Design, synthesis and bioactivities of novel diarylthiophenes: Inhibitors of tumor necrosis factor-α (TNF-α) production

Article abstract of DOI:10.1016/S0968-0896(02)00224-9

The design, synthesis and SAR of novel diarylthiophene derivatives were performed. These compounds were designed by structural hybridization of TNF-α production inhibitors bearing 4-fluorophenyl and 4-pyridyl groups such as FR133605, FR167653 and SB210313

Full text of DOI:10.1016/S0968-0896(02)00224-9

Bioorganic & Medicinal Chemistry 10 (2002) 3113–3122
Design, Synthesis and Bioactivities of Novel Diarylthiophenes:
Inhibitors of Tumor Necrosis Factor-ꢀ (TNF-ꢀ)Production
Masakazu Fujita,* Tetsuya Hirayama and Naoko Ikeda
Pharmaceutical Research Laboratories, Nikken Chemicals Co., Ltd., 1-346, Kitabukuro-cho, Saitama-shi, Saitama 330-0835, Japan
Received 17 May 2002; accepted 14 June 2002
Abstract—The design, synthesis and SAR of novel diarylthiophene derivatives were performed. These compounds were designed by
structural hybridization of TNF-a production inhibitors bearing 4-fluorophenyl and 4-pyridyl groups such as FR133605, FR167653
and SB210313, and 6-acetyl-3-ethoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine (1) found previously by us. As a result, sev-
eral compounds were more potent in vitro than FR133605 against TNF-a production stimulated with lipopolysaccharide (LPS).
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
(1) indicate more excellent inhibitory activity for TNF-a
production by oral dosing and some excellent profiles in
adjuvant-induced arthritic model (AIA) of rat as a can-
didate for antirheumatic drugs.12 On the other hand,
recently, a number of excellent TNF-a production inhi-
bitors bearing 4-fluorophenyl and 4-pyridyl groups such
as FR133605,13 FR16765314 and SB21031315 have been
reported (Fig. 1).11 These results and reports encour-
aged us to discover more excellent candidates. There-
fore, by structural hybridization of these TNF-a
production inhibitors and 1, we designed and synthesized
2-diethylcarbamoylamino-3-ethoxycarbonyl-4-(4-fluor-
ophenyl)-5-(4-pyridyl)thiophene (9c), further 2-ethyl-
Under normal conditions, the production of cytokines
helps clear viral or bacterial infections and damaged
cells from injured tissues. However, the overproduction
of proinflammatory cytokines such as tumor necrosis
factor-a (TNF-a), interleukin-1b (IL-1b) and inter-
leukin-6 (IL-6) is known to cause immune and inflam-
matory diseases.1 In particular, the overproduction of
TNF-a has been strongly implicated in the pathogenesis
of such diseases as septic shock,2 rheumatoid arthritis
(RA),3 inflammatory bowel disease (IBD),4 multiple
sclerosis,5 type II diabetes6 and AIDS.7 In addition,
TNF-a is a potent inducer of IL-1, IL-6, IL-8, GM-CSF
and so on.8 Therefore, agents which can inhibit the
production of TNF-a have attracted much attention as
potential therapies for the direct or indirect treatment of
these diseases. Recently, in a clinical trial, the TNF-a
antibodies, Remicade (infliximab),9 and the soluble
TNF receptor, Enbrel (etanercept)3,10 have been repor-
ted to have excellent efficacy in RA and Crohn’s
patients. However, these biopharmaceuticals are expen-
sive and inconvenient to patients. Given these back-
grounds, currently, small molecule anti-TNF-a agents
are being exploited through multiple approaches.11
In a previous paper we have found that 6-acetyl-3-
ethoxycarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine
*Corresponding author. Fax: +81-48-641-5749;
e-mail: mfujita-nikken@nifty.com
Figure 1.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00224-9

3114
M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
Figure 2.
amino-5-(4-fluorophenyl)-6-(4-pyridyl)-4H-thieno[2,3-
d][1,3]oxazin-4-one 11 and 5-(4-fluorophenyl)-6-(4-pyr-
idyl)thieno[2,3-d]pyrimidines 12–17 (Fig. 2). We report
here the design, synthesis and SAR of these compounds.
yield. 3-ethyl-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-
d]pyrimidine-2,4(1H, 3H)-dione (12) was afforded by
sodium ethoxide-promoted Dimroth rearrangement of
11 in 88% yield (Scheme 2).18
The reaction of 7c with triethyl orthoformate provided
5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-d]pyrimidin-
4(3H)-one (13a) in 86% yield. Pyrimidones 13b–c were
given by acylation of 7c followed by cyclocondensation
with sodium methoxide in 34–51% yield. Compounds
14a and 14b were obtained by methylation of 13b and
13c, respectively. Compounds 15 were prepared by
chlorination of 13 with phosphorus oxychloride. Subse-
quently, compounds 15a and 15b were converted to 16
and 17 by amination, respectively (Scheme 3).
Chemistry and Biology
A series of the compounds listed in Tables 1–3 were
synthesized by the methods illustrated in Scheme 1–3.
The syntheses of compounds 9 and 10 were prepared via
amines 7 and 8 from ketones 3 and 6, respectively, using
synthetic sequence previously reported by us.16 Com-
pounds 3 and 6 were synthesized from compounds 2
and 4, respectively, by the method of Bender et al.17
(Scheme 1).
As previously described, these compounds were eval-
uated for their ability to inhibit LPS-stimulated pro-
duction of TNF-a in rat whole blood. IC50 of TNF-a
production was determined by comparison of yield with
a control to which no test compound was added.
FR133605 that showed the most potent in vivo activity
in our research into many published TNF-a inhibitors
and Dexamethasone were used as the positive control.
Furthermore, representative compounds which possess
Oxazine (11) was provided by deesterification and
cyclocondensation of urea 9d with a mixture of tri-
fluoroacetic acid and trifluoroacetic anhydride in 96%
Table 1. In vitro inhibitory activity of TNF-a production for com-
pounds 7–10
Table 2. In vitro inhibitory activity of TNF-a production for com-
pounds 11–14
Compd
Ar1
Ar2
R1
R2
In vitro
IC50 (mM)a
7a
8a
7b
7c
7d
9a
10a
9b
9c
10b
9d
9e
4-FPh
4-Pyridyl
4-FPh
4-FPh
4-FPh
4-FPh
4-Pyridyl
4-FPh
4-FPh
4-Pyridyl
4-FPh
4-FPh
4-Pyridyl COOEt
4-FPh COOEt
4-Pyridyl COOH
4-Pyridyl CONH2
H
H
H
H
H
56
55
>100
>100
>100
36
4-Pyridyl
CN
Compd
R3
X
In vitro IC50 (mM)a
4-Pyridyl COOEt COCHMe2
4-FPh COOEt COCHMe2
4-Pyridyl CONH2 COCHMe2 >100
4-Pyridyl COOEt
4-FPh COOEt
4-Pyridyl COOtBu CONHEt
37
11
NHEt
H
Me
CHMe2
Me
CHMe2
O
NH
NH
NH
NMe
NMe
>100
>100
>100
>100
>100
>100
20
CONEt2
CONEt2
2.6
1.7
5.4
13a
13b
13c
14a
14b
12b
4-Pyridyl
CN
COOEt
CONEt2
CONEt2
24
1
FR133605
Dexamethasone
–(CH2)2N(Ac)CH2–
7.1ꢀ1.3
10.2ꢀ1.4
0.02
aIC50 of LPS-stimulated TNF-a production in rat whole blood.
aIC50 of LPS-stimulated TNF-a production in rat whole blood.
bSee Figure 2.

