2,3-Substituted quinoxalin-6-amine analogs as antiproliferatives: A structure-activity relationship study

Article abstract of DOI:10.1016/j.bmcl.2011.02.055

The quinoxaline core is considered a privileged scaffold as it is found in a variety of biologically relevant molecules. Here we report the synthesis of a quinoxalin-6-amine library, screening against a panel of cancer cell lines and a structure-activity

Full text of DOI:10.1016/j.bmcl.2011.02.055

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1929–1932
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,3-Substituted quinoxalin-6-amine analogs as antiproliferatives:
A structure–activity relationship study
Qianyi Chen a,c, , Vashti C. Bryant a,c, , Hernando Lopez a, David L. Kelly a, Xu Luo a, Amarnath Natarajan a,b,c,
⇑
a Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, United States
b Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE 68198, United States
c Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The quinoxaline core is considered a privileged scaffold as it is found in a variety of biologically relevant
molecules. Here we report the synthesis of a quinoxalin-6-amine library, screening against a panel of can-
cer cell lines and a structure–activity relationship (SAR). This resulted in the identification of a bisfura-
nylquinoxalineurea analog (7c) that has low micromolar potency against the panel of cancer cell lines.
We also show that cells treated with quinoxalineurea 7c results in caspase 3/7 activation, PARP cleavage
and Mcl-1 dependent apoptosis.
Received 8 January 2011
Revised 10 February 2011
Accepted 14 February 2011
Available online 17 February 2011
Keywords:
Ó 2011 Elsevier Ltd. All rights reserved.
Quinoxaline urea
Antiproliferative
Mcl-1 dependent apoptosis
The definition of privileged scaffold has evolved over the past
two decades from ligands for a diverse array of receptors to multi-
ple compounds with similar core structure having biological activ-
ity.1 Quinoxaline is an important class of nitrogen containing
heterocycle that is found in drugs ( Fig. 1) that aid in smoking
cessation (Varenicline), have antiglaucoma activity (Brimonidine)
and have antibacterial properties (Quinacillin).2–4 Not surprisingly,
recent high throughput screening (HTS) campaigns against a
variety of biological targets have identified quinoxaline analogs
as hits. Quinoxaline analogs have been identified as inhibitors of
heparin-induced tau fibril formation, cyclophin A, kinases (p110d
of PI3 kinase, JSP-1), phosphatases (cdc25B, MAPK phosphatase-
1) and isomerases (peptidyl-prolyl-cis–trans isomerase).5–11
Europium and Indium complexes containing substituted quinoxa-
lines show large fluorescence enhancement through ligand to
metal transfer, which has been exploited in developing imaging
agents.12 Together these studies indicate that the quinoxaline core
is a privileged scaffold.
in the onset and progression of cancer. A HTS using a fluorescence
polarization (FP) assay to identify small molecule inhibitors of the
carboxy terminus domains of BRCA1 (BRCT) resulted in the identi-
fication of a quinoxalineurea as a validated hit.10 This prompted us
to generate a focused chemical library to explore this scaffold as a
BRCT inhibitor. The library was screened using the BRCT FP assay.15
However, this did not result in the identification of either a com-
pound with improved potency or a discernable structure–activity
relationship (SAR).
Recent studies from other labs have shown that quinoxaline
analogs have antiproliferative activity with micromolar potency
against breast and prostate cancer cell lines.16,17 Based on these
studies and the privileged scaffold status of the quinoxalines we
screened our library against a panel of cancer cell lines and esti-
mated their growth inhibitory effects. We identified a compound
(7c) with anti-tumor activity against a panel of cancer cell lines.
We also show that this compound (7c) was able to induce activa-
tion of caspase 3/7, poly-ADP-ribose polymerase (PARP) cleavage
and Mcl-1 dependent apoptosis.
Our lab is focused on the identification and characterization of
small molecule inhibitors of protein–protein interactions.13,14 The
early onset of breast cancer gene 1 (BRCA1) contains multiple func-
tional domains that interact with a plethora of proteins to mediate
key signal transduction events that are critical for normal cellular
functions. Abnormalities of BRCA1 result in the dysfunction of
the corresponding signaling networks and have been implicated
⇑
Corresponding author. Tel.: +1 402 559 3793; fax: +1 402 559 8257.
E-mail address: anatarajan@unmc.edu (A. Natarajan).
These authors contributed equally to this work.
Figure 1. Drugs containing the quinoxaline core.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.055

