Design, synthesis, and X-ray crystal structures of 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.08.044

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structu

Full text of DOI:10.1016/j.bmc.2009.08.044

Bioorganic & Medicinal Chemistry 17 (2009) 7324–7336
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and X-ray crystal structures of
2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase
and dihydrofolate reductase inhibitors
a
a
a
b
b
b
Aleem Gangjee a, , Wei Li , Lu Lin , Yibin Zeng , Michael Ihnat , Linda A. Warnke , Dixy W. Green ,
*
Vivian Cody c, Jim Pace c, Sherry F. Queener d
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, The University of Oklahoma Health Science Center, Oklahoma City, OK 73104, United States
c Hauptman-Woodward Medical Research Institute, Inc., 700 Ellicott St., Buffalo, NY 14203, United States
d Department of Pharmacology and Toxicology, School of Medicine, Indiana University, Indianapolis, IN 46202, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E-
and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and
1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4 Å, respectively) with mouse
DHFR and NADPH as well as 1b with human DHFR (1.5 Å) were determined. The E- and Z-isomers adopt
different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2–
13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase
their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-dia-
mino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8–C9 unsaturated compounds 2–7 and cata-
lytic reduction gave the saturated 8–13. Homologation of the C9-methyl analog maintains DHFR
inhibitory activity. In addition, inhibition of EGFR and PDGFR-b were discovered for saturated C9-homol-
ogated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.
Received 24 June 2009
Revised 17 August 2009
Accepted 18 August 2009
Available online 22 August 2009
Keywords:
PDGFR-b
EGFR
Dihydrofolate reductase
Multitargeted inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
development, progression, and aggressiveness and metastasis of
a variety of solid tumors including head and neck cancers,7 non-
Angiogenesis, the formation of new blood vessels from pre-
existing vasculature, plays a crucial role in the growth and metas-
tasis of solid tumors.1 Angiogenesis and metastasis contribute to
the poor prognosis in patients with angiogenic solid tumors.2 In
addition, angiogenesis has been considered the key step in the
transformation of some tumor cells from the dormant state to
the malignant state. Thus, inhibition of tumor angiogenesis is an
attractive target for the development of new antitumor agents.3,4
Some of the receptors5 involved in angiogenesis are members of
the receptor tyrosine kinase (RTK) superfamily, which includes
vascular endothelial growth factor receptor (VEGFR), epidermal
growth factor receptor (EGFR), platelet-derived growth factor
receptor (PDGFR), and fibroblast growth factor receptor (FGFR)
among several others.6 These RTKs have been implicated in the
small cell lung cancer,8,9 and glioblastomas.10,11 These RTKs pro-
mote signal transduction via autophosphorylation that results in
angiogenesis.12–17
Thus, RTKs are attractive targets for the development of cancer
chemotherapeutic agents.18,19 Several small molecule inhibitors of
RTKs that are directed at the ATP site are currently used clinically
and several are in clinical trials as antitumor agents20 (Fig. 1).
Erlotinib and gefitinib inhibit EGFR that is overexpressed in tu-
mors, and are approved antitumor agents.21
VEGF and its receptor VEGFR-2 play crucial roles in vessel
sprouting and new vessel initiation in early stages of angiogene-
sis.22 Inhibition of VEGFR-2 affords excellent antitumor agents.22
Platelet-derived growth factor receptor (PDGFR) kinases also di-
rectly contribute to tumor growth by modifying the tumor
microenvironment.23
The first generation of therapeutic antiangiogenic agents were
designed to selectively inhibit specific RTKs, but angiogenesis
mechanisms can often be carried out by more than one RTK. Simul-
taneous targeting of two or more RTKs has recently emerged as a
more advantageous strategy that may circumvent resistance that
develops with inhibitors aimed at a single RTK.20 The recently
Abbreviations: RTK, receptor tyrosine kinase; VEGFR, vascular endothelial
growth factor receptor; EGFR, epidermal growth factor receptor; PDGFR, platelet-
derived growth factor receptor; FGFR, fibroblast growth factor receptor; ATP,
adenosine 50-triphosphate; DHFR, dihydrofolate reductase; MTX, methotrexate.
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.044

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7325
F
Br
NH
N
NH
N
Cl
NH
O
OCH3
OCH3
O
O
O
N
N
N
OCH3
OCH3
N
N
OCH3
Gefitinib
Erlotinib
17 (PD153035)
Br
F
H3C
NH
N
N
CH3
CH3
CH3
N
H
OCH3
N
O
N
H
N
O
N
CH3
18 (AG1295)
Semaxanib (SU5416)
Figure 1. Structures of small molecule inhibitors of RTKs.
Vandetanib (ZD6474)
approved inhibitors of multiple RTKs, sunitinib and sorafenib, are
examples of anticancer agents exploiting this strategy.
demonstrated tumor growth inhibition and a decrease of tumor
lung metastases in a mouse model of B16 melanomas.28
Dihydrofolate reductase (DHFR) is an enzyme that catalyzes the
reduction of dihydrofolate to tetrahydrofolate and provides the
precursor for the folate cofactors that transfer one carbon units
in the synthesis of metabolites, including purines and pyrimidines.
Inhibition of DHFR blocks DNA synthesis among other metabolic
pathways and causes cell death. Thus DHFR inhibitors such as
methotrexate (MTX) (Fig. 1) and the multitargeted folate metabo-
lizing enzyme inhibitor, pemetrexed (PMX) (Fig. 1), are clinically
used antitumor agents. A variety of classical antifolates (with the
The first objective of the present study was to separate and indi-
vidually evaluate the E- and Z-isomers, 1a and 1b, respectively, to
ascertain and compare the influence of the geometric isomers on
biological activities. Molecular modeling in the previous report28
predicted two different modes of binding of 1a and 1b to DHFR.
Thus, the second objective of this study was to determine the X-
ray crystal structures of the separated E- and Z-isomers 1a and
1b, respectively, with DHFR. Finally, the RTK inhibitory activity of
the previous compounds compared to standards was very good,
but the DHFR inhibitory activity was marginal. On the basis of
molecular modeling we determined that the 9-CH3 group of the
previous analogs (specifically 1b) and Val115 of human DHFR were
too distant to interact. Hence, a third objective of this study was to
increase DHFR inhibitory effects of the parent 9-CH3 compounds
via homologation of the 9-CH3 moiety to afford 2–7 (Fig. 3) a pro-
pyl, isopropyl, cyclopropyl, butyl, isobutyl, and a sec-butyl, respec-
tively, to provide hydrophobic (van der Waals) interactions with
Val115 of hDHFR. It was also necessary to maintain or improve
the RTK inhibitory activities. Molecular modeling of 2–7 using
the published X-ray crystal structure of VEGFR-2 and a sequence
homology alignment with the X-ray crystal structure of insulin
receptor kinase containing a bound ATP molecule as we reported
previously,28 allowed the superimposition of energy minimized
conformations of 2–7 on ATP in VEGFR-2. This indicated that there
was sufficient space in the area around the R substituent of 2–7 to
allow the C9-alkyl moiety to interact with Cys1045 and Val848 in
the Z- and E-isomers and hence allow activity in VEGFR-2. Since
there is no X-ray crystal structure of PDGFR-b available, homology
modeling suggested that the R substituent in 2–7 should maintain
PDGFR-b activity. In addition, it was also of interest to evaluate the
corresponding saturated analogs 8–13 (Fig. 3) to determine the
contribution, if any, of the C8–C9 double bond and its restricted
conformation to the biological activities (RTK inhibition and DHFR
inhibition).
L-glutamate as in MTX and PMX) and nonclassical antifolates (with
lipophilic aryl side chains) like piritrexim (PTX) (Fig. 1) and trime-
trexate (TMQ) (Fig. 1) have been used as cytotoxic agents in trials
for cancer treatment. The structure of these agents usually contain
a pyrimidine ring that is substituted in the 2- and 4-positions with
amino groups. This 2,4-diaminopyrimidine is usually fused to a
second ring which contains a substitution in the 5- or 6-position.
