Quinoxaline chemistry. Part 13: 3-carboxy-2-benzylamino-substituted quinoxalines and N-[4-[(3-carboxyquinoxalin-2-yl) aminomethyl]benzoyl]-L-glutamates: Synthesis and evaluation of in vitro anticancer activity

Article abstract of DOI:10.1016/S0014-827X(99)00119-6

Among a new series of 28 3-carboxy or carbethoxy quinoxalines bearing a substituted benzylamino or N-[4-(aminomethyl)benzoyl]glutamate group on position 2 of the ring and various substituents at C-6, 7 positions, 21 were selected at the National Cancer Institute for evaluation of their in vitro anticancer activity. The results obtained seem to confirm that the carboxy or carbethoxy group on position 3 is not helpful, with a few exceptions, for the anticancer activity. Copyright (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(99)00119-6

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 77–86
Quinoxaline chemistry. Part 13: 3-carboxy-2-benzylamino-
substituted quinoxalines and N-[4-[(3-carboxyquinoxalin-2-yl)
aminomethyl]benzoyl]-L-glutamates: synthesis and evaluation of in
vitro anticancer activity
Paola Corona, Gabriella Vitale, Mario Loriga, Giuseppe Paglietti *
Dipartimento Farmaco Chimico Tossicologico, Uni6ersita` di Sassari, 6ia Muroni, 23a, 07100 Sassari, Italy
Received 23 July 1999; accepted 15 October 1999
Abstract
Among
a new series of 28 3-carboxy or carbethoxy quinoxalines bearing a substituted benzylamino or N-[4-
(aminomethyl)benzoyl]glutamate group on position 2 of the ring and various substituents at C-6, 7 positions, 21 were selected at
the National Cancer Institute for evaluation of their in vitro anticancer activity. The results obtained seem to confirm that the
carboxy or carbethoxy group on position 3 is not helpful, with a few exceptions, for the anticancer activity. © 2000 Elsevier
Science S.A. All rights reserved.
Keywords: Anticancer activity; Quinoxalines derivatives; Benzylaminoquinoxalines
In particular, we have taken into account the pres-
ence of a carboxy or carbethoxy group on position 3 of
quinoxaline, which in a previous series led to the dis-
covery of compounds endowed with anticancer, anti-
HIV, anti Candida activity [2].
1. Introduction
Our continuous efforts to develop potential
chemotherapeutic anticancer drugs among quinoxaline
derivatives as analogues of both classical and non-clas-
sical antifolate agents have so far produced some inter-
esting compounds, which received attention at the
National Cancer Institute (NCI), Bethesda, USA, for in
depth in vivo investigation [1–4]. In line with these
results, we designed new quinoxalines bearing novel
combinations of substituents in order to get a closer
structure–activity relationship.
2. Chemistry
Compounds 1–11 and 23–25 of Fig. 1 were obtained
according to the sequence of reactions outlined in
Scheme 1.
Nucleophilic displacement of chloroderivatives 31a–c
with the appropriate benzylamines was carried out in
refluxing ethanol, whereas in the case of diethyl N-[4-
In this paper we report the synthesis of a series of
compounds 1–28 listed in Fig. 1, which can be consid-
ered as homologues of the previously described [2,3]
(aminomethyl)benzoyl]-L-glutamate, this reaction was
2-[anilino] and 2-[4-(aminobenzoyl)-L-glutamate]-3,6-
performed in DMF and in the presence of Cs2CO3.
It is interesting to note that in two cases (31a, b),
along with the expected compounds 4 and 8, a con-
comitant formation of the amides 29, 30 was observed.
Alkaline hydrolysis of the esters 1–11 and 23–25
gave good yields of the corresponding acids 12–22 and
26–28 (Table 1).
disubstituted quinoxalines, in order to verify the influ-
ence of the methylene linkage in this series. Preliminary
results of their anticancer activity, as well as of their
preparation, have been communicated recently [5,6].
* Corresponding author.
