Thieno[2,3-d]pyrimidine-2-carboxamides bearing a carboxybenzene group at 5-position: Highly potent, selective, and orally available MMP-13 inhibitors interacting with the S1″ binding site

Article abstract of DOI:10.1016/j.bmc.2014.07.025

On the basis of X-ray co-crystal structures of matrix metalloproteinase-13 (MMP-13) in complex with its inhibitors, our structure-based drug design (SBDD) strategy was directed to achieving high affinity through optimal protein-ligand interaction with the unique S1″ hydrophobic specificity pocket. This report details the optimization of lead compound 44 to highly potent and selective MMP-13 inhibitors based on fused pyrimidine scaffolds represented by the thienopyrimidin-4-one 26c. Furthermore, we have examined the release of collagen fragments from bovine nasal cartilage in response to a combination of IL-1 and oncostatin M.

Full text of DOI:10.1016/j.bmc.2014.07.025

Bioorganic & Medicinal Chemistry 22 (2014) 5487–5505
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Thieno[2,3-d]pyrimidine-2-carboxamides bearing a carboxybenzene
group at 5-position: Highly potent, selective, and orally available
MMP-13 inhibitors interacting with the S100 binding site
⇑
Hiroshi Nara , Kenjiro Sato, Takako Naito, Hideyuki Mototani, Hideyuki Oki, Yoshio Yamamoto,
Haruhiko Kuno, Takashi Santou, Naoyuki Kanzaki, Jun Terauchi, Osamu Uchikawa, Masakuni Kori
Pharmaceutical Research Division, Takeda Pharmaceutical Company Ltd, 26-1, Muraokahigashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of X-ray co-crystal structures of matrix metalloproteinase-13 (MMP-13) in complex with its
inhibitors, our structure-based drug design (SBDD) strategy was directed to achieving high affinity
through optimal protein–ligand interaction with the unique S100 hydrophobic specificity pocket. This
report details the optimization of lead compound 44 to highly potent and selective MMP-13 inhibitors
based on fused pyrimidine scaffolds represented by the thienopyrimidin-4-one 26c. Furthermore, we
have examined the release of collagen fragments from bovine nasal cartilage in response to a combina-
tion of IL-1 and oncostatin M.
Received 10 June 2014
Revised 15 July 2014
Accepted 16 July 2014
Available online 7 August 2014
Keywords:
Matrix metalloproteinase
MMP-13
Ó 2014 Published by Elsevier Ltd.
Thieno[2,3-d]pyrimidin-4-one
Osteoarthritis
OA
X-ray crystallography
Structure-based drug design
1. Introduction
diseases6 such as osteoarthritis (OA), rheumatoid arthritis, cancer,
inflammatory bowel diseases, periodontal disease, and corneal
Osteoarthritis is characterized by a loss of articular cartilage
resulting in chronic pain and disability. Current treatments are lim-
ited to symptomatic relief with NSAIDs or COXIBs (selective COX-2
inhibitors), intra-articular injections of hyaluronic acid conjugates
(Synvisc) or surgical joint replacement. Additionally, COXIBs’
increased cardiovascular events such as heart attacks and stroke
led to the recent withdrawal of the COX-2 inhibitor Vioxx from
the market.1,2
The matrix metalloproteinases (MMPs) are a family of zinc-
dependent enzymes involved in the degradation and remodeling
of extra-cellular matrix.3,4 The first matrix metalloproteinase
(MMP-1) was a collagenase found in metamorphosing tadpole
tail.5 Since then, three members have been added to the collage-
nase family. These four collagenases MMP-1, MMP-8, MMP-13,
and MMP-18 (a Xenopus collagenase-4) are capable of degrading
interstitial collagens I, II, and III into two segments of 1/4 and
3/4 length.4 MMPs are important therapeutic targets for various
ulceration. Among the MMP family, MMP-13 has been suggested
to be intimately involved in the destruction of joint components
in OA.7,8 Therefore, MMP-13 inhibitors are expected to be dis-
ease-modifying drugs for OA.
In the past two decades, a large number of potent MMP inhibi-
tors have been disclosed in the literature.6,9 Earlier generation
inhibitors designed based on the peptide fragment of substrates
recognized by the MMP active site were found to fill the unprimed
side of the active site by X-ray co-crystallographic studies. Most of
the inhibitors have hydroxamic acid moiety as the zinc-binding
group (ZBG).10,11 Many of these small-molecule inhibitors showed
a broad-spectrum inhibitory effect and were not selective between
the MMP isoforms. Furthermore, a number of non-selective MMP
inhibitors have been abandoned because of their musculoskeletal
side effects (MSS) characterized by joint stiffness and pain.12–14
Therefore, there is a great need for a new generation of inhibitors
that can prevent degrading activities of specific MMPs with
improved potency and reduced toxicity.
These findings prompted us to pursue selective MMP-13 inhib-
itors for the treatment of OA. Previously we have discovered a new
class of human MMP-13 selective nonpeptide inhibitors based on
Abbreviations: MMP-13, matrix metalloproteinase 13; OA, osteoarthritis.
Corresponding author. Tel.: +81 466 32 1230; fax: +81 466 29 4471.
E-mail address: hiroshi.nara@takeda.com (H. Nara).
⇑
http://dx.doi.org/10.1016/j.bmc.2014.07.025
0968-0896/Ó 2014 Published by Elsevier Ltd.

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H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
O
OH
O
N
O
O
HN
H
N
HN
H
MeO
N
S
MeO
N
O
O
44
26c
Figure 1. Chemical structures of 44 and 26c.
treatment with a combination of interleukin-1 (IL-1) and oncosta-
tin M (OSM).
(iii) P1" modification
In this report, we describe design, synthesis and SARs of a new
generation of fused pyrimidine-based nonpeptide MMP-13 selec-
tive inhibitors.
P1"
O
HN
A
2. Chemistry
MeO
N
linker
(ii) ring substitution
Several routes were investigated for the synthesis of the fused
pyrimidine-2-carboxylic acid derivatives, each of which generated
the ester 6a–m as a key intermediate for further functionalization.
Starting from 2-aminoheteroaryl carboxylic acid derivatives 1a–d,
3a–c, and 4a–c, the key intermediates 6a–m were prepared by the
standard one- or two-step procedure. The 2-ester groups of 6a–m
readily react with primary amines. Indeed, the compounds 6a–m
were easily converted to the corresponding amides 7a–m by the
general method described below in Scheme 1. Additionally, amide
formation with a variety of substituted benzylamines in a parallel
array led to a small library of the amides. The enhanced reactivity
of the 2-ester group is probably attributed to anchimeric assistance
by the neighboring 3-NH moiety. Activation of the 2-ester group
through intramolecular hydrogen bonding with the 3-NH group
readily accounts for the exquisite sensitivity of the 2-ester amida-
tion. However, several analogous amidation could not be achieved.
The ester is inert toward secondary amines in DMF at reflux tem-
perature for 12 h or anilinic amines in DMF at reflux temperature
over similar periods of time. Such a lack of reactivity in this ami-
nolysis reaction is probably due to steric hindrance or low
nucleophilicity.
(i) linker modification
Figure 2. Structural modification of fused pyrimidine MMP inhibitors.
the lead compound 4415 (Fig. 1). In a parallel effort, a series of
related fused pyrimidine analogues were also investigated.
Novel non-zinc chelating MMP-13 inhibitors were pursued with
the goals of improving potency and selectivity for MMP-13 and
physicochemical profile. As described in Figure 2, optimization
studies were focused on (i) modification of the left-hand linker;
(ii) modification/replacement of the ring A; (iii) modification of
the right-hand P100 group that could fit into the S100 pocket.16
Optimization of this chemotype was conducted utilizing struc-
ture-based drug design (SBDD). The X-ray co-crystallographic anal-
ysis of 44 suggests that there is a unique and MMP-13-specific S100
hydrophobic pocket16 adjacent to the S10 site accommodating the
quinazolinone template (Fig. 3). To achieve an effective interaction
with the S100 binding site, the computer-aided analysis led to the
design of inhibitors bearing a benzoic acid P100 group, which can
form a salt bridge with Lys140 in the S100 site, tethered via a linker
to the ring A, providing highly potent inhibitor thienopyrimidin-4-
one 26c (Fig. 1), which could potentially improve physicochemical
properties such as water solubility and hence could improve the
oral bioavailability. Furthermore, 26c significantly inhibited degra-
dation of explants of bovine nasal cartilage (BNC) induced by the
Reaction of commercially available
9 with 2-(3-methoxy-
phenyl)acetyl chloride in the presence of triethylamine provided
reverse amide-linked analogue 10 in 71% yield (Scheme 2).
As shown in Scheme 3, reaction of methylanthranilic acid 11
with chloroacetonitrile in methanol in the presence of sodium
methoxide at 80 °C provided the 2-(chloromethyl)quinazolinone
12 in 67% yield. Replacement of the chlorine atom of 12 by
(A)
(B)
S1" site
Zn
S1' site
Figure 3. Crystal structure of the complex of 44 and MMP-13 (PDB code: 3WV2). (A) Surface representation of MMP-13 illustrating the binding cavity. The inhibitor is buried
deeply into the S10 pocket. (B) Schematic representation of the binding mode of 44 and MMP-13.

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5489
O
O
O
H2N
H2N
1a-d
MeO, EtO
HO
Het
Het
Het
H2N
H2N
3a-c
4a-c
g
d
a or b
e
f
O
O
3
O
N
H2N
O
HN
O
O
c
h or i or j
Het
Het
Het
EtO
EtO
N
H
N
2
EtO
O
O
5a-c
6a-m
2a,b
k or l
O
HN
N
H
N
Het
MeO
O
7a-m
Compound
Compound
Het
Het
1
2
3
6
7
3
4
5
6
7
Me
S
1a 2a
-
6a 7a
3a
-
-
6h 7h
S
Me
-
-
-
-
-
-
-
-
-
-
-
-
6b 7b
6c 7c
6d 7d
6e 7e
3b
3c
-
-
-
-
-
6i
6j
7i
7j
Me
Me
S
N
H
Me
Me
Me
S
N
Me
Me
4a 5a
4b 5b
6k 7k
S
S
Me
1b 2b
-
6l
7l
O
N
Me
1c
1d
-
-
6f
7f
-
4c
-
6m 7m
N
O
N
Me
6g 7g
N
N
Me
Scheme 1. Synthesis of fused pyrimidine-2-carboxamide derivatives 7a–m. Reagents and conditions: (a) ethyl chloroglyoxylate, Et3N, THF, 0 °C to rt; (b) (1) diethyl oxalate,
EtONa, EtOH, reflux, (2) oxalyl chloride, DMF, THF, rt, (3) EtOH, pyridine, THF, rt; (c) p-TsOH, toluene or xylene, reflux; (d) diethyl oxalate, EtONa, EtOH, reflux; (e) CNCO2Et,
HCl, AcOH, 80 °C; (f) ethyl chloroglyoxylate, pyridine, rt to 50 °C [for 4a,b]; (g) (1) ethyl chloroglyoxylate, pyridine, rt, (2) oxalyl chloride, DMF, THF, 0 °C to rt [for 4c]; (h)
NH4OAc, EtOH, reflux [for 5a]; (i) NH4OAc, AcOH, EtOH, reflux [for 5b]; (j) (1) NH3, EtOH, THF, 0 °C, (2) EtONa, EtOH, 0 °C to rt [for 5c]; (k) 3-methoxybenzylamine, DMF or
EtOH, 80–90 °C [for 6a–h, j–m]; (l) 3-methoxybenzylamine, N-ethyldiisopropylamine, DMA, 80–90 °C [for 6i].

5490
H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
3-methoxybenzylamine gave 13 in 41% yield by using potassium
carbonate in DMF.
6b using N-bromosuccinimide (NBS) in chlorobenzene provided
24 (Scheme 5). The ether analogues 26a,b,d,e derived from the bro-
mide 24 were prepared by etherification with benzylalcohols and
NaH in THF, followed by aminolysis of the resulting ester 25a–d
with 3-methoxybenzylmine. During the etherification step, partial
hydrolysis of the ester occasionally occurred to give a carboxylic
acid as a byproduct. The carboxylic acid was cleanly re-esterified
under standard conditions to recover the ethyl ester, which upon
treatment with 3-methoxybenzylamine was converted to the tar-
get amide. The synthesis of 26f and 26g containing the N-methyl
or thioether spacers was achieved in a similar manner.
Alternately, the bromine atom of 24 can be displaced by
nucleophiles such as sodium azide or cyanide to generate the
corresponding azide analogue 27a and cyano analogue 30. The 5-
[(phenylcarbamoyl)methyl] derivatives 29a–c were prepared from
the 5-bromomethyl intermediate 24 according to Scheme 5. The
bromide 24 was converted by displacement with sodium azide,
reduction of the resulting azide to the primary amine, and acyla-
tion to give 28a,b. Aminolysis of the esters 28a,b with 3-methoxy-
benzylamine in ethanol produced 29a,b. The ester 29b was next
converted into the desired carboxylic acid 29c by alkaline
hydrolysis.
The general synthetic pathway to the 2-[3-(3-methoxy-
phenyl)propanoyl]-6-methyl-3,4-dihydroquinazolin-4-one 23 is
shown in Scheme 4. The benzamide 14 was cyclized to the
6-methyl-3,4-dihydroquinazolin-4-one 15 by reaction with
trimethyl orthoformate. Subsequently, the 3-nitrogen atom of
compound 15 was easily protected by Boc group to give 16, which
upon treatment with LDA, was reacted with 3-(3-methoxy-
phenyl)propanal 19 prepared from carboxylic acid 17 to furnish
hydroxyl derivative 20. After deprotection of the Boc group of
20, the alcohol function of 21 was then subjected to Swern
oxidation using oxalyl chloride and dimethylsulfoxide with
concomitant protection of the 4-carbonyl of the quinazoline ring
as methylthiomethyl (MTM) ether to give the O-MTM compound
22. Aqueous acidic hydrolysis of the MTM group afforded the
target compound 23.
The synthesis of the 3-methoxybenzylamide derivatives bear-
ing para-substituted phenyl group via various 3-atom spacers
linked to the 5-carbon of thieno[2,3-d]pyrimidine are described
in Scheme 5.
Starting with commercially available ester 6b, the six thieno-
pyrimidine-2-carboxylic acid ethyl esters bearing various substitu-
ents at the 5-position 25a–f have been prepared by a modified
Williamson procedure. Radical bromination of the C5-methyl of
The bromomethyl intermediate 24 was converted by means of
sodium cyanide to the cyanomethyl compound 30, which was
subjected to aminolysis of the ethyl ester to give 31a. Cyano
O
O
Me
Me
O HN
HN
H2N
O
a
MeO
N
H
N
N
MeO
Cl
8
9
10
Scheme 2. Synthesis of reverse amide derivative 10. Reagents and conditions: (a) TEA, THF, DMA, 90 °C, 71%.
O
O
N
O
Me
Me
Me
a
b
HO
H2N
HN
HN
H
N
Cl
MeO
N
11
12
13
Scheme 3. Synthesis of amine derivative 13. Reagents and conditions: (a) chloroacetonitrile, MeONa, MeOH, rt to 80 °C, 67%; (b) 3-methoxybenzylamine, K2CO3, THF, 40 °C,
41%.
O
O
N
O
N
b
Me
a
Me
Boc
d
Me
H2N
H2N
HN
N
O
N
Boc
Me
e
N
15
16
14
MeO
OH
c
OH
OH
20
H
MeO
MeO
MeO
O
O
17
19
18
O
OMTM
N
O
Me
Me
Me
f
g
h
HN
N
HN
MeO
N
MeO
MeO
N
OH
O
O
21
22
23
Scheme 4. Synthesis of ketone derivative 23. Reagents and conditions: (a) CH(OMe)3, concd HCl, 0 °C to rt, 81%; (b) Boc2O, NaH, THF, 0 °C to rt, 91%; (c) (1) oxalyl chloride,
DMF, THF, rt, (2) NaBH4, THF, reflux, 92%; (d) TPAP, NMO, MS4A, CH2Cl2, rt, 63%; (e) (1) 16, LDA, THF, ꢀ78 °C, (2) 19, ꢀ78 °C to rt, 44%; (f) TFA, CH2Cl2, rt, 45%; (g) DMSO, oxalyl
chloride, TEA, ꢀ78 °C to rt, 41%; (h) TFA, H2O, CH2Cl2, rt, 64%.

