Quinoxaline chemistry. Part 16. 4-Substituted anilino and 4-substituted phenoxymethyl pyrrolo[1,2-a]quinoxalines and N-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)amino and hydroxymethyl]benzoyl glutamates. Synthesis and evaluation of in vitro biological activity

Article abstract of DOI:10.1016/S0014-827X(03)00101-0

Twenty eight pyrrolo[1,2-a]quinoxalines bearing at position 4 various substituents related to the moieties present in classical and non classical antifolic agents were prepared and evaluated in vitro for antiproliferative activity. In an in vitro screening performed at NCI, several compounds emerged as potent antiproliferative agents at concentrations ranging between 10 and 100 μM. Interestingly, some of these compounds proved active also against bovine and murine DHFR (Farmaco 53 (1998) 480). More recently, a compound of classical antifolate type has been reported to be a potent inhibitor of hDHFR in vitro (Farmaco 58 (2003) 51). We then synthesized new derivatives that, in our hands, were endowed with in vitro antiproliferative activities as low as 3.4 μM against a panel of cell lines derived from hematological and solid tumours. In addition, a complete screening of cytotoxicity, antiretroviral HIV-1 and antimicrobial activity has been carried out.

Full text of DOI:10.1016/S0014-827X(03)00101-0

Il Farmaco 58 (2003) 639ꢀ650
/
www.elsevier.com/locate/farmac
Quinoxaline chemistry. Part 16. 4-Substituted anilino and 4-
substituted phenoxymethyl pyrrolo[1,2-a]quinoxalines and N-[4-
(pyrrolo[1,2-a]quinoxalin-4-yl)amino and hydroxymethyl]benzoyl
glutamates. Synthesis and evaluation of in vitro biological activity
Sergio Alleca a, Paola Corona a, Mario Loriga a, Giuseppe Paglietti a,*,
Roberta Loddo b, Valeria Mascia b, Bernardetta Busonera b, Paolo La Colla b
a Dipartimento Farmaco Chimico Tossicologico, University of Sassari, Via Muroni, 23-07100 Sassari, Italy
b Dipartimento di Biologia Sperimentale-Sezione di Microbiologia, University of Cagliari, Cittadella Universitaria Strada Statale 554, 09042 Cagliari,
Italy
Received 9 August 2002; accepted 13 February 2003
Abstract
Twenty eight pyrrolo[1,2-a]quinoxalines bearing at position 4 various substituents related to the moieties present in classical and
non classical antifolic agents were prepared and evaluated in vitro for antiproliferative activity. In an in vitro screening performed at
NCI, several compounds emerged as potent antiproliferative agents at concentrations ranging between 10 and 100 mM. Interestingly,
some of these compounds proved active also against bovine and murine DHFR (Farmaco 53 (1998) 480). More recently, a
compound of classical antifolate type has been reported to be a potent inhibitor of hDHFR in vitro (Farmaco 58 (2003) 51). We
then synthesized new derivatives that, in our hands, were endowed with in vitro antiproliferative activities as low as 3.4 mM against a
panel of cell lines derived from hematological and solid tumours. In addition, a complete screening of cytotoxicity, antiretroviral
HIV-1 and antimicrobial activity has been carried out.
´
# 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Potential antifolate agents; Antiproliferative agents; Pyrrolo[1,2-a]quinoxalines
1. Introduction
proliferative agents at concentrations ranging between
10 and 100 mM. Interestingly, some of these compounds
proved active also against bovine and murine DHFR[7].
More recently, a compound of classical antifolate type
has been reported to be a potent inhibitor of hDHFR in
vitro[11]. These encouraging results prompted us to
develop new structure-based compounds with the aim of
finding more selective antifolic agents.
In an attempt to develop new anticancer agents
targeted at human dihydrofolate reductase (DHFR)
and tymidylate synthase (TS), two specific enzymes
involved in the biosynthesis of the nucleic acids, we
designed and synthesized more than three hundred
quinoxaline derivatives [1ꢀ11]. This has been done on
/
Thus, we took into account the preparation of
the ground that the quinoxaline ring can bioisosterically
replace the pteridine ring in both classical and non
classical antifolates. In an in vitro screening performed
at NCI, several compounds emerged as potent anti-
compounds 1ꢀ28 (Fig. 1), which are structurally related
/
to the previously described 6(7)-trifluoromethylquinox-
alines bearing at position 2 both classical and non-
classical antifolic moieties [1ꢀ11]. In particular, we
/
considered the annulation of the pyrrole ring at the 3,4
positions of quinoxaline in order to mimic the lipophilic
character, or the steric hindrance, of the phenyl group at
* Corresponding author.
E-mail address: paglietti@uniss.it (G. Paglietti).
´
0014-827X/03/$ - see front matter # 2003 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0014-827X(03)00101-0

640
S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ650
/
position 3 which characterized the previously reported
compounds endowed with the best antiproliferative
activity. A number of the above compounds were
selected and evaluated at NCI for anticancer activity
in vitro. In addition, a complete screening for cytotoxi-
city, antiretroviral (HIV-1) and antimicrobial activity,
was carried out in our laboratories.
3. Antiproliferative, anti-HIV-1 and antimicrobial assays
All the compounds of Fig. 1 and 29c,d were submitted
to NCI. Its scientific committee incontrovertibly selected
the following 1, 2, 4, 5, 6, 10, 11, 15, 21, 25 for the
evaluation of their antiproliferative activity according to
the in vitro disease oriented antitumour screening
program, against a panel of 60 tumour cell-lines [15].
The activity of each compound, calculated from dose-
response curves provided by NCI, is shown in Tables 2
and 3.
2. Chemistry
In Table 3 the response parameters refer to drug
concentrations that produced 50% growth inhibition
The preparation of the compounds was accomplished
according to the reactions described in Scheme 1.
The chloropyrrolo[1,2-a]quinoxalines 29a,b, prepared
as described later, underwent nucleophilic attack by the
corresponding anilines 30, 32 of Fig. 1 in refluxing 1-
propanol to give the anilinopyrrolo[1,2-a]quinoxalines
(ꢁ
/
log GI50), total growth inhibition (ꢁlog TGI) and
/
50% cytotoxicity (ꢁ
/
log LC50) respectively, and were
1ꢀ
/
8 and 21ꢀ
/
22. Compounds 11ꢀ18 and 25, 26 were
/
instead obtained by nucleophilic displacement of chlor-
omethyl derivatives 29c,d with the corresponding phe-
nols 31ꢀ33 of Fig. 1 carrying out the reaction at 70 8C in
/
DMF and in the presence of one mole equivalent of
Cs2CO3 for 6 h. In the case of 14, 18, 25 and 26 cesium
hydrogen carbonate was alternatively used and heating
at 70 8C was prolonged for 2 h. The desired acids 9, 10,
19, 20, 23, 24, 27, 28 were obtained on alkaline
hydrolysis of the parent esters in good yields. The
intermediate halides 29aꢀc were known compounds
/
and have been purposely prepared according to the
indications of Rault et al. [12]. The synthesis of 29d was
instead achieved adapting the Rault’s indications for an
intramolecular ring closure of the new amide 36 (Scheme
2), in refluxing phosphorous oxychloride. The latter was
obtained from the unknown intermediate 35 in turn
prepared via reduction with palladised charcoal and
hydrazine hydrate in ethanol of 34 previously described
by Campiani et al. [13]. Elucidation of the compounds’
structures was achieved by the whole of the elemental
analyses and spectroscopic data (Table 1). The UV
spectra in ethanol showed very fine structure, as
reported by Cheeseman and Tuck [14]. In particular, it
is important to note that compounds 1ꢀ
which do not bear any substituent at position 7,
exhibited a set of six maxima in the region of 360ꢀ260
/4, 9, 19, 21, 23,
/
nm endowed with hyperchromic effect when in para
position to 4-anilino group exists a possibility of
conjugation with a carbonyl group as in the case of
compounds 4, 9, 19, 21, 23. This fine structure collapses
when an electrowithdrawing group as trifluoromethyl
was present in position 7 of pyrroloquinoxaline ring. All
compounds which bear a CH2Oꢀ
showed a flat maximum around 340ꢀ
most intense peaks between 260 and 250 nm.
/
Ph at position 4
/320 nm with the
Fig. 1.