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(99)00119-6

Table 1
Melting points, yields, analytical and spectroscopic (IR, UV, 1H NMR) data of the compounds of Fig. 1
Comp. m.p. (°C) a Yield (%) Analysis for
IR (Nujol)
UV (EtOH)
(umax, nm)
1H NMR, lH (J in Hz) b
(wmax, cm−1
)
1
2
118–120 (a) 70
115–116 (a) 75
C21H23N3O5
C20H27N3O4
3380, 1680
3380, 1680
416, 311, 262, [A] 8.31 (1H, s, NHCH2), 8.00 (1H, d, J=8.4 Hz, arom.), 7.78–7.62 (2H, m, arom.) 7.50–7.38 (1H, m,
207
arom), 6.70 (2H, s, H–2%,6%), 4.75 (2H, d, J=5.2 Hz, CH2NH), 4.53 (2H, q, CH2CH3), 3.86 (6H, s,
3%,5%-OCH3), 3.84 (3H, s, 4%-OCH3), 1.49 (3H, t, CH3CH2)
417, 311, 262, [A] 8.28 (1H, t, NHCH2), 7.99 (1H, d, J=8.4 Hz, arom.), 7.76–7.61 (2H, m, arom.), 7.44–7.36 (1H, m,
219, 204
arom.), 7.02 (1H, s, H-2%) 7.01 (1H, dd, H 6%,5%=8.8 and J6%,5%=2.0 Hz, H-6%), 6.85 (1H, d, J5%,6%=8.8
Hz, H-5%), 4.75 (2H, d, J=5.4 Hz, CH2NH), 4.52 (2H, q, CH2CH3), 3.89 (3H, s, OCH3), 3.88 (3H,
s, OCH3), 1.48 (3H, t, CH3CH2)
3
4
5
6
7
121–123 (a) 40.82
143–145 (a) 32
C19H19N3O3
3380, 1680
418, 311, 262, [A] 8.26 (1H, t, NHCH2), 7.95 (1H, d, J=8.4 Hz, arom.), 7.74–7.60 (2H, m, arom.), 7.45–7.40 (1H, m,
219, 201
arom.), 7.37 (2H, d, J=8.2 Hz, H-3%,5%), 6.89 (2H, d, J=8.2 Hz, H-2%,6%), 4.75 (2H, d, J=4.8 Hz,
CH2NH), 4.51 (2H, q, CH2CH3), 3.80 (3H, s, OCH3), 1.47 (3H, t, CH3CH2)
C18H15Cl2N3O2 3380, 1680
412, 311, 261, [A] 8.41 (1H, t, NHCH2), 8.01 (1H, d, J=8.4 Hz, arom.), 7.70–7.64 (2H, m, arom.), 7.53 (1H, d,
218, 203
J2%,6%=2.0 Hz, H-2%), 7.67–7.56 (2H, m, arom.), 7.28 (1H, dd, J6%,5%=8.8 and J6%,2%=2.0 Hz, H-6%),
4.79 (2H, d, J=6.0 Hz, CH2NH), 4.55 (2H, q, CH2CH3), 1.52 (3H, t, CH3CH2)
143–145 (a) 73.17
125–127 (a) 65.2
C18H16FN3O2
3400, 1720
415, 311, 261, [A] 8.33 (1H, t, NHCH2), 8.00 (1H, d, J=8.2 Hz, arom.) 7.75–7.60 (2H, m, arom.), 7.44–7.36 (2H, m,
219
H-3%, 5%, and 1H arom.), 7.07–6.99 (2H, m, H-2%,6%), 4.79 (2H, d, J=5.4, CH2NH), 4.53 (2H, q,
CH2CH3), 1.49 (3H, t, CH3CH2)
/
C22H22F3N3O5 3380, 1720
C21H20F3N3O4 3380, 1680
419, 263, 208 [A] 8.42 (1H, t, NHCH2), 8.11 (1H, d, J5,6=8.6 Hz, H-5), 8.00 (1H, d, J8,6=2.0 Hz, H-8), 7.57 (1H,
dd, J6,5=8.6 and J6,8=2.0 Hz, H-6), 6.68 (2H, s, H-2%,6%), 4.75 (2H, d, J=5.4 Hz, CH2NH), 4.54
(2H, q, CH2CH3), 3.87 (6H, s, 3%,5%-OCH3), 3.85 (3H, s, 4%-OCH3), 1.50 (3H, t, CH3CH2)
95–96 (a)
81.4
420, 304, 262, [A] 8.40 (1H, t, NHCH2), 8.06 (1H, d, J5,6=8.6 Hz, H-5), 8.00 (1H, d, J8,6=2.0 Hz, H-8), 7.56 (1H,
215, 204
dd, J6,5=8.6 and J6,8=2.0 Hz, H-6), 7.00 (1H, d, J5%,6%=8.8 Hz, H-5%), 6.98 (1H, s, H-2%), 6.86 (1H,
dd, J6%,5%=8.8 and J6%,2%=2.0 Hz, H-6%), 4.74 (2H, d, J=5.4 Hz, CH2NH), 4.54 (2H, q, CH2CH3),
3.89 (3H, s, 4%-OCH3), 3.88 (3H, s, 3%-OCH3), 1.49 (3H, t, CH3CH2)
)
8
9
106–107 (a) 52
C20H18F3N3O3 3380, 1680
420, 263, 216, [A] 8.39 (1H, t, NHCH2), 8.09 (1H, d, J5,6=8.2 Hz, H-5), 8.00 (1H, s, H-8), 7.55 (1H, dd, J6,5=8.2
and J6,8=1.8 Hz, H-6), 7.37 (2H, d, J=8.6 Hz, H-3%, 5%), 6.90 (2H, d, J=8.6 Hz, H-2%, 6%), 4.74
(2H, d, J=5.6 Hz, CH2NH), 4.53 (2H, q, CH2CH3), 3.81 (3H, s, 4%-OCH3), 1.49 (3H, t, CH3CH2)
414, 261, 215, [A] 8.53 (1H, t, NHCH2), 8.11 (1H, d, J5,6=8.8 Hz, H-5), 7.97 (1H, d, J8,6=1.8 Hz, H-8), 7.58 (1H,
139–141 (a) 45.45
