Novel tricyclic indeno[2,1-d]pyrimidines with dual antiangiogenic and cytotoxic activities as potent antitumor agents

Article abstract of DOI:10.1016/j.bmc.2012.05.068

We designed, synthesized and evaluated 13 novel tricyclic indeno[2,1-d]pyrimidines as RTK inhibitors. These analogues were synthesized via a Dieckmann condensation of 1,2-phenylenediacetonitrile followed by cyclocondensation with guanidine carbonate to af

Full text of DOI:10.1016/j.bmc.2012.05.068

Bioorganic & Medicinal Chemistry 20 (2012) 4217–4225
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel tricyclic indeno[2,1-d]pyrimidines with dual antiangiogenic
and cytotoxic activities as potent antitumor agents
Aleem Gangjee a, , Ying Zhao a, Michael A. Ihnat b, Jessica E. Thorpe b, Lora C. Bailey-Downs b,
⇑
Roy L. Kisliuk c
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, United States
b Department of Cell Biology, School of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, United States
c Department of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed, synthesized and evaluated 13 novel tricyclic indeno[2,1-d]pyrimidines as RTK inhibitors.
Received 30 January 2012
Revised 21 May 2012
Accepted 29 May 2012
Available online 6 June 2012
These analogues were synthesized via
a Dieckmann condensation of 1,2-phenylenediacetonitrile
followed by cyclocondensation with guanidine carbonate to afford the 2-amino-3,9-dihydro-
indeno[2,1-d]pyrimidin-4-one. Sulfonation of the 4-position followed by displacement with appropri-
ately substituted anilines afforded the target compounds. These compounds were potent inhibitors of
platelet-derived growth factor receptor b (PDGFRb) and inhibited angiogenesis in the chicken embryo
chorioallantonic membrane (CAM) assay compared to standards. In addition, compound 7 had a two digit
nanomolar GI50 against nine tumor cell lines, a submicromolar GI50 against 29 of other tumor cell lines in
the preclinical NCI 60 tumor cell line panel. Compound 7 also demonstrated significant in vivo inhibition
of tumor growth and angiogenesis in a B16-F10 syngeneic mouse melanoma model.
Keywords:
Indeno[2,1-d]pyrimidines
Receptor tyrosine kinase inhibitors
Antiangiogenic agents
Antitumor agents
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
and angiogenesis5 Dysfunctional, hyperactive growth factor RTKs
have been associated with several tumors and play a pivotal role
Angiogenesis is the formation of new blood vessels from pre-
existing vasculature.1 Based on Folkman’s seminal observation,2
in order to grow beyond a few millimeters in diameter, solid tu-
mors depend on angiogenesis for the transport of nutrients and re-
moval of metabolite waste from tumor cells. It is now well
established that angiogenesis plays a key role in the growth of solid
tumors, tumor invasion and metastasis.3 Angiogenesis is primarily
a receptor-mediated process by growth factors that cause signal
transduction, for the most part, via receptor tyrosine kinases
(RTK). These RTK, including platelet-derived growth factor receptor
(PDGFR), fibroblast growth factor receptor (FGFR), vascular endo-
thelial growth factor receptor (VEGFR), insulin-like growth factor
receptor (IGFR) and epidermal growth factor receptor (EGFR)
among several others.4 The catalytic tyrosine kinase domain of
RTKs contains binding sites for both ATP and substrates, allowing
for autophosphorylation, which is critical for signal transduction
in tumor angiogenesis.5,6 Abrogation of angiogenesis via RTK inhi-
bition provides a viable approach for the treatment of cancer.7
In the early stage of RTK inhibitor development, the majority of
the effort was focused on targeting a single RTK by small mole-
cules. Examples of such clinically used agents include gefitinib
(specific EGFR inhibitor; approved for limited use for the treatment
of non small cell lung cancer)8 and erlotinib (specific EGFR inhibi-
tor; approved for the treatment of non small cell lung cancer)
(Fig. 1).9 Since there are redundant signaling pathways for angio-
genesis, tumors often survive through alternative signaling path-
ways and develop resistance to agents that target single RTK.
Currently, the paradigm for RTK inhibitors in cancer chemotherapy
is the inhibition of multiple, rather than single, RTKs to block po-
tential ‘escape routes’ from single RTK inhibition.10 Clinical studies
have recently shown that the inhibition of multiple kinases either
by single-agents or with combinations of two or more agents have
the potential to increase antitumor activity. Sunitinib (SU11248,
inhibiting PDGFR, VEGFR, Kit, Fms-related tyrosine kinase 3)
(Fig. 1) was the first multitargeted RTK inhibitor approved by
FDA for the treatment of renal cell carcinoma (RCC) and imatinib
resistant gastrointestinal stromal tumor (GIST).11 Sorafenib
(Fig. 1) is another multi-targeted inhibitor of PDGF, VEGFR-2 and
-3 kinases and is approved by the FDA for the treatment of
advanced hepatocellular carcinoma (primary liver cancer) and
Abbreviations: RTK, receptor tyrosine kinase; PDGFRb, platelet-derived growth
factor receptor b; FGFR, fibroblast growth factor receptor; VEGFR, vascular
epidermal growth factor receptor; IGFR, insulin growth factor receptor; EGFR,
epidermal growth factor receptor; CAM, chicken embryo chorioallantonic mem-
brane; PCC, Pearson correlation coefficients; DHFR, dihydrofolate reductase;
DHODH, dihydroorotate dehydrogenase.
⇑
Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396 5593.
E-mail address: gangjee@duq.edu (A. Gangjee).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.068

4218
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
CH3
CH3
N
O
F
H3C
NH
HN
HN
Cl
O
O
O
CH3
N
N
H
N
OCH3
OCH3
N
F
O
N
O
N
OCH3
N
H
Sunitinib (SU11248)
Gefitinib
Erlotinib
H
H
N
N
CF3
Cl
H3C
O
O
N
Br
NH
CH3
N
OCH3
OCH3
H
N
H
N
O
N
H
CH3
N
O
Semaxanib (SU5416)
18 (PD153035)
Sorafenib
COOH
H3C
CH3
NC
HN
O
CF3
N
NH3
NH3
Cl
Cl
CH3
Pt
CH3
N
H3C
H
O
OH
O
N
H
N
H
19 (SU4312)
20 (A77-1726)
Figure 1.