S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ
/
650
641
Scheme 1. i: PropOH, reflux, 2-10h; ii:EtOH, 1M NaOH, reflux, 4h; iii: Cs2CO3, DMF, 70 8C, 6h; iv:CsHCO3, 2h; v: EtOH, 1M NaOH, 70 8C, 4h
expressed as mean graph midpoints, according to the
data provided by NCI. Table 2 reports the activities of
those compounds which showed a growth inhibition
greater than 40% on subpanel cell lines at both 10ꢁ4 and
10ꢁ5 M. Based on the significant cytotoxicity for MT-4
cells shown in antiviral assays (Table 4) by compounds
2, 10, 13, 19, 29c,d, we studied in detail their potential
antiproliferative activity against a panel of cell lines
derived from hematological (Table 5) and solid tumours
(Table 6). In addition to the antiretroviral activity
against HIV-1 (Table 4), title compounds were evaluated
in vitro against representative strains of Gram-positive
and Gram-negative bacteria (S. aureus, Salmonella
spp.), of mycobacteria (M. fortuitum, M. smegmatis
ATCC 19420 and M. tuberculosis ATCC 27294), of
yeast and moulds (C. albicans ATCC 10231 and A.
fumigatus), but their results have been omitted because
none of them resulted active.
4. Results and discussion
From the data reported in Table 3 we can deduce that
the average inhibitory activity of test compounds at
Scheme 2. i: NH2-NH2 H2O, Pd/C 10%, EtOH, reflux, 1.5h ii:
ClCOCH2Cl, pyridine, dioxane, reflux, 4h iii: POCl3, reflux, 4h

Table 1
Comp. M.p.
(8C) a
Yield
(%)
Analysis for
IR (Nujol)
UV (EtOH)
1H NMR, dH (J in Hz)
Solvent: [A]ꢂCDCl3 [B]ꢂ
nmax (cmꢁ1)
lmax (nm)
/
/
CDCl3:DMSO-d6 (3:1) [C]ꢂCDCl3:DMSO-d6 (1:1)
/
1
2
3
4
5
6
7
8
218ꢀ
/
220 b 82
C20H19N3O3
3485, 1633
350, 334, 320 (sh), 309, 264, [A] 11.31 (1H, br s, NH), 7.99ꢀ
237, 208 8), 6.73ꢀ6.67 (3H, m, H-3,ꢃH-2?6?), 6.20ꢀ
5?-OCH3)
335 (sh), 313, 266, 231, 216, [A] 11.19 (1H, br s, NH), 7.97 (1H, d, J2?,6?ꢂ
205 (2H, m, H-8,7), 6.99ꢀ6.92 (2H, m, H-6?,5?), 6.68-6.65 (1H, m, H-3), 6.04ꢀ
s, OCH3), 3.86 (3H, s, OCH3)
348 (sh), 334, 308, 297, 264 [B] 11.40 (1H, br s, NH), 8.24 (1H, s, H-1), 8.01ꢀ
(sh), 236, 212 (sh), 202 7.38 (4H, m, H-7,8ꢃ3?,5?), 7.07ꢀ7.03 (1H, m, H-3), 7.05 (2H, d, Jꢂ
m, H-2), 3.89 (3H, s, OCH3)
3443, 1709, 1629 356, 342, 328, 313, 302, 241, [A] 11.60 (1H, br s, NH), 8.15 (2H, d, Jꢂ
/
7.95 (1H, m, H-1), 7.82ꢀ
/
7.77 (2H, m, H-6,9), 7.48ꢀ7.43 (2H, m, H-7,
/
/
/
/6.17 (1H, m, H-2), 3.93 (3H, s, 4?-OCH3), 3.63 (6H, s, 3?,
215ꢀ
/
218 c 87
C19H17N3O2
3281, 1630
3446, 1630
/1.6 Hz, H-2?), 7.82ꢀ
/
7.55 (3H, m, H-6, 9, 1), 7.46-7.42
6.02 (1H, m, H-2), 3.98 (3H,
/
/
230ꢀ
/
232 d 77
C18H15N3O
/7.97 (1H, m, H-8), 7.80ꢀ
/
7.76 (1H, m, H-9), 7.46ꢀ
/
/
/
/
8.2 Hz, H-2?, 6?), 6.88-6.85 (1H,
125ꢀ
/
127 d 73
C20H17N3O2
/
8.4 Hz, H-3?,5?), 8.03ꢀ
8.2 Hz, H-2?6?), 7.48-7.44 (2H, m, H-7,8), 6.70ꢀ
(1H, m, H-2), 4.42 (2H, q, CH2CH3), 1.44 (3H, t, CH3CH2)
[A] 8.09ꢀ8.00 (2H, m, H-1,6), 7.84 (1H, d, J9,8 8.4 Hz, H-9), 7.70 (1H, dd, J8,9
1.6 Hz, H-8), 6.80ꢀ6.75 (1H, m, H-3), 6.67 (2H, s, H-2?6?), 6.28ꢀ6.20 (1H, m, H-2), 3.93 (3H, s, 4?-
OCH3), 3.84 (6H, s, 3?, 5?-OCH3)
350 (sh), 340, 313, 240, 216, [A] 11.42 (1H, br s, NH), 8.04ꢀ8.03 (2H, m, H-7,8), 7.90 (1H, d, J9,8
202 J8,9 8.0 Hz and J8,6 2.4 Hz, H-8), 7.08ꢀ6.90 (3H, m, H-2?,5?,6?), 6.80ꢀ
6.05 (1H, m, H-2), 3.98 (3H, s, OCH3), 3.86 (3H, s, OCH3)
350 (sh), 338, 303, 243, 221, [A] 11.40 (1H, br s, NH), 8.05ꢀ8.01 (2H, m, H-1,6), 7.85 (1H, dd, J8,9
210 8), 7.65 (1H, d, J9,8 8.0 Hz, H-9), 7.34 (2H, d, Jꢂ8.8 Hz, H-3?,5?), 7.01 (2H, d, Jꢂ
6.78-6.69 (1H, m, H-3), 6.18-6.00 (1H, m, H-2)
C21H16F3N3O2 3092, 1728, 1604 356, 340, 311, 246, 216, 203 [A] 11.84 (1H, br s, NH), 8.18 (2H, d, Jꢂ8.6 Hz, H-3?,5?), 8.10ꢀ
2.8 Hz, H-6), 7.91 (1H, d, J9,8 8.8 Hz, H-9), 7.70 (1H, dd, J8,9
(2H, d, Jꢂ8.6 Hz, H-2?,6?), 6.80-6.75 (1H, m, H-3), 6.36ꢀ6.30 (1H, m, H-2), 4.43 (2H, q, CH2CH3),
1.44 (3H, t, CH3CH2)
3400, 1725, 1620 356, 340, 326, 303, 312, 240, [C] 11.40 (1H, br s, NH), 8.42ꢀ
212, 202 H-3), 7.93ꢀ7.78 (2H, m, arom.), 7.80 (2H, d, Jꢂ
6.86 (1H, m, H-2)
C19H12F3N3O2 3435, 1701, 1604 355, 340, 311, 246, 216, 203 [B] 9.52 (1H, br s, NH), 8.30ꢀ
1,3), 6.91ꢀ6.85 (1H, m, H-2)
346, 262, 248, 237 (sh), 225, [A] 8.00ꢀ7.90 (2H, m, H-1,6), 7.90ꢀ
205 H-3), 6.92ꢀ6.89 (1H, m, H-2), 6.39 (2H, s, H-2?, 6?), 5.38 (2H, s, CH2), 3.81 (6H, s, 3?, 5?-OCH3), 3.76
(3H, s, 4?-OCH3)
346, 263 (sh), 248, 225, 207 [A] 8.00ꢀ7.86 (2H, m, H-1,6), 7.85 (1H, dd, J9,8
H-7,8), 7.14ꢀ7.10 (1H, m, H-3), 6.92ꢀ6.86 (1H, m, H-2), 6.29 (2H, d, J2?,4?ꢂ
6.18 (1H, m, H-4?), 5.36 (2H, s, CH2), 3.75 (6H, s, 3?,5?-OCH3)
337, 320 (sh), 264 (sh), 248, [A] 8.02ꢀ7.92 (2H, m, H-1,6), 7.76 (1H, dd, J9,8 8.0 Hz and J9,7
226, 204 H-7,8), 7.18ꢀ7.15 (1H, m, H-3), 7.02 (2H, d, Jꢂ9.0 Hz, H-2?6?), 6.90ꢀ
Jꢂ9.2 Hz, H-3?,5?), 5.36 (2H, s, CH2), 3.75 (3H, s, OCH3)
336, 257 (sh), 249, 226, 205, [A] 8.06ꢀ7.91 (2H, m, H-1,6), 7.98 (2H, d, Jꢂ9.0 Hz, H-3?,5?), 7.85 (1H, dd, J9,8
198 1.6 Hz, H-9), 7.60ꢀ7.40 (2H, m, H-7,8), 7.10 (2H, d, Jꢂ9.0 Hz, H-2?,6?), 7.15ꢀ
6.88 (1H, t, H-2), 5.45 (2H, s, CH2 ), 4.32 (2H, q, CH2CH3), 1.35 (3H, t, CH3CH2)
/
8.02 (1H, m, H-1), 7.84ꢀ
/
7.80 (2H, m,
213, 202
H-6,9), 7.52 (2H, d, Jꢂ
/
/6.68 (1H, m, H-3), 6.25ꢀ
/
6.23
194ꢀ
/
196 52
C21H18F3N3O3 3090, 1633
C20H16F3N3O2 3086, 1632
C19H14F3N3O 3087, 1634
354, 336, 303, 240, 209
/
ꢂ/
ꢂ/8.4 Hz and J8,6
ꢂ/
/
/
131ꢀ
/
133 b 77
/
ꢂ/
8.0 Hz, H-9), 7.66 (1H, dd,
ꢂ/
ꢂ/
/
/
6.73 (1H, m, H-3), 6.09ꢀ
/
202ꢀ
/
204 d 76
/
ꢂ/8.0 Hz and J8,6
ꢂ2.4 Hz, H-
/
ꢂ/
/
/
8.8 Hz, H-2?, 6?),
223ꢀ
/
226 b 41
/
/8.05 (1H, m, H-1), 8.03 (1H, d, J6,8
ꢂ/
ꢂ/
ꢂ/
8.8 Hz and J8,6 2.8 Hz, H-8), 7.58
ꢂ/
/
/
9
278ꢀ
/
281 85
300 71
C18H13N3O2
/
8.40 (1H, m, H-1), 8.13 (2H, d, Jꢂ/8.8 Hz, H-3?,5?), 7.93ꢀ7.91 (1H, m,
/
/
/8.8 Hz, H-2?, 6?), 7.54ꢀ
/
7.36 (2H, m, arom.), 6.96ꢀ
/
10
11
ꢀ
/
/8.10 (4H, m, arom.), 8.03ꢀ/7.90 (3H, m, arom.), 7.61ꢀ
/7.48 (2H, m, H-
/
135ꢀ
/
137 e 64
C21H20N2O4
C20H18N2O3
C19H16N2O2
C21H18N2O3
1606
/
/7.82 (1H, m, H-9), 7.60ꢀ/7.40 (2H, m, H-7,8), 7.19ꢀ7.13 (1H, m,
/
/
12
13
14
90ꢀ
/
92 f 86
80 f 62
92 f 49
1599
/
ꢂ/8.0 Hz and J9,7
ꢂ/
1.6 Hz, H-9), 7.58ꢀ
/7.38 (2H, m,
/
/
/
2.2 Hz, H-2?, 6?), 6.22ꢀ
/
78ꢀ
/
1612
/
ꢂ/
ꢂ/1.6 Hz, H-9), 7.60ꢀ
/7.40 (2H, m,
/
/
/6.86 (1H, m, H-2), 6.82 (2H, d,
/
91ꢀ
/
1710, 1608
/
/
ꢂ/7.8 Hz and J9,7
ꢂ/
/
/
/7.00 (1H, m, H-3),

