Synthesis and structure activity relationship studies of benzothieno[3,2-b]furan derivatives as a novel class of IKKβ inhibitors

Article abstract of DOI:10.1248/cpb.55.613

As a novel class of IKKβ inhibitors, a series of tricyclic furan derivatives was designed and synthesized based on the structure of known thiophene IKKβ inhibitors. Among the various fused furan derivatives synthesized, a benzothieno[3,2-b]furan derivative 13a displayed potent inhibitory activity towards IKKβ in enzymatic and cellular assays. The potent inhibitory activity originates from an intramolecular non-bonded S...O interaction which was confirmed by the X-ray structure of JNK3 with 16k. The introduction of further substituents on the core structure led to the discovery of the 6-alkoxy derivatives, which possessed a comparable IKKβ inhibitory activity to 13a and an improved metabolic stability. Among these, appropriately lipophilic compounds 16a, h, i, and 13g (logD>2) were found to possess good oral bioavailability.

Full text of DOI:10.1248/cpb.55.613

April 2007
Chem. Pharm. Bull. 55(4) 613—624 (2007)
613
Synthesis and Structure Activity Relationship Studies of Benzothieno[3,2-
b
b]furan Derivatives as a Novel Class of IKK Inhibitors
Hideyuki SUGIYAMA,* Masato YOSHIDA, Kouji MORI, Tomohiro KAWAMOTO, Satoshi SOGABE,
Terufumi TAKAGI, Hideyuki OKI, Toshimasa TANAKA, Hiroyuki KIMURA, and Yoshinori IKEURA
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited; 2–17–85 Jusohonmachi, Yodogawa-ku,
Osaka 532–8686, Japan. Received December 22, 2006; accepted January 18, 2007; published online January 22, 2007
As a novel class of IKKb inhibitors, a series of tricyclic furan derivatives was designed and synthesized
based on the structure of known thiophene IKKb inhibitors. Among the various fused furan derivatives synthe-
sized, a benzothieno[3,2-b]furan derivative 13a displayed potent inhibitory activity towards IKKb in enzymatic
…
and cellular assays. The potent inhibitory activity originates from an intramolecular non-bonded S O interac-
tion which was confirmed by the X-ray structure of JNK3 with 16k. The introduction of further substituents on
the core structure led to the discovery of the 6-alkoxy derivatives, which possessed a comparable IKKb in-
hibitory activity to 13a and an improved metabolic stability. Among these, appropriately lipophilic compounds
16a, h, i, and 13g (log Dꢀ2) were found to possess good oral bioavailability.
Key words IKK; kinase; inhibitor; benzothieno[3,2-b]furan; non-bonded interaction
IkB kinase (IKK) has been identified as a serine–threonine sidered to be coplanar to the thiophene ring in it’s active con-
kinase complex, which phosphorylates the inhibitor of NF- formation,11) it seemed that the relative orientation of the
kB (IkB).1—3) This complex is composed of three subunits, urea, carbamoyl, and benzene was important for IKKb inhi-
i.e. the catalytic subunits IKKa and IKKb, and the regula- bition. Thus, it was also thought that constraining the termi-
tory subunit IKKg, also called IKK-1, IKK-2, and NEMO, nal benzene to be in the active conformation could improve
respectively. The complex plays a critical role in a signaling activity. From this point of view and taking into considera-
pathway leading to NF-kB (nuclear factor-kB) activation.4) tion of patentability issues, we designed tricyclic fused furan
The activation of IKK by cytokine signals (tumor necrosis derivatives for the development of novel IKK inhibitors. In
factor (TNF)-a, interleukin (IL)-1 etc.), lipopolysaccharide this manuscript, we describe the synthesis and the SAR of
(LPS), and stress leads to phosphorylation of IkB, which the series of tricyclic furan derivatives.
triggers poly-ubiquitinylation and proteasome-dependent
degradation of IkB, resulting in transcriptional activation of Chemistry
NF-kB. It has been recognized that the kinase IKKb plays a
The synthesis of benzothieno[3,2-b]furans 13a—i and
major role in activation of NF-kB via classical (canonical) 16a—o having a 2-carbamoyl and 3-urea group, is outlined
pathway, and activation of the classical pathway promotes the in Chart 1. The substituted thiophenols 8b—g were synthe-
production of TNF-a, IL-1, intercellular adhesion molecule sized from the substituted salicylic acids 3 or anthranilic
(ICAM)-1, and cyclooxygenase (COX)-2, which indicates acids 6 according to the known procedures.12—14) These thio-
that this pathway is important for inflammation and the in- phenol derivatives were alkylated with 2-bromoacetonitrile
nate immune system. Therefore, IKKb inhibitors are ex- and cyclized using sodium ethoxide to afford benzothiophene
pected to be useful for the treatment of acute and chronic in- derivatives 10a—g. The bromo substituted derivatives 10f, g
flammatory diseases and autoimmune disorders, such as were coupled with phenylboronic acid to afford 10h, i. These
rheumatoid arthritis (RA).5) Alternatively, IKKb inhibitors phenol derivatives 10a—i were alkylated with 2-bromo- (or
are also thought to be useful for the treatment of type 2 dia- 2-iodo-) acetamide followed by cyclization to afford the
betes because activation or overexpression of the IKKb at- benzothieno[3,2-b]furan 12a—i. Finally, the amino group of
tenuated insulin signaling.6) In addition, IKKb inhibitors may 12a—i was converted into an urea group using potassium
also have application for the treatment of asthma, chronic ob- cyanate or trichloroacetyl isocyante to afford 13a—i. To
structive pulmonary disease (COPD),7) and cancer.8) Because allow further modification of 6-alkoxy derivatives, 12c was
of the key role played by the IKK complex in the NF-kB me- demethylated, and selective alkylation using various alkylat-
diated transcription, and of the druggability of kinases as a ing reagents with sodium hydride was carried out to give
target class, numerous companies have been pursuing discov- 15a—j. These 3-amino derivatives were converted to the
ery programs aimed at identifying small molecule inhibitors urea derivatives 16a—j in a similar mannar. The tert-butoxy-
of this enzyme.9,10)
carbonyl (Boc) group of 16a, b was cleaved to give amines
As part of our program to discover new IKK inhibitors, we 16k, m, respectively, and the acetylation afforded acetamide
identified 1 (MAYBRIDGE) as a potent hit compound. How- 16l. The ester derivatives 16d, e were converted to the corre-
ever, AstraZeneca has previously reported that the struc- sponding carboxylic acid 16n and alcohol 16o, respectively.
turally-analogous urea derivative 2,11) which could be derived
A 5-phenyl furan derivative 21 was synthesized as shown
from the compound 1, exhibited excellent inhibitory activity. in Chart 2. The amino group of the known furan derivative
The introduction of a urea group resulted in the improvement 1715) was protected with Boc group, and the resulting ester
of activity by as much as 100-fold compared with the amino 18 was converted to amide 19 by alkaline hydrolysis and
derivative 1 (Fig. 1). Because the benzene ring of 2 was con- subsequent amidation using 1-ethyl-3-(3-dimethylamino-
∗ To whom correspondence should be addressed. e-mail: Sugiyama_Hideyuki@takeda.co.jp
© 2007 Pharmaceutical Society of Japan

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Vol. 55, No. 4
Fig. 1. Drug Design of the Tricyclic Furan Derivative
Fig. 3. Predicted Binding Model Orientated to Interact with Asn150
Fig. 2. Predicted Binding Mode of 13a with IKKb
propyl)carbodiimide hydrochloride (WSC) and ammonium
1H-1,2,3-benzotriazol-1-olate (HOBt·NH3). N-Deprotection
of 19 by treatment with trifluoroacetic acid and subsequent
acylation of the resulting amine 20 provided urea 21.