Several comprehensive structure–activity reviews of DHFR inhibi-
tors are available in the literature.24–26 X-ray crystal structures of
DHFR with 2,4-diamino substituted fused pyrimidine rings demon-
strate that this fused 2,4-diaminopyrimidine ring is protonated at
physiological pHs and forms an ionic bond with a conserved gluta-
mate or aspartate (Glu30 in hDHFR).27 X-ray crystal structures
show that the fused pyrimidine ring of these 2,4-diamino analogs
is rotated 180° compared to that of folate when bound to DHFR.27
RTK inhibitors and antiangiogenic agents are generally cyto-
static and the combinations of such agents with standard cytotoxic
chemotherapeutic agents, including antifolates such as MTX and
PMX among others significantly improves clinical response in tri-
als. We proposed to combine RTK inhibitory activity (cytostatic)
with DHFR inhibitory activity (cytotoxic) in a single molecule to
gain several advantages. The dual targets of RTKs and DHFR should
retard development of resistance and single molecules simplify the
pharmacokinetics and toxicity issues compared to two or more
separate agents. Our initial attempt at providing both cytostatic
activity (via RTK inhibition) and cytotoxic or tumoricidal activity
(via DHFR inhibition) in single molecules involved 5-substituted
2,4-diaminofuro[2,3-d]pyrimidines including the E-isomer 1a and
the Z-isomer 1b28 (Fig. 2). These compounds demonstrated excel-
lent dual RTK inhibitory activities (VEGFR-2 and PDGFR-b) along
with modest DHFR inhibition. In addition, some of the analogs
1.1. HPLC separation and isolation of the Z-isomer 1b
We obtained the pure E-isomer 1a through silica gel chroma-
tography.28 Purification of the Z-isomer 1b required reverse phase
HPLC (WatersÒ 4000 system equipped with an X-BridgeÒ C-18

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A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
NH2 CH3
OCH3
OCH3
CO-L-glu
O
N
NH2
5
9
10
N
CH3
N
4
N
HN
H2N
3 N
6
H2N
N
N
N
H
CO-L-glu
H2N
N
1
N
8
7
2
MTX
PMX
PTX
OCH3
CH3
OCH3
OCH3
NH2
N
NH2 CH3
9
6
OCH3
CH3
8
NH2
N
4
5
N
N
H
OCH3
N
2
3
O
H2N
H2N
N
O
7
H2N
N
1
TMQ
E-isomer 1a
Figure 2. Structures of antifolates MTX, PMX, PTX, TMQ, and E- and Z-isomers 1a and 1b.
Z-isomer 1b
2.
R = Propyl
8.
9.
R = Propyl
R
R
*
OMe
3. R = Isopropyl
4. R = Cyclopropyl
5. R = Butyl
NH2
N
OMe
R = Isopropyl
NH2
10. R = Cyclopropyl
11. R = Butyl
N
N
6. R = Isobutyl
O
H2N
O
12.
13.
R = Isobutyl
H2N
N
7.
R = sec-butyl
R = sec-butyl
2-7
8-13
Figure 3. Target compounds.
19 ꢀ 50 mm column, WatersÒ 2487 Dual k Absorbance Detector).
Multiple variations of conditions were evaluated, including both
isocratic and gradient mobile phase compositions. The solvent sys-
tem with 75% water and 25% acetonitrile (for 0–1 min, 10 mL/min;
for 1 min and beyond, 35 mL/min) was found to be the most effi-
cient. Retention times for the Z- and E-isomers are 6.580 min and
11.453 min, respectively. Purity was confirmed by the same re-
verse phase HPLC system.
1.2. Confirmation of the separation and structure of the E- and
Z-isomers by 1H NMR
In the 1H NMR, the 6-H of the Z-isomer of the furo[2,3-d]pyrim-
idine ring occurs at d 6.06, due to a shielding effect of the phenyl
ring, which is significantly different from the 6-H of the E-isomers,
at d 7.4724 where there is no shielding of the phenyl ring.
1.3. X-ray crystal structure
Figure 4. View of the 2Fo ꢁ Fc difference density map for mDHFR-NADPH-1a,
contoured at 1.2 (blue). These data show a ternary complex with the E-isomer
r
Structural data for the inhibitors 1a and 1b in complex with
NADPH and mouse (m) DHFR, as well as inhibitor 1b with human
(h) DHFR, were determined to validate the binding orientation of
these isomers in the active site of DHFR. As illustrated in Figure
4, the binding of the E-isomer 1a is observed in a ‘flipped’ mode
in mDHFR such that the furo oxygen of the furo[2,3-d]pyrimidine
of 1a occupies the 4-amino position of 2,4-diaminopyrimidine
antifolates such as MTX,27,29 and also as observed for the Z-isomer,
1b for hDHFR and mDHFR (Fig. 5). Compound 1a is the first exam-
ple of a furo[2,3-d]pyrimidine antifolate binding in a ‘flipped’
orientation.
bound with a ‘flipped’ furo[2,3-d]pyrimidine orientation with the furo oxygen of the
furo[2,3-d]pyrimidine occupying the position of the 4-amino group of antifolates
MTX, PTX, etc. The configuration about the double bond is
E
with the 20-
methoxyphenyl ring trans to the furo[2,3-d]pryimidine ring. The 20-methoxyphenyl
ring is tilted nearly 90° from the rest of the structure.
hydrogen bonds with Glu30 (Fig. 6), and is similar to that observed
for folic acid (FA). In this orientation the 7-oxo group of the
furo[2,3-d]pyrimidine of 1a interacts with the backbone functional
groups of Ile7, Val115, the hydroxyl of Tyr121, and the nicotin-
amide carboxamide group of NADPH.32 Similar to the MTX binding
mode,30 the 4-amino group of the Z-isomer 1b makes a number of
hydrogen bonding interactions with the backbone functional
groups of Ile7 (3.0 Å) and Val115 (2.9 Å) and the side chains of
Tyr121 (3.4 Å) and the nicotinamide of NADPH (3.7 Å) that contrib-
The overall structure of the ternary mDHFR and hDHFR com-
plexes with NADPH and 1a or 1b are similar to previously reported
structures.30,31 The furo[2,3-d]pyrimidine ring in the E-isomer 1a
binds with the 2-amino and N3 of the pyrimidine interacting via

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7327
places the 9-methyl group in the E-configuration (the methyl
group is cis and coplanar with the furo ring system (Figs. 4 and
5). In both the E-isomer 1a and Z-isomer 1b mDHFR and hDHFR
structure, the C9-methyl and the 20-methoxyphenyl group occupy
the same position in the active site of mouse DHFR (Figs. 7 and 8).
In fact, modeling studies show that if either 1a or 1b were to
bind with the alternate furo[2,3-d]pyrimidine orientation, that is
if either molecule were to be rotated by 180° about the C2–NH2
bond, then the 20-methoxyphenyl ring would make unfavorable
contacts to residues Thr56, Ile60, and Leu67 in the active site.
These data demonstrate the influence of conformational flexibility
of DHFR in adapting different binding modes in the active site. In
addition, these results indicated that both mDHFR and hDHFR
show a slight but not statistically significant preference for the Z-
isomer within the active site binding pocket.
On the basis of molecular modeling Gangjee et al.28 had pre-
dicted that the Z-isomers of these furo[2,3-d]pyrimidines could
bind in the normal mode and the E-isomers could bind in the
flipped mode. The X-ray crystal studies of the individual E- and
Z-isomers do indeed confirm the predicted orientations of the E-
and Z-isomer binding to DHFR.
1.4. Molecular modeling
Figure 5. View of the 2Fo ꢁ Fc difference electron density map for mDHFR-NADPH-
1b, contoured at 1.2 (blue). The red X represents water molecules. These data
r
show a ternary complex with the inhibitor 1b bound with a normal antifolate
orientation with N1 and the 2-amino group interacting with Glu30 (E30 in Figs. 4
and 5). The configuration about the double bond is Z with the 20-methoxyphenyl
ring cis to the furo[2,3-d]pyrimidine ring.
Molecular modeling using SYBYL 8.034 and superimposition of the
E-isomer 3 (Fig. 3) on 1a crystallized with mDHFR indicated that,
like compound 1a, the E-isomer of 3 can bind in the mDHFR bind-
ing pocket with a ‘flipped’ orientation (Fig. 9) such that the furo
oxygen of the furo[2,3-d]pyrimidine occupies the 4-amino position
observed in the binding of 2,4-diaminopyrimidine antifolates like
MTX.30 In addition, the C9 isopropyl moiety of the E-isomer of 3
makes hydrophobic contact with Val115 as does the C9 methyl
moiety of 1a. The van der Waals distance of the methyl of the
C9-isopropyl group of E-3 from Val115 is 3.48 and 4.10 Å for the
methyl and methylene of Val115, respectively, which is much clo-
ser than that for the C9-methyl group of 1a and was expected to
improve the hDHFR inhibitory activity. The other methyl group
of the C9-isopropyl moiety of E-3 interacts with the methyl and
methylene groups of Ile60 as shown in Figure 9.
ute to the tight binding of antifolates.30 The Z-isomer 1b places the
2-methoxyphenyl ring cis to the furo[2,3-d]pyrimidine ring. In this
orientation, the 9-methyl and the 20-methoxy methyl make van der
Waals contacts with the methyl of Thr56 (3.6 and 3.8 Å, respec-
tively). On the other hand, in the ‘flipped’ orientation, the furo oxy-
gen of 1a makes much weaker contacts to Ile7 (3.2 Å), Val115
(3.4 Å), Tyr121 (3.4 Å), and the nicotinamide of NADPH (3.7 Å).
