2-triazenopyrroles: Synthesis and biological activity

Article abstract of DOI:10.1016/S0223-5234(99)80085-3

2-Triazenopyrroles were synthesized by coupling the corresponding 2- diazopyrroles with secondary amines and tested for antiproliferative, antifungal, antiviral and antibacterial activities. Derivative 9m was the most cytotoxic, showing, against leukaemia, lymphoma and carcinoma cell lines, IC₅₀ 3.9-21 μM and inhibited Cox-B2 and VSV with EC₅₀ 10 μM. Derivative 9j, instead, was active against C. albicans with a selectivity index higher than that of miconazole.

Full text of DOI:10.1016/S0223-5234(99)80085-3

Eur. J. Med. Chem. 34 (1999) 353−360
© Elsevier, Paris
353
Short communication
2-Triazenopyrroles: synthesis and biological activity
Patrizia Dianaa, Paola Barrajaa, Antonino Lauriaa, Anna Maria Almericoa,
Girolamo Cirrincionea*, Anna Giulia Loib, Chiara Musiub, Alessandra Panib,
Paolo La Collab*, Maria Elena Marongiub
aIstituto Farmacochimico dell’Università, Via Archirafi 32, 90123 Palermo, Italy
bDipartimento Biologia Sperimentale, Sezione Microbiologia, Università degli Studi, V.le Regina Margherita 45, 09124 Cagliari, Italy
(Received 8 October 1998; accepted 9 December 1998)
Abstract – 2-Triazenopyrroles were synthesized by coupling the corresponding 2-diazopyrroles with secondary amines and tested for
antiproliferative, antifungal, antiviral and antibacterial activities. Derivative 9m was the most cytotoxic, showing, against leukaemia,
lymphoma and carcinoma cell lines, IC50 3.9–21 µM and inhibited Cox-B2 and VSV with EC50 10 µM. Derivative 9j, instead, was active
against C. albicans with a selectivity index higher than that of miconazole. © Elsevier, Paris
2-triazenopyrroles / synthesis / cytotoxic activity / antifungal activity
1. Introduction
N7- and O6-methylguanine in the DNA of cells from
cancer patients treated with dacarbazine [7].
Dacarbazine, 5-(3,3-dimethyl-1-triazenyl)imidazole-4-
carboxamide (1, DTIC), is the only triazene derivative
used in anticancer chemotherapy and is the most active
drug available for treatment of malignant melanomas and
Hodgkin tumours resistant to MOPP therapy, a combina-
tion of mechlorethamine, oncovinet (vincristine), pred-
nisone and procarbazine [1, 2].
However, DTIC is not the sole dialkyltriazene having
antineoplastic activity. In fact, among compounds of type
2 which have been examined extensively in the search for
analogues more potent than DTIC [3], 1-(4-carboxy-
phenyl)-3,3-dimethyltriazene (2, R = COOH, R1 = Me)
emerged [4].
A widely accepted mode of action for dimethyltriaz-
enes encompasses the following steps: a) oxidative me-
tabolism, in the liver, of one of the methyl groups at N-3
leading to α-hydroxymethyltriazenes of type 3; b) loss of
the α-hydroxymethyl group as formic aldehyde generates
monomethyltriazenes of type 5 which tautomerise to
unstable 3-aryl-1-alkyltriazene forms 6 [5]; c) DNA alky-
lation by the latter [6], as demonstrated by high levels of
More recently, imidazotetrazinone 4 (temozolomide),
has been developed. At present this compound is in phase
II clinical trials due to its efficacy in patients affected by
malignant melanomas, mycosis fungoides and brain tu-
mours [8–11]. Although 4 itself is not an acyclic triazene,
its mode of action has been shown to involve ring
opening of the tetrazinone moiety following nucleophilic
attack at C-4 by a molecule of water to afford the same
monomethyltriazene of type 5 produced by dacar-
bazine [12–15].