Synthesis of the 4,5-dihydronaphtho[1,2-b]furan 26a
and 5,6-dihydro-4H-benz[6,7]cyclohepta[1,2-b]furan 26b is
shown in Chart 3. The Mitsunobu reaction of the known
cyanoketones 22a,16) b17) and ethyl glycolate afforded esters
23a, b, which were converted to amides 24a, b, respectively,
by the aminolysis using aqueous ammonia. The resulting
amides were converted to 26a, b in a manner similar to that
Fig. 4. The X-Ray Structure of JNK3 with 16k
described for the synthesis of benzothieno[3,2-b]furans in with the addition of 0.1 mg/ml bovine serum albumin (BSA)
Chart 1.
to reduce false positive activity.18) To examine the secondary
The naphthofuran derivative 32 was synthesized as shown cellular efficacy assays of IKKb inhibition, the inhibitory ac-
in Chart 4. Naphthoic acid 27 was converted to the amide 28, tivity of TNF-a production in LPS-stimulated THP-1 cells
which after alkylation of the phenolic hydroxy group fol- was evaluated.
lowed by the treatment with thionyl chloride gave the naph-
The effect of altering the central core of the furan deriva-
thonitrile 30. Cyclization using sodium ethoxide afforded the tives on inhibitory activity was evaluated using 3-aminofu-
naphthofuran 31, in which the cyano group was simultane- ran-2-carboxamide derivatives, which were either substituted
ously hydrolyzed to the amide. Finally, the amino group of or fused with other rings (Table 1). The benzothieno[3,2-
31 was converted to an urea group to afford 32.
b]furan 12a exhibited moderate inhibitory activity
The furo[2ꢁ,3ꢁ:4,5]thieno[2,3-b]pyridine 39, which pos- (IC50ꢂ5500 nM). The 5-phenyl furan 20 was as potent as 12a,
sesses a pyridine ring instead of a benzene ring in 13a, was furthermore, 25a, b, and 31 which contain a saturated or un-
synthesized as shown in Chart 5. Introduction of a sulfur saturated ring in their central core had the comparable po-
atom to 2-chloropyridine 33 was carried out using sodium tency to 12a. The thienopyridine derivative 38 was also as
hydrogen sulfide, and the resulting thiol 34 was instantly sub- potent as 12a. Next, we examined the 3-ureido derivatives of
jected to the alkylation to give 35 because 34 was easily oxi- the 3-aminofurans. All of the 3-ureido compounds exhibited
dized when isolated. The obtained 35 was converted to 39 an improvement in activity compared to the correspond-
using a method similar to that described in Chart 1.
ing 3-amino compounds. The effect was significant on the
thiophene-fused furans 13a and 39. Notably, benzothieno-
[3,2-b]furan 13a exhibited excellent potency (IC50ꢂ45 nM),
Results and Discussion
The compounds were evaluated in vitro for inhibitory po- which was 100-fold greater than the corresponding 3-amino
tency against the recombinant IKKb activity in the non-RI derivative 12a (IC50ꢂ5500 nM), while the increase on
kinase assay using a Kinase-Glo reagent (Promega, U.S.A.) furo[2ꢁ,3ꢁ:4,5]thieno[2,3-b]pyridine 39 was 72-fold. The ob-

April 2007
615
Reagents and conditions: (a) SOCl2, MeOH, reflux; (b) Me2NCSCl, DBU, DMF; (c) 210 °C; (d) aq. NaOH, reflux; (e) i) NaNO2, aq. HCl, 0 °C; ii) KSCSOEt, 85 °C; iii) aq.
NaOH, 80 °C; (f) conc. H2SO4, MeOH, reflux; (g) HCl/MeOH, reflux; (h) bromoacetonitrile, K2CO3, THF; (i) NaOEt, EtOH, reflux; (j) phenylboronic acid, Pd(Ph3P)4, K2CO3, aq.
THF, reflux; (k) 2-iodoacetamide, K2CO3, THF, reflux; (l) NaOEt, EtOH, reflux; (m) KNCO, AcOH, reflux; (n) Cl3CCONCO, THF, ꢃ78 to ꢃ30 °C then 2 N NH3 in MeOH; (o)
BBr3, CH2Cl2, reflux; (p) NaH, R2X (XꢂI, Br, Cl), (NaI), DMF, rt to 60 °C; (q) aq. HCl, MeOH, 60 °C; (r) i) TFA, CHCl3, rt, ii) Ac2O, Et3N, DMF, rt; (s) 2 N aq. NaOH, MeOH, rt;
the positional numbers of R1 are based on the [1]benzothieno[3,2-b]furan structure.
Chart 1. Synthesis of Benzothieno[3,2-b]furans
Reagents and conditions: (a) Boc2O, THF; (b) 8 N aq. NaOH, EtOH; (c) WSC, HOBt·NH3, CH3CN–DMF; (d) TFA, CHCl3; (e) Cl3CCONCO, THF, ꢃ78 to 0 °C, then 2 N NH3 in
MeOH.
Chart 2. Synthesis of 5-Phenyl Furan Derivative
Reagents and conditions: (a) ethyl glycolate, Ph3P, DEAD, THF, 0 °C; (b) 28% aq. NH3, MeOH; (c) NaOEt, EtOH, reflux; (d) Cl3CCONCO, THF, ꢃ78 to ꢃ30 °C then 2 N NH3
in MeOH.
Chart 3. Synthesis of Tricyclic Furans Having Saturated Hydrocarbon Ring
Reagents and conditions: (a) WSC, HOBt·NH3, DMF; (b) BrCH2CN, K2CO3, THF; (c) SOCl2, DMF; (d) NaOEt, EtOH, reflux; (e) KCNO, AcOH, reflux.