M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
3115
excellent in vitro activity were tested in vivo after oral
administration. TNF-a inhibitory activity was assessed
by in vivo inhibition of serum TNF-a production in the
rat.16
Table 3. In vitro inhibitory activity of TNF-a production for com-
pounds 15–17
Results and Discussion
According to our previous report, it suggests that che-
mical modification of the pyridine moiety of 4,5,6,7-tet-
rahydrothieno[2,3-c]pyridine analogues makes little
difference to the activity.12 Therefore, we designed 9c by
structural hybridization, that is, changing the pyridine
moiety to 4-fluorophenyl and 4-pyridyl groups. Fur-
thermore, bicyclic compounds 11–17, which were pro-
vided by cyclization of the analogues of 9c, were
synthesized and evaluated for their ability to inhibit
lipopolysaccharide (LPS)-stimulated production of
TNF-a in rat whole blood.
Compd
R3
R4
In vitro IC50 (mM)a
15a
15b
15c
16a
16b
17a
16c
16d
16e
17b
16f
17c
16g
16h
17d
16i
H
Me
CHMe2
H
H
Me
H
H
H
Me
H
Me
H
H
Me
H
H
H
H
H
H
Cl
Cl
Cl
NHEt
NHCH2Ph
NHCH2Ph
2.9
14
15
40
14
15
66
29
29
17
10
20
NHCH2(4-Cl-Ph)
NH-Piperonyl
NH(CH2)2OH
NH(CH2)2OH
NH(CH2)2OEt
NH(CH2)2OEt
NHCH2CH(OEt)2
NH(CH2)2O(CH2)2OH
NH(CH2)2O(CH2)2OH
NH(CH2)2NH2
NHCH2COOH
NHCH2COOEt
NHCH(Me)COOEt
NEt2
>100
10
13
As we expected, compound 9c exhibited 4-fold more
potent activity than FR133605. Based on our previous
SAR of 4,5,6,7-tetrahydrothieno[2,3-c]pyridines, we
initially investigated in vitro SAR of 9c (Table 1).
Compound having basic amino group at the 2-position
was weaker active. Further, carboxylic acid, carbox-
amide and cyanide analogues were inactive (see 7a–d).
In the case of acylation of their amino groups, the car-
bamide analogue was similarly inactive, while the cya-
nide analogue indicated increased activity (7c vs 9b and
7d vs 9e). Isobutyrylation of 7a and 8a showed slightly
more potent activity (9a and 10a). tert-Butylester 9d
showed excellent in vitro activity, but was essentially
inactive in the in vivo activity (data not shown). This
modification in the above cases afforded very similar
88
16j
16k
16l
16m
16n
>100
>100
>100
>100
40
N((CH2)2OEt)2
aIC50 of LPS-stimulated TNF-a production in rat whole blood.
results as the previous. On the other hand, the in vitro
activities for regioisomers were found to be equipotent
(7a, 9a, 9c vs 8a, 10a, 10b, respectively). However, as
these compounds were tested in vivo after oral admin-
istration, the inhibitions of 9a, 10a, 9c and 10b at 50 mg/
Scheme 1. Reagents and conditions: (a) 4-FPhCOOEt, LHMDS, THF, 0 ꢁC, quant.; (b) NCCH2R1, S, base/EtOH or pyridine, 60 ꢁC, 39–72%; (c)
4-FPhCH2CN, NaOEt, EtOH, reflux; (d) 48% HBr, reflux, 23% (two steps); (e) for 9a–b and 10a: Me2CHCOCl, pyridine, 12%–quant. For 9c–e
and 10b: Et2NH or EtNH2, triphosgene, Et3N, CH2Cl2, 0 ꢁC–rt, 47%–quant.
Scheme 2. Reagents and conditions: (a) EtNH2, triphosgene, Et3N, CH2Cl2, 71%; (b) (CF3CO)2O, TFA, 0 ꢁC–rt, 96%; (c) NaOEt, EtOH, reflux,
88%.

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M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
Scheme 3. Reagents and conditions: (a) CH(OEt)3, PhMe, reflux, 86%; (b) (i) AcCl or Me2CHCOCl, pyridine; (ii) NaOMe, MeOH, reflux, 34–51%;
(c) MeI, NaH, DMF, 40–79% (d) POCl3, 100 ꢁC, 71–93%; (e) amines, Et3N/CH2Cl2 or NaOH/THF, rt–reflux, 65%–quant.
kg were 22.1, 12.3, 53.1 and 34.2%, respectively.
Namely, it demonstrates that 4-(4-fluorophenyl)-5-(4-
pyridyl)thiophene analogues are desirable.
the hydroxyl with an ethyl groupand repeating them led
to increased potency up to 10 mM (16f and 16h). N-dis-
ubstituted amino groups had diminished potency (16f vs
16n and 16a vs 16m). Incorporation of an acid sub-
stituent (16j), a basic substituent (16i) or polar groups
such as ethoxycarbonyl (16k–l) and diethylacetal (16g)
groups were found to be deleterious toward activity.
Aryl residues showed the decrease in activity in the
order of 4-Cl-benzyl < piperonyl < benzyl (16b–d).
Next, we investigated the activities for bicyclic com-
pounds 11–17.
Oxazinone 11 and a series of pyrimidones 13–14 were
inactive despite pyrimidinedione 12 revealed weaker
activity (Table 2).
Representative compounds that possess excellent in
vitro activity were tested in vivo after oral administra-
tion at 50 mg/kg (Table 4). TNF-a inhibitory activity
was assessed by in vivo inhibition of serum TNF-a
production in the rat.16 As a result, compound 17b was
slightly weaker than 1.
The results of pyrimidines 15–17 are summarized in
Table 3. Keeping the substituents at the 4-position,
modification of 2-substituents suggests that hydrogen
atom is preferred to methyl and isopropyl groups (e.g.,
15a vs 15b vs 15c). Next, to see the substituent effect of
the 4-position, the introductions of N-substituted amino
groups were examined. Ethylamino group had an IC50
value of 40 mM (16a). A hydroxyl groupwas oriented to
the rear of the ethyl, resulting in increased activity (16e).
Further, extending the alkyl group outward by capping
Conclusion
We performed the design, synthesis and SAR of novel
diarylthiophene derivatives. Several compounds exhib-
ited more potent in vitro activity than 1 and FR133605
but they could not exceed the two by oral dosing at 50
mg/kg. However, these results suggest that further
SAR of novel diarylthiophene derivatives lead to the
discovery of orally excellent agents. At present, a
number of novel diaryl substituted heterocyclic com-
pounds that possess more potent TNF-a inhibitory
activity than 1 and FR133605 are synthesized, and it
continues. In addition, interestingly, although diaryl
substituted heterocyclic inhibitors of p38 MAP kinase
have been reported,19 fortunately, many of our com-
pounds have not almost shown inhibition of p38 MAP
kinase. On the other hand, it is very important to
understand the action mechanism of them. Therefore,
our studies are currently in progress toward the goal
and a complete report containing them will appear
elsewhere.
Table 4. In vivo inhibitory activities of TNF-a production for
representative compounds
Compd
Inhibition (%) at 50 mg/kg, poa
9c
9d
53.1
4.8
10b
15a
15b
15c
16b
16f
34.2
20.1
15.8
17.1
69.2
50.1
16h
17a
17b
17d
1
58.9
58.1
78.3
45.1
85.1ꢀ2.8
FR133605
88.1ꢀ3.5
aInhibition of LPS-stimulated serum TNF-a production in the rat.

M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
3117
Experimental
was added and the solution was extracted with AcOEt.