Although aryl and heteroaryl rings can be considered
carrier groups for the triazene moiety; and in spite of the
fact that structure-activity relationships are not straight-
forward (in fact, efficacy parallels toxicity so closely that
the latter often masks the antineoplastic activity [16]), in
the triazenoazole series the activity increases as the rings
become more electron-rich, although at the expense of
stability. Therefore, triazenopyrroles, which contain the
most electron-rich azole ring could be expected to display
good antineoplastic activity.
Here we report on the biological activity of triazenopy-
rroles which were synthesised to verify the above as-
sumption.
*Correspondence and reprints

354
Scheme 1.
2. Chemistry
Approaching the synthesis of 2-triazenopyrroles, diffi-
culties were connected with the availability of
2-diazopyrroles. In fact, in the past, synthesis, of
2-diazopyrroles in preparative yield were unknown and
the very few 2-diazopyrroles known were prepared by
direct introduction of the diazo group into the pyrrole
ring, having the 2-position unsubstituted, in very low
yields (2–6%) [19]. Only recently we reported a synthesis
of 2-diazopyrroles, in preparative yield, by diazotization
of the corresponding 2-aminopyrroles [20].
Triazenoazoles are prepared by a coupling reaction of
diazoazoles and suitable amines. Actually the conjugate
acids of the diazoazoles, i.e. the azole diazonium salts
which show the typical reactivity of aromatic diazonium
salts, are much more reactive than the neutral species.
However, in the case of coupling with amines it is
impossible to react the diazonium species because, in the
presence of the amine, the latter lose the imine proton
becoming diazo compounds. It was observed that some
diazo forms are not sufficiently electrophilic to undergo
coupling reactions even with the most reactive nucleo-
philes. This reactivity failure was observed in the diaz-
oazoles lacking a ring nitrogen adjacent to the reactive
centre, i.e. 3-diazopyrroles, 3-diazoindoles and 4-diazo-
pyrazoles [17].
Such a synthesis was achieved following studies dem-
onstrating that 2- and 3-aminopyrroles behave similarly
towards the protonation [21] and that 2-aminopyrroles do
react as primary aromatic amines towards nucleo-
philes [22] and do not behave like enamines as previously
reported [23]. Thus, it was assumed that the failure of the
2-aminopyrroles in giving the pyrrole diazonium salts
upon diazotization was due to the instability of the final
products, rather than to lack of reactivity as primary
aromatic amines. In fact under suitable reaction condi-
However it was possible to obtain 3-triazenopyrroles
using an inert solvent in which the species were not
extensively solvated and the coupling reaction could take
place more easily [18].

355
Scheme 2.
tions it was possible to obtain the 2-diazopyrroles in
40–76% yield.
yield by reacting, in the dark, at room temperature,
2-diazopyrroles 7 with a large excess of secondary
amines 8e and f (ratio 1:10) in dry dichloromethane, or
with bubbling anhydrous dimethylamine 8d. Disappear-
ance of the diazo band at ca. 2 110 cm–1 indicated the
completeness of the reaction (2–3 h). The structure of the
Once
obtained
in
preparative
yields
the
2-diazopyrroles, the coupling reaction with secondary
amines was carried out without any difficulty. In fact
2-triazenopyrroles 9g–m were isolated in quantitative

356
Table I. Antiproliferative activity of 9m and Cis-platinum.
aIC50[µM]
2-triazenopyrroles was confirmed by IR and NMR spec-
tral data. Infrared spectra, in fact, showed at
3 436–3 447 cm–1 and 3 235–3 287 cm–1 the free and
associated pyrrole NH stretching bands respectively,
while the N=N band was observed at 1 599–1 605 cm–1.
The 1H NMR spectra, beside the signals due to the
pyrrole substituents, showed the imine proton at
8.25–11.74 ppm testifying the conversion of the 2H-
pyrrole structure of the starting 2-diazopyrroles to the
1H-pyrrole structure of the 2-triazeno derivatives. The
1H-pyrrole structure was confirmed by 13C NMR spectra.
