2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.11.029

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR.

Full text of DOI:10.1016/j.bmc.2009.11.029

Bioorganic & Medicinal Chemistry 18 (2010) 953–961
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3-d]pyrimidines as novel
classical and nonclassical antifolates as potential dual thymidylate
synthase and dihydrofolate reductase inhibitors q
a
a
a
b
c
Aleem Gangjee a, , Hiteshkumar D. Jain , Jaclyn Phan , Xin Guo , Sherry F. Queener , Roy L. Kisliuk
*
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States
b Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN 46202, United States
c Department of Biochemistry, Tufts University School of Medicine, Boston, MA 02111, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A
novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-L-
Received 2 September 2009
Revised 10 November 2009
Accepted 12 November 2009
Available online 26 December 2009
glutamic acid 5 and 11 nonclassical antifolates 6–16 were designed, synthesized, and evaluated as
inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds
6–16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide
coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound
5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that
has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar
inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR
at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6–16
were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine
scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity
for pathogen DHFR.
Keywords:
Furo[2,3-d]pyrimidines
Thymidylate synthase
Dihydrofolate reductase
Dual inhibitors
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
quently phosphorylated to 20-deoxythymidine-50-diphosphate
(dTDP) and 20-deoxythymidine-50-triphosphate (dTTP) and finally
Thymidylate synthase (TS) catalyzes the de novo synthesis of 20-
deoxythymidine-50-monophosphate (dTMP) from 20-deoxyuridine-
50-monophosphate (dUMP) by transferring a methyl group from
5,10-methylenetetrahydrofolate polyglutamates (5,10-CH2-H4Pte-
Glun) to the C-5 position of the pyrimidine ring of the substrate
dUMP. During the TS catalyzed reaction, 5,10-CH2-H4PteGlun spe-
cies are converted to the corresponding 7,8-dihydrofolate (7,8-
H2PteGlun). Dihydrofolate reductase (DHFR) carries out the first
step in the regeneration of 5,10-CH2-H4PteGlun, that is, formation
of 5,6,7,8-tetrahydrofolate utilizing NADPH as the reductant.2
dTMP is the product of the TS catalyzed reaction and is subse-
incorporated into DNA via DNA polymerase. Thus, both TS and
DHFR play a crucial role in DNA biosynthesis and cell proliferation
and hence both enzymes have been extensively targeted for devel-
oping cancer chemotherapeutic and antiopportunistic infection
agents.3 Raltitrexed4 ( Fig. 1) and pemetrexed5 are folate based
inhibitors of TS and represent important examples of clinically
used antitumor agents, while methotrexate (MTX) (Fig. 1) a DHFR
inhibitor is a mainstay in single agent and combination cancer
chemotherapy.6
Many cultured tumor cell lines show synergistic cytotoxicity
when grown in a medium containing a combination of a DHFR
inhibitor with either a TS or glycinamide ribonucleotide formyl-
transferase (GARFT) antifolate.7 This effect is termed the ‘Kisliuk
effect’.8 In addition, synergistic growth inhibition has also been ob-
served by combining a DHFR inhibitor with a TS inhibitor, against
cell cultures of Lactobacillus casei,9,10 rat hepatoma cells,11,12 and
human lymphoma cells.9,13,14 Numerous examples of antifolates
have been reported that possess dual TS and DHFR activity.5,15–18
It has also been suggested15 that the chemotherapeutic utility of
such analogs would be viable if the inhibitory activity against
DHFR is similar to that against TS. Pemetrexed5 represents an
important example of a classical antifolate that has reported dual
Abbreviations: TS, thymidylate synthase; dTMP, 20-deoxythymidine-50-mono-
phosphate; dUMP, 20-deoxyuridine-50-monophosphate; DHFR, dihydrofolate
reductase; dTDP, 20-deoxythymidine-50-diphosphate; dTTP, 20-deoxythymidine-50-
triphosphate; MTX, methotrexate; GARFT, glycinamide ribonucleotide
formyltransferase; AICARFT, 5-aminoimidazole-4-carboxamide ribonucleotide
formyltransferase; FPGS, folyl poly-c-glutamate synthetase; PTX, piritrexim;
P. carinii, Pneumocystis carinii; T. gondii, Toxoplasma gondii; M. avium, Mycobacterium
avium; MAC, Mycobacterium avium complex.
q
See Refs. 1a,b.
* 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.2009.11.029

954
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
NH2
N
CH3
N
O
C N
O
N
COOH
COOH
O
C N
COOH
S
H
N
N
N
CH3
N
HN
H3C
H
H2N
COOH
Methotrexate (MTX)
Raltitrexed
(ZD1694, TomudexTM
)
O
N
NH2
N
O
H
O
C N
COOH
COOH
COOH
COOH
H
C N
X
HN
H2N
N
N
H
H2N
O
1
X = NH
2 X = NCH3
Pemetrexed
3
X = CH2
(LY231514, AlimtaTM
)
N
NH2
O
N
O
N
S
O
OH
OH
O
HO
HO
CH3
O
S
N
HN
H2N
O
OH
N
H3C
AG331
Nolatrexed (AG337, Thymitaq)
NH2
N
O
C
COOH
NH2
N
CH3
CH3
S
N
N
H
COOH
N
R
H2N
X
S
H2N
O
6 16
-
4
X = NH
5 X = O
6
7
8
9
11
R = 2',3'-(CH)4
R = 3',4'-(CH)4
R = 2',6'-diCH3
R = 4'-OCH3
12 R = 4'-CF3-2',3',5',6'-tetraF
13
R = 2'-
OCH3
diCl
R = 2',6'-
14 R = 3'-OCH3
10 R = 2',5'-diOCH3 15 R = 3',4'-diOCH3
16
R = 2'-CH3-6'-isopropyl
Figure 1.
TS and DHFR inhibitory activity. In addition, pemetrexed and its
polyglutamylated metabolites are also reported as inhibitors of
several other important folate-dependent enzymes including GAR-
FT and 5-aminoimidazole-4-carboxamide ribonucleotide formyl-
transferase (AICARFT).19 The clinical success of pemetrexed has
generated a renewed interest in designing single agents that could
function as multi targeted inhibitors of folate metabolizing en-
zymes. Such inhibitors could also circumvent the pharmacokinetic
disadvantages of administering separate agents in combination
chemotherapy protocols.