The combined organic layers were washed with brine,
dried with MgSO4 and evaporated. The residue was
purified by column chromatography on silica gel eluting
with a mixture of CHCl3/MeOH (15:1 v/v) followed by
crystallization from Et2O afforded 7a (3.7 g, y. 39%);
mp: 210–213 ꢁC; 1H NMR (CDCl3) d: 1.02 (3H, t,
J=7.2 Hz, CH3(Et)), 4.07 (2H, q, J=7.2 Hz, CH2(Et)),
5.74 (2H, br s, NH2), 6.09 (2H, t, J=8.8 Hz, Ph), 7.16
(2H, dd, J=4.4, 8.8 Hz, Ph), 7.28 (2H, dd, J=1.6, 4.4
Hz, Pyridyl), 8.62 (2H, dd, J=1.6, 4.4 Hz, Pyridyl);
HRMS (FAB+) m/z exact mass calcd for
C18H15FN2O2S 342.0838, found 342.0841.
Chemistry
General procedures. Infrared (IR) spectra were recorded
on a Perkin-Elmer FT-IR SPECTRUM 2000 using KBr
pellets. 1H NMR spectra were obtained on a Jeol
JNM-AL 400. Chemical shifts are reported in parts per
million relative to TMS as internal standard. Melting
points were determined using Yanaco MP-500D and are
uncorrected. Mass spectra (MS) and high-resolution
mass spectra (HRMS) were measured on a Thermo-
Quest FINNIGAN AQA and a Micromass VG-70VSE,
respectively. When air- or moisture-sensitive reagents
were used, reactions were run under argon. All reagents
and solvents were of commercial quality and used with-
out further purification unless indicated otherwise. Col-
umn chromatography was performed on columns of E.
Merck silica gel 60 (230–400 mesh).
2-Amino-4-(4-fluorophenyl)-3-hydroxycarbonyl-5-(4-pyri-
dyl)thiophene (7b). This was prepared as described in the
synthesis of 7a; mp: 221–223 ꢁC; H NMR (CDCl3) d:
1
6.75 (2H, d, J=5.6 Hz, Pyridyl), 7.12–7.25 (4H, m, Ph),
7.79 (2H, br s, NH2), 8.25 (2H, d, J=5.6 Hz, Pyridyl),
11.91 (1H, br s, COOH); HRMS (FAB+) m/z exact
mass calcd for C16H11FN2O2S 314.0525, found
314.0520.
1-(4-Fluorophenyl)-2-(4-pyridyl)ethanone (3). To a mix-
ture of 4-methylpyridine (2) (5.0 g, 53.7 mmol) and ethyl
4-fluorobenzoate (9.0 g, 53.5 mmol) in THF (40 mL)
was added dropwise lithium bis(trimethylsilyl)amide
(53.5 mL, 1.0 M solution in THF) at 0 ꢁC. After stirring
for 1 h, the reaction mixture was allowed to warm to
room temperature, triturated with hexane (138 mL) and
filtered. The solid was dissolved in 3 N HCl (50 mL).
The solution was neutralized with saturated aqueous
NaHCO3 and extracted with AcOEt. The combined
organic layers were washed with H2O followed by brine,
dried with MgSO4 and evaporated to afford 3 (10.5 g, y.
2-Amino-3-carbamoyl-4-(4-fluorophenyl)-5-(4-pyridyl)-
thiophene (7c). This was prepared as described in the
ꢁ
1
synthesis of 7a; mp: 243–246 C; H NMR (CDCl3) d:
5.02 (2H, br s, NH2), 6.17 (2H, br s, CONH2), 7.04 (2H,
t, J=8.8 Hz, Ph), 7.23–7.28 (2H, m, Ph), 7.54 (2H, dd,
J=1.6, 4.4 Hz, Pyridyl), 8.91 (2H, dd, J=1.6, 4.4 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C16H12FN3OS 313.0685, found 313.0692.
1
91%) as a light yellow solid; H NMR (CDCl3) d: 4.27
(2H, s, CH2), 7.11–7.21 (4H, m, Pyridyl+Ph), 8.00–8.05
(2H, m, Ph), 8.57 (2H, dd, J=2.0, 4.8 Hz, Pyridyl).
2-Amino-3-cyano-4-(4-fluorophenyl)-5-(4-pyridyl)thio-
phene (7d). To a mixture of 3 (4.0g, 18.6 mmol), sulfur
(0.8 g, 25.0 mmol) and malononitrile (1.4 g, 21.2 mmol)
in pyridine (40 mL) was added morpholine (0.8 g, 9.2
mmol). After stirring at 60 ꢁC for 3 days, the reaction
mixture was evaporated. Then H2O was added and the
solution was extracted with AcOEt. The combined
organic layers were washed with brine, dried with
MgSO4 and evaporated. The residue was purified by
column chromatography on silica gel eluting with a
mixture of CHCl3/MeOH (100:1 followed by 15:1 v/v)
to afford 7d (2.1 g, y. 39% ); mp: 254–256 ꢁC; 1H NMR
(CDCl3) d: 6.97 (2H, d, J=4.8 Hz, Pyridyl), 7.11 (2H, t,
J=8.8 Hz, Ph), 7.24–7.33 (2H, m, Ph), 8.33 (2H, d,
J=4.8 Hz, Pyridyl); HRMS (FAB+) m/z exact mass
calcd for C16H10FN3S 295.0579, found 295.0571.
2-(4-Fluorophenyl)-1-(4-pyridyl)ethanone (6). To a mix-
ture of ethyl isonicotinate (4) (99.5 g, 0.66 mol) and 4-
fluorophenylacetonitrile (100 g, 0.74 mol) in EtOH (300
mL) was added sodium ethoxide (59 g, 0.87 mol) at
room temperature. After reflux for 24 h, the cooled
reaction mixture was neutralized with concd. HCl and
extracted with AcOEt. The combined organic layers
were washed with H2O followed by brine, dried with
MgSO4 and evaporated to afford 2-(4-fluorophenyl)-3-
hydroxy-3-(4-pyridyl)acrylonitrile (5). A solution of 5 in
concd. HBr (100 mL) was heated to reflux for 4 h. The
reaction solution was kept at room temperature for 1 h,
neutralized with K2CO3 and extracted with AcOEt. The
combined organic layers were washed with H2O, dried
with MgSO4 and evaporated. The residue was purified
by column chromatography on silica gel eluting with a
mixture of CHCl3/MeOH (50:1 v/v) to afford 6 (35.7 g,
2-Amino-3-ethoxycarbonyl-5-(4-fluorophenyl)-4-(4-pyri-
dyl)thiophene (8a). This was prepared as described in
the synthesis of 7a with 6 instead of 3; mp: 161–163 ꢁC;
1H NMR (CDCl3) d: 0.98 (3H, t, J=7.6 Hz, CH3 (Et)),
4.05 (2H, q, J=7.6 Hz, CH2 (Et)), 6.24 (2H, s, NH2),
7.00–7.04 (2H, m, Harom), 7.13 (2H, t, J=8.4 Hz, Ph),
7.27–7.40 (2H, m, Harom), 8.35 (2H, dd, J=1.6, 4.8 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C18H15FN2O2S 342.0838, found 342.0837.
1
y. 23%) as a light yellow solid; H NMR (CDCl3) d:
4.26 (2H, s, CH2), 6.98–7.04 (2H, m, Ph), 7.17–7.22 (2H,
m, Ph), 7.74 (2H, dd, J=2.0, 4.4 Hz, Pyridyl), 8.78 (2H,
dd, J=2.0, 4.4 Hz, Pyridyl).
