Synthesis and biological activity of 5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular endothelial growth factor receptor-2 inhibitors and antiangiogenic agents

Article abstract of DOI:10.1016/j.bmc.2013.01.040

Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-N⁴-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)- 2,2-dimethylpropanamide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and semaxinib.

Full text of DOI:10.1016/j.bmc.2013.01.040

Bioorganic & Medicinal Chemistry 21 (2013) 1857–1864
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological activity of 5-chloro-N4-substituted
phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as vascular
endothelial growth factor receptor-2 inhibitors and antiangiogenic
agents q
a
a
b
b
b
Aleem Gangjee a, , Nilesh Zaware , Sudhir Raghavan , Bryan C. Disch , Jessica E. Thorpe , Anja Bastian ,
⇑
Michael A. Ihnat b
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, United States
b Department of Pharmaceutical Sciences, University of Oklahoma, College of Pharmacy, Oklahoma City, OK 73117, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibition of receptor tyrosine kinase (RTK) signaling pathways is an important area for the development
of novel anticancer agents. Numerous multikinase inhibitors (MKIs) have been recently approved for the
treatment of cancer. Vascular endothelial growth factor receptor-2 (VEGFR-2) is the principal mediator of
tumor angiogenesis. In an effort to develop ATP-competitive VEGFR-2 selective inhibitors the 5-chloro-
N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold was designed. The synthesis of
the target compounds involved N-(4,5-dichloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-dimethylpropana-
mide) as a common intermediate. A nucleophilic displacement of the 4-chloro group of the common
intermediate by appropriately substituted anilines afforded the target compounds. Biological evaluation
indicated that compound 5 is a potent and selective VEGFR-2 inhibitor comparable to sunitinib and
semaxinib.
Received 24 August 2012
Revised 10 January 2013
Accepted 18 January 2013
Available online 31 January 2013
Keywords:
Receptor tryosine kinase inhibitors
Pyrimido[4,5-b]indol synthesis
Cytotoxicity
VEGFR-2 inhibitors
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
for a solid tumor to grow beyond 1–2 mm. As a tumor grows in
size, it becomes increasingly hypoxic, leading to induction of
Protein tyrosine kinases are a key means of signal transduction
in eukaryotic cells and control processes such as cell proliferation,
differentiation, migration, survival, and cell cycle progression. Dys-
regulation of these tightly regulated processes through overex-
pression of kinases is implicated in numerous disease states
including cancer.1,2
growth factors including vascular endothelial growth factor
(VEGF), and other important proangiogenic regulators like epider-
mal growth factor (EGF), platelet derived growth factor (PDGF),
fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-
1), transforming growth factors (TGF
a and b), and tumor necrosis
factor- (TNF-
a
a
) among others.1,3 The growth factors function by
Angiogenesis is the process by which new blood vessels are
formed from pre-existing vasculature and this process is crucial
binding to receptor tyrosine kinases (RTKs), a super family of trans-
membrane proteins. Subsequent to binding of the growth factor,
RTKs dimerize and undergo autophosphorylation, initiating angio-
genesis.4 Anti-angiogenic therapy targets non-tumor cells (endo-
thelial cells) which may be less prone to mutations and hence to
resistance compared to tumor cells.5
One of the approaches to circumvent angiogensis is the inhibi-
tion of the key RTKs involved in angiogenesis. Several reports on
multikinase inhibitors (MKIs) have appeared in the recent litera-
ture, some of which have afforded clinically approved agents.
Imatinib (Fig. 1) was the first RTK inhibitor approved in 2001 for
chronic myelogenous leukemia (CML).6,7 Imatinib inhibits Abelson
Abbreviations: ATP, adenosine 50triphosphate; VEGF, vascular endothelial
growth factor; EGF, epidermal growth factor; PDGF, platelet derived growth factor;
RTK, receptor tyrosine kinase; VEGFR-2, vascular endothelial growth factor recep-
tor-2; VEGFR-1, vascular endothelial growth factor receptor-1; PDGFR-b, platelet
derived growth factor receptor-b; FGF, fibroblast growth factor; IGF-1, insulin-like
growth factor-1; TGFa and b, transforming growth factors; TNF-a, tumor necrosis
factor- ; MKIs, multikinase inhibitors; CML, chronic myelogenous leukemia; Abl,
a
Abelson tyrosine kinase; CD117, c-kit protein; FLT3, FMS-like tyrosine kinase 3.
q
Taken in part from the dissertation submitted by N.Z. to the Graduate School of
Pharmaceutical Sciences, Duquesne University, in partial fulfillment of the require-
ments for the degree of Doctor of Philosophy, July 2006; Presented in part at the
237th American Chemical Society National Meeting, Salt Lake City, UT, United
States, March 22–26, 2009, MEDI-226.
Tyrosine Kinase (Abl), c-kit protein (CD117), PDGFR-a and PDGFR-
b (See Table 1).
Sorafenib, approved in 2005 for the treatment of hepatocellular
carcinoma and renal cell carcinoma, inhibits Raf kinase, VEGFR-2,
⇑
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 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.040

1858
A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
O
N
H
N
CF3
O
N
N
H
N
H
N
Cl
O
N
N
O
N
H
N
H
F
N
N
O
N
H
N
H
O
N
H
Imatinib
Sorafenib
Sunitinib
N
Cl
HN
N
N
O
N
HN
N
N
N
N
O
H
N
O
HN
N
N
H
N
S
N
NH
O
S
HO
N
F
O
O
Cl
CF3
Nilotinib
Dasatinib
Lapatinib
Figure 1. Various RTK inhibitors.