Antifolate inhibitors of TS are characterized by the presence of a
2-substituted-4-oxo moiety in the pyrimidine ring as exemplified
by pemetrexed and raltitrexed.20,21 In contrast, inhibitors of DHFR
are characterized by the presence of a 2,4-diamino substitution in
the pyrimidine ring20,21 as represented by MTX.
proposed flipped mode22,23 (Fig. 2) the 5-position side chain substi-
tuent lies between the 6- and 7-carbon atoms of a 6–6 fused ring sys-
tem and may account for its lower or lack of TS inhibitory activity.
Compounds 1–3 were found to be substrates for folyl poly-c-gluta-
mate synthetase (FPGS) and demonstrated significant tumor cell
growth inhibitory activity. As an extension of our ongoing program
compound 5 (Fig. 1) was synthesized as a potential dual inhibitor
of TS and DHFR. Compound 5 is a furo[2,3-d]pyrimidine analog of
4 and was designed to bind to human DHFR in the normal 2,4-diami-
no bindingmode (Fig. 2) and to human TS in the flipped mode where-
in the furo 7-O could mimic the 4-oxo of classical 2-substituted-4-
oxo pyrimidine moiety containing antifolate.
We17 recently reported the design and synthesis of the 2,4-dia-
mino 6-substituted pyrrolo[2,3-d]pyrimidine 4 (Fig. 1) as a dual
inhibitor of human DHFR and human TS. The 5-methyl group
was incorporated to provide hydrophobic interaction with
Val115 in human DHFR. Compound 4 was initially designed as a
potential nonpolyglutamylatable DHFR inhibitor. However, unex-
pectedly 4 was found to be a dual inhibitor of human DHFR
(IC50 = 210 nM) and human TS (IC50 = 540 nM) with reasonable
FPGS substrate activity. Molecular modeling using SYBYL 6.824 indi-
cated that 4 binds to human DHFR in the normal 2,4-diamino mode
while it could bind to human TS in the flipped mode similar to its
The 2,4-diaminofuro[2,3-d]pyrimidine ring system represents an
ideal scaffold that could function as both a 2,4-diamino and 2-ami-
no-4-oxo substituted system. To test this hypothesis we22,23
reported compounds 1–3 (Fig. 1) as potential dual DHFR-TS inhibi-
tors. Compounds 1–3 inhibited human DHFR with an IC50 in the
0.22–1.0
>200 M) human TS inhibitory activity. One probable reason for this
is that when these 5-substituted analogs bind to human TS in the
lM range however they did not possess any (IC50
l

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
955
R
S
CH3
NH2
N
O
N
CH3
rotation 180 o
N
N
S
R
H2N
O
H2N
NH2
2,4-Diamino (DHFR) Binding Mode
2-Amino-4-Oxo (TS) Binding Mode
'Normal'
'Flipped'
Figure 2.
furo[2,3-d]pyrimidine counterpart 5. In addition, molecular model-
ing also indicated that the 5-CH3 group in 4 could have a hydro-
phobic interaction with Trp109 in human TS.
Asp218 of human TS in a salt bridge interaction much like that
seen with the N1 and 2NH2 of MTX with Glu30 in human DHFR.26
The 2NH2 could also interact with the backbone CO of Ala312 and
the 4NH2 of 5 could H-bond with Arg50 and/or Asn112 mimicking
the N1 and N7 of pemetrexed.25 The 5-CH3 of 5 interacts with
Trp109 and with Ile108. The phenyl ring of 5 makes hydrophobic
contact with Phe225 much like the phenyl ring of pemetrexed.25
There is no direct H-bonding with the 4-oxo moiety of pemetrexed
and none with the O7 of 5 in its ‘flipped’ mode (Fig. 2). Thus molec-
ular modeling (Fig. 3) predicts that 5 would bind to human TS and
afford inhibition of human TS.
Energy minimized (SYBYL 8.1) 5 (yellow) (Fig. 4) was superim-
posed with its pyrimidine ring on the pyrimidine ring of MTX
(green) in the X-ray crystal structure of MTX in human DHFR.26
Compound 5 superimposes very well onto MTX in Figure 4. The
N1 and 2NH2 of 5 are protonated, much like MTX26 and form a salt
bridge with Glu30. In addition, the 4NH2 moiety is hydrogen
bonded to the backbone CO of Ile7 and Val115. The phenyl ring
of 5 makes hydrophobic contacts with Phe34, Phe31, Ile60, Pro61
Thus, compound 5 was synthesized as an oxygen (furo) isostere
of 4, and to also determine the importance of the pyrrole 7-NH in 4
for binding to both human TS and human DHFR. The replacement
of an ‘NH’ of a pyrrolo[2,3-d]pyrimidine with an ‘O’ to afford the
furo[2,3-d]pyrimidine was also anticipated to evaluate the impor-
tance of a ‘hydrogen donor’ (NH) versus a ‘hydrogen acceptor’ (O).
Molecular modeling (see below) allows a prediction of activity
depending on the mode of binding of the molecule at the enzyme
active-site. Thus N-[4-[(2,4-diamino-5-methylfuro[2,3-d]pyrimi-
din-6-yl)thio]benzoyl]-L-glutamic acid 5 ( Fig. 1) was designed
and synthesized as a potential dual DHFR-TS inhibitor and as a po-
tential antitumor agent. Since the 6-substitution of 4 afforded dual
inhibitory (TS and DHFR) activity and the 5-substitution of
furo[2,3-d]pyrimidines 1–3 did not afford dual TS/DHFR inhibitory
activity, it was anticipated, on the basis of molecular modeling,
that the transposed side chain from the 5-position (1–3) to the
6-position in 5 would afford dual TS/DHFR inhibitory activity akin
to 4. Compound 5 was expected to possess FPGS substrate activity
similar to its pyrrolo[2,3-d]pyrimidine counterpart 4.