2-Amino-3-ethoxycarbonyl-4-(4-fluorophenyl)-5-(4-pyri-
dyl)thiophene (7a). To a mixture of 3 (6.0 g, 27.9
mmol), sulfur (1.1 g, 34.3 mmol) and ethyl cyanoacetate
(4.1 g, 36.2 mmol) in EtOH (50 mL) was added triethyl-
amine (5.6 g, 55.3 mmol). After stirring at 60 ꢁC for 3
days, the reaction mixture was evaporated. Then H2O
3-Ethoxycarbonyl-2-isobutyrylamino-4-(4-fluorophenyl)-
5-(4-pyridyl)thiophene (9a). To a solution of 7a (1.0 g,
2.92 mmol) in pyridine (20 mL) was added dropwise
isobutyryl chloride (0.34 mL, 3.25 mmol) at room

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M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
temperature. After stirring for 2 h at room temperature,
MeOH (10 mL) was added. The reaction mixture was
stirred for 30 min at room temperature and evaporated.
Then saturated aqueous NaHCO3 was added and the
solution was extracted with AcOEt. The combined
organic layers were washed with H2O followed by brine,
dried with MgSO4 and evaporated. The residue was
purified by column chromatography on silica gel eluting
with a mixture of CHCl3/MeOH (30:1 v/v) followed by
crystallization from Et2O afforded 9a (190 mg, y. 16%);
mp: 167–169 ꢁC; 1H NMR (CDCl3) d: 1.01 (3H, t,
J=7.2 Hz, CH3 (Et)), 1.33 (6H, d, J=6.8 Hz, CH3 (i-
Bu)ꢂ2), 2.61–2.75 (1H, m, CH (i-Bu)) 4.10 (2H, q,
J=7.2 Hz, CH2 (Et)), 6.92 (2H, t, J=8.8 Hz, Ph), 7.20–
7.40 (4H, m, Ph and Pyridyl),8.66 (2H, d, J=5.2 Hz,
Pyridyl), 9.85 (1H, br s, NH); HRMS (FAB+) m/z exact
mass calcd for C22H21FN2O3S 412.1257, found
412.1260.
3-Cyano-2-diethylcarbamoylamino-4-(4-fluorophenyl)-5-
(4-pyridyl)thiophene (9e). This was prepared as descri-
bed in the synthesis of 9c with 7d instead of 7a; mp :
147–149 ꢁC; H NMR (CDCl3) d: 1.28 (6H, t, J=7.2
1
Hz, CH3 (Et)ꢂ2), 3.46 (4H, q, J=7.2 Hz, CH2 (Et)ꢂ2),
7.05 (2H, dd, J=1.6, 4.4 Hz, Pyridyl), 7.10 (2H, t,
J=8.8 Hz, Ph), 7.26–7.35 (2H, m, Ph), 7.70 (1H, s,
NH), 8.45 (2H, dd, J=1.6, 4.4 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C21H19FN4OS
394.1264, found 394.1277.
3-Ethoxycarbonyl-2-isobutyrylamino-5-(4-fluorophenyl)-
4-(4-pyridyl)thiophene (10a). This was prepared as
described in the synthesis of 9a with 8a instead of 7a;
mp: 154–156 ꢁC; 1H NMR (CDCl3) d: 1.00 (3H, t,
J=7.2 Hz, CH3 (Et)), 1.34 (6H, d, J=6.8 Hz, CH3 (i-
Bu)ꢂ2), 2.68–2.73 (1H, m, CH (i-Bu)) 4.09 (2H, q,
J=7.2 Hz, CH2 (Et)), 7.12–7.21 (4H, m, Harom), 7.26–
7.33 (2H, m, Harom), 8.42 (2H, d, J=6.4 Hz, Pyridyl),
9.93 (1H, s, NH); HRMS (FAB+) m/z exact mass calcd
for C22H21FN2O3S 412.1257, found 412.1250.
3-Carbamoyl-2-isobutyrylamino-4-(4-fluorophenyl)-5-(4-
pyridyl)thiophene (9b). This was prepared as described
in the synthesis of 9a with 7c instead of 7a; mp: 243–
ꢁ
1
246 C; H NMR (CDCl3) d: 1.32 (6H, d, J=6.8 Hz,
CH3 (i-Bu)ꢂ2), 2.60–2.71 (1H, m, CH (i-Bu)), 7.05 (2H,
t, J=8.4 Hz, Ph), 7.31–7.42 (4H, m, Ph and Pyridyl),
8.08–8.25 (1H, m, NH), 8.68 (2H, d, J=4.4 Hz, Pyr-
idyl); HRMS (FAB+) m/z exact mass calcd for
C20H18FN3O2S 383.1104, found 383.1094.
2-Diethylcarbamoylamino-3-ethoxycarbonyl-5-(4-fluoro-
phenyl)-4-(4-pyridyl)thiophene (10b). This was prepared
as described in the synthesis of 9c with 8a instead of 7a;
mp: 140–142 ꢁC; 1H NMR (CDCl3) d: 0.97 (3H, t, J=6.8
Hz, CH3CH2O), 1.30 (6H, t, J=6.8 Hz, (CH3CH2)2N),
3.47 (4H, q, J=6.8 Hz, (CH3CH2)2N), 4.06 (2H, q,
J=6.8 Hz, CH3CH2O), 7.10–7.19 (4H, m, Harom), 7.26–
7.33 (2H, m, Harom),8.38 (2H, dd, J=2.0, 4.8 Hz, Pyr-
idyl), 9.65 (1H, s, NH); HRMS (FAB+) m/z exact mass
calcd for C23H24FN3O3S 441.1522, found 441.1521.
2-Diethylcarbamoylamino-3-ethoxycarbonyl-4-(4-fluoro-
phenyl)-5-(4-pyridyl)thiophene (9c). To a solution of 7a
(140 mg, 0.41 mmol) in CH2Cl2 (7 mL) was added
dropwise triphosgene (122 mg, 0.41 mmol), followed by
dropwise triethylamine (0.09 mL, 0.65 mmol) at 0 ꢁC.
After stirring for 2 h at room temperature, diethylamine
(0.34 mL, 3.28 mmol) was added. In addition, after
stirring for 1 h, H2O was added. The solution was
extracted with CH2Cl2. The combined organic layers
were washed with brine, dried with Na2SO4 and evapo-
rated. The residue was recrystallized from hexane/Et2O
to afford 9c (151 mg, y. 84%); mp: 141–143 ꢁC; 1H
NMR (CDCl3) d: 0.98 (3H, t, J=6.8 Hz, CH3CH2O),
1.30 (6H, t, J=7.2 Hz, (CH3CH2)2N), 3.45 (4H, q,
J=7.2 Hz, (CH3CH2)2N), 4.08 (2H, q, J=6.8 Hz,
CH3CH2O), 6.90 (2H, t, J=8.4 Hz, Ph), 7.26–7.33 (4H,
m, Ph and Pyridyl), 8.64 (2H, dd, J=1.6, 4.8 Hz, Pyr-
idyl), 9.55 (1H, br s, NH); HRMS (FAB+) m/z exact
mass calcd for C23H24FN3O3S 441.1522, found
441.1513.