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5491
R
R
O
O
N
O
N
O
N
Br
X
X
Me
a
b
c
HN
HN
HN
HN
H
N
EtO
EtO
EtO
S
S
S
S
N
X
MeO
X
R
O
O
O
O
R
6b
24
O
O
O
O
O
26a
26b
26c
26d
26e
26f
H
O
O
O
O
H
25a
25b
25c
25d
25e
25f
COOEt
COOEt
F
CN
g
COOH
F
h
d
CN
H
NMe H
NMe
S
S
H
26g
H
R
O
R
O
O
R
O
NH
O
N
NH
f
c
HN
HN
HN
EtO
H
N
S
N
EtO
S
S
MeO
N
O
a
O
O
R = H
29a
R = N3
NH2 HCl
27a
27b
28a
28b
R = H
COOMe
e
COOMe 29b
g
COOH
29c
R
HN
O
N
O
N
R
CN
i
O
N
c
O
HN
HN
H
N
HN
H
EtO
S
S
MeO
R
N
S
g
MeO
O
O
O
R = CN
COOH 31b
31a
30
g
R = H
COOEt 32b
COOH
32a
32c
R
O
O
O
O
N
NH
O
N
NH
R
j
c
HN
HN
HN
H
EtO
EtO
N
S
S
S
N
MeO
O
O
R = H
O
R = H
35a
35b
35c
R = CHBr2 33a
CHO 33b
34a
COOEt 34b
k
l
COOEt
COOH
g
COOH 33c
c
COOEt
COOH
O
N
O
O
N
R
HN
S
HN
f
O
O
HN
HN
HN
H
H
N
H
N
N
+
S
S
MeO
MeO
N
MeO
O
O
O
37
38
36a
36b
R = COOH
m
NH2
Scheme 5. Synthesis of thieno[2,3-d]pyrimidines 26a–g, 29a–c, 32a–c, 35a–c, 37, and 38. Reagents and conditions: (a) 1.2 equiv NBS, AIBN, chlorobenzene, 80 °C, 54% [for
24]; 2.3 equiv NBS, AIBN, carbon tetrachloride, 80 °C, 85% [for 33a]; (b) (1) benzyl alcohol, NaH, THF, (2) oxalyl chloride, DMF, THF, (3) EtOH, THF, 16% [for 25a], (1) 4-
hydroxymethylbenzoic acid ethyl ester, NaH, THF, (2) oxalyl chloride, DMF, THF, (3) EtOH, pyridine, THF, 75% [for 25b], (1) (4-fluorophenyl)methanol, THF, NaH, (2) EtOH, EDC
HCl, DMAP, THF, 8% [for 25c], (1) 4-(hydroxymethyl)benzonitrile, THF, NaH, (2) EtOH, EDC HCl, DMAP, THF, 52% [for 25d]; N-methyl-1-phenylmethanamine, Et3N, THF, 52%
[for 25e]; phenylmethanethiol, Et3N, DMA, 30% [for 25f]; (c) 1-[3-(methyloxy)phenyl]methanamine, EtOH, 80–90 °C, 60% [for 26a]; 90% [for 26b]; 81% [for 26d]; 79% [for
26e]; 58% [for 26f]; 67% [for 26d]; 82% [for 29a]; 89% [for 29b]; 76% [for 31a]; 85% [for 35a]; 91% [for 35b]; 1-[3-(methyloxy)phenyl]methanamine, ethyldiisopropylamine,
EtOH, 90 °C, 67% [for 36a]; (d) NaN3, DMF, 76%; (e) H2, HCl, Pd/C, EtOH–THF, 94%; (f) benzoyl chloride, triethylamine, THF, 72% [for 28a]; 4-chlorocarbonylbenzoicacidmethyl
ester (prepared from monomethyl terephthalate, oxalyl chloride, DMF, THF), Et3N, THF, 82% [for 28b], (1) ethyl 4-(2-chloro-2-oxoethyl)benzoate (prepared from 2-[4-
(ethoxycarbonyl)phenyl]acetic acid, oxalyl chloride, DMF, THF), pyridine, THF, (2) aqueous NaOH solution, EtOH/THF, 6% [for 37]; 17% [for 38]; (g) aqueous NaOH solution,
EtOH or THF, MeOH, 80–100 °C, quant. [for 26c]; 98% [for 29c]; 62% [for 31b]; 86% [for 32c]; 62% [for 31b]; 86% [for 32c]; 94% [for 35c]; (h) NaCN, DMF/H2O, 0 °C to rt, 69%; (i)
(1) oxalyl chloride, THF, (2) aniline, pyridine, THF, 64% [for 32a], (1) oxalyl chloride, THF, (2) ethyl 4-aminobenzoate, pyridine, THF, 76% [for 32b]; (j) (1) oxalyl chloride, DMF,
THF (2) benzylamine, THF, 70% [for 34a], (1) oxalyl chloride, DMF, THF (2) ethyl 4-(aminomethyl)benzoate hydrochloride, THF, 80% [for 34b]; (k) 1 N HCl, THF, MeOH, 60 °C,
69%; (l) NaClO2, MeCN, H2O, 57%; (m) (1) DPPA, Et3N, toluene, 100 °C, (2) tert-butyl alcohol, 100 °C, (3) HCl, AcOEt, (4) aqueous NaOH, THF, MeOH, 80 °C, 26%.
compound 31a was hydrolyzed with aqueous sodium hydroxide to
afford carboxylic acid 31b. The carboxylic acid was converted to
the acid chloride, which was then condensed with anilines to give
32a,b. Ester 32b was converted by the treatment of sodium
hydroxide to give compound 32c.
Bromination of 6b with 2.3 equiv of NBS in the presence of AIBN
afforded gem-dibrominated product 33a. Hydrolysis of the gem-
dibromo compound gave the aldehyde 33b. Oxidation of the
resulting aldehyde intermediate to the corresponding carboxylic
acid 33c was achieved using sodium chlorite in aqueous acetoni-
trile. Conversion of the carboxylic acid moiety to acid chloride
followed by coupling with benzylamines and subsequent
aminolysis by 3-methoxybenzylamine provided 35a,b. The ethyl
ester of 35b was hydrolyzed to give the corresponding carboxylic
acid 35c in 94% yield.
The synthesis of 5-substituted 4-oxo-3H,4H-thieno[2,3-
d]pyrimidine derivatives 37 and 38 was performed according to
Scheme 5. Reaction of the ester 33c with 3-methoxybenzylamine
proceeded at the 2-position to provide the amide 36a. Heating a
solution of 36a with diphenyl phosphoryl azide (DPPA) and trieth-
ylamine in toluene generated the isocyanate in situ, which could
be trapped with tert-butanol to afford tert-butyl carbamate

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H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
accompanied by a side product, as identified by LCMS spectra17 of
the crude reaction mixture. This is interpreted as a result of intra-
molecular cyclization via O-4 attack on the isocyanate. The mixture
was treated with HCl–EtOAc to remove the Boc protecting group
and subsequently treated with aqueous sodium hydroxide to
hydrolyze the cyclized byproduct17. Neutralization the reaction
mixture afforded the free primary amine 36b. Acylation of amine
36b with ethyl 4-(2-chloro-2-oxoethyl)benzoate readily afforded
the 5-substituted thienopyrimidine derivatives that was subjected
to alkaline hydrolysis to provide a separable mixture of unreacted
37 and hydrolyzed product 38.
As shown in Scheme 6, bromination followed by nitration with
potassium nitrate in sulfuric acid exclusively provided ethyl 6-
bromo-5-nitro-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate
39b. Hydrogenation in the presence of palladium catalyst produced
a simultaneous reduction of the nitro group and hydrogenolysis of
the bromo group to give 39c. Acylation of 39c with phenylacetyl
chloride, followed by aminolysis with 3-methoxybenzylamine
provided the diamide compound 41.
carboxylic acid 26c with methylamine hydrochloride using
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDC) as a coupling agent in the presence of DMAP. The acid
chloride prepared from 26c was converted to the corresponding
primary alcohol 42c by reduction with sodium borohydride in
DMA. The alcohol 42c was reacted with methanesulfonyl
chloride in THF and the resulting mesylate was then converted
to the corresponding methyl ether 42d by displacement with
sodium methoxide.
Disodium salt of 26c was prepared as shown in Scheme 8. The
precipitate, obtained by addition of 2.0 equivalent of aqueous
sodium hydrogen carbonate to a solution of 26c in THF–EtOH,
was separated from the solution by filtration. Reprecipitation
was carried out in EtOH at 90 °C to obtain the disodium salt 43.
3. Results and discussion
The high-throughput screening first identified compound 44 as
a lead compound of MMP-13 inhibitor. The compound exhibited
potent inhibitory activity with an IC50 value of 12 nM and selective
properties by inhibiting MMP-13 showing weak inhibitory activity
towards MMP-1, 3, 7, 9, 14, and TACE (see Table 4).
The carboxylic acid 26c was converted to carboxamide 42a by
reaction with oxalyl chloride followed by ammonia (Scheme 7).
N-Methylamide 42b was prepared by condensation of the
O
O
N
O
N
O
N
R1
HN
S
HN
S
O
O
e
a
d
HN
HN
HN
HN
H
N
R2
EtO
EtO
EtO
S
S
N
MeO
O
O
O
O
40
41
39a
39b
39c
R1, R2 = H, Br
NO2, Br
NH2, H
6a
b
c
Scheme 6. Synthesis of 5-(2-phenylacetamido)thieno[2,3-d]pyrimidine 41. Reagents and conditions: (a) Br2, AcOH, 93%; (b) concd H2SO4, NaNO3, 0 °C, 80%; (c) H2, Pd/C,
EtOH/THF, 36%; (d) phenylacetyl chloride, Et3N, THF, 77%; (e) 3-methoxybenzylamine, EtOH, 90 °C, 93%.
O
R
OH
O
N
O
O
N
O
a
HN
HN
H
N
H
N
S
S
MeO
MeO
O
O
R = CONH2
42a
26c
CONHMe 42b
CH2OH
CH2OMe
42c
42d
b
Scheme 7. Synthesis of compounds 42a, 42b, 42c, and 42d. Reagents and conditions: (a) (1) oxalyl chloride, DMF, THF, (2) 28% aqueous ammonia solution, THF, 87% [for 42a];
methylamine hydrochloride, EDC, DMAP, THF, 67% [for 42b], (1) oxalyl chloride, DMF, THF, (2) NaBH4, DMA, 65% [for 42c]; (b) (1) MsCl, Et3N, THF, (2) sodium methoxide,
MeOH/THF, 80 °C, 66% [for 42d].
O
O
OH
O-Na+
O
N
O
O
N
O
Na+
-N
HN
a
H
N
H
N
S
S
MeO
MeO
O
O
43
26c
Scheme 8. Synthesis of disodium salt 43. Reagents and conditions: (a) (1) THF, EtOH, aqueous NaHCO3, (2) EtOH, 90 °C, 91%.

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5493
In an effort to optimize the left-hand portion of 44 in an effi-
cient fashion, a small library of its analogues was synthesized by
an automated high throughput approach. The biological activity
of the first set of substituted benzyl derivatives on the terminal
aryl ring in the left hand side portion of the molecule demonstrated
that the C-3, 4 positions were promising for introduction of small
substituents. Among them, 3-methoxy group was found to be
one of the best substituent of the terminal aryl group. We therefore
carried out further optimization of compound bearing 3-methoxy
phenyl group to explore the SAR.
To confirm the pivotal importance of the linkers between the
quinazoline and 3-methoxybenzene rings, compounds bearing
modified linkers were initially investigated. Table 1 shows the
SAR of the three-atom linker analogues. Replacement of the amide
carbonyl with a methylene group (13) gave a large decrease in
activity, and the same was observed in the reverse amide linker
analogue 10. In contrast, replacement of the amide nitrogen with
a methylene group retained moderate activity (23), demonstrating
the importance of the carbonyl group which is assumed to interact
with Tyr244 and Thr245 from X-ray crystallographic analysis
(Fig. 4). Also, reduction of the carbonyl group of 23 to a secondary
hydroxyl group (21) resulted in a drastic drop in potency. These
results revealed that the original amide linker (44, 45, and 46) is
crucial for potent enzyme inhibition. The addition of a methyl
group at the 5- or 6-position of the quinazoline (45 and 46) was
found to be acceptable, since both amide analogues retained good
inhibitory activity.
Figure 4. X-ray co-crystal structure of prototype compound 7a in complex with
MMP-13 catalytic domain (PDB code: 3WV3) in a schematic representation. The
ligand interacts in a similar fashion as 44 with MMP-13 residues of the specific S10
pocket (dotted curved line). Molecular modeling suggested that attachment of the
P100 substituent via
a linker to the thiophene 5-position of the 4-oxo-3H,4H-
thieno[2,3-d]pyrimidine scaffold would afford a target molecule. An additional
possible interaction of P100 with Lys140 residue at the bottom of the S100 pocket is
present in the protein.
7b is more than 20-fold more potent than the corresponding 5-
methylquinazoline analogue 45 (see Table 1). Replacement of the
thiophene ring with the other heteroaromatic rings such as pyrrol-
o-, furo-, pyrazolo-, isoxazolo-, and pyridopyrimidines generally
decreased activity. Specifically, even replacing the thiophene with
a furan caused a more than 10-fold loss of potency (7d vs 7e), and
replacement of the thiophene with a six-membered heteroaryl
group such as pyridine led to a more than 100-fold reduction in
potency (7a vs 7l). Because of its activity, 4-oxo-3H,4H-thie-
no[2,3-d]pyrimidine ring system was chosen for systematic
exploration.
As mentioned earlier, the X-ray co-crystallographic analysis
revealed that compound 44 does not interact with the catalytic
zinc atom of MMP-13 and binds deeply in the S10 pocket. The anal-
ysis showed an important finding that an MMP-13-specific hydro-
phobic pocket, which is referred to as the S100 pocket, exists
adjacent to the S10 pocket (see Figs. 2 and 4). As shown in Figure 4,
the thiophene congener 7a proved to have a similar binding mode,
suggesting that the 5-position of the 4-oxo-3H,4H-thieno[2,
3-d]pyrimidine scaffold can be suitable for incorporation of a
substituent binding to the S100 pocket (P100 substituent). The co-
crystallographic analysis indicates that the S100 pocket is mainly
hydrophobic and provides sufficient binding space accommodating
a substituted phenyl group via a linker length of three atoms. In
addition, the structural studies have shown that an additional
hydrogen bond or ionic interaction can be exploited through inter-
action of P100 substituent with Lys140 residue at the bottom of the
S100 pocket present in the protein. Additionally, the phenyl group of
the 3-methoxybenzyl amide of compound 7a could participate in a
nearly co-planar stacking interaction with His222.
Replacement of the benzene ring of the core quinazoline with a
thiophene ring, a known bioisostere, was studied (Table 2). Methyl-
substituted compound 7b–c exhibited approximately 4- to 10-fold
improved activity compared to the original inhibitor 44 (see
Table 1). It should be noted that the 5-methylthienopyrimidine
Table 1
Effect of linker variation within 3,4-dihydroquinazolin-4-one series
O
R1
5
R2
6
4
HN
N
MeO
3
linker
Compound
44
Linker
R1
H
R2
H
IC50 (nM)a
12 1.5
H
N
O
O
H
N
45
Me
H
29 3.4
H
N
46
H
H
Me
Me
26 3.1
>10,000
O
21b
OH
H
N
H
H
Me
Me
>10,000
220 25
To improve the overall profile such as MMP-13 inhibitory activ-
ity, aqueous solubility, and pharmacokinetic property, we utilized
a strategy of incorporation of hydrogen bond donor/acceptor
groups at the 4-position of the P100 phenyl group. Molecular mod-
eling suggested that attachment of the phenyl group via a three-
atom linker to the 5-position of the thienopyrimidine core would
afford a target molecule which could bind to MMP-13 with little
conformational strain.
13
23
O
O
H
Me
>10,000
10
N
H
a
IC50 (nM) represents the average IC50 value in nanomolars for the inhibition
We therefore set out to explore favorable P100 substituents. The
effects of the substituent of the terminal phenyl group at the 5-
position and the three-atom linker between the phenyl group
assay of the compound using the human MMP-13 catalytic domain enzyme. Data
are expressed as mean IC50 SD, n = 3.
b
Tested as a racemic mixture.

