carbonyldii-
3
1H, t, J=7.9 Hz, Ph), 7.24–7.52 (9H, m, Ph), 8.67
1
midazole (1 mmol) was added. The mixtures, stirred at
rt, after 48 h TLC analysis showed no pyrrolote-
trazinone but a very complex sequence of spots due to
the decomposition of the starting triazene.
3
1H, s, NH), 8.98 (1H, s, NH), 11.44 (1H, s, NH);
C
NMR d 10.9 (q, CH ), 84.4 (s, C-3), 115.4 (s, CN),
3
1
5
15.9 (s, C-4), 118.4 (d, Ph), 122.3 (d, Ph), 123.8 (s, C-
), 126.4(d, Ph), 128.6 (d, Ph), 128.7 (d, Ph), 128.8 (d,
Ph), 131.5 (s, C-2), 135.0 (s, Ph), 139.7 (s, Ph), 152.3 (s,
CO). Anal. calcd for C H N O (MW 316.36): C,
Acknowledgements
1
9
16
4
7
1
2.14; H, 5.10; N, 17.71%; found: C, 71.88; H, 5,31; N,
7.92%.
This work was financially supported by Ministero
dell’Istruzione dell’Universita e della Ricerca. A pre-
`
2
nylpyrrole (11b). Yield 88%; mp 234–238 C; IR 3356
-Amino-3-cyano-5-methyl-1-(N-phenylcarbamoyl)-4-phe-
ꢀ
liminary account of this work was presented the First
Italian-Hungarian-Polish Joint Meeting on Medicinal
Chemistry, Taormina September 1999. We thank the
National Cancer Institute (Bethesda, MD) and espe-
cially Dr V.L. Narayanan and his team for the anti-
tumor tests reported in this paper.
(
(
br, NH and NH ), 2208 (CN), 1694 (CO), 758 and 695
2
ꢁ
1 1
Ar CH) cm ; H NMR d 2.23 (3H, s, CH ), 6.96 (1H,
3
t, J=7.8 Hz, Ph), 7.28 (2H, t, J=7.8 Hz, Ph), 7.45 (2H,
d, J=7.8 Hz, Ph), 8.64 (5H, bs, Ph), 8.98 (1H, s, NH),
1
3
9
.16 (1H, s, NH), 11.52(1H, s, NH); C NMR d 11.3
(
q, CH ), 78.4 (s, C-3), 116.6 (s, CN), 118.1 (d, Ph),
3
References and Notes
1
1
1
15.9 (s, C-4), 121.7 (d, Ph), 120.9 (s, C-5), 126.2 (d, Ph),
28.1 (d, Ph), 128.4 (d, Ph), 128.7 (d, Ph), 133.5 (s, C-2),
35.0 (s, Ph), 139.6 (s, Ph), 152.4 (s, CO). Anal. calcd
1
. Stevens, M. F. G.; Hickman, J. A.; Stone, R.; Gibson,
N. W.; Baig, G. U.; Lunt, E.; Newton, C. G. J. Med. Chem.
984, 27, 196.
. (a) Hickman, J. A.; Stevens, M. F. G.; Gibson, N. W.;
for C H N O (MW 316.36): C, 72.14; H, 5.10; N,
1
1
9
16
4
1
2
7.71%; found: C, 72.07; H, 4.99; N, 17.77%.
Langdon, S. P.; Fizames, C.; Lavelle, F.; Atassi, G.; Lunt, E.;
Tilson, R. M. Cancer Res. 1985, 45, 3008. (b) Fodstad, O.;
Aamdal, S.; Pihl, A.; Boyd, M. R. Cancer Res. 1985, 45, 1778.
3. (a) Newlands, E. S.; Blackledge, G. R. P.; Slack, J. A.;
Nitrosation of 2-amino-1-carbamoylpyrroles (11a,b). To
an ice cooled solution of 2-amino-1-carbamoyl-pyrroles
1
1a,b (2mmol) in acetic acid (80%, 20 mL), sodium

2380
P. Diana et al. / Bioorg. Med. Chem. 11 (2003) 2371–2380
Goddard, C.; Brindley, C. J.; Holden, L.; Stevens, M. F. G.
Cancer Treat. Rep. 1985, 69, 801. (b) Harding, M.; Northcott,
D.; Smyth, J.; Stuart, N. S. A.; Green, J. A.; Newlands, E. S.
Br. J. Cancer 1988, 57, 113. (c) Stevens, M. F. G.; Newlands,
E. S. Eur. J. Cancer 1993, 29A, 1045.
13. Wheelhouse, R. T.; Wilman, D. E. V.; Thomson, W.; Ste-
vens, M. F. G. J. Chem. Soc., Perkin Trans. 1 1995, 249.
14. Diana, P. unpublished results.
15. Traynor, J. R.; Wibberly, D. G. J. Chem. Soc., Perkin
Trans. 1 1974, 1786.
4
. (a) Newlands, E. S.; Stevens, M. F. G.; Wedge, S. R.;
Wheelhouse, R. T.; Brock, C. Cancer Treat. Rep. 1997, 23, 35.
b) Bleehen, N. M.; Newlands, E. S.; Lee, S. M.; Thatcher, N.;
16. Mattson, R. J.; Sowell, J. W. Synthesis 1979, 217.
17. (a) Grever, M. R.; Sherpartz, S. A.; Chabner, B. A. Semin.
Oncol. 1992, 19, 622. (b) Monks, A. P.; Scudiero, D. A.; Ske-
han, P.; Shoemaker, R.; Paull, K. D.; Vistica, D.; Hose, C.;
Langley, J.; Croniste, P.; Vaigro-Woiff, A.; Gray-Goodrich,
M.; Campbell, H.; Mayo, J.; Boyd, M. R. J. Natl. Cancer Inst.