1930
Q. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1929–1932
Scheme 1. Synthesis of 2,3-substituted quinoxalin-6-amine analogs. Reagents and conditions: (i) ethanol, reflux, 36–48 h; (ii) Pd/C, H2, ethanol, room temperature, 6–8 h;
(iii) R2NCO, DIPEA, DCM 24–72 h; (iv) R2COCl, DCM, 4 h; (v) TsCl, TEA, DCM 6 h; (vi) R5NCS (2 equiv), DCM, reflux; (vii) triphosgene, DIPEA, DCM, 4 h; amine, DCM, 8–24 h;
(viii) R6C6H4NCO (1.5 equiv), DIPEA, DCM, 12–24 h.
A small focused library with substitutions at the 2,3- and
6-positions on the quinoxaline core was envisioned to probe the
scaffold for biological activity (Scheme 1). The various analogs
and yields for the final step are summarized in Table 1. The varia-
tions at the R1 position were methyl, furan, thiophene and phenyl
groups. At the 6-position acetyl, phenylurea, and tolylsulfonamide
were explored in the 5 compound series. The compounds (5a–l)
were generated from 2,3-substituted-6-aminoquinoxaline analogs
(4, Scheme 1)11 with the amino group functionalized with acetyl
chloride, phenylisocyanate and tosylchloride respectively. The
synthesis of acetylated and phenyl urea analogs (5a–h) was
accomplished using reported methods. However reaction of the
quinoxalineamine analogs (4) with tosyl chloride yielded rear-
ranged, monosubstituted and disubstituted products depending
on the substitution at the 2,3-positions (5i–l). The amines (4a–d)
were also reacted with isothiocyanates to generate the correspond-
ing quinoxalinylthiourea analogs (6a–f). In order to generate quin-
oxaline urea compounds (6g–m) with secondary amines, 2,3-
substituted quinoxaline isocyanates were generated in situ using
triphosgene and reacted with the corresponding secondary amine.
The compounds were screened in a growth inhibition assay at
20 lM over a 72 h period in a panel of cancer cell lines (lung-
A549; pancreatic-Aspc1; colon-HT29; breast-MDAMB231; pros-
tate-PC3; ovarian-SKOV3 and bone-U2OS) and the results are
summarized in Table 2. In the 5 compound series only 5a, 5b,
and 5f inhibited growth of the various cancer cell lines. This sug-
gests that the furan substitutions at the 2,3-positions are clearly
better than the other three. It also suggests that sulfonamide sub-
stitution at the 6-position is not suitable for the growth inhibitory
activity. The screening results in the 6 compound series again show
that all the furan compounds (6a, 6d, 6h, 6k, and 6m) were active.
It also shows that substitution at the 4-position of the phenyl thio-
ureas plays a role in the biological activity (6b and 6c vs 6e and 6f).
This size effect was also observed with the urea compounds from
