Design, synthesis, biological evaluation and X-ray crystal structure of novel classical 6,5,6-tricyclic benzo[4,5]thieno[2,3-d]pyrimidines as dual thymidylate synthase and dihydrofolate reductase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.03.067

Classical antifolates (4-7) with a tricyclic benzo[4,5]thieno[2,3-d] pyrimidine scaffold and a flexible and rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors. Oxidative aromatization of

Full text of DOI:10.1016/j.bmc.2011.03.067

Bioorganic & Medicinal Chemistry 19 (2011) 3585–3594
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, biological evaluation and X-ray crystal structure of novel
classical 6,5,6-tricyclic benzo[4,5]thieno[2,3-d]pyrimidines as dual
thymidylate synthase and dihydrofolate reductase inhibitors
Xin Zhang a, Xilin Zhou a, Roy L. Kisliuk b, Jennifer Piraino c, Vivian Cody c, Aleem Gangjee a,
⇑
a Division of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Duquesne University, Pittsburgh, PA 15282, United States
b University School of Medicine, Boston, MA 02111, United States
c Hauptman-Woodward Medical Research Institute, 700 Ellicott Street, Buffalo, NY 14203, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Classical antifolates (4–7) with a tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold and a flexible and
rigid benzoylglutamate were synthesized as dual thymidylate synthase (TS) and dihydrofolate reductase
(DHFR) inhibitors. Oxidative aromatization of ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-benzothio-
phene-3-carboxylate ( )-9 to ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate 10 with 10%
Received 10 February 2011
Revised 25 March 2011
Accepted 30 March 2011
Available online 9 April 2011
Pd/C was a key synthetic step. Compounds with 2-CH3 substituents inhibited human (h) TS (IC50
0.26–0.8 M), but not hDHFR. Substitution of the 2-CH3 with a 2-NH2 increases hTS inhibition by more
than 10-fold and also affords excellent hDHFR inhibition (IC50 = 0.09–0.1 M). This study shows that
=
l
Keywords:
DHFR
TS
Dual
Inhibitor
l
the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine scaffold is highly conducive to single hTS or dual hTS-
hDHFR inhibition depending on the 2-position substituents. The X-ray crystal structures of 6 and 7 with
hDHFR reveal, for the first time, that tricyclics 6 and 7 bind with the benzo[4,5]thieno[2,3-d]pyrimidine
ring in the folate binding mode with the thieno S mimicking the 4-amino of methotrexate.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
combination with cisplatin, was approved for the treatment of
malignant pleural mesothelioma and also for non-small cell lung
Because of its critical importance in the biosynthesis of purine
and pyrimidine nucleic acids, folate metabolism is an attractive
target for chemotherapy. Antifolates that target folate metabolism
have found clinical utility as antitumor, antimicrobial, and antipro-
tozoal agents.1–5 Among the folate dependent enzymes, thymidyl-
ate synthase (TS) and dihydrofolate reductase (DHFR) have been of
particular interest. TS is a key enzyme in the de novo synthesis of
20-deoxythymidine-50-monophosphate (dTMP) from 20-deoxyuri-
dine-50-monophosphate (dUMP).6,7 The reaction requires 5,10-
methylenetetrahydrofolate (5,10-CH2THF) as a cofactor for one
carbon unit transfer and represents the only de novo pathway for
intracellular dTMP synthesis. DHFR catalyzes the reduction of
dihydrofolate to tetrahydrofolate, and is indirectly responsible for
dTMP synthesis by maintaining the reduced folate pool.8
cancer. With TS inhibition as the primary mechanism of action,
PMX was reported to inhibit several other folate-dependent en-
zymes including DHFR, glycinamide ribonucleotide formyltransfer-
ase (GARFTase), and aminoimidazole carboxamideribonucleotide
formyltransferase (AICARFTase).10 Similar to RTX, polyglutamyl-
tion by FPGS is essential for the cytotoxicity of PMX.
Classical antifolates, such as RTX and PMX, that have an N-ben-
zoyl-L-glutamic acid side chain usually function as substrates for
FPGS, which leads to high intracellular concentrations of these
antitumor agents and increases TS inhibitory activity for some
antifolates (RTX, 60-fold and PMX 130-fold) compared to their
monoglutamate forms.9,10,14,15 Although polyglutamylation of cer-
tain antifolates (such as RTX and PMX) is necessary for their cyto-
toxic activity, it has also been implicated in toxicity to host cells,
because of the longer cellular retention time of such polyanionic
poly-glutamate metabolites.16 In addition, reduced expression of
FPGS in tumor cells can lead to resistance to FPGS dependent clas-
sical antifolates such as PMX.17–19
To circumvent the potential tumor resistance problems
associated with FPGS, classical antifolates should have high en-
zyme inhibitory potency as their monoglutamate forms and
not require polyglutamylation by FPGS to exert their antitumor
activity.20,21
Raltitrexed (RTX),9 pemetrexed (PMX)10 and methotrexate
(MTX)11 (Fig. 1) are examples of TS and/or DHFR inhibitors used
in the clinic. RTX, approved in several European countries, Austra-
lia, Canada, and Japan for the treatment of advanced colorectal
cancer, is a TS inhibitor that undergoes rapid polyglutamylation
by the enzyme folylpolyglutamate synthetase12,13 (FPGS). PMX, in
⇑
Corresponding author. Tel.: +1 412 396 6070.
E-mail address: gangjee@duq.edu (A. Gangjee).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.067

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
Figure 1. Antifolates.
It has been our long-standing goal not only to design potent TS
for binding to hFPGS. The fact that PMX, a pyrrolo[2,3-d]pyrimi-
dine, much like 3, lacks a 6-methyl group and is a substrate for
FPGS lends credence to the involvement of the 6-methyl moiety
in preventing FPGS substrate activity in 3.
and DHFR inhibitors but also to design and synthesize single
agents that have potent dual TS and DHFR inhibitory activity
simultaneously. Such dual inhibitors could act at two different
sites (TS and DHFR) and might be capable of providing ‘combina-
tion chemotherapy’ in single agents without the pharmacokinetic,
overlapping toxicities and other disadvantages of two separate
agents.20 This strategy may also lead to an improved toxicity
profile.
Typically, antifolates that contain a 2-methyl-4-oxo substitu-
tion in the pyrimidine ring (such as RTX) are TS inhibitors, while
2-amino-4-oxo substitution in the pyrimidine ring (such as PMX)
may provide affinity for both TS and DHFR. 2,4-Diamino substitu-
tions on the pyrimidine ring of antifolates is usually associated
with DHFR inhibitory activity.
