2-[N-Alkyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines as anticancer agents inhibitors of folate enzymes

Article abstract of DOI:10.1016/j.ejmech.2014.01.048

Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60 human tumors cell lines at National Cancer Institute of Bethesda (NCI), we have synthesized a novel series of thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1-31). The lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR enzyme, with a Ki of 0.2 μM. Docking studies were performed on compound 1 and here reported to predict its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1-31) were assayed versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds inhibited hDHFR with Ki values included between 0.2 and 11 μM, while only a few (6, 21, 24, 27, 29) showed great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50 equal to -5.49 and log LC50 equal to -4.19 and maintained significant percent of growth inhibition on seven cancer cell lines at the concentration of 1 μM. Compound 17 was the second most active and moreover showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257, Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100-0.01 μM).

Full text of DOI:10.1016/j.ejmech.2014.01.048

European Journal of Medicinal Chemistry 75 (2014) 169e183
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
2-[N-Alkyl(R-phenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxalines as anticancer agents inhibitors of folate
enzymes
,
a
a
a
a
Sandra Piras a , Antonio Carta , Irene Briguglio , Paola Corona , Giuseppe Paglietti ,
*
Rosaria Luciani b, Maria Paola Costi b, Stefania Ferrari b
a Dipartimento di Chimica e Farmacia, Università degli Studi di Sassari, Via Muroni 23/a, 07100 Sassari, Italy
b Dipartimento di Scienze Farmaceutiche, Università di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on our previous results on the ascertained potent growth inhibition effect against a panel of 60
human tumors cell lines at National Cancer Institute of Bethesda (NCI), wehave synthesized a novel series of
thirty-one 2-[N-methyl(R-phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines (1e31). The
lead compound 1 was previously reported to be endowed with significant inhibition against hDHFR
enzyme, with a Ki of 0.2 mM. Docking studies were performed on compound 1 and here reported to predict
its binding conformation to human dihydrofolate reductase (hDHFR). All compounds (1e31) were assayed
Received 17 September 2013
Received in revised form
13 January 2014
Accepted 16 January 2014
Available online 30 January 2014
versus hDHFR and human thymidylate synthase (hTS). From the screening emerged that all compounds
inhibited hDHFR with Ki values included between 0.2 and 11 mM, while only a few (6, 21, 24, 27, 29) showed
Keywords:
Trifluoromehylquinoxaline
Anticancer agents
Enzyme inhibition
Dihydrofolate reductase
Thymidylate synthase
great activity and selectivity towards hTS. Evaluation of the anticancer activity was performed by NCI, first
against the three cell line panel, and only the most active compounds (17, 21, 24, 26, 27) were evaluated on a
panel of 60 human tumor cell lines. Compound 21 was the most active against all cell lines with log GI50
equal toꢀ5.49 and log LC50 equal toꢀ4.19 and maintained significant percentof growth inhibition onseven
cancer cell lines at the concentration of 1 mM. Compound 17 was the second most active and moreover
showed interesting selectivity against some cell lines (Lung cancer: A549/ATCC, Melanoma: UACC-257,
Ovarian Cancer: ovcar-8 and Renal cancer: RXF 393) at all concentration examined (100e0.01 M).
m
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
produced by most bacteria and plants, are essential for the syn-
thesis of certain amino acids, whiles some bacteria and mammalian
cells rely on the use of preformed folates and have salvage path-
ways for reduced folates, purines, and pyrimidines. For this reason
various antifolate agents, which interfere with this pathway, are
currently used in therapy. Some representative drugs which block
dihydrofolate reductase like methotrexate, trimetrexate and the
most recent pralatrexate, and others agents which are selective
thymidylate synthase inhibitors like raltitrexed, pemetrexed and
plevitrexed (that is assessed in several phase II clinical studies for
treatment of various solid cancers) were reported (Fig. 1).
Consequently, the discovery of novel antifolate agents with
improved properties continues to be an active area of research,
both in academia and the pharmaceutical industry [4e6].
With this in mind, in the last twenty years our research team has
analyzed the structural analogy between quinoxaline and both
pteridine and quinazoline which are the rings characterizing the
most representative antifolate drugs.
Folates are vitamins belonging to B group which act as one-
carbon donors in multiple essential biosynthetic pathways
including methylation reactions, de novo biosynthesis of purines
and thymidylate, and amino acid metabolism. In the last sixty years,
drugs targeting the folate pathway have significantly impacted
disease treatment as cancer [1], bacterial infections [2] and against
certain parasitic diseases, notably malaria [3]. These drugs act
through the inhibition of the human enzymes dihydrofolate
reductase (hDHFR) and thymidylate synthase (hTS). DHFR is an
enzyme that reduces dihydrofolic acid to tetrahydrofolic acid, while
TS is the enzyme used to generate deoxythymidine mono-
phosphate (dTMP). Both enzymes are essential for the nucleic acid
precursor’s synthesis and repair. Furthermore, folate cofactors,
We have reported the anticancer activity data supplied by Na-
tional Cancer Institute (NCI of Bethesda, USA) concerning a wide
* Corresponding author.
E-mail address: piras@uniss.it (S. Piras).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.048

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S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
Fig. 1. Chemical structure of some classical and non classical anti-folates.
amount of different quinoxaline series [7e23] and among these, the
most interesting compounds were selected for in depth in vivo
investigation [8,10,11,15]. In Figs. 2 and 3 we depicted the chemical
structures of the all compounds synthesized with the purpose to
identify potent and selective inhibitors.
By these studies we have demonstrated that several of these
compounds which showed in vitro anticancer activity were able to
inhibit either the DHFR or TS [14,18,20e22].
In the series showed in Fig. 2, it was considered interesting
introduce on position 2 some of the substituents present in both
classical and non classical antifolate agents as previously reported
by other authors [24]. In addiction, on position 3 we considered
some groups able to modulate both steric hindrance and lip-
ophilicity (R ¼ H, CH3, Ph, Th, COOR0, CF3). Finally, on the benzene
counterpart of the quinoxaline ring were introduced tri-
fluoromethyl and amino groups to evaluate the influence of elec-
tron withdrawing or electron donor substituents.
The wide range of compounds synthesized and evaluated as
anti-tumor agents at the NCI allowed us to establish some
structureeactivity relationships (SAR), which can be summa-
rized as follows: (a) Comparation of the biological data showed
that the hypothesized bioisosterism between quinoxaline and
pteridine rings is corrected. (b) Compounds bearing on qui-
noxaline ring a trifluoromethyl group at position 6 or 7 and a
phenyl group at position 3 resulted more active than the
unsubstituted ones. (c) The concomitant presence of both tri-
fluoromethyl group at position 6 and amine group at position 8,
whatever is the substituent present at positions 2 and 3, in-
creases the activity. This is further enhanced by the simulta-
neous presence of two amino groups in the same position. (d)
Trimethoxyphenyl on the side chain resulted the best substitu-
ent in almost all the series. (e) Methylenamino and benzylamino
linkers resulted the most effective in comparison to an amino or
oxygen bridge [7e23].

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
171
Fig. 2. Quinoxalines analogs of classical anti-folate pteridines.
On the contrary, quinoxalines of the series depicted in Fig. 3,
which are analogous of quinazoline type antifolates, were generally
less active than quinoxalines analogs of pteridines. As regard the
SAR of this series of derivatives, the best results were obtained
when in the pyrazine ring were present a phenyl and an amino
groups, at position 2 and 3 respectively. Also for this series the
trimethoxyphenyl group resulted the most profitable substituent,
while a benzoylglutamate determined a reduced activity.
In this paper we report the synthesis of a series of compounds
(1e31) listed in Fig. 4, keeping in position 2 the methyleneanilino
linker, the CF3 substituent in the benzene moiety and the phenyl in
position 3, that previously demonstrated the ability to implement
the antiproliferative activity, with the aim to fulfill the ongoing SAR
analysis.
In fact in the past we reported the biological activity, but not the
chemical characteristics, of the diethyl N-[4-(3-phenyl-7-
As far as concerns the bridge, linker as methylenoxy or meth-
ylenamino does not seem to be crucial for the biological activity, in
fact the percentage of cell growth inhibition was of the same order
of magnitude for both the linkers.
From the analysis of anti-cancer activity values of more than 400
synthesized compounds, belonging at both the above described
series, it was evident that most derivatives depicted in Fig. 2 were
active against the 60 human tumor cell lines tested, showing
trifluoromethylquinoxalin-2-yl)methyl]-N-methyl-benzoyl-
glutamate (1) [18], which was selectively active against hDHFR with
Ki ¼ 0.2 M.
L-
m
On these bases we have made two type of further modifications:
the first one concerns the substituent on nitrogen of methyl-
eneamino group, where the methyl group of the compound 1 was
also replaced by bigger ones as ethyl or propargyl, the last one
presents in powerful antifolate as CB 3717 and ZD 9331 [24]. The
second one considers the substitution of the aminoglutamic moiety
with various substituent as CH3, Cl, F, OCH3, OCF3, CH2COOC2H5,
possessing different steric hindrance or lipophilic characteristics.
The most synthesizes compounds are structurally related to non
classical antifolate derivatives, because they proved to be, among
the previously tested quinoxalines, the most active as anticancer
agents.
inhibitory activities between 10 and 0.1
Furthermore some compounds exhibited high activity against
several cell lines also at 0.01 M concentration. As regard the de-
rivatives depicted in Fig. 3 unfortunately, the highest percent tumor
growth inhibitions were between 100 and 10 M.
mM concentrations.
m
m
In order to extend the study of antifolate activities of our com-
pounds we have tested some of them, against a panel of protozoal
enzymes as the pteridine reductase (PTR1) of Leishmania major and
Tripanosoma cruzii. This has allowed us to discover a potent class of
PTR1 inhibitors to direct antiparasitic drug development [25].
All the synthesized compounds (1e31) were evaluate towards
hDHFR and hTS enzymes and the inhibition constant (Ki) values are
reported.
Fig. 3. Quinoxalines analogs of non classical anti-folate quinazolines.