5494
H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
Table 2
In vitro data for ring A substituted N-[(3-methoxyphenyl)methyl]-6-oxo-1,6-dihydropyrimidine-2-carboxamide derivatives
O
HN
H
N
A
MeO
N
O
Compound
7b
A
IC50 (nM)a
1.1 0.043
Compound
7k
A
IC50 (nM)a
29 5.2
Me
S
S
Me
Me
Me
Me
N
7d
1.2 0.069
7g
65 10
S
N
Me
7c
7h
3.1 0.29
12 1.6
7m
7f
200 38
760 95
N
S
Me
Me
S
N
O
Me
Me
Me
7j
14 2.2
7j
7l
1,100 220
3,200 300
N
Me
Me
N
H
7e
7a
16 2.1
24 3.7
N
O
S
a
IC50 (nM) represents the average IC50 value in nanomolars for the inhibition assay of the compound using the human MMP-13 catalytic domain enzyme. Data are
expressed as mean IC50 SD, n = 3.
and thieno[2,3-d]pyrimidine core were investigated (Table 3). Con-
sidering synthetic feasibility, a benzyloxy group was initially incor-
porated into the 5-methyl group of 7b (see Table 2), resulting in an
about 6-fold increase in potency (26a). Indeed, the benzyloxym-
ethyl analogue 26a exhibited subnanomolar potency (IC50 = 0.19 -
nM). The para position of the terminal phenyl group of 26a was
then substituted with hydrogen bonding groups. Incorporation of
On the basis of the activities and pharmacokinetic properties of
the carboxylic acid derivatives 26c, 32c, 29c, 35c, and 38, the 4-
carboxybenzyloxymethyl analogue 26c was selected for further
investigation.
Compound 26c that has the most favorable profile was subse-
quently evaluated for selectivity against MMP-1, -2, -3, -7, -8, -9,
-10, -14, and tumor necrosis factor-alpha converting enzyme
(TACE). As shown in Table 4, the hydroxamic acid-based MMP
a
carboxylic acid moiety to the para position dramatically
enhanced the activity, affording the extremely potent inhibitor
26c with an IC50 value of 6.9 pM. Other substitution such as fluoro,
hydroxymethyl, methoxymethyl, cyano, carboxamide, and N-
methylcarboxamide gave less potent inhibition than the carboxy
substituent 26c although the potencies were enhanced compared
to the corresponding non-substituted compounds.
Modification of the linker with or without a carboxylic acid at
the terminal phenyl group was next examined (Table 3). When
compared with compounds possessing non-substituted phenyl
group, replacement of the ether linker with a thioether or tertiary
amine linker significantly decreased potency. In contrast, replace-
ment of the ether linker with amide-containing linkers increased
the potency by more than 3-fold. The compounds with amide link-
ers (32a, 29a, 35a, and 41) displayed two-digit pM order potency,
indicating that there is little preference of the positions of the car-
bonyl and NH groups. Incorporation of a carboxylic acid moiety
into these compounds gave an additional boost in activity, afford-
ing picomolar inhibitors (32c, 29c, and 35c).
inhibitor 47 (RS-130,83018
) reported by Roche Biosciences
research group did not exhibit sufficient selectivity. For instance,
its selectivity for MMP-13 over the other metalloenzymes tested
proved to be less than 3-fold. On the other hand, the thieno[2,3-
d]pyrimidine analogue 26c showed improved selectivity over all
other related metalloenzymes tested by approximately 100-fold
relative to 44. As a consequence, 26c showed more than 2600-fold
selectivity over the other related metalloenzymes. Furthermore,
26c was ca. 1700-fold more potent than the lead compound 44.
The pharmacokinetics of 26c have been studied in guinea pigs,
dogs, and monkeys. As shown in Table 5, oral administration of the
disodium salt formulations of 26c (43) to guinea pigs resulted in
significant increases in AUC (8357 ng h/mL) and Cmax (1445 ng/
mL) compared with those of the free acid 26c (AUC = 6478 ng h/
mL and Cmax = 911 ng/mL). The compound was well absorbed in
all species at the oral dose of 10–20 mg/kg. The oral AUC values
of 43 in dogs and monkeys were found to be 27136 and
82360 ngꢁh/mL, respectively.

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5495
Table 3
Inhibition of MMP-13 by derivatives with different linkers and their pharmacokinetic parameters in male rats
R
linker
O
5
HN
P1"
H
N
S
MeO
N
O
Linker
IC50 (nM)a
Cpd
Cpd
Linker
R
H
F
IC50 (nM)a
AUCb,c Vd,ssb,d CL b,e
R
H
AUC b,c Vd,ssb,dCL b,e
311 867 1037
201 1417
H
N
0.028 0.0033
0.005 0.0006
0. 19 0.021
0. 13 0.016
82
ND f
< 1
< 1
42
1061 2179
ND f ND f
1285 5216
960 1946
813 961
874 2472
780 2722
878 693
32a
32c
26a
26d
42c
42d
26e
42a
42b
26c
COOH
1362
O
O
CH2OH 0.032 0.0031
CH2OMe 0.080 0.0063
0.018 0.0038
29a
29c
H
2289
22
314
214
N
H
O
0.0022 0.00073
CN
0.018 0.0032
COOH
871 1218
CONH2 0.015 0.0013
CONHMe 0.029 0.0012
6
O
0.026 0.0022
47
35a
35c
H
1685
238
153
865
N
H
0.0077 0.00095
COOH
0.0069 0.00078 366
COOH
304 1072
H
N
0.053 0.0075
0.021 0.0018
H
H
0.66 0.075
39 8.3
12
1175 1624
767 778
41
38
H
2705
1044
315
773
120
501
26d
26f
S
411
COOH
N
Me
O
a IC50 (nM) represents the average IC50 value in nanomolars for the inhibition assay of the compound using the human MMP-13 catalytic domain enzyme. Data are expressed
as mean IC50 SE, n = 3.
b
Data are given as mean values of triplicates.
Rat AUC (ng h/mL) following a single 1 mg/kg oral cassette gavage dose in rats.
Rat volume of distribution at steady state (Vd,ss) (mL/kg) at an intravenous dose of 0.1 mg/kg.
Rat total body clearance (mL/h/kg) at an intravenous dose of 0.1 mg/kg.
c
d
e
f
Not determined.
To assess the in vitro efficacy of 26c in tissues, bovine nasal sep-
tum cartilage explants method was used.19,20 The cartilage
explants were treated with a combination of two cytokines, IL-1b
and oncostatin M, to induce production of endogenous MMPs from
chondrocytes, resulting in the degradation and the release of type
II collagen fragments into the tissue culture fluid. Compound 26c
significantly inhibited the breakdown of collagen (87.4% inhibition
of P100 with Lys140 residue at the bottom of the S100 pocket led to
the discovery of the highly potent and selective MMP13 inhibitor
26c with an IC50 value of 6.9 pM and more than 2600-fold selectiv-
ity over the other related metalloenzymes. Furthermore, the inhib-
itor was shown to be active in bovine nasal cartilage explants assay
and possesses favorable ADME and safety profiles.
at 0.1 lM) in IL-1b and oncostatin M stimulated cartilage (as mea-
sured by hydroxyproline release), which is comparable to broad
spectrum MMP inhibitor 47 (RS-130,830) (76.3% inhibition at
5. Experimental section
5.1. Chemistry
0.1
l
M) (Table 6).
Furthermore, compound 43 was evaluated for 2-week repeated
dose oral toxicity study in rats and a no observed adverse effect
level of 60 mg/kg/day was established.
Melting points were determined in open capillary tubes on a
Büchi melting point apparatus B545 and are uncorrected. 1H
NMR spectra were recorded on a Bruker DPX-300 (300 MHz) spec-
trometer and are reported in parts per million (d) relative to tetra-
methylsilane (TMS: d 0.00 ppm). Data are reported as follows:
chemical shift, integration, multiplicity (s = singlet, d = doublet,
t = triplet, q = quartet, m = multiplet, dd = doublet of doublet,
dt = doublet of triplet, tt = triplet of triplet, bs = broad singlet),
and coupling constants (J, Hz). Unless otherwise specified, all sol-
vents and reagents were obtained from commercial suppliers and
used without further purification. Column chromatography was
performed using Merck silica gel 60 (70–230 mesh). Thin-layer
chromatography (TLC) was performed on Merck silica gel plates
60F254. LC-MS analysis was performed on a Shiseido CAPCELL
4. Conclusion
On the basis of the X-ray co-crystal structure of MMP-13 in
complex with 44, we designed a new structural class of MMP-13
selective inhibitors employing a thieno[2,3-d]pyrimidine as the
central scaffold. Our strategy to identify highly selective and potent
inhibitors is based on the assumption that additional interaction
with an unexplored selectivity binding site (S100) of MMP-13 could
improve the potency and selectivity.
Using the X-ray structural analyses and computer modeling
studies, we optimized the inhibitory activity and selectivity against
MMP-13 of these compounds. The thienopyrimidine-based inhibi-
tors that could bind to the unprimed side of MMP-13 form a tight
b-sheet type interaction between the 3, 4 position of the pyrimi-
dine and Thr245, 247 residues from the enzyme’s backbone span-
ning the S10 and S100 pockets (see Fig. 4). Attachment of the P100
substituent via a linker aimed to form hydrogen bond interactions
PACK C-18 UG120 S-3 column (1.5 mm
U
ꢂ 35 mm) in a Waters
Alliance 2795 or an Agilent 1100 LC system equipped with a
Waters 2487 absorbance detector and a Micromass ZQ2000 mass
spectrometer. Analytes were eluted using a linear gradient of water
(0.05% TFA)/acetonitrile (0.04% TFA) from 90:10 to 0:100 over
4 min at a flow rate of 0.5 mL/min. UV detection was at 220 nm.
Preparative HPLC was performed on a Shiseido CAPCELL PACK

5496
H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
Table 4
IC50 values for inhibition of MMPs and TACE
O
OH
O
O
O
O
O
S
HO
HN
HN
N
H
H
N
H
N
Cl
O
S
MeO
N
MeO
N
O
O
O
O
47
44
26c
RS-130,830
IC50 (nM)a
MMP-1
Compound
MMP-13
MMP-2
MMP-3
MMP-7
210 15
>10,000
>10,000
47
0.010 0.0016
12 1.5
34 16
>10,000
>10,000
0.029 0.0036
300 9.0
0.30 0.051
>10,000
44
26c
0.0069 0.00078
18 3.6
600 220
IC50 (nM)a
Compound
MMP-8
MMP-9
MMP-10
MMP-14
1.1 0.16
>10,000
>10,000
TACE
47
0.097 0.024
1,100 150
780 290
0.11 0.0013
>10,000
0.54 0.076
3,400 1,000
160 35
14 1.3
>10,000
>10,000
44
26c
>10,000
a Values are shown as the mean IC50 SD of triplicates.
Table 5
Pharmacokinetics of 26c and its disodium salt 43 in guinea pig, dog, and monkey
Compound
Cmax ng/mL(po)
Tmax h (po)
AUC ngꢁh/mL (po)
Vd,sse mL/kg (iv)
CLf mL/h/kg (iv)
% F
Guinea piga
Guinea pigb
Dogc
26c
43
911
1445
2438
6607
0.83
0.67
2.0
6478
8357
923
—
431
—
28
—
29
—
43
27136
82360
395
—
111
—
Monkeyd
43
3.0
a
b
c
d
e
f
iv 1 mg/kg, p.o. 10 mg/kg male (n = 3).
p.o. 10.9 mg/kg male (n = 3).
iv 1.09 mg/kg, p.o. 10.9 mg/kg male (n = 3).
p.o. 20 mg/kg male (n = 2) and female (n = 2).
Volume of distribution at steady state.
Total body clearance.
Table 6
C-18 UG120 S-5 column (20 mm
U
ꢂ 50 mm), eluting at a flow rate
Inhibition activity in bovine nasal cartilage assay
of 25 mL/min with a linear gradient of water (0.1% TFA)/acetoni-
trile (0.1% TFA) from 90:10 to 0:100 over 10 min. UV detection
was at 220 nm. Elemental analyses were performed by Takeda
Analytical Research Laboratories, Ltd.
Compound
Concentration (
lM)
Inhibitiona (%)
0.01
0.1
1
ꢀ4.7 10.83
76.3 18.54*
102.3 0.61*
47
(RS-130,830)
0.01
0.1
1
22.6 24.18
87.4 7.64*
100.3 1.09*
5.1.1. Ethyl 4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate
(6a)
A mixture of compound 2a (2.00 g, 8.26 mmol) and p-toluene-
sulfonic acid monohydrate (514 mg, 2.70 mmol) in xylene
(50 mL) was heated under reflux for 11 h. The reaction mixture
26c
a
Data are represented as means SEM (n = 6).
Denotes P <0.025 by one-tailed Williams’ test.
*