The 9-methyl and 20-methoxy methyl contacts with Thr56 are
3.9 and 4.6 Å, respectively. The 4-amino group of the E-isomer 1a
interacts with a water molecule but does not make close contact
to Glu30.
Similarly, molecular modeling (SYBYL 8.0) and superimposition of
the Z-isomer of 3 onto 1b in the mDHFR X-ray crystal structure
showed that like 1b, the Z-isomer of 3 could also bind with mDHFR
Inspection of the electron density profile for the inhibitor fur-
ther reveals that the configuration about the bridging double bond
Figure 6. PyMOL33 stereoview of the active site of mDHFR ternary complex with NADPH and 1a highlighting the contacts to the furo[2,3-d]pyrimidine ring oxygen. The
closest contacts are with the carbonyl of Ile7 and the hydroxyl of Tyr121 and the nicotinamide group of NADPH. These data show that inhibitor 1a binds with furo[2,3-
d]pyrimidine ring flipped from the normal orientation observed for most 2,4-diaminopyrimidine inhibitors.

7328
A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
Figure 7. (PyMOL)33 Superimposition of 1a (cyan) and 1b (green) in the active site of mDHFR highlighting the different modes of binding for the E- and Z-isomers of the
furo[2,3-d]pyrimidine analogs.
Figure 8. (PyMol)33 Superimposition of 1b in human DHFR (green) and 1b in mouse DHFR (violet) highlighting those resides in the active site that make contact with the
inhibitor.
using the ‘normal’ 2,4-diaminopyrimidine ring binding geometry
with the furo oxygen of the furo[2,3-d]pyrimidine near Glu30
and the 4-amino interacting near the cofactor nicotinamide ring
(Fig. 10).
In addition, the C9-isopropyl moiety of the Z-isomer of 3 makes
better hydrophobic contact with Val115 than the C9-methyl moi-
ety of 1b. The van der Waals distance from one of the methyl moi-
eties of the C9-isopropyl group of the Z-isomer 3 to the methyl and
methylene of Val115 is 3.45 and 3.72 Å, respectively ( Fig. 10),
which is much closer than that for the C9-methyl group of 1b. In
addition the other methyl group of the C9-isopropyl moiety of Z-
3 interacts with Ile60 at 3.53 Å (Fig. 10). Thus, it was anticipated
that both the E- and Z-isomers of 3 would have better DHFR inhib-
itory activity compared to 1a and 1b. Further molecular modeling
34
Figure 9. Stereoview (SYBYL
)
E-isomer of 3 (green) superimposed on the crystal
also indicated that larger alkyl substitutions such as cyclopropyl, n-
butyl, isobutyl, sec-butyl could also be accommodated into this
binding pocket at the C9-position. These molecular modeling stud-
structure of the E-isomer 1a (not shown) bound to the ternary complex of mouse
DHFR and NADPH, showing contacts of the C9-isopropyl of 3 with residues Val115
and Ile60 of mouse DHFR.

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7329
2–7 were E/Z-mixtures which were difficult to separate using flash
or column chromatography. Hydrogenation of compounds 2–7 in
CH3OH/CHCl3 afforded the desired reduced compounds 8–13 as
racemic mixtures with yields that varied from 35% to 65%.
3. Biological evaluation and discussion
Kinase activity of the compounds 1b and 2–13 were evaluated
using human tumor cells known to express high levels of VEGFR-
2 (Flk-1, KDR), EGFR, and PDGFR-b using a phosphotyrosine
ELISA.40,41 The effect of the compounds on cell proliferation was
measured using A431 human epithelial carcinoma cells that over-
express EGFR. Finally, the effect of selected compounds on blood
vessel formation was assessed using the chicken embryo chorioa-
llantoic membrane (CAM) assay, a standard test for angiogenesis.42
All the experimental methods for evaluation were as previously
described by Gangjee et al.43 Since the IC50 values of the same com-
pound vary under different assay conditions, we used a standard
(control) compound in each of the evaluations to provide a valid
comparison with the synthesized analogs. For VEGFR-2 the stan-
dard was semaxanib ( Fig. 1); for EGFR the standard was 17
(PD153035, Fig. 1); for PDGFR-b the standard was 6,7-dimethyl-
2-phenyl quinoxaline ( Fig. 1, AG1295) 18; for the cytotoxicity
study against the growth of A431 cells in culture the standard
was 17. Semaxanib was also used as the standard for the CAM
assay.
As shown in Table 1 the E-isomer 1a was more potent in all of
the assays listed compared to the Z-isomer 1b, thus indicating that
the E-configuration is preferred for RTK inhibition. In the EGFR as-
say (Table 1), most of the unsaturated compounds 2–7 were inac-
tive. In contrast to the previous report of the C9-methyl analogs,28
the saturated analogs of the homologated series (8–11) demon-
strated good inhibitory activity against EGFR. Among these com-
pounds the propyl analog 8, showed an IC50 value significantly
better than 1a and only ninefold less than the standard 17. A mod-
erate EGFR inhibitory activity was obtained for 9 and 11 (Table 1).
Most of the side chain saturated analogs were more potent than
the unsaturated analogs. These results indicate that the restricted
C8@C9 conformation is not conducive to potent EGFR inhibitory
activity and depends more on the size of the C9-alkyl substitution.
Against VEGFR-2 the most potent compound was 13, which was
34
Figure 10. Stereoview (SYBYL
)
Z-isomer of compound 3 (green) superimposed on
the crystal structure of the Z-isomer 1b (not shown) bound to ternary complex of
mDHFR and NADPH, showing contacts of the C9-isopropyl of 3 with residues Ile60
and Val115 of mDHFR.
ies afford credence to our original hypothesis that homologating
the 9-CH3 moiety should increase hDHFR inhibition.
2. Chemistry
The previously described method28 was modified for the syn-
thesis of 5-(chloromethyl)furo[2,3-d]pyrimidine-2,4-diamine, 16
(Scheme 1). Reactions of 16 with the corresponding ketones using
Wittig type condensations under basic conditions afforded the de-
sired target compounds 2–7. The required alkyl 2-methoxyphenyl
ketones were synthesized according to literature methods.35–38
The crucial intermediate, 5-(chloromethyl)furo[2,3-d]pyrimidine-
2,4-diamine 16, was readily obtained by the condensation of 2,6-
diaminopyrimidin-4-ol 14 and 1,3-dichloroacetone 15 in DMF at
room temperature.39 Displacement of the chloride with tributyl-
phosphine followed by NaH in anhydrous DMSO afforded the semi-
stable ylide. This ylide was immediately reacted with the
appropriate ketone to afford the desired olefinic compounds 2–7
in 18–45% yield usually as mixtures of E- and Z-isomers. The yields
of the Wittig reaction were lower than those reported28 for the
lead compounds 1a and 1b. The reason for the lower yields is prob-
ably the steric effect of the larger 9-substitution. Final compounds
NH2
NH2
N
Cl
O
a
b
N
N
Cl
Cl
+
O
H2N
N
OH
H2N
16
14
15
OCH3
OCH3
c
NH2
N
NH2
N
*
R
R
N
N
O
O
H2N
H2N
2-7
2. R = Propyl
3. R = Isopropyl
4. R = Cyclopropyl
5. R = Butyl
6. R = Isobutyl
7. R = sec-butyl
8-13
8. R = Propyl
9. R = Isopropyl
10. R = Cyclopropyl
11. R = Butyl
12. R = Isobutyl
13. R = sec-butyl
Scheme 1. Reagents and conditions: (a) DMF, rt, 24 h; (b) NaH, tributylphosphine, alkyl-2-methoxyphenyl ketones, DMF, 60 °C, 3 h; (c) Pd/C, H2, 55 psi, MeOH/CHCl3.