Molecular modeling of 5 (SYBYL 8.1) predicted dual human TS/
DHFR inhibitory activity for 5. Compound 5 (yellow in Fig. 3) in
the flipped orientation was minimized and superimposed onto
pemetrexed (green) in the X-ray crystal structure of pemetrexed
in human TS.25
Like pemetrexed, compound 5 is anchored in the active-site of
human TS by aromatic stacking between the furo[2,3-d]pyrimidine
ring of 5 and the dUMP (314 in Fig. 3) pyrimidine ring. In addition,
the N1 and 2NH2 of 5 are probably protonated much like the 2,4-
diNH2 substitution of MTX that allows a protonation of the N1
and 2NH2.26 This protonated N1 and 2NH2 of 5 could interact with
and Leu67. The a-COOH of 5 makes a salt bridge with Arg70. The
N8 of MTX forms H-bonding via a conserved active-site water mol-
ecule (H2O257, not shown in Fig. 4) to the indole ring of Trp24 and
the COOH of Glu30. In Figure 4 the O7 of the furo ring of 5 mimics
the N8 of MTX and could also H-bond via H2O257 to Trp84 and
Glu30. Thus the interaction of 5 with human DHFR mimics those
of MTX and 5 was expected to provide human DHFR inhibitory
activity. Hence on the basis of molecular modeling, 5 was antici-
pated to possess dual human DHFR and TS inhibitory activity.
As mentioned above, inhibition of either DHFR or TS can result
in cytotoxicity and TS and DHFR inhibitors can be combined in can-
cer chemotherapy to achieve synergistic effect.27,7 Nonclassical
(compounds lacking a
L-glutamate side-chain) DHFR inhibitor such
as piritrexim (PTX)28 and TS inhibitors such as N6-[4-(morphol-
Figure 3. Stereoview 5 (yellow) superimposed on pemetrexed (green) in the X-ray crystal structure of pemetrexed in human TS (PDB: 1JUJ)25 using SYBYL 8.1.

956
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
Figure 4. Stereoview of minimized 5 (yellow) superimposed onto MTX (green) in the X-ray crystal structure of MTX in human DHFR. (PDB: 1U72).26
inosulfonyl)benzyl]-N6-methyl-2,6-diaminobenz-[c,d]-indole glu-
curonate29 and nolatrexed30 ( Fig. 1) are also potent antitumor
agents. Hence, nonclassical antifolates that display dual TS-DHFR
inhibition are attractive antitumor agents and like their classical
counterparts could afford ‘combination therapy’ in a single agent.
Thus in addition to the antitumor activity of 5, we were also inter-
ested in the antitumor activity of the nonclassical analogs 6–16,
which could perhaps function as dual TS-DHFR inhibitors.
1. 2,6-Diamino-4-hydroxypyrimidine (17) was condensed with
chloroacetone (18) to afford the known 2,4-diamino-5-methylf-
uro[2,3-d]pyrimidine (20)34 which was reacted with appropriately
substituted thiophenols in the presence of iodine to afford 6–16.
Compound 20 was prepared by a modified procedure reported
by Secrist and Liu.34 As previously reported,34 the cycloaddition
of 2,6-diamino-4-hydroxypyrimidine (17) and chloroacetone (18)
in DMF at 50–60 °C gave two products, that is, 2-amino-4-oxo-6-
methylpyrrolo[2,3-d]pyrimidine (19) and 2,4-diamino-5-methylf-
uro[2,3-d]pyrimidine (20) in a 2:1 ratio, the former being the major
product. Chromatographic separation afforded 20. The yield of the
furo[2,3-d]pyrimidine 20 by this reported method was 20%. In our
hands with careful protection of the reaction from light and mois-
ture, the yield of 20 was increased to 25%. The 1H NMR spectrum of
20 was identical to that reported in the literature34 except that
both the 5-CH3 peak and the 6-CH peak were doublets with a cou-
pling constant of J = 2.0 Hz rather than two singlets as reported in
the literature.34 This was due to the higher resolution of the cur-
rent 1H NMR spectrometer (300 MHz compared to 70 MHz in the
literature),34 which readily detected the allylic coupling between
the two sets of protons.
With 20 in hand the key step was to add appropriately substi-
tuted aryl thiols to the 6-position of the furo[2,3-d]pyrimidine. In
1971, Beveridge and Harris35 reported the oxidative coupling of
thiols to pyrroles and indoles in the presence of I2. The proposed
mechanism of this oxidative coupling was that first the I2 reacts
with the thiol to form a sulfenyl iodide species, which then couples
with the pyrroles or indoles via an electrophilic substitution reac-
tion. To the best of our knowledge, no oxidative thiolation has been
reported for the substitution of furans. Since furans and pyrroles
are both five-membered aromatic ring systems, with a heteroatom
contributing one lone pair of electrons to its aromaticity, it was
envisioned that the oxidative coupling reaction could also be
adopted for substitution at the 6-position of 20 to afford target
molecules 6–16. We17,18,36,37 have extensively utilized the oxida-
tive thiolation procedure in their synthesis of both 5- and 6-aryl-
thio substituted pyrrolo[2,3-d]pyrimidine containing antifolates.
For the synthesis of 6 and 7, 20 in EtOH/water was treated with
iodine followed by the appropriate 1-naphthalene thiol or 2-naph-
thalene thiol to afford on workup and chromatographic purifica-
tion 6 and 7, respectively. The 1H NMR of the products showed
Another aspect of our interest in nonclassical dual TS-DHFR
inhibitors lies in the treatment of opportunistic infections in im-
mune-compromised patients, especially those with AIDS.31,32
Some of the most prevalent opportunistic infections which are
the major cause of morbidity and mortality in patients with AIDS
and in other immune-compromised patients, such as heart and
other transplant patients, are caused by Pneumocystis jirovecii (P.
jirovecii) formerly Pneumocystis carinii (P. carinii)31,32 and Toxo-
plasma gondii (T. gondii).31,32 A third infection, though not usually
fatal, is Mycobacterium avium (M. avium) complex (MAC) a group
of organisms responsible for disseminated infections in AIDS
patients, that is responsible for a decrease in the quality of life of
the patients.31,32 Several nonclassical pyrrolo[2,3-d]pyrimidines,
and furo[2,3-d]pyrimidines have been evaluated as inhibitors of
P. carinii, T. gondii, and M. avium DHFR.21,33 Some of these have
been found to be selective for DHFR from these pathogens. Thus,
we were also interested in evaluating the nonclassical compounds
6–16 as inhibitors of P. carinii DHFR, T. gondii DHFR, and M. avium
DHFR. The phenyl ring substitutions in 6–16 are based on
similar substitutions that have provided potent and/or selective
agents.21,33 The nonclassical analogs 6–16 were anticipated to inhi-
bit DHFR and/or TS from P. carinii and/or T. gondii, and/or M. avium
and perhaps provide selective inhibitors against these pathogens.
P. carinii, T. gondii and M. avium lack the transport system(s) re-
quired for classical antifolates; however, the lipophilic nonclassical
compounds 6–16 were anticipated to gain access to the pathogenic
cells by passive diffusion.