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5497
was diluted with ethyl acetate, washed with water and brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography
(67% ethyl acetate/hexane) to give 6a as a pale yellow powder
(531 mg, 29%). 1H NMR (200 MHz, DMSO-d6) d 1.35 (3H, t,
J = 7.2 Hz), 4.38 (2H, q, J = 7.2 Hz), 7.49 (1H, d, J = 5.6 Hz), 7.81
(1H, d, J = 5.6 Hz).
5.1.7. Ethyl 4-oxo-3H,4H-thieno[3,2-d]pyrimidine-2-carboxylate
(6k)
A mixture of compound 5a (2.50 g, 11.1 mmol) and ammo-
nium acetate (941 mg, 12.2 mmol) in ethanol (30 mL) was
heated under reflux for 1 h. The reaction mixture was concen-
trated under reduced pressure. The residue was diluted with
ethyl acetate, washed with water and brine, dried over anhy-
drous Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
(67–80% ethyl acetate/hexane) to give 6k as a brown powder
(478 mg, 19%). 1H NMR (200 MHz, DMSO-d6) d 1.35 (3H, t,
J = 7.2 Hz), 4.38 (2H, q, J = 7.2 Hz), 7.56 (1H, d, J = 5.6 Hz), 8.28
(1H, d, J = 5.6 Hz).
5.1.2. Ethyl 5,6-dimethyl-4-oxo-3H,4H-furo[2,3-d]pyrimidine-2-
carboxylate (6e)
A mixture of compound 2b (280 mg, 1.10 mmol) and p-toluene-
sulfonic acid monohydrate (105 mg, 0.550 mmol) in toluene
(20 mL) was heated under reflux for 4 h. The reaction mixture
was partitioned between ethyl acetate and water. The organic layer
was washed with brine, dried over anhydrous Na2SO4, and concen-
trated under reduced pressure. The residue was crystallized from
ethanol to give 6e as a pale yellow powder (150 mg, 0.635 mmol,
58%). 1H NMR (300 MHz, DMSO-d6) d 1.33 (3H, t, J = 7.2 Hz), 2.19
(3H, s), 2.34 (3H, s), 4.35 (2H, q, J = 7.2 Hz), 12.8 (1H, bs).
5.1.8. Ethyl 4-oxo-3H,4H-pyrido[2,3-d]pyrimidine-2-carboxylate
(6l)
A mixture of compound 5b (2.20 g, 9.99 mmol), ammonium
acetate (770 mg, 9.99 mmol) and acetic acid (240 mg, 4.00 mmol)
in ethanol (30 mL) was heated under reflux for 1 h. After the reac-
tion mixture was cooled to room temperature, the precipitated
solid was collected by filtration and washed with ethanol to give
6l as a pale yellow powder (1.11 g, 51%). 1H NMR (200 MHz,
DMSO-d6) d 1.38 (3H, t, J = 7.0 Hz), 4.41 (2H, q, J = 7.0 Hz), 7.65
(1H, dd, J = 7.8, 4.4 Hz), 8.56 (1H, dd, J = 7.8, 1.8 Hz), 9.04 (1H, dd,
J = 4.4, 1.8 Hz).
5.1.3. Ethyl 1,3-dimethyl-4-oxo-1H,4H,5H-pyrazolo[3,4-d]-
pyrimidine-6-carboxylate (6g)
To a solution of 5-amino-1,3-dimethyl-1H-pyrazole-4-carbox-
amide21 (1d) (3.00 g, 19.5 mmol, synthesized by the method of
Cheng et al.) and diethyl oxalate (11.4 g, 77.8 mmol) in ethanol
(500 mL) was added sodium ethylate (33.1 g, 97.3 mmol) at 0 °C,
and the mixture was heated under reflux for 18 h. The reaction
mixture was allowed to cool to room temperature and poured into
1 M hydrochloric acid (100 mL). The mixture was concentrated
under reduced pressure and the residue was suspended with
water. The precipitated solid was collected by filtration and
washed with water and ethanol to give 6g as a pale yellow powder
5.1.9. Ethyl 4-oxo-3H,4H-pyrido[3,4-d]pyrimidine-2-carboxylate
(6m)
Step 1. To a suspension of commercially available 3-aminopyri-
dine-4-carboxylic acid (4.84 g, 35.1 mmol) in pyridine (60 mL) was
added dropwise ethyl chloroglyoxylate (9.58 g, 70.2 mmol) at 0 °C,
and the mixture was stirred at room temperature for 15 h. The
reaction mixture was concentrated under reduced pressure and
the residue was suspended with water. The resulting precipitate
was collected by filtration to give 3-(2-ethoxy-2-oxoacetami-
do)pyridine-4-carboxylic acid as a pale yellow powder (3.74 g,
45%). 1H NMR (200 MHz, DMSO-d6) d 1.33 (3H, t, J = 7.0 Hz), 4.33
(2H, q, J = 7.0 Hz), 7.88 (1H, d, J = 5.0 Hz), 8.53 (1H, d, J = 5.0 Hz),
9.71 (1H, s), 12.10 (1H, s).
Step 2. To a solution of 3-(2-ethoxy-2-oxoacetamido)pyridine-
4-carboxylic acid (100 mg, 0.420 mmol) and DMF (0.030 mL) in
THF (3 mL) was added dropwise oxalyl chloride (0.040 mL,
0.460 mmol) at 0 °C, and the mixture was stirred at room
temperature for 2 h. The reaction mixture was cooled to 0 °C
followed by the addition of 2 M ammonia in ethanol (0.693 mL,
1.39 mmol). The mixture was stirred at 0 °C for 1 h and then
partitioned between ethyl acetate and water. The organic layer
was washed with brine and dried over anhydrous Na2SO4. The
filtrate was concentrated under reduced pressure to give ethyl
[(4-carbamoylpyridin-3-yl)carbamoyl]formate as a white powder
(76 mg, 0.320 mmol, 76%). 1H NMR (200 MHz, DMSO-d6) d 1.32
(3H, t, J = 7.0 Hz), 4.31 (2H, q, J = 7.4 Hz), 7.78 (1H, d,
J = 5.2 Hz), 8.17 (1H, bs), 8.50 (1H, d, J = 5.2 Hz), 8.60 (1H, bs),
9.68 (1H, s).
(2.52 g, 55%). 1H NMR (300 MHz, DMSO-d6)
d 1.35 (3H, t,
J = 7.2 Hz), 2.44 (3H, s), 3.86 (3H, s), 4.38 (2H, q, J = 7.2 Hz), 12.4
(1H, bs).
5.1.4. Ethyl 5-methyl-4-oxo-3H,4H-thieno[3,4-d]pyrimidine-2-
carboxylate (6h)
A mixture of methyl 4-amino-2-methylthiophene-3-carboxyl-
ate hydrochloride22 (3a) (1.90 g, 9.15 mmol, synthesized by the
method of Barker et al.), ethyl cyanoformate (1.36 g, 13.7 mmol)
and 1 M hydrochloric acid in acetic acid (40 mL) was stirred at
80 °C for 2 h. After removal of the solvent, the residue was sus-
pended with water. The resulting precipitate was collected and
washed with water and diethyl ether to give 6h as a brown powder
(1.48 g, 68%). 1H NMR (300 MHz, DMSO-d6)
d 1.33 (3H, d,
J = 7.2 Hz), 2.88 (3H, s), 4.34 (2H, q, J = 7.2 Hz), 7.79 (1H, s), 11.7
(1H, bs).
5.1.5. Ethyl 6-methyl-4-oxo-3H,4H,7H-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (6i)
Compound 6i was prepared from ethyl 2-amino-5-methyl-1H-
pyrrole-3-carboxylate23 (3b) (synthesized by a method of Toja
et al.) with a similar procedure as described for 6h (white powder,
50%). 1H NMR (300 MHz, DMSO-d6) d 1.33 (3H, t, J = 7.2 Hz), 2.31
(3H, s), 4.33 (2H, q, J = 7.2 Hz), 6.26 (1H, s), 12.0 (1H, bs), 12.1
(1H, bs).
Step 3. To
a suspension of ethyl [(4-carbamoylpyridin-3-
yl)carbamoyl]formate (76 mg, 0.320 mmol) in ethanol (4 mL)
was added dropwise sodium ethylate (20% ethanol solution,
120 mg, 0.350 mmol) at 0 °C, and the mixture was stirred at
room temperature for 1 h. The reaction was quenched with
1 M hydrochloric acid (0.5 mL) and the resulting mixture
was neutralized with aqueous NaHCO3 solution. The mixture
was extracted with ethyl acetate, washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pres-
sure to give 6m as a white amorphous form (22 mg, 31%).
The crude 6m was used for the next reaction without
purification.
5.1.6. Ethyl 6,7-dimethyl-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidine-2-carboxylate (6j)
Compound 6j was prepared from ethyl 2-amino-1,5-dimethyl-
1H-pyrrole-3-carboxylate23 (3c, synthesized by a method of Toja
et al.) with a similar procedure as described for 6h (white powder,
26%). 1H NMR (300 MHz, DMSO-d6) d 1.34 (3H, t, J = 6.9 Hz),
2.36 (3H, s), 3.66 (3H, s), 4.36 (2H, q, J = 7.2 Hz), 6.37 (1H, s), 12.1
(1H, bs).

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H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5.1.10. N-[(3-Methoxyphenyl)methyl]-4-oxo-3H,4H-thieno[2,3-
d]pyrimidine-2-carboxamide (7a)
5.1.16. N-[(3-Methoxyphenyl)methyl]-1,3-dimethyl-4-oxo-
1H,4H,5H-pyrazolo[3,4-d]pyrimidine-6-carboxamide (7g)
Compound 7g was prepared from compound 6g with a similar
procedure as described for 7a (white powder, 90%). mp 186–
188 °C. 1H NMR (300 MHz, DMSO-d6) d 2.43 (3H, s), 3.73 (3H, s),
3.90 (3H, s), 4.45 (2H, d, J = 6.6 Hz), 6.80–6.84 (1H, m), 6.88–6.92
(2H, m), 7.24 (1H, t, J = 8.1 Hz), 9.56 (1H, t, J = 6.6 Hz), 11.9 (1H,
bs). Anal. Calcd for C16H17N5O3: C, 58.71; H, 5.23; N, 21.39. Found:
C, 58.41; H, 5.20; N, 21.23.
A
mixture of compound 6a (240 mg, 4.00 mmol) and 3-
methoxybenzylamine (138 mg, 1.00 mmol) in DMF (3 mL) was
heated at 90 °C for 5 h. The reaction mixture was concentrated
under reduced pressure. The residue was diluted with ethyl ace-
tate, washed with brine, dried over anhydrous Na2SO4 and concen-
trated under reduced pressure. The crude product was purified by
preparative HPLC and crystallized from ethanol-diethyl ether to
give 7a as a beige powder (88.5 mg, 42%). mp 179–182 °C. 1H
NMR (200 MHz, DMSO-d6) d 3.73 (3H, s), 4.42 (2H, d, J = 6.6 Hz),
6.80–6.92 (3H, m), 7.24 (1H, t, J = 8.0 Hz), 7.43 (1H, d, J = 5.6 Hz),
7.66 (1H, d, J = 5.6 Hz), 9.56 (1H, m), 1H hidden. Anal. Calcd for
5.1.17. N-[(3-Methoxyphenyl)methyl]-5-methyl-4-oxo-3H,4H-
thieno[3,4-d]pyrimidine-2-carboxamide (7h)
Compound 7h was prepared from compound 6h with a similar
procedure as described for 7a (beige powder, 76%). mp 168–170 °C.
1H NMR (300 MHz, DMSO-d6) d 2.88 (3H, s), 3.73 (3H, s), 4.41 (2H,
d, J = 6.0 Hz), 6.81–6.91 (3H, m), 7.24 (1H, t, J = 8.1 Hz), 7.64 (1H, s),
9.43 (1H, t, J = 6.0 Hz), 11.3 (1H, bs). Anal. Calcd for C16H15N3O3S: C,
58.34; H, 4.59; N, 12.76. Found: C, 58.24; H, 4.69; N, 12.49.
C
15H13N3O3Sꢁ0.2CF3CO2Hꢁ0.6H2O: C, 53.00; H, 4.16; N, 12.04.
Found: C, 53.05; H, 4.13; N, 11.85.
5.1.11. N-[(3-Methoxyphenyl)methyl]-5-methyl-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxamide (7b)
Compound 7b was prepared from ethyl 5-methyl-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxylate24 with a similar procedure
as described for 7a (white powder, 86%). mp 148–151 °C. 1H
NMR (300 MHz, CDCl3) d 2.60 (3H, s), 3.81 (3H, s), 4.60 (2H, d,
J = 6.0 Hz), 6.83–6.92 (4H, m), 7.25–7.32 (1H, m), 7.91 (1H, br).
Anal. Calcd for C16H15N3O3Sꢁ0.60H2O: C, 56.49; H, 4.80; N, 12.35.
Found: C, 56.47; H, 4.62; N 12.44.
5.1.18. N-[(3-Methoxyphenyl)methyl]-6-methyl-4-oxo-
3H,4H,7H-pyrrolo[2,3-d]pyrimidine-2-carboxamide (7i)
Compound 7i was prepared from compound 6i with a similar
procedure as described for 7a (white powder, 86%). mp 255–
256 °C. 1H NMR (300 MHz, DMSO-d6) d 2.32 (3H, s), 3.73 (3H, s),
4.44 (2H, d, J = 6.3 Hz), 6.25 (1H, s), 6.81–6.91 (3H, m), 7.24 (1H,
t, J = 8.1 Hz), 9.14 (1H, t, J = 6.6 Hz), 11.5 (1H, bs), 11.8 (1H, bs).
Anal. Calcd for C16H16N4O3: C, 61.53; H, 5.16; N, 17.94. Found: C,
61.44; H, 5.16; N, 17.89.
5.1.12. N-[(3-Methoxyphenyl)methyl]-6-methyl-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxamide (7c)
Compound 7c was prepared from ethyl 6-methyl-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxylate25 with a similar procedure
as described for 7a (white powder, 84%). mp 187 °C. 1H NMR
(300 MHz, DMSO-d6) d 2.55 (3H, d, J = 1.1 Hz), 3.73 (3H, s), 4.41
(2H, d, J = 6.4 Hz), 6.78–6.94 (3H, m), 7.13–7.30 (2H, m), 9.63
(1H, t, J = 6.3 Hz), 12.4 (1H, s). Anal. Calcd for C16H15N3O3S: C,
58.34; H, 4.59; N, 12.76. Found: C, 58.14; H, 4.61; N, 12.73.
5.1.19. N-[(3-Methoxyphenyl)methyl]-6,7-dimethyl-4-oxo-
3H,4H,7H-pyrrolo[2,3-d]pyrimidine-2-carboxamide (7j)
Compound 7j was prepared from compound 6j with a similar
procedure as described for 7a (white powder, 31%). mp 205–
207 °C. 1H NMR (300 MHz, DMSO-d6) d 2.35 (3H, s), 3.72 (3H, s),
3.73 (3H, s), 4.46 (2H, d, J = 6.3 Hz), 6.33 (1H, s), 6.80–6.85 (1H,
m), 6.88–6.92 (2H, m), 7.25 (1H, t, J = 8.1 Hz), 9.47 (1H, t,
J = 6.3 Hz), 11.6 (1H, bs). Anal. Calcd for C17H18N4O3ꢁH2O: C,
59.45; H, 5.50; N, 16.30. Found: C, 59.29; H, 5.85; N, 16.27.
5.1.13. N-[(3-Methoxyphenyl)methyl]-5,6-dimethyl-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide (7d)
Compound 7d was prepared from ethyl 5,6-dimethyl-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate25 with
a
similar
5.1.20. N-[(3-Methoxyphenyl)methyl]-4-oxo-3H,4H-thieno[3,2-
d]pyrimidine-2-carboxamide (7k)
procedure as described for 7a (white powder, 84%). mp 194 °C.
1H NMR (300 MHz, DMSO-d6) d 2.40 (3H, s), 2.42 (3H, s), 3.73
(3H, s), 4.40 (2H, d, J = 6.4 Hz), 6.78–6.93 (3H, m), 7.18–7.29
(1H, m), 9.60 (1H, t, J = 6.3 Hz), 12.2 (1H, s). Anal. Calcd for
Compound 7k was prepared from compound 6k with a similar
procedure as described for 7a (pale yellow powder, 36%). mp 201–
202 °C. 1H NMR (200 MHz, DMSO-d6) d 3.73 (3H, s), 4.44 (2H, d,
J = 6.6 Hz), 6.80–6.93 (3H, m), 7.24 (1H, t, J = 8.0 Hz), 7.48 (1H, d,
J = 5.4 Hz), 8.27 (1H, d, J = 5.4 Hz), 9.57 (1H, t, J = 6.6 Hz), 1H hid-
den. Anal. Calcd for C15H13N3O3S: C, 57.13; H, 4.16; N, 13.33.
Found: C, 56.95; H, 4.14; N, 13.08.
C
17H17N3O3Sꢁ0.25H2O: C, 58.69; H, 5.07; N, 12.08. Found: C,
58.77; H, 4.93; N, 11.94.
5.1.14. N-[(3-Methoxyphenyl)methyl]-5,6-dimethyl-4-oxo-
3H,4H-furo[2,3-d]pyrimidine-2-carboxamide (7e)
Compound 7e was prepared from compound 6e with a similar
procedure as described for 7a (white powder, 26%). mp 178–
180 °C. 1H NMR (300 MHz, DMSO-d6) d 2.18 (3H, s), 2.32 (3H, s),
3.73 (3H, s), 4.41 (2H, d, J = 6.3 Hz), 6.80–6.84 (1H, m), 6.88–6.92
(2H, m), 7.24 (1H, t, J = 8.1 Hz), 9.55 (1H, t, J = 6.0 Hz), 12.3 (1H,
bs). Anal. Calcd for C17H17N3O4: C, 62.38; H, 5.23; N, 12.84. Found:
C, 62.16; H, 5.29; N, 12.77.
5.1.21. N-[(3-Methoxyphenyl)methyl]-4-oxo-3H,4H-pyrido[2,3-
d]pyrimidine-2-carboxamide (7l)
Compound 7l was prepared from compound 6l with a similar
procedure as described for 7a (white powder, 76%). mp 181–
183 °C. 1H NMR (200 MHz, DMSO-d6) d 3.74 (3H, s), 4.46 (2H, d,
J = 6.2 Hz), 6.80–6.95 (3H, m), 7.25 (1H, t, J = 8.0 Hz), 7.62 (1H,
dd, J = 8.0, 4.6 Hz), 8.55 (1H, dd, J = 8.2, 2.2 Hz), 9.02 (1H,
dd, J = 4.6, 2.0 Hz), 9.71 (1H, m), 1H hidden. Anal. Calcd for
5.1.15. N-[(3-Methoxyphenyl)methyl]-3-methyl-4-oxo-4H,5H-
[1,2]oxazolo[5,4-d]pyrimidine-6-carboxamide (7f)
C
16H14N4O3ꢁ0.1AcOEt: C, 61.72; H, 4.67; N, 17.56. Found: C,
61.64; H, 4.56; N, 17.52.
Compound 7f was prepared from compound 6f26 with a similar
procedure as described for 7a (pale pink powder, 71%). mp 236–
237 °C. 1H NMR (300 MHz, DMSO-d6) d 2.49 (3H, m), 3.73 (3H, s),
4.42 (2H, d, J = 6.3 Hz), 6.80–6.84 (1H, m), 6.89–6.92 (2H, m),
7.24 (1H, t, J = 8.1 Hz), 9.80 (1H, t, J = 6.3 Hz), 13.0 (1H, bs). Anal.
Calcd for C15H14N4O4: C, 57.32; H, 4.49; N, 17.83. Found: C,
57.35; H, 4.47; N, 17.81.
5.1.22. N-[(3-Methoxyphenyl)methyl]-4-oxo-3H,4H-pyrido[3,4-
d]pyrimidine-2-carboxamide (7m)
Compound 7m was prepared from compound 6m with a similar
procedure as described for 7a (white powder, 65%). mp 231–
233 °C. 1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.45 (2H, d,
J = 6.3 Hz), 6.80–6.84 (1H, m), 6.90–6.93 (2H, m), 7.24 (1H, t,