21 (SU6668)
Cisplatin
renal cell carcinoma (primary kidney cancer).12 RTK inhibitors are
generally cytostatic against tumors.13–18 Thus the combination of
RTK inhibitors with standard cytotoxic chemotherapeutic agents
is the subject of several clinical trials to improve long-term sur-
vival in cancer patients. We19 reasoned that the combination of
RTK inhibition along with cytotoxic activity in single molecules
would provide single agent(s) with ‘combination chemotherapeu-
tic potential.’ Such agents would have both cytotoxic and antian-
giogenic activity. These single agents would be tumoricidal and
may have much lower cytotoxic activity and hence toxicity than
a chemotherapeutic agent used in combination with a RTK inhibi-
tor. In addition, such single agents could circumvent or delay the
emergence of resistance and simplify the pharmacokinetics and
toxicity issues compared to two or more separate agents.19
increase in inhibitory activities over those reported by Showalter
et al.20 We22 have shown that inclusion of the 2-NH2 moiety does,
in some cases, result in better inhibitory activity for RTK. In this
study we report novel tricyclic indeno[2,1-d]pyrimidines 1–13
(Fig. 2) with different anilino substitutions at the 4-position of
the pyrimidine ring. The anilino ring of the molecule is expected
to be involved in RTK binding at the Hydrophobic Region 121 and
influence inhibition, selectivity as well as the antitumor activity.
Thus, the variations in the anilino ring include electron donating
and withdrawing groups.
2. Chemistry
The synthesis of the target compounds was accomplished as
shown in Scheme 1. 2-Oxo-indan-1-carboxylic acid ethyl ester
15 was obtained by hydrolysis of 1,2-phenylenediacetonitrile 14
with conc. H2SO4 followed by Dieckmann condensation (42%
yield for two steps). Reaction of 15 with guanidine in the pres-
ence of potassium t-butoxide at 150 °C in a microwave reactor
gave 16 (43% yield). The usual synthetic methodology for similar
4-anilino substituted compounds is to chlorinate the 4-oxo moi-
ety of 16 to the corresponding 4-chloro followed by displacement
with appropriate anilines.21 However, several attempts at chlori-
nation of 16 with POCl3 with variation in time and temperature,
even at reflux, were unsuccessful. This failure of chlorination
could be attributed to the lower aromaticity of the tricyclic ring
system of 16. Thus, the 4-oxo was first converted to the 4-sulfo-
nate which is a better leaving group for SNAr reactions. Treat-
ment of 16 with 4-nitrobenzensulfonyl chloride afforded 17
(36% yield). Nucleophilic displacement of 17 with the appropriate
anilines at reflux for 12–18 h afforded target compounds 1–13
(yields 18% to 59%).
In 1999, Showalter et al.20 reported that tricyclics with a third
ring fused to the 6,7-positions of general bicyclic RTK inhibitors
could moderately enhance RTK inhibitory activity over the parent
compounds. We have shown that similar tricyclic molecules could
bind to VEGFR-2 as well as to PDGFRb19 We hypothesized that the
introduction of a 2-NH2 group on the pyrimidine ring could form
additional hydrogen-bonds in the Hinge region19,21 with the back-
bone carbonyl of the ATP binding sites to perhaps afford an
8
1
R = 3-Br
R = H
R
9
R = 4-Br
2 R = 2-CH(CH3)2
10
11
3
4
R = 4-CF3
R = 3-OCH3
R = 4-CH(CH3)2
R = 3-F
6
8
HN
5
4
7
3
12 R = 4-F, 3-Cl
5 R = 4-F
N
13
6
7
R = 2-F, 4-Cl
R = 3-Cl
R = 4-Cl
2
H2N
N
9
1
Figure 2. Target compounds.

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
4219
NO2
O
S
O
a
b
c
CN
CN
O
O
N
O
HN
H2N
N
COOEt
N
H2N
14
15
16
17
R
HN
d
N
H2N
N
1-13
Scheme 1. Reagents and conditions: (a) (1) Conc. H2SO4, EtOH, reflux; (2) toluene, Na, reflux, 3 h, (42% for 2 steps); (b) guanidine carbonate, t-butanol, potassium t-butoxide,
lW, 4 h (34%); (c) 4-nitrobenzenesulfonylchloride, DMAP, Et3N, dichloromethane, rt (36%); (d) 1,4-dioxane, substituted aniline, reflux, 12–18 h (18–59%).
than erlotinib and 4.5-fold more potent than the standard PDGFRb
inhibitor, 19. The 2-fluoro, 4-chloro analogue 13 was 23-fold more
potent than sunitinib, 4-fold more potent than erlotinib and equi-
potent with the standard 19. Compounds 3–5 and 12 were about
10-times more potent than sunitinib, 1.5-fold more potent than
erlotinib and one half as potent as the standard 19. In general,
these modifications indicate that an electron-withdrawing group
at the 4-position (except 4-CF3) is preferred for PDGFRb activity
and an electron-donating group is detrimental for inhibitory activ-
ity. The 4-position is the optimum position for substitution for
PDGFRb inhibition (5, 7 and 13).
Cytotoxicity studies against the growth of A431 cells in culture
provided interesting results. The standard compound, cisplatin, is a
cytotoxic chemotherapeutic agent and not a kinase inhibitor. In the
current study, the most potent analogue 1 was 2-fold more potent
than the standard, cisplatin. Compounds 4, 7, 11 and 13 were equi-
potent against A431 compared with cisplatin. In keeping with the
fact that EGFR is overexpressed in A431 cells, compounds 7 and 13
were also potent against EGFR kinase, however 4 and 11 do not
show potent inhibition of EGFR kinase. That the IC50 values for
A431 cytotoxicity are lower than for EGFR kinase suggests that
there could be other mechanism(s) which could account for the po-
tent inhibitory activity of these compounds against A431 cells in
culture.11
3. Biological evaluation and discussion
The RTK inhibitory activities of the compounds were deter-
mined using human tumor cells known to express high levels of
EGFR, VEGFR-2 and PDGFRb, respectively using ELISA assay.
Cytotoxicity studies against the growth of A431 cells, which over-
express EGFR, in culture, were also carried out for these com-
pounds. In addition, the effect of 1–13 on blood vessel formation
(angiogenesis) was assessed using the chicken embryo chorioallan-
tonic membrane (CAM) assay, a standard test for angiogenesis.
