Novel 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines as multireceptor tyrosine kinase and dihydrofolate reductase inhibitors with antiangiogenic and antitumor activity

Article abstract of DOI:10.1016/j.bmc.2005.04.087

Recent evidence suggests that combination therapy of cancer with receptor tyrosine kinase (RTK) inhibitors, which are usually cytostatic, with conventional chemotherapeutic agents, which are usually cytotoxic, provide an improved treatment option. We have

Full text of DOI:10.1016/j.bmc.2005.04.087

Bioorganic & Medicinal Chemistry 13 (2005) 5475–5491
Novel 5-substituted, 2,4-diaminofuro[2,3-d]pyrimidines as
multireceptor tyrosine kinase and dihydrofolate reductase
inhibitors with antiangiogenic and antitumor activityq
Aleem Gangjee,a,* Yibin Zeng,a,ꢀ Michael Ihnat,b Linda A. Warnke,b Dixy W. Green,b
Roy L. Kisliukc and Fu-Tyan Lind
aDivision of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
600 Forbes Avenue, Pittsburgh, PA 15282, USA
bDepartment of Cell Biology, The University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA
cDepartment of Biochemistry, School of Medicine, Tufts University, Boston, MA 02111, USA
dDepartment of Chemistry, University of Pittsburgh, Pittsburgh, PA 15213, USA
Received 7 June 2004; accepted 25 April 2005
Available online 21 July 2005
Abstract—Recent evidence suggests that combination therapy of cancer with receptor tyrosine kinase (RTK) inhibitors, which are
usually cytostatic, with conventional chemotherapeutic agents, which are usually cytotoxic, provide an improved treatment option.
We have designed, synthesized, and evaluated a series of novel 2,4-diamino-5-substituted furo[2,3-d]pyrimidines with RTK and
dihydrofolate reductase (DHFR) inhibitory activity in single molecules, as potential cytostatic and cytotoxic agents with antitumor
activity. These compounds were synthesized from 2,4-diamino-5-chloromethyl furo[2,3-d]pyrimidine and aryl methyl ketones using
the Wittig reaction to afford the C-8–C-9 unsaturated analogs followed by catalytic reduction to the corresponding saturated com-
pounds. The saturated and unsaturated C-8–C-9 bridged compounds were evaluated as inhibitors of vascular endothelial growth
factor receptor (VEGFR-2, Flk, KDR), epidermal growth factor receptor, and platelet-derived growth factor receptor-b
(PDGFR-b). Selected analogs were also evaluated as antiangiogenic agents in the chicken embryo chorioallantoic membrane
(CAM) assay. The compounds were also evaluated as inhibitors of human (h) DHFR and Toxoplasma gondii (tg) DHFR. In each
evaluation, a known standard compound was used as a comparison. Of the compounds evaluated, compound 32 was as potent as
the standard compounds against VEGFR-2 and PDGFR-b, showing dual inhibitory activity against RTK. This analog was also
highly effective in the CAM assay. A second analog 18 also demonstrated dual VEGFR-2 and PDGFR-b inhibitory activity as well
as potent antiangiogenic activity in the CAM assay. Four additional analogs were also effective against PDGFR-b and in the CAM
assay. An unsaturated C-8–C-9 moiety was necessary for RTK inhibitory activity. Compound 32 also showed inhibitory activity
against hDHFR and tgDHFR, illustrating the multitarget inhibitory potential of these analogs. The biological activity of these ana-
logs also suggests the necessity of an unsaturated C-8–C-9 bridge for dual RTK and DHFR inhibitory activity. Compounds 18 and
32 were also evaluated in a B16 melanoma mouse model and were found to be more active as antitumor agents than methotrexate.
In addition, both 18 and 32 were also active in decreasing lung metastases in a mouse model of B16 melanomas.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
The formation of new blood vessels from existing vascu-
lature is termed angiogenesis.1 Angiogenesis plays a cru-
cial role in the growth and metastasis of solid tumors.2–4
Solid tumors require angiogenesis to grow beyond
q
Presented in part at the 218th American Chemical Society National
Meeting, New Orleans, LA, August 1999, MEDI 223; and the 94th
1–2 mm in diameter and metastasis requires the presence
American Association for Cancer Research National Meeting,
of blood vessels to allow access to the circulation and to
Washington, DC, July 2003, Abstract number 101368.
form tumors at distal sites to the primary tumor. Angi-
ogenesis and metastasis contribute to the poor prognosis
in patients with angiogenic solid tumors.5 Thus, agents
that inhibit the angiogenic process have afforded new
*
Corresponding author. Tel.: +1 412 396 6070; fax: +1 412 396
5593; e-mail: gangjee@duq.edu
ꢀ
Present address: Department of Medicinal Chemistry, University of
Kansas, Lawrence, KS 66045, USA.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.04.087

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A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
paradigms for the treatment of tumors.6,7 Angiogenesis
is primarily a receptor-mediated process by growth fac-
tors that cause signal transduction, for the most part, via
receptor tyrosine kinases (RTK). RTKs consist of fam-
ilies of growth factor receptors such as vascular endo-
thelial growth factor receptor (VEGFR), epidermal
growth factor receptor (EGFR), platelet-derived growth
factor receptor (PDGFR), and fibroblast growth factor
receptor (FGFR) among several others.8 Aberrant
expression or overexpression of EGFR and PDGFR,
both of which are directly or indirectly involved in angi-
ogenesis, have been implicated in the development, pro-
gression, and aggressiveness of a variety of solid tumors.
These include head and neck cancers,9 non-small cell
lung cancer,10,11 glial tumors,12 and glioblastomas.12–14
VEGF is a known survival factor for endothelial cells
of tumor vessels.15–23
Since RTK inhibitors are generally cytostatic against tu-
mors and the combination of RTK inhibitors with cyto-
toxic agents, currently in clinical trials, are anticipated
to provide better efficacy against tumors, we reasoned
that the combination of an RTK inhibitor along with
cytotoxic activity in a single molecule could provide
single agents with ꢁcombination chemotherapeutic
potentialꢂ with both cytotoxic and cytostatic activity.
Because of our long-standing interest in dihydrofolate
reductase (DHFR) inhibitors we elected to use DHFR
as the cytotoxic target.
Dihydrofolate reductase (DHFR) carries out the reduc-
tion of dihydrofolate to tetrahydrofolate (THF), which
is utilized by serinehydroxymethyltransferase to afford
5,10-methylene-tetrahydrofolate (5,10-CH2THF). The
cofactor 5,10-CH2THF serves as the source of the meth-
yl group in the conversion of deoxyuridine monophos-
phate (dUMP) to thymidylate (dTMP) catalyzed by
thymidylate synthase (TS). DHFR inhibitors are well-
established cytotoxic agents used in cancer chemothera-
py.32,33 Methotrexate (MTX) and trimetrexate (TMQ)
(Fig. 1) are examples of such classical and non-classical
antifolates, respectively.34
RTKs are generally transmembrane receptors consisting
of an extracellular growth factor binding domain, a
hydrophobic transmembrane domain, and a cytoplas-
mic domain. The cytoplasmic domain contains regulato-
ry regions and the catalytic tyrosine kinase domain with
binding sites for both ATP and substrates allowing for
autophosphorylation critical for signal transduction
and angiogenesis.7,8 Thus, RTKs are attractive targets
for cancer chemotherapeutic agents. Since RTKs are
present in endothelial cells (VEGFR, PDGFR), tumor
cells (FGFR, PDGFR), and pericytes/smooth muscle
cells (FGFR, PDGFR), inhibition of more than one
RTK could provide synergistic inhibitory effects against
solid tumors.24–26 Several small molecule RTK inhibi-
tors are currently in clinical use and in clinical trials as
antitumor agents (Fig. 1). The majority of these are tar-
geted at the ATP binding sites of tyrosine kinases. The
recent approval of Tarceva (OSI774) and Iressa
(ZD1839), both of which are EGFR kinase inhibitors,
for clinical use against non-small cell lung cancer as well
as Gleevac (STI571) against chronic myeloid leukemia
and gastrointestinal stromal tumors has established
small molecule RTK inhibitors in the clinic as antitumor
agents.
Since inhibition of two or more RTKs have also been
proposed to provide synergistic effects when used in
combination, it was of interest to structurally engineer
single molecules that would possess inhibitory activity
against two or more RTKs along with DHFR inhibi-
tion. Such analogs were expected to display both cyto-
static (RTK inhibition) as well as cytotoxic (DHFR
inhibition) activity. Single agents would overcome the
pharmacokinetic and pharmacodynamic drawbacks as
well as combined toxicities of using two or more sepa-
rate agents. Recently, VEGFR-2 and PDGFR-b, two
RTKs, have been implicated in controlling angiogenesis
at two different stages of the angiogenic process, and it
has been recently shown that inhibition of VEGFR-2
and PDGFR-b with two separate inhibitors, SU5416
and SU6668, respectively, produces a synergistic effect
in early stage as well as late stage pancreatic islet cancer
in mouse models by attacking the angiogenic process at
two different sites.35,36 Thus, we elected to design our
multitargeted inhibitor(s) to inhibit DHFR and VEG-
FR-2 and to evaluate our compounds against
PDGFR-b as well.
There has been considerable discussion in the literature
regarding the use of RTK inhibitors as monotherapy
for cancer or the combination of multiple RTK inhib-
itors either as single agents or in combination. In addi-
tion, combinations of antiangiogenic agents or RTK
inhibitors with conventional cytotoxic cancer chemo-
therapeutic agents have also been discussed.5,22–30 Clin-
ical trials of such combinations are in progress (see
website, Cancer Trials at NCI). On the basis of pre-
clinical evidence it appears that the primary effect of
antiangiogenic agents is cytostatic and that removal
of the agent leads to a regrowth of the tumor. In pre-
clinical studies, inhibition of multiple RTKs has shown
increase in survival in mice.24 There is agreement in
the literature that the use of RTK inhibitors (or multi-
ple RTK inhibitors) along with a cytotoxic or conven-
tional cancer chemotherapeutic agents and/or radiation
should enhance the efficacy of the overall antitumor
therapy and prevent regrowth following cessation of
therapy.30,31
2,4-Diamino-5-substituted furo[2,3-d]pyrimidines 1–16
(Fig. 2), by virtue of their structural analogy to TMQ
and molecular modeling, were designed as potential
DHFR inhibitors. These analogs have a C–C bridge
between the furo[2,3-d]pyrimidine and the side chain
phenyl ring. The Wittig reaction of 2,4-diamino-5-chlo-
romethyl furo[2,3-d]pyrimidine with appropriate aryl
methyl ketones was expected to also afford the unsatu-
rated precursors 17–35 (Fig. 2) to the target compounds.
Similar unsaturated analogs37a,b have shown DHFR
inhibition and would allow the control of side chain
conformation in the E- and Z-isomers for biological
activity against the target enzymes. The unsaturated pre-
cursors on reduction were to afford the target DHFR
inhibitors. Since we desired to combine DHFR and

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5477
Figure 1. RTK inhibitors and DHFR inhibitors.
RTK inhibitory activity in single molecules we modeled
the potential 2,4-diamino-5-substituted furo[2,3-d]
pyrimidine DHFR inhibitors into the VEGFR-2 model
with ATP (see below). Both the target compounds and
the unsaturated precursors were used in the modeling
studies.
2. Molecular modeling
The X-ray crystal structure of VEGFR-2 tyrosine kinase
has been published.38 Using this crystal structure and
sequence homology alignment (SYBYL 6.9)39 with the
X-ray crystal structure of insulin receptor kinase
(IRK) 40 containing an ATP bound molecule, it was pos-
sible to place the ATP in the VEGFR-2 tyrosine kinase.
Compound 32, in its energy minimized conformation
(SYBYL 6.9) of both the Z- and E-isomers serves as
the prototype and were aligned on the ATP in VEG-
FR-2 such that the 4-aminopyrimidine moieties of
ATP and 32 were superimposed. This allowed a possible
binding mode of 32 in VEGFR-2. Figures 3 and 4 indi-
cate that both the Z- and E-isomers in their energy min-
Figure 2. Target compounds.