2-Ethylamino-5-(4-fluorophenyl)-6-(4-pyridyl)-4H-thieno
[2,3-d][1,3]oxazin-4-one (11). 9d (300 mg, 0.68 mmol)
was treated dropwise with a mixture of trifluoroacetic
acid (3 mL) and trifluoroacetic anhydride (140 mL, 1.02
mmol) at 0 ꢁC. The reaction mixture was stirred for 30
min at 0 ꢁC and for an additional 16 h at room tem-
perature. The mixture was diluted with saturated
aqueous NaHCO3 and extracted with CHCl3. The
combined organic layers were washed with H2O fol-
lowed by brine, dried with MgSO4 and evaporated to an
oil. Trituration from AcOEt afford 11 (239 mg, y. 96%);
mp: 219–221 ꢁC; IR (KBr, cmꢃ1): 1743 (C¼O); 1H
NMR (CDCl3) d: 1.31 (3H, t, J=7.2 Hz, CH3), 3.45–
3.61 (2H, m, CH2), 5.18 (1H, br s, NH), 7.01 (2H, d,
J=6.0 Hz, Pyridyl), 7.07 (2H, t, J=8.8 Hz, Ph), 7.20–
7.30 (2H, m, Ph), 8.44 (2H, d, J=6.0 Hz, Pyridyl);
HRMS (FAB+) m/z exact mass calcd for
C19H14FN3O2S 367.0791, found 367.0799.
3-tert-Butoxycarbonyl-2-ethylcarbamoylamino-4-(4-fluoro-
phenyl)-5-(4-pyridyl)thiophene (9d). 7d was prepared as
described in the synthesis of 7a with t-butyl cyanoace-
tate instead of ethyl cyanoacetate. Then 9d was pre-
pared as described in the synthesis of 9c with ethylamine
instead of diethylamine; mp: 223–225 ꢁC; 1H NMR
(CDCl3) d: 1.16 (9H, s, CH3 (t-Bu)ꢂ3), 1.24 (3H, t,
J=7.2 Hz, CH3 (Et)), 3.36–3.44 (2H, m, CH2 (Et)), 4.91
(1H, br s, NH), 6.90 (2H, dd, J=1.6, 4.4 Hz, Pyridyl),
7.03 (2H,dt, J=2.0, 8.8 Hz, Ph), 7.10–7.53 (2H, m, Ph),
8.33 (2H, dd, J=1.6, 4.4 Hz, Pyridyl); HRMS (FAB+)
m/z exact mass calcd for C23H24FN3O3S 441.1522,
found 441.1525.
3-Ethyl-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-d]pyri-
midine-2,4(1H,3H)-dione (12). To a solution of 11 (200
mg, 0.54 mmol) in EtOH (20 mL) was added sodium
ethoxide (37 mg, 0.54 mmol) at room temperature.
After reflux for 1 h, the reaction mixture was allowed to
cool to room temperature. Furthermore, the reaction
mixture was cooled, neutralized with 1 N HCl and eva-
porated. The residue was diluted with H2O and extrac-
ted with AcOEt. The combined organic layers were
washed with brine, dried with MgSO4 and evaporated.
The residue was purified by column chromatography on

M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
3119
silica gel eluting with a mixture of CHCl3/MeOH (30:1
v/v) to afford 12 (175 mg, y. 88%) as a yellow solid; mp:
291–293 ꢁC; IR (KBr, cmꢃ1): 1668, 1714 (C¼O); 1H
NMR (CDCl3) d: 1.23 (3H, br t, J=7.2 Hz, CH3), 4.04
(2H, br q, J=7.2 Hz, CH2), 5.30 (1H, s, NH), 6.99 (2H,
d, J=5.2 Hz, Pyridyl), 7.09 (2H, t, J=8.4 Hz, Ph), 7.20–
7.31 (2H, m, Ph), 8.46 (2H, br d, J=5.2 Hz, Pyridyl);
HRMS (FAB+) m/z exact mass calcd for
C19H14FN3O2S 367.0791, found 367.0790.
iodide (140 mL, 2.25 mmol) was added at 0 ꢁC. The
reaction mixture was stirred for 2 h at room tempera-
ture and after adding H2O for an additional 30 min at
room temperature. Then the solution was extracted with
AcOEt. The combined organic layers were washed with
H2O followed by brine, dried with MgSO4 and evapo-
rated. The residue was purified by column chromato-
graphy on silica gel eluting with a mixture of CHCl3/
MeOH (30:1 v/v) followed by crystallization from Et2O
afforded 14a (341 mg, y. 66%); mp: 238–240 ꢁC; 1H
NMR (CDCl3) d: 2.69 (3H, s, CH3), 3.61 (3H, s, CH3N),
7.03 (2H, t, J=8.4 Hz, Ph), 7.42–7.52 (4H, m, Ph and
Pyridyl), 8.66 (2H, d, J=5.6 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C19H14FN3OS
351.0842, found 351.0847.
5-(4-Fluorophenyl)-6-(4-pyridyl)thieno[2,3- d]pyrimidin-
4(3H)-one (13a). To a solution of 7c (1.0 g, 3.2 mmol)
in toluene (20 mL) was added dropwise triethyl ortho-
formate (2.4 g, 16.2 mmol) at room temperature. After
reflux for 6 h, the reaction mixture was allowed to cool
to room temperature. The resulting precipitate was col-
lected, washed with AcOEt, CHCl3 followed by MeOH
and dried in vacuo to afford 13a (887 mg, y. 86%) as a
white solid; mp: 325–327 ꢁC; 1H NMR (CDCl3+
CD3OD) d: 7.07 (2H, t, J=8.8 Hz, Ph), 7.41–7.62 (4H,
m, Ph and Pyridyl), 8.00 (1H, s, Harom), 8.58 (2H, d,
J=6.0 Hz, Pyridyl); HRMS (FAB+) m/z exact mass
calcd for C17H10FN3OS 323.0529, found 323.0541.
5-(4-Fluorophenyl)-2-isopropyl-3-methyl-6-(4-pyridyl)thieno
[2,3-d]pyrimidin-4(3H)-one (14b). This was prepared as
described in the synthesis of 14a with 13c instead of 13b;
mp: 223–225 ꢁC; 1H NMR (CDCl3) d: 1.36 (6H, d,
J=7.2 Hz, CH3 ꢂ 2), 3.10–3.19 (1H, m, CH), 3.62 (3H,
s, CH3N), 7.03 (2H, t, J=8.4 Hz, Ph), 7.43–7.55 (4H, m,
Ph and Pyridyl), 8.65 (2H, dd, J=1.6, 4.8 Hz, Pyridyl);
HRMS (FAB+) m/z exact mass calcd for C21H18FN3OS
379.1155, found 379.1156.
5 - (4 - Fluorophenyl)- 2 - methyl - 6 - (4 -pyridyl)thieno[2,3-
d]pyrimidin-4(3H)-one (13b). To a solution of 7c (5.0 g,
16.0 mmol) in pyridine (20 mL) was added dropwise
acetyl chloride (1.25 mL, 17.6 mmol) at room tempera-
ture. After stirring for 3 h at room temperature, the
reaction mixture was evaporated. The residue was dilu-
ted with MeOH (100 mL). To the solution was added
sodium methoxide (1.7 g, 31.5 mmol) at room tempera-
ture. After reflux for 4 h, the reaction mixture was
allowed to cool to room temperature. Furthermore, the
reaction mixture was cooled, neutralized with concd.
HCl and evaporated. The residue was diluted with H2O
and extracted with AcOEt. The combined organic layers
were washed with brine, dried with MgSO4 and evapo-
rated. The residue was purified by column chromato-
graphy on silica gel eluting with a mixture of CHCl3/
MeOH (30:1 v/v) followed by crystallization from Et2O
afforded 13b (2.1 g, y. 40%); mp: 319–321 ꢁC; 1H NMR
(CDCl3) d: 2.55 (3H, s, CH3), 7.05 (2H, t, J=8.4 Hz,
Ph), 7.48–7.53 (4H, m, Ph and Pyridyl), 8.66 (2H, d,
J=6.0 Hz, Pyridyl), 11.38 (1H, br s, NH); HRMS
(FAB+) m/z exact mass calcd for C18H12FN3OS
337.0685, found 337.0680.