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5499
J = 8.1 Hz), 7.99 (1H, d, J = 5.1 Hz), 8.72 (1H, d, J = 5.4 Hz), 9.12 (1H,
s), 9.63 (1H, t, J = 6.3 Hz), 12.7 (1H, bs). Anal. Calcd for C16H14N4O3-
ꢁ0.2H2O: C, 61.22; H, 4.62; N, 17.85. Found: C, 61.04; H, 4.42; N,
17.88.
anhydrous Na2SO4 and concentrated under reduced pressure. The
crude product was purified by silica gel column chromatography
(25–33% ethyl acetate/hexane) to give 20 as a yellow oil (815 mg,
44%). 1H NMR (300 MHz, DMSO-d6) d 1.40 (9H, s), 2.14–2.27 (2H,
m), 2.44 (3H, s), 2.64–2.76 (2H, s), 3.71 (3H, s), 5.17–5.22 (1H,
m), 6.65–6.81 (3H, m), 7.14–7.21 (1H, m), 7.52–7.55 (1H, m),
7.61–7.65 (1H, m), 7.89 (1H, s).
5.1.23. 2-(3-Methoxyphenyl)-N-(6-methyl-4-oxo-3,4-
dihydroquinazolin-2-yl)acetamide (10)
A mixture of commercially available 2-amino-6-methyl-3,4-
dihydroquinazolin-4-one (9, 150 mg, 0.856 mmol), compound 8
(380 mg, 2.06 mmol), triethylamine (260 mg, 2.56 mmol), THF
(6 mL), and DMF (4 mL) was stirred at 90 °C for 4 h. The insoluble
materials were filtered off and the filtrate was concentrated under
reduced pressure. The residue was suspended with ethanol and the
resulting precipitate was collected by filtration. The solid was
washed with ethanol and dried to give 10 as a white powder
(198 mg, 72%). mp 208–210 °C. 1H NMR (200 MHz, DMSO-d6) d
2.41 (3H, s), 3.70–7.80 (5H, m), 6.82–6.94 (3H, m), 7.26 (1H, t,
J = 8.0 Hz), 7.40 (1H, d, J = 8.4 Hz), 7.57–7.62 (1H, m), 7.85 (1H, s),
11.8 (1H, bs), 11.9 (1H, bs). Anal. Calcd for C18H17N3O3ꢁ0.1H2O: C,
66.49; H, 5.33; N, 12.92. Found: C, 66.26; H, 5.43; N, 13.14.
5.1.27. 2-[1-Hydroxy-3-(3-methoxyphenyl)propyl]-6-methyl-
3,4-dihydroquinazolin-4-one (21)
A mixture of compound 20 (360 mg, 0.848 mmol), trifluoro-
acetic acid (3 mL), and CH2Cl2 (6 mL) was stirred at room tem-
perature for 1 h. The reaction mixture was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (60% ethyl acetate/hexane) and crystallized
from diethyl ether to give 21 as a white powder (125 mg,
0.385 mmol, 45%). mp 149–151 °C. 1H NMR (300 MHz, DMSO-
d6) d 2.00–2.09 (2H, m), 2.43 (3H, s), 2.57–2.72 (2H, m), 3.72
(3H, s), 4.38–4.44 (1H, m), 5.76 (1H, t, J = 4.8 Hz), 6.72–6.81
(3H, m), 7.18 (1H, t, J = 7.8 Hz), 7.54 (1H, d, J = 8.4 Hz), 7.62
(1H, d, J = 7.8 Hz), 7.90 (1H, s), 11.7 (1H, bs). Anal. Calcd for
5.1.24. 2-(Chloromethyl)-6-methylquinazolin-4(3H)-one (12)27
Sodium methylate (357 mg, 6.62 mmol) was added to a solution
of chloroacetonitrile (2.75 g, 36.4 mmol) in methanol (75 mL) at
0 °C and the mixture was stirred at room temperature for 1 h. In
another flask, commercially available 2-amino-5-methylbenzoic
acid 11 (5.00 g, 33.1 mmol) was added to a solution of sodium
methylate (179 mg, 3.31 mmol) in methanol (75 mL) and this solu-
tion was added to the above solution of chloroacetoimidate at
room temperature. The reaction mixture was stirred at room tem-
perature for 1 h and then heated at 80 °C for 2 h. After the mixture
was cooled to room temperature, the precipitated solid was col-
lected and washed with methanol to give 12 as a pale gray powder
(4.60 g, 67%). 1H NMR (300 MHz, DMSO-d6) d 2.45 (3H, s), 4.54 (2H,
s), 7.58 (1H, d, J = 8.1 Hz), 7.66 (1H, dd, J = 8.1, 1.8 Hz), 7.92 (1H, d,
J = 0.6 Hz), 12.5 (1H, bs).
C
19H20N2O3ꢁ0.1H2O: C, 69.96; H, 6.24; N, 8.59. Found: C, 69.67;
H, 6.18; N, 8.51.
5.1.28. 2-[3-(3-Methoxyphenyl)propanoyl]-6-methyl-3,4-
dihydroquinazolin-4-one (23)
Dimethylsulfoxide (0.011 mL, 0.154 mmol) was added to a
solution of oxalyl chloride (0.013 mL, 0.154 mmol) in CH2Cl2
(4 mL) at ꢀ78 °C under nitrogen atmosphere and the mixture
was stirred at ꢀ78 °C for 2 min. A solution of compound 21
(25 mg, 0.077 mmol) and dimethylsulfoxide (0.020 mL) in CH2Cl2
(2 mL) was added and the mixture was stirred at ꢀ78 °C for 1 h.
After triethylamine (0.107 mL, 0.771 mmol) was added, the
resulting mixture was allowed to warm to room temperature
and stirred at room temperature for 2 h. The reaction mixture
was partitioned between ethyl acetate and water. The organic
layer was washed with brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The crude product
was purified by silica gel column chromatography (25% ethyl
acetate/hexane) to give compound 22 (1-(6-methyl-4-{[(methyl-
thio)methyl]oxy}quinazolin-2-yl)-3-[3-(methyloxy)phenyl]pro-
pan-1-one) as a pale yellow oil (12 mg). To a solution of the pale
yellow oil obtained above in CH2Cl2 (1 mL) was added 90% aque-
ous trifluoroacetic acid (0.5 mL), and the mixture was stirred at
room temperature for 10 min. The reaction mixture was concen-
trated under reduced pressure and the residue was purified by
preparative HPLC to give 23 as a white powder (6.5 mg, 26%).
1H NMR (300 MHz, CDCl3) d 2.53 (3H, s), 3.03–3.08 (2H, m),
3.54–3.59 (2H, m), 3.80 (3H, s), 6.73–6.86 (3H, m), 7.22 (1H, t,
J = 8.1 Hz), 7.65 (1H, dd, J = 8.4, 2.1 Hz), 7.75 (1H, d, J = 8.4 Hz),
8.13–8.14 (1H, m), 10.1 (1H, bs).
5.1.25. 2-({[(3-Methoxyphenyl)methyl]amino}methyl)-6-
methyl-3,4-dihydroquinazolin-4-one (13)
A mixture of compound 12 (200 mg, 0.959 mmol), 3-methoxy-
benzylamine (263 mg, 1.92 mmol), and K2CO3 (132 mg,
0.959 mmol) in THF (6 mL) was stirred at 40 °C for 15 h. The reac-
tion mixture was concentrated under reduced pressure. The resi-
due was diluted with ethyl acetate, washed with brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
The crude product was purified by preparative HPLC and crystal-
lized from diethyl ether to give 13 as a white powder (120 mg,
41%). mp 172–174 °C. 1H NMR (300 MHz, DMSO-d6) d 2.43 (3H,
s), 3.64 (2H, s), 3.71 (2H, s), 3.73 (3H, s), 6.76–6.80 (1H, m),
6.89–6.93 (2H, m), 7.21 (1H, t, J = 7.8 Hz), 7.53 (1H, d, J = 8.1 Hz),
7.61 (1H, dd, J = 8.4, 2.1 Hz), 7.89 (1H, d, J = 0.6 Hz). Anal. Calcd
for C18H19N3O2ꢁ0.4H2O: C, 68.29; H, 6.30; N, 13.27. Found: C,
68.23; H, 6.07; N, 13.23.
5.1.29. Ethyl 5-[(benzyloxy)methyl]-4-oxo-3H,4H-thieno[2,3-
d]pyrimidine-2-carboxylate (25a)
5.1.26. tert-Butyl 2-[1-hydroxy-3-(3-methoxyphenyl)propyl]-6-
methyl-4-oxo-3,4-dihydroquinazoline-3-carboxylate (20)
To a solution of compound 16 (500 mg, 1.92 mmol) in THF
(20 mL) was added dropwise lithium diisopropylamide (1.8 M in
a mixed solvent of heptane, THF and ethylbenzene, 1.28 mL,
2.30 mmol) at ꢀ78 °C, and the mixture was stirred at ꢀ78 °C for
10 min. A solution of compound 19 (631 mg, 3.84 mmol) in THF
(5 mL) was added at ꢀ78 °C and the reaction mixture was allowed
to warm to room temperature followed by stirring at room tem-
perature for 1 h. The reaction was quenched 1 M hydrochloric acid
and the resulting mixture was partitioned between ethyl acetate
and H2O. The organic layer was washed with brine, dried over
To a solution of benzyl alcohol (0.157 mL) in THF (10 mL) was
gradually added 60% sodium hydride (116 mg, 3.03 mmol), and
the mixture was stirred at room temperature for 10 min. Com-
pound 24 (400 mg, 1.26 mmol) was added at once, and the mixture
was stirred at room temperature for 1 h. The solvent was evapo-
rated under reduced pressure and the residue was extracted with
ethyl acetate. The mixture was washed with 1 M hydrochloric acid
and a 1:1 mixture of 1 M hydrochloric acid-saturated brine, and
after drying over anhydrous Na2SO4, the solvent was evaporated.
The residue was suspended in diethyl ether, filtrated, dried and
suspended in THF (5 mL). Oxalyl chloride (0.550 mL, 6.31 mmol)
and DMF (one drop) were added, and the mixture was stirred