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A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
Figure 3. Stereoview of compound Z-32 modeled in VEGFR2.
Figure 4. Stereoview of compound E-32 modeled in VEGFR2.
imized conformations, respectively, could bind to the
ATP domain of VEGFR-2. The 4-NH2 of 32 along with
the ring N3 makes hydrogen bonds in the hinge region,
with the backbone peptide residues Glu917 (carbonyl)
and Cys919 (NH), respectively, similar to that seen in
ATP. In addition, the 2-NH2 forms an additional
H-bond with Cys919 (carbonyl) which is absent in
ATP, since ATP does not contain a 2-NH2 moiety.
We have previously reported 2-NH2 containing pyrrolo-
[2,3-d]pyrimidines as RTK inhibitors with antitumor
activity.41 In the Z-isomer (Fig. 3), the 2-naphthyl
moiety makes hydrophobic contact with Leu1035 and
the C-9-Me moiety is close to Cys1045. In the E-isomer
(Fig. 4), the 4-NH2, N3, and 2-NH2 interact like the
Z-isomer, while the 2-naphthyl moiety lies in a hydro-
phobic pocket consisting of Leu901, Val916, Leu889,
Phe1047, and the C-9-Me moiety makes hydrophobic
contact with Val848. Thus, on the basis of molecular

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5479
modeling both Z- and E-isomers of 32 can be accommo-
dated in the ATP binding domain of VEGFR-2 and
were expected to afford VEGFR-2 inhibitory activity.
3. Results and discussion
A Wittig type condensation of 2,4-diamino-5-chloro
methylfuro[2,3-d]pyrimidine (38) with the appropriately
substituted aryl methyl ketones was anticipated to afford
the desired 9-methyl furo[2,3-d]pyrimidine nucleus
required for the target compounds. The crucial interme-
diate, 5-chloromethylfuro[2,3-d]pyrimidine 38, was
readily obtained by the condensation of 2,6-diaminopyr-
imidin-4-one (36) and dichloroacetone (37) in DMF at
room temperature as previously reported (Scheme 1).37a
Displacement of the chloride with tributylphosphine fol-
lowed by NaH in anhydrous DMSO afforded the semi-
stable ylide. This ylide was immediately condensed with
the corresponding aryl methyl ketones to afford the
desired olefinic compounds (17–35) in 18–63% yield
usually as mixtures of E- and Z-isomers. The yield of
the reaction was dependent on the nature and position
of the substituents in the aryl methyl ketones. In general,
electron withdrawing groups such as chloro afforded
higher yields (33–63%) than electron donating moieties
such as methoxy (18–42%). In addition, steric effects also
played a role in the yield. Ortho substituted aryl methyl
ketones gave lower yields (20, 24, and 27) than the
corresponding para substituted analogs (22, 25, and
28). E- and Z-isomers were chromatographically separat-
ed in two instances and their structures determined by 1H
NMR, 13C NMR, NOESY, and COSY. These NMR
data were used to establish the proton positions in the
E/Z mixture. The chemical shift of the C6–H of E-isomers
was around 7.4–7.5 ppm which was about 0.8 ppm
deshielded as compared with the corresponding Z-iso-
mers (6.5–6.8 ppm). NOESY experiments demonstrated
the correlation of the C6–H with the C-9-CH3 in the
E-isomers; while in the Z-isomers, the C6–H correlated
with the side chain aromatic ring protons.
Molecular modeling of compound 32 was also carried
out using human (h) DHFR. The pyrimidine ring of
32 in its energy minimized Z-isomer was superimposed
on the pyrimidine ring of previously reported X-ray
crystal structures of hDHFR inhibitors complexed with
hDHFR.42 In this model, shown in Figure 5, the Glu30
of hDHFR interacts with the 2-NH2 and N1 of the
pyrimidine like other 2,4-diamino pyrimidine containing
DHFR inhibitors.42 The side chain 2-naphthyl moiety
sits in a hydrophobic pocket composed of Phe31,
Phe34, and Ile60. In addition, the C-9-methyl group
˚
interacts with Val115 (3.9 A). Thus, the Z-isomer of 32
was expected to inhibit hDHFR. The E-isomer of 32
could not be accommodated in hDHFR with the
furo[2,3-d]pyrimidine ring binding as shown for the
Z-isomer in Figure 5. However, a 180ꢁ rotation of
the E-isomer of 32 about the C-2–NH2 bond such that
the 4-NH2 moiety and the pyrrole N7 switch positions
provides an orientation for the E-32 in which it can be
accommodated in hDHFR. Figure 6 shows the bonding
of E-32 in hDHFR.
The saturated analogs 1–16 as well as the unsaturated
analogs 17–31 and 33–35 were similarly modeled and
these studies indicated somewhat similar binding to
both VEGFR-2 and hDHFR as described for 32.
Thus, on the basis of molecular modeling described
above for 32 as a prototype we designed and synthe-
sized compounds 1–16 and the corresponding
C-8@C-9
E- and Z-isomers (17–35) as potential multitargeted
conformationally
restricted
precursor
antitumor agents.
Figure 5. Stereoview of compound Z-32 modeled in hDHFR.

5480
A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
Figure 6. Stereoview of compound E-32 modeled in hDHFR in an alternate binding mode to that of Z-32.
Scheme 1.
The semistable ylide derived from the 5-chloromethyl
furo[2,3-d]pyrimidine theoretically ensures a mixture of
E- and Z-isomers for the resulting alkenes,43 thus, it
was difficult to synthetically control the E/Z ratio. Using
lithium free, aprotic, polar DMSO as the solvent at
room temperature afforded the E-isomer (18–40%) in
greater amount than the Z-isomer (0–24%).44a In gener-
al, attempts to separate the E/Z mixtures were extremely
tedious. Repeated flash chromatographic separations
followed by recrystallization resulted, in some cases, in
partial separation of the pure E-isomer. The Z-isomer
either could not be obtained free of E-isomer or was ob-
tained in very poor yields (<5%). In one instance for the
E- and Z-20,50-dichlorophenyl analogs (27 and 27a) and
in the other instance for the E- and Z-2,6-dimethoxy-
phenyl analogs only analytic amounts were obtained.
tion times and/or hydrogen pressures resulted in further
reduction of the 5,6-bond of the furan ring.
Compounds 1–35 were evaluated as inhibitors of VEG-
FR-2 (Flk-1, KDR), EGFR, and PDGFR-b . Selected
analogs were also evaluated in the chicken embryo cho-
rioallantoic membrane (CAM) assay for antiangiogenic
activity (Table 1). The experimental methods for evalu-
ation were as described by Gangjee et al.41 Since the
IC50 values of the same compound varies under different
biological assay conditions,44b we used a standard (con-
trol) compound in each of the evaluations to provide a
valid comparison with the synthesized analogs. For
VEGFR-2, the standard was SU5416, for EGFR
the standard was PD153035, and for PDGFR-b the stan-
dard was AG1295. SU5416 was also used as the
standard for the antiangiogenic activity in the CAM
assay.
Hydrogenation of compounds 17–35 in CH3OH/CHCl3
afforded the desired target compounds 1–16 as racemic
mixtures with yields that varied from 50 to 80%.
However, for compounds 21, 23, and 31, due to the for-
mation of the overreduced 5,6-dihydro byproducts, dur-
ing reduction, they could not be obtained in the pure
form without byproduct contamination, even after sev-
eral rounds of column chromatography. Increased reac-
The saturated compounds 1–16 were inactive in the RTK
assays. Interestingly, the corresponding C-8@C-9 con-
formationally restricted precursor E- and Z-isomers,
analogs 18, 21, 30b, and 32 demonstrated inhibitory
activity against VEGFR-2 and 18, 29, and 32 were active
against PDGFR-b . In addition, these compounds were