In fact the pyrrole ring carbon resonances were found in
the usual range for an aromatic structure, in particular, the
C-2 carbon resonances were found at 143.9–148.1 ppm,
typical of a pyrrole α-carbon affected by the deshielding
effect of ca. 22 ppm of the triazene function [24], and the
C-3 carbon resonances were found, with the exception of
the ethoxycarbonyl derivative 9m, at 81.9–84.9 due to the
shielding effect of ca. 16 ppm of the cyano group [25].
The NMR spectra also indicate a substantial contribution
of the form A to the resonance hybrid of the triazene in
which the N-2-N-3 bond has a strong character of double
bond hindering the free rotation and generating two sets
of signals for the substituents on the N-3 nitrogen of the
triazene chain. For example, in the 3-cyano-5-methyl
series, such sets of signals are only present in the 13C
NMR spectra, whereas, in the 3-cyano-5-phenyl series,
due to a higher delocalization of the negative charge of
the N-1 nitrogen because of the phenyl ring, the contri-
bution of the form A is higher and the sets of signals are
Cell lines
9m
Cis-platinum
Leukemia/Lymphoma
Wil2-NS
CCRF-SB
Raji
CCRF-CEM
MOLT-4
MT-4
9.1
6.4
10.0
12.4
19.3
11.4
32.0
1.3
4.8
1.9
32.6
14.8
Carcinoma
HT-29
Hela
ACHN
5637
19.4
5.7
3.9
ND
1.3
4.7
8.3
21.2
aCompound concentration required to reduce cell multiplication by
50% under conditions allowing untreated controls to undergo at
least three consecutive rounds of multiplication.
Wil2-NS, human splenic B-lymphoblastoid cells; CCRF-SB, hu-
man acute B-lymphoblastic leukaemia; Raji, human Burkitt lym-
phoma; CCRF-CEM and MOLT-4, human acute T-lymphoblastic
leukaemia; MT-4, human CD4+ T-cells containing an integrated
HTLV-1 genome; HT-29, human colon adenocarcinoma; HeLa,
human cervix carcinoma; ACHN, human renal adenocarcinoma;
5637, human bladder carcinoma.
concentrations between 6.2 and 25 µM (MIC/MFC in
table II). Interestingly, derivative 9j was active against C.
albicans at concentrations slightly lower than those of
miconazole (3.7 µM) and because of its lower cytotox-
icity against Vero cells it also showed a more favourable
selectivity index (SI = CC50/MIC). On the contrary, none
of the derivatives was significantly active against Gram-
negative and Gram-positive bacteria (data not shown).
Furthermore, plaque reduction assays revealed that
most compounds were inactive against representative
DNA and RNA viruses. The sole exception was 9m,
which inhibited Cox-B2 and VSV at non-cytotoxic con-
centrations (table III). However, when evaluated for anti-
retroviral activity, none of the compounds was capable of
protecting MT-4 cells from the cytophatic effect induced
by HIV-1 (data not shown).
1
also present in the H NMR spectra.
3. Biological results and discussion
When title compounds were screened in MT-4 cells for
antiproliferative activity, 9m was the most potent deriva-
tive (data not shown). Therefore, it was further tested
against a panel of leukaemia, lymphoma and carcinoma
cell lines. As shown in table I, 9m prevented cell growth
concentrations ranging from 3.9–21 µM.
Due to the fact that dacarbazine is inactive in vitro, the
above results indicate that the antiproliferative activity of
2-triazenopyrroles could be related to the easier breakage
of the N-2-N-3 bond. This result is consistent with our
findings on the related classes of 3-triazenopyrroles and
3-triazenoindoles which exhibited in vitro anti-leukaemic
activity (IC50s were in the range of 1.1–3.1 µM and
0.05–0.34 µM respectively [26, 27]).