Tricyclic 2 (Fig. 1) is a classical TS inhibitor with Ki = 0.09
nM.23,24 Although this compound is an excellent substrate for
FPGS, it is subject to the addition of only one additional glutamic
acid. Moreover, the high potency of 2 does not rely on polygluta-
mation as the monoglutamate form is equi-potent with the diglu-
tamate form.25 Compound 2 is a noncompetitive TS inhibitor and
its activity is not affected by the concentration of 5,10-CH2THF.23,24
In addition, it has been demonstrated that overexpression of the
multidrug resistance proteins, MRP1 and MRP2, can confer tumor
resistance to short term (4 h), but not long term (72 h), exposure
of 2.26
In an attempt to prevent or minimize the potential problems
associated with FPGS, including tumor resistance, and to develop
dual TS and DHFR inhibitors, Gangjee et al.20 reported the synthesis
In the course of our structure-based drug design program it was
of interest to synthesize classical antifolates 4–7 with a tricyclic
benzo[4,5]thieno[2,3-d]pyrimidine scaffold (Fig. 2), as structural
hybrides of 2 and 3. Compounds 4 and 5, similar to 2, have a 2-
methyl-4-oxo pyrimidine ring which is generally associated with
TS inhibition. In contrast, 6 and 7 have a 2-amino-4-oxo substitu-
ent, which could afford dual TS and DHFR inhibition, as observed
for 3 and PMX. The 2-substitutions on 4–7 would assess the
of
a
classical antifolate N-{4-[(2-amino-6-methyl-4-oxo-4,7-
-glu-
dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl}-
L
tamic acid, 3 (Fig. 1), as a potent inhibitor of isolated hTS
(IC50 = 42 nM) with a reasonable inhibition of human recombinant
DHFR (IC50 = 2.2 lM) in its monoglutamate form thus providing
dual inhibitory activity of TS and DHFR. Compound 3 was equipo-
tent with 1 (Fig. 1), a potent TS inhibitor, against hTS and was more
potent than the clinically used RTX and PMX against isolated hTS in
their monoglutamate forms. Molecular modeling (SYBYL 6.91)22
suggested that the 6-methyl group in compound 3 makes impor-
tant hydrophobic contacts with Trp109 in hTS and also serves to
lock the 5-position side chain into favorable, low energy conforma-
tions. Both these factors probably contribute to the high inhibitory
activity of 3 in its monoglutamate form against hTS.
A potential advantage of compound 3 over RTX and PMX is that
it is not a substrate for hFPGS, from CCRF-CEM cells, at concentra-
tions up to 1045
was attributed, in part, to the presence of the 6-methyl group on
the pyrrolo[2,3-d]pyrimidine of 3. The 6-methyl group of
l
M.20 The lack of hFPGS substrate activity of 3
3
probably creates steric hindrance in its binding to the active site
of hFPGS. Alternatively, the 6-methyl group of 3 may force the
5-position side chain into a conformation, that is, not conducive
Figure 2. Target classical antifolates with the tricyclic benzo[4,5]thieno[2,3-
d]pyrimidine scaffold.

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
3587
importance of hydrogen-bonding at this position (6, 7) versus
hydrophobic binding (4, 5) to biological activity. The size of a sulfur
atom in 4–7 is larger than a nitrogen atom and smaller than two
carbon atoms, thus the thiophene B-ring in benzo[4,5]thieno[2,3-
d]pyrimidines 4–7 mimics both the smaller pyrrolo B-ring in 3
and the larger quinazoline B-ring in 2 (Fig. 3).
Similar to the C-ring in 2 and the 6-methyl group in 3, the benzo
C-ring in 4–7 makes hydrophobic contacts with Trp109 in hTS and
restricts the side chain to a conformation conducive for potent TS
activity but perhaps not for FPGS substrate activity.
To explore the effects of side chain flexibility on biological activ-
ity, 4 and 6 have the same benzoylglutamate side chain as 3, while
5 and 7 have a more conformationally restricted 2-isoindolinylglu-
tamate side chain like 2. Unlike other classical TS inhibitors, the
glutamate side chain in 2 is part of an isoindolinone system, which
restricts the side chain conformation. The crystal structure of ter-
nary complex TS-dUMP-2 (PDB: 1SYN)27 revealed that the binding
of 2 and the nucleotide induced a closed conformation of the TS
protein, similar to other antifolates. Surprisingly, however, the
binding surface of 2 includes a hydrophobic patch from Val 77, that
is, normally buried.27–30
Figure 4. Stereoview of compound 5 (blue) superimposed on 2 (purple) in ecTS
(green). Figure prepared with MOE 2008.10.31
As shown in Figure 4, molecular modeling using MOE 2008.1031
revealed that when the central ring in 2 is truncated to a five-
member ring in the benzo[4,5]thieno[2,3-d]pyrimidine 5, and the
substitution is moved from the 6-position to the 5-position, the
resulting compound could bind to TS in a manner similar to 2.
agent to effect aromatization. Reaction of ( )-10 with DDQ at reflux
in dioxane for up to 24 h afforded no new product (TLC). Other sol-
vents with different boiling points were also attempted at reflux
and microwave conditions. Trace amounts of a new product was
observed under certain conditions, however, the yields were poor
and precluded characterization.
The poor solubility of ( )-10 in organic solvents could, in part,
be responsible for the failure of aromatization. Thus, the 2-amino
group in ( )-10 was protected with a pivaloyl group at reflux with
the anhydride (Piv)2O (Scheme 1) to give ( )-11, which was then
subjected to DDQ oxidation under different reaction conditions.
Unfortunately, no desired product was obtained.
2. Chemistry
It was envisioned that target compounds 4–7 would be synthe-
sized via the coupling between the benzo[4,5]thieno[2,3-d]pyrim-
idine scaffold and the glutamate side chain.
The synthesis of benzo[4,5]thieno[2,3-d]pyrimidines started
The failure of the previous strategy prompted us to explore an
alternate method, where the bicylic scaffold was aromatized first
(Scheme 2). Bicyclic intermediate ( )-9 showed good solubility in
most organic solvents. With toluene as the solvent and MnO2,
SeO2 or DDQ as the oxidant, under bench-top conditions or micro-
wave irradiation no desired product was obtained. A literature
search revealed Pd/C oxidation.37,38 This allowed the conversion
of ( )-9 to the fully aromatized 14. The solvent and time of the
reaction were optimized for the aromatization with the optimal
conditions being mesitylene as solvent at reflux for 48 h. Compared
with ( )-9, the 1H NMR of 14 showed the disappearance of protons
at d 1.54–3.17 ppm and the appearance of three aromatic protons
at d 6.98–7.43 ppm, which confirmed aromatization. In addition,
the appearance of benzylic protons at d 2.38 as singlet also
confirmed aromatization. With 14 in hand, cyclization was carried
out to afford the tricyclic scaffold. The substitution at the 2-posi-
tion of the benzo[4,5]thieno[2,3-d]pyrimidine are predicated by
the reactant. Cyclization of 14 (Scheme 2) with chloroformamidine
hydrochloride afforded the 2-amino-4-oxo benzo[4,5]thieno
[2,3-d]pyrimidine 12 in 60% yield. Pivaloylation of 12 afforded
13. The reaction of 14 in acetonitrile with gaseous hydrogen
chloride afforded the 2-methyl-4-oxo product 15 in 57% yield.