2. Chemistry
The synthesis of nonclassical 2,4-diamino-5-methyl-6-substi-
tuted arylthio-furo[2,3-d]pyrimidines (6–16) is shown in Scheme

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
957
O
N
O
N
Cl
a
HN
HN
+
NH
O
CH3
H2N
N
H
CH3
H2N
2
18
19
17
+
NH2
CH3
e/f
6-16
N
H2N
N
O
20
b
NH2
NH2
N
O
COOR
CH3
S
CH3
c
COOH
N
N
N
H
H2N
N
O
H2N
O
d
S
COOR
21
22
5
R=Et
R=H
Scheme 1. Reagents and conditions: (a) DMF, 50–60 °C, 72 h; (b) 4-mercaptobenzoic acid, EtOH/H2O (2:1), 100–110 °C, I2; (c) 2-chloro-4,6-dimethoxy-1,3,5-triazine, N-
methylmorpholine, diethyl- -glutamate hydrochloride, 0 °C to rt; (d) 1 N NaOH, 0 °C to rt; (e) ArSH, I2, 80–90 °C, 4 h (6 and 7); (f) ArSH, 100–110 °C, I2 (8–16).
L
Table 1
the disappearance of the 6-CH (d 7.09) of the starting material and
new peaks corresponding to the protons of the aromatic side
chains at d 7.10–8.30. The structure of the products was confirmed
by elemental analysis. Recently, we38,39 reported an improved oxi-
dative thiolation procedure, which involved the addition of I2 after
the thiophenols rather than before, resulting in significant
improvement in the yields. Thus, compounds 8–16 were prepared
via the modified method in 10–25% yields. The yields did not cor-
relate with the extent of furo[2,3-d]pyrimidine substitution or the
electron donating or withdrawing effects of substituents in the
thiophenol.
Inhibitory concentrations (IC50
,
l
M) against isolated TS and DHFRa
TS DHFR
Humanb E. colib T. gondiic Humand E. colie T. gondiic
Compd
4
5
0.54
5.4
0.38
>180
230
5.7
76
90
1.8
27
0.9
2.8
18
0.21
4.0
21
6.6
0.022
0.016
0.020
23
0.17
2.2
2.3
Raltitrexedf
Pemetrexedg 9.5
230
0.46
MTX
29
0.0066 0.011
Dihydrofolate reductase (DHFR) assay:41 All enzymes were assayed spetrophoto-
metrically in a solution containing 50 M dihydrofolate, 80 M NADPH, 0.05 M
l
l
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/min.
For the synthesis of the classical analog 5, 4-mercaptobenzoic
acid was oxidatively added to the 6-position of 20 in the presence
of I2 in H2O/EtOH mixture at reflux (Scheme 1). The desired inter-
mediate 21 was contaminated with the disulfide of 4-mercapto-
benzoic acid as indicated by its 1H NMR. This crude product was
purified by washing with a mixture of ethyl acetate and ethyl alco-
hol (v/v 2:1) to afford pure 21 in 87% yield.
Thymidylate synthase (TS) assay: TS was assayed spectrophotometrically at 30 °C
and pH 7.4 in a mixture containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-
tetrahydrofolate, 0.012 M formaldehyde, 0.02 M MgCl2, 0.001 M dUMP, 0.04 M
Tris–HCl, and 0.00075 M NaEDTA. This was the assay described by Wahba and
Friedkin,42 except that the dUMP concentration was increased 25-fold according to
the method of Davisson et al.43 The reaction was initiated by the addition of an
amount of enzyme yielding a change in absorbance at 340 nm of 0.016/min in the
absence of inhibitor.
This method gave 21 in one step with high yield, which signif-
icantly simplified the procedure for classical compounds compared
to the previously reported methods18,36 (Scheme 1), where the
pteroate esters were first synthesized followed by saponification
to afford the free acid.
a
The percent inhibition was determined at a minimum of four inhibitor con-
centrations within 20% of the 50% point. The standard deviations for determination
of 50% points are within 10% of the value given.
b
Kindly provided by Dr. Frank Maley, New York State Department of Health,
Peptide coupling40 of the acid 21 with diethyl
L-glutamate using
Albany, NY.
c
Kindly provided by Dr. K. Anderson, Yale University.
Kindly provided by Dr. J. H. Freisheim, Medical College of Ohio, Toledo, OH.
Kindly provided by Dr. R. L. Blakley, St. Jude Children’s hospital, Memphis TN.
Kindly provided by Dr. Ann Jackman, Institute of Cancer Research, Sutton,
2,6-dimethoxy-4-chlorotriazine and N-methyl morpholine, fol-
lowed by chromatographic purification gave the coupled product
22 in 60% yield. Hydrolysis18,36 of the diester 22 at room tempera-
ture, followed by acidification gave 5 in 86% yield.
d
e
f
Surrey, UK.
g
Kindly provided by Dr. Chuan Shih, Eli Lilly and Co.
3. Biological evaluation and discussion
Compound 5 was evaluated as an inhibitor of human, Esche-
richia coli (E. coli), and T. gondii TS and DHFR. The inhibitory po-
tency (IC50) values are listed in Table 1 and compared with 4,
raltitrexed, pemetrexed and MTX. Compound 5 was about twofold
more potent as a human TS inhibitor than pemetrexed and about
14-fold less potent than raltitrexed. Against human DHFR 5 was
1.5-fold more potent than pemetrexed and fivefold more potent
than raltitrexed. Thus, compound 5 is a novel dual DHFR-TS

958
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
inhibitor. To the best of our knowledge this is the first example of a
classical 2,4-diamino furo[2,3-d]pyrimidine antifolate that possess
dual DHFR-TS inhibitory activity that is equal to or better than the
clinically used pemetrexed.
agents.21,33 Biological evaluation of compounds 6–16 indicated
that they were moderately potent inhibitors of P. carinii, T. gondii
and M. avium DHFR. The most potent and selective compound
against P. carinii DHFR was 6 containing a 1-naphthyl side chain.
The most potent and selective compound against T. gondii DHFR
was 16 containing a 2-isopropyl-6-methyl phenyl side chain.