Since the IC50 values of compounds vary under different assay
conditions, we used a standard (control) compound in each of
the evaluations. Compound 18 along with erlotinib (Fig. 1) were
used as standards for EGFR inhibition. (Z)-3-[4-(dimethyl-
amino)benzylidenyl]indolin-2-one (SU4312, Fig. 1) 19, sunitinib
and erlotinib were used as standards for PDGFRb inhibition.
Semaxanib, sunitinib and erlotinib (Fig. 1) were used as standards
for VEGFR-2 inhibition, and semaxanib for the CAM assay, and
cisplatin (Fig. 1) was used as a standard for the A431 cytotoxicity
assay. The results are reported in Table 1. We have used whole cell
assays in preference to isolated enzyme assays since whole cell
assay results are usually more amenable to translation to in vivo
studies than isolated enzyme results.
In the EGFR assay (Table 1), all analogues had comparatively
lower or no inhibitory activity compared to the standard 18.
Among these analogues, the most potent compound was the 2-F,
In the CAM angiogenesis assay, 7 was the most potent compound
with an IC50 value of 0.8 lM. The next most potent compounds were
2 and 13. These three analogues 2, 7 and 13 had no inhibitory activ-
ity against VEGFR-2, the principle mediator of angiogenesis, how-
ever 7 and 13 were the most potent analogues against PDGFRb
which has also shown importance in angiogenesis.23
4-Cl analogue 13 (IC50 = 11.7 lM) and it was about 50-fold less po-
tent than the standard 18. The 4-Cl analogue 7 was comparable to
13 and both analogs were the most potent of the series in the A431
assay.
Some of the analogues demonstrated dual RTK inhibitory activ-
ity. Compounds 3–5 all had good VEGFR-2 and PDGFRb activity.
Compound 7 was chosen by the National Cancer Institute24 for
evaluation in its preclinical 60 tumor cell line panel. Compound 7
had a two digit nanomolar GI50 against nine tumor cell lines, a sub-
micromolar GI50 against twenty nine of the other tumor cell lines
and micromolar GI50 against 16 tumor cell lines (Table 2). These re-
sults indicate selectivity for tumors within a class. For example,
compound 7 was 8- to 10-fold more potent against CCRF-CEM
and MOLT-4 leukemia cells than against other leukemia cells in
culture (Table 2).
Against VEGFR-2 the most potent compound was the 4-isopro-
pyl analogue 3, and it was equipotent with the standard, semaxa-
nib and better than sunitinib and erlotinib. The smaller 3-fluoro
(electron-withdrawing group), 4 was 4.5-fold less potent than
semaxanib and also less potent than sunitinib but more potent
than erlotinib. Larger halogen atoms such as chlorine or bromine
at the 3-position resulted in a lower, or loss of, inhibitory activity
against VEGFR-2. Electron-donating substituent (3-OCH3) in 11
was 4-fold less potent than semaxanib, about 2-fold less potent
than sunitinib but 3-fold better than erlotinib. Large hydrophobic
groups at the 4-postion (3 and 9) were most conducive to VEG-
FR-2 inhibition.
Since RTK inhibition in most instances is only a cytostatic effect,
it is interesting that compound 7 showed potent cytotoxic activity
against the NCI 60 tumor cell lines. The mechanism of action of the
potent tumor cytotoxicity of 7 must be due to mechanism(s) in
Six of the analogues showed excellent activity against PDGFRb.
The 4-chloro analogue 7 was the most potent compound and was
about 100-fold more potent than sunitinib, 15-fold more potent

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A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
Table 1
IC50 values (lM) of kinase inhibition, A431 cytotoxicity and inhibition of the CAM assay
R
HN
N
H2N
N
R
EGFR kinase
VEGFR-2 kinase
PDGFRb kinase
A431 cytotoxicity
CAM angiogenesis
1
2
3
4
5
6
7
8
H
>200
>200
42.6 5.8
>300
276.1 30.2
19.6 2.7
26.0 3.2
24.1 3.1
>300
>300
>200
>200
11.7 2.1
0.23 0.03
171.2 18.0
>200
43.1 4.7
21.7 2.6
7.8 0.9
7.1 1.0
5.9 0.7
>500
0.8 0.08
126.3 15.0
38.9 4.0
23.1 0.38
75.5 9.1
6.4 0.7
3.6 0.6
4.9 0.7
50.8 6.7
2.12 0.18
14.8 1.6
78.3 8.3
46.7 5.9
ND
0.82 0.09
104.3 14.2
19.1 2.0
8.1 1.4
36.4 3.9
ND
2-CH(CH3)2
4-CH(CH3)2
3-F
4-F
3-Cl
4-Cl
3-Br
4-Br
4-CF3
3-OCH3
4-F,3-Cl
2-F, 4-Cl
126.4 18.0
126.4 13.9
9.8 1.3
50.3 7.0
235.0 29.1
9.7 1.1
11.0 1.8
55.7 7.0
89.3 9.2
133.9 16.8
>200
>200
30.1 4.8
>200
>500
9
103.1 14.8
306.3 40.1
13.9 1.6
158.1 17.9
9.7 0.8
10
11
12
13
18
19
43.0 5.9
72.9 0.9
197.1 2.6
3.67 0.4
3.75 0.06
Semaxanib
Sunitinib
Erlotinib
Cisplatin
12.9 2.7
18.9 2.7
124.7 18.2
0.04 0.009
172.1 19.4
1.2 0.2
83.1 10.1
12.2 1.9
10.6 2.9
ND. Not determined.
Table 2
Tumor cell line inhibitory activity GI50 (nM) of 7(NCI).