The 2-methyl and 5-methyl groups of 15 occur in the 1H NMR at
d 2.35 and 2.95, respectively, akin to similar dimethyl substituted
quinazolines and benzoquinazolines.39,23,40,41 Free radical bromin-
ation of 13 and 15 with N-bromosuccinimide and a catalytic
amount of benzoyl peroxide afforded intermediates 16 and 17
respectively.39,23,40 By limiting the amount of the NBS to just over
1 equiv, the 5-methyl moiety of 15 was selectively brominated to
afford 17, similar to that reported for quinazolines and benzoqui-
nazolines.39,23,40,41 The 2-methyl moiety of 17 occurs in the 1H
NMR at d 2.69 and the 5-methylene protons occur at d 5.84 similar
to the corresponding quinazolines and benzoquinazolines.39,23,40,41
from commercially available a-methyl cyclohexanone ( )-8 to the
thiophene intermediate ( )-9 (Scheme 1) via a Gewald reaction32
in 81% yield. The reaction was attempted in various solvents and
baseswiththeoptimizedresultsobtainedwithethanolandmorpho-
line. Cyclization of ( )-9 via the partially aromatized tricyclic inter-
mediate should afford benzo[4,5]thieno[2,3-d]pyrimidines. To
explore this strategy, ( )-10 (Scheme 1) was synthesized via the con-
densation of ( )-9 and chloroformamidine hydrochloride.33 Aroma-
tization of ( )-10 was expected to afford 12 (Scheme 1).
Rosowsky et al.34 reported aromatization of tetracyclic thieno-
[2,3-d]pyrimidine via SeO2 in acetic acid at reflux. Attempts at this
reaction for the conversion of ( )-10 to 12 were unsuccessful.
Gangjee et al.35 reported the oxidation of dihydropyrrolo[2,3-
d]pyrimidines to their aromatic congeners via MnO2 oxidation.
However, MnO2 oxidation for the aromatization of ( )-10 was also
unsuccessful. DDQ is reported36 to serve as a dehydrogenation
Figure 3. Superimposition of benzo[4,5]thieno[2,3-d]pyrimidine (red), pyrolo[2,3-
d]pyrimidine (green) and benzo[f]quinazoline (cyan).

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
Scheme 1. Synthesis of tricyclic thieno[2,3-d]pyrimidines 10 and 11. Reagents and conditions: (a) ethylcyanoacetate, morpholine, sulfur, ethanol, 45 °C to rt, 12 h; (b)
chloroformamidine hydrochloride, DMSO2, 150 °C; (c) (Piv)2O, reflux, 3 h.
activities of PMX and lead compounds 2 and 3 are also provided.
The classical benzo[4,5]thieno[2,3-d]pyrimidines 6–7 showed
potent hTS inhibitory activity comparable to that of 1, 2 and 3
which supports the hypothesis that a 6-5-6 tricyclic system with
a 5-substitution can inhibit TS in a similar fashion as the 6-6-6 tri-
cyclic system with a 6-substitution. Compound 7 is the most po-
tent hTS inhibitor in the series and is two-fold more potent than
compound 1 and equipotent with compound 2. Against isolated
hTS, all four compounds 4–7 showed 50 to 100-fold greater activity
Scheme 2. Synthesis of tricyclic thieno[2,3-d]pyrimidines 16 and 17. Reagents and
than PMX, which relies on FPGS for its potent hTS inhibitory activ-
ity. Thus compounds 4–7, and in particular 6 and 7, have the po-
tential to overcome the tumor resistance problems of PMX
associated with lower levels of FPGS. With identical side chain sub-
stitution, the 2-amino analogs are more potent than the 2-methyl
compounds. This indicates the importance of hydrogen bonding
ability at the 2-position. Side chain flexibility could also play a role
in enzyme inhibition. When the 2-substitution is methyl, the more
rigid side chain analog 5 is three-fold less potent than the more
flexible 4 against hTS. However, this trend is reversed when the
2-substitution is an amino group, where the more rigid compound
7 is twice as potent as the flexible 6. These data suggest that the
conformation of side chain glutamate along with the 2-substitu-
tion pattern dictates enzyme binding.
Compounds 4 and 5 are inactive against DHFR from different
sources, while 6 and 7 are about equally potent against hDHFR
and are about five-times less potent than MTX. Compounds 6
and 7 also have different selectivity profile than MTX. MTX is
three-times more selective for E. coli (ec) DHFR than hDHFR, while
6 and 7 are more selective for hDHFR and the selectivity index is
about 4. Inhibition of DHFR by 6 and 7 confirms our hypothesis
that the replacement of the 2-methyl group by 2-amino group in
benzo[4,5]thieno[2,3-d]pyrimidines can provide dual TS and DHFR
inhibition. The inactivity of 4 and 5 against hDHFR is probably due
to the absence of a salt bridge with Glu30 of hDHFR at the N1 and
2NH2. While compounds 6 and 7 can bind just like folate (PDB:
1U72) in which the 2-amino-4-oxo group binds to the enzyme
with hydrogen bonding and the heterocycle and the benzoyl moi-
conditions: (a) Pd/C, toluene, reflux; (b) HCl (g), acetonitrile, ammonium hydoxide;
(c) chloroformamidine hydrochloride, DMSO2, 150 °C; (d) (Piv)2O, reflux, 3 h, NBS,
Bz2O2.
The benzoylglutamate side chain for 4 and 6 is commercially
available, however the 2-isoindolinylglutamate side chain for 5
and 7 was synthesized via a literature method (Scheme 3).39
Esterification of 18 with SOCl2 in methanol afforded 19 in 91%
yield. Radical bromination of 19 with N-bromosuccinimide and a
catalytic amount of benzoyl peroxide afforded 20 in 48% yield.
The 1H NMR of 20 showed the disappearance of protons at d
2.69 ppm (CH3) and appearance of protons at d 4.86 ppm (CH2Br).
Treatment of 20 with excess diethyl L-glutamate hydrochloride and
K2CO3 in DMF at room temperature afforded isoindoline 21 as an
orange oil in 56% yield. The 1H NMR of 21 showed the appearance
of protons at
d
4.51–4.83 ppm (isoindoline CH2) and
d
5.09–5.14 ppm (Glu
a-CH). Reduction of the nitro group in 21 affor-
ded the amine 22 in 92.5% yield.
As shown in Scheme 4, N-alkylation42 of 16 and 17 followed by
hydrolysis of the ethyl ester (and the removal of pivaloyl protect-
ing group in 6–7) with 1 N NaOH and subsequent acid workup
afforded target compounds 4–7. The presence of glutamate in the
side chain was confirmed from 1H NMR. The expected NH at d
6.74–7.40 ppm, exchanged with D2O, and the benzylic protons oc-
curred as a doublet at d 5.13–5.24 ppm, which converts to a singlet
upon D2O exchange, indicated the success of the coupling reaction.
High resolution MS (HRMS) and the presence of the requisite pro-
tons of the side chain via NMR confirmed the structure of 4–7.
eties bind to Phe31, Phe34 and Ile 60. The a-carboxylic acid of the
glutamate makes ionic contact with Arg70. A second mode of bind-
ing would involve a 180° rotation about the C-2, NH2 bond (Fig. 5),
whereby the sulfur of the thiophene ring is now superimposed on
the 4-oxo group of folate, with all other interactions being the
same. It is important to note that binding of 6 and 7 in the flip
mode (Fig. 5) also allows the sulfur of the thiophene ring to mimic
the 4-amino of MTX. To determine which of these two modes of
3. Biological evaluation and discussion
Compounds 4–7 were evaluated as inhibitors of TS and
DHFR (Table 1) from three different sources; human, Escherichia
coli and Toxoplasma gondii. Compound 1 (PDDF) was used as
the standard compound for TS inhibition and MTX was used as
the standard compound for DHFR inhibition. For comparison, the
Scheme 3. Synthesis of side-chain 22. Reagents and conditions: (a) SOCl2, MeOH; (b) NBS, Bz2O2; (c) diethyl glutamate (d) H2, Pd/C.