Against M. avium DHFR compound 15 was the most potent and
selective and contained an 3,4-dimethoxy phenyl side chain. Most
of the compounds tested against all the three pathogen DHFR
lacked the high potency and selectivity of compound 5. A possible
explanation for the lack of DHFR inhibitory activity of the nonclas-
sical analogs is that the single atom 6-S bridge between the
furo[2,3-d]pyrimidine and the substituted phenyl side chain is
Compound 5 was 10-fold and 19-fold less potent than its pyr-
rolo[2,3-d]pyrimidine counterpart 4, against human TS and human
DHFR, respectively. These results indicate that the pyrrole 7-NH in
4 plays an important role in binding and that a hydrogen donor 7-
NH as in the pyrrolo[2,3-d]pyrimidine 4 is more conducive for
binding to both human DHFR and human TS than the hydrogen
acceptor 7-O in the furo[2,3-d]pyrimidine 5. Surprisingly, the non-
classical analogs 6–16 (data not included) were inactive against
both human and E. coli TS and DHFR with IC50 values >2 ꢀ 10
ꢁ5 M. A possible reason for the inactivity of the nonclassical ana-
logs 6–16 could be that the 6-substituted single atom sulfur bridge
is perhaps too short to allow appropriate interactions with the en-
zymes in the absence of the glutamate side substituent present in
5. Studies are currently underway to increase the bridge length and
to provide other substitutions on the side chain phenyl ring to af-
ford better inhibitors of TS and/or DHFR.
Compounds 5–16 were also evaluated as inhibitors of P. carinii,
T. gondii, M. avium, and rat liver DHFR in a slightly different assay
system than that reported in Table 1. The inhibitory potencies
(IC50) and selectivity ratios are listed in Table 2 along with that
for trimetrexate (TMQ) and trimethoprim (TMP). Compound 5 dis-
played a two digit nanomolar potency against P. carinii DHFR and
single digit nanomolar potency against M. avium DHFR. In addition
compound 5 displayed a 263-fold selectivity for P. carinii DHFR and
a remarkable 2100-fold selectivity for M. avium DHFR, compared
with the mammalian standard rat liver DHFR. Compound 5 would
not be expected to be useful against P. carinii and M. avium infec-
tions in immunocompromized patients because these organisms
lack the transport mechanisms necessary for classical antifolates
like 5. However the potent inhibitory activity along with the
remarkable selectivity of 5 against these pathogen DHFRs provides
useful information on structural features that afford both high po-
tency and high selectivity and served as a template for the design
of lipophilic nonclassical analogs 6–16 containing various lipo-
too short and lacks a a-COOH and does not permit sufficient inter-
action of the substituted phenyl ring with appropriate hydrophobic
(Phe31, Phe34, Ile60, Pro61, Leu67) and/or ionic (Arg70) portions of
the DHFR enzyme. Alternatively the side chain could be held in a
conformation that is not conducive to optimum binding in the ab-
sence of the
classical analog a potent inhibitor of P. carinii DHFR and M. avium
DHFR? The answer probably lies in the -carboxylic acid moiety
of the -glutamate of 5 which should make ionic (salt bridge) inter-
a-COOH of the L-glutamate as in 5. Why then was the
a
L
actions with an invariant Arg in DHFR. Thus, the classical analog 5
has additional binding sites which appropriately orients the mole-
cule for optimum hydrophobic and ionic interactions with DHFR
and translates into better inhibitory activity.
In summary we have reported the synthesis and biological eval-
uation of a classical 2,4-diamino-5-methyl-6-substituted furo[2,3-
d]pyrimidine 5 along with eleven nonclassical antifolate analogs
6–16. The classical compound 5 is the first analog with the
furo[2,3-d]pyrimidine scaffold to demonstrate dual human TS
and DHFR inhibitory activity. In addition against P. carinii DHFR
and M. avium DHFR compound 5 demonstrated high potency
(IC50 = 78 nM; P. carinii DHFR and IC50 = 9.73 nM; M. avium DHFR)
and selectivity (263-fold for P. carinii DHFR and 2100-fold for M.
avium DHFR). Since P. carinii and M. avium lack the transport sys-
tem(s) for classical folates compound 5 would not enter these
pathogen cells.
philic substituents in the side chains and lacking the polar
mate moiety.
Lipophilic nonclassical analogs containing pyrrolo[2,3-d]pyrim-
idine or furo[2,3-d]pyrimidine scaffolds have been extensively syn-
thesized as potential antibacterial, antiprotozoan and antifungal
L
-gluta-
The nonclassical analogs 6–16 had moderate potency and
lacked appreciable selectivity for pathogen DHFR. This study pro-
vides the first example of a classical 2,4-diamino furo[2,3-d]pyrim-
idine scaffold with dual human TS-DHFR activity and affords a
scaffold, the furo[2,3-d]pyrimidine, that with appropriate bridge
Table 2
Inhibition concentrations (IC50, l
M) against isolated DHFRa and selectivity ratiosb
Compound
P. carinii
Rat liver
rl/pcb
T. gondii
rl/tgb
M. avium
rl/mab
2106.9
5
6
7
8
9
10
11
12
13
14
15
16
TMQ
TMP
0.078
8.6
20.5
>83
>12
262.8
>10
ND
ND
0.2
1.8
1.8
ND
2.7
2.0
12
1.7
NDc
ND
ND
0.6
1.1
0.9
0.5
1.3
1.1
1.3
1.5
0.3
65
0.00973
>83
>12
19.7
25.5
38.4
69.4
81.8
44.2
45.9
51.7
16.2
0.010
2.8
>12
20 (27%)d
60
196 (41%)
14.4
42.2
61.9
39.1
55.6
51.3
66.6
24.3
0.003
180
196 (20%)
56.2
61.9
25.5
31 (8%)
23.9
28.4
16.3
23.4
0.0015
0.3
ND
0.26
0.68
2.43
ND
2.33
1.81
4.09
1.04
2.0
24
34.7
31 (39%)
20.3
25.2
36.4
36.5
0.042
12
1.8
0.7
0.07
14
610
a
These assays were carried out at 37 °C under conditions of saturation with substrate (90
l
M dihydrofolic acid) and cofactor (119
lM NADPH) in the presence of 150 mM
KCl.
b
Selectivity ratios, rl/pc = IC50 rat liver dihydrofolate reductase/IC50 P. carinii dihydrofolate reductase; rl/tg = IC50 rat liver dihydrofolate reductase/IC50 T. gondii dihy-
drofolate reductase; rl/ma = IC50 rat liver dihydrofolate reductase/ IC50 M. avium dihydrofolate reductase.
c
ND = not determined.