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
Table 1. RTKs inhibitory data and antiangiogenic activity IC50 (lM)41
Compound E/Z ratio EGFR kinase inhibition VEGFR-2 kinase inhibition PDGFR-b kinase inhibition CAM angiogenesis inhibition
5481
18
21
E-isomer >50
E-isomer >50
12.8
35.8
10.3
>50
0.08
0.19
22
24
4:1
>50
>50
>50
>50
>50
>50
>50
>50
E-isomer >50
E-isomer >50
E-isomer >50
Z-isomer >50
>50
>50
>50
>50
>50
>50
>50
26
27
27a
28
3:2
2:1
>50
>50
29
30a
31.8
0.37
E-isomer >50
>50
>50
30b
32
4:1
2:1
2:1
>50
>50
>50
35.8
2.8
>50
>50
0.46
0.04
8.6
34
35
>50
>50
E-isomer >50
0.2
PD153035
SU5416
AG1295
2.4
0.03
6.2
also active in the CAM assay. Notable among these com-
pounds are the E-2-methoxyphenyl analog 18 and the
(2:1) E/Z mixture, 2-naphthyl analog 32. Both of these
analogs demonstrated potent VEGFR-2 as well as
PDGFR-b kinase inhibitory activity with IC50 values
comparable to the standards SU5416 and AG1295,
respectively. The most potent inhibitors of angiogenesis
(in the CAM assay) were the 2:1 E/Z mixture 32 and
the pure E-18 with IC50 values of 40 and 80 nM, respec-
tively, similar to that of the standard SU5416 (IC50
30 nM). The difference in potency of 18 and 32 against
the RTK (IC50 in lM) and in the CAM assay (IC50
40–80 nM) is identical to that found for the standard
SU5416. This underscores the fact that angiogenesis is
a multifaceted, multicellular, multikinase, and multipro-
tein activated process and that perhaps other RTKs
involved may also be inhibited, thus providing a syner-
gistic effect on the overall angiogenesis process.
Analogs 1–35 were also evaluated as inhibitors of re-
combinant human (rh) DHFR and recombinant Toxo-
plasma gondii (tg) DHFR, and the results are reported
in Table 2. The methods of evaluation were as previ-
ously reported.45,46 In general, 1–35 were moderately
Table 2. Inhibitory concentration (IC50 lM) and selectivity ratios against tgDHFR vs hDHFRa
Compound (CH@C(Me))
E/Z ratio
hDHFR
tgDHFR
h/tg
Compound (CH2CH(Me))
hDHFR
TgDHFR
h/tg
17
18
19
20
21
22
23
24
25
26
27
27a
28
29
30a
30b
31
32
33
34
35
2:1
E-isomer
2:1
40
30
11
1.3
>3
1
2
3
4
>37 (35%)
>33 (31%)
>34 (22%)
31
37
33
28
11
NDb
NDb
NDb
2.8
>34
34
10
12
3.4
>3
E-isomer
E-isomer
4:1
>33
33
1.5
11
22
35
30
3.2
2.7
>2
5
>33 (26%)
40
NDb
E-isomer
E-isomer
2:1
11
15
>31
20
6
7
8
9
>30 (38%)
30
29
15
10
2.9
15
NDb
3
27
10
0.74
3.5
3
E-isomer
E-isomer
Z-isomer
3:2
35
30
10
NDb
10
14
>10 (39%)
25
20
1.8
11.8
1.1
ND
14.7
100
33
1.7
25
10
11
12
30
270
5
27
5
6
10
NDb
2:1
E-isomer
4:1
28
>27
25
27
>10 (37%)
1.7
0.3
0.3
0.3
0.3
NDb
E-isomer
2:1
2:1
30
10
13
14
16
>28 (22%)
11
28
7
1.5
NDb
3.6
2.3
>20
1
25
20
NDb
83
67
NDb
2:1
E-isomer
15
16
25
23
10
17
0.02
TMP
MTX
>350 (28%)
0.02
DHFR assay conditions: all enzymes were assayed spectrophotometrically in a solution containing 50 M dihydrofolate, 80 M NADPH, 0.05 M Tris–
HCl, 0.001 M 2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 and 30 ꢁC. The reaction was initiated with an amount of enzyme yielding a change
in OD at 340 nM of 0.015 minꢀ1
.
a Recombinant hDHFR was provided by Dr. J.H. Freisheim. Recombinant tgDHFR was provided by Dr. D.V. Santi.
b ND, not determined.

5482
A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
active as inhibitors of rhDHFR and tgDHFR with
IC50 values in the range 10ꢀ7–10ꢀ6 M. Interestingly,
they inhibited tgDHFR to a greater extent than
rhDHFR. Some of the geometrically restricted, bicyclic
side chain analogs 31–34 showed higher potency and
remarkable selectivity ratios (IC50 h/tg) of 33–100
against tgDHFR. The most potent rhDHFR inhibitor
was analog 32 with an IC50 of 10 lM. There was no
major difference in the E- and Z-isomers in DHFR
inhibitory potency where the E- and Z-isomers were
separated (compound 27). The results suggest that,
for
2,4-diamino-5-substituted-furo[2,3-d]pyrimidines,
the side chain substitution pattern may be more impor-
tant than the geometric orientation for DHFR
inhibition.
To examine whether compounds 18 and 32 were effec-
tive in vivo in reducing tumor volume and metastasis,
a syngeneic mouse tumor model was used. This model
is a widely accepted model for testing tumor growth
and metastases.47 The B16 model of tumor growth
produces almost quantitative (100%) tumor take in a
very short time after administration,48,49 so animals
are not wasted. The B16 tumor cells are skin-derived,
so subcutaneous implantation is adequate and result-
ing tumors are superficial and easily measured, and
the B16-F10 variant used in these studies is highly met-
astatic and most if not all metastases go to the lung,
making metastasis evaluation very facile.48–51 Unlike
most human tumor xenograft models, the B16 model
produces highly vascularized tumors so that the effect
of tumor-mediated angiogenesis can be evaluated.48–51
For these studies, animals were implanted with green
fluorescent protein (GFP) tagged B16-F10 cells.47
One week after implantation, animals were treated
biweekly with 25 mg/kg methotrexate or 25 mg/kg
compounds 18 or 32 intraperitoneally. Primary tumor
volume was measured and the tumor rates were calcu-
lated as in the Section 5. The results of the compounds
on primary tumor growth rates are shown in Figure 7.
Methotrexate, compound 18, and compound 32 all
resulted in a significant (P < 0.005) decrease in primary
tumor growth rate as compared to untreated animals.
In addition, compounds 18 and 32 resulted in a signif-
icant (P < 0.05) reduction in growth rate as compared
to methotrexate treatment. Lung metastases was mea-
sured by staining for GFP, an exogenous protein,
and the results are shown in Figure 8. All three drugs
(methotrexate, 18, and 32) resulted in a significant
(P < 0.05) decrease in the number of lung metastases
per lobe. In this case, however, there was no significant
decrease in lung metastases with 18 or 32 as compared
with methotrexate, although a trend toward signifi-
cance was present. The increased activity of 18 and
32 in the in vivo antitumor evaluation, compared to
methotrexate could be the result of a synergistic effect
of RTK inhibition along with DHFR inhibitory activ-
ity. For a synergistic effect the individual drugs need
not be highly potent. Thus, even the low hDHFR
inhibitory activity of both 18 and 32 perhaps act in
concert with the RTK inhibitory activity to provide
Figure 7. Effect of methotrexate, compound 18, and compound 32 on
growth of B16-10 primary tumors. Mice bearing B16-F10 GFP-tagged
tumors were treated biweekly with 25 mg/kg drug and tumors
measured biweekly as in Section 5. Tumor growth rates were
calculated using linear regression analysis in Prism 4.0 software. Data
represent means SEM from 5 to 9 animals. *P < 0.05, **P < 0.005 as
compared to untreated animals; a = P < 0.005, b = P < 0.05 as com-
pared with methotrexate-treated animals using two-way ANOVA with
Dunnettꢂs post-test.
Figure 8. Effect of methotrexate, compound 18, and compound 32 on
growth of B16-10 lung metastases. Mice bearing B16-F10 GFP-tagged
tumors were treated biweekly with 25 mg/kg drug and tumors
measured biweekly as in Section 5. Tumor growth rates were
calculated using linear regression analysis in Prism 4.0 software. Data
represent means SEM from 5 to 9 animals. *P < 0.05, **P < 0.005 as
compared to untreated animals using one-way ANOVA with Bon-
feronniꢂs multiple comparison post-test.
4. Conclusion
In summary, a series of 5-substituted, 2,4-diamino-
furo[2,3-d]pyrimidines were designed and synthesized
as a novel class of multitarget inhibitors of RTKs, which
also exhibit DHFR inhibitory activity. These analogs
were designed with the potential to possess both cyto-
static activity (RTK inhibition) and cytotoxic activity
(DHFR inhibition) in a single molecule. Several of the
unsaturated bridge analogs synthesized not only possess
good inhibitory activity against VEGFR-2 and
PDGFR-b that were similar to that of the standard
a viable antitumor effect in vivo against B16
melanoma.