Table 1 (Continued)
Comp. M.p.
(8C) a
Yield
(%)
Analysis for
IR (Nujol)
UV (EtOH)
1H NMR, dH (J in Hz)
Solvent: [A]ꢂCDCl3 [B]ꢂ
nmax (cmꢁ1)
lmax (nm)
/
/
CDCl3:DMSO-d6 (3:1) [C]ꢂCDCl3:DMSO-d6 (1:1)
/
15
16
17
18
19
20
21
137ꢀ
/
139 e 51
C22H19F3N2O4 1588
C21H17F3N2O3 1595
C20H15F3N2O2 1630
C22H17F3N2O3 1708, 1607
338, 270 (sh), 252, 230, 207 [A] 8.26ꢀ
H-8), 7.28ꢀ
(6H, s, 3?,5?,-OCH3), 3.76 (3H, s, 4?-OCH3)
332, 266 (sh), 252, 229, 206 [A] 8.29ꢀ8.22 (1H, m, H-1), 7.98ꢀ7.90 (2H, m, H-6,9), 7.75 (1H, dd, J8,9
H-8), 7.20ꢀ7.18 (1H, m, H-3), 6.96ꢀ6.90 (1H, m, H-2), 6.27 (2H, d, J2?,4?ꢂ
d, J4?,2?ꢂ1.6 Hz, H-4?), 5.37 (2H, s, CH2), 3.75 (6H, s, 3?,5?ꢀOCH3)
389, 321, 254 (sh), 236, 214 [A] 8.26 (1H, a s, H-1), 7.95 (1H, d, J6,8 2.8 Hz, H-6), 7.92 (1H, a s, H-9), 7.75 (1H, dd, J8,9
and J8,6 1.8 Hz, H-8), 7.26ꢀ7.20 (1H, m, H-3), 7.01 (2H, d, Jꢂ9.0 Hz, H-2?, 6?), 6.93 (1H, t, H-2),
8.15 (2H, d, Jꢂ9.2 Hz, H-3?,5?), 5.36 (2H, s, CH2), 3.74 (3H, s, OCH3)
[A] 8.27 (1H, a s, H-1), 8.01-7.93 (2H, m, H-6,9), 7.99 (2H, d, Jꢂ8.6 Hz, H-3?,5?), 7.78 (1H, dd,
J8,9 8.4 Hz and J8,6 1.6 Hz, H-8), 7.21ꢀ7.19 (1H, m, H-3), 7.10 (2H, d, Jꢂ8.8 Hz, H-2?, 6?), 6.96
(1H, t, H-2), 5.47 (2H, s, CH2), 4.32 (2H, q, CH2CH3), 1.36 (3H, t, CH3CH2)
[C] 8.93ꢀ8.86 (1H, m, H-1), 8.47 (1H, dd, J6,7 8.2 Hz and J6,8 1.6 Hz, H-6), 8.29 (1H, dd, J9,8
8.4 Hz and J9,7 1.6 Hz, H-9), 7.96 (2H, d, Jꢂ8.8 Hz, H-3?,5?), 7.84ꢀ7.70 (1H, m, H-7), 7.70ꢀ7.64
(1H, m, H-8), 7.25 (2H, d, Jꢂ8.6 Hz, H-2?, 6?), 5.89 (2H, s, CH2), 4.67 (1H, br s, COOH)
[C] 8.48 (1H, d, J6,8 1.6 Hz, H-6), 8.39 (1H, d, J9,8 8.6 Hz, H-9), 8.24 (1H, a s, H-1), 7.93 (2H, d,
Jꢂ8.8 Hz, H-3?,5?), 7.64 (1H, dd, J8,9 8.8 Hz and J8,6 1.6 Hz, H-8), 7.34ꢀ7.32 (1H, m, H-3), 7.14
(2H, d, Jꢂ8.8 Hz, H-2?, 6?), 7.02 (1H, t, H-2), 5.57 (2H, s, CH2)
3324, 1758, 1738, 356, 340, 312, 303, 272, 240, [A] 11.57 (1H, br s, NH), 8.02ꢀ8.00 (1H, m, H-1), 7.97 (2H, d, Jꢂ8.4 Hz, H-3?,5?), 7.86ꢀ
1601 210, 202 H-7,8), 7.64 (1H, d, Jꢂ7.2 Hz, NHCO), 7.49 (2H, d, Jꢂ8.4 Hz, H-2?, 6?), 7.46ꢀ7.43 (2H, m, H-6,9),
6.71ꢀ6.69 (1H, m, H-3), 6.45ꢀ6.35 (1H, m, H-2), 4.80ꢀ4.65 (1H, m, NHCHCH2), 4.24 (2H, q,
CH2CH3), 4.14 (2H, q, CH2CH3), 2.60ꢀ2.20 (4H, m, CH2CH2), 1.32 (3H, t, CH3CH2), 1.25 (3H, t,
CH3CH2)
C28H27F3N4O5 3299, 1727, 1712, 340, 356, 311, 247, 216, 203 [A] 11.78 (1H, br s, NH), 8.12ꢀ
1606 m, NHCO), 7.91ꢀ7.64 (2H, m, H-6,9), 7.49 (2H, d, Jꢂ
4.81ꢀ4.60 (1H, m, NHCHCH2), 4.24 (2H, q, CH2CH3), 4.14 (2H, q, CH2CH3), 2.61ꢀ
CH2CH2), 1.31 (3H, t, CH3CH2), 1.25 (3H, t, CH3CH2)
3397, 3311, 1733, 355, 340, 326, 312, 270, 241, [C] 11.58 (1H, br s, NH), 8.65 (1H, d, Jꢂ7.4 Hz, NHCO), 8.48 (1H, a s, H-1), 8.21ꢀ
1704, 1600 212, 202 6), 8.15 (2H, d, Jꢂ7.8 Hz, H-3?,5?), 7.95ꢀ7.93 (1H, m, H-3), 7.84ꢀ7.78 (1H, m, H-9), 7.72 (2H, d,
Jꢂ8.2 Hz, H-2?, 6?), 7.58ꢀ7.38 (2H, m, H-7,8), 6.97 (1H, t, H-2), 4.59ꢀ4.51 (1H, m, NHCHCH2),
2.48ꢀ2.00 (4H, m, CH2CH2)
C24H19F3N4O5 1794, 1716, 1603 355, 339, 311, 247, 216, 204 [B] 10.85 (1H, br s, NH), 8.42ꢀ
8.05 (2H, d, Jꢂ8.2 Hz, H-3?,5?), 7.93 (2H, d, Jꢂ
(1H, m, H-8), 6.93 (1H, t, H-2), 4.78 (2H, a s, 2×COOH), 4.70ꢀ
(4H, m, CH2CH2)
3290, 1731, 1607 341, 259 (sh), 249, 227, 205 [A] 8.01ꢀ7.93 (2H, m, H-1,6), 7.88 (1H, dd, J9,8
Hz, H-3?,5?), 7.56ꢀ7.47 (2H, m, H-7,8), 7.12 (2H, d, Jꢂ
6.91ꢀ6.88 (1H, m, H-2), 5.46 (2H, s, CH2), 4.80ꢀ4.70 (1H, m, NHCHCH2), 4.22 (2H, q, CH2CH3),
4.09 (2H, q, CH2CH3), 2.50ꢀ2.00 (4H, m, CH2CH2), 1.29 (3H, t, CH3CH2), 1.20 (3H, t, CH3CH2)
[A] 8.26 (1H, a s, H-1), 8.19 (1H, d, J6,8 2.8 Hz, H-6), 7.96 (1H, d, J9,8 10.4 Hz, H-9), 7.77 (2H, d,
Jꢂ8.6 Hz, H-3?,5?), 7.80ꢀ7.73 (1H, m, H-8), 7.19 (1H, d, Jꢂ4.2 Hz, NHCO), 7.11 (2H, d, Jꢂ8.8
Hz, H-2?6?), 6.96ꢀ6.92 (2H, m, H-2,3), 5.46 (2H, s, CH2), 4.80ꢀ4.70 (1H, m, NHCHCH2), 4.22 (2H,
q, CH2CH3), 4.10 (2H, q, CH2CH3), 2.48ꢀ2.00 (4H, m, CH2CH2), 1.29 (3H, t, CH3CH2), 1.20 (3H, t,
CH3CH2)
/
8.22 (1H, m, H-1), 8.03ꢀ
/
7.90 (2H, m, H-6,9), 7.74 (1H, dd, J8,9
ꢂ
/
8.8 Hz and J8,6
ꢂ
/
1.6 Hz,
/7.18 (1H, m, H-3), 7.00ꢀ
/6.92 (1H, m, H-2), 6.67 (2H, s, H-2?, 6?), 5.38 (2H, s, CH2), 3.82
129ꢀ
/
130 76
/
/
ꢂ
/
8.8 Hz and J8,6
ꢂ/
1.6 Hz,
/
/
/1.6 Hz, H-2?, 6?), 6.10 (1H,
/
/
90ꢀ
/
91 21
ꢂ/
ꢂ8.8 Hz
/
ꢂ
/
/
/
/
96ꢀ
/
97 g 23
336, 253, 232, 207
336, 249, 226, 205
321, 236, 215
/
ꢂ
/
ꢂ/
/
/
228ꢀ
/
230 g 54
C19H14N2O3
1694, 1605
/
ꢂ/
ꢂ/
ꢂ/
ꢂ/
/
/
/
/
218ꢀ
/
220 e 75
C20H13F3N2O3 1702, 1604
ꢂ/
ꢂ
/
/
ꢂ/
ꢂ/
/
/
168ꢀ
/
170 d 75
C27H28N4O5
/
/
/
7.79 (2H, m,
/
/
/
/
/
/
/
22
23
24
25
26
126ꢀ
/
128 b 59
/
8.06 (2H, m, H-1,8), 7.92 (2H, d, Jꢂ
/
7.8 Hz, H-3?,5?), 7.98ꢀ
6.60 (2H, m, H-2,3),
2.05 (4H, m,
/7.80 (1H,
/
/
8.2 Hz, H-2?,6?), 6.80ꢀ
/
/
/
174ꢀ
/
179 85
C23H20N4O5
/
/8.17 (1H, m, H-
/
/
/
/
/
/
/
183ꢀ
/
186 g 72
/
8.38 (1H, m, H-1), 8.33 (1H, a s, H-6), 8.19 (1H, d, J9,8
8.8 Hz, H-2?6?), 7.88ꢀ7.86 (1H, m, H-3), 7.72ꢀ
4.55 (1H, m, NHCHCH2), 2.47-2.00
ꢂ/
8.8 Hz, H-9),
/
/
/
/
7.58
/
/
72ꢀ
/
73 g 78
C28H29N3O6
/
ꢂ/7.2 Hz and J9,7
ꢂ/1.2 Hz, H-9), 7.77 (2H, d, Jꢂ8.4
/
/
/8.0 Hz, H-2?6?), 7.20-7.15 (1H, m, H-3),
/
/
/
68ꢀ
/
70 35
C29H28F3N3O6 3310, 1733, 1605 332, 252, 231, 206
ꢂ/
ꢂ
/
/
/
/
/
/
/
/