Table 1
IC50 values (lM) of kinase inhibiton and A431 cytotoxicity for compounds 3–14
Compound
VEGFR-2 kinase inhibition
EGFR kinase inhibition
PDGFR-b kinase inhibition
A431 cytotoxicity
3
4
5
6
7
8
9
10
11
12
13
14
123.8 29.0
30.3 5.0
13.2 1.7
38.9 6.0
86.1 7.8
70.1 10.1
192.0 16.2
47.8 10.1
21.3 4.0
89.1 10.9
31.2 4.5
41.0 7.2
12.0 2.7
>200
>200
>200
193.1 29.7
45.1 10.1
44.1
128.3 20.2
184.1 22.1
167.0 38.6
185.2 20.1
>250
55.0
>250
>250
>250
42 10.5
63.6 8.1
38.9 6.0
>200
82.9 19.2
114.7 30.2
39.2 7.8
58.7 7.2
>200
196.2 22.4
>200
172.2 30.1
50.5 7.2
70.6 10.7
>200
>200
170.8 19.6
>200
30.8 7.0
130.5 32.8
196.2 30.0
Semaxanib (20)
PD153035 (21)
DMBI (22)
Sunitinib
Erlotinib
Cisplatin (23)
0.2 0.004
3.7 0.06
83.1 10.1
12.2 1.9
18.9 2.7
124.7 18.2
172.1 19.4
1.2 0.2
10.6 2.9
VEGFR-3, PDGFR-b, and c-kit.8 Sunitinib, approved in 2006 for the
treatment of renal cell carcinoma and imatinib-resistant gastroin-
testinal stromal tumors, inhibits VEGFR, PDGFR, c-kit, and FMS-like
tyrosine kinase 3 (FLT3).9
Where a large number of closely related kinase isozymes exist,
some of which may be crucial for normal cellular function, it is crit-
ical to avoid the off-target activity of MKIs as it might translate into
undesirable biology. This has been exemplified by the approved
MKIs sorafenib and sunitinib which have been reported to be car-
diotoxic.16,17 In addition, imatinib has been reported to have mech-
anism-based cardiotoxic effects, and nilotinib carries a black box
warning for possible heart complications.18 Hence, specific inhibi-
tors of kinases critical in tumor survival like VEGFR-2 or other
RTKs, with minimal off-target activity, are also of considerable
interest.
Two drugs—dasatinib10 and nilotinib11 were approved in 2006
and 2007, respectively, specifically for imatinib-resistant or unre-
sponsive tumors in CML. Dasatinib inhibits Abl, c-kit, PDGFR, and
Src. Nilotinib inihibits Abl, c-kit, PDGFR-b, Src, and Ephrin. Lapati-
nib, approved in 2007 for advanced metastatic breast cancer in
conjunction with chemotherapy, inhibits EGFR and erythroblastic
leukemia viral oncogene homolog-2 (ErbB-2).12
Although the approval of numerous MKIs for cancer therapy at-
tests to the importance of this approach in cancer chemotherapy,
gaining selectivity for a limited subset of kinases for MKIs is a
widely recognized challenge facing medicinal chemists in the ki-
nase area.13 The risk of a MKI approach is that such compounds
are by their very nature more likely to hit a diverse spectrum of ki-
nases. A recent report studied the selectivity of approved kinase
inhibitors and candidates across 317 different kinases.14 The most
selective of the currently approved MKIs was lapatinib, and the
least selective was sunitinib; the latter bound > 15% of kinases
tested with Kd < 100 nM. Bamborough et al.15 screened 577 diverse
compounds versus 203 protein kinases and found that two-thirds
of the compounds bound to more than 10 kinases, hence clearly
demonstrating the extent of the selectivity challenge in the kinase
area.
In 1996, Traxler et al.19 reported a series of N4-substituted phe-
nyl-9H-pyrimido[4,5-b]indoles as inhibitors of EGFR. From this ser-
ies, compound 1 (Fig. 2) emerged as a single digit nanomolar
inhibitors of EGFR (IC50 = 6 nM). Showalter et al.20 reported several
elaborations of similar anilino pyrimidine inhibitors of which com-
pound
2
emerged as another potent inhibitor of EGFR
(IC50 = 147 nM). Compounds 1 and 2 were not tested in VEGFR-2,
which has been implicated as the principal mediator of angiogen-
esis21–24 and VEGFR-2 stimulation alone is enough to initiate tu-
mor growth and metastases.24 It was of interest to structurally
design a scaffold to afford either dual EGFR/VEGFR-2 inhibition
or perhaps a selective VEGFR-2 inhibition.
On the basis of 1 and 2, we designed general scaffold A
5-chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-dia-
mine (Fig. 2). A systematic conformational search (5° increments)

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
1859
3'
4'
R
2'
1'
Cl
6
5'
Cl
NH
N
NH
N
NH
4
5
6'
7
3
N
N
N
8
N
H
N
H
9
N
H
H2N
H2N
N
2
1
A
1
2
Figure 2. Design of scaffold (I).
