Synthesis of classical and nonclassical 2-amino-4-oxo-6-benzylthieno-[2,3- d]pyrimidines as potential thymidylate synthase inhibitors

Article abstract of DOI:10.1002/jhet.5570410613

A series of seven nonclassical 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines 2-8 and one classical N-[4-(2-amino-4-oxo-3,4- dihydrothieno[2,3-d]pyrimidin-6-ylmethyl)benzoyl]-L-glutamic acid 9 (Table I) were designed as the first in a series of 6-substituted 6-5 fused ring analogs as potential thymidylate synthase (TS) inhibitors and as antitumor agents. The target compounds were synthesized via a Heck coupling of appropriately substituted iodobenzenes and allyl alcohol followed by cyclization using cyanoacetate and sulfur powder to afford substituted thiophenes. The resulting thiophenes were then cyclocondensed with chloroformamidine hydrochloride to afford 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines 2-8 and 26. Hydrolysis of 26 followed by coupling with diethyl L-glutamate afforded 28. The classical analog 9 was obtained by hydrolysis of 28. None of the target compounds inhibited human recombinant thymidylate synthase at 23 μM except 9 for which the IC₅₀ value was 100 μM.

Full text of DOI:10.1002/jhet.5570410613

Nov-Dec 2004
941
Synthesis of Classical and Nonclassical 2-Amino-4-oxo-6-benzylthieno-
[2,3-d]pyrimidines as Potential Thymidylate Synthase Inhibitors
,a
a
b
Aleem Gangjee,* Yibin Qiu and Roy L. Kisliuk
a
Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University,
Pittsburgh, PA, 15282
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111
b
Received June 8, 2004
A series of seven nonclassical 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines 2-8 and one classi-
cal N-[4-(2-amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-ylmethyl)benzoyl]-L-glutamic acid 9 (Table
I) were designed as the first in a series of 6-substituted 6-5 fused ring analogs as potential thymidylate syn-
thase (TS) inhibitors and as antitumor agents. The target compounds were synthesized via a Heck coupling
of appropriately substituted iodobenzenes and allyl alcohol followed by cyclization using cyanoacetate and
sulfur powder to afford substituted thiophenes. The resulting thiophenes were then cyclocondensed with
chloroformamidine hydrochloride to afford 2-amino-4-oxo-6-substituted thieno[2,3-d]pyrimidines 2-8 and
26. Hydrolysis of 26 followed by coupling with diethyl L-glutamate afforded 28. The classical analog 9 was
obtained by hydrolysis of 28. None of the target compounds inhibited human recombinant thymidylate syn-
thase at 23 µM except 9 for which the IC value was 100 µM.
50
J. Heterocyclic Chem., 41, 941 (2004).
Thymidylate synthase (TS) catalyzes the reductive
inhibitors and as antitumor agents [4]. Several TS
inhibitors have entered clinical trails as antitumor agents,
notable among them are CB3717 (PDDF) [5], ZD1694
(Tomudex) [6] and LY231514 (Alimta) (Figure 1) [7].
Alimta has been recently approved for treatment of lung
cancer [8]. These compounds are "classical" antifolates,
i.e. they contain a benzoyl-L-glutamate side chain, which
makes them substrates for the enzyme folylpolyglutamate
synthetase (FPGS). Although polyglutamylation of certain
classical antifolates appears necessary for cytotoxicity, it
has also been implicated as a possible cause of toxicity to
host cells and in the development of resistance. In addi-
tion, classical antifolates require folate uptake systems to
gain access to cells. Thus, lipophilic, nonclassical TS
inhibitors that lack the benzoyl glutamate are not sub-
strates for FPGS and do not require that folate uptake sys-
tems could be useful where resistance is due to decreased
FPGS activity and/or inefficient uptake [9].
methylation of deoxyuridylate (dUMP) to deoxythymidy-
late (dTMP) utilizing 5,10-methylenetetrahydrofolate
(5,10-CH -FH ) as the source of the methyl group as well
2
4
as the reductant [1]. This represents the sole de novo
source of dTMP and hence inhibition of TS, in the absence
of salvage, leads to "thymineless cell death" [2,3], and is
an attractive target for the development of antitumor
agents.
TS inhibitors that are clinically used as antitumor agent
include 5-fluorouracil (5-FU), which is a mechanism-
based inhibitor. Since different types of cancer either do
not respond to 5-FU or develop resistance to it, there is a
pressing need to design novel analogs as potential TS
TM
Nolatrexate dihydrochloride (Thymitaq , AG337), a
lipophilic TS inhibitor, was designed using X-ray crystal-
lographic structures and molecular modeling, and is the
first potent lipophilic TS inhibitor currently in clinical tri-
als [9]. Molecular modeling of 6-5 ring-fused analogs such
as pyrrolo[2,3-d]pyrimidines, furo[2,3-d]pyrimidines and
thieno[2,3-d]pyrimidines superimposed on 6-6 ring-fused
analogs such as AG337, indicate that the 5-substituents are
closely positioned in both ring systems and the 6-sub-
stituent of the 6-5 system lies in between the 6- and 7-sub-
stituent of the 6-6 system (Figure 2) [10]. An example of
potent TS inhibitors in the 6-6 system with a 5-substituent
is AG337 [10], and an example of a 5-substituted 6-5 sys-
tem is LY231514 [7]. The 6-6 fused systems that have been
found to be potent inhibitors of TS are usually 6-substi-
tuted [4]. A recent report by Gangjee et al. [11] showed
that a series of 5-substituted, nonclassical, 6-5 fused
pyrrolo[2,3-d]pyrimidines 1 were inactive against TS and
Figure 1

942
A. Gangjee, Y. Qiu and R. L. Kisliuk
Vol. 41
Table 1
commercially available iodobenzenes and allyl alcohol.
Heck arylation of allyl alcohol with appropriate iodoben-
zenes catalyzed by palladium (II) acetate in water for 24
to 30 hours at 80-90 °C afforded 3-phenylpropionalde-
hydes 10-17 in 70-85% yields [12]. The substituted thio-
phenes 18-25 were synthesized from the appropriate
3-phenylpropionaldehyde, cyanoacetate, triethylamine
and sulfur powder in DMF, [13] in yields ranging from
30-40%. The thieno[2,3-d]pyrimidines 2-8 and 26 were
obtained by chloroformamidine cyclization of the thio-
phenes 18-25 in dimethylsulfone in yields ranging from
50-60%.
Figure 2
were also poor inhibitors of dihydrofolate reductase
(DHFR) from rat liver and Pneumocystis carinii. It was
thus of interest to synthesize 6-substituted thieno[2,3-d]-
pyrimidines as potential inhibitors of TS. The sulfur atom
of the thieno ring is larger than the nitrogen of a pyrrole
and approximates a 6-6 ring system such as AG337. With
these objectives in mind, we synthesized the nonclassical
analogs 2-8 and the classical analog 9 (Table I).
The synthetic strategy of nonclassical analogs 2-8 and
26 is shown in Scheme I. The synthesis commenced from
The synthetic strategy for the classical analog 9 is shown
in Scheme II. The synthesis of 9 started with compound 26
Scheme I

Nov-Dec 2004
Synthesis of Classical and Nonclassical
943
2-Amino-4-oxo-6-benzylthieno[2,3-d]pyrimidines
Scheme II
matography was carried out using Merck silica gel 60 (200-400
mesh). The amount (weight) of silica gel for column chromatog-
raphy was in the range of 50-100 times the amount (weight) of
the crude compounds being separated. Columns were dry packed
unless specified otherwise. Solvent systems are reported as vol-
ume percent mixture. Melting points were determined on a Mel-
Temp II melting point apparatus with a digital thermometer and
which was obtained in Scheme I. Compound 26 was
hydrolyzed with aqueous sodium hydroxide and then neu-
tralized with aqueous hydrogen chloride to afford the acid
27. Coupling of the acid 27 with diethyl L-glutamate using
the mixed anhydride method [14] with isobutyl chlorofor-
mate and triethylamine, followed by a repeated cycle of
activation (Scheme II), afforded, after chromatographic
purification, the coupled product 28 in 51% yield.
Hydrolysis of the diester 28 with aqueous sodium hydrox-
ide at room temperature, followed by acidification gave
the desired compound 9 in 93% yield.
1
are uncorrected. H nmr spectra were recorded on a Bruker WH-
300 (300 MHz) nmr spectrometer. The chemical shift (δ) values
are reported as parts per million (ppm) relative to tetramethylsi-
lane as internal standard; s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, dd = doublet of doublet, bs = broad sin-
glet, exch = protons exchangeable by addition of D O. Elemental
2
Compounds 2-9 were evaluated as inhibitors of human
analyses were performed by Atlantic Microlab, Inc., Norcross,
GA. Elemental compositions were 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
removed despite 24 hours of drying in vacuo and were con-
recombinant thymidylate synthase. Accurate IC values
50
could not be obtained for any of the target compounds
except 9. None of the target compounds inhibited human
recombinant thymidylate synthase at 23 µM except 9 for
1
firmed, where possible, by their presence in the H nmr spectrum.
which the IC value was 100 µM. This clearly indicated
50
All solvents and chemicals were purchased from Aldrich
Chemical Co. and Fisher Scientific and were used as received
except anhydrous solvents, which were freshly dried in the labo-
ratory.
that 6-substituted one-carbon bridged classical and non-
classical 2-amino-4-oxo-6-benzylthieno[2,3-d]pyrimidine
systems are not conducive to thymidylate synthase
inhibitory activity.
General Procedure for the Synthesis of Compounds 10-17.
To a 250 ml round-bottomed flask, fitted with a magnetic stir
bar and a reflux condenser, were placed palladium diacetate
(134.7 mg, 0.6 mmol), the appropriate substituted iodobenzenes
(30 mmol), allyl alcohol (5.23 g, 90 mmol), sodium bicarbonate
(6.3 g, 75 mmol), tetrabutylammonium chloride water (0.84 g, 3
mmol), and water (90 ml). The mixture was stirred vigorously at
80 °C for 24-30 hours under nitrogen. The reaction mixture was
cooled and ethyl acetate (50 ml) was added to afford a solution.
The ethyl acetate solution was washed with water (4 x 30 ml),
dried over anhydrous sodium sulfate, and concentrated under
EXPERIMENTAL
All evaporations were carried out in vacuo with a rotary evap-
orator. Analytical samples were dried in vacuo (0.2 mmHg) in an
Abderhalden drying apparatus over P O . Thin layer chromatog-
2
5
raphy (TLC) was performed on silica gel plates 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 systems. Purification by column and flash chro-

944
A. Gangjee, Y. Qiu and R. L. Kisliuk
Vol. 41
reduced pressure. The residue was loaded on column packed with
silica gel and eluted with 5% ethyl acetate in hexanes and frac-
tions containing the desired product (tlc) were pooled and evapo-
rated to afford the products.
30 ml), dried over anhydrous sodium sulfate, and concentrated
under reduced pressure to afford yellow oil. The residue was
loaded on a column packed with silica gel and eluted with 5%
ethyl acetate in hexanes and fractions containing the desired
product (tlc) were pooled and evaporated to afford the products.
3-(2'-Methylphenyl)propionaldehyde (11).
2-Amino-5-benzylthiophene-3-carboxylic Acid Ethyl Ester (18).
This compound was obtained as a yellow liquid (3.18 g,
1
71.5%); R = 0.75 (hexanes: ethyl acetate = 3:1); H nmr
This compound was obtained as a red oil (1.6 g, 31%); R = 0.6
f
f
1
(DMSO-d ): δ 2.37 (3H, s, 2'-CH ), 2.51-2.62 (2H, q,
(hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.20-1.25
6
3
6
CH CH CHO), 2.69-2.80 (2H, t, CH CH CHO), 7.01-7.19 (4H,
(3H, t, COOCH CH ), 3.86 (2H, s, C H -CH ), 4.10-4.17 (2H, q,
2
2
2
2
2 3 6 5 2
m, C H ), 9.49 (1H, s, CHO).
COOCH CH ), 6.56 (1H, s, 4-H), 7.13 (2H, s, NH , exch), 7.20-
2 3 2
6
4
7.32 (5H, m, C H ).
6
5
3-(2'-Chlorophenyl)propionaldehyde (12).
2-Amino-5-(2'-methylbenzyl)thiophene-3-carboxylic Acid Ethyl
Ester (19).
This compound was obtained as a yellow liquid (3.5 g, 69%);
1
R = 0.77 (hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ): δ
f
6
2.58-2.63 (2H, q, CH CH CHO), 2.82-2.87 (2H, t,
This compound was obtained as a red oil (1.7 g, 31%); R = 0.6
2
2
f
1
CH CH CHO), 6.83-7.40 (4H, m, C H ), 9.48 (1H, s, CHO).
(hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.30-1.34
2
2
6
4
6
(3H, t, COOCH CH ), 2.33 (3H, s, C H -CH ), 3.97 (2H, s,
2
3
6
4
3
3-(2'-Methoxyphenyl)propionaldehyde (13).
C H -CH ), 4.28-4.35 (2H, q, COOCH CH ), 5.85 (2H, s, NH ,
6
4
2
2
3
2
This compound was obtained as a yellow liquid (4.1 g, 83%);
exch), 6.69 (1H, s, 4-H), 7.11-7.24 (4H, m, C H ).
6
4
1
R = 0.70 (hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ): δ
f
6
2-Amino-5-(2'-chlorobenzyl)thiophene-3-carboxylic Acid Ethyl
Ester (20).
2.54-2.59 (2H, q, CH CH CHO), 2.66-2.71 (2H, t,
2
2
CH CH CHO), 3.81 (3H, s, 2'-OCH ), 7.06-7.20 (4H, m, C H ),
2
2
3
6 4
9.42 (1H, s, CHO).
This compound was obtained as a red oil (3.6 g, 49%) under
1.25 equivalent of the general procedure; R = 0.65
f
3-(2',4'-Dimethylphenyl)propionaldehyde (14).
1
(hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.19-1.24
6
This compound was obtained as a yellow liquid (3.7 g, 76%);
(3H, t, COOCH CH ), 3.96 (2H, s, C H -CH ), 4.12-4.17 (2H, q,
2
3
6
4
2
1
R = 0.71 (hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ):
COOCH CH ), 6.54 (1H, s, 4-H), 7.14 (2H, s, NH , exch), 7.27-
f
6
2
3
2
δ2.30 (6H, s, CH ); 2.67 (2H, t, CH CH CHO); 2.88 (2H, t,
7.45 (4H, m, C H ).
3
2
2
6
4
CH CH CHO); 6.90-7.10 (3H, m, C H ); 9.72 (1H, s, CHO).
2
2
6 4
2-Amino-5-(2'-methoxybenzyl)thiophene-3-carboxylic acid
ethyl ester (21).
3-(2',4'-Dichlorophenyl)propionaldehyde (15).
This compound was obtained as a yellow liquid (3.9 g, 64%);
This compound was obtained as a red oil (3 g, 41%) under 1.25
1
R = 0.74 (hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ): δ
equivalent of the general procedure; R = 0.55 (hexanes:ethyl
f
6
f
1
2.54-2.61 (2H, q, CH CH CHO), 2.81-2.86 (2H, t,
acetate = 3:1); H nmr (DMSO-d ): δ 1.20-1.24 (3H, t,
2
2
6
CH CH CHO), 6.77-7.48 (3H, m, C H ), 9.47 (1H, s, CHO).
COOCH CH ), 3.79 (3H, s, C H -OCH ), 4.04 (2H, s, C H -
2
2
6 4
2
3
6
4
3
6 4
CH ), 4.12-4.17 (2H, q, COOCH CH ), 6.52 (1H, s, 4-H), 6.85-
2
2
3
3-(2',4'-Dimethoxyphenyl)propionaldehyde (16).
7.24 (4H, m, C H ), 7.08 (2H, s, NH , exch).
6
4
2
This compound was obtained as a yellow liquid (4.8 g, 82%);
2-Amino-5-(2',4'-dimethylbenzyl)thiophene-3-carboxylic Acid
Ethyl ester (22).
1
R = 0.71(hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ): δ
f
6
2.57 (2H, q, CH CH CHO), 2.59 (2H, t, CH CH CHO), 3.69
2
2
2
2
(3H, s, 2'-OCH ), 3.71 (3H, s, 4'-OCH ), 6.30-6.98 (3H, m,
This compound was obtained as a red oil (2.3 g, 40%); R = 0.6
3
3
f
1
C H ), 9.38 (1H, s, CHO).
(hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.19-1.23
6
4
6
(3H, t, COOCH CH ), 2.19 (3H, s, C H -CH ), 2.24 (3H, s,
2
3
6
3
3
4-(3'-Oxo-propyl)benzoic Acid Ethyl Ester (17).
C H -CH ), 3.79 (2H, s, C H CH ), 4.11-4.14 (2H, q,
6
3
3
6
3-
2
This compound was obtained as a yellow liquid (5.2 g, 84%);
COOCH CH ), 6.41 (1H, s, 4-H), 6.96-7.05 (3H, m, C H ), 7.11
2
3
6 3
1
R = 0.78 (hexanes: ethyl acetate = 3:1); H nmr (DMSO-d ): δ
(2H, s, NH , exch).
f
6
2
1.28-1.29 (3H, t, COOCH CH ), 2.70-2.72 (2H, t,
2
3
2-Amino-5-(2',4'-dichlorobenzyl)thiophene-3-carboxylic acid
ethyl ester (23).
CH CH CHO), 2.80-2.825 (2H, t, CH CH CHO), 4.25-4.29
2
2
2
2
(2H, t, COOCH CH ), 6.84-7.85 (4H, m, C H ), 9.43 (1H, s,
2
3
6 4
CHO).
This compound was obtained as a red oil (2.3 g, 35%); R
=
f
1
0.6 (hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.18-
6
General Procedure for the Synthesis of Compounds 18-25.
1.23 (3H, t, COOCH CH ), 3.995 (2H, s, C H -CH ), 4.11-4.18
2
3
6
3
2
Triethylamine (1.2 g, 0.01 mol) was added to a vigorously
stirred mixture of ethylcyanoacetate (2.26 g, 0.02 mol), sulfur
powder (0.64 g, 0.02 mol) and DMF (10 ml). The appropriately
substituted phenylpropionaldehyde (0.02 mol) was added drop-
wise to this suspension, while maintaining the temperature at 50
°C. When the addition was complete, the reaction mixture was
allowed to cool to room temperature and then stirred for 2 hours.
To the mixture was added water and then ether was added to
afford a solution. The ether solution was washed with water (4 x
(2H, q, COOCH CH ), 6.55 (1H, s, 4-H), 7.165 (2H, s, NH ,
2
3
2
exch), 7.33-7.59 (3H, m, C H ).
6
3
2-Amino-5-(2',4'-dimethoxybenzyl)thiophene-3-carboxylic Acid
Ethyl Ester (24).
This compound was obtained as a red oil (2.1 g, 33%); R =
f
1
0.64 (hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.19-
6
1.23 (3H, t, COOCH CH ), 3.70 (2H, s, C H -CH ), 3.73 (3H, s,
2
3
6
3
2
C H -OCH ), 3.77 (3H, s, C H -OCH ), 4.09-4.16 (2H, q,
6
3
3
6
3
3

Nov-Dec 2004
Synthesis of Classical and Nonclassical
945
2-Amino-4-oxo-6-benzylthieno[2,3-d]pyrimidines
COOCH CH ), 6.43 (1H, s, 4-H), 6.46-6.53 (2H, d, 5', 6'-H),
2-Amino-4-oxo-6-(2'-methoxybenzyl)thieno[2,3-d]pyrimidine
(5).
2
3
7.03, 7.05 (1H, d, 3'-H), 7.05 (2H, s, NH , exch).
2
Compound 5 (1.7 g, 59%) was obtained from 21 (2.9 g, 10
2-Amino-5-(4'-ethoxycarbonyl-benzyl)thiophene-3-carboxylic
mmol) and chloroformamidine hydrochloride (1.7 g, 15 mmol).
Acid Ethyl ester (25).
1
R = 0.83, (chloroform:methanol = 7:1); mp 291-293 °C; H nmr
f
This compound was obtained as a red oil (2.2 g, 33%); R =
0.65 (hexanes:ethyl acetate = 3:1); H nmr (DMSO-d ): δ 1.19-
1.23 (3H, t, COOCH CH ), 1.30 (3H, t, COOCH CH ), 4.00
f
(DMSO-d ): δ 3.79 (3H, s, C H -OCH ), 3.95 (2H, s, C H -
6
6
4
3
6 4
1
6
CH ), 6.44 (2H, s, 2-NH , exch), 6.74 (1H, s, 5-H), 6.88-7.20
2
2
2
3
2
3
(4H, m, C H ); 10.81 (1H, s, 3-NH, exch).
6
4
(2H, s, C H -CH ), 4.12-4.16 (2H, q, COOCH CH ), 4.28-4.30
6
4
2
2
3
Anal. Calcd. for C
H N O S·0.2H O: C, 57.80; H, 4.64; N,
14 13 3 2 2
(2H, q, COOCH CH ), 6.60 (1H, s, 4-H), 7.15 (2H, s, NH ,
2
3
2
14.44; S, 11.02. Found: C, 57.75; H, 4.58; N, 14.44; S, 11.07.
exch), 7.36-7.95 (4H, m, C H ).
6
4
2-Amino-4-oxo-6-(2',4'-dimethylbenzyl)thieno[2,3-d]pyrimidine
(6).
General Procedure for the Synthesis of Compounds 2-8 and 26.
A solution of hydrogen chloride in diethyl ether (100 ml, 0.1
mol) was cooled in an ice-bath. To this vigorously stirred solution
was added a solution of cyanamide (1.8 g, 0.05 mol) in diethyl
ether (34.5 ml) over a 15 minute period. The reaction mixture was
then allowed to stir for 15 minutes. A white, solid precipitate of
chloroformamidine hydrochloride that formed was collected by
filtration and washed with diethyl ether. An intimate mixture of
appropriate thiophenes and chloroformamidine hydrochloride
(1:1.5) in dimethylsulfone (4 g) was heated at 120-130° C for 30
minutes. The mixture was cooled to room temperature, 10 ml of
water was added and ammonium hydroxide was used to neutralize
the suspension. The greenish brown solid obtained was collected
by filtration, washed with water and dried. The crude product was
dissolved in DMF and silica gel was added followed by evapora-
tion of the DMF to afford a dry silica gel plug. The plug was then
loaded onto a silica gel column and eluted with 3% methanol in
chloroform. The fractions containing the desired product (tlc)
were pooled and the solvent evaporated to afford the product.
Compound 6 (1.4 g, 61%) was obtained from 22 (2.3 g, 8
mmol) and chloroformamidine hydrochloride (1.38 g, 12 mmol).
1
R = 0.7, (chloroform:methanol = 7:1); mp 297-300 °C; H nmr
f
(DMSO-d ): δ 2.19 (3H, s, C H -CH ), 2.24 (3H, s, C H -CH ),
6
6
3
3
6
3
3
3.97 (2H, s, C H -CH ), 6.49 (2H, s, 2-NH , exch), 6.67 (1H, s,
6
3
2
2
5-H), 6.94-7.09 (3H, dd, C H ); 10.84 (1H, s, 3-NH, exch).
6
3
Anal. Calcd. for C
H N OS · 0.2H O: C, 62.35; H, 5.37; N,
15 15 3 2
14.54; S, 11.10. Found: C, 62.26; H, 5.34; N, 14.60; S, 11.16.
2-Amino-4-oxo-6-(2',4'-dichlorobenzyl)thieno[2,3-d]pyrimidine
(7).
Compound 7 (1.1 g, 56%) was obtained from 23 (2.0 g, 6
mmol) and chloroformamidine hydrochloride (1.0 g, 9 mmol). R
= 0.7, (chloroform:methanol = 7:1); mp 298-301 °C; H nmr
f
1
(DMSO-d ): δ 4.14 (2H, s, C H -CH ), 6.52 (2H, s, 2-NH ,
6
6
3
2
2
exch), 6.80 (1H, s, 5-H), 7.43-7.63 (3H, m, C H ); 10.87 (1H, s,
6
3
3-NH, exch).
Anal. Calcd. for C H N OSCl : C, 47.60; H, 2.83; N, 12.81;
13
9
3
2
S, 9.78; Cl, 21.62. Found: C, 47.41; H, 2.99; N, 12.78; S, 9.63;
Cl, 21.31.
2-Amino-4-oxo-6-benzylthieno[2,3-d]pyrimidine (2).
Compound 2 (0.54 g, 34%) was obtained from 18 (1.6 g, 6 mmol)
2-Amino-4-oxo-6-(2',4'-dimethoxybenzyl)thieno[2,3-d]pyrimi-
dine (8).
and chloroformamidine hydrochloride (1.0 g, 9 mmol). R = 0.63,
f
1
(chloroform: methanol = 7:1); mp 306-309 °C; H nmr (DMSO-d ):
6
δ 4.03 (2H, s, C H -CH ), 6.48 (2H, s, 2-NH , exch), 6.83 (1H, s, 5-
6
5
2
2
Compound 8 (1.2 g, 63%) was obtained from 24 (1.9 g, 6
H), 7.22-7.34 (5H, m, C H ); 10.83 (1H, s, 3-NH, exch).
6
5
mmol) and chloroformamidine hydrochloride (1.0 g, 9 mmol). R
= 0.72, (chloroform:methanol = 7:1); mp 262-265° C; H nmr
f
Anal. Calcd. for C H N OS: C, 60.68; H, 4.31; N, 16.33; S,
1
13 11
3
12.46. Found: C, 60.75; H, 4.38; N, 16.41; S, 12.56.
(DMSO-d ): δ 3.74 (3H, s, C H -OCH ), 3.78 (3H, s, C H -
6
6
3
3
6 3
OCH ), 3.88 (2H, s, C H -CH ), 6.43 (2H, s, 2-NH , exch), 6.48
3
6
3
2
2
2-Amino-4-oxo-6-(2'-methylbenzyl)thieno[2,3-d]pyrimidine (3).
Compound 3 (1.6 g, 65%) was obtained from 19 (2.57 g, 9
(1H, s, 5-H), 6.56-7.10 (3H, m, C H ); 10.79 (1H, s, 3-NH,
6
3
exch).
Anal. Calcd. for C
mmol) and chloroformamidine hydrochloride (1.7 g, 14 mmol).
H
N O S: C, 56.77; H, 4.76; N, 13.24; S,
3 3
15 15
1
R = 0.60, (chloroform:methanol = 7:1); mp 295-297 °C; H nmr
f
10.10. Found: C, 56.62; H, 4.87; N, 13.26; S, 10.16.
(DMSO-d ): δ 2.25 (3H, s, C H -CH ), 4.03 (2H, s, C H -CH ),
6
6
4
3
6
4
2
4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-
ylmethyl)benzoic Acid Ethyl Ester (26).
6.47 (2H, s, 2-NH , exch), 6.69 (1H, s, 5-H), 7.165 (4H, m,
2
C H ); 10.82 (1H, s, 3-NH, exch).
6
4
Anal. Calcd. for C
H N OS · 0.6H O: C, 59.60; H, 5.07; N,
14 13 3 2
Compound 26 (1.0 g, 51%) was obtained from 25 (2.0 g, 6
14.89; S, 11.36. Found: C, 59.35; H, 4.74; N, 14.68; S, 11.13.
2-Amino-4-oxo-6-(2'-chlorobenzyl)thieno[2,3-d]pyrimidine (4).
Compound 4 (0.85 g, 24%) was obtained from 20 (3.6 g, 12
mmol) and chloroformamidine hydrochloride (1.0 g, 9 mmol). R
= 0.6, (chloroform:methanol = 7:1); mp 193-198 °C; H nmr
f
1
(DMSO-d ): δ 1.15-1.17 (3H, t, COOCH CH ), 4.12 (2H, s,
6
2
3
C H -CH ), 4.25-4.32 (2H, q, COOCH CH ), 6.51 (2H, s, 2-
6
4
2
2
3
mmol) and chloroformamidine hydrochloride (2.1 g, 18 mmol).
NH , exch), 6.88 (1H, s, 5-H), 7.24-7.91 (4H, dd, C H ); 10.54
2
6 4
1
R = 0.73, (chloroform:methanol = 7:1); mp 290-294 °C; H nmr
f
(1H, s, 3-NH, exch).
(DMSO-d ): δ 4.15 (2H, s, C H -CH ), 6.50 (2H, s, 2-NH ,
6
6
4
2
2
4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-
ylmethyl)benzoic Acid (27).
exch), 6.78 (1H, s, 5-H), 7.31-7.44 (4H, dd, C H ); 10.85 (1H, s,
6
4
3-NH, exch).
Anal. Calcd. for C
S, 10.99; Cl, 12.15. Found: C, 53.26; H, 3.54; N, 14.26; S, 10.87;
Cl, 12.43.
H N OSCl: C, 53.52; H, 3.45; N, 14.40;
13 10 3
To a solution of 26 in ethanol (10 ml) was added aqueous 1 N
NaOH (1ml) and the reaction mixture stirred at room temperature
for 18 hours and carefully acidified to pH 4 with drop wise

946
A. Gangjee, Y. Qiu and R. L. Kisliuk
Vol. 41
addition of 3 N HCl. The resulting suspension was left at 0 °C for
2 hours at which time the residue was collected by filtration,
washed with water (10 ml), acetone, and ethyl ether and dried
drop wise addition of 3 N HCl. This suspension was left at 0-5 °C
for 24 hours and then filtered. The residue was washed well with
water and dried over P O /vacuum to afford 9 (0.21 g, 92.8%) as
2
5
1
over P O /vacuum at 78° C to afford the free acid 27 (0.8 g, 87%)
a white solid. mp 214-217 °C. H nmr (DMSO-d ): δ1.94-2.06
2
5
6
1
as a light yellow solid. mp 296-300° C. H nmr (DMSO-d ): δ
(m, 2H, Gluβ-CH ); 2.32-2.49 (t, 2H, Gluγ-CH , J = 7.2 Hz);
2 2
6
4.11 (2H, s, C H -CH ); 6.51 (2H, s, NH , exch); 6.88 (1H, s, 5-
4.11 (s, 2H, C H CH ); 4.38 (m, 1 H, Gluα-CH), 6.50 (s, 2 H, 2-
6
4
2
2
6 4 2
H); 7.36-7.90 (4H, m, C H ); 10.79 (1H, s, NH, exch).
NH , exch); 6.87 (s, 1H, 5-H); 7.35,7.38-7.81,7.84 (dd, 4H,
2
6
4
C H , J = 8.1 Hz), 8.568-8.593 (d, 1 H, CONH, J = 7.5 Hz, exch),
6
4
Diethyl 2-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrim-
idin-6-ylmethyl)benzoyl]-L-glutamate (28).
10.86 (s, 1 H, 3-NH, exch).
Anal. Calcd. For C N O S·0.2H O: C, 52.58; H, 4.27; N,
H
19 18
4
6
2
To a suspension of the acid 27 (0.302 g, 1 mmol) in anhydrous
DMF (10 ml) under nitrogen was added triethylamine (0.418 ml,
3 mmol) and the suspension heated to 80 °C to form a solution.
The solution was cooled to 0 °C and isobutyl chloroformate (0.26
ml, 2 mmol) was added, followed, after 15 mintues, with diethyl
L-glutamate hydrochloride (0.36 g, 1.5 mmol) and immediately
followed by triethylamine (0.418 ml, 3 mmol). The reaction mix-
ture was warmed slowly to room temperature and stirred for 12
hours. At this time the reaction mixture was cooled to 0 °C and
the activation steps described above were repeated using triethy-
lamine (0.21 ml, 1.5 mmol), followed by isobutyl chloroformate
(0.13 ml, 1 mmol). After stirring for 15 mintues at 0 °C diethyl L-
glutamate hydrochloride (0.18 g, 0.76 mmol) was added followed
immediately by triethylamine (0.21 ml, 1.5 mmol). The reaction
mixture was stirred for another 24 hours at room temperature and
to this solution was added silica gel (1.2 g) and the suspension
evaporated to dryness under reduced pressure. The silica gel plug
obtained was loaded on a dry silica gel column which was rapidly
eluted with chloroform and then stepwise with 1-5% methanol in
chloroform. The fractions showing a single spot (tlc) were pooled
and evaporated to afford compound 28 (0.25 g, 51.4%) as an off-
12.91; S, 7.39. Found: C, 52.21; H, 4.47; N, 12.74; S, 7.33.
Acknowledgement.
This work was supported, in part, by grants from the National
Institutes of Health, National Cancer Institute CA 89300.
REFERENCES AND NOTES
[1] C. W. Carreras and D. V. Santi, Annu. Rev. Biochem., 64, 721
(1995).
[2] K. T. Douglas, Med. Res. Rev., 7, 441 (1987).
[3] E. M. Berman and L. M. Werbel, J. Med. Chem., 34, 479
(1991).
[4] A. Gangjee, E. Elzein, M. Kothare and A. Vasudevan, Curr.
Pharm. Design., 2, 263 (1996).
[5] T. R.Jones, A. H. Calvert, A. L. Jackman, S. J. Brown, M.
Jones and K. R. Harrap, Eur. J. Cancer., 17, 11 (1981).
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J. Barredo, M. Jannatipour, and R. G. Moran, J. Med. Chem., 35, 4450
(1992).
white solid. R = 0.50 (chloroform: methanol = 7:1). mp 186-
f
1
187.5 °C. H nmr (DMSO-d ): δ 1.03-1.21 (m, 6 H,
6
COOCH CH ); 1.95-2.12 (m, 2H, Gluβ-CH ); 2.41 (t, 2H, Gluγ-
2
3
2
[8] R. Zhao and I. D. Goldman, The Oncologist, 9, 242-244
(2004).
CH , J = 7.2 Hz); 4.06 (m, 4 H, COOCH CH ); 4.14 (s, 2H,
2
2
3
C H -CH ); 4.35-4.43 (m, 1 H, Gluα-CH), 6.49 (s, 2 H, 2-NH ,
6
4
2
2
[9] S. E. Webber, T. M. Bleckman, J. Attard, J. G. Deal, V.
Katherdekar, K. M. Welsh, S. Webber, C. A. Janson, D. A. Matthews, W.
W. Smith, S. T. Freer, S. R. Jordan, R. J. Bacquet, E. F. Howland, C. L. J.
Booth, R. W. Ward, S. M. Hermann, J. White, C. A. Morse, J. A. Hilliard,
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exch); 6.88 (s, 1H, 5-H); 7.36,7.39-7.81,7.84 (dd, 4H, C H , J =
6
4
8.1 Hz), 8.67-8.69 (d, 1 H, CONH, J = 7.5 Hz, exch), 10.84 (s, 1
H, 3-NH, exch).
Anal. Calcd. For C
H N O S: C, 56.78; H, 5.39; N, 11.52;
23 26 4 6
S, 6.59. Found: C, 56.89; H, 5.40; N, 11.59; S, 6.55.
N-[4-(2-Amino-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-6-
ylmethyl)benzoyl]-L-glutamic Acid (9).
To a solution of 28 (0.25 g, 0.5 mmol) in ethanol (15 ml) was
added 1 N NaOH (1 ml) and the solution stirred at room tempera-
ture for 24 hours. The ethanol was evaporated under reduced
pressure, the residue was dissolved in water (15 ml), and the solu-
tion was stirred for a further 24 hours. The solution was then
cooled in an ice-bath and carefully acidified to pH 4.0 with the