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H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
at room temperature for 2.5 h. The solvent was evaporated
under reduced pressure. The obtained residue was dissolved in
EtOH–THF (1:1) solution, and the mixture was stirred at room tem-
perature for 12 h. The solvent was evaporated under reduced pres-
sure and the residue was extracted with ethyl acetate, washed
with water and saturated brine, and dried over anhydrous Na2SO4.
The solvent was evaporated under reduced pressure. The obtained
residue was purified by silica gel column chromatography (20–40%
ethyl acetate/hexane) to give 25a (68.7 mg, 16%) as a colorless
powder. mp 155–156 °C. 1H NMR (300 MHz, DMSO-d6) d 1.34
(3H, t, J = 7.1 Hz), 4.36 (2H, q, J = 7.0 Hz), 4.66 (2H, s), 4.86 (2H, d,
J = 0.9 Hz), 7.13–7.51 (5H, m), 7.66 (1H, s), 12.9 (1H, s). Anal. Calcd
for C17H16N2O4Sꢁ0.25H2O: C, 58.52; H, 4.77; N, 8.03. Found: C,
58.30; H, 4.53; N, 8.30.
5.1.34. Ethyl 5-[(benzylsulfanyl)methyl]-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxylate (25f)
To a mixture of compound 24 (600 mg, 1.89 mmol) and DMA
(12 mL) were added phenylmethanethiol (0.244 mL, 2.08 mmol)
and triethylamine (0.527 mL, 3.78 mmol) at room temperature
and the mixture was stirred for 1 h. The reaction mixture was con-
centrated under reduced pressure, and ethyl acetate was added to
the obtained residue. The mixture was washed with 1 M hydro-
chloric acid and saturated brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The concentrated resi-
due was purified by silica gel column chromatography (5–50%
ethyl acetate/hexane). The obtained crude crystals were recrystal-
lized from ethyl acetate–hexane to give 25f as a white powder
(207 mg, 30%). mp 171 °C. 1H NMR (300 MHz, DMSO-d6) d 1.35
(3H, t, J = 7.2 Hz), 3.73 (2H, s), 3.99 (2H, s), 4.37 (2H, q,
J = 7.1 Hz), 7.15–7.34 (5H, m), 7.57 (1H, s), 12.8 (1H, s). Anal. Calcd
for C17H16N2O3S2ꢁ0.20H2O: C, 56.09; H, 4.54; N, 7.69. Found: C,
55.93; H, 4.41; N, 7.96.
5.1.30. Ethyl 5-({[4-(ethoxycarbonyl)phenyl]methoxy}methyl)-
4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate (25b)
Compound 25b was prepared from compound 24 and ethyl 4-
(hydroxymethyl)benzoate with a similar procedure as described
for 25a (pale yellow powder, 75%). mp 181 °C. 1H NMR
(300 MHz, DMSO-d6) d 1.33 (6H, q, J = 7.0 Hz), 4.27–4.41 (4H, m),
4.75 (2H, s), 4.89 (2H, d, J = 1.1 Hz), 7.54 (2H, d, J = 8.5 Hz),
7.70 (1H, s), 7.91–7.99 (2H, m), 12.9 (1H, s). Anal. Calcd for
5.1.35. 5-[(Benzyloxy)methyl]-N-[(3-methoxyphenyl)methyl]-4-
oxo-3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide (26a)
Compound 26a was prepared from compound 25a with a similar
procedure as described for 7a (white powder, 60%). mp 145 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.41 (2H, d, J = 6.4 Hz),
4.66 (2H, s), 4.85 (2H, d, J = 0.8 Hz), 6.82 (1H, dd, J = 8.3, 2.3 Hz),
6.87–6.95 (2H, m), 7.18–7.45 (6H, m), 7.60 (1H, s), 9.66 (1H, t,
J = 6.5 Hz), 12.4 (1H, s). Anal. Calcd for C23H21N3O4Sꢁ0.25H2O: C,
62.78; H, 4.93; N, 9.55. Found: C, 62.54; H, 4.90; N, 9.88.
C
20H20N2O6Sꢁ0.05H2O: C, 57.56; H, 4.85; N, 6.71. Found: C, 57.55;
H, 4.82; N, 6.70.
5.1.31. Ethyl 5-{[(4-fluorophenyl)methoxy]methyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate (25c)
Compound 25c was prepared from compound 24 and ethyl (4-
fluorophenyl)methanol with a similar procedure as described for
25a (pale yellow powder, 8%). mp 195–196 °C. 1H NMR
5.1.36. Ethyl 4-{[(2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-
oxo-3H,4H-thieno[2,3-d]pyrimidin-5-yl)methoxy]methyl}-
benzoate (26b)
(300 MHz, DMSO-d6)
d 1.34 (3H, t, J = 7.1 Hz), 4.37 (2H, q,
J = 7.0 Hz), 4.64 (2H, s), 4.85 (2H, d, J = 1.3 Hz), 7.18 (2H, t,
J = 9.0 Hz), 7.44 (2H, dd, J = 8.7, 5.7 Hz), 7.66 (1H, t, J = 1.2 Hz),
12.9 (1H, s). Anal. Calcd for C17H15FN2O4Sꢁ0.25H2O: C, 55.65; H,
4.26; N, 7.64. Found: C, 55.51; H, 4.13; N, 7.84.
Compound 26b was prepared from compound 25b with a similar
procedure as described for 7a (white powder, 90%). mp 173 °C. 1H
NMR (300 MHz, DMSO-d6) d 1.32 (3H, t, J = 7.1 Hz), 3.73 (3H, s),
4.31 (2H, q, J = 7.2 Hz), 4.42 (2H, d, J = 6.2 Hz), 4.75 (2H, s), 4.89
(2H, d, J = 0.9 Hz), 6.79–6.85 (1H, m), 6.87–6.93 (2H, m), 7.24 (1H,
t, J = 8.1 Hz), 7.54 (2H, d, J = 8.5 Hz), 7.63 (1H, s), 7.88–8.03 (2H, m),
9.65 (1H, t, J = 6.5 Hz), 12.4 (1H, s). Anal. Calcd for C26H25N3O6
Sꢁ0.10H2O:C, 61.31;H, 4.99;N, 8.25. Found:C, 61.17;H, 4.94;N, 8.42.
5.1.32. Ethyl 5-{[(4-cyanophenyl)methoxy]methyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2-carboxylate (25d)
Compound 25d was prepared from compound 24 and 4-
(hydroxymethyl)benzonitrile with
a
similar procedure as
described for 25a (pale yellow powder, 52%). mp 236–237 °C. 1H
NMR (300 MHz, DMSO-d6) d 1.34 (3H, t, J = 7.1 Hz), 4.37 (2H, q,
J = 7.1 Hz), 4.76 (2H, s), 4.90 (2H, d, J = 1.1 Hz), 7.59 (2H, d,
J = 8.5 Hz), 7.71 (1H, t, J = 1.1 Hz), 7.77–7.89 (2H, m), 12.9 (1H, s).
Anal. Calcd for C18H15N3O4Sꢁ0.25H2O: C, 57.82; H, 4.18; N, 11.24.
Found: C, 57.68; H, 4.09; N, 11.44.
5.1.37. 4-{[(2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidin-5-yl)methoxy]methyl}benzoic
acid (26c)
A mixture of 26b (126 g, 249 mmol) and 8 M NaOH (77.7 mL,
622 mmol) in a mixture of MeOH (600 mL), THF (600 mL) and
water (600 mL) was stirred at 90 °C for 1 h. The mixture was con-
centrated in vacuo. After acidification with 1 M hydrochloric acid
(933 mL, 933 mmol), the crude materials were collected by filtra-
tion, washed with water (7 ꢂ 500 mL) and MeOH (6ꢂ500 mL) to
give a white powder. The crude product was suspended in MeOH
(2500 mL) at refluxed temperature for 1.5 h, cooled to room tem-
perature, and collected by filtration, washed with MeOH and air
dried to give 26c as a white powder (122 g, quant.). mp 229 °C.
5.1.33. Ethyl 5-{[benzyl(methyl)amino]methyl}-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-2-carboxylate (25e)
To a mixture of compound 24 (600 mg, 1.89 mmol) obtained
and THF (12 mL) were added N-methyl-1-phenylmethanamine
(0.269 mL, 2.08 mmol) and triethylamine (0.527 mL, 3.78 mmol)
at room temperature and the mixture was stirred for 1 h. The
reaction mixture was concentrated under reduced pressure
and ethyl acetate was added to the residue. The mixture was
washed with saturated brine, dried over anhydrous Na2SO4
and concentrated under reduced pressure. The concentrated
residue was purified by silica gel column chromatography (1–8%
methanol/ethyl acetate). The obtained crude crystals were
recrystallized from ethyl acetate–hexane to give 25e as a white
powder (351 mg, 52%). mp 129 °C. 1H NMR (300 MHz, DMSO-
d6) d 1.33 (3H, t, J = 7.2 Hz), 2.32 (3H, s), 4.15 (4H, s), 4.32
(2H, q, J = 7.0 Hz), 7.28–7.51 (5H, m), 7.64 (1H, s). Anal. Calcd
for C18H19N3O3Sꢁ0.05H2O: C, 60.33; H, 5.37; N, 11.73. Found:
C, 60.37; H, 5.28; N, 11.74.
1H NMR (300 MHz, DMSO-d6)
d 3.73 (3H, s), 4.41 (2H, d,
J = 6.0 Hz), 4.74 (2H, s), 4.89 (2H, s), 6.82 (1H, dd, J = 8.5, 1.9 Hz),
6.86–6.97 (2H, m), 7.24 (1H, t, J = 8.1 Hz), 7.51 (2H, d, J = 8.1 Hz),
7.63 (1H, s), 7.93 (2H, d, J = 8.3 Hz), 9.66 (1H, t, J = 6.4 Hz), 12.5
(1H, s), 12.9 (1H, s). Anal. Calcd for C24H21N3O6S: C, 60.12; H,
4.41; N, 8.76. Found: C, 60.30; H, 4.53; N, 8.61.
5.1.38. 5-{[(4-Fluorophenyl)methoxy]methyl}-N-[(3-methoxy-
phenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-
carboxamide (26d)
Compound 26d was prepared from compound 25c with a sim-
ilar procedure as described for 7a (white powder, 81%). mp 166 °C.

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5501
1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.41 (2H, d, J = 6.0 Hz),
4.64 (2H, s), 4.85 (2H, d, J = 0.8 Hz), 6.74–6.94 (3H, m), 7.11–7.30
(3H, m), 7.38–7.49 (2H, m), 7.59 (1H, s), 9.64 (1H, s), 12.4 (1H, s).
Anal. Calcd for C23H20FN3O4Sꢁ0.15H2O: C, 60.56; H, 4.49; N, 9.21.
Found: C, 60.48; H, 4.44; N, 9.27.
carbonate solution (twice), brine, 1 M hydrochloric acid, brine,
dried over Na2SO4 and concentrated in vacuo. The residue was
crystallized from ethyl acetate to give 28b (588 mg, 82%) as a white
powder. mp 236 °C. 1H NMR (300 MHz, DMSO-d6) d 1.35 (3H, t,
J = 7.2 Hz), 3.89 (3H, s), 4.37 (2H, q, J = 7.2 Hz), 4.82 (2H, d,
J = 5.5 Hz), 7.52 (1H, s), 7.77–8.22 (4H, m), 9.23 (1H, s), 12.9 (1H,
s). Anal. Calcd for C19H17N3O6S: C, 54.93; H, 4.12; N, 10.12. Found:
C, 54.65; H, 4.24; N, 9.86.
5.1.39. 5-{[(4-Cyanophenyl)methoxy]methyl}-N-[(3-methoxy-
phenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-
carboxamide (26e)
Compound 26e was prepared from compound 25d with a sim-
ilar procedure as described for 7a (white powder, 79%). mp 205 °C.
1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.42 (2H, d, J = 6.4 Hz),
4.76 (2H, s), 4.89 (2H, d, J = 1.1 Hz), 6.79–6.93 (3H, m), 7.24 (1H, t,
J = 8.1 Hz), 7.55–7.66 (3H, m), 7.77–7.90 (2H, m), 9.65 (1H, t,
J = 6.4 Hz), 12.4 (1H, s). Anal. Calcd for C24H20N4O4Sꢁ0.25H2O: C,
61.99; H, 4.44; N, 12.05. Found: C, 62.02; H, 4.36; N, 12.13.
5.1.44. 5-[(Benzoylamino)methyl]-N-(3-methoxybenzyl)-4-oxo-
3,4-dihydrothieno[2,3-d]pyrimidine-2-carboxamide (29a)
Compound 29a was prepared from compound 28a with a
similar procedure as described for 7a (white powder, 82%). mp
195–196 °C. 1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.42
(2H, d, J = 6.2 Hz), 4.80 (2H, d, J = 5.1 Hz), 6.74–6.98 (3H, m), 7.24
(1H, t, J = 8.1 Hz), 7.38–7.62 (4H, m), 7.82–7.98 (2H, m), 9.02
(1H, s), 9.64 (1H, t, J = 6.3 Hz), 12.5 (1H, s). Anal. Calcd for
5.1.40. 5-{[Benzyl(methyl)amino]methyl}-N-[(3-methoxy-
phenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-
carboxamide (26f)
C
23H20N4O4Sꢁ0.15H2O: C, 61.23; H, 4.53; N, 12.42. Found: C,
61.28; H, 4.47; N, 12.33.
Compound 26f was prepared from compound 25e with a simi-
lar procedure as described for 7a (white powder, 58%). mp 127 °C.
1H NMR (300 MHz, DMSO-d6) d 2.18 (3H, s), 3.60 (2H, s), 3.73 (3H,
s), 3.94 (2H, s), 4.42 (2H, d, J = 6.4 Hz), 6.78–6.86 (1H, m), 6.87–6.93
(2H, m), 7.17–7.44 (6H, m), 7.59 (1H, s), 9.63 (1H, s), 12.3 (1H, s).
Anal. Calcd for C24H24N4O3S: C, 64.27; H, 5.39; N, 12.49. Found:
C, 64.02; H, 5.21; N, 12.39.
5.1.45. Methyl 4-{[(2-{[(3-methoxyphenyl)methyl]carbamoyl}-
4-oxo-3H,4H-thieno[2,3-d]pyrimidin-5-yl)methyl]carbamoyl}-
benzoate (29b)
Compound 29b was prepared from compound 28b with a sim-
ilar procedure as described for 7a (white powder, 89%). mp 237 °C.
1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 3.89 (3H, s), 4.42 (2H,
d, J = 6.4 Hz), 4.80 (2H, d, J = 5.7 Hz), 6.80–6.93 (3H, m), 7.24 (1H, t,
J = 8.1 Hz), 7.41–7.46 (1H, m), 8.00–8.08 (4H, m), 9.26 (1H, s), 9.59
(1H, s), 12.5 (1H, s). Anal. Calcd for C25H22N4O6S: C, 59.28; H, 4.38;
N, 11.06. Found: C, 59.20; H, 4.41; N, 11.05.
5.1.41. 5-[(Benzylsulfanyl)methyl]-N-[(3-methoxyphenyl)-
methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide
(26g)
Compound 26g was prepared from compound 25f with a simi-
lar procedure as described for 7a (white powder, 67%). mp 126 °C.
1H NMR (300 MHz, DMSO-d6) d 3.73 (2H, s), 3.74 (3H, s), 3.99 (2H,
s), 4.42 (2H, d, J = 6.4 Hz), 6.79–6.93 (3H, m), 7.19–7.32 (6H, m),
7.51 (1H, s), 9.64 (1H, t, J = 6.1 Hz), 12.4 (1H, s). Anal. Calcd for
5.1.46. 4-{[(2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidin-5-yl)methyl]carbamoyl}benzoic
acid (29c)
A mixture of compound 29b (275 mg, 0.543 mmol) and 12 M
NaOH (0.113 mL, 1.36 mmol) in THF–MeOH–H2O (1:1:1) (6.0 mL)
was stirred at 90 °C for 1 h. After evaporation in vacuo, the residue
was dissolved in ethyl acetate–THF (1:1) (700 mL) and the organic
layer was washed with 1 M hydrochloric acid, brine, dried over
Na2SO4, and concentrated in vacuo. The residue was crystallized
from ethyl acetate to give 29c as a white powder (263 mg, 98%).
mp 285–286 °C. 1H NMR (300 MHz, DMSO-d6) d 3.74 (3H, s), 4.42
(2H, d, J = 6.2 Hz), 4.81 (2H, d, J = 5.3 Hz), 6.80–6.93 (3H, m),
7.21–7.27 (1H, m), 7.46 (1H, s), 7.98–8.06 (4H, m), 9.15 (1H, t,
J = 5.8 Hz), 9.66 (1H, t, J = 6.3 Hz), 12.5 (1H, s), 13.1 (1H, s). Anal.
Calcd for C24H20N4O6Sꢁ0.35H2O: C, 57.79; H, 4.18; N, 11.23. Found:
C, 57.86; H, 4.12; N, 11.14.
C
23H21N3O3S2ꢁ0.45H2O: C, 60.10; H, 4.80; N, 9.14. Found: C,
59.95; H, 4.57; N, 9.38.
5.1.42. Ethyl 5-[(benzoylamino)methyl]-4-oxo-3,4-dihydro-
thieno[2,3-d]pyrimidine-2-carboxylate (28a)
To a mixture of compound 27b (250 mg, 0.863 mmol) in THF
(3.0 mL) were added benzoyl chloride (0.110 mL, 0.949 mmol)
and triethylamine (0.253 mL, 1.81 mmol). After being stirred for
1 h at room temperature, the mixture was concentrated in vacuo.
The residue was taken up in ethyl acetate, washed with saturated
sodium hydrogen carbonate solution (twice), brine, 1 M hydrochlo-
ric acid, brine, dried over Na2SO4 and concentrated in vacuo. The
residue was crystallized from ethyl acetate to give 28a (223 mg,
72%) as a white powder. mp 204 °C. 1H NMR (300 MHz, DMSO-
d6) d 1.35 (3H, t, J = 7.1 Hz), 4.38 (2H, q, J = 7.0 Hz), 4.80 (2H, d,
J = 4.9 Hz), 7.43–7.60 (4H, m), 7.86–7.94 (2H, m), 9.03 (1H, t,
J = 5.8 Hz), 12.9 (1H, s). Anal. Calcd for C17H15N3O4S: C, 57.13; H,
4.23; N, 11.76. Found: C, 57.28; H, 4.27; N, 11.56.
5.1.47. 5-(Cyanomethyl)-N-[(3-methoxyphenyl)methyl]-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide (31a)
Compound 31a was prepared from compound 30 with a similar
procedure as described for 7a (brown powder, 76%). mp 217 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.27 (2H, d, J = 0.9 Hz),
4.42 (2H, d, J = 6.4 Hz), 6.78–6.94 (3H, m), 7.24 (1H, t, J = 8.1 Hz),
7.69 (1H, t, J = 0.9 Hz), 9.66 (1H, t, J = 6.4 Hz), 12.6 (1H, s). Anal.
Calcd for C17H14N4O3Sꢁ0.20H2O: C, 57.04; H, 4.05; N, 15.65. Found:
C, 57.23; H, 4.08; N, 15.44.
5.1.43. Ethyl 5-({[4-(methoxycarbonyl)benzoyl]amino}methyl)-
4-oxo-3,4-dihydrothieno[2,3-d]pyrimidine-2-carboxylate (28b)
To a mixture of monomethyl terephthalate (373 mg, 2.07
mmol) in THF (5.0 mL) were added oxalyl chloride (0.450 mL,
5.18 mmol) and N,N-dimethylformamide (1 drop). After being stir-
red for 1 h at room temperature, the mixture was concentrated in
vacuo. The residue was resuspended in THF (5.0 mL), and to the
mixture were added 27b (500 mg, 1.73 mmol) and triethylamine
(0.960 mL, 6.90 mmol). After being stirred for 1 h at room temper-
ature, the mixture was concentrated in vacuo. The residue was
taken up in ethyl acetate, washed with saturated sodium hydrogen
5.1.48. 2-(2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidin-5-yl)acetic acid (31b)
A mixture of compound 30 (200 mg, 0.564 mmol), 2 M aqueous
sodium hydroxide solution (4 mL, 8 mmol) and ethanol (2 mL) was
stirred at 100 °C for 1 h. The reaction mixture was concentrated
under reduced pressure, and ethyl acetate was added to the
obtained residue. The organic layer was washed with 1 M hydro-
chloric acid and saturated brine, dried over anhydrous Na2SO4