C19H14Cl2F3N3O 3380, 1700
204
dd, J6,5=8.8 and J6,5=1.8 Hz, H-6), 7.51 (1H, d, J2%,6%=1.6 Hz, H-2%), 7.42 (1H, d, J5%,6%=8.2 Hz,
H-5%), 7.26 (1H, dd, J6%,5%=8.2 and J6%,2%=1.6 Hz, H-6%), 4.79 (2H, d, J=5.8 Hz, CH2NH), 4.56 (2H,
q, CH2CH3), 1.51 (3H, t, CH3CH2)
2
10
11
12
13
106–108 (a) 65
C19H15F4N3O2 3360, 1700
C21H21F2N3O5 3360, 1680
417, 304, 261, [A] 8.45 (1H, t, NHCH2), 8.10 (1H, d, J5,6=8.8 Hz, H-5), 7.98 (1H, d, J8,6=1.8 Hz, H-8), 7.56 (1H,
216
dd, J6,5=8.8 and J6,8=1.8 Hz, H-6), 7.48–7.37 (2H, m, H-3%,5%), 7.13–7.01 (2H, m, H-2%,6%), 4.79
(2H, d, J=5.6 Hz, CH2NH), 4.54 (2H, q, CH2CH3), 1.50 (3H, t, CH3CH2)
148–150 (a) 68.75
155–158 (a) 81.08
125–128 (a) 74.07
413, 304, 258, [B] 8.41 (1H, t, NHCH2), 7.85-7.70 (1H, m, H-5), 7.55–7.40 (1H, m, H-8), 6.72 (2H, H-2%,6%), 4.70 (2H,
208
d, NHCH2), 4.45 (2H, q, CH2CH3), 3.83 (6H, s, 3%,5%-OCH3), 3.76 (3H, s, 4%-OCH3), 1.46 (3H, t,
CH3CH2)
C19H19N3O5
C18H17N3O4
3400, 1680
3340, 1680
386, 300, 258, [A] 8.38 (1H, t, NHCH2), 7.86 (1H, d, J=8.2 Hz, arom.), 7.82–7.68 (2H, m, arom.), 7.52–7.42 (1H, m,
208
arom.), 6.69 (2H, s, H-2%,6%), 4.78 (2H, d, J=5.6 Hz, CH2NH), 3.86 (6H, s, 3%,5%-OCH3), 3.84 (3H, s,
4%-OCH3)
388, 304, 259, [A] 8.33 (1H, t, NHCH2), 7.86 (1H, d, J=8.8 Hz, arom.), 7.80–7.65 (2H, m, arom.), 7.50–7.40 (1H, m,
204
arom.), 7.01 (1H, d, J2%,6%=1.6 Hz, H-2%), 6.99 (1H, dd, J6%,5%=6.4 and J6%,2%=1.8 Hz, H-6%), 6.84 (1H,
d, J=8.8 Hz, H-5%), 4.78 (2H, d, J=5.6 Hz, CH2NH), 3.88 (3H, s, OCH3), 3.87 (3H, s, OCH3)

Table 1 (Continued)
162–164 (a) 94.6
14
15
16
17
18
19
20
C17H15N3O3
3300, 1720
387, 304, 259, [B] 8.52 (1H, t, NHCH2), 7.93 (1H, d, J=8.4 Hz, arom.), 7.75–7.62 (2H, m, arom.), 7.45–7.38 (1H, m,
215
arom.), 7.36 (2H, d, J=8.6 Hz, H-3%,5%), 6.87 (2H, d, J=8.6 Hz, H-2%,6%), 4.74 (2H, d, J=5.0 Hz,
CH2NH), 3.80 (3H, s, OCH3)
159–161 (a) 55
C16H11Cl2N3O2 3340, 1700
382, 304, 258, [B] 8.63 (1H, t, NHCH2), 7.96 (1H, d, J=8.4 Hz, H-5), 7.70–7.60 (2H, m, arom.), 7.55 (1H, s, H-2%),
213, 205
7.50–7.36 (2H, m, arom.), 7.33 (1H, dd, J6%,5%=8.4 and J6%,2%=1.8 Hz, H-6%), 4.80 (2H, d, J=6.0 Hz,
CH2NH), 4.35 (1H, brs, COOH c)
175–177 (a) 74.07
143–145 (a) 96.43
127–128 (a) 96
C16H12FN3O2
3400, 1700
388, 304, 259, [B] 8.56 (1H, t, NHCH2), 7.94 (1H, dd, J5,6=8.6 and J5,7=1 Hz, H-5), 7.67–7.64 (2H, m, arom.),
214
7.47–7.38 (3H, m, arom.), 7.07–6.98 (2H, m, arom.), 5.15 (1H, brs, COOH c), 4.79 (2H, d, J=5.4 Hz,
CH2NH)
C20H18F3N3O5 3440, 3360,
1680
393, 332, 258, [A] 8.52 (1H, t, NHCH2), 8.05 (1H, s, H-8), 7.99 (1H, d, J5,6=8.4 Hz, H-5), 7.60 (1H, dd, J6,5=8.4 and
209 6,8=1.8 Hz, H-6), 6.68 (2H, s, H-2%,6%), 4.77 (2H, d, J=5.4, CH2NH), 3.87 (6H, s, 3%,5%-OCH3), 3.84
(3H, s, 4%-OCH3)
393, 258, 204 [A] 8.48 (1H, t, NHCH2), 8.05 (1H, s, H-8), 7.98 (1H, d, J5,6=8.8 Hz, H-5), 7.59 (1H, dd, J6,5=8.8 and
6,8=1.8 Hz, H-6), 7.10–6.98 (2H, m, H-2%,6%), 6.85 (1H, d, J5%,6%=8.0 Hz, H-5%), 4.76 (2H, d, J=5.4
J
C19H16F3N3O4 3520, 3360,
1760
J
Hz, CH2NH), 3.89 (3H, s, OCH3), 3.88 (3H, s, OCH3)
116–118 (a) 85.71
C18H14F3N3O3 3360, 1700
393, 258, 213 [A] 8.48 (1H, t, NHCH2), 8.05 (1H, s, H-8), 7.97 (1H, d, J5,6=8.4 Hz, H-5), 7.58 (1H, dd, J6,5=8.8 and
J
