The contribution of a 2-amino group on receptor tyrosine kinase inhibition and antiangiogenic activity in 4-anilinosubstituted pyrrolo[2,3-d]pyrimidines

Article abstract of DOI:10.1016/j.bmcl.2010.03.064

Comparison between a series of pyrrolo[2,3-d]pyrimidines with and without the 2-amino group is presented in order to determine the validity of our hypothesis that inclusion of this group improves potency against receptor tyrosine kinases (RTK). The 2-amino analogs were better against epidermal growth factor receptor (EGFR) and platelet derived growth factor-β (PDGFR-β) in whole cell inhibition assays and in the A431 cytotoxicity assay compared to the 2-desamino analogs. However, the 2-desamino analogs were more potent inhibitors against vascular endothelial growth factor-2 (VEGFR-2) than the corresponding 2-amino compounds. In addition, none of the 2-desamino compounds exhibited better anti-angiogenic activity in the chorioallantoic membrane (CAM) assay as compared to the standard and were only micromolar inhibitors. This study validates our original hypothesis that the inclusion of a 2-amino group in pyrrolo[2,3-d]pyrimidines improves multiple RTK inhibition and antiangiogenic activity.

Full text of DOI:10.1016/j.bmcl.2010.03.064

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3177–3181
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The contribution of a 2-amino group on receptor tyrosine kinase inhibition
and antiangiogenic activity in 4-anilinosubstituted pyrrolo[2,3-d]pyrimidines
a
b
b
Aleem Gangjee a, , Ojas A. Namjoshi , Michael A. Ihnat , Aaron Buchanan
*
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, 600 Forbes Avenue, Pittsburgh, PA 15282, USA
b Department of Cell Biology, School of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73126, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Comparison between a series of pyrrolo[2,3-d]pyrimidines with and without the 2-amino group is pre-
sented in order to determine the validity of our hypothesis that inclusion of this group improves potency
against receptor tyrosine kinases (RTK). The 2-amino analogs were better against epidermal growth fac-
tor receptor (EGFR) and platelet derived growth factor-b (PDGFR-b) in whole cell inhibition assays and in
the A431 cytotoxicity assay compared to the 2-desamino analogs. However, the 2-desamino analogs were
more potent inhibitors against vascular endothelial growth factor-2 (VEGFR-2) than the corresponding
2-amino compounds. In addition, none of the 2-desamino compounds exhibited better anti-angiogenic
activity in the chorioallantoic membrane (CAM) assay as compared to the standard and were only micro-
molar inhibitors. This study validates our original hypothesis that the inclusion of a 2-amino group in
pyrrolo[2,3-d]pyrimidines improves multiple RTK inhibition and antiangiogenic activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 December 2009
Revised 12 March 2010
Accepted 12 March 2010
Available online 24 March 2010
Keywords:
Tyrosine kinase inhibitors
Antitumor agents
Structure–activity relationship
Molecular modeling
Receptor tyrosine kinases (RTKs) are a subfamily of protein
tyrosine kinases, which play key roles in tumor growth, survival
and dissemination.1 A variety of growth factors particularly vascu-
lar endothelial growth factor (VEGF), epidermal growth factor
(EGF), platelet derived growth factor (PDGF) and their receptors
are overexpressed in several tumors. These growth factors and
their receptors are directly or indirectly involved in the growth
and metastasis of tumors.2
Angiogenesis is the formation of new blood vessels from exist-
ing vasculature and is essential for both physiological and patho-
logical processes. It is a complex cascade that is tightly regulated
by proangiogenic and antiangiogenic factors.3 Members of the
VEGF family are the predominant stimulators of angiogenesis
and mediation of VEGF expression is one of the main mechanisms
by which tissue vasculature is controlled under normal physiologic
conditions.4–6 In majority of cancers, in addition to this PDGF
subfamilies appear to play essential roles in all stages of tumor
angiogenesis and are able to form autocrine loops, which mediate
cancer cell growth and survival, and drive hematologic malignan-
cies.7 Angiogenesis is a pivotal step in the transition of some solid
tumors from a dormant state to a malignant state; it also provides
a metastatic pathway for solid tumors.8 In addition, angiogenesis
contributes to the development of hematologic malignancies, par-
ticularly multiple myeloma, leukemia, and lymphoma. Inhibition
of tumor angiogenesis affords attractive targets for the develop-
ment of antitumor agents.
A multifaceted approach that targets multiple signaling path-
ways has been shown to be more effective than the inhibition of
a single target.9–11 The most important consequence of inhibiting
multiple RTKs would be to retard tumor resistance by blocking po-
tential ‘escape routes’.12
Several small molecule inhibitors of RTK targeting the ATP bind-
ing site of tyrosine kinases are currently used or are in clinical trials
as antitumor agents (Fig. 1).13,14
Gangjee et al.15 designed a series of 2-amino-4-(m-bromoanili-
no)-6-substituted pyrrolo[2,3-d]pyrimidines as multiple RTK
inhibitors and antiangiogenic agents. A key aspect of this study
was the inclusion of a 2-amino group on the pyrrolo[2,3-d]pyrim-
idine scaffold which was anticipated to utilize an additional hydro-
gen-bond binding site in the Hinge region (ATP binding site)
compared to 2-desamino analogs. ATP, which lacks this 2-NH2
moiety, does not use this site. The RTK inhibitors in the literature,
which contain 6–6 and 6–5 bicyclic ring scaffolds usually do not
have a 2-amino group to exploit this H-bonding site.
Abbreviations: RTK, receptor tyrosine kinase; EGF, epidermal growth factor
receptor; VEGF, vascular endothelial growth factor; PDGF, platelet derived growth
factor; EGFR, epidermal growth factor receptor; VEGFR, vascular endothelial growth
factor receptor; PDGFR, platelet derived growth factor receptor; CAM, chorioallan-
toic membrane; ATP, adenine triphosphate.
Recently Gangjee et al.16 reported a series of N4-(substituted-
phenyl)-6-(2-phenylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-dia-
mines (compounds 12–18, Fig. 2) as RTK inhibitors. In this series
* Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396 5593.
E-mail address: gangjee@duq.edu (A. Gangjee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.064