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5483
compounds SU5416 and AG1295, respectively, but also
maintain moderate DHFR inhibitory activity. Several of
these compounds also showed potent antiangiogenic
activity in the CAM assay similar to the standard
SU5416. The results suggest that the nature of the side
chain aromatic substituent plays an important role in
the inhibitory activity against RTKs and DHFR. Com-
pound 32 serves as a lead analog for rationally designed
RTK inhibitory activity combined with DHFR inhibito-
ry activity in a single molecule. Efforts are currently
underway to improve the DHFR inhibitory activity
using 32 as the lead analog. The in vivo antitumor eval-
uation of 18 and 32 suggest that both compounds 18 and
32 are active as antitumor agents in vivo, both against
primary tumors and metastases, and are both at least
as active against B16 melanomas as the standard drug,
methotrexate.
sodium hydride (90% dispersion in mineral oil, 0.2 g,
6 mmol), followed by the desired commercially available
aryl methyl ketone (5.5 mmol). The reaction mixture
was stirred at room temperature for 24–32 h. TLC
showed the disappearance of the starting 2,4-diamino-
5-(chloromethyl)- furo[2,3-d]pyrimidine and the forma-
tion of two (olefinic) spots. 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 6 g of silica gel and CHCl3 (25 mL) and the
slurry was loaded onto a 4 · 20 cm dry silica gel column
and flash chromatographed initially with CHCl3
(300 mL), then sequentially with 2% MeOH in CHCl3
(250 mL), 5% CH3OH in CHCl3 (300 mL), and 10%
CH3OH in CHCl3 (250 mL). Fractions which showed
the desired spot on TLC were pooled and evaporated
to dryness and the residue was recrystallized from ethy-
lacetate to afford the desired olefinic targets 17–35.
5. Experimental
5.1.1. E/Z-2,4-Diamino-5-[2-(phenyl)propenyl]furo[2,3-d]-
pyrimidine (17). Compound 38 (1.0 g, 5 mmol) and ace-
tophenone (660 mg, 5.5 mmol) for 24 h afforded 17
(400 mg, 30%) as yellow crystals: mp 230.8–233.8 ꢁC;
Rf = 0.59 and 0.54 (CHCl3/CH3OH 5:1); 1H NMR
(DMSO-d6) (E/Z = 2:1) E-isomer d 2.22 (s, 3H, 9-
CH3), 6.07 (s, 2H, 4-NH2), 6.45 (s, 2H, 2-NH2), 6.88
(s, 1H, 8-CH), 7.29–7.40 (m, 3H, C6H5), 7.46 (s, 1H,
6-CH), 7.62–7.65 (d, 2H, C6H5); Z-isomer d 2.10 (s,
3H, 9-CH3), 5.99 (s, 2H, 4-NH2), 6.23 (s, 1H, C-8-
CH), 6.49 (s, 2H, 2-NH2), 6.59 (s, 1H, 6-CH), 7.18–
7.40 (m, 5H, C6H5). Anal. (C15H14N4O) C, H, N.
All evaporations were carried out in vacuo with a rotary
evaporator. 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 magnetic resonance spectra for proton (1H
NMR) were recorded on a Bruker WH-300 (300 MHz)
spectrometer. The chemical 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
integrals of peak areas agreed with those expected for
the assigned structures. NOESY, COSY experiments
were performed on a Bruker DRX500 (500 MHz) spec-
trometer. Thin-layer chromatography (TLC) was per-
formed on POLYGRAM Sil G/UV254 silica gel plates
with a fluorescent indicator, and the spots were visual-
ized under 254 and 366 nm illumination. Proportions
of solvents used for TLC are by volume. Column chro-
matography was performed on a 230–400 mesh silica gel
purchased from Aldrich, Milwaukee, WI. 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
5.1.2. E-2,4-Diamino-5-[2-(20-methoxyphenyl)propenyl]-
furo[2,3-d]pyrimidine (18). Compound 38 (1.0 g, 5 mmol)
and 20-methoxyacetophenone (830 mg, 5.5 mmol) for
32 h afforded 18 (350 mg, 25%) as orange crystals: mp
191.3–193.3 ꢁC; Rf = 0.54 (CHCl3/CH3OH 5:1); 1H
NMR (DMSO-d6): d 2.07 (s, 3H, 9-CH3), 3.80 (s, 3H,
OMe), 6.08 (s, 2H, 4-NH2), 6.42 (s, 2H, 2-NH2), 6.48
(s, 1H, 8-CH), 6.93–7.11 (m, 2H, C6H4), 7.26–7.42 (d,
2H, C6H4), 7.47 (s, 1H, 6-CH). Anal. (C16H16N4O2) C,
H, N.
5.1.3. E/Z-2,4-Diamino-5-[2-(30-methoxyphenyl)propenyl]-
furo[2,3-d]pyrimidine (19). Compound 38 (1.0 g, 5 mmol)
and 30-methoxyacetophenone (830 mg, 5.5 mmol) for
30 h afforded 19 (400 mg, 27%) as yellow needles: mp
193.2–197.8 ꢁC; Rf = 0.55 and 0.50 (CHCl3/CH3OH
1
1
possible by their presence in the H NMR spectra. All
5:1); H NMR (DMSO-d6) (E/Z = 2:1) E-isomer d 2.20
solvents and chemicals were purchased from Aldrich
Chemical Co. or Fisher Scientific and were used as
received.
(s, 3H, 9-CH3), 3.80 (s, 3H, OMe), 6.07 (s, 2H, 4-
NH2), 6.45 (s, 2H, 2-NH2), 6.88–6.89 (t, 2H, C-8-CH
and C6H4), 7.18–7.32 (d, 3H, C6H4), 7.45 (s, 1H, 6-
CH); Z-isomer d 2.17 (s, 3H, 9-CH3), 3.75 (s, 3H,
OMe), 6.00 (s, 2H, 4-NH2), 6.36 (s, 1H, C-8-CH), 6.52
(s,1H, 6-CH), 6.57 (s, 2H, 2-NH2), 6.73–6.89 (m, 3H,
5.1. General procedure for the synthesis of 17–35
To a solution of 2,4-diamino-5-(chloromethyl)furo[2,3-
d]pyrimidine, 38, (1.0 g, 5 mmol) in anhydrous DMSO
(15 mL) was added tributylphosphine (92%, 1.7 g
7.5 mmol), and the resulting mixture was stirred at
60 ꢁC in an oil bath for 3 h under N2 to form the phos-
phonium salt. The deep orange solution was then cooled
to room temperature. To this solution was added
C6H4),
7.14–7.18
(m,
1H,
C6H4).
Anal.
(C16H16N4O2Æ0.25H2O) C, H, N.
5.1.4. E-2,4-Diamino-5-[2-(20-chlorophenyl)propenyl]furo-
[2,3-d]pyrimidine (20). Compound 38 (1.0 g, 5 mmol) and
20-chloroacetophenone (850 mg, 5.5 mmol) for 24 h
afforded 20 (500 mg, 33%) as yellow crystals: mp

5484
A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
234.5–236.4 ꢁC (dec); Rf = 0.58 (CHCl3/CH3OH 5:1); 1H
NMR (DMSO-d6) E-isomer d 2.20 (s, 3H, 9-CH3), 6.15 (s,
2H, 4-NH2), 6.50 (s, 2H, 2-NH2), 6.66 (s, 1H, 8-CH),
7.39–7.54 (m, 4H, C6H4), 7.58 (s, 1H, 6-CH). Anal.
(C15H13N4OCl) C, H, N, Cl.
(m, 2H, C6H3), 6.95–6.97 (d, 1H, C6H3), 7.41(s, 1H, 6-
CH); Anal. (C17H18N4O3) C, H, N.
A (5:2) E/Z mixture (200 mg) was also obtained: mp
170.9–179.9 ꢁC; Rf = 0.68 and 0.65 (CHCl3/CH3OH
1
5:1); total yield 28%. H NMR (DMSO-d6) E-isomer d
5.1.5. E-2,4-Diamino-5-[2-(30-chlorophenyl)propenyl]furo-
[2,3-d]pyrimidine (21). Compound 38 (1.0 g, 5 mmol) and
30-chloroacetophenone (850 mg, 5.5 mmol) for 24 h
afforded 21 along with its Z-isomer (550 mg, 37%) as yel-
low needles: mp 210.6–215.7 ꢁC (dec); Rf = 0.58 and 0.54
(CHCl3/CH3OH 5:1); 1H NMR (DMSO-d6) (E/Z = 2:1);
E-isomer d 2.21 (s, 3H, 9-CH3), 6.08 (s, 2H, 4-NH2), 6.54
(s, 2H, 2-NH2), 6.92 (s, 1H, 8-CH), 7.35–7.43 (m, 2H,
C6H4), 7.48(s, 1H, 6-CH), 7.57–7.60 (d, 1H, C6H4),
7.73 (s, 1H, C6H4); Z-isomer d 2.17 (s, 3H, 9-CH3),
6.02 (s, 2H, 4-NH2), 6.36 (s, 2H, 2-NH2), 6.54 (s, 1H,
8-CH), 6.62 (s, 1H, 6-CH), 7.43–7.13 (m, 4H, C6H4).
Anal. (C15H13N4OCl) C, H, N, Cl.
2.06 (s, 3H, 9-CH3), 3.72 (s, 3H, OMe), 3.74 (s, 3H,
OMe), 6.08 (s, 2H, 4-NH2), 6.44 (s, 2H, 2-NH2), 6.51
(s, 1H, 8-CH), 6.82–6.85 (m, 2H, C6H3), 6.95–6.97 (d,
1H, C6H3), 7.41(s, 1H, 6-CH); Z-isomer d 2.10 (s, 3H,
9-CH3), 3.65 (s, 6H, OMe), 5.98 (s, 2H, 4-NH2), 6.17
(s, 1H, 8-CH), 6.44 (s, 2H, 2-NH2), 6.55–6.56 (d, 1H,
C6H3), 6.60 (s, 1H, 6-CH), 6.82–6.86 (dd, 2H, C6H3).
Anal. (C17H18N4O3) C, H, N.
5.1.9. E/Z-2,4-Diamino-5-[2-(30,40-dimethoxyphenyl)pro-
penyl]furo[2,3-d]pyrimidine (25). Compound 38 (1.0 g,
5 mmol) and 30,40-dimethoxyacetophenone (1.0 g,
5.5 mmol) for 32 h afforded 25 (480 mg, 29%) as a white
powder: mp 232–251 ꢁC (dec); Rf = 0.52 and 0.49
(CHCl3/CH3OH 5:1); 1H NMR (DMSO-d6) (E/Z =
2:1) E-isomer d 2.18 (s, 3H, 9-CH3), 3.77 (s, 3H,
OMe), 3.82 (s, 3H, OMe), 6.06 (s, 2H, 4-NH2), 6.47 (s,
2H, 2-NH2), 6.79 (s, 1H, 8-CH), 7.13–7.16 (d, 2H,
C6H3), 7.20 (s, 1H, C6H3), 7.41 (s,1H, 6-CH); Z-isomer
d 2.16 (s, 3H, 9-CH3), 3.59 (s, 3H, OMe), 3.72 (s, 3H,
OMe), 5.98 (s, 2H, 4-NH2), 6.42 (s, 2H, 2-NH2), 6.50
(s, 1H, 8-CH), 6.76 (s, 1H, 6-CH), 6.86–6.95 (m, 3H,
C6H3). Anal. (C17H18N4O3) C, H, N.
The above mixture was reloaded for another round of
flash column chromatography and afforded the pure
E-isomer 21 (150 mg, 11%) as light yellow needles: mp
215.6–218.7 ꢁC; Rf = 0.58 (CHCl3/CH3OH 5:1); 1H
NMR (DMSO-d6): d 2.21 (s, 3H, 9-CH3), 6.08 (s, 2H,
4-NH2), 6.54 (s, 2H, 2-NH2), 6.92 (s, 1H, 8-CH), 7.35–
7.43 (m, 2H, C6H4), 7.48 (s, 1H, 6-CH), 7.57–7.60 (d,
1H, C6H4), 7.73 (s, 1H, C6H4). Anal. (C15H13N4OCl)
C, H, N, Cl.
5.1.6. E/Z-2,4-Diamino-5-[2-(40-chlorophenyl)propenyl]-
furo[2,3-d]pyrimidine (22). Compound 38 (1.0 g, 5 mmol)
and 40-chloroacetophenone (850 mg, 5.5 mmol) for 24 h
afforded 22 (600 mg, 40%) as yellow needles: mp 247.5–
5.1.10. E-2,4-Diamino-5-[2-(20,40-dichlorophenyl)prope-
nyl]furo[2,3-d]pyrimidine (26). Compound 38 (1.0 g,
5 mmol) and 20,40-dichloroacetophenone (1.05 g,
5.5 mmol) for 24 h afforded 26 (850 mg, 50%) as yellow
needles: mp 220.8–223.2 ꢁC; Rf = 0.63 (CH3Cl/CH3OH
1
252.5 ꢁC; Rf = 0.61 and 0.58 (CHCl3/CH3OH 5:1); H
1
NMR (DMSO-d6) (E/Z = 4:1) E-isomer d 2.20 (s, 3H,
9-CH3), 6.09 (s, 2H, 4-NH2), 6.52 (s, 2H, 2-NH2), 6.88
(s, 1H, 8-CH), 7.44–7.39 (d, 2H, C6H4), 7.47 (s, 1H, 6-
CH), 7.66–7.69 (d, 2H, C6H4); Z-isomer d 2.17 (s, 3H,
9-CH3), 6.02 (s, 2H, 4-NH2), 6.38 (s, 1H, 8-CH) 6.44
(s, 2H, 2-NH2), 6.61 (s, 1H, 6-CH), 7.20–7.23 (d, 2H,
C6H5), 7.35–7.38 (d, 2H, C6H4). Anal. (C15H13N4OCl)
C, H, N, Cl.
5:1); H NMR (DMSO-d6): d 2.12 (s, 3H, 9-CH3), 6.09
(s, 2H, 4-NH2), 6.49 (s, 2H, 2-NH2), 6.61 (s, 1H, 8-
CH), 7.69–7.43 (m, 4H, C6H3 and C6-CH). Anal.
(C15H12N4OCl2) C, H, N,Cl.
5.1.11. E-2,4-Diamino-5-[2-(20,50-dichlorophenyl)prope-
nyl]furo[2,3-d]pyrimidine (27). Compound 38 (1.0 g,
5 mmol) and 20,50-dichloroacetophenone (1.05 g,
5.5 mmol) for 24 h afforded 27 (410 mg, 24%) as yellow
needles: mp 229.5–231.5 ꢁ C; Rf = 0.69 (CHCl3/CH3OH
5.1.7. E-2,4-Diamino-5-[2-(20,40-dimethoxyphenyl)prope-
nyl]furo[2,3-d]pyrimidine (23). Compound 38 (1.0 g,
5 mmol) and 20,40-dimethoxyacetophenone (1.0 g,
5.5 mmol) for 32 h afforded 23 (300 mg, 18%) as yellow
needles: mp 210.3–211.6 ꢁC; Rf = 5.7 (EtOH/EtOAc
1
5:1); H NMR (DMSO-d6): d 2.13 (s, 3H, 9-CH3), 6.09
(s, 2H, 4-NH2), 6.51 (s, 2H, 2-NH2), 6.64 (s, 1H,
8-CH), 7.39–7.42 (dd, 1H, C6H3), 7.50–7.54 (m, 3H,
6-CH and C6H3). Anal. (C15H12N4OCl2) C, H, N, Cl.
1
1:2); H NMR (DMSO-d6): d 2.04 (s, 3H, 9-CH3), 3.77
(s, 3H, 40-OCH3), 3.79 (s, 3H, 20-OCH3), 6.08 (s, 2H,
4-NH2), 6.39 (s, 2H, 2-NH2), 6.44 (s, 1H, C6H3), 6.58
(s, 1H, C6H3), 6.59 (s, 1H, 8-CH), 7.19 (s, 1H, C6H3),
7.45 (s, 1H, 6-CH). Anal. (C17H18N4O3) C, H, N.
A mixture of 27 and its Z-isomer27a (270 mg, total) was
also obtained as yellow crystals: mp 225–232.8 ꢁC;
Rf = 0.69 and 0.62 (CHCl3/CH3OH 5:1); 1H NMR
(DMSO-d6) E-isomer d 2.13 (s, 3H, 9-CH3), 6.09 (s,
2H, 4-NH2), 6.51 (s, 2H, 2-NH2), 6.64 (s, 1H, 8-CH),
7.39–7.42 (dd, 1H, C6H3), 7.50–7.54 (m, 3H, 6-CH and
C6H3); Z-isomer d 2.13 (s, 3H, 9-CH3), 6.03 (s, 2H, 4-
NH2), 6.10 (s, 1H, 8-CH), 6.63 (s, 2H, 2-NH2), 6.80 (s,
1H, 6-CH), 7.33–7.53 (m, 3H, C6H3); total yield 41%.
5.1.8. E-2,4-Diamino-5-[2-(20,50-dimethoxyphenyl)prope-
nyl]furo[2,3-d]pyrimidine (24). Compound 38 (1.0 g,
5 mmol) and 20,50-dimethoxyacetophenone (1.0 g,
5.5 mmol) for 32 h afforded 24 (250 mg, 15%) as yellow
needles: mp 178.5–179.9 ꢁC; Rf = 0.68 (CHCl3/CH3OH
1
5:1); H NMR (DMSO-d6): d 2.06 (s, 3H, 9-CH3), 3.72
5.1.12. Z-2,4-Diamino-5-[2-(20,50-dichlorophenyl)prope-
nyl]furo[2,3-d]pyrimidine (27a). The above mixture was
reloaded for additional two rounds of flash column
(s, 3H, OMe), 3.74 (s, 3H, OMe), 6.08 (s, 2H, 4-NH2),
6.44 (s, 2H, 2-NH2), 6.51 (s, 1H, 8-CH), 6.82–6.85