Figure 3. Stereoview of docked pose of compound 3 in VEGFR-2 (PDB code:1YWN) showing the key interactions.
performed using Sybyl X 1.325 indicated a lower number of confor-
mations (24) about the C4–N bond with the pyrimidine ring for
5-chloro-N4-phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine
3
(Fig. 5) compared to its corresponding 5-deschloro analog, 2
(41). Hence, the addition of a 5-Cl substitution was expected
to provide conformational restriction through steric hindrance
to rotation and this may allow for specificity against some ki-
nases over others and perhaps allow potent inhibition of a dif-
ferent spectrum of RTKs.
Molecular modeling was carried out to gain some insight to the
binding mode of 3 in the VEGFR-2 active site. The best scored pose
on docking compound 3 using Lead IT 2.1.026 is depicted in Figures
3 and 4. The binding site of ATP competitive inhibitors in RTKs
consists of a Hinge region, and a hydrophobic binding site (Hydro-
phobic Region I) among others (Fig. 3).27,20,28,29 The pyrimido[4,5-
b]indole ring of 3 occupies the adenine binding portion of the ATP
binding site. The 1-N and the 9-NH are involved in hydrogen bonds
with Cys917 in the Hinge region. Hydrophobic interactions of the
indole ring with Val914, Phe916, Cys917, Leu1033, Cys1043,
Leu838 and Val846 can stabilize the docked pose. In addition, the
aniline phenyl extends towards Hydrophobic region I and is in-
volved in interactions with Val846, Ala864, Lys866, Val897,
Val914 and Leu1033. The 2-amino group could potentially form a
hydrogen bond with Glu915 in the hinge region.
Figure 4. Ligand interaction plot of the docked pose of compound 3 in VEGFR-2
(PDB code:1YWN) showing the key interactions.
A series of six compounds 3–8 (Fig. 5) was designed to study
scaffold A with respect to inhibition of receptor tyrosine kinases.
The selection of the substituents on the 4-position are rationalized
below. The 40-isopropyl group in 4 was included to obtain informa-
tion about bulk tolerance in this position in the RTK active site.
15 (Fig. 5). Thus compound 5 with the 40-Cl aniline was proposed
with the purpose of obtaining potent inhibition of VEGFR-2 and/
or VEGFR-1. The 30-OMe aniline substitution in 8 is similar to com-
pounds 16 and 17 (Fig. 5) which are potent inhibitors of EGFR.31 In
Compound 15 (bearing
a 4-chloroaniline) reported by Bold
et al.30 demonstrated potent VEGFR-2 and VEGFR-1 inhibition in

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A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
Figure 5. 5-Chloro-N4-substituted phenyl-9H-pyrimido[4,5-b]indole-2,4-diamines as potential multiple RTK inhibitors with lead compounds.
order to obtain further SAR information at this position, the 30-OMe
group (8) was replaced with the 30-F (7) which would afford infor-
mation regarding electron withdrawing groups at the 30-position.
Compound 6 with the 20-F, 40-Cl aniline substituted ring is similar
to that in 1832 and was included with the purpose of obtaining po-
tent inhibition of VEGFR-2 and VEGFR-1 as reported for 18. The
unsubstituted phenyl ring (3) served as a comparison with the
substituted compounds.
time (1–3 days) and lower yields for 3 (52%) and 4 (49%) (This
method afforded 6 in 74% yield). Reaction time was significantly
decreased when 1 N NaOH in isopropanol was used as the reaction
system; 13 and 14 were converted to 7 (70%) and 8 (92%), respec-
tively in 2–14 h. Isopropanol was more efficient, probably because
of the enhanced solubility of the starting material (13, 14) as well
as the nucleophile (hydroxide ions).
The synthesis of 3–8 was designed from the corresponding
2-amino pivaloyl (2,2-dimethylpropan-1-one) protected precur-
sors 9–14. It was decided to biologically evaluate intermediates
9–14 to study the effect of the 2-amino pivaloyl groups on binding
to RTKs. It was anticipated that the pivaloyl moieties of 9–14 could
interact at hydrophobic region II33 and perhaps afford increased
activity against some RTK.
3. Biological evaluation and discussion
Compounds 3–14 were evaluated as RTK inhibitors using hu-
man tumor cells known to express high levels of VEGFR-2, EGFR
and PDFGR-b using
a
phosphotyrosine ELISA cytoblot (1).34
Whole cell assays were used for RTK inhibitory activity since
these assays afford more meaningful results for translation to
in vivo studies. The effect of compounds on cell proliferation
was measured using A431 cancer cells, known to overexpress
EGFR. EGFR is known to play a role in the overall survival of
A431 cells.34
Since the IC50 values of compounds vary under different assay
conditions (e.g., ATP concentrations), standard compounds (Fig. 6)
were used as controls in each of the evaluations. The standard
compounds used were semaxanib (SU5416), 20 for VEGFR-2;35
4-[(3-bromophenyl)amino]-6,7-dimethoxyquinazoline (PD153035),
21 for EGFR;36 3-(4-dimethylamino-benzylidenyl)-2-indolinone
(DMBI), 22 for PDGFR-b.37 and cisplatin, 23 for A431 cytotoxicity.
Approved agents sunitinib (a multitargeted RTK inhibitor including
VEGFR-2, PDGFRs) and erlotinib38 (a specific EGFR inhibitor ap-
proved for non small cell lung cancer) were also used as standard
agents.
2. Chemistry
Compounds 3–14 were synthesized as described in Scheme 1.