Chart 4. Synthesis of Naphthofuran Derivative

616
Vol. 55, No. 4
Reagents and conditions: (a) NaSH, EtOH, reflux; (b) chloroacetonitrile, NaI, K2CO3, THF; (c) NaOEt, EtOH, reflux; (d) 2-iodoacetamide, K2CO3, THF, reflux; (e) NaOEt,
EtOH, reflux; (f) Cl3CCONCO, THF, ꢃ78 to ꢃ30 °C then 2 N NH3 in MeOH.
Chart 5. Synthesis of Furo[2ꢁ,3ꢁ:4,5]thieno[2,3-b]pyridine Derivative
Table 1. Evaluation of Furan-2-carboxamide Derivatives with Various Skeletons
IKKb
IC50 (nM)
IKKb
IC50 (nM)
No.
Structure
No.
Structure
12a: R3ꢂꢄ
5500
45
25b: R3ꢂꢄ
13000
3400
13a: R3ꢂCONH2
26b: R3ꢂCONH2
20: R3ꢂH
2100
120
31: R3ꢂH
3500
160
21: R3ꢂCONH2
32: R3ꢂCONH2
25a: R3ꢂH
7100
150
38: R3ꢂH
7200
100
26a: R3ꢂCONH2
39: R3ꢂCONH2
Table 2. Profile of 13a
In vitro
PK
Rat cassette (1 mg/kg, p.o.)a)
Metabolic
stability
Solubility (mg/ml)
THP-1
(TNF-a)
IC50 (nM)
(ml/min/mg)b)
IKKb
IC50 (nM)
AUC
MRT
BA
JP1
3.4
JP2
JP2ꢅGCDC
ꢀ63
(ng·h/ml)
(h)
(%)
Rat
71
Human
55
19—45
310
ND
ND
0
3.5
a) Administration substrate: i.v., dimethyl sulfoxide; p.o., 0.5% methyl cellulose. b) Liver microsomes clearance.
served increase of the other 3-ureido compounds 21, 26a, b,
Table 3. Selectivity Profile of 13a for Representative Kinases
and 32 was smaller than that of 13a. This effect could be ex-
plained by the following hypothesis of the intramolecular
non-bonded S···O interaction.
The docking model of 13a with IKKb (using
SCWRL2.9,19,20) based on the crystal structure of PKA, PDB
ID, 1ATP21)) is shown in Fig. 2. In this model, 13a can form
hydrogen bonds with the kinase back bone via the urea and
carbamoyl moieties. Furthermore, it is likely that the urea
group of the tricyclic furan is constrained to lie in-plane with
the benzothienofuran ring by two intramolecular interactions:
Kinases
IC50 (mM)a)
Kinases
IC50 (mM)a)
IKKb
0.022
1.2
1.6
ꢀ100
0.97
ERK1
p38a
PKCq
GSK3b
15
ꢀ100
18
ASK1
TAK1
MEKK1
JNK1
1.6
a) RI-method.
i) a hydrogen bond between the urea proton and the amide ity of TNF-a production in THP-1 cell line, IC50ꢂ310 nM),
carbonyl and ii) a non-bonded S···O interaction22) between and high selectivity more than 44-fold for IKKb against sev-
the oxygen atom of urea carbonyl and the sulfur atom on the eral other serine/threonine kinases. However, 13a was not de-
thiophene ring. The later is particularly important to increase tected in rat plasma after oral administration, may be due to
IKKb inhibition, which is confirmed by the reduced potency it’s metabolic instability, although it showed acceptable solu-
observed when the intramolecular non-bonded S···O interac- bility at pH 6.8 with glycochenodeoxycholic acid (GCDC).
tion is lost by conversion of the thiophene to other rings (13a
The result of introduction of substituents on the terminal
vs. 21, 26a, b, and 32). This hypothetical S···O interaction benzene ring to improve the bioavailability of 13a is shown
was confirmed by X-ray crystallography of a complex be- in Table 4. In these derivatives, substitution at the 5- or 6-po-
tween one of the benzothienofuran derivatives 16k and the sition (13b, c) was well tolerated without reduction of the ac-
related kinase (JNK3, see below).
tivity. In contrast, introduction of substituents at the 8-posi-
The result of further profiling of the potent benzothi- tion caused decrease of activity (13e). Although introduction
eno[3,2-b]furan 13a is shown in Tables 2 and 3. The com- of a bromine at the 6- or 7-position was tolerated (13f, g), di-
pound 13a exhibited good cellular activity (inhibitory activ- rect attachment of a phenyl group to the benzene ring caused

April 2007
617
a decrease in activity (13h, i). In the cellular assay, the 6- tency was observed. The other compounds having a basic
MeO (13c) and 7-MeO (13d) derivatives had comparable po- moiety (16c, g, m) showed only moderate inhibition of the
tency to 13a, and 7-bromo derivative 13g (IC50ꢂ120 nM) was enzyme (ꢀ10-fold less potent than 16k), and introduction of
more potent than 13a. These results suggested that only hydrophobic substituents caused a decrease in enzymatic ac-
small substituents are tolerated at 7-position, and the 6-posi- tivities (16f, j). Interestingly, enzymatic potency was retained
tion is most favorable for further modification.
by carboxylic acid 16n and alcohol 16o. The carbamate 16a
With the aim of improving activity, we investigated the po- and amide 16l exhibited good enzyme inhibition, and also
tential of interaction with Asn150 of IKKb. This residue is showed moderate inhibition of TNF-a production (10ꢃ7 M of
almost completely conserved throughout the kinase family, IC50’s). Enzymatic and cellular activities were also retained
and coordinated with Mg2ꢅ in the phosphate binding site.21) after introduction of nitrogen-containing aromatic sub-
Therefore, it was considered that the basic moieties would in- stituents (16h, i). These results suggested that the presence of
teract with Asn, and the modeling study suggested that intro- certain hetero atoms is essential for enzymatic potency be-
duction of an aminoethoxy group at the 6-position of the tri- cause these substituents are located in a hydrophilic phos-
cyclic furan would provide a suitable distance to interact with phate binding site.
Asn150 (Fig. 3, nꢂ1).
A crystal structure of IKKb is not available yet, however,
According to this hypothesis, we introduced various kinds it was possible to obtain structural data from the related ki-
of basic substituents on the 6-position, including the nase, i.e. JNK3. From the X-ray structure of a complex of
aminoethoxy group, and SAR are shown in Table 5. JNK3 and 16k (Fig. 4; 2.0 Å resolution), the binding mode of
Aminoethoxy derivative 16k exhibited slightly increased ac- 16k to the ATP binding site was confirmed as predicted by
tivity (IC50ꢂ11 nM), however, a large decrease of cellular po- the binding model (Fig. 3) with the two hydrogen bonds.