4. Experimental
4.1. Chemistry
All melting points were taken on a Buchi-Tottoli
capillary apparatus and are uncorrected; IR spectra were
determined in bromoform with a Jasco FT/IR 5 300
In order to investigate whether title compounds were
endowed with a broader biological activity, they were
tested against eukaryotic and prokaryotic micro-
organisms. They were found active against fungi at
1
spectrophotometer; H and 13C NMR spectra were mea-
sured at 200 and 50.3 MHz respectively in DMSO-d6

357
Table II. Antifungal activity of 2-triazenopyrroles.
aCC50
bMIC/MFC
Compound
Vero
C. albicans
C. parapsilosis
C. paratropicalis C. neoformans
A. fumigatus
9g
9h
9i
9j
9k
9l
100
100
50
> 100
> 100
> 100
33
25
25
25
6.2
25
12.5
100
3.7
50
50
50
25
25
25
100
0.9
50
25
25
12.5
25
12.5
100
3.7
25
25
25
12.5
25
25
66
0.9
50
25
25
25
25
25
> 100
3.7
9m
Miconazole
18
aCompound concentration (µM) required to reduce the viability of Vero cells by 50%. bMinimum inhibitory concentration (µM). cMinimum
fungicidal concentration (µM).
MFC = MIC.
solution, unless otherwise specified, using a Bruker AC-E
series 200 MHz spectrometer (TMS as internal refer-
ence); column chromatography was performed with
Merck silica gel 230–400 Mesh ASTM. For all new
compounds analyses indicated by the symbols of the
elements or functions were within ± 0.4% of theoretical
values.
dried over sodium sulphate and evaporated under reduced
pressure to give the diazo compounds 7 which were
purified by chromatography on a column of silica gel
using dichloromethane:ethyl acetate (9:1) as eluant.
4.1.1.1. 2-Diazo-4-methyl-5-phenylpyrrole-3-carboxylic
acid ethyl ester 7c
4.1.1. Preparation of 2-Diazopyrroles 7a–c
Yield 77%, m.p. 77–80 °C; IR: 2 112 (N≡N), 1 713
(CO) cm–1; 1H NMR ppm: 1.34 (3H, t, J = 7.4 Hz, CH3),
2.55 (3H, s, CH3), 4.34 (2H, q, J = 7.4 Hz, CH2), 7.43
(2H, dt, J = 7.4, 1.8 Hz, H-3≠ and H-5≠), 7.51 (1H, dt, J
= 7.4, 1.8 Hz, H-4≠), 7.71 (2H, dd, J = 7.4, 1.8 Hz, H-2≠
and H-6≠); 13C NMR ppm: 12.9 (q, CH3), 13. 9 (q,
OCH2CH3), 60.6 (t, CH2), 75.4 (s, C-2), 127.9 (d, C-3≠
and C-5≠), 128.3 (d, C-4≠), 128.5 (d, C-2≠ and C-6≠),
130.5 (s, C-1≠), 135.9 (s, C-4), 153.9 (s, C-3), 155.3 (s,
C-5), 172.0 (s, CO). Anal. (C14H13N3O2) C, H, N.
2-Diazopyrroles were prepared according to the proce-
dure described previously for 7a and b [19] from the
corresponding 2-aminopyrroles [28, 29] (3 mmol) dis-
solved in glacial acetic acid (6 mL) by addition (dropwise
at 0 °C under a nitrogen atmosphere) of a solution of
sodium nitrite (3 mmol) in a small amount of water
(1 mL). The mixture was neutralised at 0 °C with a
saturated aqueous solution of sodium carbonate and
extracted with dichloromethane. The organic layer was
Table III. Antiviral activity of 2-triazenopyrroles.
aCC50
bEC50
Compourd
Vero
SB
HSV-1
COxs-B2
VSV
9g
9h
9i
9j
9k
9l
9m
100
100
50
> 100
> 100
> 100
33
>100
37.5
37.5
>100
>100
>100
ND
100
> 100
> 50
> 100
> 100
> 100
> 33
> 100
50
50
> 100
50
22.2
10
25
25
25
> 100
50
> 100
10
ACG
Guanidine
> 100
> 100
> 100
30
0.04
> 100
> 100
40
> 100
> 100
b
aCompound concentration (µM) required to reduce the viability of Vero cells by 50%, as determined by Plaque reduction test. Compound
concentration (µM) required to achieve 50% protection of Vero cells from the viral induced cytopathogenicity as determined by PRT method.