N-(4,5-Dichloro-9H-pyrimido[4,5-b]indol-2-yl)pivalamide 19 was
synthesized by a method developed by Gangjee et al.33 Compound
19 was condensed with appropriately substituted anilines in iso-
propanol at reflux, in the presence of three drops of conc. HCl, to
afford regiospecifically 9–14. Deprotection of the 2-pivaloyl group
in 11 by reaction with 15% KOH in 1,4-dioxane at reflux afforded 5.
Compounds 9, 10 and 12 were more soluble in a 1:1 mixture of
methanol to methylene chloride than in 1,4-dioxane. However,
when 1 N NaOH and the 1:1 methanol/methylene chloride solvent
system were used for the deprotection reaction of 9, 10 and 12 to
obtain 3, 4 and 6, respectively, the result was an increased reaction
b
Ar
Ar
5
Cl
NH
NH
Cl
Cl
Cl
N
N
N
OR
c
a
HN
HN
H2N
O
O
N
N
N
N
N
N
3
4 6
,
,
H
H
H
19
9-14
3-8
OR
d
Ar
Ar
Ph (49%)
4-IsopropylPh (52%)
4-ClPh (95%)
2-F, 4-ClPh (74%)
3-FPh (70%)
3-OMePh (92%)
9
Ph (86%)
3
4
5
6
7
8
7
,
8
10
4-IsopropylPh (75%)
11 4-ClPh (48%)
12
13
2-F, 4-ClPh (74%)
3-FPh (95%)
14 3-OMePh (91%)
Scheme 1. Reagents and conditions: (a) ArNH2, isopropanol, 3 drops of concd HCl, reflux, 1.5–6 h (48–95%); (b) 15% KOH, 1,4-dioxane, reflux, 14 h (95%); (c) 1 N NaOH,
methanol/CH2Cl2, reflux, 1–3 d (49–74%); (d) 1 N NaOH, isopropanol, reflux, 2–14 h (70–92%).

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
1861
Br
N
HN
HN
N
H
Cl
O
O
OMe
N
O
O
N
H2N Pt Cl
O
O
NH2
N
H
N
N
N
OMe
H
20, Semaxanib (SU5416)
21, PD153035
23, Cisplatin
22, DMBI
Figure 6.
Erlotinib
In the VEGFR-2 kinase inhibition assay, of the target compounds
3–8, the 4-chlorophenyl substituted compound 5 was the most
active and equipotent to the standard 20. Compound 5 was also
1.4- and 9.4-fold more potent than sunitinib and erlotinib, respec-
tively, in the VEGFR-2 inhibition assay. The next most potent
compounds were the 4-isopropylphenyl substituted 4, and the
2-fluoro-4-chlorophenyl substituted 6 which were 2.5-fold less
active and 3-fold less active, respectively, than 20. VEGFR-2 inhibi-
tion decreased on moving the phenyl substitution from the 4- to
the 3-position, as exemplified by the 3-fluorophenyl substituted
7 and the 3-methoxy substituted 8, which were 6- and 7-fold less
potent then 20, respectively.
For the 2-amino pivaloyl substituted compounds 9–14, the
4-chlorophenyl substituted compound 11 was the most active
and was 2-fold less potent than the standard 20 in the VEGFR-2
inhibition assay. The next most potent compounds included the
3-fluorophenyl substituted 13, 3-methoxyphenyl substituted 14,
and the 4-isopropylphenyl substituted 10, which were about
2.5-, 3.5- and 4-fold less active than 20, respectively. The increased
activity of the 2-pivaloyl analogs 13–14 in the VEGFR-2 assay over
the corresponding despivaloyl analogs 7 and 8, respectively indi-
cates a possible interaction with hydrophobic site II of the pivaloyl
moiety in VEGFR-2.
A trend in activity was observed for 3–5, however in that these
compounds were about 1.5-fold more active than their 2-amino
pivaloyl derivatives. However, a similar trend was not observed
for 7 and 8 which were about 2.8- and 1.7-fold less active than
the corresponding 2-amino pivaloyl compounds 13 and 14. Thus
subtle differences in binding orientation of 3–5 compared with
7–8 perhaps accounts for the differences in their respective piva-
loyl derivatives.
The most potent compound against EGFR was the 2-amino
pivaloyl, 3-fluorophenyl substituted 13 which was 150-fold less
potent than the standard 21. However compound 13 was 5.5-
fold more active then sunitinib, and 25-fold less active than erl-
otinib in the EGFR inhibition assay. The presence of a 5-chloro
group is detrimental to cell-based EGFR inhibition relative to
the 5-deschloro lead compound 2 (Fig. 2) which is a reported
nanomolar EGFR inhibitor in an isolated enzyme assay. The most
potent PDGFR-b inhibitor in this study was the 3-methoxyphenyl
substituted 8, which is about 13.5-fold less active than the stan-
dard 22. Compound 8 was 1.6-fold more potent than sunitinib
and 4-fold less active than erlotinib in the PDGFR-b inhibition
assay.
In summary, compounds 3–14 were synthesized to develop a
novel scaffold with either selective VEGFR-2 and/or VEGFR-1 inhi-
bition or EGFR/VEGFR-2 dual inhibition. The cellular inhibition as-
says demonstrated that the compounds were indeed selective for
VEGFR-2, relative to EGFR and PDGFR-b. The 4-chlorophenyl
substituted 5 was the most potent inhibitor in the VEGFR-2 inhibi-
tion assay and was equipotent with the standard compound 20,
and was 1.4- and 9.4-fold more potent than sunitinib and erlotinib,
respectively. The results of this study indicate that the 5-chloro-
N4-substituted
phenyl-9H-pyrimido[4,5-b]indole-2,4-diamine
scaffold is highly selective for VEGFR-2 inihibition with minimal
off-target kinase inhibition in the limited number of RTKs these
compounds were evaluated against and serves as a new lead ana-
log that is equipotent to sunitinib and almost 10ꢀ more potent
that erlotinib in the VEGFR-2 inhibition whole cell assay.