Panel/cell line
GI50 (nM)
Panel/cell line
GI50 (nM)
Panel/cell line
GI50 (nM)
Panel/cell line
GI50 (nM)
Leukemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
NSCLC
A549/ATCC
EKVX
Colon Cancer
COLO 205
HCT-116
HCT-15
KM12
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
Melanoma
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROVI
Renal Cancer
786 - 0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK10
UO-31
Breast Cancer
MCF7
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
BT-549
31.2
150
366
66.1
489
484
638
38.1
434
330
63.0
308
>10000
431
558
1510
513
514
2490
3420
1080
2910
189
86.2
547
381
113
5030
184
84.5
641
2150
790
43.4
2710
654
18100
2140
565
1350
19.3
622
HOP-62
HOP-92
287
216
2090
216
2090
73.4
787
641
2220
554
2830
2900
OVCAR-3
OVCAR-4
OVCAR-8
SK-OV-3
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
U251
Prostate Cancer
PC-3
715
212
DU-145
T-47D
addition to its RTK inhibitory activity, since most tumor cells in
culture are not angiogenic. Thus, the NCI COMPARE analysis25
was performed for 7 to elucidate possible mechanism(s) of action
of 7 by the similarity responses of the 60 cell lines to known com-
pounds. The five compounds whose cell type selectivity profile
showed the highest Pearson correlation coefficients (PCC)26 (Table
3) with 7 were all well known dihydrofolate reductase (DHFR) or
dihydroorotate dehydrogenase (DHODH) inhibitors. Thus it was
necessary to evaluate 7 and selected analogues for inhibitory activ-
ities against DHFR and DHODH.
Table 3
COMPARE analysis data for compound 7
Drug
Correlation coefficient
Biphenquinate (DHODH inhibitor)
Trimetrexate (DHFR inhibitor)
Pyrazofurin (OMP decarboxylase inhibitor)
Acivicin (
Triazinate (DHFR inhibitor)
0.745
0.716
0.682
0.645
0.635
c-glutamyl transpeptidase inhibitor)
Compounds 3, 7, 8, 12 and 13 were evaluated against DHFR27
and thymidylate synthase (TS)28 from human, Eshcerichia coli
(E. coli) and Toxoplasma gondii (T. gondii) and the results are in-
cluded in Table 4. None of these compounds showed activity
against either DHFR or TS. The IC50 values of these compounds
dium falciparum (Pf).29 None of the analogues inhibited hDHODH or
PfDHODH at IC50 less than 200 M compared to the standard
compound N-(4-trifluoromethylphenyl)-2-cyano-3-hydroxycro-
tonamide-2-cyano-3-hydroxy-N-[4-(trifluoromethyl)phenyl]-2-
butenamide (A77-1726, Fig. 1) 20 (data not shown). Thus the cyto-
toxic mechanism of action of 7 is currently under investigation.
l
were all greater than 10
lM. Compounds 1, 4, 7, 8, 11 and 13 were
also evaluated as inhibitors of DHODH from human (h) and Plasmo-

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
4221
Table 4
IC50 value (M) of DHFR and TS inhibition for selected compounds
DHFR (IC50 M)
Human
TS (IC50 M)
Human
E. coli
T. gondii
E. coli
T. gondii
3
7
8
12
13
MTX
Trimethoprim
Pyrimethamine
PDDF
Raltitrexed
Pemetrexed
>1.6 Â 10À5
>1.6 Â 10À5
>1.4 Â 10À5
>1.6 Â 10À5
>1.6 Â 10À5
2.0 Â 10À8
>3.4 Â 10À4
6.0 Â 10À6
>1.6 Â 10À5
>1.6 Â 10À5
>1.4 Â 10À5
>1.6 Â 10À5
>1.6 Â 10À5
8.8x10À9
>1.6 Â 10À5
>1.6 Â 10À5
>1.4x10À5
>1.6 Â 10À5
>1.6 Â 10À5
3.3 Â 10À8
6.8 Â 10À6
2.0 Â 10À7
>1.4 Â 10À5
>1.4 Â 10À5
>1.2 Â 10À5
>1.3 Â 10À5
>1.3 Â 10À5
>1.4 Â 10À5
>1.4 Â 10À5
>1.2 Â 10À5
>1.3 Â 10À5
>1.3 Â 10À5
>1.4 Â 10À5
>1.4 Â 10À5
>1.2 Â 10À5
>1.3 Â 10À5
>1.3 Â 10À5
1.0 Â 10À8
6.6 Â 10À6
8.5 Â 10À8
2.9 Â 10À7
2.9 Â 10À5
1.9 Â 10À8
2.3 Â 10À6
1.5 Â 10À5
4.3 Â 10À7
4.8 Â 10À7
1.4 Â 10À5
Compound 7 with its promising in vitro PDGFRb inhibitory
activity, CAM assay results and results from the preclinical NCI
60 tumor cell line panel, was evaluated in vivo for the tumor
growth inhibition, antiangiogenic effects and metastasis of primary
B16-F10 mouse melanoma tumor cells in athymic mice at a dose of
35 mg/kg 3Â weekly (M, W, F) for two weeks. (Z)-3-(2,4-Dimethyl-
5-(2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-1H-pyrrol-3-yl)-
propionic acid 21 (Fig. 1), a multi-targeted RTK inhibitor, was used
as a standard. Compound 7 was equipotent with the standard 21
both in decreasing the growth rate of B16-F10 tumor cells
(Fig. 3A) and inhibiting tumor angiogenesis (Fig. 3B). However, un-
like standard 21, compound 7 did not decrease tumor metastasis
activity compared to control (Fig. 3C).
In summary a novel series of 4-anilino substituted tricyclic in-
deno[2,1-d]pyrimidines 1–13 were synthesized and biologically
evaluated. Most of the analogues showed potent inhibitory activ-
ity against PDGFRb compared to a standard 19. Compound 7 was
about 4.5-fold more potent against PDGFRb than 19. Compound 3
was equipotent with the standard semaxanib against VEGFR-2.
Compound 3 was also a dual inhibitor of VEGFR-2 and PDGFRb.
Some analogues (1, 4, 7, 11, and 13) exhibited potent A431 cyto-
toxicity as well, compared to the standard, cisplatin. In addition,
compound 7 inhibited most of the NCI 60 tumor cell lines with
GI50s at nanomolar to micromolar levels. Thus, compound 7 pos-
sess both antiangiogenic and cytotoxic activity and could be used
alone or in combination in cancer chemotherapy. The cytotoxic
mechanism(s) of action of 7 is currently under investigation.
The in vivo results showed that 7 at 35 mg/kg decreased tumor
growth rate and inhibited angiogenesis similar to the standard
compound 21 and much more than in the untreated control
animals.