the secondary amine (6j vs 6l and 6k vs 6m).
Table 1
Isolated yields of 2,3-substituted quinoxalin-6-amine analogs
Entry
R1
R2
R3
R4
% Isolated yield
5a
5b
5c
5d
5e
5f
5g
5h
5i
Methyl
–COCH3
–COCH3
–COCH3
–COCH3
–C(@O)–NHPh
–C(@O)–NHPh
–C(@O)–NHPh
–C(@O)–NHPh
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
–H
64
84
77
70
83
77
70
80
65
66
69
65
2-Furanyl
2-Thienyl
Phenyl
Methyl
These results prompted us to synthesize four additional com-
pounds to probe the size effect at the 4-position on a phenyl urea
(7a–d). Evaluation of these analogs in the growth inhibition assay
clearly showed a size effect and compound 7c with bromo substi-
tution at the 4-position was identified as the best compound. A
2-Furanyl
2-Thienyl
Phenyl
Methyl
–H
–SO2Tol
–H
–H
5j
5k
5l
2-Furanyl
2-Thienyl
Phenyl
–SO2Tol
–SO2Tol
–SO2Tol
–SO2Tol
–SO2Tol
–H
dose–response study with compound 7c shows low-lM GI50 val-
–H
ues against a panel of cancer cell lines (Table 3). In summary this
iterative synthesis and screening effort show that the furan substi-
tution at the 2,3-position, a urea at the 6-position and the substi-
tuent at the para-position of a phenyl urea are important for the
biological activity. These studies also resulted in the identification
R1
R5
6a
6b
6c
6d
6e
6f
6g
6h
6i
2-Furanyl
2-Thienyl
Phenyl
2-Furanyl
2-Thienyl
Phenyl
Methyl
2-Furanyl
Phenyl
Methyl
2-Furanyl
Methyl
2-Furanyl
R1
–C(@S)–NHPh
–C(@S)–NHPh
–C(@S)–NHPh
–C(@S)–NH-(4-nitro)-phenyl
–C(@S)–NH-(4-nitro)-phenyl
–C(@S)–NH–(4-nitro)-phenyl
–CO-pyrrolidine
–CO-pyrrolidine
–CO-pyrrolidine
–C(@O)–NH-(4-benzyl)-piperidine
–C(@O)-NH-(4-benzyl)-piperidine
–CO-piperdine
70
74
75
78
80
80
55
57
40
50
53
54
80
of 7c with low-lM GI50 values against a panel of cancer cell lines.
Caspases are a class of cysteine proteinases that are activated
during apoptosis and measuring caspase activity is often used to
detect activation of apoptotic signaling. To determine if the growth
inhibitory effects observed with 7c in various cancer cell lines were
a result of programmed cell death, we explored the ability of 7c to
induce caspase-3/7. Our results show that 7c induces caspase 3/7
much more rapidly compared to the positive control (Etoposide)
in MDA-MB-231 and PC3 cells (Fig. 2A and B) and the induction
is sustained for 72 h in these cell lines.
6j
6k
6l
6m
–CO-morpholine
R6
7a
7b
7c
7d
2-Furanyl
2-Furanyl
2-Furanyl
2-Furanyl
–F
–Cl
–Br
–Phenyl
80
81
76
71
Bcl-xL, Bcl-2, and Mcl-1 are antiapoptotic proteins that are
implicated in the survival of cancer cells.18,19 Bad3SA is the endog-
enous inhibitor of Bcl-xL and Bcl-2 but not Mcl-1. Using HeLa cells