1991, 83, 757. (c) Weinstein, J. N.; Meyers, T. G.; O’Connor,
P. M.; Friend, S. H.; Fornace, A. J., Jr.; Kohn, K. W.; Fojo,
T.; Bates, S. E.; Rubinstein, L. V.; Anderson, N. L.; Bou-
lamwini, J. K.; van Osdol, W. W.; Monks, A. P.; Scudiero,
D. A.; Sausville, E. A.; Zaharevitz, D. W.; Bunow, B.; Viswa-
nadhan, V. N.; Johnson, G. S.; Wittes, R. E.; Paull, K. D.
Science 1997, 275, 343.
(
Selby, P.; Calvert, A. H.; Rustin, G. J. S.; Brampton, M.;
Stevens, M. F. G. J. Clin. Oncol. 1995, 13, 910. (c) O’Reilly,
S. M.; Newlands, E. S.; Glaser, M. G.; Brampton, M.; Rice-
Edwards, J. M.; Illinworth, R. D.; Richards, P. G.; Kennard,
C.; Colquhoun, I. R.; Lewis, P.; Stevens, M. F. G. Eur. J.
Cancer 1993, 29A, 940. (d) Newlands, E. S.; Blackledge,
G. R. P.; Slack, J. A.; Rustin, G. J. S.; Smith, D. B.; Stuart,
N. S. A.; Quarterman, C. P.; Hoffman, R.; Stevens, M. F. G.;
Brampton, M. H.; Gibson, A. C. Br. J. Cancer 1992, 65, 287.
5
. (a) Stevens, M. F. G.; Hickman, J. A.; Langdon, S. P.;
Chubb, D.; Vickers, L.; Stone, R.; Baig, G.; Goddard, C. W.;
Gibson, N.; Slack, J. A.; Newton, C. G.; Lunt, E.; Fizames,
C.; Lavelle, F. Cancer Res. 1987, 47, 5846. (b) Clark, A. S.;
Stevens, M. F. G.; Sansom, C. E.; Schwalbe, C. H. Anticancer
Drug Des. 1990, 5, 63. (c) Lowe, P. R.; Sansom, C. E.;
Schwalbe, C. H.; Stevens, M. F. G.; Clark, A. S. J. Med.
Chem. 1992, 35, 3377. (d) Denny, B. J.; Wheelhouse, R. T.;
Stevens, M. F. G.; Tsang, L. L. H.; Slack, J. A. Biochemistry
18. Plowman, J.; Dykes, D. J.; Hollingshead, M.; Simpson-
Herren, L.; Alley, M. C. In Anticancer Drug Development Guide;
Teicher, B., Ed.; Humana Press: Totowa, NJ, 1997; pp 101–125.
19. Hollingshead, M.; Plowman, J.; Alley, M. C.; Mayo, J.;
Sausville, E. A. Contrib. Oncol 1999, 54, 109.
20. Lunt, E.; Newton, C. G.; Smith, C.; Stevens, G.; Stevens,
M. F. G.; Straw, C. G.; Walsh, R. J. A.; Warren, P. J.;
Fizames, C.; Lavelle, F.; Langdon, S. P.; Vickers, L. M. J.
Med. Chem. 1987, 30, 357.
21. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubistein, L.; Plowman, J.; Boyd, M. R. J.
Natl. Cancer Inst. 1989, 81, 1088.
22. (a) This N-acetyl-a-aminoketone was prepared according
to the procedure described by Dakin, H. D.; West, R. J. Biol.
Chem. 1928, 78, 91 taking care of removing the solvent by
steam distillation to avoid the formation of the N,N-diacetyl-
a-aminoketone. (b) Wiley, R. H.; Borum, O. H. J. Am. Chem.
Soc. 1948, 70, 2005.
1994, 33, 9045.
6. Ege, G.; Gilbert, K. Tetrahedron Lett. 1979, 4253.
7. Stevens, M. F. G. In New Avenues in Developmental Cancer
Chemotherapy; Harrap, K. R., Connors, T. A., Eds.; Bristol-
Myers Cancer Symposium Vol 8; Academic Press: Orlando,
Florida, USA, 1987; pp 345–354.
8. Cheng, C. C.; Elslanger, E. F.; Werbel, L. M.; Priebe, S. R.;
Leopold, W. R. J. Med. Chem. 1986, 29, 1544.
9
1
1
1
. Ege, G.; Gilbert, K.; Maurer, K. Chem. Ber. 1987, 120,
375.
0. Wang, Y.; Stevens, M. F. G. J. Org. Chem. 1997, 62, 7288.
1. Diana, P.; Barraja, P.; Lauria, A.; Almerico, A. M.; Dat-
23. This N-acetyl-a-aminoketone was prepared by reduction of
the corresponding oxime with zinc in acetic acid and acetic
anhydride taking care of removing the solvent by steam distilla-
tion to avoid the formation of the N,N-diacetyl-a-aminoketone;
24. Johnson, R. W.; Mattson, R. J.; Sowell, J. W., Sr. J. Het-
erocyclic Chem. 1977, 14, 383.
tolo, G.; Cirrincione, G. Synthesis 1999, 2082.
2. Cirrincione, G.; Almerico, A. M.; Diana, P.; Grimaudo,
S.; Barraja, P.; Mingoia, F.; Dattolo, G.; Aiello, E. Il Farmaco
996, 51, 275.
1
1