Number in parentheses indicate the percentage inhibition at the given concentration.
d

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
959
length variation and substitution(s) on the phenyl ring could afford
nonclassical antifolate inhibitors of TS and/or DHFR.
residue was washed first with EtOAc and then with EtOH to afford
21 (838 mg, 87%) as a white solid.
4.3. Diethyl N-[4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimi-
din-6-yl)thio]-benzoyl]-L-glutamate (22)
4. Experimental section
All evaporations were carried out in vacuum with a rotary
evaporator. Analytical samples were dried in vacuum (0.2 mmHg)
in an Abderhalden drying apparatus over P2O5 at 70 °C. Thin-layer
chromatography (TLC) was performed on silica gel plates (What-
To a suspension of the acid 21 (500 mg, 1.57 mmol) in anhy-
drous DMF (40 mL) under N2 was added N-methylmorpholine
(217 lL, 2.00 mmol) and the resulting suspension was cooled
to 0 °C. At this point 2-chloro-4,6-dimethoxy-1,3,5-triazine
(350 mg, 2.00 mmol) was added and the suspension was stirred
for 2 h during this time it formed a solution. The reaction mixture
man 250 lM PE SiLG/UV) with fluorescent indicator. Spots were
visualized by UV light (254 and 365 nm). All analytical samples
were homogeneous on TLC in at least two different solvent sys-
tems. Purification by column and flash chromatography was car-
ried out using Merck Silica Gel 60 (200–400 mesh). The amount
(weight) of silica gel for column chromatography was in the
range of 50–100 times the amount (weight) of the crude com-
pounds being separated. Columns were dry-packed unless speci-
fied otherwise. Solvent systems are reported as volume percent
of mixture. Melting points were determined on a Mel-Temp II
melting point apparatus and are uncorrected. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded on a Bruker
WH-300 (300 MHz) spectrometer. The chemical shift (d) values
are reported as parts per million (ppm) relative to tetramethylsil-
ane as internal standard; s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, br = broad singlet. Elemental analyses
were performed by Atlantic Microlab, Inc., Norcross, GA. Elemen-
tal compositions were within 0.4% of the calculated values. Frac-
tional moles of water or organic solvents frequently found in
some analytical samples of antifolates could not be removed de-
spite 24 h of drying in vacuum and were confirmed, where possi-
ble, by their presence in the 1H NMR spectrum. High-resolution
mass spectra (HRMS), using Electron impact (EI), were recorded
on a VG Autospec (Fisons Instruments) micromass (EBE Geome-
try) double focusing mass spectrometer. All solvents and chemi-
cals were used as received.
was again cooled to 0 °C and diethyl-
2.00 mmol) was added followed by N-methylmorpholine
(217 L, 2.00 mmol). The solution was slowly allowed to warm
L-glutamate (475 mg,
l
to room temperature with stirring and left at room temperature
for a total of 24 h. To the resulting solution was added silica gel
(5 g) and the solvent was evaporated under reduced pressure
using an oil pump. The silica gel plug was loaded on top of a
wet (CHCl3) silica gel column and eluted with a gradient of 1–
3% MeOH in CHCl3. Fractions containing the desired spot (TLC)
were pooled and evaporated to dryness under vacuum to give
475 mg (60%) of 22 as a white solid: Mp 210–217 °C; TLC
Rf = 0.61 (CHCl3/MeOH, 5:1); 1H NMR (DMSO-d6) d 1.13–1.20
(m, 6H, CH2CH3), 1.98–2.10 (m, 2H, Glu b-CH2), 2.33 (s, 3H, 5-
CH3), 2.39–2.44 (t, 2H, Glu
c-CH2), 4.00–4.13 (q, 4H, CH2CH3),
4.39–4.44 (m, 1H, Glu -CH), 6.26 (br s, 2H, 2/4-NH2), 6.72 (br
a
s, 2H, 2/4-NH2), 7.16–7.19 (d, 2H, C6H4), 7.78–7.81 (d, 2H,
C6H4), 8.68–8.70 (d, 1H, CONH). Anal. Calcd for (C23H27N5O6S) C,
H, N, S.
4.4. N-[4-[(2,4-Diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)-
thio]-benzoyl]-L-glutamic acid (5)
To a suspension of 22 (200 mg, 0.4 mmol) in EtOH (15 mL) was
added 1 N NaOH (6 mL) and the suspension stirred at 0 °C (4 h) and
then at room temperature for 24 h. The solvent was evaporated un-
der reduced pressure, the yellow oil was dissolved in water (5 mL)
and the solution was cooled in an ice-bath and acidified carefully
to pH 4.0 with drop wise addition of 3 N HCl. This suspension
was left at 5 °C for 24 h and filtered. The residue was washed well
with water and Et2O and then dried over P2O5/vacuum to afford
159 mg (86%) of 5 as a white solid: Mp 217–222 °C; 1H NMR
(DMSO-d6) d 1.98–2.10 (m, 2H, Glu b-CH2), 2.33 (s, 3H, 5-CH3),
4.1. 2,4-Diamino-5-methyl-furo[2,3-d]pyrimidine (20)
To
a suspension of 2,6-diamino-4-hydroxypyrimidine (17)
(1.0 g, 8 mmol) in anhydrous DMF (12 mL) was added chloroace-
tone (18) (0.74 g, 8 mmol). The mixture was heated at 50–60 °C
for 40 h under N2 and protected from light with aluminum foil.
Two major spots at Rf = 0.36 (corresponding to 19) and Rf = 0.56
(corresponding to 20) were visualized on TLC (CHCl3/MeOH, 4:1).
To the suspension was added silica gel (3 g) followed by MeOH
(100 mL) and the solvent evaporated under reduced pressure to
dryness to afford a dry plug. The silica gel plug was loaded on
top of a dry silica gel column and eluted with 10% MeOH in CHCl3.
Fractions containing the desired spot (TLC) were pooled and evap-
orated to dryness under vacuum to give 290 mg (26%) of 20 as a
white solid: Mp 264–266 °C (lit. >260 °C); 1H NMR (DMSO-d6); d
2.18 (d, 3H, 5-CH3, J = 1.1 Hz), 5.96 (br s, 2H, 2/4-NH2), 6.42 (br s,
2H, 2/4-NH2), 7.09 (s, 1H, 6-CH, J = 1.1 Hz).