4. Experimental section
Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-
DRY drying apparatus over P2O5 at 80 °C. Melting points were
determined on a MEL-TEMP II melting point apparatus with FLUKE
51 K/J electronic thermometer and are uncorrected. Nuclear mag-
netic resonance spectra for proton (1H NMR) were recorded on a
Bruker WH-300 (300 MHz) or Bruker WH-400 (400 MHz) spec-
trometer. The chemical shift values are expressed in ppm (parts
per million): s, singlet; d, doublet; t, triplet; q, quartet; m, multi-
plet; br, broad singlet. Thin-layer chromatography (TLC) was per-
formed on Whatman Sil G/UV254 silica gel plates with
a
fluorescent indicator, and the spots were visualized under 254
and 366 nm illumination. Proportions of solvents used for TLC
are by volume. Column chromatography was performed on a
230–400 mesh silica gel (Fisher, Somerville, NJ) column. Elemental
analyses were performed by Atlantic Microlab, Inc., Norcross, GA.
Element compositions are within 0.4% of the calculated values.
Fractional moles of water or organic solvents frequently found in
some analytical samples of antifolates could not be prevented in
spite of 24–48 h of drying in vacuo and were confirmed where pos-
sible by their presence in the 1H NMR spectra. All solvents and
chemicals were purchased from Aldrich Chemical Co. or Fisher Sci-
entific and were used as received.
4.1. General procedure for the synthesis of 9–14
To a 50 mL round bottom flask were added N-(4,5-dichloro-9H-
pyrimido[4,5-b]indol-2-yl)-2,2-dimethyl propanamide 19, various
substituted anilines, isopropanol (50 mL) followed by 3 drops of
concd HCl. The mixture was heated at reflux for 1.5–6 h. The sol-
vent was removed by evaporation in vacuo. The residue was dis-
solved in methanol and silica gel (2 g) was added followed by
evaporation in vacuo to afford a dry silica gel plug which was
loaded on top of a wet (hexane) silica gel column sequentially elut-
ing with 0%, 1% and 2% methanol in chloroform. Fractions contain-
ing desired product (TLC) were pooled and evaporated to dryness
to afford the product.
In the A431 cytotoxicity assay, the most potent compounds
were the 4-isopropylphenyl substituted 4, and the 4-chloro-
phenyl substituted 5 which were 4-fold less potent than the
standard 23. Since substitution at any other position produced
a further decrease in activity, the 4-position is optimum for
activity, and tolerates both electron withdrawing and electron
donating as well as bulky groups. Of the 2-amino pivaloyl deriv-
atives 9–14, only the 2-amino pivaloyl, 4-chlorophenyl substi-
tuted 11 produced potent inibition, which was 5-fold less than
the standard 23.

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A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
4.1.1. N-(4-Anilino-5-chloro-9H-pyrimido[4,5-b]indol-2-yl)-2,2-
dimethylpropanamide(9)
4.1.7. 5-Chloro-N4-(4-chlorophenyl)-9H-pyrimido[4,5-b]indole-
2,4-diamine (5)
Reaction of 19 (150 mg, 0.44 mmol) and aniline (310 mg,
3.33 mmol) using the general procedure described above, gave 9:
yield 86%, TLC Rf 0.6 (CHCl3/MeOH, 10:1 with 2 drops concentrated
NH4OH); mp 249–250 °C; 1H NMR (DMSO-d6) d 1.27 (s, 9H,
C(CH3)3), 7.03–7.08 (m, 1H, Ar), 7.33–7.38 (m, 4H, Ar), 7.46–7.49
(m, 1H, Ar), 8.13–8.16 (d, 2H, Ar), 9.36 (s, 1H, 2-NH, exch), 9.67
(s, 1H, 4-NH, exch), 12.25 (s, 1H, 9-NH, exch). Anal. (C21H20ClN5O)
C, H, N, Cl.
In a 50 mL flask 11 (70 mg, 0.16 mmol) was dissolved in 1,
4-dioxane 20 mL, and 2 mL of 15% KOH aqueous solution was
added. The reaction mixture was heated to reflux for 14 h. The sol-
vent was evaporated to obtain a residue. Water was added and the
mixture was extracted with chloroform. Sodium sulfate was used
to dry the extract. Silica gel was added and solvent evaporated to
make a plug. The plug was loaded on top of a wet (CHCl3) silica
gel column (25 ꢀ 3 cm) and eluted with 60:1 (CHCl3/CH3OH). Frac-
tions containing desired product were pooled and evaporated to
afford 5 in 95% yield. TLC Rf 0.35 (CHCl3/MeOH, 10:1 with 3 drops
concentrated NH4OH); mp 306.1–307 °C; 1H NMR (DMSO-d6) d
6.54 (bs, 2H, NH2, exch), 7.19–7.21 (m, 2H, Ar), 7.29–7.37 (m, 4H,
Ar), 7.95–7.98 (m, 2H, Ar), 9.12 (s, 1H, 4-NH, exch), 11.75 (s, 1H,
9-NH, exch). HRMS (ESI) [M+H]+ Calcd for C16H11Cl2N5 m/
z = 344.0470, found m/z = 344.0449.