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5485
chromatography and afforded the pure Z-27a (70 mg,
4%) as yellow crystals: mp: 253.6–255.8 ꢁC; Rf = 0.60
afforded 31 (320 mg, 22%) as yellow needles: mp
232.8–235 ꢁC (dec); Rf = 0.41 (CHCl3/CH3OH 8:1);
1H NMR (DMSO-d6) d 2.28 (s, 3H, 9-CH3), 6.08 (s,
2H, 4-NH2), 6.40 (s, 2H, 2-NH2), 6.70 (s, 1H, 8-CH),
7.44–7.54 (m, 3H, C10H7), 7.64 (s, 1H, 6-CH), 7.86–
7.95 (m, 4H, C10H7). Anal. (C19H16N4OÆ0.25H2O) C,
H, N.
1
(CHCl3/CH3OH 5:1); H NMR (DMSO-d6): d 2.13 (s,
3H, 9-CH3), 6.03 (s, 2H, 4-NH2), 6.10 (s, 1H, 8-CH),
6.63 (s, 2H, 2-NH2), 6.80 (s, 1H, 6-CH), 7.33–7.53 (m,
3H, C6H3). Anal. (C15H12N4OCl2) C, H, N, Cl.
5.1.13. E/Z-2,4-Diamino-5-[2-(30,40-dichlorophenyl)pro-
penyl]furo[2,3-d]pyrimidine (28). Compound 38 (1.0 g,
5 mmol) and 30,40-dichloroacetophenone (1.05 g,
5.5 mmol) for 24 h afforded 28 (1.12 g, 63%) as a white
powder: mp 232.6–236.6 ꢁC; Rf = 0.57 and 0.55 (CHCl3/
CH3OH 5:1); 1H NMR (DMSO-d6) (E/Z = 3:2) E-isomer
d 2.20 (s, 3H, 9-CH3), 6.08 (s, 2H, 4-NH2), 6.57 (s, 2H,
2-NH2), 6.94 (s, 1H, 8-CH), 7.49 (s,1H, 6-CH), 7.62 (s,
2H, C6H3), 7.93 (s, 1H, C6H3); Z-isomer d 2.17 (s, 3H,
9-CH3), 6.03 (s, 2H, 4-NH2), 6.51 (s, 2H, 2-NH2), 6.57
(s, 1H, 8-CH) ,6.63 (s, 1H, 6-CH), 7.16–7.15 (d, 1H,
C6H3), 7.47 (s, 1H, C6H3), 7.56–7.53 (d, 1H, C6H3). Anal.
(C15H12N4OCl2) C, H, N, Cl.
5.1.18.
E/Z-2,4-Diamino-5-[2-(20-naphthyl)propenyl]-
furo[2,3-d]pyrimidine (32). Compound 38 (1.0 g,
5 mmol) and 20-acetonaphthone (940 mg, 5.5 mmol)
for 28 h afforded 32 (480 mg, 30%) as yellow needles:
mp 238.2–247.5 ꢁC; Rf = 0.55 and 0.52 (CHCl3/
1
CH3OH 5:1); H NMR (DMSO-d6) (E/Z = 2:1) E-iso-
mer d 2.34 (s, 3H, 9-CH3), 6.09 (s, 2H, 4-NH2), 6.52 (s,
2H, 2-NH2), 7.05 (s, 1H, 8-CH), 7.51–7.49 (m, 3H, 6-
CH and C10H7), 7.96–7.89 (m, 4H, C10H7), 8.09 (s,
1H, C10H7); Z-isomer d 2.26 (s, 3H, 9-CH3), 5.99 (s,
2H, 4-NH2), 6.29 (s, 1H, 8-CH), 6.54 (s, 2H, 2-
NH2), 6.67 (s, 1H, 6-CH), 7.13–7.17 (m, 3H, C10H7),
7.70–7.89 (m, 4H, C10H7). Anal. (C19H16N4O) C, H, N.
5.1.14. E/Z-2,4-Diamino-5-[2-(30,40,50- trimethoxyphenyl)-
propenyl]furo[2,3-d]-pyrimidine (29). Compound 38 (1.0 g,
5 mmol) and 30,40,50-trimethoxyacetophenone (1.16 g,
5.5 mmol) for 28 h afforded 29 (1.0 g, 42%) as yellow
needles: mp 198–207 ꢁC; Rf = 0.48 and 0.43 (CHCl3/
CH3OH 7:1); 1H NMR (DMSO-d6) (E/Z = 2:1) E-isomer
d 2.20 (s, 3H, 9-CH3), 3.67 (s, 3H, 40-OCH3), 3.83 (s, 6H,
30,50-OCH3), 6.07 (s, 2H, 4-NH2), 6.43 (s, 2H, 2-NH2),
6.81 (s, 1H, 8-CH), 6.85 (s, 2H, C6H2), 7.44 (s, 1H,
6-CH); Z-isomer d 2.18 (s, 3H, 9-CH3), 3.57 (s, 9H,
OCH3), 5.99 (s, 2H, 4-NH2), 6.45 (s, 2H, 2-NH2), 6.50 (s,
3H, 8-CH and C6H2), 6.55 (s, 1H, 6-CH). Anal.
(C18H20N4O4) C, H, N.
5.1.19. E/Z-2,4-Diamino-5-[2-(60-methoxy-20-naphthyl)-
propenyl]furo[2,3-d]- pyrimidine (33). Compound 38
(1.0 g, 5 mmol) and 2-acetyl-6-methoxynaphthalene
(1.05 g, 5.5 mmol) for 28 h afforded 33 (540 mg, 31%)
as a white powder: mp 254.4–257.8 ꢁC (dec); Rf = 0.7
1
and 0.67 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6)
(E/Z = 2:1) E-isomer d 2.31 (s, 3H, 9-CH3), 3.88 (s,
3H, OMe), 6.07 (s, 2H, 4-NH2), 6.49 (s, 2H, 2-NH2),
7.00 (s, 1H, 8-CH), 7.24–7.32 (m, 2H, C10H6), 7.48 (s,
2H, 6-CH), 7.81–7.95 (m, 3H, C10H6), 8.09 (s, 1H,
C10H6); Z-isomer d 2.26 (s, 3H, 9-CH3), 3.93 (s, 3H,
OMe), 5.98 (s, 2H, 4-NH2), 6.29 (s, 1H, 8-CH), 6.49
(s, 2H, 2-NH2), 6.64 (s, 1H, 6-CH), 7.13–7.17 (m, 2H,
5.1.15. E-2,4-Diamino-5-[2-(20,30,40-trichlorophenyl)pro-
penyl]furo[2,3-d]pyrimidine (30a). Compound 38 (1.0 g,
5 mmol) and 20,30,40-chloroacetophenone (1.25 g,
5.5 mmol) for 28 h and after two rounds of flash column
chromatography afforded 30a (130 mg, 8%) as a white
powder: mp 265.2–266.5 ꢁC, Rf = 0.57 (CHCl3/CH3OH
5:1);1H NMR (DMSO-d6) E-isomer d 2.13 (s, 3H,
9-CH3), 6.07(s, 2H, 4-NH2), 6.49 (s, 2H, 2-NH2), 6.64
(s, 1H, 8-CH), 7.39–7.42 (d, 1H, C6H2), 7.56 (s, 1H,
6-CH), 7.65–7.68 (d, 1H, C6H2); Anal. (C15H11N4OCl3)
C, H, N, Cl.
C10H6), 7.71–7.84
(C20H18N4O2) C, H, N.
(m,
4H,
C10H6).
Anal.
5.1.20. E/Z-2,4-Diamino-5-[2-(40-biphenyl)propenyl]furo-
[2,3-d]pyrimidine (34). Compound 38 (1.0 g, 5 mmol)
and 4-phenylacetophenone (1.08 g, 5.5 mmol) for 32 h
afforded 34 (550 mg, 30%) as yellow needles: mp
239.8–245.8 ꢁC; Rf = 0.58 and 0.53 (CHCl3/CH3OH
5:1); 1H NMR (DMSO-d6) (E/Z = 2:1) E-isomer d
2.25 (s, 3H, 9-CH3), 6.08 (s, 2H, 4-NH2), 6.50 (s,
2H, 2-NH2), 6.94 (s, 1H, 8-CH), 7.30–7.47 (m, 4H,
C6H4), 7.48 (s, 1H, 6-CH), 7.65–7.73 (m, 5H, C6H5);
Z-isomer d 2.21 (s, 3H, 9-CH3), 6.01 (s, 2H, 4-NH2),
6.41 (s, 1H, 8-CH), 6.52 (s, 2H, 2-NH2), 6.62 (s, 1H,
6-CH), 7.30–7.73 (m, 9H, C12H9). Anal. (C21H18N4O)
C, H, N.
5.1.16. E/Z-2,4-Diamino-5-[2-(20,30,40-trichlorophenyl)-
propenyl]furo[2,3-d]pyrimidine (30b). The above proce-
dure also afforded 30b (800 mg, 42%) as a light yellow
powder: mp 260.6–265.6 ꢁC; Rf = 0.55 and 0.57
1
(CHCl3/CH3OH 5:1) (E/Z 4:1) H NMR (DMSO-d6)
E-isomer d 2.13 (s, 3H, 9-CH3), 6.07 (s, 2H, 4-NH2),
6.49 (s, 2H, 2-NH2), 6.64 (s, 1H, 8-CH), 7.39–7.42 (d,
1H, C6H2), 7.56 (s, 1H, 6-CH), 7.65–7.68 (d, 1H,
C6H2); Z-isomer d 2.54 (s, 3H, 9-CH3), 6.03 (s, 2H,
4-NH2), 6.51 (s, 2H, 2-NH2), 6.57 (s, 1H, 8-CH), 6.63
(s, 1H, 6-CH), 7.15–7.16 (d, 1H, C6H5), 7.53–7.56 (d,
1H, C6H2). Anal. (C15H11N4OCl3) C, H, N, Cl.
5.1.21. E-2,4-Diamino-5-[2-(20-fluorenyl)propenyl]furo-
[2,3-d]pyrimidine (35). Compound 38 (1.0 g, 5 mmol)
and 2-acetylfluorene (1.15 g, 5.5 mmol) for 32 h afforded
35 (350 mg, 23%) as a yellow powder: mp 279.8–
282.2 ꢁC (dec); Rf = 0.56 (CHCl3/CH3OH 5:1); 1H
NMR (DMSO-d6) d 2.08 (s, 3H, 9-CH3), 2.24 (s, 2H,
C13H9), 6.08 (s, 2H, 4-NH2), 6.59 (s, 2H, 2-NH2), 7.01
(s, 1H, 8-CH), 7.40-7.35 (m, 1H, C13H9), 7.49 (s, 1H,
6-CH), 7.64–7.60 (m, 2H, C13H9), 7.90–7.78 (m, 4H,
C13H9). Anal. (C22H18N4O) C, H, N.
5.1.17. E-2,4-Diamino-5-[2-(10-naphthyl)propenyl]furo-
[2,3-d]pyrimidine (31). Compound 38 (1.0 g, 5 mmol)
and 10-acetonaphthone (940 mg, 5.5 mmol) for 28 h