2.39–2.44 (t, 2H, Glu
c-CH2), 4.39–4.44 (m, 1H, Glu a-CH), 6.26
(br s, 2H, 2/4-NH2), 6.72 (br s, 2H, 2/4-NH2), 7.16–7.19 (d, 2H,
C6H4), 7.78–7.81 (d, 2H, C6H4), 8.68–8.70 (d, 1H, CONH). Anal.
Calcd for (C19H19N5SO6ꢂ1.0H2O) C, H, N, S.
4.5. 2,4-Diamino-5-methyl-6-(1-naphthylthio)furo[2,3-d]pyri-
midine (6)
A suspension of 20 (0.1 g, 0.61 mmol) in EtOH/water (5:4,
4.5 mL) was heated to 80 °C. Iodine (0.31 g, 1.22 mmol) was added,
followed by dropwise addition of a solution of 1-naphthalenethiol
(0.15 g, 0.91 mmol) in EtOH (3 mL). The reaction mixture was then
heated to 90 °C for 3 h after which a dark colored solution formed.
The reaction mixture was then allowed to cool to room tempera-
ture and the solvent was evaporated to dryness under reduced
pressure. The resulting residue was washed with a minimum
amount of EtOAc until the color of the residue became light yellow.
The solid was then suspended in cold water (3 mL) and the pH ad-
justed to 7.0 with concd NH4OH. The suspension was then soni-
cated, cooled to 5 °C and filtered. The residue was washed twice
with cold water (3 mL) and then stirred in MeOH (3 mL) for 12 h
and filtered. The solid was washed twice with MeOH (2 mL) to
4.2. 4-[(2,4-Diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-
benzoic acid (21)
To a suspension of 20 (500 mg, 3 mmol) and 4-mercaptobenzoic
acid (940 mg, 6.0 mmol) in a 250 mL round-bottom flask was
added a solution of EtOH/water (v/v 2:1, 40 mL) and heated to re-
flux. Iodine (1.5 g, 6.0 mmol) was then added to the solution at re-
flux. The reaction mixture was monitored for completion at reflux
(3 h) and then allowed to cool to room temperature. The precipi-
tate formed was filtered, and to the filtrate was added excess
Na2S2O3, and the resulting precipitate formed was filtered. The

960
A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
afford 0.10 g (52.5%) of pure 6: Mp 300–305 °C (dec.); TLC Rf = 0.48
(CHCl3/MeOH, 7:1 with one drop of NH4OH, silica gel); 1H NMR
DMSO-d6 d 2.38 (s, 3H, 5-CH3), 6.23 (s, 2H, 2/4-NH2), 6.70 (s, 2H,
2/4-NH2), 7.16 (d, 1H, 50-CH, J = 7.0 Hz), 7.44 (m, 1H, 30-CH),
7.60–7.70 (m, 2H, 60,70-CH), 7.84 (d, 1H, 40-CH, J = 8.0 Hz), 7.99 (d,
1H, 20-CH, J = 7.6 Hz), 8.29 (d, 1H, 80-CH, J = 8.2 Hz). Anal. Calcd
for (C17H14N4OSꢂ0.5H2O) C, H, N, S.
4.9. 2,4-Diamino-5-methyl-6-(20,50-dimethoxyphenylsulfanyl)-
furo[2,3-d]pyrimidine (10)
Compound 10 was synthesized as described for 8: yield 19%;
mp 292–295 °C; TLC Rf = 0.58 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.30 (s, 3H, 5-CH3), 3.58 (s, 3H,
20-OCH3), 3.80 (s, 3H, 50-OCH3), 6.08 (s, 1H, C6H3), 6.24 (s, 2H, 2/
4-NH2), 6.69 (s, 2H, 2/4-NH2), 6.72–6.75 (d, 1H, C6H3), 6.95–6.98
(d, 1H, C6H3). Anal. Calcd for (C15H16N4O3S) C, H, N, S.
4.6. 2,4-Diamino-5-methyl-6-(2-naphthylthio)furo[2,3-d]pyri-
midine (7)
4.10. 2,4-Diamino-5-methyl-6-(40-methoxyphenylsulfanyl)-
furo[2,3-d]pyrimidine (11)
A suspension of 20 (0.1 g, 0.61 mmol) in EtOH/water (5:4,
4.5 mL) was heated to 80 °C. Iodine (0.31 g, 1.22 mmol) was
added, followed by the dropwise addition of a solution of 2-
naphthalenethiol (0.15 g, 0.91 mmol) in EtOH (3 mL). The reaction
mixture was heated to 90 °C for 4 h and then allowed to cool to
room temperature. The solvent was evaporated to dryness under
reduced pressure. The resulting residue was washed with EtOAc
(1 mL) and then suspended in cold water (3 mL) and the pH
was adjusted to 7.0 with concd NH4OH. The suspension was son-
icated, cooled to 5 °C, filtered and the residue was washed twice
with cold water (3 mL) and dissolved in MeOH (100 mL). The
solution was mixed with silica gel (0.7 g) and evaporated under
reduced pressure. The residue was dried in vacuo, powdered
and poured on top of a wet silica column and eluted with 3%
MeOH in CHCl3. Fractions corresponding to a single spot of the
product were pooled and evaporated to dryness to afford
0.025 g (12.7%) of 7: Mp 225–230 °C; TLC Rf = 0.45 (CHCl3/MeOH,
7:1 with one drop of NH4OH, silica gel); 1H NMR (DMSO-d6) d
2.38 (s, 3H, 5-CH3), 6.25 (s, 2H, 2/4-NH2), 6.72 (s, 2H, 2/4-NH2),
7.25 (dd, 1H, 30-CH, 7.40–7.51 (m, 2H, C10H6), 7.68 (d, 1H, 10-
CH), 7.83–7.89 (m, 2H, C10H6). Anal. Calcd for (C17H14N4OSꢂH2O)
C, H, N, S.
Compound 11 was synthesized as described for 8: yield 16%;
mp 273.5–276 °C; TLC Rf = 0.54 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.34 (s, 3H, 5-CH3), 3.71 (s, 3H,
40-OCH3), 6.17 (s, 2H, 2/4-NH2), 6.63 (s, 2H, 2/4-NH2), 6.89–6.92
(d, 2H, C6H4), 7.15–7.18 (d, 2H, C6H4). Anal. Calcd for
(C14H14N4O2Sꢂ0.2H2O) C, H, N, S.