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S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
Fig. 4. List of the prepared compounds 1e31.
2. Results and discussion
each compound were reported as the percent of growth of the
treated cells compared to the untreated control cells (Table 2). For
the NCI criteria, compounds which reduce the growth of anyone of
the cancer cell lines to 32% or less (negative numbers indicate cell
kill) are identified as ‘active’ and subsequently are evaluated in a
full panel of 60 human cell lines over a 5-log dose range. The qui-
noxalines 17, 21, 24, 26, 27 matched these criteria and were eval-
uated for their anti-cancer activity following the known in vitro
disease-oriented anti-tumor screening program which is based
upon use of a multiple panels of 60 human cancer cell lines [26]. A
48 h drug exposure protocol was used and a sulforhodamine B
(SRB) protein assay was employed to estimate cell viability or
growth [27] The anti-cancer activity of a tested compound is given
by three parameters for each cell line: log10 GI50 value (GI50 ¼ molar
concentration of the compound that inhibits 50% net cell growth),
log10 TGI value (TGI ¼ molar concentration of the compound
leading to total inhibition of net cell growth), and log10 LC50 value
(LC50 ¼ molar concentration of the compound leading to 50% net
cell death). Furthermore, a mean graph midpoint (MG-MID) is
calculated for each of the mentioned parameters, giving an aver-
aged activity of each compound deduced from dose response
curves. The log10 GI50, log10 TGI and log10 LC50 values provided by
NCI for compounds 17, 21, 24, 26, 27 are reported in Table 5,
whereas in Tables 3 and 4 are reported the results of percent
growth inhibition (I%) recorded at 100, 10, 1, 0.1 and 0.01 micro-
Molar concentrations. The data showed that all compounds
exhibited strong activity against all human tumor cell lines at
2.1. Chemistry
Preparation of compounds 1e31 was achieved according to the
reactions outlined in Scheme 1.The known 2-phenyl-6-
trifluoromethyl-3-bromomethylquinoxaline (32) [18] was reacted
with the purposely prepared N-alkylanilines (35e66) mostly at
room temperature in anhydrous dimethylacetamide (DMA). In no
way the secondary amine 66 was able to react with compound 32 to
obtain the corresponding alkylquinoxaline. In some cases moderate
heating was necessary (Table 1). The starting anilines (33aem), all
commercially available, were alkylated with the halides (34aed) to
give in moderate yields the secondary amines (35e66) (Scheme 2).
2.2. Anti-cancer activity
Evaluation of the anti-cancer activity of the synthesized qui-
noxalines (1e31) was performed at the NCI. Only 25 compounds
from the synthesized quinoxalines were accepted by NCI, (de-
rivatives 1, 5, 11, 18, 19, 20 were rejected). First, all quinoxalines
were evaluated in primary anti-cancer assay at 10ꢀ4 M concentra-
tion against NCI-H460 (lung), MCF7 (breast), and SF-268 (CNS) cell
lines. In the assay protocol, each cell line was inoculated and pre-
incubated on a microtiter plate. Test agents were added in a single
concentration (10ꢀ4 M) and the culture was incubated for 48 h.
End-point determinations were made with alamar blue. Results for

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
173
Ovarian cancer ovcar-8 (Log10 GI50 ¼ ꢀ5.82), Renal cancer RXF 393
(Log10 GI50 ¼ ꢀ6.51) (Fig. 7). No comparison with the model 1 was
possible owing to a lack of screening of this compound by NCI.
However, it is important to note that the presence at N-10 of a
susbstituent different from hydrogen increase considerably the
cytotoxicity as it appears by comparison with the analogous com-
pounds without substituent at N-10 [18].
2.3. Docking studies
Docking studies were performed on compound 1 to predict its
binding conformation to hDHFR (Fig. 5). Compound 1 is predicted
to bind in the hDHFR active site and its binding site partly overlap
that of MTX. In fact, due to bulky phenyl and trifluoromethyl sub-
stituents at positions 3 and 7 respectively the quinoxaline ring
cannot be matched to the pteridine ring of Metotrexate (Fig. 5A). In
the binding mode predicted, the two aromatic rings at positions 2
of the quinoxaline and on the nitrogen of methylenamino group,
are involved in aromatic stacking interactions with the nicotin-
amide moiety of the cofactor and Phe31 and Phe34, respectively,
whereas the glutamate moiety, forms two H-bonds with the side-
chain of Gln35 and Lys68. The compound interacts also with
Asp21, Leu22, Pro26, Thr56, Ile60, Pro61, Asn64, Leu67, Arg70 and
with backbones of Arg28, Arg32 and Ser59 (Fig. 5B).
On the base of this predicted binding mode, all the proposed
chemical modifications of compound 1 should not induce changes
in the predicted binding mode. In particular modifications on ni-
trogen of methylenamino group should allow a better interaction
with the hydrophobic pocket formed by Phe34, Thr56, Ile60 and
Leu67 (Fig. 5C). Also the modifications on positions 2, 3, 4 and 5 of
the benzene ring are allowed since they are quite small and there is
enough available space in this part of the enzyme.
Scheme 1. Synthesis of compounds 1e31.
0.1 mM. Compound 21 has showed the highest values of cytotox-
icity (MG-MID log10 GI50 ¼ ꢀ5.49) against all cell lines. Moreover,
this compound maintained significant percent of growth inhibition
on seven cancer cell lines at the lowest concentration examined
2.4. Anti-folate activity
(1 mM) with values ranged between 41 and 109. In particular Colo
In Table 6, the inhibition constant (Ki) values for compounds 1e
31 towards human dihydrofolate reductase (hDHFR) and human
thymidylate synthase (TS) enzymes, are reported. Almost all com-
pounds were active against hDHFR. The model compound 1 was
205 (colon cancer), NCI/ADR-RES, MDA-MB-435 and T-47D (Breast
cancer) were showed the most active with I% of 108,109,104 and 90
respectively (Fig. 6). Other seven cell lines (leukemia: HL-60, MOLT-
4; lung cancer: NCI-H460; colon cancer: HCT-116, HCT-15; ovarian
cancer: OVCAR-3; breast: NCI/ADR-RES) were inhibited by com-
pound 21 with the I% around 60. In the case of compounds 24, 26,
27 appeared high activity towards all tumor cell lines at 0.1 mM
concentration with the MG-MID GI50 equal to ꢀ4.51, ꢀ4.29
and ꢀ4.35 respectively, while nothing of considerable is showed at
the lowest concentrations. Compound 17, the only one among the
selected by NCI having an ethyl group bounded to nitrogen in po-
sition 10 (all the other having a propargyl group), displayed the
highest activity at 0.1 and 0.01 mM concentrations versus all 60
human tumor cell lines (Table 4). Remarkable anti-cancer activity
resulted the most active against hDHFR with Ki of 0.2
other compounds (2e8, 10e29, 31) showed Ki values in the range
2e11 M. Only compound 9 did not inhibit the enzyme at the tested
mM whereas
m
concentration.
From the screening against hTS only a few compounds 6, 21, 24,
27, 29 emerged as active in inhibiting the enzyme. Except for
compound 29, all the others showed greater activity towards hu-
man TS in respect of hDHFR. The best compounds, 6 and 21, showed
activity at the submicromolar level with Ki values of 0.27 and
0.51 mM respectively.
and selectivity has been observed until 0.01 mM concentration to-
wards some cell lines as: Non small cell lung cancer, A549/ATCC
3. Conclusion
(Log10 GI50 ¼ ꢀ6.43), Melanoma UACC-257 (Log10 GI50 ¼ ꢀ7.60),
In summary, we have synthesized a series of 2-[N-alkyl(R-
phenyl)-aminomethyl]-3-phenyl-7-trifluoromethylquinoxalines
with the aim to produce antitumor compounds having as target
folate enzymes. All tested compounds exhibited acceptable activity
Table 1
Reaction conditions for alkylation of the primary amines.
against hDHFR, with Ki values ranged between 2 and 11
fortunately, they resulted to be less active than the lead compound
1, that shows Ki value of 0.2 M. Regrettably the NCI did not accept
compound 1 for anticancer preliminary assays, so no data for
comparison with the other compounds is available. Furthermore,
while the compound 1 had not shown any activity against hTS, a
few of the others novel derivatives were able to inhibit this enzyme.
In particular the most active compounds against hTS were 6
mM. Un-
Amines
Molar ratio (A:DIPA:RX)a
T
ꢁC
t
37
40
44
49
50
54
64
1:1:1.5
1:1:1.5
1:1:1.5
1:1:2
1:1:1.5
1:1:2
90
90
90
90
90
90
24 h
24 h
18 h
24 h
24 h
5 days
10 days
m
1:1:2
100
a
A ¼ amine; DIPA ¼ N-ethyldiisopropylamine; RX ¼ alkyl halides or ethyl sulfate.
(Ki ¼ 0.27
mM) and 21 (Ki ¼ 0.51
mM). As far as concerns the

174
S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
Scheme 2. Synthesis of the secondary amines 35e66.
anticancer activity, compounds 17, 21, 24, 26 and 27 exhibited a
was maintained at lower dosages as 1 mM for Colo 205, HCT-116,
complete antiproliferative activity on all 60 tumor cell lines at
HCT-15 (colon cancer), NCI/ADR-RES, MDA-MB-435 and T-47D,
NCI/ADR-RES (Breast cancer), HL-60, MOLT-4 (leukemia), NCI-H460
(lung cancer), OVCAR-3 (ovarian cancer) cell lines.
100
10
m
M concentration, between these 21 was the most active at
m
M on all cell lines (MG-MID log10 GI50 ¼ ꢀ5.49). The activity
In addiction compound 17 exhibited high activity (MG-MID GI50
equal to ꢀ4.95), and selectivity against: Non small cell lung cancer,
A549/ATCC (Log10 GI50 ¼ ꢀ6.43), Melanoma UACC-257 (Log10
GI50 ¼ ꢀ7.60), Ovarian cancer ovcar-8 (Log10 GI50 ¼ ꢀ5.82), Renal
cancer RXF 393 (Log10 GI50 ¼ ꢀ6.51) cell lines, and its activity was
Table 2
Prescreening assays: percent tumor cells growth inhibition recorded on three cell
lines at 10ꢀ4 or 10ꢀ5 M concentration of the accepted compounds.
Compd
Concentr.
Panel cell lines
Breast MCF7
Lung NCI-H-460
CNS SF-268
conserved also at 0.01 mM.
2
3
4
6
7
8
9
1$10ꢀ5
1$10ꢀ4
1$10ꢀ4
1$10ꢀ5
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ5
1$10ꢀ4
1$10ꢀ5
1$10ꢀ4
1$10ꢀ4
1$10ꢀ5
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
1$10ꢀ4
109
180
105
114
54
118
119
125
88
88
77
125
116
68
92
63
83
81
0
17
101
85
6
98
7
108
125
107
97
103
97
107
121
81
102
89
85
94
0
89
87
84
90
103
21
From the presented data emerges that all the novel derivatives
(2e31) are able to inhibit hDHFR, possessing Ki values of the same
order of magnitude (2e11 mM). Besides, these results are consistent
with docking studies where the predicted binding mode of the
substituents on nitrogen should allow a better interaction with
hydrophobic pocket (Fig. 5), while the modifications on positions
2,3,4 and 5 of the benzene ring are quite small. In addition, some
compounds also show activity against hTS, in particular the de-
rivatives bearing a propargyl group bonded to nitrogen of the
bridge, whereas methyl or ethyl groups result less effective. In
conclusion, our hypothesis that quinoxaline scaffold is able to
interact with the folate enzymes is confirmed.
61
105
115
46
103
54
61
73
1
58
102
67
41
82
17
40
119
64
87
106
10
12
13
14
15
16
17
21
22
23
24
25
26
27
28
29
30
31
Interestingly the majority of compounds that showed hTS-
inhibition also resulted the most active versus all 60 tumor cell
lines. In particular compound 21, which exhibit against hTS a Ki of
0.51 mM, is emerged as one of the best derivatives active versus the
tumor cell lines, while its counterparts 10 and 11, which bear
methyl and ethyl group respectively, are completely inactive.
The results of this investigation prompt us to continue the
development of novel derivatives keeping a propargyl group on the
nitrogen bridge and introducing different substituents (like an
18
80
104
61
115
95
109
102
110
111