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A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5.2. General procedure for the synthesis of 1–16
3.48–3.55 (m, 1H, 9-CH), 5.97 (s, 2H, 4-NH2), 6.45 (s,
2H, 2-NH2), 6.94 (s, 1H, 6-CH), 7.16–7.51 (m, 4H,
C6H4). Anal. (C15H15N4OClꢁ0.5 CH3OH) C, H, N,Cl.
To a solution of the olefinic intermediate (0.3–0.7 mmol)
in a mixture of CHCl3 (50 mL) and CH3OH (15 mL)
was added 5% palladium on activated carbon (0.20 g),
and the suspension was hydrogenated in a Parr appara-
tus at room temperature at 40–55 psi for 3–24 h, TLC
indicated the disappearance of the starting material
and the formation of one major spot. The reaction mix-
ture was filtered through Celite, washed with 30%
CH3OH in CHCl3 (3 · 20 mL). After evaporation of
the solvent, CH3OH (50 mL) was added to afford a solu-
tion. To this solution was added 5 g silica gel and the
mixture was evaporated under reduced pressure to dry-
ness. The silica gel plug was loaded on a dry silica gel
column (2 · 16 cm) and flash chromatographed initially
with CHCl3 (150 mL), then sequentially with 1%
CH3OH in CHCl3 (150 mL), 2% CH3OH in CHCl3
(150 mL), and 5% CH3OH in CHCl3 (150 mL). Frac-
tions which showed the major spot on TLC were pooled
and evaporated to dryness. The residue was recrystal-
lized from CH3OH or other solvent combinations as
indicated to afford the desired target compounds 1–16.
The yields varied from 50 to 80%.
5.2.5. (R,S)-2,4-Diamino-5-[2-(40-chlorophenyl)propyl]-
furo[2,3-d]pyrimidine (5). Compound 22 (100 mg,
0.3 mmol) hydrogenated at 50 psi for 5 h afforded 5
(60 mg, 60%) as white crystals: mp 249.7–252.5 ꢁC.
1
Rf = 0.57 (CHCl3/CH3OH 5:1). H NMR (DMSO-d6):
d 1.21–1.23 (d, 3H, 9-CH3), 2.90–2.92 (d, 2H, 8-CH2),
2.96–3.02 (m, 1H, 9-CH), 5.96 (s, 2H, 4-NH2), 6.41 (s,
2H, 2-NH2), 6.88 (s, 1H, 6-CH), 7.23–7.32 (dd, 4H,
C6H4). Anal. (C15H15N4OCl) C, H, N, Cl.
5.2.6. (R,S)-2,4-Diamino-5-[2-(20,50-dimethoxyphenyl)pro-
pyl]furo[2,3-d]pyrimidine (6). Compound 24 (100 mg,
0.3 mmol) hydrogenated at 45 psi for 6 h afforded 6
(50 mg, 50%) as yellow crystals: mp 171.6–173.6 ꢁC.
1
Rf = 0.56 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.29–1.31 (d,3H, 9-CH3), 2.98–3.00 (d, 2H, 8-CH2),
3.09–3.17 (m, 1H, 9-CH), 3.65 (s, 6H, OMe), 6.02 (s,
2H, 4-NH2), 6.44 (s, 2H, 2-NH2), 6.55–6.56 (d, 1H,
C6H3), 6.82–6.90 (m, 2H, C6H3), 6.92 (s, 1H, C-6-CH)
Anal. (C17H20N4O3Æ0.2H2O) C, H, N.
5.2.1. (R,S)-2,4-Diamino-5-[2-(phenyl)propyl]furo[2,3-d]-
pyrimidine (1). Compound 17 (180 mg, 0.7 mmol)
hydrogenated at 40 psi for 3 h afforded 1 (120 mg,
66%) as white needles: mp 257.6–259.6 ꢁC; Rf = 0.54
(CHCl3/CH3OH 5:1); 1H NMR (DMSO-d6): d 1.22–
1.24 (d, 3H, 9-CH3), 2.90–2.92 (d, 2H, 8-CH2), 2.93–
3.01 (m, 1H, 9-CH), 5.95 (s, 2H, 4-NH2), 6.39 (s, 2H,
2-NH2), 6.88 (s, 1 H, 6-CH), 7.13–7.30 (m, 5H, C6H5).
Anal. (C15H16N4O) C, H, N.
5.2.7. (R,S)-2,4-Diamino-5-[2-(30,40-dimethoxyphenyl)pro-
pyl]furo[2,3-d]pyrimidine (7). Compound 25 (100 mg,
0.3 mmol) hydrogenated at 45 psi for 3 h afforded 7
(50 mg, 50%) as white crystals: mp 198.7–201.2 ꢁC;
1
Rf = 0.52 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.20–1.22 (d, 3H, 9-CH3), 2.83–2.97 (m, 3H, 8-CH2,
9-CH), 3.68 (s, 3H, 40-OCH3), 3.70 (s, 3H, 30- OCH3),
5.97 (s, 2H, 4-NH2), 6.40 (s, 2H, 2-NH2), 6.69–6.72 (d,
1H, C6H3), 6.79–6.81 (t, 2H, C6H3), 6.91 (s, 1H, 6-
CH). Anal. (C17H20N4O3Æ0.5 H2O) C, H, N.
5.2.2. (R,S)-2,4-Diamino-5-[2-(20-methoxyphenyl)propyl]-
furo[2,3-d]pyrimidine (2). Compound 18 (100 mg,
0.3 mmol) hydrogenated at 45 psi for 3 h afforded 2
(70 mg, 70%) as white needles: mp 182.5–185.1 ꢁC;
5.2.8. (R,S)-2,4-Diamino-5-[2-(20,40-dichlorophenyl)pro-
pyl]furo[2,3-d]pyrimidine (8). Compound 26 (100 mg,
0.3 mmol) hydrogenated at 50 psi for 16 h afforded 8
(65 mg, 65%) as white needles (recrystallized from
Et2O/CH3OH): mp 220.5–223.1 ꢁC; Rf = 0.58 (CHCl3/
CH3OH 5:1). 1H NMR (DMSO-d6): d 1.20–1.22 (d,
3H, 9-CH3), 2.90–3.05 (d, 2H, 8-CH2), 3.30–3.35 (m,
1H, 9-CH), 5.96 (s, 2H, 4-NH2), 6.45 (s, 2H, 2-NH2),
6.86 (s, 1H, 6-CH), 7.40–7.52 (m, 3H, C6H3). Anal.
(C15H14N4OCl2Æ0.075Et2O) C, H, N,Cl.
1
Rf = 0.53 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.13–1.15 (d, 3H, 9-CH3), 2.46–2.54 (d, 2H, 8-CH2),
2.88 (m, 1H, 9-CH), 3.80 (s, 3H, OMe), 6.04 (s, 2H,
4-NH2), 6.57 (s, 2H, 2-NH2), 6.90–6.99 (m, 2H, C6H4),
7.02 (s, 1H, 6-CH), 7.16–7.27 (m, 2H, C6H4). Anal.
(C16H18N4O2.0.25 H2O) C, H, N.
5.2.3. (R,S)-2,4-Diamino-5-[2-(30-methoxyphenyl)propyl]-
furo[2,3-d]pyrimidine (3). Compound 19 (120 mg,
0.4 mmol) hydrogenated at 45 psi for 6 h afforded 3
(75 mg, 62%) as white crystals: mp 186.7–188.5 ꢁ C;
5.2.9. (R,S)-2,4-Diamino-5-[2-(20,50dichlorophenyl)pro-
pyl]furo[2,3-d]pyrimidine (9). Compound 27 (120 mg,
0.28 mmol) hydrogenated at 50 psi for 20 h afforded 9
(60 mg, 50%) as white needles (recrystallized from cyclo-
hexane/CH3OH): mp 246.7–248.6 ꢁC; Rf = 0.57 (CHCl3/
CH3OH 5:1); 1H NMR (DMSO-d6): d 1.20–1.22 (d, 3H,
9-CH3), 2.96–3.04 (br, 2H, 8-CH2), 3.17–3.19 (br, 1H, 9-
CH), 5.96 (s, 2H, 4-NH2), 6.47 (s, 2H, 2-NH2), 6.92 (s,
1H, 6-CH), 7.27–7.28 (m, 1H, C6H3), 7.38 (s, 1H,
C6H3), 7.58–7.57 (d, 1H, C6H3). Anal. (C15H14N4O-
Cl2Æ0.05C6H12) C, H, N,Cl.
1
Rf = 0.52 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.21–1.23 (d, 3H, 9-CH3), 2.90–2.99 (m, 3H, 8-CH2
and 9-CH), 3.71 (s, 3H, OMe), 5.97 (s, 2H, 4-NH2),
6.41 (s, 2H, 2-NH2), 6.71–6.81 (m, 3H, C6H4), 6.92
(s, 1H, 6-CH), 7.14–7.19 (t, 1H, C6H4). Anal.
(C16H18N4O2) C, H, N.
5.2.4. (R,S)-2,4-Diamino-5-[2-(20-chlorophenyl)propyl]-
furo[2,3-d]pyrimidine (4). Compound 20 (150 mg,
0.4 mmol) hydrogenated at 55 psi for 24 h afforded 4
(80 mg, 52%) as white needles: mp 232.2–233.9 ꢁC;
5.2.10. (R,S)-2,4-Diamino-5-[2-(30,40dichlorophenyl)pro-
pyl]furo[2,3-d]pyrimidine (10). Compound 28 (150 mg,
0.4 mmol) hydrogenated at 50 psi for 14 h afforded 10
(80 mg, 52%) as white needles: mp 187.2–189.2 ꢁC;
1
Rf = 0.53 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.20–1.22 (d, 3H, 9-CH3), 2.96–2.98 (d, 2H, 8-CH2),