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A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
Table 1
RTK inhibitory data and antiangiogenic activity IC50
(
l
M)43
VEGFR-2 kinase inhibition
Compd
E/Z ratio
EGFR kinase inhibition
PDGFR-b kinase inhibition
A431 cytotoxicity
CAM angiogenesis inhibition
1a
1b
2
3
4
5
6
7
8
9
10
11
12
13
17
E-Isomer
Z-Isomer
2:1
3:1
2:1
3:2
2:1
3:2
>50
>200
12.8 1.6
151.9
88.2 14.3
>200
>200
>200
>200
>200
>200
118.0 16.3
>200
10.3 1.2
30.3
17.8 2.4
>500
>500
>500
>500
>500
>500
5.0 0.9
13.7
ND
>250
>500
48.3 5.1
9.5 1.4
>500
18.6 3.0
12.3 2.9
>500
>500
>500
>500
27.3 5.1
106.3 17.3
12.6 1.4
0.08 0.009
17.8
160.3 20.1
111.4 18.2
>200
529.3 65.2
>200
22.3 3.7
5.9 0.7
5.0 0.6
2.0 0.3
16.7 1.8
92.2 10.5
11.7 2.3
5.0 0.7
5.3 0.6
10.6 1.3
9.3 0.8
5.6 0.61
>200
2.2 0.31
18.2 2.2
56.5 7.2
13.4 2.6
>500
>200
>200
17.4 1.5
>500
>500
>500
>200
0.2 0.03
18
6.2 0.3
Semaxanib
2.4 0.2
0.03 0.004
Data represent a sample size of 7–16 from 2 to 3 separate experiments.
similar to the parent 1a and sevenfold less than the standard VEG-
FR-2 inhibitor semaxanib. Among the side chain unsaturated ana-
logs, increasing the chain length or bulk at the C9-position beyond
a propyl drastically reduced VEGFR-2 inhibitory activity.
lation is that transport and/or other factors such as DHFR inhibition
(Table 2) may influence the cytotoxic activity against A431 cells in
culture. In the CAM assay, none of these compounds 2–13 were
more potent than lead compound 1a or the standard semaxanib.
Compounds 1a, 1b, and 2–13 were also evaluated as inhibitors
of mammalian DHFR (human (h), rat liver (rl), and mouse (m)), and
of Pneumocystis carinii (Pc) DHFR, and Toxoplasma gondii (Tg) DHFR
(Table 2). Against hDHFR, rlDHFR, and mDHFR the IC50 values of
the analogs showed a similar trend. Among the unsaturated ana-
logs 2–4 and 7 were most potent against the mammalian DHFRs,
but these compounds were not consistently active against PcDHFR
and TgDHFR; compounds 3, 4, and 7 were active against TgDHFR
but not against PcDHFR. Since the only variation in these analogs
is the alkyl C9-substituent clearly this moiety dictates both the po-
tency and the selectivity for DHFR in this series.
Among the reduced analogs 8–13 only compounds 9 and 10
showed consistently potent inhibition of mammalian DHFR; nei-
ther of these compounds was a strong inhibitor of PcDHFR but they
did inhibit TgDHFR. The similar activity of the 3 and 9 pair as well
as the 4 and 10 pair argues that for some of these C9-alkyl substi-
tuted compounds conformational restriction about the C8–C9 bond
did not play a major role in the inhibitory activity against DHFR. In
other cases, conformational restriction about the C8–C9 bond
clearly did play a role; for example in mDHFR a 20-fold decrease
in inhibition is observed on reduction of 2–8 and a sixfold decrease
is observed on reduction of 5–11.
Compound 2 maintained PDGFR-b activity, similar to 1a, and
improved hDHFR inhibitory activity compared to 1a but is about
sevenfold less potent against VEGFR-2. However, except for 2,
these C9-homologated unsaturated analogs lost their activity
against PDGFR-b and VEGFR-2 compared to 1a and 1b. On the basis
of the data against both VEGFR-2 and PDGFR-b increasing the size
and/or length of the C9-alkyl substitution beyond propyl is detri-
mental to RTK inhibitory activity for the unsaturated analogs. In
the PDGFR-b inhibitory assay, saturated compound 9 was more po-
tent than the standard compound 18 and compounds 2 and 10
were similar to the standard 18 and the parent analog 1a. The
trend indicated that increasing the chain length or bulk at C9 be-
yond an isopropyl was detrimental to PDGFR-b activity.
Cytotoxicity studies against the growth of A431 tumor cells in
culture that overexpress EGFR compared analogs 2–13 with stan-
dard compounds 17 and semaxanib. The most potent compound
was the unsaturated C9-cyclopropyl 4, which was somewhat more
active than the standards. Compounds 6, 7, and 12 were similar to
17 and semaxanib. The other analogs were much less active than
the standard compounds. For compounds 2–13 there was no corre-
lation of the EFGR inhibitory activity with cytotoxicity against
A431 cells in culture. A possible explanation for the lack of corre-
Table 2
Inhibitory concentration (IC50
lM) against DHFR and selectivity ratios
Compd
Human DHFR
rl DHFR
Mouse DHFR
Pc DHFR
rl/Pc
Tg DHFR
rl/Tg
1a
1b
2
3
4
5
6
7
8
16.4
1.9
18.5
10.19
15.2
11.9
10.34
29.9
43.9
9.64
30.8
12.6
10.1
46.7
39.3
34.5
14.5
3.9
77
30
0.24
0.34
ND
ND
0.5
1.2
0.8
0.2
0.72
0.24
ND
1.51
0.98
0.76
4.0
3.94
11.8
7.5
4.62
2.58
1.3
1.59
2.2
1.0
1.8
1.7
1.03
1.80
2.3
2.38
1.45
2.23
12.2
6.35
10.2
33.3
86.5
8.64
25.9
5.09
5.01
93.9
62.7
42.1
7.92
4.14
4.64
31.2
51.9
4.2
162
3.2
3.7
37(30%)
34(29%)
22.2
25.9
52.6
55
42.5
53.6
12.5(12%)
30.9
4.6
30.3
24.9
5.7
30
7
4.44
19.6
22.2
15.4
9
10
11
12
13
187
35.4
44.5
40
45.1
All assays contained 117
lM (saturating concentration) NADPH and saturating concentrations of DHFA, optimized for each enzyme. Final DHFA concentrations in the assays
were as follows: human DHFR 18
l
M; rat liver DHFR 90 M; mouse DHFR 18 M; Pc and Tg DHFR 90 M.
l
l
l

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7331
The saturated compounds also showed some dependence on
the alkyl C9-substituent for potency and selectivity for PcDHFR
and TgDHFR, but none of the compounds were appreciably potent
or selective against these non-mammalian DHFR.
lian DHFR, in keeping with the structure based design rationale of
increasing the length and/or bulk of the alkyl substitution on the
C9-position to afford hydrophobic interactions with Val115 in
hDHFR. In addition, compounds with comparable or better
PDGFR-b inhibitory activity than 1a and 1b were also obtained.
Conformational restriction of the C8–C9 bond was not an impor-
tant criteria for DHFR or PDGFR-b inhibitory activity. Both unsatu-
rated and saturated C8–C9 bond analogs were active. Compounds
with EGFR inhibitory activity and two (9 and 10) with dual EGFR
and PDGFR-b activity along with good hDHFR activity were discov-
ered. The parent compounds had no EGFR activity. Taken together
these results warrant the further development of both the satu-
rated and unsaturated analogs as potential antitumor agents.
Taken together these results suggest that the compounds de-
scribed in this study can serve as lead analogs for the development
of more potent dual RTK and DHFR inhibitors in single molecules.
The hDHFR inhibitory activity also has an effect on A431 cyto-
toxicity. Thus for 4 and 7 there is a direct correlation of the hDHFR
inhibitory activity (Table 2) and the A431 cytotoxicity (Table 1).
Unfortunately the other analogs do not show this correlation.
In order to more carefully compare activities of the E- and Z-iso-
mers and the analogs, we determined Ki values against all three
forms of mammalian DHFR (Table 3). First, we observed a consis-
tent but not quite statistically significant difference in the Ki values
between the E- and Z-isomers, 1a and 1b. In all cases the E-isomer
was less active than the Z-isomer and the difference ranged from
about 2.5- to 4-fold. Moreover, an artificially created E/Z-geometric
isomer (1:1) mixture showed a Ki value between the values for the
two isomers for mDHFR. Among the unsaturated series of analogs,
compounds3, 4, and 7 had Ki values against mDHFR close to that of
1b; for the saturated series the compounds closest to 1b were 9
and 10. Therefore in both series, the isopropyl and the cyclopropyl
substituents showed the best activity against mDHFR; similar pat-
terns were seen with hDHFR and rlDHFR.