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A. Gangjee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3177–3181
N
N
H
F
N
N
O
O
N
N
N
HN
N
HN
N
Cl
O
O
O
O
O
HN
N
N
.
O
CH3SO3H
Gefitinib (1)
Erlotinib (2)
Imatinib (3)
N
O
O
S
O
Cl
O
N
O
NH
N
H
HN
O
CF3
O
HN
Cl
O
F
N
F
O
N
H
N
H
N
N
H
Lapatinib (6)
Sorafenib (5)
Sunitinib (4)
HN
N
NH3
NH3
R
Cl
Cl
N
H
O
N
N
Pt
O
N
H
O
N
PD153035: R= 3'-Br (7)
CB676475: R = 2'-F,4'-Cl (8)
Semaxanib (SU5416) (9)
AG1295 (10)
Cisplatin (11)
Figure 1. Structures of RTK inhibitors and standards used in the assays.
Comp R1
R
Comp R1
R
12
13
14
15
16
17
18
NH2 3'-Br
19
20
21
22
23
24
25
H
H
H
H
H
H
H
3'-Br
HN
R
NH2 3'-ethynyl
NH2 3'-CF3
NH2 3'-Br, 4'-F
NH2 3'-CF3, 4'-F
NH2 2'-F, 4'-Cl
NH2 3',4'-(C2H3N)
3'-ethynyl
3'-CF3
3'-Br, 4'-F
3'-CF3, 4'-F
2'-F, 4'-Cl
3',4'-(C2H3N)
N
R1
N
N
H
12-25
Figure 2. 2-Amino16 and 2-desaminopyrrolo[2,3-d]pyrimidine RTK inhibitors.
the nature and substitutions on the anilino moiety determines
both the selectivity and potency against a variety of RTKs in whole
cell assays.
binding sites for ATP and ATP-competitive inhibitors and serve to
anchor the heterocyclic portion of the molecule and appropriately
orient the other parts of the molecule in the ATP binding site. In
addition to this, the 2-NH2 group of 12 (Fig. 3) is only 2.5 Å away
from the backbone carbonyl of Met769 and hence can form an
additional H-bond. For the 2-desamino analog 19 (Fig. 4), however,
there is no group at the 2-position to provide an H-bond with
EGFR. The molecular modeling studies provided further credence
to our hypothesis. The distance of the C-2 of 19 and backbone car-
bonyl of Met769 is ꢀ3.5 Å (Fig. 4). Similarly ATP does not use this
H-bonding site. Hence the inclusion of a 2-amino group in our pyr-
rolo[2,3-d]pyrimidine analogs was also expected to perhaps im-
prove selectivity. Thus 19–25 were designed so as to compare
their biological activities with 12–18.
It was important to validate our hypothesis regarding the inclu-
sion of a 2-amino group to increase binding and potency of RTK
inhibitors.17 This was especially crucial because most of the mar-
keted tyrosine kinase inhibitors such as 1 (gefitinib), 2 (erlotinib)
and 6 (lapatinib) including the substrate ATP lack any substitution
at their corresponding 2-position. Hence compounds 19–25 (Fig. 2)
were designed as 2-desamino analogs of 12–18, in order to com-
pare their activities as RTK inhibitors. Substitutions on the 4-anili-
no ring were kept same as 12–18 from our previous report16 to
allow us for one-to-one comparison between 2-amino analogs
(12–18) and 2-desamino analogs (19–25).
Superimposition of 12 (Fig. 3) and 19 (Fig. 4) on to ATP from its
crystal structure with EGFR (PDB id: 2gs619) using MOE2007.0918
showed that the pyrrolo[2,3-d]pyrimidine ring fits in the same
region as the adenine ring of ATP. The N-3 and 4-NH moieties of
both 12 and 19 are within H-bond distances (ꢀ3 Å) from the back-
bone NH of Met769 and backbone carbonyl of Gln767, respectively,
in the Hinge region of the binding pocket. These are important
The synthesis of 19–25 is shown in Scheme 1. Intermediate 2716
(obtained from phenyl propionic acid in two steps) was treated
with the free base of ethoxycarbonylacetamidine hydrochloride
2820,21 (obtained from ethyl cyanoacetate) at reflux to afford the
pyrrole 29.22 Pyrrolo[2,3-d]pyrimidine 30 was synthesized from
29 by cyclization with formamide.23 Chlorination of 30 with phos-
phorous oxychloride afforded 31. Nucleophilic displacement of the