4-Chloro-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-d]pyr-
imidine (15a). A mixture of 13a (470 mg, 1.5 mmol) and
phosphorus oxychloride (5 mL) was heated to 100 ꢁC
for 7 h, cooled to room temperature and poured into
H2O (50 mL) at 0 ꢁC. The pH was adjusted to 8 with
saturated aqueous NaHCO3. Then the solution was
extracted with AcOEt. The combined organic layers
were washed with H2O followed by brine, dried with
MgSO4 and evaporated. The residue was crystallized
from Et2O to afford 15a (459 mg, y. 93%); mp: 157–
159 ꢁC; H NMR (CDCl3) d: 7.06 (2H, t, J=8.4 Hz,
1
Ph), 7.43 (2H, d, J=5.2 Hz, Pyridyl), 7.50–7.62 (2H, m,
Ph), 8.78 (2H, d, J=5.2 Hz, Pyridyl), 8.80 (1H, s,
Harom); HRMS (FAB+) m/z exact mass calcd for
C17H9ClFN3S 341.0190, found 341.0199.
4-Chloro-5-(4-fluorophenyl)-2-methyl-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (15b). This was prepared as
described in the synthesis of 15a with 13b instead of 13a;
mp: 153–155 ꢁC; 1H NMR (CDCl3) d: 2.83 (3H, s, CH3),
7.04 (2H, dt, J=2.0, 8.4 Hz, Ph), 7.41 (2H, d, J=5.6
Hz, Pyridyl), 7.49–7.54 (2H, m, Ph), 8.77 (2H, d, J=5.6
Hz, Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C18H11ClFN3S 355.0346, found 355.0356.
5-(4-Fluorophenyl)-2-isopropyl-6-(4-pyridyl)thieno[2,3-d]
pyrimidin-4(3H)-one (13c). This was prepared by treat-
ment of 9b with sodium methoxide as described in the
ꢁ
1
synthesis of 13b; mp: 311–312 C; H NMR (CDCl3) d:
1.38 (6H, d, J=7.2 Hz, CH3ꢂ2), 2.93–3.02 (1H, m,
CH), 7.06 (2H, t, J=8.4 Hz, Ph), 7.49–7.57 (4H, m, Ph
and Pyridyl), 8.65 (2H, dd, J=1.6, 4.4 Hz, Pyridyl),
11.92 (1H, s, NH); HRMS (FAB+) m/z exact mass
calcd for C20H16FN3OS 365.0998, found 365.1001.
4-Chloro-5-(4-fluorophenyl)-2-isopropyl-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (15c). This was prepared as
described in the synthesis of 15a with 13c instead of 13a;
mp: 139–141 ꢁC; 1H NMR (CDCl3) d: 1.42 (6H, d,
J=6.8 Hz, CH3ꢂ2), 3.25–3.35 (1H, m, CH), 7.04 (2H, t,
J=8.4 Hz, Ph), 7.42 (2H, d, J=5.6 Hz, Pyridyl), 7.53
(2H, dd, J=5.6, 8.4 Hz, Ph), 8.77 (2H, d, J=5.6 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C20H15ClFN3S 383.0659, found 383.0651.
5-(4-Fluorophenyl)-2,3-dimethyl-6-(4-pyridyl)thieno[2,3-d]
pyrimidin-4(3H)-one (14a). Sodium hydride (119 mg,
60% dispersion, 3.0 mmol) was added to a stirred solu-
tion of 13b (500 mg, 1.5 mmol) in DMF (5 mL) at 0 ꢁC.
After stirring for 1 h at room temperature, methyl
4-Ethylamino-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-
d]pyrimidine (16a). To a mixture of 15a (400 mg, 1.2

3120
M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
mmol) and triethylamine (486 mg, 4.8 mmol) in CH2Cl2
(40 mL) was added ethylamine hydrochloride (196 mg,
2.4 mmol). After reflux for 10 h, the reaction mixture
was cooled to room temperature, diluted with saturated
aqueous NaHCO3 and extracted with AcOEt. The
combined organic layers were washed with brine, dried
with MgSO4 and evaporated. The residue was purified
by column chromatography on silica gel eluting with a
mixture of CHCl3/MeOH (50:1 v/v) to afford 16a (360
mg, y. 88%) as a light brown solid; mp: 181–183 ꢁC; 1H
NMR (CDCl3) d: 1.13 (3H, t, J=7.2 Hz, CH3), 3.42–
3.61 (2H, m, CH2),4.50 (1H, s, NH), 7.01 (2H, t, J=8.8
Hz, Ph), 7.40–7.51 (4H, m, Ph and Pyridyl), 8.45 (1H, s,
Harom), 8.81 (2H, d, J=6.0 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C19H15FN4S
350.1001, found 350.1003.
described in the synthesis of 16a with 2-aminoethanol
instead of ethylamine hydrochloride; mp: 191–193 ꢁC;
1H NMR (CDCl3) d: 3.64 (2H, q, J=4.8 Hz,
NHCH2CH2OH), 3.73 (1H, br s, OH), 3.77 (2H, br s,
NHCH2CH2OH), 5.15 (1H, br s, NH), 7.01 (2H, t,
J=8.4 Hz, Ph), 7.35–7.58 (4H, m, Ph and Pyridyl), 8.41
(1H, s, Harom), 8.77 (2H, d, J=5.2 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C19H15FN4OS
366.0951, found 366.0942.
4-(2-Ethoxyethylamino)-5-(4-fluorophenyl)-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (16f). This was prepared as
described in the synthesis of 16a with 2-ethoxy-
ethylamine instead of ethylamine hydrochloride; mp:
160–162 ꢁC; H NMR (CDCl3) d: 1.10 (3H, t, J=6.8
1
Hz, CH3CH2O), 3.33 (2H, q, J=6.8 Hz, CH3CH2O),
3.39 (2H, t, J=4.8 Hz, NHCH2CH2OEt), 3.58 (2H, q,
J=4.8 Hz, NHCH2CH2OEt), 5.17 (1H, br s, NH), 7.04
(2H, d, J=6.0 Hz, Pyridyl), 7.20 (2H, t, J=8.4 Hz, Ph),
7.37 (2H, dd, J=4.8, 8.4 Hz, Ph), 8.46 (2H, d, J=6.0
Hz, Pyridyl), 8.46 (1H, s, Harom); HRMS (FAB+) m/z
exact mass calcd for C21H19FN4OS 394.1264, found
394.1270.
4-Benzylamino-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-
d]pyrimidine (16b). To a mixture of 15a (200 mg, 0.59
mmol) and NaOH (48 mg, 1.2 mmol) in THF (20 mL)
was added benzylamine (129 mg, 1.2 mmol). After
reflux for 16 h, the reaction mixture was cooled to room
temperature, diluted with H2O and extracted with
AcOEt. The combined organic layers were washed with
brine, dried with MgSO4 and evaporated. The residue
was purified by column chromatography on silica gel
eluting with a mixture of CHCl3:MeOH (100:1 v/v) to
afford 16b (182 mg, y. 76%) as a white solid; mp: 199–
4-(2,2-Diethoxyethylamino)-5-(4-fluorophenyl)-6-(4-pyri-
dyl)thieno[2,3-d]pyrimidine (16g). This was prepared as
described in the synthesis of 16a with 2,2-diethoxy-
ethylamiꢁne instead of ethylamine hydrochloride; mp:
ꢁ
1
1
201 C; H NMR (CDCl3) d: 4.70 (2H, d, J=5.6 Hz,
CH2), 4.87 (1H, br, NH), 7.00 (2H, t, J=8.8 Hz, FPh),
7.18 (2H, d, J=6.8 Hz, CH2Ph), 7.22–7.35 (3H, m,
CH2Ph), 7.40 (2H, d, J=5.6 Hz, Pyridyl), 7.46 (2H, dd,
J=5.6, 8.8 Hz, FPh), 8.47 (1H, s, Harom), 8.71 (2H, d,
J=5.6 Hz, Pyridyl); HRMS (FAB+) m/z exact mass
calcd for C24H17FN4S 412.1158, found 412.1170.