One of the goals of synthesizing the analogs was to increase the
hDHFR inhibitory activity of the unsaturated C9-methyl analogs 1a
and 1b. Compounds 9 and 10 come closest to achieving this goal in
that their Ki values are very near that of 1b. In this series where
reduction of the C8–C9 double bond affords conformationally flex-
ible molecules 8–13, RTK inhibitory activity was observed beyond
that for the C9-methyl analogs which were inactive. Compounds
8–11 showed EGFR inhibition and 9 and 10 showed good PDGFR-
b inhibition as well. Thus new lead analogs 9 and 10 with dual
RTK inhibitory activity against EGFR and PDGFR-b (Table 1) along
with acceptable hDHFR activity (Table 2) were discovered.
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
the Bruker WH-300 (300 MHz) and Bruker Avance II 400
(400 MHz, used for compound 1b only) spectrometers. The chem-
ical shift values are expressed in ppm (parts per million) relative to
tetramethylsilane as an internal standard: s, singlet; d, doublet; t,
triplet; q, quartet; m, multiplet; br, broad singlet. The relative inte-
grals of peak areas agreed with those expected for the assigned
structures. Thin-layer chromatography (TLC) was performed on
POLYGRAM Sil G/UV254 silica gel plates with a fluorescent indica-
tor, and the spots were visualized under 254 and 366 nm illumina-
tion. 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 per-
formed by Atlantic Microlab, Inc., Norcross, GA. Element composi-
tions are within 0.4% of the calculated values. Fractional moles of
water or organic solvents frequently found in some analytical sam-
ples of antifolates could not be prevented in spite of 24–48 h of
drying in vacuo and were confirmed where possible by their pres-
ence in the 1H NMR spectra. All solvents and chemicals were pur-
In summary, the E- and Z-isomers of a previously reported mix-
ture were separated and evaluated as inhibitors of DHFR. In addi-
tion, the X-ray crystal structures of these analogs were
determined with mDHFR and hDHFR. Twelve 5-substituted
furo[2,3-d]pyrimidine-2,4-diamines were designed on the basis
of the X-ray crystal structures of the lead E- and Z-isomers 1a
and 1b, respectively, as multitargeted RTK inhibitors along with
hDHFR inhibition in single molecules to afford combination che-
motherapeutic potential in single agents. The biological evaluation
identified analogs3, 4, 7, 9, and 10 as strong inhibitors of mamma-
Table 3
Characterization of isomer and analog interactions with mammalian DHFR
Compd
Mouse DHFR
lM Ki
Human DHFR
l
M Ki
Rat liver DHFR lM Ki
1a
0.16 0.4 (n = 5)
1.6 0.5 (n = 3)
4.6 0.1 (n = 3)
1b
1a + 1b
2
3
4
5
6
7
0.062 0.01 (n = 4)
0.092 0.005 (n = 2)
0.14
0.07
0.08
0.55
0.91
0.07
2.8
0.06
0.06
3.3
0.62
0.78
0.5
0.5 0.26 (n = 2)
0.5
1.6
0.83
1.3
4.3
11
1.1
2.5
0.77
0.65
12
5.3
4.6
1.1
1.4
1.6
1.3
1.1
3.2
4.7
1.0
3.3
1.4
1.1
5.0
4.2
4.0
8
9
10
11
12
13
TMP
MTX
4.9 0.7 (n = 4)
0.000077 0.000017 (n = 10)
14.3 1.4 (n = 6)
0.000073 0.000006 (n = 4)
0.000003 0.0000001 (n = 2)
The Ki values were calculated as described, based on competitive inhibition except for MTX which acted as a tight binding pseudo-irreversible inhibitor. Values in the table
that appear as single values represent Ki values determined from a single, full dose response curve; for others, the n value represents the independent experiments and the
values are means SEM.

7332
A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
chased from Aldrich Chemical Co. or Fisher Scientific and were
used as received.
(MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) (E/Z 2:1) E-isomer d 0.70 (t,
3H, J = 7.6 Hz), 1.14–1.21 (m, 4H), 3.79 (s, 3H), 6.09 (s, 2H), 6.33 (s,
1H), 6.87 (s, 2H), 6.96–7.29 (m, 4H), 7.41 (s, 1H); Z-isomer d 0.72
(t, 3H, J = 7.6 Hz), 1.16–1.21 (m, 4H), 3.86 (s, 3H), 6.09 (s, 2H), 6.13
(s, 1H), 6.42 (s, 2H), 6.65 (s, 1H), 6.93–7.26 (m, 4H). Anal.
(C18H20N4O2) C, H, N.
4.1. (Z)-5-(2-(2-Methoxyphenyl)prop-1-enyl)furo[2,3-d]pyrimi-
dine-2,4-diamine 1b
To a solution of 5-(chloromethyl)furo[2,3-d]pyrimidine-2,4-dia-
mine (800 mg, 4 mmol) in DMSO (5 mL) was added tributylphos-
phine (1.2 mL, 6 mmol). After stirring at 60 °C under N2 for 3 h,
the yellowish green solution was cooled to room temperature
and sodium hydride (72 mg, 3 mmol) was added followed by 1-
(2-methoxyphenyl)ethanone (0.6 mL, 4 mmol). The reaction mix-
ture was stirred at room temperature under N2 overnight. Two
new spots (TLC) with Rf = 0.54 and 0.52 (CH3OH:CHCl3 = 1:5) ap-
peared and the reaction was quenched with methanol. Purification
with a 4 ꢀ 25 cm silica gel column was carried out initially with
chloroform and then 1–2% methanol in chloroform. The fractions
at Rf = 0.52 and 0.54 were collected to give the product as a white
powder with a yield of 40% (677 mg). The mixture containing both
E- and Z-isomers was subjected to preparative reverse phase HPLC
separation. A WatersÒ 4000 system with the X-BridgeÒ C-18
19 ꢀ 50 mm column combined with a WatersÒ 2487 Dual k Absor-
bance Detector (245 nm) was used for this purpose. An isocratic
scheme was adapted for efficient separation. Details: mobile phase
composition, 75% water and 25% acetonitrile; for 0–1 min, 10 mL/
min; for 1 min and beyond, 35 mL/min. The sample was prepared
with 5 mg of mixture dissolved in 5 mL of methanol and injection
was made at 1 mL each time. Retention times for the Z- and E-iso-
mers are 6.580 and 11.453 min, respectively. Purity was confirmed
by the same reverse phase HPLC system: mp 237–239 °C (decom-
posed), 1H NMR (DMSO-d6): d 2.09 (s, 3H, CH3), 3.70 (s, 3H, OCH3),
5.96 (s, 2H, 4-NH2, D2O exchanged), 6.06 (s, 1H, 6-H), 6.53 (s, 2H, 2-
NH2, D2O exchanged), 6.60 (s, 1H, H double bond), 6.88–6.92 (m,
1H, C6H4), 6.96–6.98 (m, 1H, C6H4), 7.02–7.04 (m, 2H, C6H4),
7.27–7.29 (m, 1H, C6H4). Rf 0.52 (CH3OH:CHCl3 = 1:5); HRMS m/z
calcd for C16H17N4O2 [M+H]+, 297.1352; found, 297.1372 [M+H]+.
4.2.2. 5-[(Z/E)-2-(20-Methoxyphenyl)-3-methylbut-1-en-1-yl]-
furo[2,3-d]pyrimidine-2,4-diamine (3)
Using the general method above compound 16 (0.50 g, 2.5 mmol)
and 1-(2-methoxyphenyl)-2-methylpropan-1-one (0.49 g, 2.75 mmol)
afforded 3 (0.16 g, 20%) as white crystals: mp 217–219 °C; TLC
Rf = 0.55 and 0.56 (MeOH/CHCl3, 1: 5); 1H NMR (DMSO-d6) (E/Z 3:1)
E-isomer d 1.05 (m, 6H), 2.73–2.89 (m, 1H), 3.68 (s, 3H), 5.96 (s,
2H), 6.41 (s, 2H), 6.53 (s, 1H), 6.88–7.27 (m, 4H), 7.32 (s, 1H); Z-isomer
1.23 (m, 6H), 2.75–2.98 (m, 1H), 3.78 (s, 3H), 5.99 (s, 2H), 6.10 (s, 1H),
6.45 (s, 2H), 6.53 (s, 1H), 6.90–7.23 (m, 4H). Anal. (C18H20N4O2) C, H, N.