358
4.1.2. Preparation of 2-Triazenopyrroles 9g–m
(1H, dt, J = 7.3, 1.2 Hz, H-4≠), 7.42 (2H, dt, J = 7.3, 1.2
Hz, H-3≠ and H-5≠), 7.53 (2H, dd, J = 7.3, 1.2 Hz, H-2≠
and H-6≠), 11.72 (1H, s, NH); 13C.NMR ppm: 11.0 (q,
CH3), 35.9 (q, NCH3), 43.1 (q, NCH3), 82.2 (s, C-3),
117.1 (s, CN), 117.4 (s, C-4), 125.2 (s, C-5), 126.3 (d,
C-4≠), 126.7 (d, C-3≠ and C-5≠), 128.4 (d, C-2≠ and
C-6≠), 131.6 (s, C-1≠), 147.3 (s, C-2). Anal. (C14H15N5)
C, H, N.
To a solution of 2-diazopyrroles 7a–c (3 mmol) in dry
dichloromethane (30 mL), a solution of secondary amines
(30 mmol) 8e and f in dry dichloromethane (50 mL) or
bubbling anhydrous dimethylamine 8d was added. The
reaction mixture was kept in the dark at room temperature
and under nitrogen atmosphere until the diazo stretching
band at 2 110 cm–1 disappeared (2–3 h). Removal of the
solvent, under reduced pressure gave the 2-triazeno-
pyrroles in quantitative yields.
The crude 2-triazenopyrroles were crystallized from
ethanol (9h–m) whereas compound 9g was further puri-
fied by column chromatography on silica gel (dichlo-
romethane:ethyl acetate, 9:1).
4.1.2.5. 2-(3,3-Diethyl-1-triazenyl)-4-methyl-5-phenyl-
pyrrole-3-carbonitrile 9k
M.p. 104–106 °C, IR: 3 437 and 3 273 (NH), 2 212
1
(CN), 1 605 (N=N) cm–1; H NMR ppm: 1.19 (3H, bs,
NCH2CH3), 1.28 (3H, bs, NCH2CH3), 2.22 (3H, s, CH3),
3.76 (2H, bs, NCH2CH3), 3.78 (2H, bs, NCH2CH3), 7.27
(1H, dt, J = 7.3, 1.0 Hz, H-4≠), 7.42 (2H, dt, J = 7.3, 1.0
Hz, H-3≠ and H-5≠), 7.50 (2H, dd, J = 7.3, 1.0 Hz, H-2≠
and H-6≠), 11.68 (1H, s, NH); 13C NMR ppm: 10.9 (q,
NCH2CH3), 11.2 (q, CH3), 14.0 (q, NCH2CH3), 41.6 (t,
NCH2CH3), 49.1 (t, NCH2CH3), 82.1 (s, C-3), 117.5 (s,
CN), 117.6 (s, C-4), 125.4 (s, C-5), 126.5 (d, C-4≠), 127.0
(d, C-3≠ and C-5≠), 128.6 (d, C-2≠ and C-6≠), 131.7 (s,
C-1≠), 148.1 (s, C-2). Anal. (C16H19N5) C, H, N.
4.1.2.1. 2-(3,3-Dimethyl-1-triazenyl)-4,5-dimethylpyrrole-
3-carbonitrile 9g
M.p. 189–190 °C, IR: 3 437 and 3 239 (NH), 2 213
(CN), 1 599 (N=N) cm–1; 1H NMR (CDCl3) ppm: 2.06
(3H, s, CH3), 2.12 (3H, s, CH3), 3.34 (6H, bs, N(CH3)2),
8.25 (1H, bs, NH); 13C NMR (CDCl3) ppm: 9.6 (q, CH3),
10.8 (q, CH3), 29.7 (q, NCH3), 30.9 (q, NCH3), 84.9 (s,
C-3), 116.7 (s, CN), 117.1 (s, C-4), 120.9 (s, C-5), 145.6
(s, C-2). Anal. (C9H13N5) C, H, N.
4.1.2.6.