However, no obvious interaction between the amine moiety
on the substituent and the Asn194 of JNK3 (Asn150 of
IKKb) was observed. On the other hand, an intramolecular
Table 4. SAR of the Substituents on the Benzene Ring of Benzo[3,2-b]fu-
rans
nonbonded S···O interaction between the urea carbonyl and
the sulfur atom on the thiophene ring predicted in the model
was clearly confirmed in this crystal structure, and the S···O
distance was 2.8 Å shorter than the corresponding van der
Waals radii (3.3 Å).
The ADMETox parameters of the selected compounds ex-
hibiting potent cellular activity (IC50ꢆ1 mM) are summarized
in Table 6. Introduction of substituents generally resulted in
the improvement of metabolic stability, thus the main site of
metabolism is presumed to be on the benzene ring. A clear
tendency was observed that the lipophilic compounds
(log Dꢀ2) 16a, h, i, and 13g possessed good oral bioavail-
ability (BA 7—32%) in rat. In contrast, the compounds 13a,
16k, and 16l with log D’s less than 2 did not show oral
bioavailability regardless of potency in cellular assay. It is as-
sumed that the membrane permeability of compounds 13a
IKKb
THP-1 (TNF-a)
No.
R1
IC50 (nM)
IC50 (nM)
13a
13b
13c
13d
13e
13f
13g
13h
13i
H
19—45
57
26
110
670
57
30
120
970
180—310
600
5-OMe
6-OMe
7-OMe
8-OMe
6-Br
7-Br
6-Ph
7-Ph
450
330
23000
1200
120
1400
2500
Table 5. SAR for Various 6-Alkoxy Tricyclic Furans
IKKb
TNF-a
IKKb
TNF-a
No.
R1
H
No.
R1
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
13a
16a
16c
16f
19—45
44
180—310
660
16i
16j
16k
25
990
11
1000
NTa)
220
NTa)
ꢀ50000
480
1900
16l
37
390
16m
110
NTa)
16g
240
34
1300
590
16n
16o
29
35
25000
1700
16h
a) NT means not tested.

618
Vol. 55, No. 4
Table 6. Pharmacokinetic Profiles of Tricyclic Furans
In vitro
PK
Metabolic
stability
Rat cassette (0.1 mg/kg, i.v., 1 mg/kg, p.o.)a)
THP-1
(ml/min/mg)c)
IKKb
(TNF-a)
Vdss
(ml/kg)
CLtotal
Cmax
AUC p.o.
MRT p.o.
BA
(%)
log D
No.
IC50 (nM)
IC50 (nM)
(ml/h/kg)
(ng/ml)
(ng·h/ml)
(h)
Rat/Human
at pH 7.4
13a
16k
16l
16a
16h
16i
19—45
11
37
44
34
180—310
ꢀ50000
390
2884
17274
394
240
856
5120
102148
1463
119
1182
643
NDb)
NDb)
NDb)
114
12
NDb)
NDb)
NDb)
736
59
NDb)
NDb)
NDb)
3.9
3.3
2.8
0
0
0
8
7
71/55
9/9
ꢃ14/2
9/21
36/21
29/27
10/14
1.44
ꢃ1.11
0.23
2.01
2.12
660
590
1000
120
25
30
677
1358
95
59
368
119
24
32
2.01
2.46
13g
2373
1.7
a) Administration substrate; i.v., dimethyl sulfoxide; p.o., 0.5% methyl cellulose. b) ND means not detected. c) Liver microsomes clearance.
and 16l is enough for cellular potency, however, they don’t
have sufficient lipophilicity for oral bioavailability.
Typical Procedure A.12,13) Methyl 2-{[(Dimethylamino)carbonothi-
oyl]oxy}-4-methoxybenzoate (4c) To a stirred solution of 3c (100 g) and
N,N-dimethylthiocarbamoyl chloride (88.2 g) in DMF (200 ml) was added
dropwise 1,8-diazabicyclo[5.4.0]undec-7-ene (88.2 g) at 0 °C, and the mix-
ture was stirred at room temperature for 3 d. The mixture was poured into
ice-water, and extracted with EtOAc. The extract was washed with 1 N aq.
HCl and brine, dried over MgSO4, and concentrated in vacuo. The residue
was crystallized EtOAc/hexane to afford the crude title compound (128 g).
This was purified by the column chromatography on silica gel with EtOAc,
and recrystallization from EtOAc/hexane afforded the title compound
Conclusion
A novel class of IKK inhibitors, tricyclic furan derivatives
were designed based on the known thiophenecarboxamide
IKK inhibitors, synthesized and evaluated for their inhibitory
activity against IKKb. Among the various fused furan deriv-
atives, the benzothieno[3,2-b]furan derivative 13a, possess-
ing an intramolecular non-bonded S···O interaction, dis-
played high inhibitory activity for IKKb in enzyme and cell-
based assays. Introduction of substituents onto the benzothi-
eno[3,2-b]furan to overcome the low metabolic stability led
to the discovery of a series of 6-alkoxy derivatives. Further-
more, it was found that the lipophilic compounds (log Dꢀ2),
16a, h, i, and 13g showed an improved oral bioavailability
with potent enzymatic inhibitory activity. Future efforts will
focus on further improvements in potency in order to evalu-
ate the therapeutic application of these IKK inhibitors.
1
(123 g, 83%) as a white solid. H-NMR (CDCl3) d: 3.40 (3H, s), 3.47 (3H,
s), 3.81 (3H, s), 3.85 (3H, s), 6.64 (1H, d, Jꢂ3 Hz), 6.82 (1H, dd, Jꢂ9,
3 Hz), 7.97 (1H, d, Jꢂ9 Hz).
Methyl 2-{[(Dimethylamino)carbonyl]thio}-4-methoxybenzoate (5c)
A compound 4c (123 g) was heated to 210 °C for 14 h, and then cooled to
room temperature. This was purified by column chromatography on silica
gel with EtOAc/hexane (1/4 to 2/3) to afford the title compound (85.4 g,
1
70%) as a reddish oil. H-NMR (CDCl3) d: 3.00—3.20 (6H, br), 3.84 (6H,
s), 6.91 (1H, dd, Jꢂ9, 2 Hz), 7.17 (1H, d, Jꢂ3 Hz), 7.93 (1H, d, Jꢂ9 Hz).
2-Mercapto-4-methoxybenzoic Acid (7c) A mixture of 5c (85.3 g) and
4 N aq. NaOH (250 ml) was heated to reflux for 1 d, and then acidified with
conc. HCl (100 ml) at 0 °C. The precipitate was collected by filtration and
washed with H2O. The moist cake was dissolved in THF/EtOAc, and the so-
lution was washed with brine, dried over Na2SO4, and concentrated in
Experimental
vacuo. The residue was recrystallized from EtOAc/hexane to afford the title
Reagents were purchased from Wako Pure Chemical Industries, Ltd., compound (51.9 g, 89%) as a pale brown solid. H-NMR (CDCl3) d: 3.79
Tokyo Chemical Industry, Aldrich Chemical Company, Inc. and Lancaster (3H, s), 6.73 (1H, dd, Jꢂ9, 2 Hz), 7.10 (1H, d, Jꢂ2 Hz), 7.87 (1H, d,
1
and used without further purification. All reagents and solvents were of
commercial quality. 1H-NMR spectra were recorded on a Varian Gemini-200
(200 MHz), a Varian Mercury-300 (300 MHz) or a Brucker 300 (300 MHz)
Jꢂ9 Hz).