6,8=1.8 Hz, H-6), 7.36 (2H, d, J=8.6 Hz, H-3%,5%), 6.89 (2H, d, J=8.6 Hz, H-2%,6%), 4.77 (2H, d,
J=5.8 Hz, CH2NH), 3.80 (3H, s, OCH3)
270 (a)
90.91
C17H10Cl2F3N3O 3380, 1750
387, 332, 257, [B] 9.13 (1H, t, NHCH2), 8.10 (1H, d, J5,6=8.2 Hz, H-5), 7.89 (1H, s, H-8), 7.56 (1H, s, H-2%), 7.54
211, 203
(1H, d, J6,5=8.2 Hz, H-6), 7.44 (1H, d, J5%,6%=8.2 Hz, H-5%), 7.35 (1H, d, J6%,5%=8.2 Hz, H-6%), 4.80
(2H, d, J=5.4 Hz, CH2NH)
/
2
21
22
162–163 (a) 69.44
169–171 (a) 91.3
C17H11F4N3O2 3360, 1760
392, 257, 212 [A] 8.52 (1H, t, NHCH2), 8.03 (1H, s, H-8), 7.99 (1H, d, J5,6=8.4 Hz, H-5), 8.60 (1H, d, J6,5=8.6 Hz,
H-6), 7.44–7.37 (2H, m, H-3%,5%), 7.08–7.00 (2H, m, H-2%,6%), 4.81 (2H, d, J=5.4 Hz, CH2NH)
390, 332, 253, [A] 8.41 (1H, t, NHCH2), 6.30 (1H, dd, J=10.2 and J=8.4 Hz, H-8), 4.80 (1H, dd, J=9.0 and J=8.0
C
19H17F2N3O5 3440, 3340,
1700
208
Hz, H-5), 6.66 (2H, s, H-2%,6%), 8.73 (2H, d, J=5.4 Hz, CH2NH), 3.86 (6H, s, 3%,5%-OCH3), 3.84 (3H,
s, 4%-OCH3)
)
23
24
25
26
182–184 (a) 48.78
160–161 (a) 50.63
169–170 (a) 32
C28H32N4O7
3400, 3300,
1730,
1700, 1630
414, 310, 261, [A] 8.43 (1H, t, NHCH2), 8.00 (1H, d, J=8.4 Hz, arom.), 7.80 (2H, d, J=8.0 Hz, H-3%,5%), 7.66 (2H, m,
219, 204
arom.), 7.51 (2H, d, J=8.0 Hz, H-2%,6%), 7.48–7.37 (1H, m, arom.), 7.02 (1H, d, J=7.4 Hz, NHCH),
4.89 (2H, d, J=5.6 Hz, CH2NH), 4.87–4.74 (1H, m, NHCHCH2), 4.54 (2H, q, CH2CH3), 4.23 (2H,
q, CH2CH3), 4.10 (2H, q, CH2CH3), 2.60–2.04 (4H, m, CH2CH2), 1.50 (3H, t, CH3CH2), 1.30 (3H, t,
CH3CH2), 1.21 (3H, t, CH3CH2)
C29H31F3N4O7 3380, 3320,
416, 304, 262, [A] 8.56 (1H, t, NHCH2), 8.11 (1H, d, J5,6=8.4 Hz, H-5), 7.97 (1H, s, H-8), 7.82 (2H, d, J=8.2 Hz,
1730,
1700, 1630
216, 204
H-3%,5%), 7.57 (1H, ddP, J6,5=8.4 and J6,8=1.6 Hz, H-6), 7.51 (2H, d, J=8.2 Hz, H-2%,6%), 7.04 (1H,
d, J=7.4 Hz, NHCO), 4.89 (2H, d, J=6.2, CH2NH), 4.79 (1H, m, NHCHCH2), 4.56 (2H, q,
CH2CH3), 4.24 (2H, q, CH2CH3), 4.11 (2H, q, CH2CH3), 2.60–2.10 (4H, m, CH2CH2), 1.51 (3H, t,
CH3CH2), 1.30 (3H, t, CH3CH2), 1.22 (3H, t, CH3CH2)
C28H30F2N4O7 3360, 3300,
412, 302, 258, [A] 8.50 (1H, t, NHCH2), 7.81 (2H, d, J=8.2 Hz, H-3%,5%), 7.60 (1H, dd, J=10.2 and J=8.6 Hz, H-8),
1730,
1700, 1630
203
7.89 (2H, d, J=8.4 Hz, H-2%,6%), 7.42 (1H, dd, J=10.2 and J=8.6 Hz, H-5), 7.04 (1H, d, J=7.4,
NHCO), 4.85 (2H, d, J=5.8 CH2NH), 4.80 (1H, m, NHCHCH2), 4.54 (2H, q, CH2CH3), 4.27 (2H,
q, CH2CH3), 4.11 (2H, q, CH2CH3), 2.60–2.10 (4H, m, CH2CH2), 1.49 (3H, t, CH3CH2), 1.30 (3H, t,
CH3CH2), 1.22 (3H, t, CH3CH2)
178–180
80
C22H20N4O7
3380, 3300,
1750,
1720, 1630
390, 304, 259, [B] 8.67 (1H, t, NHCH2), 8.30 (1H, d, J=7.4 Hz, NHCO), 7.93 (1H, d, J=8.2 Hz, arom.), 7.87 ( 2H,
216, 203
d, J=8.2 Hz, H-3%,5%), 7.80–7.60 (2H, m, arom.), 7.50 (2H, d, J=8.2 Hz, H-2%,6%), 7.45–7.38 (1H, m,
arom.), 5.52 (3H, brs, 3·COOH c), 4.87 (2H, d, J=4.8 Hz, CH2NH), 4.55 (1H, m, NHCHCH2),
2.60–2.10 (4H, m, CH2CH2)

Table 1 (Continued)
27
28
152–155
169–171
73.3
C23H19F3N4O7 3460, 3280,
1700, 1630
390, 332, 256, [B] 8.85 (1H, t, NHCH2), 8.10 (1H, d, J5,6=8.4 Hz, H-5), 8.00–7.90 (1H, m, arom.), 7.87 (2H, d, J=8.4
212
Hz, H-3%,5%), 7.56 (1H, s, H-8), 7.49 (2H, d, J=8.4 Hz, H-2%,6%), 5.35 (3H, brs, 3·COOH c), 4.87 (2H,
d, J=4.8 Hz, CH2NH), 4.62 (1H, m, NHCHCH2), 2.60–2.10 (4H, m, CH2CH2)