4. Experimental
4.1. General methods for synthesis
All evaporations were carried out in vacuo with a rotary evapo-
rator. Analytical samples were dried in vacuo (0.2 mm Hg) in an
Abderhalden drying apparatus over P2O5. Thin-layer chromatogra-
phy (TLC) was performed on silica gel plates with fluorescent indi-
cator. Spots were visualized by UV light (254 and 365 nm). All
analytical samples were homogeneous on TLC in at least two dif-
ferent solvent systems. Purification by column and flash chroma-
tography was carried out using Merck silica gel 60 (200–
400 mesh). The amount (weight) of silica gel for column chroma-
tography was in the range of 50–100 times the amount (weight)
of the crude compounds being separated. Columns were dry
packed unless specified otherwise. Solvent systems are reported
as volume percent mixture. Melting points were determined on a
Mel-Temp II melting point apparatus and are uncorrected. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded on
a Bruker WH-300 (300 MHz) spectrometer. The chemical shift (d)
values are reported as parts per million (ppm) relative to tetra-
methylsilane as internal standard; s = singlet, d = doublet, t = trip-
let, q = quartet, m = multiplet, br s = broad singlet, exch = protons
exchangeable by addition of D2O. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA. Elemental compo-
sitions were within 0.4% of the calculated values. Fractional
moles of water or organic solvents frequently found in some ana-
lytic samples could not be removed despite 24 h of drying in vacuo
and were confirmed, where possible, by their presence in the 1H
NMR spectrum. The purity of some final compounds was deter-
mined by UPLC/UV/ELSD/MS (see supporting information for
Figure 3. Growth rate of primary B16–F10 mouse melanoma tumor cells in athymic mice (A) and inhibition of tumor angiogenesis (B) and metastasis (C) in response to 21
and 7 given at 35 mg/kg 3Â weekly (M, W, F). ⁄⁄⁄ = P <0.001, ⁄⁄P <0.05 by one-way ANOVA and Neuman-Keuls post-test. N = 7–10. Average metastasis in untreated group-2.5/
lobe.

4222
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
UPLC/UV/ELSD/MS method). Average purity of UV and ELSD was
>95%.30 All solvents and chemicals were purchased from Aldrich
Chemical Co. and Fisher Scientific and were used as received ex-
cept anhydrous solvents which were freshly dried in the
laboratory.
4.5. General procedure for the synthesis of compounds 1–13
To a 50 mL round-bottom flask was added 17, the appropriate
substituted aniline and anhydrous 1,4-dioxane (10 mL), the mix-
ture was heated to reflux for 12–18 h. To the resulting solution
was added silica gel, and the solvent was evaporated to afford a
plug. The silica gel plug obtained was load onto a silica gel column
and eluted with 2:1 hexane/chloroform. Fractions containing the
product (TLC) were pooled, and the solvent was evaporated to af-
ford pure compound.
4.2. Ethyl 2-oxo-2,3-dihydro-1H-indene-1-carboxylate (15)
1,2-Phenylenediacetonitrile (3.0 g, 2 mmol) was dissolved in
ethyl alcohol (5 mL) and conc. sulfuric acid (2 mL) in a 25 mL
round bottom flask. The mixture was stirred and heated to reflux
for 6 h. After neutralization with ammonium hydroxide in the
cold, the reaction solution was extracted with ethyl acetate
(3 Â 50 mL). The organic phase was combined and dried with
anhydrous Na2SO4. Evaporation of ethyl acetate afforded a yellow
liquid. Without further purification, the yellow liquid was diluted
in toluene (100 mL) in a 250 mL round bottom flask and then to
the solution was added sodium metal (0.46 g, 2 mmol). The mix-
ture was heated to reflux for 4 h. After the reaction solution was
neutralized with dilute hydrochloric acid, the resulting solution
was extracted with ethyl acetate (3 Â 50 mL). The organic phase
was combined and dried with anhydrous Na2SO4. Concentration
of ethyl acetate afforded a brown solid. To this residue was
added silica gel (3 g) and ethyl acetate (30 mL), and the solvent
was evaporated to afford a plug. The silica gel plug obtained
was load onto a silica gel column and eluted with 10:1 hex-
ane/acetyl acetate. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford 1.64 g (42%
over two steps) of 15 as a white solid: TLC Rf 0.72 (Hexane/
EtOAc, 5:1); mp 59–61 °C; 1H NMR (DMSO-d6) d 3.64 (s, 2H,
Ar–CH2CO), 4.43 (q, 2H, OCH2), 7.60 (m, 4H, Ar-H), 11.0 (br s,
1H, OH, exch).
4.6. N4-Phenyl-9H-indeno[2,1-d]pyrimidine-2,4-diamine (1)
Compound 1 was synthesized from 17 (0.05 g, 0.13 mmol) and
aniline (24 mg, 0.26 mmol) using the general procedure described
above to afford after purification 13.8 mg (37%) as a light brown so-
lid: TLC Rf 0.37 (CHCl3/CH3OH, 10:1); mp: 215.3–216.9 °C; 1H NMR
(DMSO-d6) d 3.72 (s, 2H, CH2), 6.41 (br s, 2H, NH2, exch), 7.03–7.89
(m, 9H, Ph-H), 8.22 (s, H, NH, exch). HRMS (EI) calcd. for C17H14N4
274.1218, found, 274.1218.
4.7. N4-(2-Isopropylphenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (2)
Compound 2 was synthesized from 17 (0.1 g, 0.26 mmol) and 2-
isopropyl aniline (70 mg, 0.52 mmol) using the general procedure
described above to afford after purification 15 mg (18%) as a light
brown solid: TLC Rf 0.43 (Et3N/EtOAc/Hex, 1:3:5); mp 193.4–
195.3 °C; 1H NMR (DMSO-d6) d 1.14–1.16 (d, J = 6 Hz, 6H, 2CH3),
3.10–3.24 (m, H, CH), 3.70 (s, 2H, CH2), 6.13 (br s, 2H, NH2, exch),
7.11–7.93 (m, 8H, Ph-H), 7.91 (s, H, NH, exch). HRMS (EI) calcd
for C20H21N4 317.1766; found, 317.1751.
4.3. 2-Amino-3H-dihydro-indeno[2,1-d]pyrimidin-4(9H)-one
4.8. N4-(4-Isopropylphenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
(16)
diamine (3)
Compound 15 (0.1 g, 0.49 mmol), guanidine hydrochloride
(0.05 g, 0.52 mmol) and potassium t-butoxide (0.12 g, 1.1 mmol)
were dissolved in t-butanol (5 mL). The condition of the microwave
reaction was 150 °C for 4 h. The solid was filtered and washed with
methanol. To the filtrate was added silica gel (300 mg), and the sol-
vent was evaporated to afford a plug. The silica gel plug obtained
was load onto a silica gel column and eluted with 10:1 chloro-
form/methanol. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford 23 mg (34%) of
16 as a light yellow solid: TLC Rf 0.56 (CHCl3/CH3OH, 5:1); mp:
ꢀ330 °C (dec.); 1H NMR (DMSO-d6) d 3.65 (s, 2H, CH2), 6.71 (s,
2H, NH2, exch), 7.0–7.67 (m, 4H, Ar-H), 10.93 (br s, 1H, NH, exch).