HRMS (EI) calcd for C18H20N4O2 324.1587, found 324.1586.
4.2.3. 5-[(Z/E)-2-Cyclopropyl-2-(20-methoxyphenyl)vinyl]furo-
[2,3-d]pyrimidine-2,4-diamine (4)
Using the general method above compound 16 (0.50 g,
2.5 mmol) and cyclopropyl(2-methoxyphenyl)methanone (0.48 g,
2.7 mmol) afforded 4 (0.12 g, 15%) as white crystals: mp 198–
199 °C; TLC Rf = 0.57 and 0.58 (CH3OH/CHCl3, 1: 5); 1H NMR
(DMSO-d6) (E/Z 2:1) E-isomer d 0.60–0.63 (m, 4H), 1.84–1.96 (m,
1H), 3.84 (s, 3H), 5.96 (s, 2H), 6.45 (s, 2H), 6.53 (s, 1H), 6.88–7.32
(m, 4H), 7.45 (s, 1H); Z-isomer 0.63–0.66 (m, 4H), 1.96–1.97 (m,
1H), 6.06 (s, 2H), 6.50 (s, 1H), 6.77 (s, 2H), 6.94 (s, 1H), 7.01–7.34
(m, 4H). Anal. (C18H18N4O2) C, H, N.
4.2.4. 5-[(Z/E)-2-(20-Methoxyphenyl)hex-1-en-1-yl]furo[2,3-d]-
pyrimidine-2,4-diamine (5)
Using the general method above compound 16 (0.50 g, 2.5 mmol)
and 1-(2-methoxyphenyl)pentan-1-one (0.52 g, 2.7 mmol) afforded
5 (0.14 g, 17%) as white crystals: mp 195–198 °C; Rf = 0.57 and 0.58
(MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) (E/Z 3:2) E-isomer d 0.70 (m,
3H), 0.85 (m, 2H), 1.14 (m, 2H), 1.29 (m, 2H), 3.80 (s, 3H), 5.96 (s, 2H),
6.41 (s, 2H), 6.59 (s, 1H), 6.93–7.27 (m, 4H), 7.30 (s, 1H); Z-isomer
0.70 (m, 3H), 0.85 (m, 2H), 1.14 (m, 2H), 1.29 (m, 2H), 3.70 (s, 3H),
6.32 (s, 1H), 6.59 (s, 1H), 6.96–7.21 (m, 4H). Anal.
(C19H22N4O2ꢂ0.25H2O) C, H, N.
4.2. General procedure for the synthesis of compounds 2–7
To a solution of 5-(chloromethyl)furo[2,3-d]pyrimidine-2,4-dia-
mine 16, (0.50 g, 2.5 mmol) in anhydrous DMSO (10 mL) was
added tributylphosphine (0.85 g, 3.75 mmol), and the resulting
mixture was stirred at 60 °C in an oil bath for 3 h under N2 to form
the phosphonium salt. The deep orange solution was then cooled
to room temperature. To this solution was added sodium hydride
(95% dispersion in mineral oil, 0.10 g, 3 mmol), followed by the
corresponding aryl ketones (2.75 mmol). The reaction mixture
was stirred at room temperature for 24–32 h. TLC showed the dis-
appearance of 16 and the formation of two products (TLC). The
reaction was quenched with 20 mL methanol, washed with two
portions of 50 mL methanol, and the resulting solution was evapo-
rated under reduced pressure to dryness. To the residue was added
3 g of silica gel and CHCl3 (20 mL) and the slurry was loaded onto a
4 ꢀ 20 cm dry silica gel column and flash chromatographed ini-
tially with CHCl3 (300 mL), then sequentially with 2% MeOH in
CHCl3 (250 mL), 5% MeOH in CHCl3 (300 mL), and 8% MeOH in
CHCl3 (250 mL). Fractions which showed the desired spot on TLC
were pooled and evaporated to dryness and the residue was
recrystallized from ethylacetate to afford the desired olefinic tar-
gets 2–7.
4.2.5. 5-[(Z/E)-2-(20-Methoxyphenyl)-4-methylpent-1-en-1-yl]-
furo[2,3-d]pyrimidine-2,4-diamine (6)
Using the general method above compound 16 (0.50 g, 2.5 mmol)
and 1-(2-methoxyphenyl)-3-methylbutan-1-one (0.50 g, 2.7 mmol)
afforded 6 (0.20 g, 24%) as white crystals: mp 221–223 °C; Rf = 0.59
and 0.61 (MeOH/CHCl3, 1: 5); 1H NMR (DMSO-d6) (E/Z 2:1) E-isomer
d 0.72–0.72 (d, 6H, J = 6.4 Hz), 1.34–1.57 (m, 1H), 2.34–2.36 (m, 4H),
3.81 (s, 3H), 5.95 (s, 2H), 6.08 (s, 2H), 6.36 (s, 1H), 6.93–7.27 (m, 4H),
7.27 (s, 1H); Z-isomer 0.87 (d, 6H, J = 6.2 Hz), 1.48–1.57 (m, 1H),
2.36–2.44 (m, 4H), 6.11 (s, 2H), 6.41 (s, 2H), 6.46 (s, 1H), 6.58 (s,
1H), 6.94–7.25 (m, 4H). Anal. (C19H22N4O2ꢂ0.9H2O) C, H, N.
4.2.6. 5-[(Z/E)-2-(20-Methoxyphenyl)-3-methylpent-1-en-1-yl]-
furo[2,3-d]pyrimidine-2,4-diamine (7)
Using the general method above compound 16 (0.50 g, 2.5 mmol)
and 1-(2-methoxyphenyl)-2-methylbutan-1-one (0.50 g, 2.7 mmol)
affords 7 (0.18 g, 22%) as white crystals: mp 202–204 °C; Rf = 0.62
and 0.64 (MeOH/CHCl3, 1:5); 1H NMR (DMSO-d6) (E/Z 3:2) dE-isomer
0.77–0.82 (t, 3H, J = 7.6 Hz), 0.87–0.91 (m, 3H), 0.93–1.53 (m, 2H),
2.63 (m, 1H), 3.67 (s, 3H), 5.95 (s, 2H), 6.43 (s, 2H), 6.52 (s, 1H),
6.89–7.28 (m, 4H), 7.30 (s, 1H); Z-isomer 0.77–0.82 (t, 3H,
J = 7.6 Hz), 0.89–0.93 (m, 3H), 0.93–1.51 (m, 2H), 2.53 (m, 1H),
4.2.1. 5-[(Z/E)-2-(20-Methoxyphenyl)pent-1-en-1-yl]furo[2,3-d]-
pyrimidine-2,4-diamine (2)
Using the general method above compound 16 (0.50 g, 2.5 mmol)
and 1-(2-methoxyphenyl)butan-1-one (0.49 g, 2.75 mmol) afforded
2 (0.20 g, 25%) as white crystals: mp 222–224 °C; Rf = 0.57 and 0.58

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7333
3.78 (s, 3H), 5.95 (s, 2H), 6.09 (s, 1H), 6.52 (s, 2H), 6.92 (s, 1H), 6.89–
4.3.5. 5-[(R/S)-2-(20-Methoxyphenyl)-4-methylpentyl]furo[2,3-
7.28 (m, 4H). Anal. (C19H22N4O2ꢂ0.3H2O) C, H, N.
d]pyrimidine-2,4-diamine (12)
Using the general method above compound
6 (334 mg,
0.98 mmol) at 50 psi for 24 h afforded 12 (103 mg, 31%) as white
crystals: mp 192–194 °C; Rf = 0.57 (MeOH/CHCl3, 1:9); 1H NMR
(DMSO-d6) d 0.77–0.79 (d, 6H), 1.22–1.66 (m, 1H), 1.90–2.05 (m,
2H), 2.50–2.79 (m, 2H), 3.77(s, 3H), 4.01–4.24 (m, 1H), 5.65 (s,
2H), 5.02 (s, 2H), 6.56 (s, 1H), 6.93–7.29 (m, 4H). Anal.
(C19H24N4O2) C, H, N.