644
S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ650
/
NCI, represented as mean graph mid points, falls in the
range 10ꢁ4.85ꢀ10ꢁ4.00 molar concentration, corre-
sponding to 14ꢀ100 mM range. Compound 2 was the
/
/
most active; in fact, it is characterized by a significant
GI50 (14.12 mM) and a good correlation between TGI
(ꢁ
/
4.41ꢂ
/
38.90 mM) and LC50 (ꢁ
/
4.12ꢂ75.85 mM).
/
However, from the cytotoxicity test on mock-infected
MT-4 cells (Table 4), additional five compounds (13, 19,
22, 29c,d) exhibited lower CC50 values, which ranged
from 6 mM, for the most active (29c), to 39 mM, for the
least active (22).
The data in Table 2 are indicative of the wide
spectrum of tumour growth inhibition at 10ꢁ4 and
10ꢁ5 M. A large number of derivatives (2, 4, 5, 6, 10, 11,
15, 21, 25) exhibit a wide spectrum activity against all
subpanel cell lines (from 57 to 47 over 60 cell lines) and
some of them possess percent tumour growth inhibition
values at 10ꢁ5 M. In particular, compound 5 main-
tained these values over 31 cell lines, compound 11 over
21, compound 15 over 20, compound 6 over 16 and
compound 4 over 11 cell lines, whereas compounds 1, 2,
10, 21 and 25 showed very low percent growth inhibition
values (below 40%). It is worth noting that the cell lines
derived from leukemia, colon and breast cancer are the
most affected.
Data in Table 4 show that none of the compound
exhibited whatsoever significant anti-HIV-1 and in some
case EC50 was coincident with CC50. However, com-
pounds 13, 19, 29c,d, not tested at NCI, proved
significantly cytotoxic in MT-4 cells, prompting an
investigation of their potential antiproliferative activity
against a panel of cell lines derived from hematological
and solid tumours (Tables 5 and 6). Comparison of
mean graph mid points of 5, 6, 10 and 15 with those of
previously reported analogues bearing a phenyl group at
position 3 of quinoxaline ring [1,3,7], reported in Table
7, shows that pyrrolo[1,2-a]quinoxaline derivatives are
the most active. Structureꢀactivity relationships allow
/
to conclude that insertion of a pyrrole ring on the
quinoxaline system increases the antiproliferative activ-
ity.
5. Conclusion
In a first NCI screening, several compounds proved
active in vitro as antiproliferative agents in the concen-
tration range 10ꢀ100 mM. We then synthesized new
/
derivatives that, in our hands, were endowed with in
vitro antiproliferative activities as low as 3.4 mM against
a panel of cell lines derived from hematological and
solid tumours. In addition, a complete screening of
cytotoxicity, antiretroviral HIV-1 activity has been
carried out, but none of title compounds resulted active,
nor were the compounds tested active as antimicrobial.

Table 2
Percent tumour growth inhibition recorded on subpanel cell lines at 10ꢁ4 and 10ꢁ5 M of compounds 1, 2, 4ꢀ
/6, 10, 11, 15, 21 and 25
1
2
4
5
6
10 11
15
21
25
Molar concentration (M)
10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5
Leukemia
CCRF-CEM
HL-60(TB)
K-562
ꢀ
70
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
162
162
98
86
101
155
197
56
200
200
45
65
95
63
91
45
53
48
53
87
116
118
132
121
142
49
86
99
40
191
148
183
186
191
nt
61
58
71
93
82
nt
148
106
143
145
nt
50
42
57
86
nt
60
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
41
/
94
99
75
99
nt
69
47
43
90
nt
ꢀ/
52
56
ꢀ/
ꢀ/
ꢀ/
43
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
50
45
ꢀ/
68
113
120
111
141
76
106
98
MOLT-4
RPMI-8226
SR
129
120
145
46
44
nt
61
ꢀ/
52
ꢀ/
45
ꢀ/
50
ꢀ/
ꢀ/
123
51
90
87
51
Non small cell lung cancer
A549/ATCC
EKVX
ꢀ
ꢀ/
112
71
nt
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
145
119
180
89
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
53
87
113
71
nt
ꢀ
ꢀ/
ꢀ/
nt
/
ꢀ
ꢀ/
ꢀ/
48
nt
47
nt
/
44
126
125
146
134
nt
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
85
64
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
74
76
70
91
nt
39
44
93
144
80
ꢀ
ꢀ/
ꢀ/
45
51
56
nt
/
90
79
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
48
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ/
ꢀ/
47
nt
ꢀ/
71
63
HOP-62
HOP-92
84
61
ꢀ/
137
93
56
nt
67
78
NCI-H226
NCI-23
nt
nt
nt
ꢀ/
ꢀ/
ꢀ/
ꢀ/
nt
nt
79
nt
119
77
nt
91
nt
ꢀ/
82
49
56
nt
ꢀ/
ꢀ/
ꢀ/
ꢀ/
200
nt
ꢀ/
ꢀ/
ꢀ/
ꢀ/
59
58
53
52
ꢀ/
ꢀ/
147
nt
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
80
68
79
92
ꢀ/
43
NCI-H322M
NCI-H460
NCI-H522
nt
ꢀ/
ꢀ/
nt
85
200
97
ꢀ/
ꢀ/
185
187
ꢀ/
41
57
162
146
48
154
137
ꢀ/
43
ꢀ/
ꢀ/
44
108
ꢀ/
ꢀ/
ꢀ/
42
132
126
60
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
ꢀ
ꢀ/
ꢀ/
43
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
200
198
166
169
96
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
nt
73
46
64
56
58
67
ꢀ
ꢀ/
ꢀ/
43
42
43
51
/
41
50
196
197
179
118
176
145
89
ꢀ
ꢀ/
ꢀ/
46
53
49
/
200
139
118
94
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
90
61
ꢀ
ꢀ/
ꢀ/
63
/
113
113
158
94
ꢀ
ꢀ/
ꢀ/
46
/
68
88
93
88
99
83
56
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
ꢀ/
75
64
89
92
81
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
48
51
55
93
76
49
ꢀ/
ꢀ/
ꢀ/
57
ꢀ/
46
154
87
48
75
ꢀ/
45
61
96
78
ꢀ/
ꢀ/
ꢀ/
KM12
SW-620
177
162
ꢀ/
ꢀ/
ꢀ/
ꢀ/
180
143
ꢀ/
ꢀ/
ꢀ/
81
83
48
SNC cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
66
60
49
50
nt
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
135
152
170
95
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
79
83
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
62
/
ꢀ
71
/
92
156
176
122
135
200
ꢀ
48
/
78
98
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
66
84
91
44
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
82
96
nt
ꢀ
55
nt
/
88
116
187
81
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
65
60
76
67
99
86
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
88
67
ꢀ/
ꢀ/
ꢀ/
48
ꢀ/
ꢀ/
47
40
ꢀ/
ꢀ/
113
62
95
52
96
ꢀ/
ꢀ/
ꢀ/
nt
168
nt
147
101
48
109
136
ꢀ/
91
145
124
62
ꢀ/
ꢀ/
Melanoma
LOX IMVI
MALME-3M
M14
ꢀ
ꢀ/
ꢀ/
66
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
200
200
189
200
195
200
200
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
58
42
nt
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
45
52
200
138
174
184
96
75
154
53
49
100
81
ꢀ
ꢀ/
ꢀ/
ꢀ/
43
98
43
/
91
130
121
nt
ꢀ
ꢀ/
ꢀ/
nt
/
85
89
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
69
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ/
56
ꢀ/
48
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
109
94
87
70
59
45
43
51
50
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
77
51
58
46
ꢀ/
ꢀ/
57
45
ꢀ/
ꢀ/
ꢀ/
168
75
ꢀ/
ꢀ/
ꢀ/
69
105
140
77
72
ꢀ/
110
59
5
200
176
158
119
177
131
94
116
ꢀ/

Table 2 (Continued)
1
2
4
5
6
10
11
15
21
25
Molar concentration (M)
10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5 10ꢁ4 10ꢁ5
UACC-62
ꢀ/
ꢀ/
200
ꢀ/
65
ꢀ/
ꢀ/
40
200
ꢀ/
111
ꢀ/
123
41
82
43
137
ꢀ/
ꢀ/
ꢀ/
Ovarian cancer
IGROV1
61
ꢀ
ꢀ/
/
135
153
nt
ꢀ
ꢀ/
/
63
57
ꢀ
ꢀ/
/
ꢀ
ꢀ/
nt
47
47
/
ꢀ
ꢀ/
/
88
132
nt
ꢀ
ꢀ/
/
91
87
nt
ꢀ
ꢀ/
/
88
79
ꢀ
ꢀ/
/
82
137
91
ꢀ
51
47
/
90
45
ꢀ
ꢀ/
/
82
60
59
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
ꢀ/
nt
nt
nt
nt
40
nt
nt
nt
nt
134
41
50
nt
76
nt
ꢀ/
66
76
ꢀ/
43
83
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
124
105
103
ꢀ/
ꢀ/
ꢀ/
71
66
78
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
90
94
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
66
44
199
127
72
103
94
67
109
147
ꢀ/
ꢀ/
90
Renal cancer
786-0
51
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
164
126
115
105
159
nt
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
97
66
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
ꢀ
40
/
63
46
42
170
170
110
96
55
120
75
92
66
122
nt
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
93
55
85
97
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
132
45
ꢀ
ꢀ/
/
183
60
ꢀ
ꢀ/
ꢀ/
ꢀ/
/
55
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
A498
ꢀ/
ꢀ/
ꢀ/
110
ꢀ/
ꢀ/
ꢀ/
46
nt
41
ꢀ/
64
ACHN
CAKI-1
RXF 393
SN12C
TK-10
57
46
ꢀ/
ꢀ/
55
61
76
75
40
148
93
ꢀ/
81
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
74
128
141
84
163
nt
ꢀ/
41
157
nt
40
nt
78
ꢀ/
45
51
74
56
110
113
106
ꢀ/
ꢀ/
ꢀ/
ꢀ/
47
50
155
167
ꢀ/
ꢀ/
126
122
94
nt
121
92
UO-31
ꢀ/
nt
ꢀ/
ꢀ/
45
Prostate cancer
PC-3
ꢀ
ꢀ/
/
ꢀ
ꢀ/
/
200
171
ꢀ
ꢀ/
/
78
53
ꢀ
ꢀ/
/
48
43
171
134
47
103
69
ꢀ
ꢀ/
/
63
69
ꢀ
ꢀ/
/
94
55
ꢀ
ꢀ/
/
172
101
ꢀ
ꢀ/
/
70
56
ꢀ
ꢀ/
/
DU-145
ꢀ/
ꢀ/
ꢀ/
Breast cancer
MCF7
ꢀ
ꢀ/
89
57
/
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
200
125
147
86
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
2
72
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
51
92
/
ꢀ
54
/
42
51
51
129
130
170
97
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
82
61
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
133
67
94
100
112
78
77
50
100
140
145
96
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
70
ꢀ
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
/
MCF7/ADR-RES
MDA-MB-231/ATCC
HS 578T
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
55
ꢀ/
ꢀ/
58
54
56
70
50
81
76
ꢀ/
ꢀ/
57
51
138
65
51
57
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
51
65
93
MDA-MB-435
BT-549
T-47D
ꢀ/
59
127
200
136
169
200
99
47
161
125
133
173
82
86
192
87
97
ꢀ/
100
96
67
111
158
98
127
85
123
94
109
41
41
113
74
ꢀ/
93
78
140
41
MDA-N
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
ꢀ/
88
ꢀ/
ꢀ/
ꢀ/
ꢀ/, below 40% growth inhibition; nt, not tested at this molar concentration.

S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ
/
650
647
Table 3
log GI50, ꢁ
of in vitro inhibitory activity test for compounds 1, 2, 4ꢀ
Table 5
Activity of compounds 2, 13, 10, 19, 29c,d against hematological
tumour-derived cell lines
ꢁ/
/
log TGI, ꢁ
/
log LC50 mean graph midpoints (MG-MD)
/6, 10, 11, 15,
21 and 25
a
Comp.
IC50 (mM)
Comp.
ꢁ
/
log GI50
ꢁ
/
log TGI
ꢁ/log LC50
CCRF-CEM b
WIL-2NS c
CCRF-SB d
1
4.16
4.85
4.51
4.25
4.82
4.54
4.70
4.75
4.60
4.33
4.04
4.41
4.14
4.02
4.34
4.10
4.14
4.13
4.15
4.00
4.00
4.12
4.09
4.00
4.07
4.01
4.03
4.01
4.03
4.00
2
17.6
25.1
48.1
21.4
20.7
58.5
20.1
20.5
52.5
2
10
13
19
29c
29d
4
5
ꢀ/
100
3.4
6.8
ꢀ/
100
10.3
39.2
ꢀ/
100
3.5
19.5
6
10
11
15
21
25
a
Compound concentration required to reduce cell proliferation by
50%, as determined by the MTT method, under conditions allowing
untreated controls to undergo at least three consecutive rounds of
multiplication. Data represent mean values (9
dent determinations.
/
SD) for three indepen-
MG-MID: mean graph midpoints, the average sensitivity of all cell
lines towards the test agent from NCI.
b
CD4ꢃ human acute T-lymphoblastic leukaemia.
Human splenic B-lymphoblastoid cells.
Human acute B-lymphoblastic leukemia.
c
d
6. Experimental
Melting points were determined by a Kofler hot stage
or Digital Electrothermal apparatus and are uncor-
rected. UV spectra are qualitative and were recorded in
Table 4
Anti-HIV activity of compounds 1ꢀ28, 29c,d
/
a
b
Comp.
CC50
MT-4
EC50
(HIV-1)
nm for solutions in ethanol with a Perkinꢀ
5 spectrophotometer. Infrared spectra are for nujol
mulls and were recorded using a PerkinꢀElmer 781
/Elmer Lamba
1
ꢀ/
100
ꢀ
/
100
31
100
100
/
1
2
31
100
100
57
ꢀ
/
spectrophotometer. H NMR spectra were recorded on
a Varian XL-200 (200 MHz) instrument, using TMS as
internal standard Elemental analyses were performed by
the Laboratorio di Microanalisi, Dipartimento di
Scienze Farmaceutiche, University of Padua, Italy.
3
ꢀ
/
ꢀ
/
4
ꢂ/
ꢀ
/
5
ꢀ
/
57
27
6
27
ꢀ
/
7
ꢀ
/
100
100
100
15
ꢀ
/
100
100
100
8
ꢀ
/
ꢀ
/
The analytical results for C, H, N were within 90.4%
/
9
ꢂ/
ꢀ
/
of the theoretical values.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29c
29d
ꢀ
/
15
55
55
ꢀ
/
6.1. Chemistry
ꢀ/
100
16
ꢀ
/
100
16
100
ꢀ
/
ꢀ/
100
100
100
52
ꢀ
/
6.1.1. Intermediates
ꢀ
/
100
100
Compounds 29a,b could not be obtained according to
the data of the literature [13], but we performed their
preparation using the procedure described by Rault et
al. [12] for either 29a,b and the previously described 29c.
The p-hydroxybenzoyl-L-glutamate diethyl ester (33) is
not commercially available and was obtained according
to the procedure described in the literature [16,17].
ꢀ/
ꢀ
/
ꢀ
/
52
100
ꢀ/
100
8
ꢀ/
ꢀ8
ꢀ100
/
ꢀ/
100
48
/
ꢀ
/
48
39
39
ꢀ
/
ꢀ
/
100
100
100
68
ꢀ
/
100
100
100
100
ꢀ
/
ꢀ
/
ꢀ/
ꢀ
/
6.1.1.1. 1-(2-Amino-4-trifluoromethylphenyl)pyrrole
(35). A mixture of 1-(2-nitro-4-trifluoromethyl)pyrrole
[13] (34) (4.1 g, 16 mmol), and an excess of hydrazine
hydrate (5.13 g 160 mmol) in ethanol (100 ml) and in
presence of 10% palladised charcoal (0.4 g) was refluxed
for 3 h. On cooling, the ethanolic solution was filtered
off and evaporated in vacuo to give a crude solid, which
was purified by flash chromatography on a silica gel
column eluting with a mixture of ethyl acetate/petrol
ꢀ
/
95
ꢀ
/
95
100
ꢀ/
100
6
ꢀ
/
ꢀ
/
100
100
18
ꢀ/
a
Compound concentration (mM) required to reduce the viability of
mock-infected MT-4 cells by 50%, as determined by the MTT method.
b
Compound concentration (mM) required to achieve 50% protec-
tion of MT-4 cells from the HIV-1 induced cytopathogeneticy, as
determined by the MTT method.
ether (b.p. 40ꢀ60 8C) in the ratio of 9:1. The fast moving
/

648
S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ650
/
Table 6
Activity of compounds 2, 13, 10, 19, 29c,29d against solid tumour-derived cell lines
a
Comp.
IC50 (mM)
SK-MEL-28 b
MCF7 c
SKMES-1 d
HepG2 e
DU145 f
2
46.7
46.0
88.0
35.4
45.0
87.6
23.0
38.3
45.0
35.1
38.0
100
10
13
19
29c
29d
ꢀ
ꢀ/
/
100
ꢀ
ꢀ/
/
100
100
ꢀ
ꢀ/
/
100
ꢀ/
100
30.4
56.8
100
100
ꢀ
/
100
23.7
55.8
28.1
62.1
21.6
59.8
37.5
70.4
a
Compound concentration required to reduce cell proliferation by 50%, as determined by the MTT method [19], under conditions allowing
untreated controls to undergo at least three consecutive rounds of multiplication. Data represent mean values (9
/
SD) for three independent
determinations.
b
Human skin melanoma.
c
d
e
f
Human breast adenocarcinoma.
Human lung squamous carcinoma.
Human hepatocellular carcinoma.
Human prostate carcinoma.
removal of the solvent under reduced pressure, the
residue was taken up with water and extracted with
Table 7
Comparison based on mean graph activity between compounds 5, 6,
10, 15 and the corresponding 3-phenyl analogues derivatives 37ꢀ
/40
ethyl ether (2ꢄ20 ml). The organic extract was dried
/
previously described as cited [Ref.] reported below
over anhydrous sodium sulfate and evaporated to
dryness. The solid residue (85% yield) was recrystallized
Comp. (Ref.)
ꢁ
/
log GI50 (mM)
ꢁ
/
log TGI (mM)
ꢁ
/
log LC50 (mM)
from methanol/water, m.p. 125ꢀ
/
127 8C. IR: 3280, 1680.
UV: 250, 234, 202. H NMR (CDCl3): d 8.81 (1H, a s,
H-3?), 8.44 (1H, br s, NH), 7.50 (2H, dd, J5?,3?ꢂ8.4 Hz
and J5?,6?ꢂ1.6 Hz, H-5?), 7.44 (1H, d, J3?,4?ꢂ8.4 Hz, H-
5
4.25 (56.2)
4.31(49)
4.02 (95.5)
4.11 (77.6)
4.34 (45.7)
4.18 (66.1)
4.10 (79.4)
4.22 (60.2)
4.10 (79.4)
4.00 (100)
4.00 (100)
4.02 (95.5)
4.07 (85.1)
4.00 (100)
4.01 (97.7)
4.04 (91.2)
4.01 (97.7)
4.00 (100)
1
37 [1]
6
/
4.82 (15.1)
4.66 (21.9)
4.54 (28.8)
4.70 (19.9)
4.54 (2.88)
4.15 (70.8)
/
/
38 [1]
10
6?), 6.82 (2H, t, H-2,5), 6.46 (2H, t, H-3,4), 4.11 (2H, s,
CH2).
39 [3]
15
40 [7]
6.1.1.3. 4-Chloromethyl-7-trifluoromethylpyrrolo[1,2-
a]quinoxaline (29d). A mixture of the amide 36 (1.21 g,
4 mmol) and an excess of POCl3 (6.17 g, 40 mmol) was
refluxed for 4 h. On cooling, the solvent was removed in
vacuo and the residue was taken up with water, made
alkaline with NaHCO3 and extracted with ethyl acetate.
The organic layer was then washed out with water (24
ml) and eventually dried over anhydrous sodium sulfate.
On evaporation of the solvent in vacuo, a crude product
(68%) was obtained and recrystallized from methanol,
m.p. 99ꢀ
(CDCl3): d 8.27 (1H, d, J6,8
Jꢂ2.4 Hz, H-1), 7.95 (1H, d, J9,8
(1H, dd, J8,9 8.8 Hz and J8,6 1.6 Hz, H-8), 7.14ꢀ
(1H, m, H-3), 7.00ꢀ6.97 (1H, m, H-2), 4.86 (2H, s,
CH2Cl).
/
101 8C. UV 350, 253, 231, 209. 1H NMR
1.6 Hz, H-6), 7.80 (1H, d,
8.8 Hz, H-9), 7.77
7.12
ꢂ
/
fraction gave on evaporation compound 35 (55% yield),
m.p. 92ꢀ94 8C (MeOH). IR: 3380, 3300, 3206. UV: 333,
307, 229, 205. 1H NMR (CDCl3): d 7.24 (1H, d, J6?,5?ꢂ
8.4 Hz, H-6?), 7.04 (1H, d, J3?,5?ꢂ2.5 Hz, H-3?), 7.02
(1H, dd, J5?,6?ꢂ8.8 Hz and J5?,3?ꢂ2.5 Hz, H-5?), 6.85
/
ꢂ
/
/
ꢂ
/
ꢂ
/
/
/
/
/
/
/
(2H, t, H-2, 5), 6.38 (2H, t, H-3, 4), 3.94 (2H, br s, NH2).
6.1.2. General procedure for preparation of 4-anilino
pyrrolo[1,2-a] quinoxaline (1ꢀ8)
/
6.1.1.2. 2-Chloro-N-[(5-trifluoromethyl-2-pyrrol-1-
yl)phenyl]acetamide (36). Pyridine (0.38 g, 4.8 mmol)
was added to a solution of 1-(2-amino-4-trifluoro-
methylphenyl)pyrrole (35) (0.99 g, 4.4 mmol) in dioxane
(30 ml) followed by chloroacetyl cloride (0.50 g, 4.4
mmol). The reaction mixture was refluxed for 4 h. After
A mixture of equimolar amounts (0.5 mmol) of
chloropyrrolo[1,2-a] quinoxaline [12] 29a or 29b and
the corresponding substituted anilines 30 of Fig. 1 in 1-
propanol was refluxed for 2/10 h. After cooling the
solvent was removed under reduced pressure. The
residue was taken up with acetone and filtered off.

S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ
/
650
649
The coloured products (1ꢀ
described in Table 1 which also contains yields, melting
points, analytical and spectroscopic data.
/
8) were further purified as
aqueous solution. The solid precipitate was filtered off
and washed with water and eventually recrystallized. In
the case of compound 23, the acidic aqueous solution
was extracted with chloroform. The organic phase, dried
over anhydrous sodium sulfate and evaporated in vacuo
gave a crystalline solid. Yields, melting points, analytical
and spectroscopic data are reported in Table 1.
6.1.3. Diethyl N-[4-(pyrrolo[1,2-a]quinoxalin-4-
yl)amino]benzoyl-
trifluoromethylpyrrolo[1,2-a]quinoxalin-4-
yl)amino]benzoyl- -glutamate (22)
A solution of equimolar amounts (0.5 mmol) 29a or
29b and the p-amino-benzoyl- -glutamate (32) in 1-
L-glutamate (21) and diethyl N-[4-(7-
L
L
7. Material and methods
propanol (10 ml) was refluxed for 2 h. After cooling the
solvent was evaporated in vacuo and the oily residues
were taken up with acetone to give solid compounds.
Purification methods, yields, melting points, analytical
and spectroscopic data are reported in Table 1.
7.1. Compounds
Test compounds were solubilised in DMSO at 100
mM and then diluted into culture medium.
6.1.4. General procedure for preparation of 4-
7.2. Cells
phenoxymethylpyrrolo[1,2-a]quinoxalines (11ꢀ18)
/
A mixture of equimolar amounts (0.7 mmol) of 29c,d
and the corresponding substituted phenols 31 of Fig. 1
in anhydrous DMF (5 ml) and in the presence of one
mole equivalent of caesium carbonate, was stirred at
70 8C for 6 h. In the case of compounds 14 and 18,
caesium hydrogenocarbonate was alternatively used and
the mixture heated at 70 8C for 2 h. After cooling, water
Cell lines were purchased from American Type
Culture Collection (ATCC). Hematological tumor-de-
rived cells were grown in RPMI-1640 medium supple-
mented with 10% FCS, 100 U/ml penicillin G and 100
mg/ml streptomycin. Solid tumor-derived cells were
grown in their specific media supplemented with 10%
FCS and antibiotics. Cell cultures were incubated at
37 8C in a humidified, 5% CO2 atmosphere. The absence
of mycoplasma contamination was checked periodically
by the Hoechst staining method.
was added to complete precipitation of the solids (11ꢀ
/
18) which were filtered off and washed with water.
Purification methods, yields, melting points, analytical
and spectroscopic data are reported in Table 1.
7.3. Antiproliferative assays
6.1.5. Diethyl N-[4-(pyrrolo[1,2-a]quinoxalin-4-
yl)hydroxymethyl]benzoyl-
N-[4-(7-trifluoromethylpyrrolo[1,2-a]quinoxalin-4-
yl)hydroxymethyl]benzoyl- -glutamate (26)
A mixture of equimolar amounts (0.7 mmol) of 29c,d
and diethyl 4-hydroxy-benzoyl- -glutamate [15,16] (33),
L
-glutamate (25) and diethyl
Exponentially growing cells were resuspended in
growth medium containing serial dilutions of the drugs.
Cell viability was determined after 96 h at 37 8C by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) method [18,19].
L
L
dissolved in anhydrous DMF (6 ml) and in the presence
of one mole equivalent of caesium hydrogencarbonate,
was stirred at 70 8C for 2 h. After cooling water was
added. A cream coloured solid precipitate (25) was first
filtered then taken up with ether. In the case of
compound 26 the acidic aqueous solution was extracted
with chloroform. The organic phase, dried over anhy-
drous sodium sulfate, was evaporated in vacuo to give
an oily residue that was further purified as reported in
Table 1, in which are included yields, melting points,
analytical and spectroscopic data.
7.4. Virus
Human immunodeficiency virus type 1 (HIV-1) was
obtained from supernatants of persistently infected H9/
IIIB cells. The HIV-1 stock solution had a titre of 1.0ꢄ
/
107 50% cell culture infectious dose (CCID50)/ml.
7.5. Antiviral assays
Activity of compounds against HIV-1 was based on
inhibition of virus-induced cytopathogenicity in MT-4
cells acutely infected at a multiplicity of infection of
0.01.
6.1.6. Hydrolysis of esters 4, 8, 14, 18, 21, 22, 25 and 26
into the acids 9, 10, 19, 20, 23, 24, 27 and 28
A mixture of ester (4, 8, 14, 18, 21, 22, 25, 26) (0.6
mmol) in ethanol (10 ml) and 1 M NaOH aqueous
solution (5 ml) was refluxed for 4 h or heated at 70 8C in
the case of compound 19. On cooling, after removal of
the solvent under reduced pressure, the residue was
diluted with water and made acidic with 2 M HCl
References
[1] M. Loriga, M. Fiore, P. Sanna, G. Paglietti, Quinoxaline
chemistry. Part 4. 2-(R)-Anilinoquinoxalines as non classic

650
S. Alleca et al. / Il Farmaco 58 (2003) 639ꢀ650
/
antifolate agents. Synthesis, structure elucidation and evaluation
of in vitro anticancer activity, Farmaco 50 (1995) 289ꢀ301.
[2] M. Loriga, M. Fiore, P. Sanna, G. Paglietti, Quinoxaline
chemistry. Part 5. 2-(R)-Benzymamminoquinoxalines as non
classical antifolate agents. Synthesis and evaluation of in vitro
[9] P. Corona, G. Vitale, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part 13. 3-Carboxy-benzylamino-substituted quinox-
alines and N-[4-[3-carboxyquinoxaline-2-yl)aminomethyl]ben-
/
zoyl-L
-glutamate: synthesis and evaluation of in vitro anticancer
86.
activity, Farmaco 55 (2000) 77ꢀ
/
[10] S. Piras, M. Loriga, G. Paglietti, Quinoxaline chemistry. Part 14.
4-(2-Quinoxalylamino)-phenylacetates and 4-(2-quinoxalyla-
mino)-phenylacetyl-l-glutamates as analogues-homologues of
classical antifolate agents. Synthesis and evaluation of in vitro
anticancer activity, Farmaco 51 (1996) 559ꢀ568.
/
[3] M. Loriga, S. Piras, P. Sanna, G. Paglietti, Quinoxaline chem-
istry. Part 7. 2-[Aminobenzoates]- and 2[aminobenzoylglutamate]-
quinoxalines as classical antifolate agents. Synthesis and evalua-
tion of in vitro anticancer, anti-HIV and antifungal activity,
anticancer activity, Farmaco 57 (2002) 1ꢀ8.
/
[11] M. Loriga, S. Piras, G. Paglietti, M.P. Costi, A. Venturelli,
Quinoxaline chemistry. Part 15. 4-[2-Quinoxalylmethylenimino]-
benzoylglutamates and -benzoates, 4-[2-quinoxalylmethyl-N-
methylamino]-benzoylglutamates as analogues of classical anti-
folates agents. Synthesis, elucidation of structures and in vitro
evaluation of anticancer, anti HTS and antiDHFR activities,
Farmaco 58 (2003) 51.
Farmaco 52 (1997) 157ꢀ166.
/
[4] M. Loriga, P. Moro, P. Sanna, G. Paglietti, Quinoxaline
chemistry. Part 8. 2-[Anilino]-3-[carboxy]-6(7)-substituted quinox-
alines as non classical antifolate agents. Synthesis and evaluation
of in vitro anticancer, anti-HIV and antifungal activity, Farmaco
52 (1997) 531ꢀ537.
/
[5] G. Vitale, P. Corona, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part. 9. Quinoxaline analogues of TMQ and 10-
propargyl-5,8-dideaza folic acid and its precursors. Synthesis
and evaluation of in vitro anticancer activity, Farmaco 53
[12] J. Guillon, P. Dallemagne, B. Pfeiffer, P. Renard, D. Manechez,
A. Kervran, S. Rault, Synthesis of new pyrrolo[1,2-a]quinoxa-
lines: potential non-peptide glucagon receptor antagonist, Eur. J.
Med. Chem. 33 (1988) 293ꢀ308.
/
(1998) 139ꢀ149.
/
[13] G. Campiani, V. Nacci, F. Corelli, M. Anzini, Polycondensed
heterocycles. VII. A convenient synthesis of pyrrolo[1,2-a]qui-
noxaline derivatives by intramolecular aromatic nucleophilic
[6] G. Vitale, P. Corona, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part. 10. Quinoxaline 10-oxa analogues of TMQ and
10-propargyl-5,8-dideaza folic acid and its precursors. Synthesis
and evaluation of in vitro anticancer activity, Farmaco 53 (1998)
displacment, Synth. Commun. 21 (1991) 1567ꢀ1576.
/
[14] G.W.H. Cheeseman, B. Tuck, Further synthesis and properties of
pyrrolo[1,2-a]quinoxalines, J. Chem. Soc. (1965) 3678.
[15] M.R. Boyd, Status of the NCI preclinical antitumour drug
150ꢀ159.
/
[7] P. Corona, G. Vitale, M. Loriga, G. Paglietti, M.P. Costi,
Quinoxaline chemistry. Part. 11. 3-Phenyl-2(phenoxy-and phe-
noxymethyl)-6(7) or 6,8-substituted quinoxalines and N-(4-(6(7)-
substituted or 6,8-disubstituted-3-phenylquinoxalin-2-yl)hydroxy
or hydroxymethyl]benzoylglutamates. Synthesis and evaluation of
in vitro anticancer activity and enzymatic inhibitory activity
against dihydrofolate reductase and thymidylate synthase, Farm-
discovery screem, Princ. Proct.: Oncol. 3 (1989) 1ꢀ12.
/
[16] A.R. Robertson, R. Robinson, Experiments on the synthesis of
anthocyanins. Part. 1, J. Chem. Soc. (1926) 1713.
[17] E.I. Fairburn, B.J. Magerlein, L. Stubberfield, E. Staperrt, D.I.
Wisblat, Oxygen analogs of pteroic acid, J. Am. Chem. Soc. 76
(1954) 676.
aco 53 (1998) 480ꢀ493.
/
[18] R. Pauwels, J. Balzarini, M. Baba, R. Snoeck, D. Herdewijin, J.
Desmyster, E. De Clercq, Rapid and automated tetrazolium-
based colorimetric assay for the detection of anti-HIV com-
pounds, J. Virol. Methods 20 (1988) 309.
[8] G. Vitale, P. Corona, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part. 12. 3-Carboxy-2[phenoxy]-6(7)substituted qui-
noxalines and N-[4-(6(7) substituted-3-carboxyquinoxaline-2-
yl)hydroxy]benzoylglutamates. Synthesis and evaluation of in
[19] F. Denizot, R. Lang, Rapid colorimetric assay for cell growth and
vitro anticancer activity, Farmaco 53 (1998) 594ꢀ
/601.
survival, J. Immunol. Methods 89 (1986) 271ꢀ277.
/