Methyl 2-Mercapto-4-methoxybenzoate (8c) A mixture of 7c (10.6 g)
and 10% HCl–MeOH (200 ml) was stirred at 60 °C for 24 h, and the solvent
with tetramethylsilane (TMS) as the internal standard. Chemical shifts are was evaporated off. The residue was chromatographed on silica gel with
1
reported in parts per million (ppm) units. Coupling constants (J) are re-
EtOAc/hexane (1/4) to give the title compound as an oil (6.4 g, 56%). H-
ported in units of hertz (Hz). Splitting patterns are designated as: s, singlet;
d, doublet; t, triplet; q, quartet; m, multiplet; br, broad. Elemental analyses 2 Hz), 6.79 (1H, d, Jꢂ2 Hz), 7.98 (1H, d, Jꢂ9 Hz).
NMR (CDCl3) d: 3.83 (3H, s), 3.89 (3H, s), 5.10 (1H, s), 6.67 (1H, dd, Jꢂ9,
were determined by Takeda Analytical Research Laboratories, Ltd., Osaka,
and within ꢇ0.4% of the theoretical values for the elements indicated unless
Similarly, compounds 8b, d were prepared.
Methyl 2-Mercapto-3-methoxybenzoate (8b) Crystals (37%). 1H-
otherwise noted. When air or moisture-sensitive reagents were used, reac- NMR (CDCl3) d: 3.92 (3H, s), 3.94 (3H, s), 5.31 (1H, s), 7.00—7.02 (1H,
tions were run under argon. Column chromatography was performed on m), 7.11 (1H, d, Jꢂ8 Hz), 7.66 (1H, dd, Jꢂ8, 2 Hz).
columns of Kieselgel 60 (230—400 mesh) and Chromatorex (NH2-coated
silica gel, 100—200 mesh). The yields reported are not optimized.
Methyl 2-Mercapto-5-methoxybenzoate (8d) A pale green oil (34%).
1H-NMR (CDCl3) d: 3.81 (3H, s), 3.93 (3H, s), 4.48 (1H, s), 6.94 (1H, dd,
Abbreviations: rtꢂroom temperature, aq.ꢂaqueous, Et3Nꢂtriethylamine, Jꢂ9, 3 Hz), 7.21 (1H, d, Jꢂ9 Hz), 7.52 (1H, d, Jꢂ3 Hz).
HOBtꢂ1-hydroxy-1H-benzotriazole monohydrate, DMAPꢂ4-dimethyl-
Typical Procedure B.12,14) 5-Bromo-2-mercaptobenzoic Acid (7g)
A
aminopyridine, DMAꢂN,N-dimethylacetamide, WSCꢂ1-ethyl-3-(3-di- solution of 6g (47.5 g), NaOH (8.98 g), and NaNO2 (15.2 g) in H2O (220 ml)
methylaminopropyl)carbodiimide hydrochloride, IPEꢂdiisopropyl ether, was added dropwise to a mixture of conc. HCl (66 ml) and ice (ca. 30 g).
DMSOꢂdimethyl sulfoxide, AcOHꢂacetic acid, EtOAcꢂethyl acetate, The internal temperature was kept at 3—6 °C by the addition of ice. The
THFꢂtetrahydrofuran, TFAꢂtrifluoroacetic acid, DMFꢂdimethylform- mixture was stirred at 0 °C for 30 min, and then neutralized with potassium
amide, MeOHꢂmethanol, EtOHꢂethanol, Et2Oꢂdiethyl ether, CH3CNꢂ acetate (70 g). This solution was added to a solution of potassium O-ethyl-
acetonitrile, Phꢂphenyl.
xanthate (106 g) in H2O (200 ml) which had preheated to 90 °C. The mixture

April 2007
619
was stirred at the same temperature for 30 min, cooled to 0 °C, and acidified
with conc. HCl. The aqueous phase was decanted from the resulting semi-
solid sludge. This was dissolved in 10% aq. NaOH (200 ml), and heated to
85 °C for 2 h. To this mixture was added portionwise NaHSO3 (20 g), and
br). Anal. Calcd for C10H7NO2S: C, 58.52; H, 3.44; N, 6.82. Found: C,
58.59; H, 3.39; N, 6.73.
3-Hydroxy-4-methoxy-1-benzothiophene-2-carbonitrile (10e) A pale
purple solid (81%). mp 132—133 °C. 1H-NMR (DMSO-d6) d: 3.91 (3H, s),
the mixture was heated to 85 °C for 10 min. The mixture was filtered, cooled 6.95 (1H, d, Jꢂ7 Hz), 7.46—7.53 (2H, m), 10.88 (1H, br). Anal. Calcd for
to 0 °C, and acidified with conc. HCl. The precipitate was collected by fil- C10H7NO2S: C, 58.52; H, 3.44; N, 6.82. Found: C, 58.61; H, 3.44; N, 6.86.
trarion and washed with H2O. The moist cake was dissolved in THF/EtOAc,
6-Bromo-3-hydroxy-1-benzothiophene-2-carbonitrile (10f) A white
washed with brine, dried over Na2SO4, and concentrated in vacuo to afford solid (85%). mp 232—236 °C (decomposed). 1H-NMR (DMSO-d6) d:
crude 7g (50.3 g, 98%) as a pale brown solid. This material was used for the 7.64—7.69 (1H, m), 7.95 (1H, dd, Jꢂ8, 1 Hz), 8.33—8.34 (1H, m), 12.45
next reaction without further purification.
(1H, br s). Anal. Calcd for C9H4BrNOS: C, 42.54; H, 1.59; N, 5.51. Found:
C, 42.71; H, 1.67; N, 5.74.