4.3. General procedure for the synthesis of 8–13
To a solution of the olefinic intermediate (0.3–0.5 mmol) in a
mixture of CHCl3 (50 mL) and MeOH (15 mL) was added 5% palla-
dium on activated carbon (0.2 g), and the suspension was hydroge-
nated in a Parr apparatus at 50–55 psi for 12–24 h. TLC indicated
the disappearance of the starting material and the formation of
one major spot. The reaction mixture was filtered through Celite,
washed with 30% MeOH in CHCl3 50 mL. After evaporation of the
solvent, MeOH 50 mL was added to afford a solution. To this solu-
tion was added 5 g silica gel and the mixture was evaporated under
reduced pressure to dryness. This silica gel plug was loaded on a
dry silica gel column and flash chromatographed initially with
CHCl3 150 mL, then sequentially with 1% MeOH in CHCl3
(150 mL), 2% MeOH in CHCl3 (150 mL), and 5% MeOH in CHCl3
(150 mL). Fractions which showed the major spot on TLC were
pooled and evaporated to dryness. The residue was recrystallized
from MeOH, or other solvent combinations as indicated, to afford
the desired target compounds 8–13.
4.3.6. 5-[(R/S)-2-(20-Methoxyphenyl)-3-methylpentyl]furo[2,3-
d]pyrimidine-2,4-diamine (13)
Using the general method above compound
7 (140 mg,
0.4 mmol) at 50 psi for 24 h afforded 13 (85 mg, 60%) as white
crystals: mp 185–187 °C; Rf = 0.52 (CH3OH/CHCl3, 1:9); 1H NMR
(DMSO-d6) d 0.62–0.75 (m, 3H), 0.83–0.91(m, 3H), 1.04–1.17 (m,
2H), 1.19–2.20 (m, 1H), 2.70 (m, 1H), 3.06 (m, 2H), 3.73 (s, 3H),
5.92 (s, 2H), 6.34 (s, 2H), 6.60 (s, 1H), 6.94–7.36 (m, 4H). Anal.
(C19H24N4O2) C, H, N.
4.4. Crystallization and X-ray data collection
Recombinant mouse DHFR was expressed and purified as de-
scribed previously,44 as was the human DHFR enzyme.31 The
mDHFR protein was washed in a centricon-10 with 10 mM HEPES
buffer, pH 7.4 and concentrated to 33.2 mg/mL. The protein was
incubated with NADPH and a 10:1 molar excess of the inhibitor,
1a or 1b, for one hour over ice prior to crystallization using the
hanging drop vapor diffusion method. Protein droplets contained
170–200 mM Tris, pH 8.3, 85 mM Na cacodylate, 25% PEG 4 K,
15% glycerol. Crystals grew over several weeks time 0and are mono-
clinic, space group P21 and diffracted to 2.1 and 1.4 AÅ resolution for
the E- and Z-isomer, respectively. Data were collected at liquid N2
temperatures on a Rigaku RaxisIV imaging plate system with Max-
Flux optics and the data processed with using both Denzo45 and
Mosflm.46 Diffraction statistics are shown in Table 4 for both
complexes.
The human DHFR protein was washed in a Centricon-10 three
times with 50 mM phosphate buffer, pH 6.9 in 100 mM KCl buffer
and concentrated to 5.3 mg/mL and incubated overnight at 4 °C
with a molar excess of NADPH followed by the inhibitor 1a. Protein
droplets contained variable amounts of ammonium sulfate (60–
70%) at pH 6.9 and 3% ethanol. Crystals were grown using hanging
drop vapor diffusion on glass cover slips using experiments were
carried out at 14 °C. Data were collected at the Stanford Synchro-
tron Resource Laboratory on beamline 9–2. Diffraction statistics
for the current refinement are shown in Table 4.
4.3.1. 5-[(R/S)-2-(20-Methoxyphenyl)pentyl]furo[2,3-d]pyrimi-
dine-2,4-diamine (8)
Using the general method above compound
2 (152 mg,
0.46 mmol) at 50 psi for 22 h afforded 8 (100 mg, 45%) as white
crystals: mp 210–212 °C; Rf = 0.54 (MeOH/CHCl3, 1:9); 1H NMR
(DMSO-d6) d 0.75–0.79 (t, 3H, J = 6.9 Hz), 1.04–1.23 (m, 2H),
1.59–1.61 (m, 2H), 2.68–2.78 (m, 2H), 3.29 (m, 1H), 3.76 (s, 3H),
4.03–4.22 (m, 1H), 5.65–5.77 (s, 2H), 5.98 (s, 2H), 6.91 (s, 1H),
6.94–7.22 (m, 4H). HRMS (EI) calcd for C18H22N4O2 327.1821,
found 327.1775.
4.3.2. 5-[(R/S)-2-(2-Methoxyphenyl)-3-methylbutyl]furo[2,3-d]-
pyrimidine-2,4-diamine (9)
Using the general method above compound
3 (115 mg,
0.35 mmol) at 55 psi for 22 h. Following chromatography the frac-
tions containing the product were pooled and evaporated and the
residue recrystallized from MeOH/CH3COCH3 to afford 9 (100 mg,
65%) as white crystals: mp 210–212 °C; Rf = 0.53 (MeOH/CHCl3,
1:9); 1H NMR (DMSO-d6) d 0.95 (d, 6H, J = 6.9 Hz), 1.15 (m, 1H),
1.35–2.18 (m, 2H), 2.81 (m, 1H), 3.75 (s, 3H), 4.03–4.22 (m, 1H),
5.65 (s, 2H), 5.98 (s, 2H), 6.13 (s, 1H), 6.63–7.31 (m, 4H). Anal.
(C18H22N4O2ꢂ0.25CH3COCH3) C, H, N.
4.3.3. 5-[(R/S)-2-Cyclopropyl-2-(20-methoxyphenyl)ethyl]furo-
[2,3-d]pyrimidine-2,4-diamine (10)
The mDHFR structures were solved by molecular replacement
methods using the coordinates for mouse DHFR (2FZJ) in the pro-
gram Molref.46 Similarly, the hDHFR structure was solved by
molecular replacement methods using the coordinates for hDHFR
(1U72). Inspection of the resulting difference electron density
maps were made using the program COOT47 running on a Mac
G5 workstation and revealed density for a ternary complex with
mDHFR (Figs. 3 and 4). To monitor the refinement, a random subset
of all reflections was set aside for the calculation of R free (5%). The
model for the inhibitors E-isomer 1a and Z-isomer 1b were mod-
Using the general method above compound
4 (118 mg,
0.36 mmol) at 55 psi for 24 h afforded 10 (40 mg, 34%) as white
crystals: mp 178–179 °C; Rf = 0.57–0.58 (MeOH/CHCl3, 1:9); 1H
NMR (DMSO-d6) d 0.16 (m, 4H), 0.62 (m, 1H), 2.92 (m, 2H), 3.01
(m, 1H), 3.78 (s, 3H), 4.00–4.11 (m, 1H), 5.97 (s, 2H), 6.37 (s, 2H),
6.95 (s, 1H), 6.96–6.98 (m, 4H). Anal. (C18H20N4O2) C, H, N. HRMS
(EI) calcd for C18H20N4O2 324.1586, found 324.1577.
34
4.3.4. 5-[(R/S)-2-(20-Methoxyphenyl)hexyl]furo[2,3-d]pyrimi-
eled on those using the builder function in SYBYL and the param-
dine-2,4-diamine (11)
eter file for the inhibitors were prepared using the Dundee
PRODGR2 Server website (http://davapc1.bioch.dundee.ac.uk/pro-
grams/prodrg). The final cycles of refinement were carried out
using the program REFMAC5 in the CCP4 suite of programs.46 The
Ramachandran conformational parameters from the last cycle of
refinement generated by PROCHECK48 showed that more than
89.9% of the residues in both E- and Z-isomers mDHFR complexes
have the most favored conformation and none are in the disal-
Using the general method above compound
5 (125 mg,
0.36 mmol) at 50 psi for 19 h afforded 11 (44 mg, 36%) as white
crystals: mp 184–186 °C; Rf = 0.47 (MeOH/CHCl3, 1:9); 1H NMR
(DMSO-d6) d 0.70–0.74 (t, 3H, J = 3.6 Hz), 0.76–1.04 (m, 2H),
1.19–1.63 (m, 2H), 1.75–2.05 (m, 2H), 2.71 (m, 1H), 3.59 (m, 2H),
3.83 (s, 3H), 5.96 (s, 2H), 6.32 (s, 2H), 6.58 (s, 1H), 6.91–7.29 (m,
4H). Anal. (C19H24N4O2) C, H, N.