Q. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1929–1932
1931
Table 2
Screen results of the 2,3-substituted quinoxalin-6-amine analogs
Entry
% Growth inhibition at 20
MDA-MB-231
lM
A549
AsPC1
HT29
PC3
SKOV3
U2OS
5a
5b
5c
5d
5e
5f
5g
5h
5i
23.4 3.3
57.8 7.0
Inactive
Inactive
Inactive
54.6 2.6
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
18.0 0.1
Inactive
Inactive
19.2 8.9
Inactive
16.6 11.2
Inactive
23.3 2.9
11.0 1.3
40.3 1.9
93.1 7.8
Inactive
39.7 12.8
>100
26.2 2.8
Inactive
Inactive
Inactive
Inactive
9.6 6.6
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
55.5 25.4
Inactive
11.6 4.2
29.4 9.8
Inactive
>100
31.7 2.8
7.6 5.1
Inactive
Inactive
Inactive
52.1 11.6
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
26.1 15.0
Inactive
Inactive
42.0 1.9
23.9 27.8
81.2 0.7
Inactive
16.7 3.7
Inactive
16.3 10.1
55.2 4.1
Inactive
19.4 3.6
74.2 5.2
86.3 7.7
88.6 4.6
72.4 7.6
25.7 5.1
20.3 5.9
Inactive
Inactive
Inactive
31.3 10.7
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
19.1 12.5
Inactive
Inactive
9.0 4.7
53.3 7.2
72.6 0.7
Inactive
18.2 7.0
16.7 3.9
12.4 5.0
90.8 4.5
Inactive
10.9 5.0
38.4 2.7
92.5 6.6
>100
21.8 15.1
57.7 9.4
Inactive
Inactive
Inactive
70.6 1.3
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
56.8 1.4
Inactive
Inactive
43.4 9.3
19.5 37.4
81.0 8.5
Inactive
53.1 9.9
56.9 2.7
30.6 11.6
>100
4.0 3.6
9.0 3.4
Inactive
Inactive
Inactive
24.7 1.1
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
55.8 30.7
Inactive
16.9 4.9
18.5 4.3
8.9 5.1
47.6 2.5
Inactive
Inactive
Inactive
55.8 24.1
94.9 7.9
Inactive
31.4 3.6
Inactive
Inactive
Inactive
Inactive
59.6 1.6
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
21.4 8.4
Inactive
Inactive
32.2 0.1
50.6 3.9
76.1 0.4
Inactive
46.5 4.4
54.2 2.2
33.5 0.4
80.3 2.4
Inactive
Inactive
55.6 6.6
87.4 1.1
>100
5j
5k
5l
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
7a
7b
7c
7d
Inactive
Inactive
>100
81.1 2.0
>100
Inactive
48.9 7.5
90.7 4.1
86.4 1.1
>100
99.2 5.2
>100
13.8 9.8
Inactive
50.2 8.9
88.5 1.9
46.9 7.4
that over express Bad3SA, we explored the mechanistic basis for
the induction of apoptosis by 7c. In these cell lines expression of
Bad3SA is under the control of Doxycycline (Dox). The apoptosis
studies carried out in this cell line are summarized in Figure 3.
As with the other cancer cell lines we observe induction of caspase
3/7 and PARP cleavage by 7c (Fig. 3A and B, respectively). We next
carried out a dose–response study with 7c in untreated cells
Table 3
Dose–response growth inhibition study with 7c
Cell line
GI50 (lM)
A549
AsPC1
HT29
MDA-MB-231
PC3
6.4 3.0
17.3 0.9
12.1 7.4
8.4 0.9
[(À) Dox] and cells treated with Dox (1
lg/mL for 3 h) to induce
5.9 2.7
Bad3SA [(+) Dox]. The cells were incubated with 7c and a positive
control (DNA damaging agent Camptothecin, CPT) for 12 h. Cell
death was measured by counting the number of condensed nuclei.
SKOV3
U2OS
16.8 5.2
10.8 0.2
Figure 2. Induction of caspase 3/7 activity by 7c and Etoposide (a known chemotherapeutic agent) in MDA-MB-231 breast cancer cells (A) and PC3 prostate cancer cells (B).

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Q. Chen et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1929–1932
(7c) with low micromolar potency against the panel of cancer cell
lines. The analog 7c induces caspase 3/7 activation, PARP cleavage
and Mcl-1 dependent apoptosis. The molecular target of compound
7c is currently under investigation and will be reported in due
course.
Acknowledgments
We thank the Eppley NMR facility. Funding in part from the
National Institutes of Health (NIH R01CA127239 to A.N.) is
gratefully acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.055.
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between the 2,3-disubstituted quinoxaline and
a substituted
phenyl ring showed that a urea linker was optimal for the
antiproliferative activity. In addition, the size of the substituent
at the 4-position of the phenyl ring was important for the activity.
This led to the identification of a bisfuranylquinoxalineurea analog