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5487
Rf = 0.55 (CHCl3/CH3OH 5:1); 1H NMR (DMSO-d6): d
1.21–1.23 (d, 3H, 9-CH3), 2.89–2.91 (br, 2H, 8-CH2),
2.96–3.03 (br, 1H, 9-CH), 5.96 (s, 2H, 4-NH2), 6.45 (s,
2H, 2-NH2), 6.95 (s, 1H, 6-CH), 7.20–7.23 (m, 1H,
C6H3), 7.48–7.57 (m, 2H, C6H3). Anal. (C15H14N4O-
Cl2Æ0.05H2O) C, H, N,Cl.
5.2.16. (R,S)-2,4-diamino-5-[2-(20-fluorenyl)propyl]furo[2,3-
d]pyrimidine (16). Compound 35 (100 mg, 0.3 mmol)
hydrogenated at 50 psi for 22 h afforded 16 (50 mg,
50%) as yellow crystals (recrystallized from CH3OH/cy-
clohexane): mp 250.3–252.2 ꢁC; Rf = 0.59 (CHCl3/
CH3OH 5:1); 1H NMR (DMSO-d6): d 1.27–1.29 (d,
3H, 9-CH3), 2.95–2.97 (d, 2H, 8-CH2), 3.02–3.17 (m,
1H, 9-CH), 3.86 (s, 2H, C13H9), 5.95 (s, 2H, 4-NH2),
6.42 (s, 2H, 2-NH2), 6.92 (s, 1H, 6-CH), 7.22–7.83 (m,
7H, C13H9). Anal. (C22H20N4OÆ0.15C6H12) C, H, N.
5.2.11. (R,S)-2,4-Diamino-5-[2-(30,40,50-trimethoxyphe-
nyl)propyl]furo[2,3-d]pyrimidine (11). Compound 29
(120 mg, 0.4 mmol) hydrogenated at 40 psi for 5 h affor-
ded 11 (60 mg, 50%) as white needles: mp 201–203 ꢁC;
1
5.3. Mouse tumor studies
Rf = 0.62 (CHCl3/CH3OH 5:1); H NMR (DMSO-d6):
d 1.21 (d, 3H, 9-CH3), 2.90 (d, 2H, 8-CH2), 3.16 (m,
1H, 9-CH), 3.64 (s, 3H, 40-OCH3), 3.73 (s, 6H, 30,
50-OCH3), 5.95 (s, 2H, 4-NH2), 6.39 (s, 2H, 2-NH2),
6.52 (s, 2H, C6H2), 6.99 (s, 1H, 6-CH). Anal.
(C18H22N4O4Æ0.5 H2O).
All animal studies were conducted in accordance with
AALAC approved guidelines using a protocol approved
by the IACUC at the University of Oklahoma. Six-
week-old male NCr nu/nu mice (NCI APA breeding
stock, Frederick, MD) were housed in ventilated cages
with autoclaved chow, water, and bedding. Following
one week of acclimation, 5 · 105 B16-F10 (GFP) cells47
in 100 ll PBS/1 lM EDTA were injected subcutaneously
into the external surface at the base of the right
ear (where the B16 tumor originally derived; ATCC
web site http://www.atcc.org/). On days 7, 11, 14, 18,
21, and 25, post-implantation animals received
injections of 25 mg/kg methotrexate (Sigma Chemical)
or of compounds 18 or 32 intraperitoneally in 100 ll
sterile water/1% DMSO. Animals were assessed for the
presence of tumors every other day and tumor measure-
ment began 7 days after implantation and continued
biweekly until tumors reached approximately 8–10% of
animal body weight (day 28 post-implantation). The
length (L) and width (W) of tumors were measured
using Vernier calipers (Mitutoyo, Kawasaki Kanagawa,
Japan) and tumor volume calculated using the formula
L · W2.
5.2.12. (R,S)-2,4-Diamino-5-[2-(20,30,40dichlorophenyl)pro-
pyl]furo[2,3-d]pyrimidine (12). Compound 30 (180 mg,
0.4 mmol) hydrogenated at 55 psi for 22 h afforded 12
(120 mg, 66%) as white crystals (recrystallized from
EtOAc): mp 257.6–259.6 ꢁC; Rf = 0.54 (CHCl3/CH3OH
1
5:1); H NMR (DMSO-d6): d 1.27–1.29 (d, 3H, 9-CH3),
3.04–3.06 (d, 2 H, 8-CH2), 3.22–3.25 (m, 1H, 9-CH),
6.03 (s, 2H, 4-NH2), 6.54 (s, 2H, 2-NH2), 6.97 (s,1H, 6-
CH), 7.59–7.56 (d, 1H, C6H2), 7.70–7.67 (d, 1H, C6H2).
Anal. (C15H13N4OCl3) C, H, N, Cl.
5.2.13. (R,S)-2,4-Diamino-5-[2-(20-naphthyl)propyl]furo[2,3-
d]pyrimidine (13). Compound 32 (100 mg, 0.3 mmol)
hydrogenated at 50 psi for 5 h afforded 13 (50 mg,
50%) as white crystals: mp 230.2–232 ꢁC; Rf = 0.57
1
(EtOH/EtOAc/hexane 1:2:1); H NMR (DMSO-d6): d
1.29–1.31 (d, 3H, 9-CH3), 2.92–3.10 (m, 3H, 8-CH2
and 9-CH), 5.95 (s, 2H, 4-NH2), 6.43 (s, 2H, 2-NH2),
6.85 (s, 1H, 6-CH), 7.40–7.48 (m, 3H, C10H7), 7.65 (s,
1H, C10H7), 7.80–7.85 (m, 3H, C10H7). Anal.
(C19H18N4OÆ0.1H2O) C, H, N.
After killing mice (28 days post-implantation), the tu-
mor and lungs were excised, fixed for 8 h in neutral
buffered formalin, embedded in paraffin, sectioned,
and stained by either H&E or standard immunohisto-
chemical methods as previously described.47 Sections
were deparaffinized, incubated in 6% peroxide for
20 min, incubated in 0.1 M sodium citrate until boil-
ing, and then for 10 additional minutes on 30% per-
oxide using a 700 W microwave oven. Sections were
then blocked using Vectastain kits (Vector Laborato-
ries, Burlingame, CA) and then exposed to an anti-
body against GFP (Clontech) to examine micro-
metastases. Secondary antibody and ABC reagent
were then added and the sections were stained with
VIP peroxidase substrate (Vector Laboratories) and
counterstained with Gomori trichrome (J.T. Baker
Company).47
5.2.14. (R,S)-2,4-Diamino-5-[2-(60-methoxy-20-naphthyl)-
propyl]furo[2,3-d]pyrimidine (14). Compound 33
(100 mg, 0.4 mmol) hydrogenated at 50 psi for 22 h
afforded 14 (80 mg, 80%) as white crystals (recrystallized
from CH3OH/EtOAc): mp 244–245.9 ꢁC; Rf = 0.58
1
(EtOH/EtOAc/hexane 1:2:1); H NMR (DMSO-d6): d
1.29–1.31 (d, 3H, 9-CH3), 2.98–3.02 (d, 2H, 8-CH2),
3.09–3.17 (m, 1H, 9-CH), 3.83 (s, 3H, OMe), 5.99 (s,
2H, 4-NH2), 6.39 (s, 2H, 2-NH2), 6.88 (s, 1H, 6-CH),
7.11–7.08 (t, 1H, C10H6), 7.24 (d, 1H, C10H6), 7.37–
7.40 (d, 1H, C10H6), 7.61 (s, 1H, C10H6), 7.69–7.75
(dd, 2H, C10H6). Anal. (C20H20N4O2Æ0.25 H2O) C, H, N.
5.2.15. (R,S)-2,4-Diamino-5-[2-(40-biphenyl)propyl]furo[2,3-
d]pyrimidine (15). Compound 34 (120 mg, 0.4 mmol)
hydrogenated at 45 psi for 16 h afforded 15 (60 mg,
50%) as white crystals: mp 243.1–245.1 ꢁC; Rf = 0.52
(CHCl3/CH3OH 5:1); 1H NMR (DMSO-d6): d 1.27–
1.25 (d, 3H, 9-CH3), 2.95 (d, 2H, 8-CH2), 3.18–3.08
(m, 1H, 9-CH), 5.96 (s, 2H, 4-NH2), 6.42 (s, 2H, 2-
NH2), 6.94 (s, 1H, 6-CH), 7.64–7.32 (m, 9H, C12H9).
Anal. (C21H20N4OÆ0.4H2O) C, H, N.
5.3.1. 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 (Manassas, VA).
5.3.2. Chemicals. All growth factors (bFGF, VEGF,
EGF, and PDGF-BB) were purchased from Peprotech
(Rocky Hill, NJ). PD153035, SU5416, AG1295, and

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A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
VEGF kinase inhibitor (4-[(40-chloro-20-fluoro)phenyl-
amino]-6,7-dimethoxyquinazoline) were purchased from
Calbiochem (San Diego, CA). The CYQUANT cells
proliferation assay was from Molecular Probes (Eugene,
OR). All other chemicals were from Sigma Chemical un-
less otherwise noted.
intercalates into the DNA of cells, is added and after
5 min the fluorescence of each well measured using an
UV products BioChemi digital darkroom. A positive
control used for cytotoxicity in each experiment was
cisplatin, with an apparent average IC50 value of
8.2 0.65 lM. Data are graphed as a percent of cells
receiving growth factor alone and IC50 values estimat-
ed from two to three separate experiments (n = 6–15)
using probit plots.
5.3.3. Antibodies. The PY-HRP antibody was from BD
Transduction Laboratories (Franklin Lakes, NJ). Anti-
bodies against EGFR, PDGFR-b, FGFR-1, Flk-1,
and Flt-1 were purchased from Upstate Biotech (Fra-
mingham, MA).
5.3.6. Chorioallantoic membrane assay of angiogenesis.
The chorioallantoic membrane (CAM) assay is a
53
standard assay for testing antiangiogenic agents.
The CAM assay used in these studies was modified
5.3.4. Phosphotyrosine ELISA. Cells used were tumor
cell lines naturally expressing high levels of EGFR
(A431), Flk-1 (U251), Flt-1 (A498), and 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,
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 exclusion. Cells are then pretreated for 60 min
with 10, 3.33, 1.11, 0.37, and 0.12 lM compound fol-
lowed by 100 ng/ml EGF, VEGF, PDGF-BB, or
bFGF for 10 min. The reaction is 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, protease inhib-
itor cocktail and tyrosine phosphatase inhibitor cock-
tail. The TBS solution is then removed and cells fixed
to the plate for 30 min at 60 ꢁC and further incuba-
tion 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 luminol
ELISA substrate (Pierce Chemical, Rockford, IL) and
light emission measured using a UV products (Up-
land, CA) BioChemi digital darkroom. 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 VEGF kinase inhib-
itor (4-[(40-chloro-20-fluoro)phenylamino]-6,7- dimeth-
oxyquinazoline) 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 estimated from two to
three separate experiments (n = 8–24) using hand-
drawn probit plots. In each case, the activity of a po-
sitive control inhibitor did not deviate more than 10%
from the IC50 values listed in the text.
54
55
from a procedure by Sheu
and Brooks
and as
published previously.56 Briefly, fertile leghorn chicken
eggs (CBT Farms, Chestertown, MD) are allowed to
grow until 10 days of incubation. The proangiogenic
factors, human VEGF-165 and bFGF (100 ng each)
are then added saturation to a 6 mm microbial test-
ing disk (BBL, Cockeysville, MD) and placed onto
the CAM by breaking a small hole in the superior
surface of the egg. Antiangiogenic compounds are
added 8 h after the VEGF/bFGF at saturation to
the same microbial testing disk and embryos allowed
to incubate for an additional 40 h. After 48 h, the
CAMs are perfused with 2% paraformaldehyde/3%
glutaraldehyde containing 0.025% Triton X-100 for
20 s, excised around the area of treatment, fixed
again in 2% paraformaldehyde/3% glutaraldehyde
for 30 min, placed on petri dishes, and a digitized im-
age taken using a dissecting microscope (Wild M400;
Bannockburn, IL) at 7.5X and SPOT enhanced digi-
tal imaging system (Diagnostic Instruments, Sterling
Heights, MI). A grid is then added to the digital
CAM images and the average number of vessels
within 5–7 grids counted as a measure of vascularity.
AGM-1470 (a kind gift of the NIH Developmental
Therapeutics Program) and SU5416 are used as a po-
sitive control for antiangiogenic activity. Data are
graphed as a percent of CAMs receiving bFGF/
VEGF and IC50 values estimated from two to three
separate experiments (n = 5–11) using probit plots.
5.3.7. Statistics. All analysis was done using Prism
4.0. (GraphPad Software, San Diego, CA). Tumor
growth rates were assessed during the linear growth
period and statistical significance of tumor growth
between groups was calculated using two-way repeat-
ed measures ANOVA with treatments and days after
implantation as independent variables with Dunnettꢂs
post-test with the null hypothesis rejected when
P < 0.05. Tumor metastases were compared using
one-way ANOVA with Bonferonniꢂs multiple compar-
ison post-test with the null hypothesis rejected when
P < 0.05.
5.3.5. CYQUANT cell proliferation assay. As a measure
of cell proliferation, the CYQUANT cell counting/
proliferation assay was used as previously described.52
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
Acknowledgment
This work was supported, in part, by the National Insti-
tutes of Health, National Cancer Institute Grants
CA09885 (A.G.) and CA10914 (R.L.K.).

A. Gangjee et al. / Bioorg. Med. Chem. 13 (2005) 5475–5491
5489
Appendix A. Elemental analyses
Compound
Formula
Calcd (%)
Found (%)
C
H
N
Cl
C
H
N
Cl
1
2
3
4
5
6
7
8
C15H16N4O
C16H18N4O2Æ0.25H2O
C16H18N4O2
C15H15N4OClÆ0.5CH3OH
C15H15N4OCl
C17H20N4O3Æ0.2H2O
C17H20N4O30.5H2O
C15H14N4OCl2Æ0.075Et2O
C15H14N4OCl2Æ0.05C6H12
C15H14N4OCl2Æ0.05H2O
C18H22N4O4Æ0.5H2O
C15H13N4OCl3
C19H18N4OÆ0.1H2O
C20H20N4O2Æ0.25H2O
C21H20N4OÆ0.4H2O
C22H20N4OÆ0.15C6H12
C15H14N4O
67.15
64.14
64.41
58.40
59.51
61.50
60.46
53.61
53.82
53.24
58.83
48.48
71.27
68.01
71.73
74.52
67.65
64.37
63.82
59.91
59.91
59.91
62.57
62.57
62.57
53.75
53.75
53.75
53.75
60.66
48.74
48.74
71.08
72.13
69.35
73.67
74.14
6.01
6.08
6.08
5.33
4.99
6.19
6.22
4.34
4.28
4.17
6.47
3.53
5.68
5.81
5.96
5.91
5.30
5.67
5.48
4.36
4.36
4.36
5.56
5.56
5.56
3.61
3.61
3.61
3.61
5.66
3.00
3.00
5.19
5.10
5.24
5.30
5.01
20.88
18.78
18.78
17.57
18.51
16.87
16.60
16.34
16.40
16.56
15.07
15.08
17.49
15.87
15.93
15.18
21.04
19.71
18.61
18.36
18.36
18.36
17.17
17.17
17.17
16.72
16.72
16.72
16.72
15.72
15.16
15.16
17.45
17.71
16.17
16.36
15.76
67.28
63.74
64.39
58.49
59.36
61.59
60.81
53.92
54.00
53.44
58.91
48.40
71.18
67.90
71.76
74.34
67.36
64.23
64.15
59.97
59.91
59.89
62.47
62.29
62.38
53.60
53.83
53.77
53.49
60.37
48.61
48.63
71.10
71.96
69.44
73.37
73.97
5.97
6.04
5.99
5.41
5.02
6.11
6.22
4.24
4.24
4.56
6.28
3.68
5.60
5.63
5.97
5.63
5.38
5.72
5.56
4.41
4.42
4.37
5.57
5.53
5.59
3.62
3.60
3.73
3.69
5.72
3.07
3.09
5.20
5.04
5.29
5.23
5.23
20.92
18.44
18.88
17.53
18.32
16.80
16.67
16.67
16.69
16.59
15.13
14.92
17.54
15.92
15.81
14.85
20.98
19.66
18.58
18.62
18.57
18.45
17.03
16.96
17.21
16.63
16.60
16.64
16.67
15.60
15.00
15.03
17.36
17.52
16.30
16.41
15.61
11.12
11.71
11.29
11.91
20.69
20.77
20.96
20.54
20.38
20.59
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
27a
28
29
30a
30b
31
32
33
34
35
28.62
28.74
C16H16N4O2
C16H16N4O2Æ0.25H2O
C15H13N4OCl
C15H13N4OCl
C15H13N4OCl
C17H18N4O3
C17H18N4O3
C17H18N4O3
C15H12N4OCl2
C15H12N4OCl2
C15H12N4OCl2
C15H12N4OCl2
C18H20N4O4
C15H11N4OCl3
C15H11N4OCl3
C19H16N4OÆ0.25H2O
C19H16N4O
C20H18N4O2
C21H18N4O
C22H18N4O
11.79
11.79
11.79
11.91
11.69
11.83
21.15
21.15
21.15
21.15
20.97
21.01
21.02
20.91
28.77
28.77
28.88
28.67
6. Cherrington, J. M.; Strawn, L. M.; Shawver, L. K. New
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literature with different IC50 values for SU5416. We
believe that the IC50 value depends upon the assay and
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