4.1.2. N-{5-Chloro-4-[(4-isopropylphenyl)amino]-9H-pyrimido
[4,5-b]indol-2-yl}-2,2-dimethylpropanamide (10)
Reaction of 19 (250 mg, 0.74 mmol) and 4-isopropyl aniline
(750 mg, 5.56 mmol) using the general procedure described above,
gave 10: yield 75%, TLC Rf 0.62 (CHCl3/MeOH, 10:1 with 2 drops
concentrated NH4OH); mp 265 °C; 1H NMR (DMSO-d6) d 1.20 (s,
3H, CH3), 1.23 (s, 3H, CH3), 1.27 (s, 9H, C(CH3)3), 7.20–7.23 (m,
1H, Ar), 7.35–7.40 (m, 2H, Ar), 7.46–7.49 (m, 1H, Ar), 8.03–8.06
(d, 2H, Ar), 9.31 (s, 1H, 2-NH, exch), 9.63 (s, 1H, 4-NH, exch),
12.24 (s, 1H, 9-NH, exch). HRMS (ESI) [M+H]+ Calcd for
4.1.8. 5-Chloro-N4-(4-isopropylphenyl)-9H-pyrimido[4,5-b]
indole-2,4-diamine (4)
In a 50 mL flask 10 (20 mg, 0.044 mmol) was dissolved in
CH2Cl2/CH3OH, 1:1 (10 mL), and 2 mL of 1 N NaOH solution was
added. The reaction mixture was heated to reflux for 44 h. The sol-
vent was evaporated to obtain a residue. Water was added and the
mixture was filtered and dried. The solid was dissolved in MeOH,
silica gel was added and solvent evaporated to make a plug. The
plug was loaded on top of a wet (CHCl3) silica gel column and
eluted with 20:1 (CHCl3/MeOH). Fractions containing desired prod-
uct were pooled and evaporated to afford the 4 in 52% yield. TLC Rf
0.40 (CHCl3/MeOH, 10:1 with 3 drops concentrated NH4OH); mp
235–236 °C; 1H NMR (DMSO-d6) d 1.19–1.21 (m, 6H, CH3 ꢀ 2),
2.85–2.90 (m, 1H, CH), 6.42 (bs, 2H, NH2, exch), 7.17–7.20 (m,
4H, Ar), 7.27–7.30 (m, 2H, Ar), 7.75–7.77 (m, 2H, Ar), 9.00 (s, 1H,
4-NH, exch), 11.68 (s, 1H, 9-NH, exch). Anal. (C19H18ClN5) C, H, N,
Cl.
C24H26ClN5O m/z = 436.1904, found m/z = 436.1868.
4.1.3. N-{5-Chloro-4-[(4-chlorophenyl)amino]-9H-pyrimido
[4,5-b]indol-2-yl}-2,2-dimethylpropanamide (11)
Reaction of 19 (150 mg, 0.44 mmol) and 4-chloro aniline
(188 mg, 1.46 mmol) using the general procedure described above,
gave 11: yield 48%, TLC Rf 0.5 (CHCl3/MeOH, 10:1 with 2 drops con-
centrated NH4OH); mp 281.8–282 °C; 1H NMR (DMSO-d6) d 1.26 (s,
9H, C(CH3)3), 7.31–7.40 (m, 4H, Ar), 7.45–7.48 (m, 1H, Ar), 8.19–
8.22 (d, 2H, Ar), 9.37 (s, 1H, 2-NH, exch), 9.70 (s, 1H, 4-NH, exch),
12.25 (s, 1H, 9-NH, exch). Anal. (C21H19Cl2N5O) C, H, N, Cl.
4.1.4. N-{5-Chloro-4-[(4-chloro, 2-fluorophenyl)amino]-9H-
pyrimido[4,5-b]indol-2-yl}-2,2-dimethylpropanamide (12)
Reaction of 19 (150 mg, 0.44 mmol) and 2-fluoro-4-chloroani-
line (485 mg, 3.33 mmol) using the general procedure described
above, gave 12: yield 74%, TLC Rf 0.6 (CHCl3/MeOH, 10:1 with 2
drops concentrated NH4OH); mp 284–285 °C; 1H NMR (DMSO-d6)
d 1.28 (s, 9H, C(CH3)3), 7.23–7.26 (m, 1H, Ar), 7.33–7.40 (m, 2H,
Ar), 7.49–7.57 (m, 2H, Ar), 9.47–9.53 (m, 1H, Ar), 9.50 (s, 1H, 2-
NH, exch), 9.82 (s, 1H, 4-NH, exch), 12.34 (s, 1H, 9-NH, exch). Anal.
(C21H18Cl2FN5O 0.025 CHCl3) C, H, N, Cl, F.
4.1.9. 5-Chloro-N4-phenyl-9H-pyrimido[4,5-b]indole-2,4-
diamine (3)
Compound 3 (synthesized from 9 (130 mg, 0.33 mmol) as de-
scribed for 4; reaction time = 15 h): yield 49%; TLC Rf 0.40
(CHCl3/MeOH, 10:1 with 3 drops concentrated NH4OH); mp 271–
272 °C; 1H NMR (DMSO-d6) d 6.48 (bs, 2H, NH2, exch), 7.01–7.04
(m, 1H, Ar), 7.18–7.19 (m, 2H, Ar), 7.29–7.34 (m, 3H, Ar), 7.89–
7.91 (d, 2H, Ar), 9.08 (s, 1H, 4-NH, exch), 11.71 (s, 1H, 9-NH, exch).