2-(3,3-Tetramethylene-1-triazenyl)-4-methyl-
5-phenylpyrrole-3-carbonitrile 9l
4.1.2.2. 2-(3,3-Diethyl-1-triazenyl)-4,5-dimethylpyrrole-
3-carbonitrile 9h
M.p. 120–123 °C, IR: 3 437 and 3 246 (NH), 2 213
(CN), 1 602 (N=N) cm–1; 1H NMR ppm: 2.01 (4H, bs, 2
× CH2), 2.23 (3H, s, CH3), 3.59 (2H, bs, NCH2), 3.91
(2H, bs, NCH2), 7.27 (1H, dt, J = 7.3, 1.0 Hz, H-4≠), 7.42
(2H, dt, J = 7.3, 1.0 Hz, H-3≠ and H-5≠), 7.53 (2H, dd, J
= 7.3, 1.0 Hz, H-2≠ and H-6≠) 11.74 (1H, s, NH); 13C
NMR ppm: 11.3 (q, CH3), 23.2 (t, CH2), 23.6 (t, CH2)
47.1 (t, NCH2) 51.4 (t, NCH2), 82.4 (s, C-3), 117.4 (s,
CN), 117.6 (s, C-4), 125.4 (s, C-5), 126.5 (d, C-4≠), 126.9
(d, C-3≠ and C-5≠), 128.6 (d, C-2≠ and C-6≠), 131.9 (s,
C-1≠), 148.1 (s, C-2). Anal. (C16H17N5) C, H, N.
M.p. 169–171 °C, IR: 3 436 and 3 266 (NH), 2 211
1
(CN), 1 603 (N=N) cm–1; H NMR (CDCl3) ppm: 1.25
(6H, bs, N(CH2CH3)2), 2.05 (3H, s, CH3), 2.12 (3H, s,
CH3), 3.73 (4H, q, J = 7.3 Hz, N(CH2CH3)2), 8.30 (1H,
bs, NH); 13C NMR (CDCl3) ppm: 9.5 (q, CH3), 10.7 (q,
CH3), 11.0 (q, NCH2CH3), 14.2 (q, NCH2CH3), 41.5 (t,
NCH2CH3), 50.0 (t, NCH2CH3), 84.0 (s, C-3), 116.3 (s,
CN), 117.3 (s, C-4), 120.7 (s, C-5), 146.1 (s, C-2). Anal.
(C11H17N5) C, H, N.
4.1.2.3. 2-(3,3-Tetramethylene-1-triazenyl)-4,5-dimethyl-
pyrrole-3-carbonitrile 9i
4.1.2.7. 2-(3,3-Diethyl-1-triazenyl)-4-methyl-5-phenyl-
pyrrole-3-carboxylic acid ethyl ester 9m
M.p. 172–173 °C, IR: 3 439 and 3 235 (NH), 2 211
(CN), 1 599 (N=N) cm–1; 1H NMR ppm: 1.95 (6H, s, 2 ×
CH3), 2.04 (4H, bs, 2 × CH2), 3.52 (2H, bs, NCH2), 3.84
(2H, bs, NCH2), 11.15 (1H, s, NH); 13C NMR ppm: 9.5
(q, CH3), 10.5 (q, CH3), 23.4 (t, 2 × CH2), 46.8 (t, NCH2),
51.1 (t, NCH2), 81.9 (s, C-3) 115.1 (s, CN), 117.7 (s,
C-4), 121.9 (s, C-5), 146.3 (s, C-2). Anal. (C11H15N5) C,
H, N.
M.p. 134 °C, IR: 3 447 and 3 287 (NH), 1 680 (CO),
1
1 603 (N=N) cm–1; H NMR (CDCl3) ppm: 1.28 (6H, t,
J = 6.7 Hz, N(CH2CH3)2), 1.37 (3H, t, J = 7.2 Hz,
OCH2CH3), 2.42 (3H, s, CH3), 3.78 (4H, q, J = 6.7 Hz,
N(CH2CH3)2), 4.33 (2H, q, J = 7.2 Hz, OCH2CH3),
7.26–7.43 (5H, m, C6H5), 8.62 (1H, s, NH); 13C NMR
(CDCl3) ppm: 11.1 (q, NCH2CH3), 11.7 (q, CH3), 14.1
(q, NCH2CH3), 14.4 (q, OCH2CH3), 41.8 (t, NCH2CH3),
48.9 (t, NCH2CH3), 59.2 (t, OCH2CH3), 106.8 (s, C-3),
118.1 (s, C-4), 125.4 (s, C-5), 126.5 (d, C-4≠), 127.2 (d,
C-3≠ and C-5≠), 128.6 (d, C-2≠ and C-6≠), 132.8 (s,
C-1≠), 143.9 (s, C-2). 165.9 (s, CO). Anal. (C18H24N4O2)
C, H, N.