77.92
C22H18F2N4O7 3320, 3260,
385, 332, 252, [B] 8.73 (1H, t, NHCH2), 8.26 (1H, d, J=5.4 Hz, NHCO), 7.87 (2H, d, J=8.2 Hz, H-3%,5%), 7.74 (1H,
/
1740,
1700, 1640
205
dd, J=10.2 and J=8.2 Hz, H-8), 7.48 (2H, d, J=8.2 Hz, H-2%,6%), 7.41 (1H, dd, J=10.2 and J=8.2
Hz, H-5), 6.16 (3H, brs, 3·COOH c), 7.83 (2H, d, CH2NH), 4.56 (1H, m, NHCHCH2), 2.50–2.00 (4H,
m, CH2CH2)
29
30
154–156
90–92
15
21
C23H16Cl4N4O 3360, 3300,
408, 309, 260, [A] 9.15 (1H, t, NHCH2), 8.67 (1H, t, NHCH2), 7.80 (1H, d, J=8.4 Hz, arom.), 7.70–7.20 (9H, m,
219, 204 arom.), 4.77 (2H, d, J=5.6 Hz, CH2NH), 4.61 (2H, d, J=6.4 Hz, CH2NH)
416, 308, 261, [A] 9.35 (1H, t, NHCH2), 8.52 (1H, t, NHCH2), 7.97 (1H, d, J8,6=1.8 Hz, H-8), 7.85 (1H, d, J5,6=8.4
1670
C26H23F3N4O3 3400, 3260,
1620
(
217, 203
Hz, H-5), 7.49 (1H, dd, J6,5=8.6 and J6,8=1.8 Hz, H-6), 7.37 (2H, d, J=8.8 Hz, H-3%,5%), 7.30 (2H,
d, J=8.8 Hz, H-3%%,5%%), 6.90 (2H, d, J=8.6 Hz, H-2%,6%), 6.88 (2H, d, J=8.6 Hz, H-2%%,6%%), 4.73 (2H,
d, J=5.6 Hz, CH2NH), 4.57 (2H, d, J=5.8 Hz, CH2NH), 3.81 (3H, s, OCH3), 3.80 (3H, s, OCH3)
a Purification procedure: (a), crystallized from ethanol; P, partially obscured by other resonances.
b Solvent: [A]=CDCl3; [B]=CDCl3–DMSO-d6 (3:1).
c Exchanges with D2O.

P. Corona et al. / Il Farmaco 55 (2000) 77–86
81
3. Experimental
ously [7]. Compound 31c is a new compound and its
preparation is described below. Diethyl N-[4-
(aminomethyl)benzoyl]-L-glutamate was prepared ac-
cording to the indications of the literature [8].
3.1. Chemistry
Melting points are uncorrected and were recorded on
a Kofler or an electrothermal melting point apparatus.
UV spectra are qualitative and were recorded in
nanometres for solutions in ethanol with a Perkin–
Elmer Lambda 5 spectrophotometer. IR spectra are for
Nujol mulls and were recorded on Perkin–Elmer 781
instruments. 1H NMR spectra were recorded at 200
MHz with a Varian XL-200 instrument using TMS as
an internal standard. Elemental analyses were per-
formed at the Dipartimento di Scienze Farmaceutiche,
Laboratorio di Microanalisi, University of Padua, Italy.
The analytical results for C, H, N were within 90.4%
of the theoretical values.
3.1.2. 2-Chloro-6,7-difluoro-3-ethoxycarbonyl-
quinoxaline (31c)
A mixture of 3-ethoxycarbonyl-6,7-difluoroquinox-
alin-2(1H)one [9] (1 g, 3.93 mmol) and POCl3 (10 ml)
was heated at 70°C while stirring for 5 h. After the
removal of excess POCl3 in vacuo, the residue was
taken up with iced water and a solid was filtered off
and washed thoroughly with water to give 31c (0.9 g,
84% yield), m.p. 79–81°C. Anal. (C11H7ClF2N2O2): C,
H, N. IR: 1730 cm−1 1H NMR l: 7.95 (1H, dd,
.
J=8.0, 9.8 Hz, H-5), 7.83 (1H, dd, J=8.0, 9.8 Hz,
H-8), 4.58 (2H, q, J=7.0, CH2CH3), 1.49 (3H, t,
J=7.0 Hz, CH3CH2).
3.1.1. Intermediates
The intermediate chloroquinoxalines (31a, b) neces-
sary for this work were obtained as described previ-
3.1.3. General procedure for the preparation of
2-benzylamino-3-ethoxycarbonylquinoxalines (1–11)
A mixture of equimolar amounts (2 mmol) of 2-
chloro-quinoxalines (31a–c) and the appropriate com-
mercially available substituted benzylamines suspended
in ethanol (10 ml) was stirred under reflux for 13 h.