Anal. (C16H12ClN5 0.04 CHCl3) C, H, N, Cl.
4.1.5. N-{5-Chloro-4-[(3-fluorophenyl)amino]-9H-pyrimido[4,5-
b]indol-2-yl}-2,2-dimethylpropanamide (13)
Reaction of 19 (100 mg, 0.29 mmol) and 3-fluoroaniline
(242 mg, 2.17 mmol) using the general procedure described above,
gave 13: yield 95%, TLC Rf 0.6 (CHCl3/MeOH, 10:1 with 2 drops con-
centrated NH4OH); mp 273–274 °C; 1H NMR (DMSO-d6) d 1.28 (s,
9H, C(CH3)3), 6.82–6.89 (m, 1H, Ar), 7.33–7.39 (m, 3H, Ar), 7.48–
7.54 (s, 2H, Ar), 8.61–8.66 (d, 1H, Ar), 9.46 (s, 1H, 2-NH, exch),
9.79 (s, 1H, 4-NH, exch), 12.27 (s, 1H, 9-NH, exch). Anal.
(C21H19ClFN5O) C, H, N, Cl, F.
4.1.10. 5-Chloro-N4-(4-chloro-2-fluorophenyl)-9H-pyrimido
[4,5-b]indole-2,4-diamine (6)
Compound 6 (synthesized from 12 (100 mg, 0.22 mmol) as de-
scribed for 4; reaction time = 48 h): yield 74%; TLC Rf 0.40 (CHCl3/
MeOH, 10:1 with 3 drops concentrated NH4OH); mp 296 °C; 1H
NMR (DMSO-d6) d 6.61 (s, 2H, NH2, exch), 7.21–7.33 (m, 4H, Ar),
7.52–7.55 (m, 1H, Ar), 8.91–8.96 (m, 1H, Ar), 9.14 (s, 1H, 4-NH,
exch), 11.82 (s, 1H, 9-NH, exch). HRMS (ESI) [M+H]+ Calcd for
C16H11Cl2FN5 m/z = 362.0376, found m/z = 362.0381.
4.1.6. N-{5-Chloro-4-[(3-methoxyphenyl)amino]-9H-pyrimido
[4,5-b]indol-2-yl}-2,2-dimethylpropanamide (14)
4.1.11. 5-Chloro-N4-(3-fluorophenyl)-9H-pyrimido[4,5-b]
indole-2,4-diamine (7)
Reaction of 19 (100 mg, 0.29 mmol) and 3-methoxyaniline
(268 mg, 2.17 mmol) using the general procedure described
above, gave 14: yield 91%, TLC Rf 0.6 (CHCl3/MeOH, 10:1 with
In a 50 mL flask 13 (84 mg, 0.20 mmol) was dissolved in isopro-
panol (50 mL), and 2 mL of 1 N NaOH solution was added. The reac-
tion mixture was heated to reflux for 14 h. The solvent was
evaporated to obtain a residue. Water was added and the mixture
was filtered and dried. The solid was dissolved in CH3OH, silica gel
was added and solvent evaporated to make a plug. The plug was
loaded on top of a wet (CHCl3) silica gel column and eluted with
30:1 (CHCl3/MeOH). Fractions containing desired product were
2
drops concentrated NH4OH); mp 196–197 °C; 1H NMR
(DMSO-d6) d 1.26 (s, 9H, C(CH3)3), 3.86 (s, 3H, CH3), 6.61–6.63
(m, 1H, Ar), 7.19–7.28 (m, 2H, Ar), 7.31–7.44 (m, 2H, Ar),
7.48–7.50 (m, 1H, Ar), 8.18 (s, 1H, Ar), 9.37 (s, 1H, 2-NH, exch),
9.79 (s, 1H, 4-NH, exch), 12.26 (s, 1H, 9-NH, exch). Anal.
(C22H22ClN5O2 0.09 CHCl3) C, H, N, Cl.

A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
1863
pooled and evaporated to afford the 7 in 70% yield. TLC Rf 0.47
(CHCl3/MeOH, 10:1 with 3 drops concentrated NH4OH); mp 257–
258 °C; 1H NMR (DMSO-d6) d 6.64 (br s, 2H, NH2, exch), 6.81–
6.86 (m, 1H, Ar), 7.20–7.22 (m, 2H, Ar), 7.30–7.37 (m, 2H, Ar),
7.43–7.46 (m, 1H, Ar), 8.13–8.16 (m, 1H, Ar), 9.21 (s, 1H, 4-NH,
exch), 11.79 (s, 1H, 9-NH, exch). Anal. (C16H11ClFN5 0.5 H2O) C,
H, N, Cl, F.
in TBS, exposed to an enhanced luminal ELISA substrate (Pierce
Chemical, Rockford, IL), and light emission measured using a
UV product (Upland, CA) BioChemi digital darkroom. The known
RTK-specific kinase inhibitor, PD153035, was used as a positive
control compound for EGFR kinase inhibition; SU5416 for Flk1
kinase inhibition; AG1295 for PDGFR-b kinase inhibition; and
CB676475 (4-[(40-chloro-20-fluoro)phenylamino]-6,7-dimethoxy-
quinazoline) was used as a positive control for both Flt1 and
Flk1 kinase inhibition. Data were graphed as a percent of cells
receiving growth factor alone and IC50 values were determined
from two to three separate experiments (n = 8–24) using non-
linear regression dose-response analysis with Prism 5.0 software
(GraphPad, San Diego, CA). In each case, the activity of a positive
control inhibitor did not deviate more than 10% from the IC50
values listed in the text.