A. Gangjee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3177–3181
3179
Figure 3. Stereoview of compound 12 in the active site of EGFR [Superimposed on ATP from its crystal structure (pdb id: 2gs6)]; generated using MOE2007.0818 (Hydrogens
removed for clarity).
Figure 4. Stereoview of compound 19 in active site of EGFR [Superimposed on ATP from its crystal structure (pdb id: 2gs6)]; generated using MOE2007.08.118 (Hydrogens
removed for clarity).
4-chloro moiety of 31 with appropriate anilines 32a–g in isopropyl
alcohol at reflux for 4 h afforded 19–24 (catalytic amounts of conc
HCl were added at the start of the reaction) and 25 (no conc HCl
needed).
CYQUANTÒ, a DNA intercalating dye that has been shown to give a
linear approximation of cell number27 and are listed in Table 1. Fi-
nally, the effect of selected compounds on blood vessel formation
was assessed using the chicken embryo chorioallantoic membrane
(CAM) assay, a standard test for angiogenesis28 and are listed in Ta-
ble 1. In this assay, purified angiogenic growth factors are placed
locally on a vascularized membrane of a developing chicken em-
bryo together with possible inhibitors. Digitized images of the vas-
culature are taken at 48 h after growth factor administration and
the number of vessels per unit area evaluated as a measure of vas-
cular density.
RTK inhibitory activity of the compounds 19–25 were evaluated
using human tumor cells known to express high levels of EGFR,
VEGFR-1, VEGFR-2 and PDFGR-b using a phosphotyrosine ELISA
cytoblot24,25 and are listed in Table 1. Compounds known to inhibit
a particular RTK were used as positive controls for these assays.
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 mea-
sured using inhibition of the growth of A431 cancer cells known
to overexpress EGFR. EGFR has been shown to play a role in the
overall survival of the cells.26 Cell proliferation was assessed using
Since the IC50 values of RTK inhibitors vary under different
assay conditions, we used a standard (control) compound (Fig. 1)
in each of the evaluations. For EGFR the standard was
7
(PD153035); for VEGFR-1 the standard was 8 (CB676475); for

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A. Gangjee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3177–3181
EtOOC
H2N
COOH
a
Br
COOEt
N
H
O
HN
NH2
28
HCl
26
27
29
R
O
N
Cl
N
HN
b
c
d
HN
N
N
NH2
N
H
N
H
N
N
H
R
32a-g
30
31
19-25
Scheme 1. Synthesis of 19–25. Reagents and conditions: (a) Triethylamine, ethyl acetate, reflux, 25 min; (b) Na, MeOH, formamide, 150 °C, 12 h; (c) phosphorous
oxychloride, reflux, 3 h; (d) isopropanol, 2–3 drops concd HCl (except for 25), reflux.
Table 1
IC50 values (lM) of kinase inhibition, A431 cytotoxicity and inhibition of the CAM assay
Compd #
R
EGFR inhibitiona VEGFR-1 inhibitiona VEGFR-2 inhibitiona PDGFR-b inhibitiona A431 cytotoxicitya CAM angiogenesis inhibitiona
19
(12)
20
(13)
21
(14)
22
(15)
23
(16)
24
(17)
25
30-Br
>200
(0.3 0.042)
>200
(>200)
>200
>200
>200
(22.8 4.7)
>200
(122 30.1)
>200
129.4 30.1
(45.1 8.9)
142.3 23.5
(>200)
>200
(>200)
>200
(>200)
164.3 18.1
(>200)
>200
112.3 18.1
(>50)
49.4 8.0
(>200)
22.1 4.1
(>200)
25.2 5.2
(132.1 16.7)
57.5 7.1
(>200)
8.5 2.4
(11.8 3.7)
>500
(100.4 22.1)
>500
(>500)
192.4 20.8
(>500)
>500
(90.0 17.8)
>500
(>500)
104.2 16.7
>500
(28.6 8.2)
>500
(8.5 1.1)
>500
(4.6 0.67)
>500
(6.8 0.71)
>500
(9.8 1.0)
43.0
(1.4 0.12)
81.9 10.1
1.93 0.09
(19.5 5.1)
5.12 0.46
(1.1 0.07)
5.63 0.67
(0.03 0.004)
2.14 0.39
(5.9 0.7)
19.6 5.9
(0.12 0.12)
36.9 3.2
(8.6 7.6)
20.1 4.8
30-Ethynyl
30-CF3
30-Br, 40-F
30-CF3, 40-F
20-F, 40-Cl
40.5 9.3
(>200)
31.6 8.8
(1.32 0.09)
56.1 8.7
(>200)
223.8 41.3
30,40-
(C2H3N)
(18)
7
8
(>200)
0.2 0.04
(>200)
(24.9 4.1)
10.6 2.7
(77.4 9.1)
(5.5 1.4)
12.6 2.9
(50.6 18.8)
17.7 5.5
9
19.2 1.1
0.085 0.0031
10
11
6.5 1.3
7.65 1.4
a
Values in parentheses are for the corresponding 2-amino compounds.16 All the values are means of three experiments; standard deviation follows the mean value.
VEGFR-2 and the CAM angiogenesis assay the standard was 9
(semaxanib); for PDGFR-b the standard was 10 (AG1295); for the
cytotoxicity study against the growth of A431 cells in culture the
standard was 11 (cisplatin).
It is important to note that this study involves the use of cellular
assays since we believe that the data from such assays can be
extrapolated more accurately for the selection of candidates for
in vivo studies than isolated kinase inhibitory studies. However, di-
rect comparison of activities of different compounds becomes dif-
ficult, since these may involve other factors such as cellular
transport and metabolism.
amino analog. Compounds 21, 22 and 24 were inactive against
VEGFR-1.
In the VEGFR-2 inhibition assay, compounds 19–24 showed im-
proved activity compared to the corresponding 2-amino deriva-
tives (12–17). Compound 25 was equipotent with the
corresponding 18. Compounds 21, 22 and 25 were only 2–3-fold
less potent than the standard 9. All the compounds showed 2-digit
micromolar inhibition against VEGFR-2 except 19; which was only
10-fold less potent than the standard 9.
In the PDGFR-b inhibition assay, 20 and 23 were inactive; while
corresponding 2-amino compounds 13 and 16 exhibited 2- to 3-di-
git micromolar inhibition against PDGFR-b. Additionally com-
pounds 21 and 24 were inactive. Compound 19 was equipotent
to the corresponding 2-amino analog 12; while 22 was more po-
tent than its corresponding 2-amino analog 15.
In the A431 cytotoxicity assay all of the 2-desamino com-
pounds 19–25 showed diminished potency compared to the cor-
responding 2-amino analogs 12–18. Compounds 19–23 were
inactive in this assay. Compound 24 was the best compound in
the 2-desamino series; however it was about 300-fold less active
than the corresponding 2-amino compound 17 and sixfold less
active than the standard 11.
Removal of the 2-amino group was detrimental for the inhibi-
tory activity against EGFR (with the exception of 25). Compounds
19–24 were inactive in the EGFR inhibition assay (IC50 >200 lM);
while 25 was about 280-fold less active as compared to the stan-
dard 7. In comparison the corresponding 2-amino compounds 12
and 15–17 showed significant potency against EGFR. Compounds
19 and 24 showed significant loss of activities as compared to
the corresponding 2-amino analogs (12 and 17).
Against VEGFR-1, 20 and 23 showed slightly better potencies
than the corresponding 2-amino compounds 13, 16 and 18; while
19 showed lower VEGFR-1 inhibition than the corresponding 2-

A. Gangjee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3177–3181
3181
In the CAM angiogenesis inhibitory assay all the 2-desamino
References and notes
compounds showed one to two-digit micromolar inhibition of
angiogenesis and were significantly less active than the standard
9. Compounds 20, 21, 23 and 24 were significantly less potent
than corresponding 2-amino analogs (13, 14, 16 and 17). The big-
gest difference was seen for 21 (186-fold less potent than 14) and
for 23 (163-fold less potent than 17). Compounds 19, 22 and 25
were better in potency than corresponding 2-amino analogs (12,
15 and 18).
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.03.064.