After cooling, the yellow precipitates of compounds
1–11 were filtered off and washed with ethanol. Purifi-
cation methods, yields, melting points, analytical and
spectroscopic data are reported in Table 1. In the case
of compound 4, purification was accomplished by frac-
tional recrystallization from ethanol, which also gave
the amide 29 as a side compound in 15% yield. In the
case of 8, from the column chromatography on silica
gel eluting with a mixture of petrol ether (b.p. 40–
60°C) and ethyl acetate in the ratio of 95:5, we also
isolated the amide 30 (21%).
3.1.4. General procedure for the preparation of
2-benzylamino-3-carboxy-quinoxalines (12–22)
A suspension of the ester (1–11) (1 mmol) in a
mixture of ethanol (10 ml) and 1 M NaOH aqueous
solution (5 ml) was stirred under reflux for 1 h. On
evaporation of the solvent, the mixture was diluted with
water and made acidic with 2 M HCl aqueous solution
to precipitate a solid that was collected and washed
with water and eventually dried. Yields, melting points,
analytical and spectroscopic data of the acids 12–22 are
reported in Table 1.
3.1.5. General procedure for the preparation of diethyl
N-[4-[(3-ethoxycarbonylquinoxalin-2-yl)aminomethyl]-
benzoyl]- -glutamates (23–25)
L
A mixture of equimolar amounts (1 mmol) of 2-
chloroquinoxaline (31a–c), diethyl N-[4-(amino-
methyl)benzoyl]-L-glutamate, as prepared in Ref. [8],
Fig. 1. Compounds 1–28 obtained according to Scheme 1.
and cesium carbonate in anhydrous DMF (10 ml) was

82
P. Corona et al. / Il Farmaco 55 (2000) 77–86
Scheme 1.
stirred at 70°C for 13 h. On cooling, the mixture was
diluted with water and the precipitates formed were
collected and washed with water. The products 23–25
were further purified as indicated in Table 1, which also
reports the yields, melting points, analytical and spec-
troscopic data.
Table 2
−Log10 GI50
, −log10 TGI, −log10 LC50 mean graph midpoints
(MG-MID) a of in vitro inhibitory activity test for compounds 1–3,
6–10, 12–14, 16–21, 23, 24, 26, 27 against human tumour cell lines b
Comp.
−Log10 GI50
−Log10 TGI
−Log10 LC50
1
2
3
6
7
4.38
4.02
4.08
4.29
4.27
4.15
4.46
4.50
4.03
4.04
4.11
4.21
4.67
4.51
4.59
4.78
4.62
4.09
4.50
4.00
4.00
4.01
4.00
4.00
4.02
4.00
4.00
4.07
4.03
4.00
4.00
4.01
4.01
4.20
4.06
4.14
4.45
4.20
4.01
4.23
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
4.03
4.00
4.01
4.16
4.03
4.00
4.09
4.00
4.00
3.1.6. General procedure for the preparation of
N-[4-[(3-carboxyquinoxalin-2-yl)aminomethyl]benzoyl]-
L
-glutamic acids (26–28)
The acids 26–28 were obtained in an identical man-
8
9
ner as for compounds 12–22. Purification of the crude
products was accomplished by recrystallization using
the solvent indicated in Table 1, where yields, melting
10
12
13
14
16
17
18
19
20
21
23
24
26
27
1
points and spectroscopic data (IR, UV, H NMR) are
reported.
4. Pharmacology
Evaluation of anticancer and anti-HIV activity was
performed on 21 compounds referring to structures
1–3, 6–10, 12–14, 16–21, 23, 24, 26 and 27 of Fig. 1
and Table 1 at the NCI, following the known [10] in
vitro disease-oriented antitumour screening program,
against a panel of 60 human tumour cell lines and an
HIV drug-testing system [11]. No compound exhibited
anti-HIV activity.
a (MG-MID): mean graph midpoints, the average sensitivity of all
cell lines toward the test agent.
b From NCI.

Table 3
Percentage tumour growth inhibition recorded on subpanel cell lines at 10−4 M of compounds 1, 2, 3, 6–10, 12–14, 16–21, 23, 24, 26, 27 a
Panel/cell lines
1
2
3
6
7
8
9
10
12
13
14
16
17
18
19
20
21
23
24
26
27
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
74
92
96
95
95
81
*
*
*
*
*
*
*
*
*
*
*
nt
110
nt
57
137
119
59
66
69 100
82
53
93
49
81
*
*
99
42
45
40
*
45
50
40
54
51
51
nt
117
75
73
161
146
132
126
nt
102
nt
113
133
102
123
94
67
83
*
*
*
*
*
*
44
nt
85
56
46
96
91
84
87
89
*
*
*
*
*
83
64
59
76
67
nt
127
136
86
nt
147
153
96
nt
128
144
91
nt
nt
nt
*
*
*
*
60
*
*
*
*
87
55
99
*
*
52
40
82
97
135
159
143
*
Non-small-cell lung cancer
A549/ATCC
EKVX
HOP-62
76
67
60
70
54
41
nt
*
*
*
*
*
*
44
43
*
*
*
45
nt
54
65
58
63
44
95
*
64
85
71
79
61
78
60
59
68
86
97
69
48
50
68
42
54
70
93
98
88
90
136
145
94
78
75
74
151
74
87
69
138
89
97
nt
110
96
40
59
43
62
*
*
nt
51
57
42
*
47
82
50
57
*
65
74
47
51
65
65
79
65
67
123
127
122
nt
68
92
122
108
94
96
98
119
92
62
95
99
97
nt
102
110
131
121
73
112
115
98
200
191
173
179
144
193
171
190
nt
154
119
144
141
70
109
107
120
nt
*
*
44
47
*
41
80
176
200
127
200
97
61
nt
200
*
*
*
*
*
*
*
*
42
*
*
*
*
*
*
*
HOP-92
55
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
*
*
*
46
76
63
57
nt
nt
49
*
94
80
49
*
nt
40
nt
nt
/
*
83
*
nt
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM 12
SW-620
84
67
79
79
87
80
78
*
*
*
*
*
*
*
*
117
68
90
75
66
81
65
91
nt
51
nt
96
nt
99
82
104
95
110
111
77
87
87
46
*
*
*
62
40
69
126
45
60
103
50
79
145
60
72
136
87
200
99
nt
111
94
129
102
115
99
93
96
181
121
159
116
113
122
92
179
197
200
171
200
174
142
155
120
200
105
nt
*
*
*
57
nt
86
82
*
*
*
*
*
*
*
*
43
66
41
51
67
*
*
nt
nt
nt
78
61
92
71
71
65
53
64
76
49
*
*
*
*
*
*
*
*
*
*
40
49
44
44
54
)
*
*
*
62
86
96
89
*
87
47
64
86
SNC cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
40
68
*
*
*
43
60
48
87
45
104
nt
54
71
49
51
91
75
*
45
33
*
54
72
121
67
109
126
66
120
57
55
77
52
85
*
*
*
nt
57
49
44
41
77
*
46
51
63
63
90
*
93
156
154
129
111
185
nt
92
95
95
81
nt
95
146
95
95
99
nt
165
180
200
174
188
nt
108
154
99
103
123
*
*
nt
70
45
62
200
200
nt
200
114
200
*
*
*
*
*
*
*
*
*
*
*
*
*
nt
nt
51
82
nt
nt
83
58
nt
45
50
68
68
*
*
*
43
*
*
*
114
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
73
90
nt
nt
62
88
56
73
*
*
*
*
84
nt
50
76
74
nt
49
67
81
51
61
106
nt
93
65
57
68
64
*
51
nt
65
54
97
100
93
60
nt
93
69
93
91
81
68
63
71
89
85
90
*
*
77
nt
*
*
41
65
76
nt
43
51
94
88
172
144
98
nt
121
200
98
nt
97
141
64
156
87
149
nt
128
124
87
159
99
142
189
nt
159
nt
120
148
78
172
111
166
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
48
nt
nt
48
nt
nt
nt
45
53
40
200
150
105
69
nt
nt
nt
nt
200
180
184
188
133
187
*
*
*
*
*
*
*
*
*
*
*
*
*
50
57
85
40
48
59
nt
nt
*
*
*
nt
*
*
53
*
43
149
*
162

Table 3 (Continued)
Ovarian cancer
IGROV1
64
55
81
*
*
*
*
*
*
46
40
64
nt
59
74
57
49
56
60
130
120
143
112
75
102
60
*
44
46
40
42
41
45
45
nt
67
75
nt
nt
123
nt
112
128
70
92
167
84
175
171
132
200
95
143
*
167
62
54
nt
44
200
200
nt
48
*
*
*
*
OVCAR-3
OVCAR-4
OVCAR-5
*
*
*
*
*
*
*
*
*
*
*
*
*
71
78
102
115
OVCAR-8
SK-OV-3
61
54
*
*
*
48
46
nt
56
*
47
84
93
85
62
*
57
46
41
50
nt
68
nt
89
97
84
94
96
86
180
170
131
103
*
*
nt
96
*
*
*
*
*
Renal cancer
786-D
A498
ACHN
CAKI-1
56
101
71
*
*
*
*
46
82
75
45
70
41
66
68
*
*
*
161
104
117
133
85
nt
89
74
49
40
*
64
nt
73
123
127
115
110
91
116
98
123
135
97
200
178
187
176
200
135
98
*
*
48
nt
200
*
*
*
*
*
*
*
63
52
*
*
51
45
47
*
*
*
*
107
nt
*
*
nt
61
57
*
*
96
94
96
nt
nt
/
RXF 393
SN 12C
TK-10
88
84
*
84
98
91
45
54
115
nt
80
49
74
46
76
nt
46
61
50
108
nt
77
73
94
91
79
88
52
53
*
*
49
91
*
*
*
92
189
141
162
nt
112
nt
nt
122
nt
nt
200
nt
24
174
nt
nt
nt
56
161
105
*
51
75
*
*
*
*
60
*
*
*
nt
*
56
50
*
*
*
UO-31
*
100
68
48
84
99
149
200
192
Prostate cancer
PC-3
DU-145
)
*
*
nt
81
62
88
nt
64
nt
111
nt
83
70
43
49
63
47
50
128
85
98
85
98
99
194
187
108
*
*
111
*
*
*
*
*
*
*
*
*
*
*
Breast cancer
MCF7
MCF7/ADR- 132
96
*
*
70
57
65
51
85
74
74
56
87
69
88
95
55
49
*
*
54
98
138
nt
102
nt
123
nt
142
nt
117
nt
*
188
nt
*
*
nt
nt
85
82
44
RES
MDA-MB-
231/ATCC
HS 578T
MDA-MB-435 113
BT-549
T-47D
95
67
*
50
64
50
*
98
74
47
*
*
58
nt
94
100
174
111
nt
100
86
*
nt
nt
nt
83
84
83
88
114
49
75
69
63
74
70
50
47
60
55
41
108
132
81
74
65
105
86
nt
105
91
80
49
46
41
47
48
75
*
*
41
64
41
61
41
73
46
43
117
103
92
106
153
nt
110
89
85
93
nt
139
117
99
nt
192
187
170
190
46
108
nt
200
66
nt
*
*
*
*
*
*
*
*
*
*
126
77
126
104
100
113
nt
nt
54
nt
*
*
nt
nt
*
*
MDA-N
96
115
*
*
*
a *, below 40% growth inhibition; nt, not tested at this molar concentration.

P. Corona et al. / Il Farmaco 55 (2000) 77–86
85
Table 4
Comparison of the inhibitory activity of compounds on some cell lines at the most diluted concentrations
Cell line
Comp.
Percentage tumour growth inhibition at the indicated molar concentration
10−8
10−7
10−6
10−5
Leukaemia
RPMI-8226
HL-60(TB)
CCRF-CEM
HL-60(TB)
MOLT-4
MOLT-4
SR
RPMI-8226
6
7
48
29
29
34
33
50
29
37
54
77
21
41
34
90
40
53
54
108
17
17
20
21
23
24
45
35
46
53
24
39
71
Non-small-cell lung cancer
NCI-H522
HOP-92
HOP-92
A549-ATCC
EKVX
8
10
13
24
24
24
24
39
32
36
26
23
25
34
46
67
40
21
27
51
36
HOP62
NCI-460
SNC cancer
SNB-75
SF-268
U-251
SF-295
SF-539
SNB-19
SNB-75
9
10
23
24
24
24
24
32
41
27
37
45
44
57
69
62
56
20
Melanoma
SK-MEL-5
24
31
27
47
Ovarian cancer
OVCAR-4
IGROV1
OVCAR-3
OVCAR-3
OVCAR-8
10
18
20
21
24
46
44
54
54
52
Renal cancer
RXF-393
UO-31
1
21
24
24
42
115
76
786-O
Breast cancer
HS 578T
MDA-MB-435
MDA-MB-231/ATCC
17
23
24
47
21
40
21
49
82
63
In Table 2 the response parameters GI50, TGI
and LC50 refer to the concentration of the agent in the
assay that produced 50% growth inhibition, total
growth inhibition and 50% cytotoxicity, respectively,
and are expressed as mean graph midpoints. In Table 3,
we report the activities of those compounds that
showed percentage growth inhibition greater than 40%
growth inhibition at the most diluted concentrations
(10−8 to 10−5M).
5. Results and discussion
The data of Table 2 show that the average sensitivity
of cell lines towards the tested agent, represented as
mean graph midpoints, falls in the range 10−5–10−4 M
concentration. Mean graph midpoints for the reported
on subpanel cell lines at molar concentration of 10−4
,
whereas in Table 4, we report the activity of those
compounds that exhibited a significant percentage

86
P. Corona et al. / Il Farmaco 55 (2000) 77–86
though for most compounds in the series, the percent-
age growth inhibition was of the same order of magni-
tude with the widest non-selective anticancer activity at
10−4 M concentration.
compounds also show that GI50 was significant and in
the following decreasing order: 20\17\21\19\
18\10]24\9\1\6\7, whereas mean graph mid-
points for TGI and LC50 showed values near the
highest concentrations with the exception of com-
pounds 20 and 24, which in turn exhibited the lowest
values. The data of Table 3 are indicative of the wide
range of percentage tumour growth inhibition recorded
at 10−4 M concentration. From these it is easy to
observe that only a few compounds (2, 23, 26, 27) were
almost devoid of cell-line sensitivity. In particular, the
results for the acids 26 and 27 replicate previous obser-
vations, whereas in the in vitro test, the acidic functions
from the glutamate moiety are somewhat unable to
produce any observable activity, which on the contrary
appears well evident in the corresponding esters. Ac-
cording to the data of Table 4, we can observe that
some compounds still exhibited significant percentage
tumour growth inhibition values and in particular com-
pound 24 maintained these at the most diluted concen-
tration spanning over 13 subpanel cell lines. Compound
17 maintained significant percentage tumour growth
inhibition values against leukaemia HL-60(TB) and
CCRF-CEM cell lines whereas compound 23 did so
against leukaemia (SR), SNC (U-251) and breast
(MDA-MB435) cancer cell lines. It is evident from the
reported data that in some cases (Table 4) the percent-
age growth inhibition values recorded is not dose de-
pendent and that in most cases (Table 3) the highest
values of percentage growth inhibition (\100%) are
associated to high cytotoxicity.
Structure–activity relationships are limited to the
members of the series selected at the NCI. However,
comparison of the percentage growth inhibition values
establishes that compounds 6–8 bearing a 7-trifl-
uoromethyl group were superior to the corresponding
1–3 devoid of it, while in the 7-CF3 substituted series
the acids 17–21 exhibited a greater activity than the
parent esters 6–10.
Finally, comparison of mean graph midpoints of
compounds listed in Table 2 with the previously men-
tioned 2-anilino [2] and 2-aminobenzoylglutamate [3]
analogues clearly shows that with the exception of the
compounds 1, 6, 7, 17, the insertion of a methylene
bridge between the amino and the substituted phenyl
moiety does not increase the cell-line sensitivity even
References
[1] M. Loriga, M. Fiore, P. Sanna, G. Paglietti, Quinoxaline chem-
istry. Part 5. 2-(R)-Benzylaminoquinoxalines as non classical
antifolate agents. Synthesis and evaluation of in vitro anticancer
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