4.1.12. 5-Chloro-N4-(3-methoxyphenyl)-9H-pyrimido[4,5-b]
indole-2,4-diamine (8)
Compound 8 (synthesized from 14 (85 mg, 0.20 mmol) as de-
scribed for 7; reaction time = 2 h): yield 92%; TLC Rf 0.42 (CHCl3/
MeOH, 10:1 with 3 drops concentrated NH4OH); mp 242–243 °C;
1H NMR (DMSO-d6) d 3.78 (s, 3H, CH3), 6.48 (br s, 2H, NH2, exch),
6.59–6.62 (m, 1H, Ar), 7.18–7.23 (m, 3H, Ar), 7.27–7.31 (m, 2H,
Ar), 7.66 (m, 1H, Ar), 9.06 (s, 1H, 4-NH, exch), 11.72 (s, 1H, 9-NH,
exch). Anal. (C17H14ClN5O) C, H, N, Cl.
4.7. CYQUANT cell proliferation assay
4.3. Cells
As a measure of cell proliferation, the CYQUANT cell counting/
proliferation assay was used as previously described.34 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 a BioTek plate
reader (Biotek, Winooski, VT). A positive control used for cytotox-
icity in each experiment was cisplatin, 23. Data are graphed as a
percent of cells receiving growth factor alone and IC50 values deter-
mined from two to three separate experiments (n = 6–15) using
non-linear regression dose-response analysis with Prism 5.0 soft-
ware (GraphPad, San Diego, CA).
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).
4.4. Chemicals
All growth factors (bFGF, VEGF, EGF, and PDGF-b) were pur-
chased from Peprotech (Rocky Hill, NJ). PD153035, SU5416,
AG1295, and CB676475 (4-[(40-chloro-20-fluoro)phenylamino]-
6,7-dimethoxyquinazoline) were purchased from Calbiochem
(San Diego, CA). The CYQUANT cell proliferation assay was from
Molecular Probes (Eugene, OR). All other chemicals were from Sig-
ma Chemical unless otherwise noted.
4.8. Statistics
All analysis was done using Prism 5.0. (GraphPad Software, San
Diego, CA)
4.5. Antibodies
4.9. Molecular modeling and computational studies
The PY-HRP antibody was from BD Transduction Laboratories
(Franklin Lakes, NJ). Antibodies against EGFR, PDGFR-b, FGFR-1,
Flk-1, and Flt-1 were purchased from Upstate Biotech (Framing-
ham, MA).
The X-ray crystal structure of VEGFR2 at 1.71 Å resolution was
obtained from the protein database (PDB ID 1YWN).39 This crystal
structure contains VEGFR2 in complex with a novel 4-amino-
furo[2,3-d]pyrimidine. Docking studies were performed using Lead
IT.27 The protein was prepared in Lead IT using default settings.
Docking with Lead IT was carried out in by defining the active site
as a sphere of ꢁ6.5 Å from the X-ray crystal structure ligand. The
protonation states utilized for the proteins and the ligands were
calculated using the default settings. Water molecules in the active
site were permitted to rotate freely. Ligands for docking were pre-
pared using MOE 2010.1040 and energy minimized using the
4.6. Phosphotyrosine ELISA
Cells used were tumor cell lines naturally expressing high
levels of EGFR (A431), Flk-1 (U251), Flt-1 (A498), PDGFR-b (SF-
539), and FGFR-1 (NIH OVCAR-8). 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/mtargets/mt_index.html).
Briefly,
MMFF94X forcefield to a constant of 0.05 kcal/mol. Triangle
cells at 60–75% confluence were placed in serum-free medium
for 18 h to reduce the background of phosphorylation. Cells were
always >98% viable by Trypan blue exclusion. Cells were then
matching was used as the placement method and the docked poses
were scored using default settings. The docked poses were ex-
ported and visualized in MOE. The docking protocol was validated
by re-docking the X-ray crystal structure ligand. The best docked
pose in the re-docking study had an RMSD of 0.7686 Å. Docking
studies were performed for 3 in Lead IT using a similar procedure.
Poses from the docking experiment performed in Lead IT were ex-
ported and visualized in MOE.
pretreated for 60 min with 10, 3.33, 1.11, 0.37, and 0.12 lM
compounds followed by 100 ng/ml EGF, VEGF, or PDGF-BB for
10 min. The reaction was stopped and cells permeabilized by
quickly removing the media from the cells and adding ice-cold
Tris-buffered saline (TBS) containing 0.05% Triton X-100, prote-
ase inhibitor cocktail, and tyrosine phosphatase inhibitor cock-
tail. The TBS solution was 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 were further exposed to block
(TBS with 1% BSA) for 1h, washed, and then a horseradish perox-
idase (HRP)-conjugated phosphotyrosine (PY) antibody added
overnight. The antibody was removed, cells were washed again
Acknowledgments
This work was supported, in part, by the National Institutes of
Health and National Cancer Institute Grant CA98850 (A.G.) and
the Duquesne University Adrian Van Kaam Chair in Scholarly
Excellence (A.G.).

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A. Gangjee et al. / Bioorg. Med. Chem. 21 (2013) 1857–1864
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