Methyl 5-Bromo-2-mercaptobenzoate (8g) A mixture of crude 7g
(50.3 g) and conc. H2SO4 (20 ml) in MeOH (500 ml) was heated to reflux
overnight. The mixture concentrated in vacuo, diluted with ice-H2O, and the
5-Bromo-3-hydroxy-1-benzothiophene-2-carbonitrile (10g) A white
solid (90%). mp 262 °C (decomposed). 1H-NMR (CDCl3) d: 3.72 (2H, s),
whole mixture was extracted with EtOAc. The extract was washed with 3.93 (3H, s), 7.29 (1H, dd, Jꢂ8, 2 Hz), 7.34 (1H, d, Jꢂ2 Hz), 8.00 (1H, d,
brine, dried over Na2SO4, and concentrated in vacuo. The residue was puri- Jꢂ8 Hz). Anal. Calcd for C9H4BrNOS: C, 42.54; H, 1.59; N, 5.51. Found: C,
fied by column chromatography on silica gel with EtOAc/hexane (1/10) to 42.79; H, 1.67; N, 5.58.
afford the title compound (46.2 g, 88%) as a yellow solid. 1H-NMR (CDCl3)
d: 3.93 (3H, s), 4.78 (1H, s), 7.18 (1H, d, Jꢂ9 Hz), 7.42 (1H, dd, Jꢂ9,
3 Hz), 8.14 (1H, d, Jꢂ2 Hz).
3-Hydroxy-6-phenyl-1-benzothiophene-2-carbonitrile (10h) A mix-
ture of 10f (1.78 g, 7.0 mmol), phenylboronic acid (1.11 g), and K2CO3
(2.90 g) in THF (36.0 ml) and H2O (12.0 ml) was degassed, and N2 was in-
troduced. To this mixture was added Pd(Ph3P)4 (243 mg, 3 mol%), and the
Similarly, compounds 8e, f were prepared.
1
Methyl 2-Mercapto-6-methoxybenzoate (8e) A yellow oil (42%). H- mixture was heated to reflux for 18 h. The mixture was partitioned between
NMR (CDCl3) d: 3.69 (3H, s), 3.82 (3H, s), 3.95 (3H, s), 6.74—6.77 (1H,
m), 6.94 (1H, dd, Jꢂ8, 1 Hz), 7.21 (1H, t, Jꢂ8 Hz).
1 N aq. HCl and EtOAc. The organic phase was washed with brine,
dried over Na2SO4, filtered through the short pad of silica gel eluting with
EtOAc, and concentrated in vacuo. The residue was recrystallized from
EtOAc/hexane to afford the title compound (1.59 g, 90%) as a brown solid
(90%). mp 213—218 °C (decomposed). 1H-NMR (DMSO-d6) d: 7.40—7.54
Methyl 4-Bromo-2-mercaptobenzoate (8f) A yellow oil (38%). 1H-
NMR (CDCl3) d: 3.92 (3H, s), 4.83 (1H, s), 7.28 (1H, dd, Jꢂ14, 3 Hz), 7.49
(1H, d, Jꢂ3 Hz), 7.87 (1H, d, Jꢂ13 Hz).
Typical Procedure C. Methyl 2-[(Cyanomethyl)thio]benzoate (9a) (3H, m), 7.76—7.84 (3H, m), 8.11 (1H, t, Jꢂ8 Hz), 8.31 (1H, d, Jꢂ2 Hz),
A mixture of 8a (18.0 g), bromoacetonitrile (9.70 g), and K2CO3 (29.6 g) in 12.33 (1H, s). Anal. Calcd for C15H9NOS: C, 71.18; H, 3.66; N, 5.53. Found:
THF (200 ml) was heated to reflux for 8 h. The mixture was diluted with C, 71.06; H, 3.69; N, 5.53.
H2O, and extracted with EtOAc. The extract was washed with brine, dried 3-Hydroxy-5-phenyl-1-benzothiophene-2-carbonitrile (10i)
A
mix-
over Na2SO4, and concentrated in vacuo. The obtained solid was washed ture of 10g (152 mg, 0.60 mmol), phenylboronic acid (110 mg), and K2CO3
with EtOH/hexane to afford the title compound (17.4 g, 78%). mp 105— (250 mg) in THF (4.0 ml) and H2O (1.8 ml) was degassed, and N2 was intro-
1
106 °C. H-NMR (CDCl3) d: 3.72 (2H, s), 3.94 (3H, s), 7.34 (1H, td, Jꢂ8, duced. To this mixture was added Pd(Ph3P)4 (35.0 mg), and the mixture was
1 Hz), 7.43 (1H, d, Jꢂ8 Hz), 7.58 (1H, td, Jꢂ8, 1 Hz), 8.04 (1H, dd, Jꢂ8,
1 Hz).
Similarly, compounds 9b—g were prepared.
heated to reflux for 18 h. The mixture was partitioned between 1 N aq.
KHSO4 and EtOAc. The organic phase was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by column
chromatography on silica gel with EtOAc/hexane (20—30%), and the ob-
Methyl 2-[(Cyanomethyl)thio]-3-methoxybenzoate (9b) Crystals
1
(91%). H-NMR (CDCl3) d: 3.70 (3H, s), 3.90 (5H, s), 6.79 (1H, dd, Jꢂ8, tained solid was recrystallized from EtOAc/hexane to afford the title com-
2 Hz), 6.87 (1H, d, Jꢂ2 Hz), 8.04 (1H, d, Jꢂ8 Hz).
pound (103 mg, 68%) as a white solid (68%). mp 181—181 °C. 1H-NMR
Methyl 2-[(Cyanomethyl)thio]-4-methoxybenzoate (9c) Crystals
(81%). H-NMR (CDCl3) d: 3.70 (3H, s), 3.90 (5H, s), 6.79 (1H, dd, Jꢂ9, 8.09 (1H, t, Jꢂ1 Hz). Anal. Calcd for C15H9NOS: C, 71.69; H, 3.61; N, 5.57.
2 Hz), 6.87 (1H, d, Jꢂ2 Hz), 8.04 (1H, d, Jꢂ9 Hz).
(CDCl3) d: 7.36—7.50 (3H, m), 7.62—7.66 (2H, m), 7.74—7.81 (2H, m),
1
Found: C, 71.53; H, 3.66; N, 5.50.
Methyl 2-[(Cyanomethyl)thio]-5-methoxybenzoate (9d) A white solid
(98%). H-NMR (CDCl3) d: 3.69 (2H, s), 3.86 (3H, s), 3.94 (3H, s), 7.09 (11a) A mixture of 10a (2.21 g), bromoacetamide (2.09 g), and K2CO3
(1H, dd, Jꢂ9, 3 Hz), 7.44 (1H, d, Jꢂ3 Hz), 7.54 (1H, d, Jꢂ9 Hz).