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
3589
Scheme 4. Synthesis of classical analogs 4–7. Reagents and conditions: (a) DMF, reflux; (b) 1 N NaOH 60 °C.
Table 1
Inhibitory concentrations (IC50 in
U
M) Against TS and DHFRa
Compound
TS (l
M)
DHFR (lM)
Humanb E. colib
T.
Humand E. colie
T.
gondiic
gondiic
1f
0.064
0.032
0.048
0.023
0.072
0.027
11
11
20
0.26
0.71
2g
>20
(16)
2.1
>20
(35)
>20
(24)
0.09
0.1
3h
4
0.053
0.26
0.21
0.82
0.09
1.7
22
15
0.23
1.4
5
0.8
0.85
3.7
>20
(38)
0.4
0.4
2300
0.0044
1.8
6
7
0.068
0.034
9.5
>18
(29)
0.017
0.05
76
>18
(14)
0.14
0.17
2.8
0.02
0.01
0.43
0.022
Figure 6. Stereoview of the X-ray crystal structures of 6 (green) and 7 (cyan)
superimposed in the active site of hDHFR and NADPH ternary complex..
PMXh
MTX
6.6
>18 (36) 0.022
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 were within 10% of the value given.
b
Kindly provided by Dr. Frank Maley, New York State Department of Health.
Kindly provided by Dr. Karen Anderson, Yale University, New Haven, CT.
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. M.G. Nair, University of South Alabama.
Kindly provided by Dr. J.J. McGuire, Roswell Park Cancer Institute, Buffalo, NY.
Kindly provided by Dr. Chuan Shih, Eli Lilly and Co.
c
d
e
f
g
h
binding the molecule adopts to bind hDHFR, 6 and 7 were each
cocrystalized with isolated hDHFR.
Figure 7. Stereo comparison of the structure of 6 (cyan) with that of folate (yellow)
4. X-ray crystal structures
in hDHFR. Figure prepared with PyMol.45
The X-ray crystal structures of the ternary complexes of tricyclic
compounds 6 an0d 7 and NADPH with hDHFR were determined and
refined to 1.35 ÅA resolution. These data show, for the first time,
that the tricyclic benzo[4,5]thieno[2,3-d]pyrimidine antifolates 6
and 7 bind in a folate orientation such that the 2-NH2 and N3
interact with Glu30 and the thienosulfur occupies the N8 position
observed in the binding of folic acid (Figs. 6 and 7). These results
are similar to those observed for the bicyclic structure of the N-
{4-[2-amino-6-methyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
5-yl)thio]benzoyl}-L
-glutamic acid43 in complex with active site
mutants of hDHFR in which the thieno[2,3-d]pyrimidine ring sulfur
in both complexes makes intermolecular contacts with the
Figure 5. Proposed binding mode with DHFR.

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X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
Replacement of the 2-CH3 moiety with a 2-NH2 gave increased hu-
man TS inhibition and also human DHFR inhibition affording dual
hTS/hDFHR inhibitors. The X-ray crystal structures of 6 and 7 show,
for the first time, that tricyclics 6 and 7 bind in the folate rather
than the MTX mode.
6. Experimental section
All evaporations were carried out in vacuo with a rotary evapo-
rator. Analytical samples were dried in vacuo (0.2 mmHg) in a
CHEM-DRY drying apparatus over P2O5 at 80 °C. Melting points
were determined on a MEL-TEMP II melting point apparatus with
a FLUKE 51 K/J electronic thermometer and are uncorrected. Nucle-
ar magnetic resonance spectra for proton (1H NMR) were recorded
on either a Bruker WH-400 (400 MHz) spectrometer or 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; exch, D2O exchangeable protons.
Mass spectra were recorded on a VG-7070 double-focusingmass
spectrometer or in a LKB-9000 instrument in the electron ioniza-
tion (EI) mode. Chemical names follow IUPAC nomenclature.
Thin-layer chromatography (TLC) was performed on Whatman Sil
G/UV254 silica gel plates with a fluorescent indicator, and the
spots were visualized under 254 and 365 nm illumination. All ana-
lytical samples were homogeneous on TLC in three different sol-
vent systems. Proportions of solvents used for TLC are by
Figure 8. Model of 2 (violet) from ecTS28 structure modeled to 7 in hDHFR (green).
volume. Column chromatography was performed on
a 230–
400 mesh silica gel (Fisher, Somerville, NJ) column. Elemental anal-
yses were performed by Atlantic Microlab, Inc., Norcross, GA. Ele-
ment compositions are within 0.4% of the calculated values.
Fractional moles of water frequently found in the analytical sample
of antifolates could not be prevented in spite of 24–48 h of drying
in vacuo and was confirmed where possible by the presence in the
1H NMR spectra. All solvents and chemicals were purchased from
Aldrich Chemical Co. or Fisher Scientific and were used as received.
For compounds 4–7, a single spot on TLC in three different solvent
systems with three different Rf values confirmed >95% purity.
Figure 9. Stereoview of ecTS-228 (green) with compound 7 (cyan) from the crystal
structure of hDHFR fit to the TS ligand. Also highlighted is residue Val77 in TS.
0
0
carbonyl of Ile7 (3.5 ÅA), Val115 (3.7 ÅA), the hydroxyl of0 Tyr121
0
(3.8 ÅA) and the carbonyl of the nicotinamide ring (3.6 ÅA). Thus
the steric addition of the benzo fused ring does not prevent the thi-
eno S from making the same contacts with hDHFR as that of the 4-
NH2 of MTX.44
Although molecular modeling studies suggest that both a
flipped and folate orientation were possible for these structures
(Fig. 5), the observed crystal structures reveal, for the first time,
only binding in the normal mode of folate orientation, as illus-
trated in Figure 7 that compares the binding of 6 with folate. The
binding orientation of the 6-5-6-ring system is the same in 6 and
7 while the added ring in the isoindoline ring system of 7 shows,
also for the first time, that the fused isoindolinyl ring system side
chain of 7 is flipped compared to that of the normal p-amin-
obenzoylglutamate of 6 as illustrated in Figure 6. Despite this
6.1. Ethyl 2-amino-4-methyl-4,5,6,7-tetrahydro-1-
benzothiophene-3-carboxylate (9)
A mixture of sulfur (1.1 g, 36 mmol), 2-methylcyclohexanone
(4.04 g, 36 mmol), ethyl cyanoactetate (4.07 g, 36 mmol) and EtOH
(150 mL) were placed in a round bottom flask and warmed to 45 °C
and treated dropwise with morpholine (3.1 g, 36 mmol) over
15 min. The mixture was stirred for 5 h at 45 °C and 24 h at room
temperature. Unreacted sulfur was removed by filtration, and the
filtrate was concentrated under reduced pressure to afford an or-
ange solid. The residue was loaded on a silica gel column packed
with silica gel and eluted with 10% ethyl acetate in hexane. The
fractions containing the desired product (TLC) were pooled and
evaporated to afford 9 (6.97 g, 80.9%) as an orange solid; mp
69.9–71 °C; Rf 0.44 (hexane/EtOAc 3:1); 1H NMR (DMSO-d6): d
1.08–1.10 (d, 3H, J = 6.6 Hz, 4-CH3), 1.25–1.28 (t, 3H, J = 6.8 Hz,
COOCH2CH3), 1.57–1.78 (m, 4H), 2.39–2.43 (m, 2H), 3.15–3.17
(m, 1H), 4.10–4.24 (q, 2H, J = 6.8 Hz, COOCH2CH3), 7.23 (s, 2H,
NH2 exch). Titled compound was used directly for next step with-
out further characterization.
change, the functional groups of the L-glutamate side chain still oc-
cupy similar positions in the binding site. To our knowledge this is
the first time that the fused conformationally restricted isoindoli-
nyl ring system side chain has been compared side-by-side in a
X-ray structure with the benzoyl ring in hDHFR.
Comparison of binding of 2 from the structure of ecTS28 mod-
eled into the binding site of 7 in hDHFR (from the X-ray crystal
structure) shows that there are changes in the p-aminobenzoyl
bridge conformation (Fig. 8) that result from the displacement of
the 6-6-6-ring system of 2 compared to the 6-5-6-ring system of
7. Similarly, a fit of 7 to that of 2 in ecTS (Fig. 9) shows that contact
with Val 77 is the same for both 7 and 2 (Fig. 9).
5. Conclusion
6.2. 2-Amino-5-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d
]pyrimidin-4(3H)-one (10)
Compounds 4–7 were synthesized as classical antifolates with
both rigid and flexible benzoylblutamates attached to the tricyclic
benzo[4,5]thieno[2,3-d]pyrimidine scaffold. Compounds with a
2-CH3 moiety inhibited human TS but not human DHFR.
A mixture of 9 (0.74 g, 3.28 mmol) and chloroformamidine
hydrochloride (1.51 g, 13.12 mmol) in dimethyl sulfone (DMSO2)

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
3591
(4 g) was heated at 140° C for 4 h. The mixture was cooled to room
temperature and water (15 mL) was added and ammonium
hydroxide was used to neutralize the suspension. The brown solid,
obtained by filtration, was washed with water and dried over P2O5
in vacuum. The solid was dissolved in methanol and silica gel was
added. A dry silica gel plug was obtained after evaporation of the
solvent. The plug was loaded on to a silica gel column and eluted
with 5% methanol in chloroform. The fractions containing the de-
sired product (TLC) were pooled and evaporated to afford 10
(0.46 g, 59.7%) as a light yellow solid; mp >300 °C; Rf 0.5 (MeOH/
CHCl3, 1:6); 1H NMR (DMSO-d6) d 1.18–1.20 (d, 3H, J = 6.8 Hz,
CH3), 1.59–1.61 (m, 1H), 1.71–1.83 (m, 3H), 2.54–2.64 (m, 2H),
3.15–3.18 (m, 1H), 6.39 (s, 2H, 2-NH2 exch), 10.73 (s, 1H, 3-NH
exch); Anal. (C11H13N3SOꢀ0.2H2O): C, H, N, S.
Titled compound was used directly for next step without further
characterization.
6.6. 2,2-Dimethyl-N-(5-methyl-4-oxo-3,4-
dihydro[1]benzothieno[2,3-d]pyrimidin-2-yl)propanamide (13)
To a 100 mL round-bottomed flask was added 12 (1.34 g,
5.8 mmol) and excess Piv2O (4 equiv). The mixture was kept at re-
flux for 2 h. The excess Piv2O was removed under vacuum and the
residue was made into silica gel plug. The silica gel plug obtained
was loaded onto a silica gel column and eluted with 1:8 ethyl ace-
tate/hexane to afford 13 (1.299 g, 70.9%) as a white solid, mp
178.3–179.5 °C; Rf 0.35 (hexane/EtOAc 3:1); 1H NMR (CDCl3) d
1.35 (s, 9H, 3 CH3 of Piv), 3.05 (s, 3H, CH3), 7.28 (m, 2H, C6H3),
7.56, 7.59 (d, 1H, J = 8.0 Hz C6H3), 8.14 (s, 1H, NH exch), 11.86 (s,
1H, NH exch). Titled compound was used directly for next step
without further characterization.
6.3. 2,2-Dimethyl-N-(5-methyl-4-oxo-3,4,5,6,7,8-
hexahydro[1]benzothieno[2,3-d]pyrimidin-2-yl)propanamide
(11)
6.7. 2,5-Dimethyl[1]benzothieno[2,3-d]pyrimidin-4(3H)-one
To a 100 mL round-bottomed flask was added 10 (0.706 g,
3 mmol) and excess Piv2O (10 mL). The mixture was kept at reflux
for 2 h. The excess Piv2O was removed under vacuum and the res-
idue was made into silica gel plug. The silica gel plug obtained was
loaded onto a silica gel column and eluted with 1:8 ethyl acetate/
hexane. The fraction containing the desired product were pooled
afford 11 (0.956 g, 70.3%) as a white solid; mp 236.6–237.8 °C; Rf
0.31 (hexane/EtOAc 3:1); 1H NMR (DMSO-d6) d 1.22 (s, 12H, 4
CH3), 1.62–1.79 (m, 4H), 2.58–2.73 (m, 2H), 3.23 (br s, 1H), 11.07
(s, 1H, NH exch), 12.02 (s, 1H, NH exch). Titled compound was used
directly for next step without further characterization.
(15)
To a 100 mL round flask were added 14 (2.35 g, 10 mmol) and
CH3CN (50 mL). Vigorous stirring afforded, a clear solution. Anhy-
drous HCl gas was bubbled into the solution for 1 h to give a thick
precipitate, which then redissolved into the acid solution. Anhy-
drous HCl gas was added for an additional 3 h after which the reac-
tion mixture became clear. Evaporation of the solvent under
reduced pressure afforded a residue that was dissolved in water.
Concentrated aqueous NH4OH was added to afford a suspension
at pH 8. The precipitate was collected by filtration, washed with
water and dried over P2O5 in a vacuum to afford 15 (1.0 g, 57%)
as a yellow solid; mp >300 °C; Rf 0.58 (MeOH/CHCl3, 1:6); 1H
NMR (DMSO-d6) d 2.35 (s, 3H, 2-CH3), 2.95 (s, 3H, 5-CH3), 7.29
(m, 2H, C6H3), 7.78 (s, 1H, C6H3), 12.54 (s, 1H, 3-NH exch). HRMS
calcd for C12H10N2OS 231.0592, found 231.0584.
6.4. Ethyl 2-amino-4-methyl-1-benzothiophene-3-carboxylate
(14)
A mixture of 9 (0.239 g, 1 mmol), 10% Pd/C (0.239 g) and
mesitylene (50 mL) were placed in a round bottom flask and kept
at reflux for 1–2 days. The mixture was cooled to room tempera-
ture and Pd/C was removed by filtration, and the filtrate was con-
centrated under reduced pressure to afford a brown oil. The
residue was loaded on a silica gel column and eluted with 10%
ethyl acetate in hexane. The fractions containing the desired
product (TLC) were pooled and evaporated to afford 14 (0.123 g,
52.1%) as an orange semi solid; Rf 0.38 (hexane/EtOAc 3:1); 1H
NMR (DMSO-d6): d 1.29–1.32 (t, 3H, J = 6.8 Hz, COOCH2CH3),
2.38 (s, 3H, CH3), 4.25–4.30 (q, 2H, J = 6.8 Hz,COOCH2CH3), 6.98–
7.04 (m, 2H, C6H3), 7.44, 7.45 (d, 1H, J = 4 Hz, C6H3), 7.43 (s, 2H,
NH2 exch). Titled compound was used directly for next step with-
out further characterization.
6.8. 5-(Bromomethyl)-2-methyl[1]benzothieno[2,3-
d]pyrimidin-4(3H)-one (17)
To a 100 mL flask were added 15 (0.736 g, 3.2 mmol) and ben-
zene (30 mL). The suspension was stirred at 60 °C for 30 min to af-
ford
a
clear solution, followed by the addition of N-
bromosuccinimide (0.620 g, 3.49 mmol) and benzoyl peroxide
(50 mg). The mixture was maintained at reflux for 4 h and then
cooled to room temperature and washed with water, and evapo-
rated to afford a yellow solid. The solid was dissolved in methanol
and silica gel (1.5 g) was added. A dry silica gel plug was obtained
after evaporation of the solvent. The plug was loaded on to a silica
gel column and eluted with 6% ethyl acetate in hexane to afford 17
(393 mg, 39%) as a white solid; mp 217.8–219.1 °C; Rf 0.3 (hexane/
EtOAc 3:1); 1H NMR (CDCl3) d 2.69 (s, 3H, 2-CH3), 5.84 (s, 2H,
CH2Br), 7.44–7.47 (t, 1H, J = 7.2, C6H3), 7.57–7.58 (d, 1H,
J = 7.2 Hz, C6H3), 7.80–7.82 (d, 1H, J = 7.2 Hz, C6H3). HRMS calcd
for C12H9N2OSBr 307.9619, found 307.9613.
6.5. 2-Amino-5-methyl[1]benzothieno[2,3-d]pyrimidin-4(3H)-
one (12)
A mixture of 14 (0.49 g, 2.07 mmol) and chloroformamidine
hydrochloride (1.19 g, 10.37 mmol) in DMSO2 (2 g) was heated at
140° C for 2 h. The mixture was cooled to room temperature and
water (15 mL) was added and ammonium hydroxide was used to
neutralize the suspension. The brown solid, obtained by filtration,
was washed with water and dried over P2O5 vacuum. The solid was
dissolved in methanol and silica gel (1.0 g) was added. A dry silica
gel plug was obtained after evaporation of the solvent. The plug
was loaded on to a silica gel column and eluted with 5% methanol
in chloroform. The fractions containing the desired product (TLC)
were pooled and evaporated to afford 12 (0.29 g, 60.7%) as a yellow
solid; mp >300 °C; Rf 0.41 (MeOH/CHCl3, 1:6); 1H NMR (DMSO-d6)
d 2.87 (s, 3H, CH3), 6.82 (s, 2H, 2-NH2 exch), 7.13- 7.17 (m, 2H,
C6H3), 7.57, 7.59 (d, 1H, J = 8.0 Hz, C6H3), 10.92 (s, 1H, 3-NH exch).
6.9. 2,2-Dimethyl-N-(5-methyl-4-oxo-3,4-
dihydro[1]benzothieno[2,3-d]pyrimidin-2-yl)propanamide (16)
A solution of 13 (1.1 g, 3.49 mmol) in 1,2-dichloroethane
(50 mL) was treated with N-bromosuccinimide (0.62 g, 3.49 mmol)
and benzoyl peroxide (50 mg), and the mixture was maintained at
reflux for 1 day. The mixture was cooled to room temperature and
washed with water, and evaporated to an orange solid. The solid
was dissolved in methanol and silica gel (1.5 g) was added. A dry
silica gel plug was obtained after evaporation of the solvent. The
plug was loaded on to a silica gel column and eluted with 6% ethyl

3592
X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
acetate in hexane to afford 16 (0.577 g, 41.9%) as an orange solid;
mp 217.8–219.1 °C; Rf 0.3 (hexane/EtOAc 3:1); 1H NMR (CDCl3) d
1.27 (s, 9H, 3 CH3 of Piv), 5.71 (s, 2H, CH2Br), 7.30, 7.33 (d, 1H,
C6H3), 7.43, 7.46 (d, 1H, J = 7.6 Hz, C6H3), 7.64, 7.66 (d, 1H,
J = 4 Hz, C6H3), 8.07 (s, 1H, NH exch), 11.99 (s, 1H, NH exch). Titled
compound was used directly for next step without further
characterization.
6.14. General procedure for the synthesis of compounds 4–7
stirred solution of the tricyclic bromide 16 or 17
A
(0.25 mmol) in dry DMF (5 mL) was treated with the appropriate
amine 22 or 23 (1 mmol) and K2CO3 (95 mg, 0.69 mmol). The
solution was stirred for 1 h at 80 °C under argon. The cooled reac-
tion mixture was filtered and the filtrate was evaporated to ob-
tain an orange solid. The solid was dissolved in methanol and
silica gel was added. A dry silica gel plug was obtained after evap-
oration of the solvent. The plug was loaded on to a silica gel col-
umn and eluted with ethyl acetate/hexane (1:1). The fractions
containing the desired product (TLC) were pooled and evaporated
to afford a solid, to which a combined solution of aqueous 1 N
NaOH (3 mL) and methanol (12 mL) was added. The mixture
was kept at reflux for 12 h. The methanol was evaporated under
reduced pressure and the residue was dissolved in water
(5 mL). The solution was cooled to 0 °C and carefully acidified
to pH 3 with dropwise addition of 1 N HCl. The resulting suspen-
sion was left at 0 °C for 2 h and the precipitate was collected by
filtration, washed with water (5 mL) and dried over P2O5/vacuum
at 50 °C to afford target compounds 4–7.
6.10. Methyl 2-methyl-4-nitrobenzoate (19)
Thionyl chloride (4.3 g, 36.45 mmol) was added dropwise to a
stirred solution of 18 (3 g, 16.57 mmol) in MeOH (25 mL) while
maintaining the internal temperature below 12 °C. When the addi-
tion was complete the mixture was left to stand at room tempera-
ture for 12 h to result a white precipitation. The mixture was
filtered and the filtrate was concentrated under reduced pressure
to afford white solid. The solid was washed with hexane and ethyl
ether to afford 19 (2.95 g, 91.3%); mp 153.7–154.4 °C (lit.39 mp
153–154 °C); Rf 0.43 (hexane/EtOAc 3:1); 1H NMR (CDCl3) d 2.69
(s, 3H, CH3), 3.95 (s, 3H, COOCH3), 8.02–8.11 (m, 3H, C6H3).
6.11. Methyl 2-(bromomethyl)-4-nitrobenzoate (20)
6.14.1. N-(4-{[(2-methyl-4-oxo-3,4-dihydro[1]benzothieno[2,3-
A solution of 19 (2.57 g, 13.19 mmol) in 1,2-dichloroethane
(100 mL) was treated with N-bromosuccinimide (2.3 g,
13.19 mmol) and benzoyl peroxide (0.26 g), and the mixture was
kept at reflux for 2 days, then cooled, washed with water, and
evaporated to a yellow oil (3.52 g). The oil was dissolved in acetone
and silica gel (4.0 g) was added. A dry silica gel plug was obtained
after evaporation of the solvent. The plug was loaded on to a silica
gel column and eluted with 10% ethyl acetate in hexane to afford
20 (1.73 g, 48.2%) as a yellow oil; Rf 0.53 (hexane/EtOAc 3:1); 1H
NMR (CDCl3) d 3.89 (s, 3H, COOCH3), 4.86 (s, 2H, CH2Br), 7.98–
8.23 (m, 3H, C6H3).
d]pyrimidin-5-yl)methyl]amino}benzoyl)-L-glutamic acid (4)
Using the general procedure described above with 17 and 23
afforded 4 (37 mg, 37%) as a yellow solid; mp 219.6–221.3 °C; Rf
0.28 (MeOH/EtOAc, 1:6); Rf 0.32 (MeOH/CHCl3, 1:6 + 1 drop of
NEt3); Rf 0.34 (MeOH/CHCl3, 1:6 + 1 drop of gl. HOAc); 1H NMR
(DMSO-d6): d 1.22 (s, 1H, 2-CH3), 1.88–2.01 (m, 2H, Glu
c-CH2CH2),
2.29 (m, 2H, Glu -CH2CH2), 4.31 (s, 1H, Glu -CH), 5.20 (s, 2H, Ben-
c
a
zylic CH2), 6.56–6.58 (d, 2H, J = 9.0 Hz), 7.38–7.40 (d, 1H,
J = 7.5 Hz), 7.49 (d, 2H, 2-NH2 exch), 7.58–7.59 (d, 2H, J = 9.0 Hz),
7.88 (d, 1H, J = 7.5 Hz) 8.02 (s, 1H, CONH), 8.08 (d, 1H), 12.71 (s,
1H, 3-NH exch); HRMS (ESI, pos mode) m/z [M+H+] calcd for
C24H23N4O6S 495.1338, found 495.1345.
6.12. Diethyl (2S)-2-(5-nitro-1-oxo-1,3-dihydro-2H-isoindol-2-
yl)pentanedioate (21)
6.14.2. (2S)-2-(5-{[(2-methyl-4-oxo-3,4-dihydro[1]benzothieno-
[2,3-d]pyrimidin-5-yl)methyl]amino}-1-oxo-1,3-dihydro-2H-
isoindol-2-yl)pentanedioic acid (5)
Using the general procedure described above with 17 and 22
afforded 5 (35 mg, 34%) as a yellow solid; mp 236.4–237.7 °C; Rf
0.29 (MeOH/EtOAc, 1:6); Rf 0.32 (MeOH/CHCl3, 1:6 + 1 drop of
NEt3); Rf 0.36 (MeOH/CHCl3, 1:6 + 1 drop of gl. HOAc);1H NMR
(DMSO-d6): d 1.22 (s, 3H, 2-CH3), 1.95 (m, 2H, Glub-CH2), 2.18
The oil 20 (0.85 g, 3.1 mmol) was stirred for 16 h with diethyl
glutamate hydrochloride (1.54 g, 6.4 mmol) and powdered K2CO3
(1.7 g, 12 mmol) in DMA (3 mL) under argon. The reaction mixture
was diluted with water (20 mL) and extracted with ethyl acetate
(3 ꢁ 20 mL). The combined ethyl acetate solutions were washed
twice with brine, dried, and evaporated to an orange oil. The oil
was dissolved in methanol and silica gel was added. A dry silica
gel plug was obtained after evaporation of the solvent. The plug
was loaded on to a silica gel column and eluted with 25% ethyl ace-
tate in hexane to afford 21 (0.63 g, 55.7%) as an orange oil; Rf 0.44
(m, 2H, Gluc-CH2), 4.22 (s, 2H, isoindolinyl CH2), 4.67–4.69 (m,
1H, Glu -CH), 5.21 (s, 2H, Benzylic CH2), 6.65 (s, 1H, CH), 6.81 (s,
a
1H, NH exch), 7.30–7.32 (d, 1H, J = 6.0 Hz), 7.37–7.41 (d, 1H,
J = 12 Hz), 7.49–7.51 (d, 1H, J = 6.0 Hz), 7.88–7.90 (d, 1H,
J = 6.0 Hz), 8.03 (s, 1H),12.76 (s, 1H, 3-NH exch), HRMS (ESI, pos
mode) m/z [M+H+] calcd for C25H23N4O6S 507.1333, found
507.1362.
(hexane/EtOAc 1:1); 1H NMR (CDCl3)
d 1.17–1.22 (t, 3H,
COOCH2CH3), 1.26–1.31 (t, 3H, COOCH2CH3), 2.19–2.49 (m, 4H,
CHCH2CH2COOEt), 4.02–4.23 (2q, 4H, COOCH2CH3), 4.51–4.83 (dd,
2H, –CH2–), 5.09–5.14 (m, 1H, CHCH2CH2COOEt), 8.00–8.03 (d,
1H, C6H3), 8.36 (br s, 2H, C6H3).
6.14.3. N-(4-{[(2-Amino-4-oxo-3,4-dihydro[1]benzothieno[2,3-d
]pyrimidin-5-yl)methyl]amino}benzoyl)-L-glutamic acid (6)
6.13. Diethyl (2S)-2-(5-amino-1-oxo-1,3-dihydro-2H-isoindol-2-
yl)pentanedioate (22)
Using the general procedure described above with 16 and 23
afforded 6 (39 g, 32%) as an orange solid; mp >300 °C; Rf 0.30
(MeOH/EtOAc, 1:6); Rf 0.34 (MeOH/CHCl3, 1:6 + 1 drop of NEt3);
Rf 0.34 (MeOH/CHCl3, 1:6 + 1 drop of gl. HOAc);1H NMR (DMSO-
To
a Parr hydrogenation bottle was added 21 (0.55 g,
1.51 mmol), 10% Pd/C (0.09 g) and acetyl acetate (30 mL). Hydroge-
nation was carried out at 55 psi for 12 h. After filtration, the organ-
ic phase was evaporated at vacuum to afford 22 (0.467 g, 92.5%) as
a orange oil; Rf 0.19 (hexane/EtOAc 1:1); 1H NMR (CDCl3) d 1.17–
1.19 (t, 3H,COOCH2CH3), 1.20–1.25 (t, 3H, COOCH2CH3), 2.20–2.51
(m, 4H, CHCH2CH2COOEt), 4.02–4.28 (m, 6H, 2 COOCH2CH3, NH2
exch), 4.22–4.51 (dd, 2H, –CH2–), 5.03–5.07 (m, 1H,
CHCH2CH2COOEt), 6.69–6.73 (m, 2H, C6H3), 7.60, 7.63 (d, 1H, C6H3).
d6): d 1.85–1.98 (m, 2H, Glu
CH2CH2),4.26–4.34 (m, 1H, Glu
c
-CH2CH2), 2.27–2.32 (m, 2H, Glu
c-
a-CH), 5.10–5.14 (d, 2H, Benzylic
CH2), 6.55 (s,H, CH2NH), 6.55–6.58 (d, 2H, J = 9.0 Hz, 2 CH), 6.94
(br s, 2H, 2-NH2 exch), 7.19–7.24 (t, 1H, J = 7.5 Hz), 7.38–7.40 (d,
1H, J = 7.5 Hz), 7.56–7.59 (d, 2H, J = 9.0 Hz), 7.68–7.70 (d, 1H,
J = 7.5 Hz), 7.97–8.00 (d, 1H, J = 6.9 Hz, NH exch), 11.19 (s, 1H, 3-
NH exch), 12.66 (s, 2H, 2COOH exch); HRMS (ESI, pos mode) m/z
[M+H+] calcd for C23H22N5O6S 496.1291, found 496.1316.

X. Zhang et al. / Bioorg. Med. Chem. 19 (2011) 3585–3594
3593
Table 2
Data were collected to 1.35 Å resolution for both complexes using
the remote access robot46–48 at liquid N2 temperatures on
beamline 9–2 at the Stanford Synchrotron Research Laboratory
(SSRL) imaging plate system. The data were processed using
Mosflm.49 The diffraction statistics are shown in Table 2 for the
two complexes.
The structures were solved by molecular replacement methods
using the coordinates for hDHFR (u072) in the program Molref.49
Inspection of the resulting difference electron density maps made
using the program COOT50 running on a MacG5 workstation re-
vealed density for the ternary complex in both crystals. The final
cycles of refinement were carried out using the program Refmac5
in the CCP4 suite of programs. The Ramachandran conformational
parameters from the last cycle of refinement generated by PRO-
CHECK51 showed that more than 98% of the residues have the most
favored conformation and none are in the disallowed regions.
Coordinated for these structures have been deposited with the Pro-
tein Data Bank (3ntz, 3nu0).
Data collection and refinement statistics for hDHFR-NADPH-6 and
complexes
7 ternary
Data collection
hDHFR
NADPH-6
hDHFR
NADPH-7
PDB accession #
Space group
3ntz
H3
84.29 77.79
3nu0
H3
0
84.39 78.19
Cell dimensions (ÅA)
Beamline
Resolution (Á)
Wavelength (Á)
Rsym (%)a,b
SSRL 9-2
1.35
0.975
0.061 (0.72)
0.053 (0.62)
100.0 (100.0)
169,016
45,260
15.3 (1.6)
3.7 (3.7)
SSRL 9-2
1.35
0.975
0.066 (0.45)
0.063 (0.39)
100.0 (100.0)
569,239
45,587
27.0 (8.6)
12.5 (12.1)
Rmerge
Completeness (%)a
Observed reflections
Unique reflections
I/ (I)
Multiplicitya
Refinement and model quality
Resolution range (Á)
No. of reflections
26.6–1.35
45,259
18.6
33.1–1.35
45,587
18.0
R-factorc
6.16. Dihydrofolate reductase (DHFR) assay52
R
free-factord
20.5
20.9
Total protein atoms
Total water atoms
Average B-factor (Á2)
1817
164
18.9
1868
187
15.7
All enzymes were assayed spectrophotometrically in a solution
containing 50
UM dihydrofolate, 80 UM NADPH, 50 mM Tris–HCl,
0.001 M 2-mercaptoethanol, and 0.001 M EDTA at pH 7.4 at 30E C.
The reaction was initiated with an amount of enzymes yielding a
change in optical density at 340 nm of 0.015 units/min.
Rms deviation from ideal
Bond lengths (Á)
Bond angles (°)
Luzzati
0.031
2.84
0.159
0.035
2.99
0.145
Ramachandran plot
6.17. Thymidylate synthase (TS) assay
Most favored regions (%)
Additional allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
97.8
2.2
0.0
97.8
2.2
0.0
TS was assayed spectrophotometrically at 30E C and pH 7.4 in a
mixture containing 0.1 M 2-mercaptoethanol, 0.0003 M (6R,S)-tet-
rahydrofolate, 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 as-
say described by Wahba and Friedkin53 except that the dUMP con-
centration was increased 25-fold according to the method of
Davisson et al.54 The reaction was initiated by the addition of an
amount of enzyme yielding a change in absorbance at 340 nm of
0.016 units/min in the absence of inhibitor.
0.0
0.0
a
The values in parentheses refer to data in the highest resolution shell.
b
Rsym
=
RhRi|Ih,i ꢂ <Ih>|/RhRi|Ih,i|, where <Ih> is the mean intensity of a set of
equivalent reflections.
c
R-factor =
lated structure factor amplitudes.
R
|Fobsd ꢂ Fcalcd|/
RFobsd, where Fobsd and Fcalc are observed and calcu-
d
Rfree-factor was calculated for R-factor for a random 5% subset of all reflections.
6.14.4. (2S)-2-(5-{[(2-Amino-4-oxo-3,4-dihydro[1]benzothieno-
[2,3-d]pyrimidin-5-yl)methyl]amino}-1-oxo-1,3-dihydro-2H-
isoindol-2-yl)pentanedioic acid (7)
Using the general procedure described above with 16 and 22
afforded 7 (40 mg, 32%) as a orange solid; mp >300 °C; Rf 0.27
(MeOH/EtOAc, 1:6); Rf 0.30 (MeOH/CHCl3, 1:6 + 1 drop of NEt3);
Rf 0.33 (MeOH/CHCl3, 1:6 + 1 drop of gl. HOAc);1H NMR (DMSO-
Acknowledgments
This work is supported by National of Institute of Health,
National Institute of Allergy and Infectious Diseases grant
A1069966 (A.G.), National Cancer Institute grant CA 125153
(A.G.) and General Medical Institute grant GM051670 (V.C.). The
National Science Foundation, CHE0614785, is acknowledged for
NMR facilities.
d6): d 1.97–2.20 (m, 4H, Glub-CH2, Glu
isoindolinyl CH2), 4.68–4.71 (m, 1H, Glu
c
-CH2), 4.21–4.25 (m, 2H,
-CH), 5.13, 5.14 (d, 2H,
a
benzylic CH2), 6.64 (br s, 2H, C6H3), 6.74 (s, 1H, NH exch), 6.89 (s,
2H, 2-NH2 exch), 7.20–7.26 (t, 1H, J = 7.5 Hz, C6H3), 7.30–7.33 (d,
1H, J = 8.0 Hz, C6H3), 7.39–7.42 (dd, 1H, C6H3), 7.69–7.72 (d, 1H,
J = 8.0 Hz, C6H3), 11.11 (s, 1H, 3-NH exch), 12.78 (s, 2H, 2COOH
exch). HRMS (ESI, pos mode) m/z [M+H+] calcd for C24H21N5O6S,
508.1285; found, 508.1321.
Supplementary data
Supplementary data (Elemental analysis and High-Resolution
Mass Spectra (HRMS) (EI)) associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2011.03.067.
References and notes
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