Anal. (C11H9N3O.0.1H2O): C, H, N.
Compound 3 was synthesized from 17 (75 mg, 0.2 mmol) and 4-
isopropyl aniline (54 mg, 0.4 mmol) using the general procedure
described above to afford after purification 36.4 mg (59%) as a light
brown solid: TLC Rf 0.30 (CHCl3/CH3OH, 10:1); mp 178.5–180.4 °C;
1H NMR (DMSO-d6) d 1.16–1.18 (d, J = 6 Hz, 6H, 2CH3), 2.64–2.87
(m, H, CH), 3.72 (s, 2H, CH2), 6.30 (br s, 2H, NH2, exch), 7.16–7.84
(m, 8H, Ph-H), 8.10 (s, H, NH, exch). HRMS (EI) calcd for C20H20N4
316.1688; found, 316.1704.
4.9. N4-(3-Fluorophenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (4)
Compound 4 was synthesized from 17 (0.15 g, 0.39 mmol) and
3-fluoroaniline (87 mg, 0.78 mmol) using the general procedure
described above to afford after purification 15 mg (13%) as a light
brown solid: TLC Rf 0.42 (CHCl3/CH3OH, 10:1); mp 198.7–
200.1 °C; 1H NMR (DMSO-d6) d 3.78 (s, 2H, CH2), 6.56 (br s, 2H,
NH2, exch), 7.11–7.89 (m, 8H, Ph-H), 8.37 (s, H, NH, exch). HRMS
(EI) calcd. for C17H13FN4 292.1124; found, 292.1123.
4.4. 2-Amino-9H-indeno[2,1-d]pyrimidin-4-yl 4-
nitrobenzenesulfonate (17)
A solution of 16 (0.3 g, 1.5 mmol), triethylamine (0.42 mL,
3 mmol), DMAP (20 mg) and 4-nitrobenzenesulfonyl chloride
(0.67 g, 3 mmol) in dichloromethane (40 mL) was stirred at room
temperature for 4 h. To this solution was added silica gel (1.5 g),
and the solvent was evaporated to afford a plug. The silica gel plug
obtained was load onto a silica gel column and eluted with 2:1
hexane/chloroform. Fractions containing the product (TLC) were
pooled, and the solvent was evaporated to afford 0.21 g (36%) of
17 as a yellow solid: TLC Rf 0.47 (CHCl3/CH3OH, 10:1); mp:
ꢀ189.6 °C (dec); 1H NMR (DMSO-d6) d 3.92 (s, 2H, CH2), 7.18 (br
s, 2H, NH2, exch), 7.15–7.64 (m, 4H, Ph-H), 8.40–8.60 (m, 4H, 4-
NO2-Ph-H). HRMS (EI) cacd for C17H13N4O5S 385.0607; found,
385.0584.
4.10. N4-(4-Fluorophenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (5)
Compound 5 was synthesized from 17 (0.12 g, 0.3 mmol) and 4-
fluoroaniline (67 mg, 0.6 mmol) using the general procedure de-
scribed above to afford after purification 46 mg (50%) as a light
brown solid: TLC Rf 0.54 (CHCl3/CH3OH, 10:1); mp 204.7–
205.8 °C; 1H NMR (DMSO-d6) d 3.72 (s, 2H, CH2), 6.38 (br s, 2H,
NH2, exch), 7.11–7.89 (m, 8H, Ph-H), d 8.19 (s, H, NH, exch). Anal
(C17H13FN4. 0.4H2O): C, H, N, F.

A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
4223
4.11. N4-(3-Chlorophenyl)-9H-indeno[2,1-d] pyrimidine-2,4-
diamine (6)
4.17. N4-(3-Chloro-4-Fluorophenyl)-9H-indeno[2,1-
d]pyrimidine-2,4-diamine (12)
Compound 6 was synthesized from 17 (0.1 g, 0.26 mmol) and 3-
Compound 12 was synthesized from 17 (0.12 g, 0.31 mmol) and
3-chloro-4-fluoroaniline (0.09 g, 0.62 mmol) using the general pro-
cedure described above to afford after purification 53 mg (52%) as a
light brown solid: TLC Rf 0.43 (CHCl3/CH3OH, 10:1); mp 231.7–
232.8 °C; 1HNMR (DMSO-d6) d 3.74 (s, 2H, CH2), 6.47 (br s, 2H,
NH2, exch), 7.14–8.02 (m, 7H, Ph-H), 8.28 (s, H, NH, exch). HRMS
(EI) calcd. for C17H12ClFN4 326.0735, found, 326.0744.
chloroaniline (66 mg, 0.52 mmol) using the general procedure de-
scribed above to afford after purification 23 mg (35%) as a light
brown solid: TLC Rf 0.65 (CHCl3/CH3OH, 10:1); mp 224.1–
225.2 °C; 1H NMR (DMSO-d6) d 3.73 (s, 2H, CH2), 6.48 (br s, 2H,
NH2, exch), 7.06–7.84 (m, 8H, Ph-H), 8.32 (s, H, NH, exch). HRMS
(EI) calcd. for C17H13ClN4 308.0829; found, 308.0838.
4.12. N4-(4-Chlorophenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (7)
4.18. N4-(4-Chloro-2-fluorophenyl)-9H-indeno[2,1-
d]pyrimidine-2,4-diamine (13)
Compound 7 was synthesized from 17 (0.10 g, 0.26 mmol) and
4-chloroaniline (0.07 g, 0.78 mmol) using the general procedure
described above to afford after purification 30 mg (38%) as a light
brown solid: TLC Rf 0.33 (CHCl3/CH3OH, 10:1); mp 215.9–
216.8 °C; 1H NMR (DMSO-d6) d 3.76 (s, 2H, CH2), 6.50 (br s, 2H,
NH2, exch), 7.14–7.92 (m, 8H, Ph-H), 8.33 (s, H, NH, exch). Anal
(C17H13ClN4): C, H, N, Cl.