161–163 C; H NMR (CDCl3) d: 1.13 (6H, t, J=6.8
Hz, CH3CH2Oꢂ2), 3.43–3.50 (2H, m, NHCH2CH),
3.58–3.67 (4H, m, CH3CH2Oꢂ2), 4.52 (1H, t, J=5.2
Hz, NHCH2CH), 4.91 (1H, br t, NH), 7.00 (2H, t,
J=8.4 Hz, Ph), 7.42–7.48 (4H, m, Ph and Pyridyl), 8.43
(1H, s, Harom), 8.79 (2H, d, J=5.6 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C23H23FN4O2S
438.1526, found 438.1519.
4-(4-Chlorobenzylamino)-5-(4-fluorophenyl)-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (16c). This was prepared as
described in the synthesis of 16a with 4-chlorobenzyl-
amine instead of ethylamine hydrochloride; mp: 198–
5-(4-Fluorophenyl)-4-[2-(2-hydroxyethoxy)ethylamino]-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (16h). This was pre-
pared as described in the synthesis of 16a with 2-(2-
aminoethoxy)ethanol instead of ethylamine hydrochlo-
ride; mp: 161–163 C; H NMR (CDCl3) d: 1.87 (1H, t,
J=4.4 Hz, OH), 3.43 (2H, t, J=4.8 Hz, NHCH2CH2O),
3.49 (2H, t, J=4.8 Hz, NHCH2CH2O), 3.55–3.75 (4H,
m, OCH2CH2OH), 5.09 (1H, s, NH), 7.04 (2H, d,
J=6.0 Hz, Pyridyl), 7.22 (2H, t, J=8.4 Hz, Ph), 7.37
(2H, dd, J=5.2, 8.4 Hz, Ph), 8.46 (2H, d, J=6.0 Hz,
Pyridyl), 8.49 (1H, s, Harom); HRMS (FAB+) m/z exact
mass calcd for C21H19FN4O2S 410.1213, found
410.1217.
201 ꢁC; H NMR (CDCl3) d: 4.67 (2H, d, J=6.4 Hz,
1
ꢁ
1
CH2), 4.83 (1H, br t, NH), 7.01 (2H, t, J=5.2 Hz, Ph),
7.14 (2H, d, J=8.4 Hz, Ph), 7.27–7.29 (2H, m, Ph), 7.40
(2H, dd, J=1.6, 4.4 Hz, Pyridyl), 7.41–7.48 (2H, m, Ph),
8.50 (1H, s, Harom), 8.74 (2H, dd, J=1.6, 4.4 Hz, Pyr-
idyl); HRMS (FAB+) m/z exact mass calcd for
C24H16ClFN4S 446.0768, found 446.0776.
5 - (4 - Fluorophenyl)- 4 - piperonylamino - 6 - (4 - pyridyl)-
thieno[2,3-d]pyrimidine (16d). This was prepared as
described in the synthesis of 16a with piperonylamine
instead of ethylamine hydrochloride; mp: 164–166 ꢁC;
1H NMR (CDCl3) d: 4.42 (2H, d, J=4.8 Hz, CH2Ph),
4.78 (1H, br, NH), 5.96 (2H, s, OCH2O), 6.52 (1H, d,
J=7.6 Hz, Ph), 6.53 (1H, s, Ph), 6.72 (1H, d, J=7.6 Hz,
Ph), 7.06 (2H, d, J=4.8 Hz, Pyridyl), 7.09 (2H, t, J=8.4
Hz, FPh), 7.28–7.35 (2H, m, FPh), 8.46 (2H, d, J=4.8
Hz, Pyridyl), 8.53 (1H, s, Harom); HRMS (FAB+) m/z
exact mass calcd for C25H17FN4O2S 456.1056, found
456.1050.
4-(2-Aminoethylamino)-5-(4-fluorophenyl)-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (16i). This was prepared
as described in the synthesis of 16a with ethylene-
diamine instead of ethylamine hydrochloride; mp:
167–169 ꢁC; 1H NMR (CDCl3) d: 1.26 (2H, br,
NHCH2CH2NH2), 2.84 (2H, t, J=5.2 Hz, NHCH2-
CH2NH2), 3.49 (2H, t, J=5.2 Hz, NHCH2CH2NH2),
5.34 (1H, s, NHCH2CH2NH2), 7.01 (2H, t, J=8.4 Hz,
Pyridyl), 7.43–7.49 (4H, m, Ph), 8.43 (1H, s, Harom),
8.77 (2H, d, J=8.4 Hz, Pyridyl); HRMS (FAB+) m/z
exact mass calcd for C19H16FN5S 365.1110, found
365.1121.
5-(4-Fluorophenyl)-4-(2-hydroxyethylamino)-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (16e). This was prepared as

M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
3121
5-(4-Fluorophenyl)-4-[(hydroxycarbonylmethyl)amino]-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (16j). This was pre-
pared as described in the synthesis of 16b with glycine
ethyl ester hydrochloride and 2 N NaOH instead of
benzylamine and NaOH, respectively; mp: 235–238 ꢁC;
1H NMR (DMSO-d6) d: 4.05 (2H, d, J=5.6 Hz, CH2),
6.00 (1H, t, J=5.6 Hz, NH), 7.25 (2H, t, J=8.8 Hz,
Ph), 7.42–7.46 (2H, m, Ph), 7.51 (2H, dd, J=1.6, 4.4
Hz, Pyridyl), 8.36 (1H, s, Harom), 8.72 (2H, dd, J=1.6,
4.4 Hz, Pyridyl), 11.05 (1H, br, COOH); HRMS
(FAB+) m/z exact mass calcd for C19H13FN4O2S
380.0743, found 380.0741.
(4H, m, Ph), 8.47 (1H, s, Harom), 8.68 (2H, d, J=8.4 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C25H27FN4O2S 466.1839, found 466.1848.
4-Benzylamino-5-(4-fluorophenyl)-2-methyl-6-(4-pyridyl)-
thieno[2,3-d]pyrimidine (17a). This was prepared as
described in the synthesis of 16b with 15b instead of 15a;
mp: 194–197 ꢁC; 1H NMR (CDCl3) d: 2.60 (3H, s, CH3),
4.72 (2H, d, J=5.6 Hz, CH2), 4.82 (1H, br, NH), 7.01
(2H, t, J=8.8 Hz, FPh), 7.18 (2H, d, J=6.8 Hz, Ph),
7.22–7.37 (3H, m, Ph), 7.40 (2H, d, J=5.6 Hz, Pyridyl),
7.46 (2H, dd, J=5.6, 8.8 Hz, FPh), 8.71 (2H, d, J=5.6
Hz, Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C25H19FN4S 426.1314, found 426.1313.