4.1.2.4. 2-(3,3-Diethyl-1-triazenyl)-4-methyl-5-phenyl-
pyrrole-3-carbonitrile 9j
M.p. 123–125 °C, IR: 3 437 and 3 270 (NH), 2 213
(CN), 1 605 (N=N) cm–1;1H NMR ppm: 2.22 (3H, s,
CH3), 3.19 (3H, bs, NCH3), 3.53 (3H, bs, NCH3), 7.27

359
4.2. Biology
MIC was defined as the compound concentration at
which no macroscopic signs of fungal growth were
detected. The minimal germicidal concentration (MBC or
MFC) was determined by subcultivating in Sabouraud
dextrose agar samples from cultures with no apparent
growth.
4.2.1. Compounds
Test compounds were dissolved in DMSO at an initial
concentration of 200 mM and then were serially diluted
in culture medium.
4.2.2. Cells
4.2.7. Antiproliferative assays
Cell lines were from American Type Culture Collection
(ATCC); bacterial and fungal strains were either clinical
isolates (obtained from Clinica Dermosifilopatica, Uni-
versity of Cagliari) or collection strains from ATCC.
H9/IIIB, MT-4 and C8166 cells (grown in RPMI 1 640
containing 10% foetal calf serum (FCS), 100 UI/mL
penicillin G and 100 µg/mL streptomycin) were used for
anti-HIV-1 assays. Cell cultures were checked periodi-
cally for the absence of mycoplasma contamination with
a MycoTect Kit (Gibco).
Exponentially growing leukaemia and lymphoma cells
were resuspended at a density of 1 × 105 cells/mL in
RPMI containing serial dilutions of the drugs. Cell
viability was determined after 4 d at 37 °C by the MTT
method [31]. Activity against cell lines derived from solid
tumours was evaluated in exponentially growing cultures
seeded at 5 × 104 cells/mL which were allowed to adhere
for 16 h to culture plates before addition of the drugs.
Cell viability was determined by the MTT method 4 d
later.
4.2.3. Viruses
4.2.8. Linear regression analysis
Polio-virus (SB), Herpes-virus (HSV-1), Coxakie-B2-
virus (Coxs-B2), vesicular stomatitis-virus (VSV) and
HIV-1 (strain IIIB) were ATCC strains.
Viral, microbial and tumour cell growth at each drug
concentration was expressed as the percentage of un-
treated controls, and the concentration resulting in 50%
(EC50, IC50) growth inhibition was determined by linear
regression analysis.
4.2.4. Antiviral assays
The activity of test compounds against Polio-virus,
Herpes-virus and Coxakie-B2-virus and vesicular
stomatitis-virus were tested in classical plaque reduction
assays [30], whereas the anti-HIV-1 activity was evalu-
ated in MTT assays [31].
Acknowledgements
This work was financially supported by Assessorato
dei Beni Culturali ed Ambientali e della Pubblica Istruzi-
one delle Regioni Sicilia (Cap 77504 E.F. 1993) e
Sardegna, by Ministero dell’Università e della Ricerca
Scientifica (60% and 40%).
4.2.5. Antibacterial assays
Staphylococcus aureus, group D Streptococcus, Shi-
gella spp. and Salmonella spp. were recent clinical
isolates. Assays were carried out in nutrient broth, pH 7.2,
with an inoculum of 103 bacterial cells/tube. Minimum
inhibitory concentrations (MIC) were determined after
incubation at 37 °C for 18 h in the presence of serial
dilutions of the test compounds.
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