4.11. 2,4-Diamino-5-methyl-6-(20,30,50,60-tetrafluoro-40-trifluo-
romethylphenylsulfanyl)-furo[2,3-d]pyrimidine (12)
Compound 12 was synthesized as described for 8: yield 10%;
mp 305–307 °C; TLC Rf = 0.52 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.37 (s, 3H, 5-CH3), 6.27 (s, 2H,
2/4-NH2), 6.72 (s, 2H, 2/4-NH2). Anal. Calcd for (C14H7OSN4F7) C,
H, N, S, F.
4.12. 2,4-Diamino-5-methyl-6-(20-methoxyphenylsulfanyl)-
furo[2,3-d]pyrimidine (13)
Compound 13 was synthesized as described for 8: yield 21%;
mp >280 °C (dec.); TLC Rf 0.63 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.30 (s, 3H, 5-CH3), 3.86 (s, 3H,
20-OCH3), 6.22 (s, 2H, 2/4-NH2), 6.59–6.62 (d, 1H, C6H4), 6.67 (s,
2H, 2/4-NH2), 6.84–6.88 (t, 1H, C6H4), 7.01–7.04 (d, 1H, C6H4),
7.14–7.19 (t, 1H, C6H4). Anal. Calcd for (C14H14N4O2Sꢂ0.1H2O) C,
H, N, S.
4.7. 2,4-Diamino-5-methyl-6-(20,60-dimethylphenylsulfanyl)-
furo[2,3-d]pyrimidine (8)
To a 250 mL flask containing 20 (0.2 g, 1.22 mmol) and 2,6-dim-
ethylphenylthiol (0.337 g, 2.44 mmol) was added a mixture of
EtOH/water (2:1, 25 mL) and the reaction mixture was heated to
100–110 °C. Upon reflux I2 (0.619 g, 2.44 mmol) was added and
the heating continued with stirring for a total of 3 h. To this mix-
ture was added excess Na2S2O3, and the mixture was concentrated
under reduced pressure. To the resulting residue was added silica
gel (5 g) and MeOH (50 mL), and the solution was evaporated to
dryness under reduced pressure to afford a dry silica gel plug
which was loaded on top of a wet (CHCl3) silica gel column and
eluted with a gradient of 0–3% MeOH in CHCl3. Fractions contain-
ing the desired spot (TLC) were pooled and evaporated to dryness.
The resulting residue was washed with MeOH, filtered, and dried
to yield 0.055 g (15%) of 8 as a pale white solid: Mp 305–311 °C;
TLC Rf = 0.60 (CHCl3/MeOH, 5:1, with two drops of NH4OH); 1H
NMR (DMSO-d6) d 2.33 (s, 3H, 5-CH3), 2.50 (s, 6H, 20,60-diCH3),
6.05 (s, 2H, 2/4-NH2), 6.54 (s, 2H, 2/4-NH2), 7.15 (s, 3H, C6H3). Anal.
Calcd for (C15H16N4OS) C, H, N, S.
4.13. 2,4-Diamino-5-methyl-6-(30-methoxyphenylsulfanyl)-
furo[2,3-d]pyrimidine (14)
Compound 14 was synthesized as described for 8: yield 25%;
mp 288–295 °C; TLC Rf = 0.58 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.33 (s, 3H, 5-CH3), 3.71 (s, 3H,
30-OCH3), 6.23 (s, 2H, 2/4-NH2), 6.63–6.68 (m, 4H, 2/4-NH2 and
C6H4), 6.77–6.80 (d, 2H, C6H4), 7.21–7.26 (t, 1H, C6H4). Anal. Calcd
for (C14H14N4O2Sꢂ0.2H2O) C, H, N, S.
4.14. 2,4-Diamino-5-methyl-6-(30,40-dimethoxyphenylsulfa-
nyl)-furo[2,3-d]pyrimidine (15)
Compound 15 was synthesized as described for 8: yield 22%;
mp 291–293 °C; TLC Rf = 0.60 (CHCl3/MeOH, 5:1, with two drops
of NH4OH); 1H NMR (DMSO-d6) d 2.36 (s, 3H, 5-CH3), 3.72 (s, 6H,
30,40-diOCH3), 6.19 (s, 2H, 2/4-NH2), 6.64 (s, 2H, 2/4-NH2), 6.68–
6.70 (d, 1H, C6H3), 6.85 (s, 1H, C6H3), 6.90–6.93 (d, 1H, C6H3). Anal.
Calcd for (C15H16N4O3Sꢂ0.3H2O) C, H, N, S.
4.8. 2,4-Diamino-5-methyl-6-(20,60-dichlorophenylsulfanyl)-
furo[2,3-d]pyrimidine (9)
4.15. 2,4-Diamino-5-methyl-6-(20-isopropyl-60-methylphenyl-
sulfanyl)-furo[2,3-d]pyrimidine (16)
Compound 9 was synthesized as described for 8: yield 12%;
mp >305 °C (dec.); TLC Rf = 0.55 (CHCl3/MeOH, 5:1, with two
drops of NH4OH); 1H NMR (DMSO-d6) d 2.34 (s, 3H, 5-CH3),
6.14 (s, 2H, 2/4-NH2), 6.62 (s, 2H, 2/4-NH2), 7.38–7.44 (t, 1H,
C6H3), 7.56–7.59 (d, 2H, C6H3). Anal. Calcd for (C13H10N4OSCl2)
C, H, N, S, Cl.
Compound 16 was synthesized as described for 8: yield 12%;
mp 275–277 °C; TLC Rf = 0.59 (CHCl3/MeOH, 5:1, with two drops
of concentrated NH4OH); 1H NMR (DMSO-d6) d 1.18–1.20 (d, 6H,

A. Gangjee et al. / Bioorg. Med. Chem. 18 (2010) 953–961
961
CH(CH3)2), 2.35 (s, 3H, 5-CH3), 2.50 (s, 3H, 60-CH3), 4.03–4.07 (m,
1H, CH(CH3)2), 6.07 (s, 2H, 2/4-NH2), 6.53 (s, 2H, 2/4-NH2), 7.13–
7.29 (m, 3H, C6H3). Anal. Calcd for (C17H20N4OSꢂ0.2H2O) C, H, N, S.
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Acknowledgments
This work was supported in part by the National Institutes of
Health, Grants AI41743 (A.G.); AI44661 (A.G.), CA89300 (A.G.),
and AI47759 (A.G.) from the National Institute of Allergy and Infec-
tions Diseases.
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