7334
A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
Table 4
Crystal and refinement parameters for the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines 1a and 1b as a NADPH ternary
complex with mouse DHFR and for the E-isomer 1a with human DHFR
Inhibitor
E-Isomer 1a mDHFR
Z-Isomer 1b mDHFR
E-Isomer 1a hDHFR
Space group
Lattice
P21
P21
P212121
39.91 57.48 74.97
41.43 61.01 43.22
b = 117.33
3d7y
41.64 61.15 43.56
b = 117.33
3d7x
PDB entry
Data collection
Data collection
Low resolution limit
High resolution limit
R merge
Total number of observations
Total number unique
Mean (I)/sd (I)
Overall
RaxisIV
21.85
2.10
0.063
29811
10812
14.6
Outer shell
Overall
RaxisIV
17.82
1.64
0.074
28759
24297
14.6
Outer shell
Overall
SSRL beamline 9-1
45.64
1.50
0.071
Outer shell
2.05
2.05
0.320
2138
773
2.6
1.60
1.60
0.396
2236
788
3.3
95.3
1.8
1.40
1.40
0.109
33058
13.7
95.6
9.8
4606
1.2
93.1
9.4
Completeness
Multiplicity
94.8
2.8
92.8
2.8
98.5
2.5
R factor (%)
R free weighted
Protein atoms
Water molecules
Ramachandran (Procheck)
B factor (protein average)
Rms bonds
16.9
24.7
1553
133
89.9
31.4
0.24
2.60
19.6
24.2
1553
185
90.6
33.8
0.14
1.84
26.0
31.9
1557
64
91.8
24.0
0.13
1.81
0.13
30.9
Rms angles
Rms chiral
0.17
0.12
lowed regions. Coordinates for the mDHFR complexes have been
deposited with the Protein Data Bank (PDB code 3d7y and 3d7x).
Coordinates for the hDHFR complex will be deposited after further
refinement.
other over a range of concentrations. The Km value was determined
by fitting the data to the Michaelis–Menton equation using nonlin-
ear regression methods (Prism 4.0). The value of kcat was deter-
mined from the Vmax value and the protein concentration
(kcat = Vmax[Etot]).
4.5. Dihydrofolate reductase evaluations
Ki values were determined by measuring inhibition of the reac-
tion at two or more concentrations of substrate (DHFA). For the
competitive inhibitors in this study, Ki could be calculated from
the equation:
4.5.1. Kinetic properties
Kinetic properties of the recombinant mouse DHFR were deter-
mined and compared to those of recombinant human DHFR and
native rat liver DHFR (Table 5). The kinetic properties of the recom-
binant mouse DHFR used in these studies are comparable to those
published previously by others. Values for kcat were reported by
Dicker et al.49 to be 2.2 sꢁ1 (measured at 25 °C) and by Thillet
et al.50 as 7 sꢁ1 (measured at 30 °C); our value of 29 sꢁ1 compares
well, considering that the current measurements were made at
Ki ¼ IC50=ð1 þ S=Km
Þ
All determinations of IC50 were made by fitting percent inhibi-
tion data to a single site sigmoidal model with variable slope, using
nonlinear regression methods (Prism 4.0). All inhibition curves re-
quired at least four points within the central segment of the sig-
moidal curve. Statistical comparisons were preformed with InStat
2.03, using the nonparametric Welch t test because variances
among groups could not be shown to be equal; this test is more
conservative that the standard test.
37 °C. Values for the Km for NADPH of 14
(19) do not agree particularly well with each other but the value
reported herein (2.4 M) lies between the two reported values.
The value of 0.32 M we report of the Km for DHFA agrees well
with the value of 0.36
M reported by Dicker et al.49
lM (20) and 0.54 lM
l
l
l
4.6. Receptor tyrosine kinase evaluations
Standard DHFR assays were conducted at 37 °C with continuous
recording of change of OD at 340 nM. The assay contained 41 nM
sodium phosphate buffer at pH 7.4, 2-mercaptoethanol 8.9 mM,
150 mM KCl, and saturating concentrations of NADPH and dihy-
drofolic acid (DHFA). With mouse DHFR, the standard DHFA con-
4.6.1. 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).
centration was 18
l
M, but for the human enzyme standard
M DFHA; 117 M NADPH was used for
conditions included 90
l
l
both DHFR forms. Initial rates of enzyme activity were measured;
rates were linear under standard conditions for about 2 min for
mDHFR and for over 5 min for hDHFR. Activity was linearly related
to protein concentration under these conditions of assay.
Km values were determined by holding either substrate or
cofactor at a constant, saturating concentration and varying the
4.6.2. Chemicals
All growth factors (bFGF, VEGF, EGF, PDGF-BB) were purchased
from Peprotech (Rocky Hill, NJ). PD153035, SU5416, AG1295, and
VEGF kinase inhibitor (4-[40-chloro-20-fluoro)phenylamino]-6,7-
dimethoxyquinazoline) were purchased from Calbiochem (San
Diego, CA). The CYQUANT cells proliferation assay was from Molec-
ular Probes (Eugene, OR). All other chemicals were from Sigma
Chemical unless otherwise noted.
Table 5
Kinetic characterization of mammalian DHFRs
Kinetic parameter Mouse DHFR
Km DHFA ( M) 0.3 0.05 (n = 2)
Km NADPH ( M) 2.42 0.05 (n = 2) 4.0 1.8 (n = 2) 23 (Ref)
kcat (sꢁ1
29 5 (n = 4) 40 2 (n = 8)
Human DHFR
Rat liver DHFR
l
2.7 0.5 (n = 4) 10.8 3.4 (n = 4)
4.6.3. Antibodies
The PY-HRP antibody was from BD Transduction Laboratories
(Franklin Lakes, NJ). Antibodies against EGFR, PDGFR-b, FGFR-1,
l
)

A. Gangjee et al. / Bioorg. Med. Chem. 17 (2009) 7324–7336
7335
Flk-1, and Flt-1 were purchased from Upstate Biotech (Framing-
ham, MA).
(Wild M400; Bannockburn, IL) at 7.5ꢀ and SPOT Enhanced digital
imaging system (Diagnostic Instruments, Sterling Heights, MI). A
grid is then added to the digital CAM images and the average num-
ber of vessels within 5–7 grids counted as a measure of vascularity.
AGM-1470 (a kind gift of the NIH Developmental Therapeutics Pro-
gram) and SU5416 are used as a positive control for antiangiogenic
activity. Data are graphed as a percent of CAMs receiving bFGF/
VEGF and IC50 values estimated from 2 to 3 separate experiments
(n = 5–11) using Probit plots.
4.6.4. Phosphotyrosine cytoblot
Cells used were tumor cell lines naturally expressing high levels
of EGFR (A431), Flk-1 (U251), Flt-1 (A498), 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/mtar-
gets/mt_index.html). Briefly, cells at 60–75% confluence are placed
in serum-free medium for 18 h to reduce the background of phos-
phorylation. Cells were always >98% viable by Trypan blue exclu-
sion. Cells are then pretreated for 60 min with 10, 3.33, 1.11,
Acknowledgments
This work was supported, in part, by the National Institute of
Health Grants, National Cancer Institute CA09885 (A.G.), National
Institute of Allergy and Infectious Diseases AI069966 (A.G.), and
the National Institute of General Medical Sciences GM51670
(V.C.). V.C. acknowledges Jennifer Makin, Jennifer Piraino, and Jes-
sica Nowak for their efforts on the cloning, expression and crystal-
lization of the human DHFR complex. The National Science
Foundation, CHE 0614785, is acknowledged for NMR facilities.
0.37, and 0.12 lM compound followed by 100 ng/mL EGF, VEGF,
PDGF-BB, or bFGF for 10 min. The reaction is stopped and cells per-
meabilized by quickly removing the media from the cells and add-
ing ice-cold Tris-buffered saline (TBS) containing 0.05% Triton X-
100, protease inhibitor cocktail and tyrosine phosphatase inhibitor
cocktail. The TBS solution 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, 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 luminal ELISA substrate (Pierce Chemical, Rockford,
IL) and light emission measured using a plate reader (BMG Labtech,
Durham, NC). The known RTK-specific kinase inhibitor PD153035
was used as a positive control compound for EGFR kinase inhibi-
tion; SU5416 for Flk1 kinase inhibition; AG1295 for PDGFR-b
kinase inhibition; and CB676475 (4-[(40-chloro-20-fluoro)phenyla-
mino]-6,7- dimethoxyquinazoline) 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
calculated from two to three separate experiments (n = 8–17)
using Prism 5.0 (GraphPad Software, San Diego CA).
Supplementary data
Supplementary data (elemental analysis and high-resolution
mass spectra (HRMS) (EI) and HPLC separation. This material is
available free of charge via the Internet at http://pubs.acs.org)
associated with this article can be found, in the online version, at
doi:10.1016/j.bmc.2009.08.044.
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