5502
H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
and concentrated under reduced pressure. The concentrated resi-
due was crystallized from ethyl acetate to give 31b as a brown
powder (131 mg, 62%). mp 228–229 °C. 1H NMR (300 MHz,
DMSO-d6) d 3.73 (3H, s), 3.91 (2H, s), 4.42 (2H, d, J = 6.2 Hz),
6.77–6.93 (3H, m), 7.24 (1H, t, J = 8.1 Hz), 7.49 (1H, s), 9.64
(1H, t, J = 6.3 Hz), 12.3 (1H, s), 12.4 (1H, s). Anal. Calcd for
(1H, s), 12.5 (2H, s). Anal. Calcd for C24H20N4O6Sꢁ0.50H2O: C,
57.48; H, 4.22; N, 11.17. Found: C, 57.57; H, 4.11; N, 10.96.
5.1.52. Ethyl 5-(benzylcarbamoyl)-4-oxo-3H,4H-thieno[2,3-
d]pyrimidine-2-carboxylate (34a)
To a mixture of 33c (400 mg, 1.49 mmol) in THF (10 mL) were
added oxalyl chloride (0.390 mL, 4.47 mmol) and N,N-dimethyl-
formamide (1 drop). After being stirred for 1 h at room tempera-
ture, the mixture was concentrated in vacuo. The residue was
resuspended in THF (10 mL), and to the mixture was added benzyl-
amine (0.326 mL, 2.98 mmol). After being stirred for 12 h at room
temperature, the mixture was concentrated in vacuo. The residue
was taken up in ethyl acetate, washed with 1 M hydrochloric acid
(ꢂ2), saturated sodium hydrogen carbonate solution, 1 M hydro-
chloric acid, brine, dried over Na2SO4 and concentrated in vacuo.
The residue was crystallized from ethyl acetate to give 34a
(373 mg, 70%) as a white powder. mp 215–216 °C. 1H NMR
C
17H15N3O5Sꢁ0.35H2O: C, 53.78; H, 4.17; N, 11.07. Found: C,
53.62; H, 4.15; N, 11.32.
5.1.49. N-[(3-Methoxyphenyl)methyl]-4-oxo-5-[(phenylcarba-
moyl)methyl]-3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide
(32a)
To a mixture of compound 31b (200 mg, 0.536 mmol) in THF
(3.0 mL) was added oxalyl chloride (0.140 mL, 1.61 mmol). After
being stirred at room temperature for 15 h, the mixture was con-
centrated in vacuo, and the resulting residue was dissolved in
THF (3.0 mL). To this solution was added aniline (0.146 mL,
1.61 mmol) and pyridine (0.217 mL, 2.68 mmol). After being stir-
red for 1 h at room temperature, the mixture was concentrated
in vacuo, and the residue was taken up in ethyl acetate. The organic
layer was washed with saturated NaHCO3 (ꢂ3), 1 M hydrochloric
acid (ꢂ2), and brine and dried over Na2SO4. The solvent was
removed, and the residue was crystallized from ethyl acetate to
give 32a as a pale yellow powder (154 mg, 64%). mp 194–195 °C.
1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.04 (2H, s), 4.42
(2H, d, J = 6.2 Hz), 6.78–6.93 (3H, m), 7.02 (1H, t, J = 7.3 Hz),
7.20–7.33 (3H, m), 7.52 (1H, s), 7.58 (2H, d, J = 7.5 Hz), 9.66 (1H,
t, J = 6.5 Hz), 10.1 (1H, s), 12.4 (1H, s). Anal. Calcd for C23H20N4O4S:
C, 61.59; H, 4.49; N, 12.49. Found: C, 61.29; H, 4.47; N, 12.47.
(300 MHz, DMSO-d6)
d 1.35 (3H, t, J = 7.2 Hz), 4.39 (2H, q,
J = 7.1 Hz), 4.56 (2H, d, J = 5.5 Hz), 7.13–7.53 (5H, m), 8.56 (1H, s),
11.2 (1H, s), 13.5 (1H, s). Anal. Calcd for C17H15N3O4S: C, 57.13;
H, 4.23; N, 11.76. Found: C, 57.08; H, 4.19; N, 11.83.
5.1.53. Ethyl 5-({[4-(ethoxycarbonyl)phenyl]methyl}-
carbamoyl)-4-oxo-3H,4H-thieno[2,3-d]pyrimidine-2-
carboxylate (34b)
To a mixture of compound 33c (600 mg, 2.24 mmol) in THF
(6.0 mL) were added oxalyl chloride (0.590 mL, 6.72 mmol) and
N,N-dimethylformamide (1 drop). After being stirred for 1 h at
room temperature, the mixture was concentrated in vacuo. The
residue was resuspended in THF (6.0 mL), and to the mixture were
added ethyl 4-(aminomethyl)benzoate hydrochloride (965 mg,
4.48 mmol) and triethylamine (1.20 mL, 8.96 mmol). After being
stirred for 12 h at room temperature, the mixture was concen-
trated in vacuo. The residue was taken up in ethyl acetate, washed
with 1 M hydrochloric acid (ꢂ2), saturated sodium hydrogen car-
bonate solution, 1 M hydrochloric acid, brine, dried over Na2SO4
and concentrated in vacuo. The residue was crystallized from ethyl
acetate to give 34b (771 mg, 80%) as a white powder. mp 227–
228 °C. 1H NMR (300 MHz, DMSO-d6) d 1.31 (3H, t, J = 6.2 Hz),
1.36 (3H, t, J = 6.2 Hz), 4.31 (2H, q, J = 7.1 Hz), 4.40 (2H, q,
J = 7.1 Hz), 4.65 (2H, d, J = 5.5 Hz), 7.50 (2H, d, J = 8.5 Hz), 7.93
(2H, d, J = 8.5 Hz), 8.57 (1H, s), 11.3 (1H, s), 13.5 (1H, s). Anal. Calcd
for C20H19N3O6Sꢁ0.20H2O: C, 55.47; H, 4.52; N, 9.70. Found: C,
55.50; H, 4.39; N, 9.73.
5.1.50. Ethyl 4-[2-(2-{[(3-methoxyphenyl)methyl]carbamoyl}-
4-oxo-3H,4H-thieno[2,3-d]pyrimidin-5-yl)acetamido]benzoate
(32b)
To a mixture of compound 31b (350 mg, 0.937 mmol) in THF
(3.0 mL) was added oxalyl chloride (0.245 mL, 2.81 mmol). After
being stirred at room temperature for 15 h, the mixture was con-
centrated in vacuo, and the resulting residue was dissolved in
THF (3.0 mL). To this solution was added ethyl 4-aminobenzoate
(465 mg, 2.81 mmol) and pyridine (0.379 mL, 4.69 mmol). After
being stirred for 1 h at room temperature, the mixture was concen-
trated in vacuo, and the residue was taken up in ethyl acetate. The
organic layer was washed with saturated NaHCO3 (ꢂ3), 1 M hydro-
chloric acid (ꢂ4), and brine and dried over Na2SO4. The solvent was
removed, and the residue was crystallized from ethyl acetate to
give 32b as a pale yellow powder (370 mg, 76%). mp 222–223 °C.
1H NMR (300 MHz, DMSO-d6) d 1.3 (3H, t, J = 7.2 Hz), 3.73 (3H, s),
4.07 (2H, s), 4.28 (2H, q, J = 7.1 Hz), 4.42 (2H, d, J = 6.2 Hz), 6.77–
6.95 (3H, m), 7.24 (1H, t, J = 8.1 Hz), 7.52 (1H, s), 7.71 (2H, d,
J = 8.9 Hz), 7.9 (2H, d, J = 8.7 Hz), 9.64 (1H, s), 10.5 (1H, s), 12.4
(1H, s). Anal. Calcd for C26H24N4O6Sꢁ0.15H2O: C, 59.68; H, 4.68;
N, 10.71. Found: C, 59.90; H, 4.69; N, 10.43.
5.1.54. N5-Benzyl-N2-[(3-methoxyphenyl)methyl]-4-oxo-
3H,4H-thieno[2,3-d]pyrimidine-2,5-dicarboxamide (35a)
Compound 35a was prepared from compound 34a with a sim-
ilar procedure as described for 7a (white powder, 85%). mp
217 °C. 1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.43 (2H, d,
J = 6. 2 Hz), 4.56 (2H, d, J = 5.5 Hz), 6.80–6.85 (1H, m), 6.89–6.93
(2H, m), 7.21–7.29 (2H, m), 7.30–7.38 (4H, m), 8.52 (1H, s), 9.74
(1H, s), 11.3 (1H, s), 13.2 (1H, s). Anal. Calcd for C23H20N4O4S: C,
61.59; H, 4.49; N, 12.49. Found: C, 61.48; H, 4.48; N, 12.41.
5.1.51. 4-[2-(2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-oxo-
3H,4H-thieno[2,3-d]pyrimidin-5-yl)acetamido]benzoic acid
(32c)
A mixture of 32b (230 mg, 0.442 mmol) and 12 M NaOH
(0.092 mL, 1.1 mmol) in THF–MeOH–H2O (1:1:1) (3.0 mL) was stir-
red at 80 °C for 1 h. After evaporation in vacuo, the residue was dis-
solved in ethyl acetate and the organic layer was washed with 1 M
hydrochloric acid (ꢂ2), brine, dried over Na2SO4, and concentrated
in vacuo. The residue was crystallized from ethyl acetate to give
32c as a pale yellow powder (188 mg, 86%). mp 279 °C. 1H NMR
(300 MHz, DMSO-d6) d 3.73 (3H, s), 4.07 (2H, s), 4.42 (2H, d,
J = 6.4 Hz), 6.73–6.96 (3H, m), 7.24 (1H, t, J = 8.1 Hz), 7.52 (1H, s),
7.69 (2H, d, J = 8.9 Hz), 7.87 (2H, d, J = 8.9 Hz), 9.61 (1H, s), 10.5
5.1.55. Ethyl 4-{[({2-[(3-Methoxybenzyl)carbamoyl]-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidin-5-yl}carbonyl)amino]methyl}-
benzoate (35b)
Compound 35b was prepared from compound 34b with a sim-
ilar procedure as described for 7a (white powder, 91%). mp 229–
230 °C. 1H NMR (300 MHz, DMSO-d6) d 1.31 (3H, t, J = 7.1 Hz),
3.73 (3H, s), 4.31 (2H, q, J = 7.1 Hz), 4.44 (2H, d, J = 6.2 Hz), 4.64
(2H, d, J = 5.3 Hz), 6.78–6.96 (3H, m), 7.24 (1H, t, J = 8.1 Hz), 7.49
(2H, d, J = 8.3 Hz), 7.88–7.97 (2H, m), 8.51 (1H, s), 9.73 (1H, t,
J = 6.0 Hz), 11.4 (1H, s), 13.2 (1H, s). Anal. Calcd for C26H24N4O6

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5503
Sꢁ0.20H2O: C, 59.58; H, 4.69; N, 10.69. Found: C, 59.41; H, 4.46; N,
under reduced pressure. Aqueous sodium hydroxide solution
(1 M, 2 mL), ethanol (1 mL) and THF (1 mL) were added to the con-
centrated residue and the reaction mixture was stirred at room
temperature for 12 h. The reaction mixture was concentrated
under reduced pressure, and ethyl acetate was added to the
obtained residue. The organic layer was washed with water and
saturated brine, dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The concentrated residue was crystallized
from ethyl acetate to give 37 as a brown powder (29.6 mg, 5.5%).
mp 218–219 °C. 1H NMR (300 MHz, DMSO-d6) d 1.32 (3H, t,
J = 7.1 Hz), 3.73 (3H, s), 3.93 (2H, s), 4.31 (2H, q, J = 7.0 Hz), 4.41
(2H, d, J = 6.2 Hz), 6.77–6.95 (3H, m), 7.23 (1H, t, J = 8.1 Hz), 7.52
(2H, d, J = 8.3 Hz), 7.86 (1H, s), 7.95 (2H, d, J = 8.3 Hz), 9.67 (1H, t,
J = 6.1 Hz), 9.95 (1H, s), 12.8 (1H, s).
The aqueous layer was acidified with 1 M hydrochloric acid. The
mixture was extracted with ethyl acetate, and the organic layer
was washed with 1 M hydrochloric acid and saturated brine, dried
over anhydrous Na2SO4 and concentrated under reduced pressure.
The concentrated residue was crystallized from ethyl acetate to
give 38 as a brown powder (83.9 mg, 17%). mp 218–219 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 3.92 (2H, s), 4.40 (2H,
d, J = 6.4 Hz), 6.73–6.96 (3H, m), 7.23 (1H, t, J = 8.1 Hz), 7.49 (2H,
d, J = 8.3 Hz), 7.87 (1H, s), 7.93 (2H, d, J = 8.3 Hz), 9.67 (1H, t,
J = 6.5 Hz), 9.94 (1H, s), 12.8 (1H, s), 12.9 (1H, s). Anal. Calcd for
10.61.
5.1.56. 4-{[({2-[(3-Methoxybenzyl)carbamoyl]-4-oxo-3,4-
dihydrothieno[2,3-d]pyrimidin-5-yl}carbonyl)amino]methyl}-
benzoic acid (35c)
A mixture of 35b (330 mg, 0.634 mmol) and 12 M NaOH
(0.132 mL, 1.59 mmol) in a mixture of THF–MeOH–H2O (2.0 mL–
2.0 mL–2.0 mL) was refluxed at 90 °C for 1 h. The mixture was con-
centrated in vacuo to give a residue, which was taken up in ethyl
acetate–THF (1:1, ca. 1L), washed with 1 M hydrochloric acid and
brine, and dried over Na2SO4. The organic extract was concentrated
in vacuo, and the residue was crystallized from ethyl acetate to
give 35c (294 mg, 94%) as a white powder. mp 254–255 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.44 (2H, d, J = 6.4 Hz),
4.64 (2H, d, J = 5.5 Hz), 6.74–6.97 (3H, m), 7.24 (1H, t, J = 8.1 Hz),
7.47 (2H, d, J = 8.3 Hz), 7.91 (2H, d, J = 8.3 Hz), 8.52 (1H, s), 9.76
(1H, t, J = 6.4 Hz), 11.3 (1H, t, J = 5.6 Hz), 13.2 (1H, s). Anal. Calcd
for C24H20N4O6Sꢁ2.0H2O: C, 54.54; H, 4.58; N, 10.60. Found: C,
54.19; H, 4.31; N, 10.45.
5.1.57. 2-{[(3-Methoxyphenyl)methyl]carbamoyl}-4-oxo-3H,4H-
thieno[2,3-d]pyrimidine-5-carboxylic acid (36a)
Compound 36a was prepared from compound 33c with a simi-
lar procedure as described for 7a (white powder, 81%). mp 236–
237 °C. 1H NMR (300 MHz, DMSO-d6) d 3.74 (3H, s), 4.45 (2H, d,
J = 6.4 Hz), 6.76–6.96 (3H, m), 7.25 (1H, t, J = 8.1 Hz), 8.65 (1H, s),
9.82 (1H, t, J = 6.3 Hz), 13.9 (1H, s), 15.4 (1H, s). Anal. Calcd for
C
24H20N4O6Sꢁ0.30H2O: C, 57.89; H, 4.17; N, 11.25. Found: C,
57.81; H, 4.23; N, 11.52.
5.1.59. Ethyl 4-oxo-5-(2-phenylacetamido)-3H,4H-thieno[2,3-
d]pyrimidine-2-carboxylate (40)
C
16H13N3O5Sꢁ0.30AcOEt: C, 53.55; H, 4.02; N, 10.89. Found: C,
53.86; H, 3.85; N, 11.07.
To a mixture of 39c (160 mg, 0.500 mmol) and THF (2 mL) were
added phenylacetyl chloride (0.0727 mL, 0.550 mmol) and triethyl-
amine (0.146 mL, 1.05 mmol) at room temperature. The mixture
was stirred for 1 h at room temperature and concentrated under
reduced pressure. Ethyl acetate was added to the obtained residue,
and the organic layer was washed with 1 M hydrochloric acid,
water and saturated brine, dried over anhydrous Na2SO4 and con-
centrated under reduced pressure. The concentrated residue was
crystallized from ethyl acetate to give 40 as a pale purple powder
(138 mg, 77%). mp 237 °C. 1H NMR (300 MHz, DMSO-d6) d 1.33
(3H, t, J = 7.1 Hz), 3.81 (2H, s), 4.36 (2H, q, J = 7.2 Hz), 7.22–7.41
(5H, m), 7.92 (1H, s), 9.89 (1H, s), 13.1 (1H, s). Anal. Calcd for
5.1.58. Ethyl 4-{[(2-{[(3-methoxyphenyl)methyl]carbamoyl}-4-
oxo-3H,4H-thieno[2,3-d]pyrimidin-5-yl)carbamoyl]methyl}-
benzoate (37) and 5.1.76. 4-{[(2-{[(3-Methoxyphenyl)methyl]-
carbamoyl}-4-oxo-3H,4H-thieno[2,3-d]pyrimidin-5-yl)-
carbamoyl]methyl}benzoic acid (38)
To a suspension of 36a (4.45 g, 12.4 mmol) in toluene (45 mL)
were added triethylamine (10.0 mL, 74.3 mmol) and DPPA
(6.90 mL, 32.2 mmol). The mixture was stirred for 2 min at 100 °C,
followed by addition of tert-butyl alcohol (12.0 mL, 124 mmol),
and heated at 100 °C for 12 h. After the removal of solvent, the res-
idue was taken up in ethyl acetate. The organic layer was extracted
with saturated NaHCO3 (ꢂ2), and brine and dried over Na2SO4. The
solvent was removed, and the residue was dried in vacuo to give a
residue, which was used in the next step without further purifica-
tion. A mixture of the above residue in 4 M hydrochloric acid/ethyl
acetate (45 mL) was stirred overnight at room temperature. The
resulting solid was filtered and washed with ethyl acetate and dried
to provide a brown powder (3.14 g). A mixture of above powder and
12 M NaOH (4.10 mL, 49.5 mmol) in THF–MeOH–H2O (1:1:1,
45 mL) was stirred at 80 °C for 2 h. After neutralization with 1 M
hydrochloric acid, the mixture was concentrated in vacuo. The
residue was taken up in ethyl acetate, washed with water and brine
and concentrated in vacuo to give a brown foam (1.18 g, 26%). The
crude product 36b was used without purification.
C
17H15N3O4Sꢁ0.25H2O: C, 56.42; H, 4.32; N, 11.61. Found: C,
56.58; H, 4.30; N, 11.72.
5.1.60. N-[(3-Methoxyphenyl)methyl]-4-oxo-5-(2-phenylace-
tamido)-3H,4H-thieno[2,3-d]pyrimidine-2-carboxamide (41)
Compound 41 was prepared from compound 40 with a similar
procedure as described for 7a (white powder, 93%). mp 201 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 3.80 (2H, s), 4.41 (2H, d,
J = 6.2 Hz), 6.77–6.94 (3H, m), 7.18–7.42 (6H, m), 7.86 (1H, s),
9.67 (1H, t, J = 6.4 Hz), 9.93 (1H, s), 12.8 (1H, s). Anal. Calcd for
C
23H20N4O4Sꢁ0.10AcOEtꢁ0.25H2O: C, 60.86; H, 4.65; N, 12.13.
Found: C, 60.99; H, 4.62; N, 12.21.
5.1.61. 5-{[(4-Carbamoylphenyl)methoxy]methyl}-N-[(3-
methoxyphenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimi-
dine-2-carboxamide (42a)
To
a mixture of {4-[(ethyloxy)carbonyl]phenyl}acetic acid
(215 mg, 1.033 mmol) and THF (3 mL) were added oxalyl chloride
(0.183 mL, 2.10 mmol) and DMF (1 drop), and the mixture was
stirred at room temperature for 2 h. The reaction mixture was
concentrated under reduced pressure, THF (3 mL), 36b (276 mg,
0.837 mmol) and pyridine (0.418 mL, 5.17 mmol) were added to
the obtained residue, and the mixture was stirred at room temper-
ature for 2 h. The reaction mixture was concentrated under
reduced pressure and ethyl acetate was added to the obtained res-
idue. The organic layer was washed with 1 M hydrochloric acid and
saturated brine, dried over anhydrous Na2SO4 and concentrated
To a mixture of 26c (200 mg, 0.417 mmol) and THF (2 mL) were
added oxalyl chloride (0.0500 mL, 0.573 mmol) and DMF (1 drop)
and the mixture was stirred at room temperature for 1 h. The reac-
tion mixture was concentrated under reduced pressure. To a sus-
pension of the concentrated residue in THF (3 mL) was added
28% aqueous ammonia (2 mL), and the mixture was stirred at room
temperature for 10 min. The reaction mixture was concentrated
under reduced pressure, and ethyl acetate and THF were added
to the obtained residue. The organic layer was washed with 1 M