Typical Procedure E. 2-[(2-Cyano-1-benzothien-3-yl)oxy]acetamide
1
(3.48 g) in THF (50.0 ml) was heated to reflux for 14 h. The mixture was di-
Methyl 2-[(Cyanomethyl)thio]-6-methoxybenzoate (9e) A yellow oil luted with H2O, and extracted with EtOAc. The extract was washed with sat-
1
(100%). H-NMR (CDCl3) d: 3.59 (2H, s), 3.86 (3H, s), 3.95 (3H, s), 7.02 urated brine, dried over Na2SO4, and concentrated in vacuo. The residue was
(1H, dd, Jꢂ8, 1 Hz), 7.32 (1H, dd, Jꢂ7, 1 Hz), 7.42 (1H, t, Jꢂ8 Hz).
recrystallized from THF/hexane to afford the title compound (1.82 g, 62%)
1
Methyl 4-Bromo-2-[(cyanomethyl)thio]benzoate (9f) A white solid as crystals. mp 165—166 °C. H-NMR (DMSO-d6) d: 5.07 (2H, s), 7.50—
(91%). mp 129—130 °C. 1H-NMR (CDCl3) d: 3.72 (2H, s), 3.93 (3H, s),
7.44—7.48 (1H, m), 7.49 (1H, d, Jꢂ2 Hz), 7.89—7.92 (1H, m).
7.55 (2H, m), 7.64 (1H, t, Jꢂ8 Hz), 7.74 (1H, s), 8.01 (1H, d, Jꢂ8 Hz), 8.09
(1H, d, Jꢂ8 Hz).
Methyl 5-Bromo-2-[(cyanomethyl)thio]benzoate (9g) A white solid
(40%). mp 161—162 °C. 1H-NMR (CDCl3) d: 3.70 (2H, s), 3.94 (3H, s),
7.29 (1H, d, Jꢂ9 Hz), 7.66—7.70 (1H, m), 8.15 (1H, d, Jꢂ2 Hz). Anal.
Similarly, compounds 11b—i were prepared.
2-[(2-Cyano-7-methoxy-1-benzothien-3-yl)oxy]acetamide (11b) Crys-
tals (75%). mp 255—257 °C. 1H-NMR (DMSO-d6) d: 3.99 (3H, s), 5.07 (2H,
Calcd for C10H8BrNO2S: C, 41.97; H, 2.82; N, 4.89. Found: C, 41.89; H, s), 7.23 (1H, d, Jꢂ8 Hz), 7.51 (1H, t, Jꢂ8 Hz), 7.53 (1H, s), 7.69 (1H, d,
2.78; N, 4.88.
Jꢂ8 Hz), 7.74 (1H, s).
Typical Procedure D. 3-Hydroxy-1-benzothiophene-2-carbonitrile
(10a) A mixture of 9a (17.4 g) and sodium ethoxide (8.6 g) in EtOH
(200 ml) was refluxed 2 h, and the solvent was evaporated off. The residue Jꢂ9, 2 Hz), 7.54 (1H, s), 7.59 (1H, d, Jꢂ2 Hz), 7.73 (1H, s), 7.98 (1H, d,
2-[(2-Cyano-6-methoxy-1-benzothien-3-yl)oxy]acetamide (11c) Crys-
tals (43%). 1H-NMR (DMSO-d6) d: 3.85 (3H, s), 5.03 (2H, s), 7.11 (1H, dd,
was diluted with EtOAc/THF, and acidified with 1 N aq. HCl. The organic
layer was washed with brine, dried over MgSO4, and concentrated in vacuo
to afford the title compound (8.36 g, 57%) as crystals. mp 173—174 °C. 1H-
NMR (CDCl3) d: 7.48 (1H, t, Jꢂ8 Hz), 7.58 (1H, t, Jꢂ7 Hz), 7.96 (1H, d, 5.07 (2H, s), 7.28 (1H, dd, Jꢂ9, 3 Hz), 7.52 (1H, d, Jꢂ3 Hz), 7.58 (1H, br),
Jꢂ8 Hz), 8.01 (1H, d, Jꢂ8 Hz), 12.25 (1H, s).
Jꢂ9 Hz).
2-[(2-Cyano-5-methoxy-1-benzothien-3-yl)oxy]acetamide (11d)
white solid (87%). mp 181—182 °C. H-NMR (DMSO-d6) d: 3.86 (3H, s),
A
1
7.79 (1H, br), 7.91 (1H, d, Jꢂ9 Hz). Anal. Calcd for C12H10N2O3S: C, 54.95;
Similarly, compounds 10b—g were prepared.
H, 3.84; N, 10.68. Found: C, 54.85; H, 3.81; N, 10.67.
3-Hydroxy-7-methoxy-1-benzothiophene-2-carbonitrile (10b) Crys-
2-[(2-Cyano-4-methoxy-1-benzothien-3-yl)oxy]acetamide (11e)
A
1
tals (75%). 1H-NMR (DMSO-d6) d: 3.97 (3H, s), 7.17 (1H, d, Jꢂ8 Hz), white solid (68%). mp 200—202 °C. H-NMR (DMSO-d6) d: 3.92 (3H, s),
7.44—7.51 (1H, m), 7.63 (1H, d, Jꢂ8 Hz), 12.28 (1H, s).
3-Hydroxy-6-methoxy-1-benzothiophene-2-carbonitrile (10c) Crys-
tals (75%). 1H-NMR (CDCl3) d: 3.89 (3H, s), 7.05 (1H, d, Jꢂ9 Hz), 7.13
(1H, s), 7.76 (1H, d, Jꢂ9 Hz).
4.85 (2H, s), 7.02—7.06 (1H, m), 7.37 (1H, br), 7.56—7.58 (2H, m), 7.64
(1H, br). Anal. Calcd for C12H10N2O3S: C, 54.95; H, 3.84; N, 10.68. Found:
C, 54.67; H, 3.64; N, 10.60.
2-[(6-Bromo-2-cyano-1-benzothien-3-yl)oxy]acetamide (11f) A pale
1
3-Hydroxy-5-methoxy-1-benzothiophene-2-carbonitrile (10d) A pale
brown solid (57%). mp 228—232 °C. H-NMR (DMSO-d6) d: 5.07 (2H, s),
1
brown solid (95%). mp 195—196 °C. H-NMR (DMSO-d6) d: 3.83 (3H, s),
7.57 (1H, br), 7.70 (1H, dd, Jꢂ9, 2 Hz), 8.06 (1H, d, Jꢂ9 Hz), 8.38 (1H, d,
Jꢂ2 Hz). Anal. Calcd for C11H7BrN2O2S: C, 42.46; H, 2.27; N, 9.00. Found:
7.22—7.25 (1H, m), 7.53 (1H, d, Jꢂ2 Hz), 7.86 (1H, d, Jꢂ9 Hz), 12.16 (1H,

620
Vol. 55, No. 4
silica gel with THF/toluene to afford the title compound (40 mg, 15%).
C, 42.54; H, 2.22; N, 9.10.