4-[(Ethoxycarbonylmethyl)amino]-5-(4-fluorophenyl)-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (16k). This was pre-
pared as described in the synthesis of 16a with glycine
ethyl ester hydrochloride instead of ethylamine hydro-
chloride; mp: 136–138 ꢁC; 1H NMR (CDCl3) d: 1.27
(3H, t, J=7.2 Hz, CH3CH2O), 4.15–4.26 (4H, m,
CH3CH2O and NHCH2), 5.20 (1H, br t, NH), 7.02 (2H,
t, J=8.4 Hz, Ph), 7.47–7.51 (4H, m, Ph and Pyridyl),
8.45 (1H, s, Harom), 8.82 (2H, d, J=7.2 Hz, Pyridyl);
HRMS (FAB+) m/z exact mass calcd for
C21H17FN4O2S 408.1056, found 408.1055.
5-(4-Fluorophenyl)-4-(2-hydroxyethylamino)-2-methyl-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (17b). This was pre-
pared as described in the synthesis of 16e with 15b
ꢁ
1
instead of 15a; mp: 177–180 C; H NMR (CDCl3) d:
2.62 (3H, s, CH3), 3.62 (2H, dt, J=4.4, 5.2 Hz,
NHCH2CH2OH),
3.77
(2H,
t,
J=4.4
Hz,
NHCH2CH2OH), 4.44 (1H, br s, OH), 5.04 (1H, t like,
NH), 7.00 (2H, t, J=8.4 Hz, Ph), 7.35–7.55 (4H, m, Ph
and Pyridyl), 8.77 (2H, d, J=6.0 Hz, Pyridyl); HRMS
(FAB+) m/z exact mass calcd for C20H17FN4OS
380.1107, found 380.1100.
4-[(1-Ethoxycarbonylethyl)amino]-5-(4-fluorophenyl)-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (16l). This was pre-
pared as described in the synthesis of 16a with alanine
ethyl ester hydrochloride instead of ethylamine hydro-
chloride; mp: 171–173 ꢁC; 1H NMR (CDCl3) d: 1.26
(3H, t, J=7.2 Hz, CH3CH2O), 1.40 (3H, d, J=7.2 Hz,
CH3CH), 4.18 (2H, q, J=7.2 Hz, CH3CH2O), 4.84 (1H,
quint, J=7.2 Hz, NHCH), 5.28 (1H, d, J=7.2 Hz, NH),
7.02 (2H, t, J=8.4 Hz, Ph), 7.43–7.51 (4H, m, Ph and
Pyridyl), 8.43 (1H, s, Harom), 8.82 (2H, d, J=5.6 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C22H19FN4O2S 422.1213, found 422.1201.
4-(2-Ethoxyethylamino)-5-(4-fluorophenyl)-2-methyl-6-
(4-pyridyl)thieno[2,3-d]pyrimidine (17c). This was pre-
pared as described in the synthesis of 16f with 15b
instead of 15a; mp: 153–155 ꢁC; H NMR (CDCl3) d:
1
1.10 (3H, t, J=6.8 Hz, CH3CH2O), 2.62 (3H, s, CH3),
3.32 (2H, q, J=6.8 Hz, CH3CH2O), 3.37 (2H, t, J=4.8
Hz, NHCH2CH2OEt), 3.58 (2H, q, J=4.8 Hz,
NHCH2CH2OEt), 5.11 (1H, br s, NH), 7.04 (2H, d,
J=6.0 Hz, Pyridyl), 7.20 (2H, t, J=8.4 Hz, Ph), 7.37
(2H, dd, J=4.8, 8.4 Hz, Ph), 8.46 (2H, d, J=6.0 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C22H21FN4OS 408.1420, found 408.1433.
4-Diethylamino-5-(4-fluorophenyl)-6-(4-pyridyl)thieno[2,3-
d]pyrimidine (16m). To a solution of 15a (130 mg, 0.38
mmol) in CH2Cl2 (20 mL) was added diethylamine (139
mg, 1.9 mmol). After reflux for 1 day, the reaction mix-
ture was cooled to room temperature, diluted with H2O
and extracted with AcOEt. The combined organic layers
were washed with brine, dried with MgSO4 and evapo-
rated. The residue was purified by column chromato-
5-(4-Fluorophenyl)-4-[2-(2-hydroxyethoxy)ethylamino]-2-
methyl-6-(4-pyridyl)thieno[2,3-d]pyrimidine (17d). This
was prepared as described in the synthesis of 16h with
15b instead of 15a; mp: 158–160 ꢁC; H NMR (CDCl3)
1
d: 1.92 (1H, t, J=4.4 Hz, OH), 2.61 (3H, s, CH3), 3.40
(2H, t, J=4.8 Hz, NHCH2CH2O), 3.49 (2H, t, J=4.8
Hz, NHCH2CH2O), 3.56–3.75 (4H, m, OCH2CH2OH),
5.01 (1H, s, NH), 7.03 (2H, d, J=6.0 Hz, Pyridyl), 7.22
(2H, t, J=8.4 Hz, Ph), 7.38 (2H, dd, J=5.2, 8.4 Hz,
Ph), 8.46 (2H, d, J=6.0 Hz, Pyridyl); HRMS (FAB+)
m/z exact mass calcd for C22H21FN4O2S 424.1369,
found 424.1363.
graphy on silica gel eluting with a mixture of
CHCl3:MeOH (15:1 v/v) followed by crystallization
from Et2O/hexane afforded 16m (115 mg, y. 80%); mp:
175–177 ꢁC; H NMR (CDCl3) d: 0.92 (6H, t, J=7.2
1
Hz, CH3CH2ꢂ2), 3.20 (4H, q, J=7.2 Hz, CH3CH2ꢂ2),
7.00 (2H, t, J=8.4 Hz, Ph), 7.34–7.41 (4H, m, Ph and
Pyridyl), 8.46 (1H, s, Harom), 8.72 (2H, d, J=6.0 Hz,
Pyridyl); HRMS (FAB+) m/z exact mass calcd for
C21H19FN4S 378.1314, found 378.1310.
Biological methods
In vitro TNF-ꢀ inhibition assay.13,20 Whole blood from
male SD rats (Charles River Laboratories) was collected
into 2% heparinized vacuum tubes. The blood was sus-
pended in RPMI-1640 supplemented with 100 mg/mL
streptomycin and 100 U/mL penicillin at a concentra-
tion of 50%, and seeded into 24-well plate (950 mL).
Vehicle (DMSO) or test compounds in DMSO was
added to each well, and the plate was incubated at 37 ꢁC
4-Bis(2-ethoxyethyl)amino-5-(4-fluorophenyl)-6-(4-pyri-
dyl)thieno[2,3-d]pyrimidine (16n). This was prepared as
described in the synthesis of 16a with bis(2-ethoxy-
ethyl)amine instead of ethylamine hydrochloride; mp:
70–72 ꢁC; H NMR (CDCl3) d: 1.09 (6H, t, J=6.8 Hz,
1
CH3CH2ꢂ2), 3.33–3.40 (12H, m, CH3CH2ꢂ2 and
(CH2)2Oꢂ2), 7.02 (2H, t, J=8.4 Hz, Pyridyl), 7.41–7.45

3122
M. Fujita et al. / Bioorg. Med. Chem. 10 (2002) 3113–3122
in 5% CO2 for 30 min. Finally, 10 mg/mL LPS (Escher-
ichia coli B055:B5) in RPMI-1640 was added, and plates
were incubated at 37 ꢁC in 5% CO2 for 4 h. Super-
natants were then harvested, and assayed for TNF-a
using ELISA kits. IC50 of TNF-a production was
determined by comparison of yield with a control to
which no test compound was added (n=2).
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Acknowledgements
We thank Ms. Mayumi Iino for providing biological
data.
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