5504
H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
hydrochloric acid and saturated brine, dried over anhydrous
Na2SO4 and concentrated under reduced pressure. The concen-
trated residue was crystallized from ethyl acetate–THF to give
42a as a white powder (173 mg, 87%). mp 224 °C. 1H NMR
(300 MHz, DMSO-d6) d 3.73 (3H, s), 4.42 (2H, d, J = 6.2 Hz), 4.71
(2H, s), 4.87 (2H, d, J = 1.1 Hz), 6.76–6.95 (3H, m), 7.18–7.28 (1H,
m), 7.34 (1H, s), 7.47 (2H, t, J = 7.6 Hz), 7.62 (1H, s), 7.86 (2H, d,
J = 8.3 Hz), 7.95 (1H, s), 9.64 (1H, t, J = 6.4 Hz), 12.4 (1H, s). Anal.
Calcd for C24H22N4O5Sꢁ0.50H2O: C, 59.13; H, 4.76; N, 11.49. Found:
C, 59.06; H, 4.72; N, 11.22.
under reduced pressure. To a mixture of the concentrated residue
and sodium methylate (58.1 mg, 1.08 mmol) were added methanol
(2 mL) and THF (2 mL) and the mixture was stirred at 80 °C for 1 h.
The reaction mixture was concentrated under reduced pressure,
and ethyl acetate was added to the obtained residue. The organic
layer was washed with 1 M hydrochloric acid and saturated brine,
dried over anhydrous Na2SO4 and concentrated under reduced pres-
sure. The concentrated residue was crystallized from ethyl acetate
to give 42d as a white powder (68.5 mg, 66%). mp 156–157 °C. 1H
NMR (300 MHz, DMSO-d6) d 3.28 (3H, s), 3.73 (3H, s), 4.34–4.48
(4H, m), 4.65 (2H, s), 4.85 (2H, d, J = 0.9 Hz), 6.77–6.95 (3H, m),
7.18–7.40 (5H, m), 7.59 (1H, s), 9.64 (1H, t, J = 6.0 Hz), 12.4 (1H, s).
Anal. Calcd for C25H25N3O5Sꢁ0.25H2O: C, 62.03; H, 5.31; N, 8.68.
Found: C, 61.98; H, 5.32; N, 8.63.
5.1.62. N-[(3-Methoxyphenyl)methyl]-5-({[4-(methylcarba-
moyl)phenyl]methoxy}methyl)-4-oxo-3H,4H-thieno[2,3-d]-
pyrimidine-2-carboxamide (42b)
To
a
mixture of 26c (110 mg, 0.229 mmol), 1-ethyl-3-(3-
hydrochloride (65.9 mg,
dimethylaminopropyl)carbodiimide
5.1.65. Disodium 4-[({2-[(3-Methoxybenzyl)carbamoyl]-4-oxo-
4H-thieno[2,3-d]pyrimidin-3-id-5-yl}methoxy)methyl]-
benzoate (43)
0.344 mol), 4-dimethylaminopyridine (283 mg, 2.31 mol) and
THF (5 mL) was added methylamine hydrochloride (155 mg,
2.29 mmol). The mixture was stirred at room temperature for
12 h. The reaction mixture was concentrated under reduced pres-
sure, and ethyl acetate was added to the obtained residue. The
organic layer was washed with 1 M hydrochloric acid and satu-
rated brine, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The concentrated residue was crystallized from
ethyl acetate to give 42b as a white powder (75.9 mg, 67%). mp
To a mixed solution of 26c (150 mg, 0.313 mmol) in THF
(24 mL) and ethanol (6 mL) was added an aqueous solution
(3 mL) of sodium hydrogen carbonate (52.6 mg, 0.626 mmol) and
the mixture was stirred for 30 min. The reaction mixture was con-
centrated under reduced pressure, ethanol (18 mL) was added to
the obtained residue, and the ethanol suspension was stirred at
90 °C for 30 min. The mixture was allowed to cool to room temper-
ature, and the precipitated solid was collected by filtration, washed
with ethanol and dried to give 43 as a white powder (149 mg, 91%).
mp >300 °C. 1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.40 (2H,
d, J = 6.4 Hz), 4.63 (2H, s), 4.88 (2H, d, J = 1.3 Hz), 6.74–6.94 (3H, m),
6.98 (1H, t, J = 1.3 Hz), 7.14–7.33 (3H, m), 7.84 (2H, d, J = 8.1 Hz),
9.03 (1H, t, J = 6.4 Hz). Anal. Calcd for C24H19N3Na2O6Sꢁ1.0H2O: C,
53.23; H, 3.91; N, 7.76. Found: C, 53.51; H, 4.09; N, 7.83.
190–191 °C. 1H NMR (300 MHz, DMSO-d6)
d 2.78 (3H, d,
J = 4.5 Hz), 3.73 (3H, s), 4.42 (2H, d, J = 6.4 Hz), 4.71 (2H, s), 4.87
(2H, d, J = 0.8 Hz), 6.82 (1H, dd, J = 9.0, 1.8 Hz), 6.86–6.97 (2H, m),
7.24 (1H, t, J = 8.1 Hz), 7.46 (2H, d, J = 8.1 Hz), 7.62 (1H, s), 7.82
(2H, d, J = 8.1 Hz), 8.42 (1H, d, J = 4.5 Hz), 9.65 (1H, t, J = 6.4 Hz),
12.4 (1H, s). Anal. Calcd for C25H24N4O5Sꢁ0.80H2O: C, 59.23; H,
5.09; N, 11.05. Found: C, 59.33; H, 5.04; N, 11.00.
5.1.63. 5-({[4-(Hydroxymethyl)phenyl]methoxy}methyl)-N-[(3-
methoxyphenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimi-
dine-2-carboxamide (42c)
5.2. MMPs and TACE enzyme inhibition assay
Human recombinant MMP precursors were purchased from
Genzyme-Techne (MMP-1, 2, 7, 8, 9, 10, 13, and TACE) or Biogene-
sis (MMP-3). Human recombinant GST-MMP-14 was prepared as
described by Sato et al.28 The MMP assay buffer consisted of
50 mM Tris–HCl (pH 7.5), 10 mM CaCl2, 150 mM NaCl, and 0.05%
Brij-35. The pro-MMPs were activated by preincubation with
1 mM aminophenylmercuric acetate (APMA) in assay buffer at
37 °C for 2 h (MMP-1, 2, 7, 8, 10, and 13) or 18 h (MMP-3 and 9).
The TACE assay buffer consisted of 25 mM Tris–HCl (pH 9.0),
2.5 mM ZnCl2, and 0.005% Brij-35. The pro-MMPs were activated
by preincubation with 1 mM aminophenylmercuric acetate
(APMA) in assay buffer at 37 °C for 2 h (MMP-1, 2, 7, 8, 10, and
13) or 18 h (MMP-3 and 9). Enzyme inhibition assays were per-
formed in an assay buffer containing enzymes and fluorescence
peptide (Cy3-PLGLK(Cy5Q)AR-NH2 for MMPs, Cy3-PLAQAV(Cy5Q-
L-2,3-diaminopropionic acid)-RSSSR-NH2 for TACE, Amersham
Biosciences) in the presence of the various concentrations of inhib-
itors. Following incubation at 37 °C for 40 min, the reaction was
terminated by addition of EDTA (pH 8.0). The increase in fluores-
cence was measured by Farcyte spectrofluorimeter (Amersham
Bioscience, kem 535 nm, kex 595 nm). Enzyme activity (%) was
determined as following equation: Enzyme activity (%) = (X ꢀ C)/
(T ꢀ C) ꢂ 100, where X = the fluorescence count with inhibitor,
T = the fluorescence count without inhibitor and C = the fluores-
cence count with EDTA. IC50 values of inhibitors were obtained
with iterative fitting package (GraphPad Prism software).
To a mixture of 26c (1200 mg, 2.50 mmol) and THF (12 mL)
were added oxalyl chloride (0.419 mL, 4.80 mmol) and DMF (1
drop), and the mixture was stirred at room temperature for 1.5 h.
The reaction mixture was concentrated under reduced pressure.
A Mixture of the concentrated residue and sodium borohydride
(189 mg, 5.01 mmol) was stirred in DMA (15 mL) at room temper-
ature for 5 min. The reaction mixture was stirred until generation
of gas ceased and concentrated under reduced pressure. Ethyl ace-
tate was added to the obtained residue. The organic layer was
washed with water, 1 M hydrochloric acid, water and saturated
brine, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The concentrated residue was crystallized from
ethyl acetate to give 42c as a white powder (753 mg, 65%). mp
156–157 °C. 1H NMR (300 MHz, DMSO-d6) d 3.73 (3H, s), 4.42
(2H, d, J = 6.4 Hz), 4.49 (2H, d, J = 5.7 Hz), 4.64 (2H, s), 4.83 (2H,
d, J = 1.1 Hz), 5.16 (1H, t, J = 5.8 Hz), 6.75–6.95 (3H, m), 7.19–7.28
(1H, m), 7.28–7.39 (4H, m), 7.58 (1H, s), 9.64 (1H, t, J = 6.5 Hz),
12.4 (1H, s). Anal. Calcd for C24H23N3O5Sꢁ0.50H2O: C, 60.75; H,
5.10; N, 8.86. Found: C, 60.72; H, 4.98; N, 8.89.
5.1.64. 5-({[4-(Methoxymethyl)phenyl]methoxy}methyl)-N-[(3-
methoxyphenyl)methyl]-4-oxo-3H,4H-thieno[2,3-d]pyrimi-
dine-2-carboxamide (42d)
To a mixture of 26c (100 mg, 0.215 mmol) and THF (2 mL) were
added methanesulfonyl chloride (0.0266 mL, 0.344 mmol) and tri-
ethylamine (0.0929 mL, 0.667 mmol), and the mixture was stirred
at room temperature for 1 h. The reaction mixture was concentrated
under reduced pressure, and ethyl acetate was added to the
obtained residue. The organic layer was washed with water and
saturated brine, dried over anhydrous Na2SO4 and concentrated
5.3. Assay for inhibitory activity against collagen degradation
Bovine nasal septum cartilage was sliced, and the slices were
maintained in the medium of a 1:1 (v/v) mixture of Dulbecco’s

H. Nara et al. / Bioorg. Med. Chem. 22 (2014) 5487–5505
5505
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supplemented with 10 ng/mL IL-1b and 50 ng/mL oncostatin M in
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release in the media from each explant was determined as a mea-
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O
Acknowledgements
O
N
NH
We thank Mr. Hiroaki Omae and Ms. Yoko Tanaka for the prep-
aration of the catalytic domain of human MMP-13 for X-ray struc-
ture analysis, Ms. Mika Murabayashi for hit identification by NMR
measurements, and Mr. Akira Fujishima for supporting crystallo-
graphic study. The authors thank our supervisors: Dr. Shigenori
Ohkawa, Dr. Yuji Ishihara, Dr. Yoshinori Ikeura, and Dr. Masayuki
Takizawa, for valuable discussions and critical comments through-
out this work. We thank Dr. Keiji Kamiyama and Dr. Nobuo Cho for
careful reading of the manuscript. We gratefully acknowledge
fruitful discussions with Mr. Akira Kaieda. We also thank the mem-
bers of drug metabolism & pharmacokinetics research laboratories,
biomolecular research laboratories, and analytical development
laboratories for pharmacokinetic and physicochemical studies,
the members of drug safety research laboratories for toxicokinetic
and toxicological studies, and the members of the Takeda analyti-
cal research laboratories, Ltd. for elemental analyses.
N
H
N
S
MeO
O
I
18. Abbenante, G.; Fairlie, D. P. Med. Chem. 2005, 1, 71.
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Lahm, H. W.; Lawton, G.; Merritt, J. E.; Nixon, J. S.; Rothlisberger, U.; Whittle, A.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.07.025.
25. Temple, J., Davis L. U.S. 4054656.
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