2-[(5-Bromo-2-cyano-1-benzothien-3-yl)oxy]acetamide (11g) A white
solid (81%). mp 191—192 °C. 1H-NMR (DMSO-d6) d: 5.07 (2H, s), 7.60
(1H, br s), 7.79 (1H, dd, Jꢂ9, 2 Hz), 7.87 (1H, br s), 8.01 (1H, d, Jꢂ9 Hz),
Method B: To a solution of 12a (300 mg) in THF (8.0 ml) was added
Cl3CCONCO (365 mg) at ꢃ60 °C. After stirring at ꢃ30 °C for 1 h, 2 N
NH3–EtOH (5.0 ml) was added to the reaction mixture. After stirring at rt
8.41 (1H, d, Jꢂ2 Hz). Anal. Calcd for C11H7BrN2O2S·0.1H2O: C, 42.36; H, overnight, the reaction mixture was poured into H2O. The precipitate was
2.48; N, 8.82. Found: C, 42.45; H, 2.52; N, 8.63. collected by filtration and washed with H2O and EtOH to give the title com-
2-[(2-Cyano-6-phenyl-1-benzothien-3-yl)oxy]acetamide (11h) A pale pound (200 mg, 56%). mp ꢀ300 °C. H-NMR (DMSO-d6) d: 6.87 (2H, s),
1
1
brown solid (84%). mp 201—203 °C. H-NMR (DMSO-d6) d: 5.09 (2H, s),
7.38—7.52 (2H, m), 7.46 (2H, s), 7.60—7.80 (2H, br s), 7.86 (1H, d,
7.41—7.57 (4H, m), 7.77—7.81 (3H, m), 7.85 (1H, d, Jꢂ8 Hz), 8.18 (1H, d, Jꢂ8 Hz), 7.98 (1H, d, Jꢂ8 Hz), 9.24 (1H, s). Anal. Calcd for C12H9N3O3S:
Jꢂ8 Hz), 8.36 (1H, s). Anal. Calcd for C17H12N2O2S: C, 66.22; H, 3.92; N, C, 52.36; H, 3.30; N, 15.26. Found: C, 52.01; H, 3.42; N, 15.01.
9.08. Found: C, 65.94; H, 3.94; N, 9.17.
Compound 13c was prepared according to the typical procedure G
(method A), and compounds 13b, 13d—i were prepared according to the
typical procedure G (method B).
2-[(2-Cyano-5-phenyl-1-benzothien-3-yl)oxy]acetamide (11i) A pale
yellow solid (86%). mp 186—187 °C. 1H-NMR (DMSO-d6) d: 5.11 (2H, s),
7.39—7.44 (1H, m), 7.49—7.54 (2H, m), 7.61 (1H, br), 7.78 (2H, d,
Jꢂ7 Hz), 7.87 (1H, br), 7.94—7.98 (1H, m), 8.12 (1H, d, Jꢂ9 Hz), 8.35
3-[(Aminocarbonyl)amino]-5-methoxy[1]benzothieno[3,2-b]furan-2-
carboxamide (13b) Crystals (39%). mp ꢀ300 °C. 1H-NMR (DMSO-d6)
(1H, d, Jꢂ9 Hz). Anal. Calcd for C17H12N2O2S: C, 66.22; H, 3.92; N, 9.08. d: 3.98 (3H, s), 6.90 (2H, br s), 7.05 (1H, s), 7.46 (2H, s), 7.56 (1H, br s),
Found: C, 66.01; H, 3.93; N, 9.02. 7.80 (1H, br s), 9.25 (1H, s). Anal. Calcd for C13H11N3O4S·0.25H2O: C,
Typical Procedure F. 3-Amino[1]benzothieno[3,2-b]furan-2-carbox- 50.40; H, 3.74; N, 13.56. Found: C, 50.56; H, 3.71; N, 13.42.
amide (12a) A mixture of 11a (1.0 g) and sodium ethoxide (440 mg) in
EtOH (50.0 ml) was heated to reflux for 3 h, and then concentrated in vacuo.
3-[(Aminocarbonyl)amino]-6-methoxy[1]benzothieno[3,2-b]furan-2-
carboxamide (13c) Crystals (11%). mp >300 °C. 1H-NMR (DMSO-d6) d:
The residue was washed with H2O, and purified by column chromatography 3.85 (3H, s), 6.85 (2H, s), 7.08 (1H, dd, Jꢂ9, 2 Hz), 7.40—7.70 (3H, m),
on silica gel with EtOAc to afford the title compound (420 mg, 42%) as 7.73 (1H, d, Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C13H11N3O4S: C, 51.14;
1
crystals. mp 222—224 °C. H-NMR (DMSO-d6) d: 6.12 (2H, s), 7.19 (2H, H, 3.63; N, 13.76. Found: C, 51.05; H, 3.93; N, 13.88.
s), 7.41—7.53 (2H, m), 7.87 (1H, d, Jꢂ8 Hz), 8.05 (1H, d, Jꢂ8 Hz).
Similarly, compounds 12b—i were prepared.
3-[(Aminocarbonyl)amino]-7-methoxy[1]benzothieno[3,2-b]furan-2-
carboxamide (13d) A pale pink solid (88%). mp ꢀ300 °C. 1H-NMR
3-Amino-5-methoxy[1]benzothieno[3,2-b]furan-2-carboxamide (12b) (DMSO-d6) d: 3.85 (3H, s), 6.86 (2H, br), 7.05 (1H, dd, Jꢂ9, 2 Hz), 7.32
Crystals (22%). mp 239—241 °C. 1H-NMR (DMSO-d6) d: 4.00 (3H, s),
6.11 (2H, s), 7.05—7.08 (1H, m), 7.19 (2H, s), 7.46—7.48 (2H, m).
(1H, d, Jꢂ2 Hz), 7.55 (1H, br), 7.76 (1H, br), 7.85 (1H, d, Jꢂ9 Hz), 9.22
(1H, s). Anal. Calcd for C13H11N3O4S·0.6H2O: C, 49.39; H, 3.89; N, 13.29.
3-Amino-6-methoxy[1]benzothieno[3,2-b]furan-2-carboxamide (12c) Found: C, 49.30; H, 4.12; N, 13.47.
Crystals (43%). mp 231—233 °C. 1H-NMR (DMSO-d6) d: 3.84 (3H, s),
3-[(Aminocarbonyl)amino]-8-methoxy[1]benzothieno[3,2-b]furan-2-
6.06 (2H, s), 7.09—7.12 (3H, m), 7.66 (1H, d, Jꢂ2 Hz), 7.73 (1H, d, Jꢂ carboxamide (13e) A gray solid (55%). mp ꢀ300 °C. H-NMR (DMSO-
9 Hz).
1
d6) d: 3.98 (3H, s), 6.88 (2H, br s), 6.97 (1H, d, Jꢂ8 Hz), 7.35 (1H, t, Jꢂ
3-Amino-7-methoxy[1]benzothieno[3,2-b]furan-2-carboxamide (12d) 8 Hz), 7.48—7.51 (3H, m), 9.25 (1H, s). Anal. