Quinoxaline chemistry. Part 14. 4-(2-Quinoxalylamino)-phenylacetates and 4-(2-quinoxalylamino)-phenylacetyl-L-glutamates as analogues-homologues of classical antifolate agents. Synthesis and evaluation of in vitro anticancer activity

Article abstract of DOI:10.1016/S0014-827X(01)01159-4

Among a new series of 26 4-(3-substituted-2-quinoxalylamino)phenylacetates and 4-(3-substituted-2-quinoxalylamino)-phenylacetyl-L-glutamates, eight were selected at NCI for evaluation of their in vitro anticancer activity. The results obtained in comparison with the corresponding nor-compounds series seem to indicate that this type of homologation is not helpful.

Full text of DOI:10.1016/S0014-827X(01)01159-4

Il Farmaco 57 (2002) 1–8
www.elsevier.com/locate/farmac
Quinoxaline chemistry. Part 14.
4-(2-Quinoxalylamino)-phenylacetates and
4-(2-quinoxalylamino)-phenylacetyl- -glutamates as
L
analogues–homologues of classical antifolate agents. Synthesis and
evaluation of in vitro anticancer activity
Sandra Piras, Mario Loriga, Giuseppe Paglietti *
Dipartimento Chimico Tossicologico, Uni6ersita` di Sassari, Via Muroni, 23, 07100 Sassari, Italy
Received 3 March 2001; accepted 25 July 2001
Abstract
Among
phenylacetyl-
a
new series of 26 4-(3-substituted-2-quinoxalylamino)phenylacetates and 4-(3-substituted-2-quinoxalylamino)-
-glutamates, eight were selected at NCI for evaluation of their in vitro anticancer activity. The results obtained in
L
comparison with the corresponding nor-compounds series seem to indicate that this type of homologation is not helpful. © 2002
Elsevier Science S.A. All rights reserved.
Keywords: Anticancer activity; Quinoxalines derivatives; (Quinoxalylamino)-phenylacetates and (quinoxalylamino)-phenylacetyl-L-glutamate
1. Introduction
2. Chemistry
Bioisosteric replacement of the pteridine ring in the
classical antifolates with quinoxaline has proved to be a
good substrate for the biological activity as both in
vitro anticancer and anti-DHFR [1–8]. Among the
various types of quinoxalines designed for this purpose
we have considered the effect of homologation in both
para positions of the benzoyl-glutamate moiety. The
series of quinoxalines bearing at 2 position the
aminomethylbenzoylglutamate moiety has just ap-
peared [9]. In this report we describe the synthesis of
compounds 3–28 where homologation takes place on
the side of the carboxylic moiety. In the hetero-
cyclic ring we have placed the usual substitutions
that in previous cases had shown the most similarities
with the classical antifolates and they are reported in
Fig. 1.
The starting material for the preparation of com-
pounds 3–28 is represented by the chloroquinoxalines
1a–f of Scheme 1, which were all obtained according to
previously reported procedures [1,3,9–12]. Nucleophilic
attack by ethyl p-aminophenylacetate gave compounds
3–8 in good yields. Conversion by alkaline hydrolysis
into the acids 9–14 was necessary for elongation of side
chain with diethyl
L-glutamate in the presence of di-
ethylcyanophosphonate and triethylamine to give the
esters 15–20. In the case of the esters 6,7 and 8
saponification occurred also at the ester group in C-3 to
give the acids 12, 13 and 14 which only in the case of 12
and 13 underwent further amidification with diethyl
L
-glutamate at carboxy group in position 3 to give the
ester amides 27, 28. In the end the desired acids 21–26
were obtained by alkaline hydrolysis in hydroalcoholic
medium.
3. Experimental
Melting points are uncorrected and were recorded on
a Kofler or an Electrothermal melting point apparatus.
* Correspondence and reprints.
E-mail address: paglieti@ssmain.uniss.it (G. Paglietti).
0014-827X/02/$ - see front matter © 2002 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(01)01159-4

2
S. Piras et al. / Il Farmaco 57 (2002) 1–8
UV spectra are qualitative and were recorded in nm in
ethanol solution with a Perkin–Elmer Lambda 5 spec-
trophotometer. IR spectra (Nujol mulls) were recorded
3.1.2. General procedure for the preparation of the
esters 3–8
A mixture of equimolar amounts (2 mmol) of
chloroquinoxalines (1a–f) and commercially available
(Aldrich) ethyl 4-aminophenylacetate in ethanol (10 ml)
was refluxed for 13 h (compounds 5–7), for 24 h
(compounds 4, 8) and for 72 h (compound 3). On
cooling the yellow–orange precipitates were collected
and washed with ethanol to give 3–8 as crude products
which were recrystallized from ethanol. Yields, m.p.
values, analytical and spectroscopic (IR, UV, 1H
NMR) data are reported in Table 1.
1
with Perkin–Elmer 781 instrument. H NMR spectra
were recorded at 200 MHz with a Varian XL-200
instrument using TMS as internal standard. Elemental
analyses were performed at Laboratorio di Microanal-
isi, Dipartimento di Scienze Farmaceutiche, University
of Padua. The analytical results for C, H, and N were
within 90.4% of the theoretical values.
3.1. Chemistry
3.1.1. Intermediates
3.1.3. General procedure for the preparation of the
acids 9–14
The intermediate chloroquinoxalines necessary for
this work were known and prepared according to the
data of the literature as follow: 1a [10], 1b [11], 1c [3],
1d [12], 1e [9], and 1f [1].
A mixture of the ester (3–8) (0.7 mmol) in ethanol
(10 ml) and 2M NaOH (5 ml) in the case of compounds
3, 4 or 1M NaOH (5 ml) in the case of compounds 5–8
was stirred at room temperature for 2.5 h (3, 4) and
under reflux for 4 h (5–8). On evaporation of the
solvent, the mixture was taken up with water and made
acidic with 2 M HCl. The red–orange products (9–14)
were collected and washed with water. Yields, m.p.
values, analytical and spectroscopic data are reported
in Table 1.
3.1.4. General procedure for the preparation of the
esters 15–20 and isolation of compounds 27, 28
An equimolar mixture (1.2 mmol) of compounds
(9–11), diethyl L-glutamate hydrochloride and diethyl
cyanophosphonate in the presence of 2 mole equiv. of
TEA, and a ratio of 1:2:2 and 4 mole equiv. of TEA in
the case of 12–14, was stirred under nitrogen at room
temperature for 2 h. The resulting solution was poured
into a mixture of ethyl acetate and benzene in 3:1 ratio
(60 ml). The organic phase was shaken with water (50
ml), then with saturated sodium carbonate aqueous
solution (60 ml), rewashed with water (50 ml) and, if
necessary, with saturated sodium chloride aqueous so-
lution. Eventually, after drying over anhydrous sodium
sulfate, on evaporation of the solvent compounds 15–
17, 27, 28 were obtained as yellow–orange solids. From
the alkaline mother liquors, on standing compounds
18–20 were separated as pure yellow solids. Yields,
m.p. values, analytical and spectroscopic data are re-
ported in Table 1.
3.1.5. General procedure for the preparation of the
acids 21–26
A suspension of the ester (15–20) (1 mmol) in a
mixture of ethanol (10 ml) and 1 M NaOH (5 ml) was
stirred at room temperature for 4 h. The red–orange
solution formed was evaporated in vacuo and taken up
with water then made acidic with 2 M HCl. A solid
from yellow to red was collected and thoroughly
washed with water and eventually dried. Yields, m.p.
Fig. 1. Compounds 3–28 obtained according to Scheme 1.

Table 1
Melting point, yield, analytical and spectroscopic (IR, UV, 1H NMR) data of the compounds of Fig. 1
Comp. m.p. (°C) a
Yield (%)
Analysis for
IR (nujol) (wmax UV (EtOH)
1H NMR, lH (J in Hz) b
cm−1
)
(umax nm)
3
4
65–68 (a)
69–71 (a)
50
51
C
24H21N3O2
3380, 1710
370, 270, 211,
193
385, 276, 196
[A] 7.99–7.77 (2H, m, arom.), 7.76–7.44 (7H, m, arom), 7.72 (2H, d, J=8.4, H-2%,6%, 7.29
(2H, d, J=8.4, H-3%,5%, 4.14 (2H, q, CH2), 3.60 (2H, s, CH2), 1.26 (3H, t, CH3)
[A] 7.98–7.92 (1H, m, arom.), 7.86–7.72 (1H, m, arom.), 7.77 (2H, d, J=8.6, H-2%,6%), 7.31
(2H, d, J=8.6, H-3%,5%), 7.29–7.25 (1H, m, arom.), 4.15 (2H, q, CH2), 3.62 (2H, s, CH2),
1.27 (3H, t, CH3)
C22H19N3O2S 3400, 1720
C18H15F2N3O2 3380, 1720
5
6
7
8
130–133 (a)
78–80 (a)
62
62
68
49
382, 332, 283,
208
[A] 8.34 (1H, s, H-3), 7.64 (1H, ddp, J=11.2 and 8.0, H-8), 7.63 (2H, d, J=8.4, H-2%,6%),
7.49 (1H, dd, J=8.0 and 11.2, H-5), 7.29 (2H, d, J=8.4, H-3%,5%), 4.19 (2H, q, CH2), 3.63
(2H, s, CH2), 1.29 (3H, t, CH3)
C
21H21N3O4
3460, 3320, 1730, 425, 290, 222,
1700 206
[A] 10.33 (1H, s, NH), 8.04 (1H, d, J=8.2, arom.), 7.88 (2H, d, J=8.4, H-2%,6%), 7.86–7.67
(2H, m, arom.), 7.52–7.45 (1H, m, arom.), 7.32 (2H, d, J=8.4, H-3%,5%), 4.60 (2H, q, CH2),
4.17 (2H, q, CH2), 3.62 (2H, s, CH2), 1.54 (3H, t, CH3), 1.27 (3H, t, CH3)
[A] 10.39 (1H, s, NH), 8.14 (1H, d, J=8.8, H-5), 8.08 (1H, s, H-8), 7.86 (2H, d, J=8.4,
H-2%,6%), 7.64 (1H, dd, J=1.8 and 1.8, H-6), 7.34 (2H, d, J=8.4, H-3%,5%), 4.61 (2H, q,
CH2), 4.17 (2H, q, CH2), 3.63 (2H, s, CH2), 1.54 (3H, t, CH3), 1.31 (3H, t, CH3)
[A] 10.34 (1H, s, NH), 7.81 (2H, d, J=8.6, H-2%,6%), 7.78 (1H, dd,p 11.0 and 8.0 H-8), 7.51
(1H, dd, J=11 and 8.0, H-5), 7.32 (2H, d, J=8.6, H-3%,5%), 4.59 (2H, q, CH2), 4.21 (2H,
q, CH2), 3.63 (2H, s, CH2), 1.53 3H, t, CH3), 1.27 (3H, t, CH3)
112–115 (a)
72–74 (a)
C
22H20F3N3O4 3340, 1725, 1690 427, 304, 287,
219
C21H19F2N3O4 3280, 1730, 1690 426, 288, 219
9
10
11
200–203
174–177
205 (d)
85
95
87
C
22H17N3O2
1720
392, 285, 206
388, 277, 196
[A] 8.00–7.72 (2H, m, arom.), 7.75 (2H, d, J=8.4, H-2%,6%), 7.70–7.35 (7H, m, arom.), 7.28
(2H, d, J=8.4, H-3%,5%), 7.09 (1H, s, NH), 3.66 (2H, s, CH2)
[A] 7.96–7.72 (2H, m, arom.), 7.78 (d, J=8.2, H-2%,6%), 7.66–7.42 (4H, m, arom.), 7.32 (2H,
d, J=8.2, H-3%,5%), 7.29–7.23 (1H, m, arom.), 3.66 (2H, s, CH2)
[B] 9.80 (1H, s, NH), 8.56 (1H, s, H-3), 7.88 (2H, d, J=8.4, H-2%,6%), 7.64 (1H, dd,
J=11.4 and 8.2, H-8), 7.51 (dd, J=11.4 and 8.2, H-5), 7.26 (2H, d, J=8.4, H-3%,5%), 4.46
(1H, br s, COOH), 3.55 (2H, s, CH2)
/
C20H15N3O2S 1720
C16H11F2N3O2 3450, 3300, 3210, 384, 281, 207
3150, 1700
5
)
12
13
14
15
239–242 (a)
178–180
95
80
93
30
C
C
17H13N3O4
3450, 3320, 1720, 401, 290, 217,
1690 208
[B] 10.59 (1H, s, NH), 8.00 (1H, d, J=8.0, arom.), 7.87 (2H, d, J=8.4, H-2%,6%), 7.82–7.70
(2H, m, arom.), 7.60–7.45 (1H, m, arom.), 7.30 (2H, d, J=8.4, H-3%,5%), 4.60 (2H, br s, 2
COOH), 3.57 (2H, s, CH2)
18H12F3N3O4 3350, 3200, 1710 403, 302, 204
[B] 10.70 (1H, s, NH), 8.14 (1H, d, J=8.4, H-5), 8.05 (1H, d, J=1.8, H-8), 7.87 (2H,d,
J=8.6, H-2%,6%), 6.65 (1H, dd, J=8.4 and 1.8, H-6), 7.32 (2H, d, J=8.6, H-3%,5%), 4.20
(2H, br s, 2 COOH), 3.59 (2H, s, CH2)
162–163
C17H11F2N3O4 3300, 1710
408, 290, 215
371, 270, 208
[B] 10.60 (1H, s, NH), 7.87 (1H, ddp, J=11.2 and 8.2, H-8), 7.80 (2H, d, J=8.2, H-2%,6%),
7.62 (1H, dd, J=11.2 and 8.2, H-5), 7.28 (2H, d, J=8.2, H-5), 7.28 (2H, d, J=8.2,
H-3%,5%), 4.74 (2H, br s, 2 COOH), 3.56 (2H, s, CH2)
140–143
C
31H32N4O5
3260, 1710
[A] 8.26–7.75 (4H, m, arom.), 7.80 (2H, d, J=8.4, H-2%,6%), 7.70–7.45 (4H, m, arom.), 7.28
(2H, d, J=8.4, H-3%,5%), 7.11 (1H, s, NH), 6.15 (1H, d, J=7.8, NH), 4.68–4.52 (1H, m,
CH), 4.14 (2H, q, CH2), 4.10 (2H, q, CH2), 3.59 (2H, s, CH2), 2.45–1.80 (4H, m,
CH2ꢀCH2), 1.29 (3H, t, CH3), 1.23 (3H, t, CH3)
[A] 8.00–7.75 (3H, m, arom.), 7.85 (2H, d, J=8.6, H-2%,6%), 7.70–7.45 (4H, m, arom.), 7.31
(2H, d, J=8.6, H-3%,5%), 6.18 (1H, d, NH), 4.68–4.56 (1H, m, CH), 4.18 (2H, q, CH2), 4.11
(2H, q, CH2), 3.61 (2H, s, CH2), 2.40–1.90 (4H, m, CH2ꢀCH2), 1.26 (3H, t, CH3), 1.23
(3H, t, CH3)
16
17
159–162 (a)
169–170 (a)
74
73
C
29H30N4O5S 3300, 1720
386, 277, 194
C25H26F2N4O5 3370, 3300, 1740, 381, 281, 204
1720, 1660
[A] 8.36 (1H, s, H-3), 7.70 (1H, ddp, J=11.4 and 8.0, H-8), 7.69 (2H, d, J=8.6, H-2%,6%),
7.50 (1H, dd, J=11.4 and 8.0, H-5), 7.28 (2H, d, J=8.6, H-3%,5%), 7.17 (1H, s, NH), 6.29
(1H, d, J=8.0, NH), 4.70–4.55 (1H, m, CH), 4.18 (2H, q, CH2), 4.12 (2H, q, CH2), 4.12
(2H, q, CH2), 3.60 (2H, s, CH2), 2.45–1.90 (4H, m, CH2ꢀCH2), 1.27 (3H, t, CH3), 1.23
(3H, t, CH3)

Table 1 (Continued)
Comp. m.p. (°C) a
Yield (%)
38
Analysis for
C26H28N4O7
IR (nujol) (wmax UV (EtOH)
1H NMR, lH (J in Hz) b
cm−1
)
(umax nm)
18
19
20
254–256 (a)
236–240
3280, 1730
401, 295, 217
[C] 13.36 (1H, s, NH), 8.51 (1H, d, J=7.6, NH), 8.01 (1H, d, J=7.8, arom.), 7.86 (2H, d,
J=8.6, H-2%,6%), 7.72–7.58 (2H, m, arom.), 7.45–7.38 (1H, m, arom.), 7.26 (2H, d, J=8.6,
H-3%,5%), 4.32–4.20 (1H, m, CH), 4.08 (2H, q, CH2), 4.05 (2H, q, CH2), 3.45 (2H, s, CH2),
2.38 (2H, t, CH2), 2.06–1.90 (2H, m, CH2), 1.17 (3H, t, CH3), 1.16 (3H, t, CH3)
[B] 13.14 (1H, s, NH), 8.27–8.16 (2H, m, H-6,8), 7.92 (2H, J=7.8, H-2%,6%), 7.52 (1H, d,
J=9.0, H-5), 7.30 (2H, d, J=7.8, H-3%,5%), 4.43–4.26 (1H, m, CH), 4.14 (2H, q, CH2), 4.10
(2H, q, CH2), 3.52 (2H, s, CH2), 2.40–1.90 (4H, m, CH2ꢀCH2), 1.25 (3H, t, CH3), 1.23 (3H,
t, CH3)
[C] 13.44 (1H, s, 1H), 8.51 (1H, d, J=7.4, NH), 8.04 (2H, d, J=10.8 and 8.0, H-8), 7.82
(2H, d, J=8.0, H-2%,6%), 7.68 (1H, dd, J=10.8 and 8.0, H-5), 7.26 (2H, d, J=8.0, H-3%,5%),
4.30–4.13 (1H, m, CH), 4.08 (2H, q, CH2), 4.04 (2H, q, CH2), 3.46 (2H, s, CH2), 2.38 (2H,
t, CH2), 2.06–1.90 (2H, m, CH2), 1.17 (6H, t, 2 CH3)
10
40
C27H27F3N4O7 1710
305, 220, 206
404, 290, 210
152–155 (a)
C
26H26F2N4O7 3280, 1730
21
22
23
24
25
26
27
160–163
95
75
83
74
61
87
20
C27H24N4O5
3380, 3240, 1700 368, 270, 208
[B] 7.68–7.58 (3H, m, arom.), 7.74 (2H, d, J=8.2, H-2%,6%), 7.72 (6H, m, arom.), 7.29 (2H, d,
J=8.2, H-3%,5%), 7.11 (1H, d, J=7.6, NH), 4.58–4.46 (1H, m, CH), 5.58 (2H, s, CH2),
2.40–1.90 (4H, m, CH2)
[A] 7.95–7.73 (2H, m, arom.), 7.77 (2H, d, J=8.8, H-2%,6%), 7.70–7.40 (4H, m, arom.), 7.32
(2H, d, J=8.8, H-3%,5%), 7.30–7.26 (1H, m, arom.), 6.54 (1H, d, J=7.6, NH), 4.60–4.50 (1H,
m, CH), 3.60 (2H, s, CH2), 2.50–1.90 (4H, m, CH2ꢀCH2)
[B] 9.70(1H, s, NH), 8.52(1H, s, H-3), 7.94(1H, J,7.8, NH), 7,85( 2H, d, J, 8.4, H-2’,6’),
7:63(1H, dd, J 10.6 and 8.6, H-8), 7.50 (1H, dd, J 11.6 and 8.2, H-5), 7.28(2H, d, J 8.4,
H-3’,5’), 4.43–4,38(1H, m, CH), 3.53(2H, s,CH2), 2.40–1.80(4H, m, CH2ꢀCH2)
122–125
C25H22N4O5S 3400, 3260, 1710 387, 276, 193
/
218–220
C21H18F2N4O5 3360, 3300, 1730, 382, 281, 204
1660
5
169–172
C22H20N4O7
3250, 1720, 1700 402, 291, 217, 205 [B] 10.93 (1H, s, NH), 8.42 (1H, s, J=5.8, NH), 8.08–7.70 (3H, m, arom.), 7.86 (2H, d,
J=6.8, H-2%,6%), 7.58–7.46 (1H, m, arom.), 7.32 (2H, d, J=6.8, H-3%,5%), 4.88 (3H, br s, 3
COOH), 4.40–4.25 (1H, m, CH), 1.45–1.80 (4H, m, CH2ꢀCH2)
0
218–222
C
23H19F3N4O7 3310, 2250, 1700 389, 296, 209
[B] 9.76 (1H, s, NH), 8.10–7.95 (1H, m, arom.), 7.92 (2H, d, J=8.2, H-2%,6%), 7.65–7.55 (2H,
m, arom.), 7.32 (2H, d, J=8.2, H-3%,5%), 4.60–4.45 (1H, m, CH), 2.50–1.90 (4H, m,
CH2ꢀCH2)
[B] 10.54 (1H, s, NH), 8.35 (1H, d, J=6.6, NH), 7.98–7.82 (2H, m, H-5,8), 7.78 (2H, d,
J=7.8, H-2%,6%), 7.30 (2H,d, J=7.8, H-3%,5%), 5.70 (3H, br s, 3 COOH), 4.40–4.24 (1H, m,
CH), 3.51 (2H, s, CH2), 2.40–1.90 (4H, m, CH2ꢀCH2)
[A] 11.20 (1H, s, NH), 8.92 (1H, d, J=7.6, NH), 7.97 (2H, d, J=8.2, H-2%,6%), 7.96–7.45
(4H, m, arom.), 7.29 (2H, d, J=8.2, H-2%,6%), 6.18 (1H, d, J=7.6, NH), 4.90–4.76 (1H, m,
CH), 4.70–4.55 (1H, m, CH), 4.29 (2H, q, CH2), 4.25–4.05 (6H, m, 3 CH2), 2.60–1.80 (4H,
m, CH2ꢀCH2), 1.34 (3H, t, CH3), 1.35–1.20 (9H, m, 3 CH3)
105–107 (dec.)
111–115 (a)
C22H18F2N4O7 1710
405, 292, 203
C35H43N5O10
3300, 1730, 1660 419, 292, 222
28
Yellow oil
10
C36H42F3N5O10 3320, 1720
400, 303, 215
[A] 11.24 (1H, s, NH), 8.40 (1H, d, J=7.8, NH), 8.02 (1H, d, J=2.0, H-8), 7.98 (1H, d,
J=8.4, H-5), 7.67 (2H, d, J=8.4, H-2%,6%), 7.57 (1H, dd, J=8.6 and 2.0, H-6), 7.25 (2H, d,
J=8.4, H-3%,5%), 6.12 (1H, d, J=7.2, NH), 4.80–4.70 (1H, m, CH), 4.60–4.50 (1H, m, CH),
4.19 (2H, q, CH2), 4.20–4.00 (6H, m, 3 CH2), 3.54 (2H, s, CH2), 2.50–1.80 (8H, m, 2
CH2ꢀCH2), 1.48 (3H, t, CH3), 1.20–1.10 (9H, m, 3 CH3)
a Purification procedure: (a), crystallized from ethanol; p, partially obscured by other resonances.
b Solvent: [A]=CDCl3; [B]=CDCl3ꢀDMSO-d6 (3:1); [C]=DMSO-d6.

S. Piras et al. / Il Farmaco 57 (2002) 1–8
5
Scheme 1.
values, analytical and spectroscopic data are reported
in Table 1.
60 human tumour cell lines. The anticancer activity of
each compound is deduced from dose–response curves
and is presented in three different Tables according to
the data provided by NCI. In Table 2 the response
parameters GI50, TGI and LC50 refer to the concentra-
tion of the agent in the assay that produced 50%
growth inhibition, total growth inhibition, 50% cyto-
toxicity, respectively, and are expressed as Mean Graph
Midpoints. In Table 3 we reported the activities of
those compounds which showed percent growth inhibi-
tion greater than 40% on subpanel cell-lines at 10−4
molar concentration. In Fig. 2 we reported the activities
4. Pharmacology
The tested compounds 6, 7, 8, 12, 13, 15, 16, and 20
of Fig. 1 and Scheme 1 were incontrovertibly selected
by National Cancer Institute of Bethesda among the 26
submitted. Evaluation of anticancer activity was per-
formed following the known [13] in vitro disease-ori-
ented antitumour screening program against a panel of

6
S. Piras et al. / Il Farmaco 57 (2002) 1–8
Table 2
panel cell lines (53 over 60 cell lines) recording an
−log GI50, −log TGI, −log LC50 mean graph midpoints (MG-
interesting selective percent growth inhibition for renal
ACHN (191%), UO-31 (183%) and breast cancer
MDA-MB-231/ATCC (144%) cell lines at 10−4 molar
concentration. Compound 15 confirmed analogous sen-
sitivity at lower level (37 over 60) and exhibited various
selectivities at 10−5 molar concentration (Fig. 2). Com-
pounds 12 and 13 which represent the products of
hydrolysis of 6 and 7 appear to be devoid of intrinsic
activity at all the concentrations examined with the
only exception for 13 that in the leukemia SR32 cell
line recorded % growth inhibition (58, 63, 68, 66)
between 10−8 and 10−5 M, respectively. The fact of the
observed inactivity for the free acids in this test is not
surprising since we have often recorded this behaviour
in similar cases [1–9], possibly because of their low
solubility in the culture medium. Comparison of the
activity as expressed as GI50 (Mean Graph-Midpoint)
of the above described compounds with those of the
previously reported analogous nor-derivatives was ap-
plicable in a few cases (6, 7, 15, 16) (Table 4). From this
table we can observe that only with the exception of
compound 6 the nor derivatives resulted more active. A
study of structure–activity relationships is excluded by
the narrow number of derivatives tested. An observa-
tion has to be marked for the case of 8 that is the
unique example of an active compound bearing two
atoms of fluorine in the 6,7-positions of quinoxaline
ring among all the compounds so far described by us.
This substitution is somewhat profitable also in com-
pound 20 with GI50 whose value was twice that of 8. In
conclusion the results obtained seem to indicate that
MID) a of in vitro inhibitory activity test for compounds 6, 7, 8, 12,
13, 15, 16, and 20 against human tumour cells lines b
Comp.
−log GI50=mM
−log TGI
−log LC50
6
7
8
12
13
15
16
20
4.26
4.20
4.40
4.00
4.07
4.35
4.26
4.15
53.70
4.03
4.02
4.08
4.00
4.00
4.05
4.01
4.02
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
61.66
38.90
100.00
85.11
43.65
53.70
70.79
a (MG-MID) mean graph midpoints, the average sensitivity of all
cell lines towards the test agent.
b From NCI.
of the compound 15 which showed percent growth
inhibition greater than 40% on subpanel cell-lines at
10−5 molar concentration.
4.1. Results of the in 6itro pharmacological anticancer
assays
The data of in vitro anticancer activity reported in
Table 2 established that the average sensitivity of all
cell lines towards the tested agent, represented as Mean
Graph Midpoints of GI50, falls in the range of 38.90–
100 mM, whereas the TGI is very close to LC50 which
was identical for all compounds at the highest concen-
tration (100.00 mM). However, from this table it is easy
to see that the most active agent is compound 8 fol-
lowed in decreasing order by 15B6=16B7B20B13.
From the data of Table 3 we can observe that com-
pound 8 exhibited the largest sensitivity upon all sub-
this type of homologation of the aminobenzoyl-L-gluta-
mate side chain and of its precursor is not profitable
under the conditions examined.
Fig. 2. Percent growth inhibition of compound 15 against single tumour cell lines at 10−5 M.

S. Piras et al. / Il Farmaco 57 (2002) 1–8
7
Table 3
Percentage tumour growth inhibition recorded on subpanel cell lines at 10−4 M of compounds 6, 7, 8, 12, 13, 15, 16, and 20 a
Panel/cell-lines
6
7
8
12
13
15
16
20
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
*
*
*
*
78
92
80
nt
82
93
*
*
*
*
*
*
*
*
*
*
41
147
141
94
83
nt
nt
nt
nt
nt
nt
nt
nt
*
*
*
*
*
*
*
*
41
61
*
54
141
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
HCT-15
77
81
85
80
91
67
54
71
54
*
*
56
132
42
122
60
122
68
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
53
56
92
144
53
84
57
62
76
nt
43
79
60
53
81
*
*
*
83
82
103
92
109
47
*
*
*
*
*
*
*
59
139
68
nt
83
82
*
nt
47
*
*
*
*
*
83
118
58
99
65
*
*
*
*
*
*
*
*
*
*
43
*
42
54
*
*
*
nt
nt
*
*
*
*
*
*
*
*
44
*
*
*
*
*
*
*
*
*
45
46
HT29
KM12
SW-620
45
74
58
SNC cancer
SF-268
SF-295
SF-539
SNB-19
72
89
82
134
163
74
78
59
1222
91
143
73
61
56
*
70
57
96
*
*
*
*
*
*
*
*
*
*
*
*
158
133
92
81
133
108
78
71
89
83
99
75
91
*
*
*
48
SNB-75
U251
117
Melanoma
LOX IMVI
MALME-3M
M14
48
82
45
52
*
*
*
92
45
89
103
59
72
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
nt
67
79
53
63
60
*
*
SK-MEL-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
Renal cancer
786-0
60
43
67
47
91
111
64
45
71
54
58
127
49
47
78
*
*
*
*
*
67
48
74
46
62
75
72
63
65
46
57
*
67
69
nt
73
65
*
96
*
*
*
*
*
*
*
*
*
*
*
*
64
50
53
51
69
90
*
*
nt
40
*
*
51
*
*
86
78
90
71
nt
75
*
*
86
105
68
56
113
44
93
92
83
85
136
48
nt
191
152
82
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
96
74
72
62
127
45
79
58
50
*
*
*
*
*
A498
ACHN
CAKI-1
RXF 393
SN12C
*
123
44
107
*
*
56
59
TK-10
UO-31
75
142
70
*
119
*
*
183
*
*
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
67
51
*
*
63
99
*
*
*
*
75
44
64
68
*
*
*
58
51
78
*
*
73
53

8
S. Piras et al. / Il Farmaco 57 (2002) 1–8
Table 3 (Continued)
Panel/cell-lines
6
7
8
12
nt
*
*
*
*
*
13
nt
*
*
*
*
*
15
59
127
107
*
67
*
*
16
57
43
73
65
46
111
47
20
MCF7/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
65
42
108
58
70
*
*
*
*
*
*
*
144
62
59
66
69
74
80
75
43
71
110
47
106
99
69
T-47D
MDA-N
*
*
*
a *, below 40% growth inhibition; nt, not tested at this molar concentration.
Table 4
evaluation of in vitro anticancer activity, Farmaco 53 (1998)
139–149.
Comparison of mean graph activity between compounds 6, 7, 15, 16
and the corresponding nor derivatives of the cited papers (Ref)
[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)
150–159.
[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, Far-
maco 53 (1998) 480–493.
Comp. (Ref)
GI50
TGI
LC50
6
4.26
4.03
4.20
4.21
4.35
5.17
4.26
4.57
4.00
4.00
4.02
4.03
4.05
4.55
4.01
4.01
4.00
4.00
4.00
4.00
4.00
4.00
4.00
4.00
12 [3]
7
14 [3]
15
29 [3]
16
7 [3]
[8] G. Vitale, P. Corona, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part.12. 3-Carboxy-2[phenoxy]-6(7)substituted quin-
oxalines and N-[4-(6(7) substituted-3-carboxyquinoxaline-2-
yl)hydroxy] benzoylglutamates. Synthesis and evaluation of in
vitro anticancer activity, Farmaco 53 (1998) 594–601.
[9] P. Corona, G. Vitale, M. Loriga, G. Paglietti, Quinoxaline
chemistry. Part 13: 3-Carboxy-2-benzylamino-substituted
quinoxalines and N-[4[(3-carboxyquinoxalin-2-yl)aminomethyl]-
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