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
175
Table 3
Table 4
Percent tumor growth inhibition recorded on sub panel cell lines at 10ꢀ4, 10ꢀ5 and
Percent tumor growth inhibition recorded on sub panel cell lines at 10ꢀ4, 10ꢀ5, 10ꢀ6
,
10ꢀ6 M concentration of compounds 21, 24, 26 and 27.
10ꢀ7 and 10ꢀ8 M concentration of compound 17.
Panel cell lines
21
24
26
27
Panel cell lines
17
10ꢀ4
10ꢀ5
10ꢀ6
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ4
10ꢀ5
10ꢀ6
10ꢀ7
10ꢀ8
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
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
HT29
KM12
SW-620
CSN cancer
SF-268
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
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
HT29
KM12
SW-620
CNS cancer
SF-268
*
*
*
94
92
89
81
117
e
98
79
78
82
107
146
153
160
122
162
145
70
83
62
44
97
56
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
105
e
85
e
88
*
66
e
67
*
e
78
79
88
85
44
84
56
55
*
71
*
45
*
56
*
*
*
123
120
e
60
89
48
109
e
107
123
*
63
51
52
e
177
112
195
184
179
158
96
79
23
18
21
32
34
*
56
*
*
42
*
*
*
*
*
*
*
*
19
*
*
*
*
*
*
*
*
199
93
145
196
180
96
106
71
31
123
94
93
91
92
62
75
76
*
60
79
78
125
17
14
*
*
*
*
62
49
90
85
134
97
164
25
28
177
e
164
e
108
e
126
e
73
104
99
47
77
75
102
77
82
70
e
92
174
199
171
97
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
189
181
200
161
79
79
66
63
*
*
*
94
97
80
124
93
15
43
17
40
*
110
99
137
82
12
*
*
*
*
112
75
82
153
164
18
76
150
77
163
180
181
50
e
*
82
68
96
84
54
85
63
49
161
*
91
74
96
52
*
SF-295
SF-539
SNB-19
SNB-75
56
*
54
*
99
181
176
148
115
198
15
18
18
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
49
92
SF-295
SF-539
SNB-19
SNB-75
118
85
*
U251
*
70
59
*
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
U251
62
178
160
186
136
200
158
110
188
54
41
95
72
71
99
*
*
*
101
129
71
102
*
76
*
77
e
Melanoma
LOX IMVI
M14
200
194
183
172
112
191
192
195
23
17
14
15
*
17
146
24
*
*
*
*
*
*
*
*
*
*
*
*
67
*
*
*
*
*
*
*
39
*
54
41
*
*
*
41
e
e
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
71
*
*
180
119
96
86
44
64
144
146
73
91
*
104
*
79
164
200
200
77
63
125
63
*
86
94
87
56
96
93
81
88
*
58
116
*
62
*
*
*
*
178
194
76
152
174
84
13
31
*
*
*
*
*
*
25
*
*
*
*
*
*
11
*
*
25
*
*
45
*
*
71
62
*
*
57
*
98
*
*
191
112
72
21
*
184
157
193
147
137
135
181
167
70
116
95
*
*
*
*
*
*
*
132
104
89
110
89
102
123
83
46
87
65
69
67
83
64
70
44
78
56
e
83
191
180
142
117
179
189
109
145
15
63
25
29
102
21
*
*
*
*
*
*
43
*
*
*
*
*
*
22
*
*
82
70
80
54
26
*
19
57
*
103
75
48
73
*
*
58
40
*
*
*
*
92
82
77
75
78
78
*
*
*
Breast cancer
MCF7
185
173
56
26
13
*
*
*
*
*
84
195
163
113
149
104
172
70
113
64
64
109
*
89
108
129
94
87
77
79
*
81
*
86
86
70
94
67
90
70
81
67
61
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
86
174
162
167
120
25
36
43
33
24
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
88
*
147
*
104
*
T-47D
78
90
90
T-47D
* ¼ below 40%; e ¼ test not done.
* ¼ below 40%.

176
S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
Table 5
Mean graph midpoint.
Varian XL-200 instrument using TMS as internal standard. Chem-
ical shift values are expressed as parts per million downfield from
TMS and J values are in hertz. Splitting patterns are indicated as s:
singlet, d: doublet, t: triplet, q: quadruplet, m: multiplet, dd: double
doublet, td: triplet of doublet. 13C NMR were determined in DMSO-
d6 and were recorded at 100 MHz with Brucher 400 Avance
Nanobay. Elemental analyses were performed on a PerkineElmer
2400 instrument at Laboratorio di Microanalisi, Dipartimento di
Chimica e Farmacia, University of Sassari. The analytical results for
C, H, and N were within ꢂ0.4%.
Compd
Log GI50
Log TGI
Log LC50
17
21
24
26
27
ꢀ4.95
ꢀ5.49
ꢀ4.51
ꢀ4.29
ꢀ4.35
ꢀ4.44
ꢀ4.64
ꢀ4.06
ꢀ4.02
ꢀ4.02
ꢀ4.13
ꢀ4.19
ꢀ4.00
ꢀ4.00
ꢀ4.00
amino group) on the quinoxaline moiety, in order to extend the SAR
analysis.
MS spectra were performed on a combined HP 5790 (GC)eHP
5970 (MS) apparatus recorded in EI.
4. Experimental
4.1.2. Intermediates
4.1. Chemistry
The bromomethylquinoxaline (32) was prepared as previously
described by some of us [18], while the starting anilines were
commercially available (Aldrich). Among the alkylanilines (35e66)
the intermediates 61, 62, 64, were unknown and are describe
below, while the remaining ones were all prepared as described
under the below indicated procedure and were identical as re-
ported in the cited literature, as follows:35 [18], 36 [28], 37 [29,30],
38 [30,31], 39 [32], 40 [32]. 41 [33], 42 [34], 43 [35], 44 [28], 45 [36],
46 [37,38], 47 [29,37], 48 [34,39,40], 49 [41], 50 [42], 51 [33,43,44],
52 [45], 53 [46], 54 [47], 55 [48], 56 [49], 57 [46], 58 [47], 59 [50], 60
[51,52], 63 [45], 65 [53], 66 [54].
4.1.1. General remarks
Melting Points are uncorrected and were recorded on a Köfler or
an electrothermal melting point apparatus. Ultraviolet (UV) spectra
are qualitative and were recorded in nanometer (nm) in ethanol
solution with
a PerkineElmer Lambda 5 spectrophotometer.
Infrared (IR) spectra were recorded as Nujol mulls or film with
PerkineElmer.
r 781 instrument and are expressed in
were determined in CDCl3 and were recorded at 200 MHz with a
n
(cmꢀ1). 1H NMR spectra
Fig. 5. Docking results. (A) Comparison of the predicted binding site of compound 1 (colored by atom, C atoms colored in yellow) and the crystallographic binding site of
Metotrexate (represented as a surface, colored by atom, C atoms in cyan) in the active site of hDHFR (represented as ribbon; NADPH colored by atom, C atoms in pink). (B, C) Details
of the predicted binding mode of compound 1 (colored by atom, C atoms colored in yellow) in the active site of hDHFR (residues colored by atom, C atoms in gray; NADPH colored by
atom, C atoms in pink). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Percent growth inhibition of compound 21 on different sub panel cell lines at 10ꢀ6 M concentration.

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
177
Fig. 7. Percent growth inhibition of compound 17 on four cell lines at 10ꢀ4e10ꢀ8 M concentration.
4.1.2.1. General procedure for the preparation of the N10-methyl,
(ethyl, propargyl) ring substituted anilines (35e66). A mixture of a
substituted aniline (33aem) (5.5 mmol), N-ethyldiisopropylamine
and alkyl halide (34aec) or ethyl sulfate (34d) in the molar ratio of
1:1:1 in toluene (21 ml) was stirred and heated at 90 ꢁC for 18 h. In
the cases listed below it was necessary to vary the conditions of
reaction as summarized in Table 1.
chromatography on a silica gel column eluting with the following
mixture of solvents: ethyl acetate/petrol ether 40e60ꢁ, in the ratio
of 6:4 (35, 42); 7:3 (52); 8:2 (41, 43, 48, 51, 53, 62, 65); 9:1 (38, 39,
45, 46, 47, 50); 95:5 (36, 37, 44, 55, 56, 57, 58, 59, 60, 61); 98:2 (64);
petrol ether 40e60ꢁ/diethyl ether 9:1 (49); toluene/ethyl acetate
95:5 (63); chloroform/methanol 95:5 (54), chloroform/methanol
98:2 (66).
The resulting suspension was taken up with ethyl acetate. The
organic phase was washed with water then dried over anhydrous
sodium sulfate. On evaporation of the solvent, in most cases a solid
or an oily residue was obtained. Purification was performed by flash
4.1.2.2. 3,4-Difluoro-N-(propargyl)aniline (61). This compound was
obtained as an oil in 52% yield, UV (EtOH): lmax 297, 238, 206 nm; IR
(neat) 3400, 2130, 1610 cmꢀ1 1H NMR (200 MHz, CDCl3):
; d 7.09e
6.90 (1H, m, H-2), 6.58e6.42 (1H, m, H-5), 6.41e6.30 (1H, m, H-6),
3.89 (2H, d, J ¼ 2.6 Hz, CH2); 2.24 (1H, t, J ¼ 2.4 Hz, CH). MS: m/z 167
(Mþ). Anal. Calcd for C9H7F2N: C, 64.67; H, 4.22; N, 8.38 Found C,
64.35; H, 4.12; N, 8.42.
Table 6
Enzymatic activity inhibition of human folate dependent enzymes.
Compd
Ki hDHFR (
m
M)
Ki hTS (
m
M)
1
2
3
4
0.2
5
5
NI
NI
NI
NI
4.1.2.3. 3,4-Dimethoxy-N-(propargyl)aniline (62). This compound
was obtained as an oil in 38% yield, UV (EtOH): lmax 298, 243,
208 nm; IR (neat): 3350, 2120, 1620 cmꢀ1 1H NMR (200 MHz,
;
3
5
6
7
8
3
4
8
5
NI
11
4
3
3
7
5
5
4
6
3
4
6
2
4
8
3
4
8
3
4
NI
0.27
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
0.51
NI
NI
2.38
NI
NI
3.56
NI
5.0
NI
NI
CDCl3):
d
6.78 (1H, d, J ¼ 8.4 Hz, H-5), 6.33 (1H, d, J ¼ 2.8 Hz, H-2),
6.25 (1H, dd, J ¼ 8.6 Hz and 2.8 Hz, H-6), 3.91 (2H, d, J ¼ 2.6 Hz, CH2),
3.85 (3H, s, OCH3), 3.82 (3H, s, OCH3), 2.23 (1H, t, J ¼ 2.4 Hz, CH).
MS: m/z 191 (Mþ). Anal. Calcd. for C11H13NO2:C, 69.09; H, 6.85; N,
7.32; Found C, 68.87; H, 6.64; N, 7.52.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
4.1.2.4. 2-Fluoro-N-(propargyl)-5-(trifluoromethyl)aniline(
64).
This compound was obtained as an oil in 38% yield, UV (EtOH): lmax
292, 242, 207 nm; IR (neat):3430, 2130, 1610; 1H NMR (200 MHz,
CDCl3):
d
7.17e6.91 (3H, m, 3H arom), 4.01 (2H, d, J ¼ 2.6 Hz, CH2),
2.27 (1H, t, J ¼ 2.4 Hz, CH). MS: m/z 217 (Mþ). Anal. Calcd. for
C10H7F4N: C, 55.31; H, 3.25; N, 6.45 Found C, 55.60; H, 3.35; N, 6.21.
4.1.3. General procedure for the preparation of the
alkylquinoxalines (1e31)
A mixture of N-alkylaniline (35e66) (2.46 mmol) and bromo-
methylquinoxaline (32) (1.28 mmol) in dry dimethylacetamide
(DMAa) (23 ml) was stirred at room temperature for 72 h. In the
case of compounds 12, 14, 15 the reaction was prolonged up to 96 h,
while in the case of 30 was necessary to heat at 60 ꢁC. (In the case of
preparation of compound 2 an equimolar amount of the reactants
was used in acetonitrile in the presence of 1.5 mmol Cs2CO3 and the
suspension was stirred under heating at 40 ꢁC for 96 h). Then water
ND
3
ND ¼ not measured; NI ¼ no enzymatic activity inhibition detected at 10
mM.

178
S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
was added to the mixture to give crude products of 1e31 as gums or
crystalline precipitates which were collected, thoroughly washed.
Purification was accomplished by silica gel column chromatog-
raphy on eluting with petrol ether 40e60 ꢁC/ethyl acetate in the
following ratio 9:1 (2, 3, 13, 19, 23, 29); 95:5 (9, 12, 15, 21, 22, 24, 25,
26, 27, 30); 8:2 (7, 8, 28, 31); 6:4 (1, 20). In the case of the derivatives
5, 6, 10, 11, 14, 16, 17, 18, further purification was carried out by
trituration with petrol ether followed by filtration. Melting point,
yields, analytical and spectroscopic data are reported below.
for C24H20F3N3O: C, 68.08; H, 4.76; N, 9.92; Found C, 67.80; H, 4.63;
N, 10.18.
4.1.3.5. 2-[N-Methyl(4-trifluoromethoxyphenyl)-aminomethyl]-3-
phenyl-7-trifluoromethyl quinoxaline (5). Yield: 73%, m.p.: 62e
64 ꢁC; UV (EtOH) lmax 238, 205 nm; IR (film) 1580 cmꢀ1; 1H NMR
y
(200 MHz, CDCl3)
d
: 8.38 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.8 Hz, H-5),
7.92 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.69e7.45 (5H, m, 5H
arom), 6.96 (2H, d, J ¼ 8.8 Hz, H-30,50), 6.50 (2H, d, J ¼ 8.8 Hz, H-
20,60), 4.85 (2H, s, CH2N), 2.99 (3H, s, CH3N). 13C NMR (100 MHz,
4.1.3.1. Diethyl
methyl]-N-methyl-benzoyl-
60 ꢁC; UV (EtOH) lmax 310, 275, 221 nm. IR (nujol)
1720, 1600 cmꢀ1 1H NMR (200 MHz, CDCl3)
: 8.33 (1H, s, H-8),
N-[4-(3-phenyl-7-trifluoromethylquinoxalin-2-yl)
-glutamate (1). Yield: 22%, m.p.:58e
3320, 1740,
DMSO-d6) d: 47.6 (CH3), 48.4 (CH3), 114.7 (2CH), 116.3 (2CH), 123.0
L
(CH), 125.3 (C), 126.3 (C), 127.5 (2CH), 128.1 (C), 129.1 (CH), 130.4
(2CH), 130.8 (C), 131.0 (CH), 131.5 (C), 132.1 (C), 139.3 (C), 141.9 (C),
142.0 (C), 147.4 (C), 155.0 (C). MS: m/z 477 (Mþ). Anal. Calcd for
y
;
d
8.25 (1H, d, J ¼ 9.40 Hz, H-5), 7.90 (1H, dd, J ¼ 9.40 Hz and 1.60, H-
6), 7.75e7.56 (7H, m, arom þ H-30,50), 6.70 (1H, d, J ¼ 7.4 Hz, NH),
6.51 (2H, d, J ¼ 9.00 Hz, H-20,60), 4.93 (2H, s, CH2eN), 4.80e4.65 (1H,
m, NeCH), 4.20 (2H, q, J ¼ 7.2 OCH2CH3), 4.06 (2H, q, J ¼ 7.2
OCH2CH3), 3.10 (3H, s, NeCH3), 2.55e2.00 (4H, m, CH2CH2), 1.27
(3H, t, J ¼ 7.4 CH2CH3), 1.18 (3H, t, J ¼ 7.4 CH2CH3). 13C NMR
C24H17F6N3O: C, 60.38; H, 3.59; N, 8.80; Found C, 59.98; H, 3.79; N,
8.85.
4.1.3.6. 2-[N-Methyl(3,4-difluorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethyl quinoxaline (6). Yield: 23%, m.p.: 103e104 ꢁC; UV
(EtOH) lmax 238, 205 nm; IR (nujol)
(200 MHz, CDCl3)
7.93 (1H, dd, J ¼ 9.0 Hz and 2.0 Hz, H-6), 7.65e7.50 (5H, m, 5H
arom), 6.98e6.79 (1H, m, H-20), 6.40e6.26 (1H, m, H-50), 6.25e6.15
(1H, m, H-60), 4.80 (2H, s, CH2N), 2.95 (3H, s, CH3N). 13C NMR
y ;
1580 cmꢀ1 1H NMR
: 8.37 (1H, s, H-8), 8.24 (1H, d, J ¼ 9.0 Hz, H-5),
(100 MHz, DMSO-d6)
d: 141.1 (2CH3), 27.0 (CH2), 31.3 (CH2), 46.7
d
(CH3), 56.3 (CH), 49.5 (CH2), 62.1 (2CH2), 112.0 (2CH), 122.9 (2CH),
125.0 (C), 127.0 (2CH), 128.9 (CH), 129.0 (CH), 129.9 (2CH), 130.9
(2CH), 131.1 (CH), 131.5 (C), 134.2 (C), 135.8 (C), 142.3 (2C), 147.6 (C),
153.7 (C), 167.9 (C), 173.0 (C), 175.5 (C). MS: m/z 622 (Mþ). Anal.
Calcd for C33H33F3N4O5: C, 66.66; H, 5.34; N, 9.00; Found C, 66.20;
H, 5.72; N, 9.14.
(100 MHz, DMSO-d6) d: 47.2 (CH3), 49.0 (CH2), 105.7 (CH), 115.5
(CH), 124.0 (C), 124.2 (CH), 128.0 (2CH), 129.1 (CH), 129.6 (CH), 130.0
(2CH), 130.8 (CH), 132.4 (C), 134.3 (C), 139.0 (C), 140.8 (C), 141.7 (C),
146.8 (C), 147.0 (C), 147.8 (C), 155.0 (C). MS: m/z 429 (Mþ). Anal.
Calcd for C23H16F5N3: C, 64.34; H, 3.76; N, 9.79; Found C, 64.67; H,
3.86; N, 9.47.
4.1.3.2. 2-[N-Methyl(4-chlorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (2). Yield: 32%, m.p.:130e131 ꢁC; UV
(EtOH) lmax 321, 257, 238, 205 nm; IR (nujol)
(200 MHz, CDCl3)
7.92 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.66e7.50 (5H, m, 5H
arom), 7.04 (2H, d, J ¼ 9.0 Hz, H-30,50), 6.47 (2H, d, J ¼ 9.0 Hz, H-
20,60), 4.84 (2H, s, CH2N), 2.98 (3H, s, CH3N). 13C NMR (100 MHz,
y
1580 cmꢀ1; 1H NMR
: 8.36 (1H, s, H-8), 8.23 (1H, d, J ¼ 8.6 Hz, H-5),
d
4.1.3.7. 2-[N-Methyl(3,4-dimethoxyphenyl)-aminomethyl]-3-phenyl-
7-trifluoromethyl quinoxaline (7). Yield: 41%, m.p.: 102e104 ꢁC; UV
(EtOH) lmax 388, 304, 222 nm;
(200 MHz, CDCl3)
7.92 (1H, dd, J ¼ 9.0 Hz and 2.0 Hz, H-6), 7.69e7.50 (5H, m, 5H
arom), 6.66 (1H, d, J ¼ 9.2 Hz, H-50), 6.27 (1H, d, J ¼ 2.8 Hz, H-20),
6.11 (1H, dd, J ¼ 8.8 Hz and 2.8 Hz, H-60), 4.77 (2H, s, CH2N), 3.76
(3H, s, OCH3), 3.73 (3H, s, OCH3), 2.87 (3H, s, CH3N). 13C NMR
y ;
(nujol) 1580 cmꢀ1 1H NMR
: 8.41 (1H, s, H-8), 8.23 (1H, d, J ¼ 8.8 Hz, H-5),
d
DMSO-d6)
d: 47.3 (CH3), 48.7 (CH2), 116.0 (2CH), 122.8 (CH), 123.3
(C), 126.0 (C), 127.5 (2CH), 128.2 (CH), 129.3 (CH), 129.9 (2CH), 130.5
(2CH), 131.0 (CH), 131.6 (2C), 132.0 (C), 140.9 (C), 142.4 (C), 148.7 (C),
155.0 (C). MS: m/z 427 (Mþ). Anal. Calcd for C23H17ClF3N3: C, 64.57;
H, 4.00; N, 9.82; Found C, 64.20; H, 4.40; N, 9.68.
(100 MHz, DMSO-d6) d: 47.3 (CH3), 48.5 (CH2), 57.0 (2CH3), 98.0
(CH), 106.2 (CH), 113.0 (CH), 122.8 (CH), 125.0 (C), 126.2 (2CH), 127.7
(CH), 128.3 (CH), 129.9 (2CH), 131.3 (CH), 132.1 (C), 133.0 (C), 138.7
(C), 139.8 (C), 141.5 (C), 143.5 (C), 147.6 (CH), 151.0 (C), 153.9 (C). MS:
m/z 453 (Mþ). Anal. Calcd for C25H22F3N3O2: C, 66.22; H, 4.89; N,
9.27; Found C, 66.55; H, 4.66; N, 9.07.
4.1.3.3. 2-[N-Methyl(4-fluorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (3). Yield: 23%, m.p.: 110e112 ꢁC; UV
(EtOH) lmax 238, 205 nm; IR (nujol)
(200 MHz, CDCl3)
y ;
1580 cmꢀ1 1H NMR
d
: 8.38 (1H, s, H-8), 8.23 (1H, d, J ¼ 9.0 Hz, H-5),
7.92 (1H, dd, J ¼ 9.0 Hz and 2.0 Hz, H-6), 7.65e7.50 (5H, m, 5H
arom), 6.88e6.75 (2H, m, H-30,50), 6.53e6.45 (2H, m, H-20,60), 4.79
4.1.3.8. 2-[N-Methyl(3,4,5-trimethoxyphenyl)-aminomethyl]-3-
(2H, s, CH2N), 2.94 (3H, s, CH3N). 13C NMR (100 MHz, DMSO-d6)
d:
phenyl-7-trifluoromethyl quinoxaline (8). Yield: 50%, m.p.: 103e
47.8 (C), 48.9 (CH2), 116.9 (2CH), 117.2 (2CH), 122.0 (CH), 123.1 (CH),
126.0 (2CH), 127.8 (CH), 128.7 (CH), 129.0 (C), 130.4 (2CH), 130.9
(CH), 131.6 (C), 134.3 (C), 140 (C), 142.2 (C), 148.7 (C), 156.0 (C), 156.9
(C). MS: m/z 411 (Mþ) Anal. Calcd for C23H17F4N3: C, 67.15; H, 4.17;
N, 10.21; Found C, 67.20; H, 4.48; N, 9.98.
105 ꢁC; UV (EtOH) lmax 236, 207 nm; IR (nujol) 1580 cmꢀ1 1H
y ;
NMR (200 MHz, CDCl3)
d
: 8.40 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.8 Hz, H-
5), 7.93 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.63e7.51 (5H, m, 5H
arom), 5.81 (2H, s, H-20,60), 4.83 (2H, s, CH2N), 3.71 (9H, s, OCH3),
2.90 (3H, s, CH3N). 13C NMR (100 MHz, DMSO-d6)
d: 46.9 (CH2), 47.3
(CH3), 56.1 (CH3), 56.7 (CH3), 61.0 (CH3), 92.0 (2CH), 122.8 (CH),
124.5 (C), 128.6 (2CH), 128.9 (CH), 129.0 (CH), 129.5 (C), 129.9 (2CH),
130.7 (CH), 131.5 (C), 134 (C), 141.0 (C), 142.0 (C), 143.2 (C), 147.1 (C),
153.1 (C), 156.0 (2C). MS: m/z 483 (Mþ) .Anal. Calcd for
C26H24F3N3O3: C, 64.59; H, 5.00; N, 8.69; found C, 64.92; H, 4.70; N,
8.79.
4.1.3.4. 2-[N-Methyl(4-methoxyphenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (4). Yield: 60%, m.p.: 91e93 ꢁC; UV
(EtOH) lmax 304, 222 nm; IR (nujol)
(200 MHz, CDCl3)
7.91 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.68e7.50 (5H, m, 5H
arom), 6.70 (2H, d, J ¼ 9.0 Hz, H-30,50), 6.56 (2H, d, J ¼ 9.2 Hz, H-
20,60), 4.74 (2H, s, CH2N), 3.71 (3H, s, OCH3), 2.89 (3H, s, CH3N). 13C
y ;
1580 cmꢀ1 1H NMR
: 8.41 (1H, s, H-8), 8.22 (1H, d, J ¼ 8.8 Hz, H-5),
d
4.1.3.9. Ethyl-N-[4-(3-phenyl-7-trifluoromethylquinoxalin-2-yl)
methyl]-N-methylaminophenylacetate (9). Yield: 9%, gum; UV
NMR (100 MHz, DMSO-d6) d: 47.6 (CH3), 48.4 (CH2), 55.2 (CH3),
116.5 (2CH),117.0 (2CH),123.1 (CH), 124.4 (C),125.3 (C),128.2 (2CH),
128.6 (CH), 129.4 (CH), 130.3 (2CH), 130.6 (C), 134.2 (C), 139.0 (C),
141.7 (C), 149.2 (C), 151.3 (C), 153 (C). MS: m/z 423 (Mþ) Anal. Calcd
(EtOH) lmax 237, 205 nm; IR (film)
(200 MHz, CDCl3)
: 8.30 (1H, s, H-8), 8.13 (1H, d, J ¼ 8.4 Hz, H-5),
7.81 (1H, dd, J ¼ 8.4 Hz and 2.4 Hz, H-6), 7.58e7.43 (5H, m, 5H
y ;
1730, 1580 cmꢀ1 1H NMR
d

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
179
arom), 6.94 (2H, d, J ¼ 8.6 Hz, H-30,50), 6.43 (2H, d, J ¼ 8.6 Hz, H-
20,60), 4.74 (2H, s, CH2N), 4.04 (2H, q, J ¼ 7.4, OCH2CH3), 3.35 (2H, s,
CH2CO), 2.90 (3H, s, CH3N), 1.17 (3H, t, J ¼ 7.4, OCH2CH3). 13C NMR
MS: m/z 425 (Mþ) Anal. Calcd for C24H19F4N3: C, 67.76; H, 4.50; N,
9.88; Found: C, 67.52; H, 4.75; N, 9.98.
(100 MHz, DMSO-d6) d: 15.3 (CH3), 41.2 (CH2), 47.2 (CH3), 50.3
4.1.3.14. 2-[N-Ethyl(4-methoxyphenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (14). Yield: 90%, m.p.:75e76 ꢁC; UV
(CH2), 62.0 (CH2), 112.8 (2CH), 122.7 (CH), 124.0 (C), 124.6 (C), 126.7
(CH), 127.7 (CH), 128.2 (CH), 129.9 (2CH), 131.0 (CH), 131.3 (2CH),
132.0 (C), 132.2 (C), 134.0 (C), 141.6 (C), 142.5 (C). 149.0 (C), 154.2 (C),
170.3 (C). MS: m/z 479 (Mþ). Anal. Calcd for C27H24F3N3O2: C, 67.63;
H, 5.05; N, 8.76; Found C, 67.32; H, 5.24; N, 8.68.
(EtOH) lmax 307, 222 nm; IR (nujol)
(200 MHz, CDCl3)
y ;
1580 cmꢀ1 1H NMR
: 8.41 (1H, s, H-8), 8.22 (1H, d, J ¼ 8.8 Hz, H-5),
d
7.91 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.70e7.43 (5H, m, 5H
arom), 6.75 (2H, d, J ¼ 9.2 Hz, H-30,50), 6.59 (2H, d, J ¼ 9.2 Hz, H-
20,60), 4.69 (2H, s, CH2N), 3.70 (3H, s, OCH3), 3.24 (2H, q, J ¼ 7.2 Hz,
CH2CH3),1.03 (3H, t, J ¼ 7.2 Hz, CH2CH3). 13C NMR (100 MHz, DMSO-
4.1.3.10. 2-[N-Methyl(4-methylphenyl)-aminomethyl]-3-phenyl-7-
trifluoromethyl quinoxaline (10). Yield: 95%, m.p.: 119e121 ꢁC; UV
d6) d: 13.2 (CH3), 43.6 (CH2), 44.6 (CH2), 56.0 (CH3), 115.0 (2CH),
(EtOH) lmax 306, 222 nm; IR (nujol)
(200 MHz, CDCl3)
7.90 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.68e7.50 (5 H, m, 5H
arom), 6.92 (2H, d, J ¼ 8.6 Hz, H-30,50), 6.50 (2H, d, J ¼ 8.6 Hz, H-
20,60), 4.80 (2H, s, CH2N), 2.96 (3H, s, CH3N), 2.19 (3H, s, CH3). 13C
y ;
1580 cmꢀ1 1H NMR
: 8.39 (1H, s, H-8), 8.20 (1H, d, J ¼ 8.6 Hz, H-5),
115.4 (2CH), 122.7 (CH), 124.9 (C), 127.0 (2CH), 127.7 (CH), 128.0
(CH),129.9 (2CH),130.7 (CH),132.0 (C),132.5 (C),140.0 (C),141.3 (C),
143.0 (C),147.9 (C),151.7 (C),155.0 (C). MS: m/z 437 (Mþ) Anal. Calcd
for C25H22F3N3O:C, 68.64; H, 5.07; N, 9.61; Found C, 68.74; H, 5.39;
N, 9.36.
d
NMR (100 MHz, DMSO-d6) d: 20.0 (CH3), 40.1 (CH3), 56.0 (CH2),
112.8 (2CH), 122.2 (CH), 124.9 (C), 127.5 (2CH), 126.9 (CH), 128.2
(CH), 129.0 (2CH), 129.9 (3CH), 131.0 (C), 131.5 (C), 132.3 (C), 133.0
(C), 141.6 (C), 146.7 (C), 155.5 (C), 157.0 (C). MS: m/z 407 (Mþ) Anal.
Calcd for C24H20F3N3: C, 70.75; H, 4.95; F, 13.99; N, 10.31; Found C,
70.55; H, 5.05; N, 10.11.
4.1.3.15. 2-[N-(Ethyltrifluoromethoxyphenyl)-aminomethyl]-3-
phenyl-7-trifluoromethylquinoxaline
73 ꢁC; UV (EtOH) lmax 304, 239, 222 nm; IR (nujol)
NMR (200 MHz, CDCl3)
(15). Yield:61%, m.p.:72e
y
1580 cmꢀ1; 1H
d
: 8.38 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.8 Hz, H-
5), 7.92 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.68e7.47 (5H, m, 5H
arom), 6.95 (2H, d, J ¼ 8.8 Hz, H-30,50), 6.50 (2H, d, J ¼ 9.2 Hz, H-
20,60), 4.79 (2H, s, CH2N), 3.39 (2H, q, J ¼ 7.2 Hz, CH2CH3), 1.13 (3H, t,
4.1.3.11. 2-[N-Ethyl(4-methylphenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (11). Yield:83%, m.p.:76e78 ꢁC; UV
J ¼ 7.4 Hz, CH2CH3). 13C NMR (100 MHz DMSO-d6)
d: 13.0 (CH3),
(EtOH) lmax 238, 206 nm; IR (nujol)
(200 MHz, CDCl3)
y ;
1580 cmꢀ1 1H NMR
: 8.39 (1H, s, H-8), 8.21 (1H, d, J ¼ 8.2 Hz, H-5),
44.7 (CH2), 46.0 (CH2), 114.7 (2CH), 115.2 (2CH), 122.2 (CH), 124.0
(C), 126.1 (2CH), 127.7 (CH), 128.0 (CH), 128.7 (2CH), 120.1 (C), 130.8
(CH), 132.2 (C), 137.7 (C), 139.3 (C), 140.2 (C), 140.9 (C), 142.5 (C),
148.6 (C), 155.1 (C). MS: m/z 491 (Mþ) Anal. Calcd. for
d
7.89 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.65e7.51 (5H, m, 5H
arom), 6.91 (2H, d, J ¼ 8.6 Hz, H-30,50), 6.51 (2H, d, J ¼ 8.8 Hz, H-
20,60), 4.75 (2H, s, CH2N), 3.34 (2H, q, J ¼ 7.2, CH2CH3), 2.18 (3H, s,
C25H19F6N3O:C, 61.10; H, 3.90; N, 8.55; Found C, 61.30; H, 4.00; N,
CH3), 1.09 (3H, t, J ¼ 7.4, CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d:
8.25.
13.0 (CH3), 20.7 (CH3), 46.0 (CH2), 45.4 (CH2), 113.2 (2CH), 122.8
(CH), 123.2 (C), 127.9 (CH), 128.0 (2CH), 128.4 (CH), 129.0 (2CH),
130.1 (3CH), 130.7 (C), 132.0 (C), 133.0 (C), 139.2 (C), 141.3 (C), 145.8
(C), 149.0 (C), 155.0 (C). MS: m/z 421 (Mþ) Anal. Calcd for
4.1.3.16. 2-[N-Ethyl(3,4-difluorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (16). Yield:82%, m.p.:65e67 ꢁC; UV
C
25H22F3N3: C, 71.25; H, 5.26; N, 9.97; Found C, 70.88; H, 5.56; N,
(EtOH) lmax 326, 238, 206 nm; IR (nujol)
(200 MHz, CDCl3)
y ;
1580 cmꢀ1 1H NMR
: 8.38 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.6 Hz, H-5),
10.04.
d
7.93 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.70e7.53 (5H, m, 5H
arom), 6.90e6.79 (1H, m, H-20), 6.45e6.29 (1H, m, H-50), 6.27e6.17
(1H, m, H-60), 4.74 (2H, s, CH2N), 3.32 (2H, q, J ¼ 7.2 Hz, CH2CH3),
4.1.3.12. 2-[N-Ethyl(4-chlorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (12). Yield:61%, m.p.: 93e95 ꢁC; UV
1.10 (3H, t, J ¼ 7.4 Hz, CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d:
(EtOH) lmax 328, 260, 237, 205 nm; IR (nujol)
(200 MHz, CDCl3)
y
1580 cmꢀ1; 1H NMR
: 8.37 (1H, s, H-8), 8.23 (1H, d, J ¼ 8.6 Hz, H-5),
12.9 (CH3), 44.2 (CH2), 46.3 (CH2), 103.9 (CH), 112.0 (CH), 114.8 (CH),
123.1 (CH), 123.8 (C), 126.5 (2CH), 127.9 (CH), 128.2 (CH), 130.0
(2CH), 131.1 (CH), 132.2 (C), 133.0 (C), 138.1 (C), 139.9 (C), 141.6 (C),
147.7 (C), 148.1 (C), 149.3 (C), 155.0 (C). MS: m/z 443 (Mþ) Anal.
Calcd. for C24H18F5N3:C, 65.01; H, 4.09; N, 9.48; Found C, 64.81; H,
4.39; N, 9.56.
d
7.91 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.70e7.46 (5H, m, 5H
arom), 7.03 (2H, d, J ¼ 9.0 Hz, H-30,50), 6.47 (2H, d, J ¼ 9.2 Hz, H-
20,60), 4.77 (2H, s, CH2N), 3.36 (2H, q, J ¼ 7.4, CH2CH3), 1.12 (3H, t,
J ¼ 7.6, CH2CH3). 13C NMR (100 MHz DMSO-d6)
d: 13.0 (CH3), 45.9
(CH2), 50.1 (CH2), 115.2 (2CH), 123.9 (CH), 124.5 (C), 127.0 (C), 128.0
(2CH), 128.6 (CH), 129.1 (CH), 129.7 (2CH), 130.2 (2CH), 131.3 (CH),
132.2 (C), 134.1 (C), 141.0 (C), 141.7 (C), 148.2 (C), 149.3 (C), 153.8 (C).
MS: m/z 441 (Mþ) Anal. Calcd for C24H19ClF3N3: C, 65.23; H, 4.33; N,
9.51; Found C, 65.33; H, 4.00; N, 9.59.
4.1.3.17. 2-[N-Ethyl(3,4-dimethoxyphenyl)-aminomethyl]-3-phenyl-
7-trifluoromethylquinoxaline (17). Yield:67%, m.p.:85e86 ꢁC; UV
(EtOH) lmax 296, 222 nm; IR (film)
CDCl3)
y
1580 cmꢀ1; 1H NMR (200 MHz,
: 8.40 (1H, s, H-8), 8.22 (1H, d, J ¼ 8.8 Hz, H-5), 7.92 (1H, dd,
d
4.1.3.13. 2-[N-Ethyl(4-fluorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethylquinoxaline (13). Yield:34%, m.p.:98e99 ꢁC; UV
J ¼ 9.0 Hz and 2.0 Hz, H-6), 7.66e7.46 (5H, m, 5H arom), 6.65 (1H, d,
J ¼ 9.0 Hz, H-50), 6.29 (1H, d, J ¼ 2.0 Hz, H-20), 6.15 (1H, dd, J ¼ 9.0 Hz
and 2.0 Hz, H-60), 4.73 (2H, s, CH2N), 3.76 (3H, s, OCH3), 3.73 (3H, s,
OCH3), 3.21 (2H, q, J ¼ 7.4 Hz, CH2CH3), 1.05 (3H, t, J ¼ 7.6 Hz,
(EtOH) lmax 238, 205 nm; IR (nujol)
(200 MHz, CDCl3)
y ;
1580 cmꢀ1 1H NMR
: 8.39 (1H, s, H-8), 8.23 (1H, d, J ¼ 8.0 Hz, H-5),
d
7.92 (1H, dd, J ¼ 8.0 Hz and 2.0 Hz, H-6), 7.69e7.49 (5H, m, 5H
arom), 6.87e6.75 (2H, m, H-30,50), 6.60e6.45 (2H, m, H-20,60), 4.74
(2H, s, CH2N), 3.30 (2H, q, J ¼ 7.6 Hz, CH2CH3), 1.07 (3H, t, J ¼ 7.6 Hz,
CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d: 12.0 (CH3), 45.8 (CH2),
55.0 (CH2), 55.9 (2CH3), 99.7 (CH), 105.0 (CH), 113.8 (CH), 125.2 (C),
125.3 (CH), 126.3 (CH), 128.3 (2CH), 129.0 (2CH), 129.3 (CH), 129.7
(C), 130.0(C), 131.0 (CH), 138.0 (C), 139.2 (C), 140.7 (C), 142.0 (C),
142.8 (C), 149.4 (C), 155.6 (C). MS: m/z 467 (Mþ) Anal. Calcd. for
CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d: 13.2 (CH3), 45.0 (CH2),
48.2 (CH2), 114.8 (2CH), 117.2 (2CH), 123.6 (CH), 124.9 (C), 127.2
(2CH), 128.3 (CH), 129.3 (CH), 130.4 (2CH), 131.0 (CH), 132.5 (C),
134.7 (C), 141.0 (C), 142.6 (C), 144.1 (C), 149.8 (C), 154.0 (C), 157.7 (C).
C26H24F3N3O2: C, 66.80; H, 5.17; N, 8.99; Found C, 66.88; H, 5.12; N,
9.00.

180
S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
4.1.3.18. 2-[N-Ethyl(3,4,5-trimethoxyphenyl)-aminomethyl]-3-
NMR (200 MHz, CDCl3)
d
: 8.47 (1H, s, H-8), 8.25 (1H, d, J ¼ 8.8 Hz, H-
phenyl-7-trifluoromethylquinoxaline (18). Yield:77%, m.p.:144e
5), 7.93 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.80e7.38 (5H, m, 5H
arom), 7.34 (1H, t, J ¼ 7.2, H-30), 7.30 (1H, t, J ¼ 7.8, H-60), 7.19 (1H, td,
J ¼ 7.4 Hz and 1.6 Hz, H-40), 7.03 (1H, td, J ¼ 7.8 Hz and 1.6 Hz, H-50),
4.68 (2H, s, CH2N), 4.10 (2H, d, J ¼ 2.6 Hz, CH2), 2.24 (1H, t,
146 ꢁC; UV (EtOH) lmax 238, 210 nm; IR (nujol)
y ;
1580 cmꢀ1 1H
NMR (200 MHz, CDCl3)
d
: 8.39 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.6 Hz, H-
5), 7.93 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.63e7.50 (5H, m, 5H
arom), 5.83 (2H, s, H-20,60), 4.78 (2H, s, CH2N), 3.70 (9H, s, OCH3),
3.26 (2H, q, J ¼ 7.4, CH2CH3),1.11 (3H, t, J ¼ 7.4 Hz, CH2CH3). 13C NMR
J ¼ 2.4 Hz, CH). 13C NMR (100 MHz, DMSO-d6)
d: 44.0 (CH2), 46.2
(CH2), 73.4 (CH), 76.0 (C), 121.8 (CH), 122.2 (CH), 124.7 (C), 125.0
(CH), 128.2 (2CH), 127.5 (2CH), 129.0 (C), 129.3 (CH), 129.5 (2CH),
129.8 (CH), 131.0 (C), 131.4 (CH), 132.5 (C), 141.1 (C), 142.0 (C), 147.1
(C), 151.2 (C), 154.0 (C). MS: m/z 451 (Mþ) Anal. Calcd. for
(100 MHz, DMSO-d6) d: 13.0 (CH3), 46.4 (CH2), 46.8 (CH2), 55.2
(2CH3), 61.0 (CH3), 91.3 (2CH), 123.1 (CH), 124.9 (C), 127.6 (C), 128.0
(2CH), 128.3 (CH), 129.2 (CH), 129.8 (2CH), 131.0 (CH), 132.2 (C),
133.1 (C), 140.3 (C), 140.8 (C), 144.0 (C), 149.4 (C), 153.9 (C), 156.2
(2C). MS: m/z 497 (Mþ) Anal. Calcd. for C27H26F3N3O3: C, 65.18; H,
5.27; N, 8.45; Found C, 65.37; H, 5.00; N, 8.65.
C25H17ClF3N3: C, 66.45; H, 3.79; N, 9.30; Found C, 66.50; H, 3.89; N,
9.55.
4.1.3.23. 2-[N-Propargyl(4-chlorophenyl)-aminomethyl]-3-phenyl-7-
4.1.3.19. Ethyl-N-[4-(3-phenyl-7-trifluoromethylquinoxalin-2-yl)
methyl]-N-ethylaminophenylacetate (19). Yield:18%, gum; UV
trifluoromethylquinoxaline (23). Yield:12%, m.p.:45e46 ꢁC; UV
(EtOH) lmax 320, 238, 204 nm; IR (nujol)
(200 MHz, CDCl3)
y
2117, 1580 cmꢀ1; 1H NMR
(EtOH) lmax 237, 205 nm; IR (nujol)
(200 MHz, CDCl3)
y
1732, 1580 cmꢀ1
;
1H NMR
d
: 8.40 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.6 Hz, H-5),
d
: 8.39 (1H, s, H-8), 8.22 (1H, d, J ¼ 8.8 Hz, H-5);
7.93 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.68e7.50 (5H, m, 5H
arom), 7.09 (2H, d, J ¼ 9.0 Hz, H-30,50), 6.62 (2H, d, J ¼ 9.0 Hz, H-
20,60), 4.87 (2H, s, CH2N), 4.08 (2H, d, J ¼ 2.6 Hz, CH2), 2.19 (1H, t,
7.89 (1H, dd, J ¼ 8.8 Hz and 2.4 Hz, H-6), 7.65e7.30 (5H, m, 5H
arom), 7.01 (2H, d, J ¼ 8.6 Hz, H-30,50), 6.52 (2H, d, J ¼ 8.8 Hz, H-
20,60), 4.78 (2H, s, CH2N), 4.09 (2H, q, J ¼ 7.4, OCH2CH3), 3.43 (2H, s,
CH2CO), 3.40 (2H, q, J ¼ 7.6 Hz, CH2CH3), 1.27e1.18 (6H, m,
J ¼ 2.4 Hz, CH). 13C NMR (100 MHz, DMSO-d6)
d: 44.3 (CH2), 45.5
(CH2), 74.0 (CH), 77.8 (C),116.1 (2CH),123.0 (CH),123.9 (C),127.0 (C),
127.2 (2CH), 127.9 (CH), 128.9 (2CH), 129.1 (CH), 130.7 (2CH), 130.9
(CH), 131.2 (C), 132.1 (C), 141.5 (C), 141.9 (C), 146.6 (C), 148.0 (C),
155.1 (C). MS: m/z 451 (Mþ) Anal. Calcd. for C25H17ClF3N3: C, 66.45;
H, 3.79; N, 9.30; Found C, 66.70; H, 3.80; N, 9.11.
OCH2CH3, CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d: 13.0 (CH3),
15.4 (CH3), 41.0 (CH2), 44.7 (CH2), 46.9 (CH2), 61.0 (CH2), 112.9
(2CH), 124.2 (CH), 125.0 (C), 125.3 (C), 127.6 (2CH), 128.2 (CH), 128.7
(CH), 129.9 (CH), 130.1 (2CH), 131.3 (2CH), 132.0 (C), 132.8 (C), 139.9
(C), 141.2 (C), 147.5 (C), 155.1 (C), 172.6 (C). MS: m/z 493 (Mþ) Anal.
Calc. for C28H26F3N3O2: C, 68.14; H, 5.31; N, 8.51; Found C, 67.93; H,
5.16; N, 8.86.
4.1.3.24. 2-[N-Propargyl(4-fluorophenyl)-aminomethyl]-3-phenyl-7-
trifluoromethyl quinoxaline (24). Yield:22%, gum; UV (EtOH) lmax
330, 238, 205 nm; IR (nujol)
CDCl3)
y
: 2118, 1580 cmꢀ1; 1H NMR (200 MHz,
: 8.42 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.8 Hz, H-5), 7.94 (1H, dd,
4.1.3.20. Diethyl-N-[4-(3-phenyl-7-trifluoromethylquinoxalin-2-yl)
d
methyl]-N-ethylaminobenzoyl-
m.p.:34e36 ꢁC; UV (EtOH) lmax 307, 236, 205 nm; IR (nujol)
1580 cmꢀ1; 1H NMR (200 MHz, CDCl3)
: 8.34 (1H, s, H-8), 8.24 (1H,
L
-glutamate
(20). Yield:45%,
J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.72e7.48 (5H, m, 5H arom), 6.92e6.79
(2H, m, H-30,50), 6.75e6.59 (2H, m, H-20,60), 4.82 (2H, s, CH2N), 4.03
(2H, d, J ¼ 2.6 Hz, CH2), 2.18 (1H, t, J ¼ 2.4 Hz, CH). 13C NMR
y
d
d, J ¼ 8.8 Hz, H-5), 7.91 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.75e
7.47 (7H, m, 5H arom and H-30,50), 6.67 (1H, d, J ¼ 7.0, NHCO), 6.51
(2H, d, J ¼ 8.4 Hz, H-20,60), 4.87 (2H, s, CH2N), 4.82e4.67 (1H, m, CH),
4.20 (2H, q, J ¼ 7.6, OCH2CH3), 4.07 (2H, q, J ¼ 7.6, OCH2CH3), 3.50
(2H, q, J ¼ 7.2 Hz, CH2CH3), 2.55e1.97 (4H, m, CH2CH2), 1.37e1.11
(100 MHz, DMSO-d6) d: 44.2 (CH2), 46.1 (CH2), 73.0 (CH), 76.8 (C),
116.1 (2CH), 117.2 (2CH), 122.9 (CH), 123.7 (C), 127.9 (CH), 128.2
(2CH), 128.4 (CH), 129.2 (2CH), 130.1 (CH), 130.9 (C), 133.1 (C), 139.3
(C), 141.0 (C), 144.8 (C), 149.7 (C), 155.0 (C), 157.2 (C). MS: m/z 435
(Mþ) Anal. Calcd. for C25H17F4N3: C, 68.96; H, 3.94; N, 9.65; Found C,
69.00; H, 3.75; N, 9.60.
(9H, m, OCH2CH3, CH2CH3). 13C NMR (100 MHz, DMSO-d6)
d: 13.0
(CH3), 14.7 (2CH3), 25.2 (CH2), 31.0 (CH2), 45.4 (CH2), 47.3 (CH2),
55.1 (CH), 61.9 (2 CH2), 111.9 (2CH), 122.4 (CH), 123.5 (C), 124.7 (C),
128.0 (2CH), 128.3 (CH), 129.0 (CH), 130.6 (2CH), 130.9 (CH), 131.2
(2CH), 132.6 (C), 133.0 (C), 140.1 (C), 142.3 (C), 150.0 (C), 152.8 (C),
154.1 (C), 166.8 (C), 170.5 (C), 172.0 (C). MS: m/z 636 (Mþ) Anal.
Calcd. for C34H35F3N4O5: C, 64.14; H, 5.54; N, 8.80; Found C, 63.96;
H, 5.44; N, 8.39.
4.1.3.25. 2-[N-Propargyl(4-methoxyphenyl)-aminomethyl]-3-phenyl-
7-trifluoromethylquinoxaline (25). Yield:68%, m.p.:102e103 ꢁC; UV
(EtOH) lmax 320, 238, 204 nm; IR (nujol)
NMR .(200 MHz, CDCl3)
y ;
2120, 1580 cmꢀ1 1H
: 8.44 (1H, s, H-8), 8.24 (1H, d, J ¼ 9.0 Hz,
d
H-5), 7.93 (1H, dd, J ¼ 9.0 Hz and 2.0, H-6), 7.69e7.47 (5H, m, 5H
arom), 6.74 (4H, s, H-20,30,50,60), 4.77 (2H, s, CH2N), 3.98 (2H, d,
J ¼ 2.6 Hz, CH2), 3.72 (3H, s, OCH3), 2.18 (1H, t, J ¼ 2.4 Hz, CH). 13
C
4.1.3.21. 2-[N-Propargyl(4-methylphenyl)-aminomethyl]-3-phenyl-
NMR (100 MHz DMSO-d6) d: 44.1 (CH2), 46.2 (CH2), 55.8 (CH3), 73.0
7-trifluoromethylquinoxaline (21). Yield:14%, gum; UV (EtOH) lmax
(CH), 76.8 (C), 114.8 (2CH), 115.1 (2CH), 122.7 (CH), 123.5 (C), 127.0
(2CH), 127.7 (CH), 128.7 (2CH), 129.9 (CH), 131.5 (C), 133.4 (C), 139.5
(C), 141.5 (C), 142.0 (C), 147.6 (C), 153.1 (C), 155.2 (C). MS: m/z 447
(Mþ) Anal. Calcd. for C26H20F3N3O: C, 69.79; H, 4.51; N, 9.39; Found
C, 69.90; H, 4.76; N, 9.29.
341, 262, 224 nm; IR (film)
CDCl3)
y
2120, 1580 cmꢀ1; 1H NMR (200 MHz,
d
: 8.42 (1H, s, H-8), 8.23 (1H, d, J ¼ 8.2 Hz, H-5), 7.91 (1H, dd,
J ¼ 8.2 Hz and 2.0 Hz, H-6), 7.70e7.50 (5H, m, 5H arom), 6.96 (2H, d,
J ¼ 8.6 Hz, H-30,50), 6.65 (2H, d, J ¼ 8.6 Hz, H-20,60), 4.84 (2H, s,
CH2N), 4.07 (2H, d, J ¼ 2.6 Hz, CH2), 2.20 (3H, s, CH3), 2.18 (1H, t,
J ¼ 2.4 Hz, CH). 13C NMR (100 MHz, DMSO-d6)
d: 22.4 (CH3), 44.3
4.1.3.26. 2-[N-Propargyl(4-trifluoromethoxyphenyl)-aminomethyl]-
3-phenyl-7-trifluoromethyl quinoxaline (26). Yield:29%, m.p.:71e
(CH2), 45.0 (CH2), 73.2 (CH), 75.9 (C), 113.9 (2CH), 123.6 (CH), 125.0
(C), 127.3 (2CH), 128.3 (CH), 129.1 (CH), 129.9 (2CH), 130.6 (3CH),
131.3 (C), 131.5 (C), 133.5 (C), 141.7 (C), 142.0 (C), 148.0 (C), 155.1 (C).
MS: m/z 431 (Mþ). Anal. Calcd. for C26H20F3N3: C, 72.38; H, 4.67; N,
9.74; Found C, 72.40; H, 4.62; N, 9.51.
72 ꢁC; UV (EtOH) lmax 320, 238, 206 nm; IR (nujol)
y
2118,
1580 cmꢀ1; 1H NMR (200 MHz, CDCl3)
d: 8.41 (1H, s, H-8), 8.25 (1H,
d, J ¼ 8.6 Hz, H-5), 7.94 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.64e
7.52 (5H, m, 5H arom), 6.99 (2H, d, J ¼ 8.6 Hz, H-30,50), 6.65 (2H, d,
J ¼ 8.6 Hz, H-20,60), 4.89 (2H, s, CH2N), 4.10 (2H, d, J ¼ 2.6 Hz, CH2),
4.1.3.22. 2-[N-Propargyl(2-chlorophenyl)-aminomethyl]-3-phenyl-7-
2.18 (1H, t, J ¼ 2.4 Hz, CH). 13C NMR (100 MHz, DMSO-d6)
d: 41.1
ꢁ
trifluoromethylquinoxaline (22). Yield:9%, m.p.:95e97 C; UV
(CH2), 55.0 (CH2), 55.1(CH), 74.8 (C), 80.3 (C), 114.1 (2CH), 116.2
(2CH), 123.3 (CH), 126.4 (CH), 128.3 (2CH), 128.9 (2CH), 129.3 (CH),
(EtOH) lmax 368, 322, 238, 206 nm; IR (nujol)
y
2117, 1580 cmꢀ1; 1H

S. Piras et al. / European Journal of Medicinal Chemistry 75 (2014) 169e183
181
129.3 (C), 130.0 (C), 130.7 (CH), 137.6 (C), 139.2 (C), 142.0 (C), 142.1
4.1.3.31. Ethyl-N-[4-(3-phenyl-7-trifluoromethylquinoxalin-2-yl)
methyl]-N-propargylaminophenyl acetate (31). Yield:22%, gum; UV
(C), 152.3 (C), 154.8 (C), 157.0 (C). MS: m/z 501 (Mþ) Anal. Calcd. for
C
26H17F6N3O: C, 62.28; H, 3.42; N, 8.38; Found C, 62.18; H, 3.30; N,
(EtOH) lmax 239, 205 nm; IR (film)
(200 MHz, CDCl3)
y
2115, 1715, 1580 cmꢀ1; 1H NMR
8.46.
d
: 8.41 (1H, s, H-8), 8.23 (1H, d, J ¼ 9.0 Hz, H-5),
7.92 (1H, dd, J ¼ 9.0 Hz and 2.0 Hz, H-6), 7.69e7.50 (5H, m, 5H
arom), 7.06 (2H, d, J ¼ 8.8 Hz, H-30,50), 6.66 (2H, d, J ¼ 8.8 Hz, H-
20,60), 4.87 (2H, s, CH2N), 4.20e4.02 (4H, m, OCH2CH3 and CH2), 3.46
(2H, s, CH2CO), 2.18 (1H, t, J ¼ 2.4 Hz, CH), 1.22 (3H, t, J ¼ 7.6,
4.1.3.27. 2-[N-Propargyl(3,4-difluorophenyl)-aminomethyl]-3-
phenyl-7-trifluoromethyl quinoxaline (27). Yield:19%, gum; UV
(EtOH) lmax 324, 238, 205 nm; IR (nujol)
NMR (200 MHz, CDCl3)
y ;
: 2118, 1580 cmꢀ1 1H
: 8.42 (1H, s, H-8), 8.25 (1H, d, J ¼ 8.6 Hz, H-
OCH2CH3). 13C NMR (100 MHz, DMSO-d6)
d: 14.2 (CH3), 40.5 (CH2),
d
44.2 (CH2), 46.7 (CH2), 61.9 (CH2), 72.9 (CH), 78.0 (C), 114.2 (2CH),
122.2 (CH), 124.0 (2C), 128.3 (2CH), 129.5 (2CH), 130.0 (2CH), 130.7
(CH), 131.0 (C), 132.2 (C), 140.6 (C), 141.5 (C), 147.3 (C), 148.2 (C),
155.6 (C), 170.3 (C). MS: m/z 503 (Mþ) Anal. Calcd. for
5), 7.92 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.75e7.45 (5H, m, 5H
arom), 7.30e6.80 (1H, m, H-20), 6.58e6.43 (1H, m, H-50), 6.42e6.30
(1H, m, H-60), 4.83 (2H, s, CH2N), 4.04 (2H, d, J ¼ 2.6 Hz, CH2), 2.20
(1H, t, J ¼ 2.4 Hz, CH). 13C NMR (100 MHz, DMSO-d6)
d: 44.5 (CH2),
C29H24F3N3O2: C, 69.18; H, 4.80; N, 8.35; Found C, 68.88; H, 4.42; N,
46.2 (CH), 73.0 (CH), 77.1 (C),105.3 (CH),111.5 (CH),115.5 (CH),122.9
(CH), 123.2 (C), 126.7 (2CH), 126.9 (CH), 127.7 (CH), 128.9 (2CH),
130.2 (CH), 131.0 (C), 132.4 (C), 138.5 (C), 140.7 (C), 141.2 (C), 147.1
(C), 148.0 (C), 148.5 (C), 155.0 (C). MS: m/z 453 (Mþ) Anal. Calcd. for
8.60.
4.2. Enzymology assays
C
25H16F5N3: C, 66.22; H, 3.56; N, 9.27; Found C, 66.00; H, 3.56; N,
9.30.
The proteins were purified as described previously [22]. The
folate cofactors and substrates were a gift from Merck Eprova; all
other substrates, cofactors, and reagents were purchased from
Sigma. DHFR, and TS activities were assessed spectrophotometri-
cally as previously described [22]. We used methotrexate as refer-
ence compound in the activity inhibition assays against hTS and
hDHFR. The Ki values obtained (data not shown) for this compound
in our test, did not differ from the values already reported in
literature [55]. All compounds showed competitive inhibition. Ki
values were obtained from the linear least-squares fit of the re-
sidual activity as a function of inhibitor concentration, using suit-
able equations for competitive inhibition [56]. Each experiment
was repeated at least three times, and no individual measurement
differed by more than 20% from the mean. Stock solutions of the
inhibitors were freshly prepared in DMSO (dimethyl sulfoxide). The
DMSO concentration was kept below the concentration affecting
enzyme activity (5% for hDHFR, 8% for hTS).
4.1.3.28. 2-[N-Propargyl(3,4-dimethoxyphenyl)-aminomethyl]-3-
phenyl-7-trifluoromethyl quinoxaline (28). Yield:52%, m.p.:76e
77 ꢁC; UV (EtOH) lmax 237, 206 nm; IR (nujol) 2115, 1580 cmꢀ1; 1H
y
NMR (200 MHz, CDCl3)
d
: 8.44 (1H, s, H-8), 8.25 (1H, d, J ¼ 8.6 Hz,
H-5), 7.92 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-6), 7.70e7.67 (5H, m, 5H
arom), 6.68 (1H, d, J ¼ 8.6 Hz, H-50), 6.42 (1H, d, J ¼ 2.6 Hz, H-20),
6.30 (1H, dd, J ¼ 8.6 Hz and 2.0 Hz, H-60), 4.80 (2H, s, CH2N), 3.97
(2H, d, J ¼ 2.6 Hz, CH2), 3.78 (3H, s, OCH3), 3.75 (3H, s, OCH3), 2.21
(1H, t, J ¼ 2.4 Hz, CH). 13C NMR (100 MHz DMSO-d6)
d: 44.7 (CH2),
46.1 (CH2), 55.8 (2CH3), 73.0 (CH), 77.1 (C), 97.3 (CH), 106.1 (CH),
113.5 (CH), 122.8 (CH), 125.2 (C), 127.0 (2CH), 127.7 (CH), 128.1 (CH),
129.9 (2CH), 130.7 (CH),131.5 (C),132.1 (C),138.0 (C),140.9 (C),141.0
(C), 143.2 (C), 147.5 (C), 151.3 (C), 154.1 (C). MS: m/z 477 (Mþ) Anal.
Calcd. for C27H22F3N3O2: C, 67.92; H, 4.64; N, 8.80; Found C, 67.72;
H, 4.84; N, 8.65.
4.3. Docking studies
4.1.3.29. 2-[N-Propargyl(3,4,5-trimethoxyphenyl)-aminomethyl]-3-
phenyl-7-trifluoromethyl quinoxaline (29). Yield:40%, gum; UV
The program AutoDock 4 [57] was used for docking. The protein
structure of hDHFR was taken from the crystal structure of the
ternary complex with NADPH and Methotrexate (PDB file ID:
1U72). The cofactor NADPH was retained in the structure during
the calculation while the ligand was deleted from the file before
starting the calculation. The 3D structures for compound 1 was
built and optimized with the ChemOffice suite of programs [58]. A
(EtOH) lmax 339, 238, 204 nm; IR (nujol)
NMR (200 MHz, CDCl3)
y ;
2120, 1580 cmꢀ1 1H
d
: 8.42 (1H, s, H-8), 8.25 (1H, d, J ¼ 8.4 Hz, H-
5), 7.94 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.69e7.50 (5H, m, 5H
arom), 5.97 (2H, s, H-20,60), 4.87 (2H, s, CH2N), 4.02 (2H, d, J ¼ 2.6 Hz,
CH2), 3.72 (9H, s, OCH3), 2.21 (1H, t, J ¼ 2.4 Hz, CH). 13C NMR
(100 MHz, DMSO-d6) d: 44.0 (CH2), 45.9 (CH2), 56.0 (2CH3), 61.1
(CH3), 72.8 (CH), 77.2 (C), 91.7 (CH), 123.9 (CH), 124.6 (C), 125.6 (C),
127.7 (2CH), 127.9 (CH), 128.5 (CH), 129.2 (2CH), 130.1 (C), 130.2 (C),
137.3 (C), 141.0 (C), 141.7 (C), 149.3 (C), 151.9 (C), 155.0 (C). MS: m/z
507 (Mþ) Anal. Calcd. for C28H24F3N3O3: C, 66.27; H, 4.77; N, 8.28;
Found C, 66.27; H, 4.78; N, 8.30.
ꢀ
grid with sides of 22.5, 24, 22.5 A long, centered on the active site of
the enzyme was used for hDHFR. The grid is large enough to cover
the active site of the enzyme. A Lamarckian genetic algorithm was
used to generate 10 bound conformations for the ligand. All single
bounds of the ligand were allowed to rotate during the calculation.
The ga_pop_size parameter was set at 150. All other setting were
kept as default. Molecular graphics and analyses were performed
with the UCSF Chimera package. Chimera is developed by the
Resource for Biocomputing, Visualization and Informatics at the
University of California, San Francisco (supported by NIGMS P41-
GM103311) [59].
4.1.3.30. 2-[N-Propargyl(2-fluoro-5-trifluoromethylphenyl)-amino-
methyl]-3-phenyl-7-trifluoromethyl quinoxaline (30). Yield:17%,
gum; UV (EtOH) lmax 323, 238, 204 nm; IR (film)
y ;
2120, 1580 cmꢀ1
1H NMR (200 MHz, CDCl3)
d
: 8.41 (1H, s, H-8), 8.24 (1H, d, J ¼ 8.8 Hz,
H-5), 7.94 (1H, dd, J ¼ 8.8 Hz and 2.0 Hz, H-6), 7.67e7.45 (5H, m, 5H
arom), 7.20e6.93 (3H, m, 3H arom), 4.80 (2H, s, CH2N), 4.09 (2H, d,
J ¼ 2.6 Hz, CH2), 2.21 (1H, t, J ¼ 2.4 Hz, CH). 13C NMR (100 MHz,
References
DMSO-d6) d: 44.5 (CH2), 46.7 (CH2), 74.1 (CH), 78.0 (C), 113.5 (CH),
117.5 (CH), 120.3 (CH), 122.1 (C), 123.0 (CH), 123.8 (2C), 126.9 (2CH),
128.0 (CH), 128.4 (CH), 129.2 (2CH), 130.2 (CH), 130.9 (C), 133.0 (C),
141.3 (C), 141.9 (C), 142.1 (C), 149.2 (C), 153.3 (C), 155.0 (C). MS: m/z
503 (Mþ) Anal. Calcd. for C26H16F7N3: C, 62.03; H, 3.20; N, 8.35.
Found C, 61.93; H, 3.22; N, 8.40.
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