Compound 13 was synthesized from 17 (50 mg, 0.59 mmol) and
4-chloro-2-fluoroaniline (0.06 mL) using the general procedure de-
scribed above to afford after purification 15 mg (18%) as a light
brown solid: TLC Rf 0.58 (Et3N/EtOAc/Hex, 1:3:5); mp 234.7–
235.7 °C; 1H NMR (DMSO-d6) d 3.98 (s, 2H, CH2), 6.37 (br s, 2H,
NH2, exch), 7.15–7.86 (m, 7H, Ph-H), 8.16 (s, H, NH, exch). Anal.
(C17H12ClFN4): C, H, N, Cl, F.
4.13. N4-(3-Bromophenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (8)
5. General methods for biological evaluations
5.1. Receptor tyrosine kinase evaluations
Compound 8 was synthesized from 17 (0.1 g, 0.26 mmol) and 3-
bromoaniline (89 mg, 0.52 mmol) using the general procedure de-
scribed above to afford after purification 46 mg (50%) as a light
brown solid: TLC Rf 0.47 (Et3N/EtOAc/Hex, 1:3:5); mp 233–
236 °C; 1H NMR (DMSO-d6) d 3.76 (s, 2H, CH2), 6.54 (br s, 2H,
NH2, exch), 7.15–7.94 (m, 8H, Ph-H), 8.35 (s, H, NH, exch). HRMS
(EI) calcd. for C17H14BrN4 353.0402; found, 353.0387.
5.1.1. Cells
All cells were maintained at 37 °C in a humidified environment
containing 5% CO2 using media from Mediatech (Hemden, NJ). A-
431 cells were from the American Type Tissue Collection (Manas-
sas, VA).
5.1.2. Chemicals
4.14. N4-(4-Bromophenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
All growth factors (VEGF, EGF, and PDGF-BB) were purchased
from Peprotech (Rocky Hill, NJ). Compound 18 and semaxanib
were purchased from Calbiochem (San Diego, CA). The CYQUANT
cells proliferation assay was from Molecular Probes (Eugene, OR).
All other chemicals were from Sigma Chemical unless otherwise
noted.
diamine (9)
Compound 9 was synthesized from 17 (0.13 g, 0.34 mmol) and
4-bromoaniline (117 mg, 0.68 mmol) using the general procedure
described above to afford after purification 72 mg (61%) as a
white-off solid: TLC Rf 0.44 (CHCl3/CH3OH, 10:1); mp 213.6–
214.8 °C; 1H NMR (DMSO-d6) d 3.75 (s, 2H, CH2), 6.45 (br s, 2H,
NH2, exch), 7.14–7.89 (m, 8H, Ph-H), 8.31 (s, H, NH, exch). Anal
(C17H13BrN4. 0.6CH3OH): C, H, N, Br.
5.1.3. Antibodies
The PY-HRP antibody was from BD Transduction Laboratories
(Franklin Lakes, NJ). Antibodies against EGFR, PDGFR-b, and VEG-
FR-2 were purchased from Upstate Biotech (Framingham, MA).
4.15. N4-(4-(Trifluoromethyl)phenyl)-9H-indeno[2,1-
d]pyrimidine-2,4-diamine (10)
5.1.4. Phosphotyrosine ELISA
Cells used were tumor cell lines naturally expressing high levels
of EGFR (A431), VEGFR-2 (U251), and PDGFR-b (SF-539).
Expression levels at the RNA level were derived from the NCI
Developmental Therapeutics Program (NCI-DTP) web site public
molecular target information (http://www.dtp.nci.nih.gov.authen-
ticate.library.duq.edu/mtargets/mt_index.htmlhttp://www.dtp.nci.
nih.gov.authenticate.library.duq.edu/mtargets/mt_index.html).
Briefly, cells at 60–75% confluence are placed in serum-free med-
ium for 18 h to reduce the background of phosphorylation. Cells
were always >98% viable by Trypan blue exclusion. Cells are then
Compound 10 was synthesized from 17 (0.22 g, 0.57 mmol) and
4-trifluoroaniline (184 mg, 1.04 mmol) using the general proce-
dure described above to afford after purification 44 mg (27%) as a
light brown solid: TLC Rf 0.48 (CHCl3/CH3OH, 10:1); mp 229.2–
230.8 °C; 1H NMR (DMSO-d6) d 3.78 (s, 2H, CH2), 6.56 (br s, 2H,
NH2, exch), 7.18–8.02 (m, 8H, Ph-H), 8.68 (s, H, NH, exch). Anal
(C18H13F3N4): C, H, N, F.
4.16. N4-(3-Methoxyphenyl)-9H-indeno[2,1-d]pyrimidine-2,4-
diamine (11)
pretreated for 60 min with 10, 3.33, 1.11, 0.37, and 0.12 lM com-
pound followed by 100 ng/mL EGF, VEGF, or PDGF-BB for 10 min.
The reaction is stopped and cells permeabilized by quickly remov-
ing the media from the cells and adding ice-cold Tris-buffered
saline (TBS) containing 0.05% Triton X-100, protease inhibitor
cocktail and tyrosine phosphatase inhibitor cocktail. The TBS solu-
tion is then removed and cells fixed to the plate for 30 min at 60 °C
and further incubation in 70% ethanol for an additional 30 min.
Cells are further exposed to block (TBS with 1% BSA) for 1 h,
Compound 11 was synthesized from 17 (75 mg, 0.76 mmol) and
m-anisidine (0.1 mL) using the general procedure described above
to afford after purification 27 mg (45%) as a light brown solid: TLC
Rf 0.47 (CHCl3/CH3OH, 10:1); mp186.6- 187.4 °C; 1H NMR (DMSO-
d6) d 3.74 (s, 2H, CH2), 3.82 (s, 3H, CH3), 6.44 (br s, 2H, NH2, exch),
6.62–7.84 (m, 8H, Ph-H), 8.08 (s, H, NH, exch). HRMS (EI) calcd. for
C18H16N4O 304.1324, found, 304.1390.

4224
A. Gangjee et al. / Bioorg. Med. Chem. 20 (2012) 4217–4225
washed, and then a horseradish peroxidase (HRP)-conjugated
phosphotyrosine (PY) antibody added overnight. The antibody is
removed, cells are washed again in TBS, exposed to an enhanced
luminol ELISA substrate (Pierce Chemical, Rockford, IL) and light
emission measured using a UV products (Upland, CA) BioChemi
digital darkroom. The known RTK-specific kinase inhibitor 18
was used as a positive control compound for EGFR kinase inhibi-
tion; semaxanib for VEGFR-2 kinase inhibition; 19 for PDGFR-b ki-
nase inhibition. Data were graphed as a percent of cells receiving
growth factor alone and IC50 values were calculated from two to
three separate experiments (n = 8–24) using non-linear regression
dose–response relation analysis.
and IC50 values compared to one another using one-way ANOVA
with Neuman–Keuls post test with the null hypothesis rejected
when P <0.05.
5.1.8. B16-F10 syngeneic mouse melanoma model
50,000 B16-F10 (lung colonizing) mouse melanoma cells were
injected orthotopically SQ just behind the ear of athymic NCr nu/
nu mal mice, 8 week in age. First, a dose-finding study was done
with 5, 10, 15, 20, 25, 35 and 50 mg/kg compound 7 given three
times weekly for 4 weeks to 3 animals per treatment group. The
dose of 35 mg/kg compound 7 was found to result in no apparent
toxicity and no significant loss in weight over the 4 week period.
Two experiments were done starting with 5–6 animals/group. Ani-
mals were monitored every other day for the presence of tumors. At
the time in which most tumors were measurable by callipers (day 9
for this experiment), animals with tumors were randomly sorted
into treatment groups and treatment with drugs begun. DMSO
stocks (30 mM) of drugs were further dissolved into sterile water
for injection and 35 mg/kg injected intraperitoneally (IP) every
Monday (AM), Wednesday (noon) and Friday (PM). Sham treated
animals received water only Monday, Wednesday and Friday. The
length (long side), width (short side) and depth of the tumors were
measured using digital Vernier Caliers each Monday, Wednesday,
and Friday. Tumor volume was calculated using the formula length
x width x depth. Tumor growth rate was calculated using a linear
regression analysis algorithm using the software GraphPad Prism
4.0.c. at the experiment’s end, animals were humanely euthanized
using carbon dioxide, tumors and lungs excised, fixed in 20% neutral
buffered formalin for 8–10 h, embedded into paraffin, and haema-
toxylin-eosin (H&E) stain of three separate tissue sections com-
pleted to span the tumor/lung. Together with the OUHSC
Department of Pathology core, metastases per lung lobe counted
using the H&E stained sections. The metastases can be seen as pur-
ple clusters of disorganized cells on the highly organized largely
pink lung. Together with the OUHSC Department of Pathology core,
blood vessels per unit area were counted in 5 fields at 100Â magni-
fication and averaged. Tumor growth rates were compared statisti-
cally using two-way ANOVA with a Repeated Measures post-test
and tumor vascularity and metastases were compared using one-
way ANOVA and a Neuman-Keuls post test with the null hypothesis
rejected when P <0.05.
5.1.5. CYQUANT cell proliferation assay
As a measure of cell proliferation, the CYQUANT cell counting/
proliferation assay was used as previously described.31 Briefly, cells
are first treated with compounds for 12 h and then allowed to grow
for an additional 36 h. The cells are then lysed and the CYQUANT
dye, which intercalates into the DNA of cells, is added and after
5 min the fluorescence of each well measured using an UV products
BioChemi digital darkroom. A positive control used for cytotoxicity
in each experiment was cisplatin, with an apparent average IC50 va-
lue about 8.2 0.65 lM. Data are graphed as a percent of cells
receiving growth factor alone and IC50 values calculated from two
to three separate experiments (n = 6–15) using non-linear regres-
sion dose-response relation analysis.
5.1.6. Chorioallantoic membrane assay of angiogenesis
The CAM assay is a standard assay for testing antiangiogenic
agents.32 The CAM assay used in these studies was modified from
a procedure by Sheu33 and Brooks34 and as published previ-
ously.35 Briefly, fertile leghorn chicken eggs (CBT Farms, Chester-
town, MD) are allowed to grow until 10 days of incubation. The
proangiogenic factors, human VEGF-165 and bFGF (100 ng each)
are then added saturation to a 6 mm microbial testing disk (BBL,
Cockeysville, MD) and placed onto the CAM by breaking a small
hole in the superior surface of the egg. Antiangiogenic compounds
are added 8 h after the VEGF/bFGF at saturation to the same
microbial testing disk and embryos allowed to incubate for an
additional 40 h. After 48 h, the CAMs are perfused with 2% para-
formaldehyde/3% glutaraldehyde containing 0.025% Triton X-100
for 20 s, excised around the area of treatment, fixed again in 2%
paraformaldehyde/3% glutaraldehyde for 30 min, placed on petri
dishes, and a digitized image taken using a dissecting microscope
(Wild M400; Bannockburn, IL) at 7.5X and SPOT enhanced digital
imaging system (Diagnostic Instruments, Sterling Heights, MI). A
grid is then added to the digital CAM images and the average
number of vessels within 5–7 grids counted as a measure of vas-
cularity. AGM-1470 (a kind gift of the NIH Developmental Thera-
peutics Program) and semaxanib are used as a positive control for
antiangiogenic activity. Data are graphed as a percent of CAMs
receiving bFGF/VEGF and IC50 values calculated from two to three
separate experiments (n = 5–11) using non-linear regression dose-
response relation analysis.
Acknowledgements
We thank Dr. Margaret Phillips (Department of Pharmacology,
University of Texas Southwestern Medical Center, Dallas, Texas)
for carrying out the DHODH assays. This work was supported, in
part, by the National Institutes of Health, National Cancer Institute
grant CA 098850 (AG) and the Duquesne University Adrian Van
Kaam Chair in Scholarly Excellence (AG).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.05.068.
5.1.7. Statistics
All analysis was done using Prism 4.0. (GraphPad Software, San
Diego, CA). Tumor growth rates were assessed during the linear
growth period and statistical significance of tumor growth be-
tween groups was calculated using two-way repeated measures
ANOVA with treatments and days after implantation as indepen-
dent variables with Dunnett’s post-test with the null hypothesis
rejected when P < 0.05. Tumor metastases were compared using
one-way ANOVA with Bonferonni’s multiple comparison post-test
with the null hypothesis rejected when P < 0.05. IC50 values were
calculated using a non-linear dose-response relation algorithm
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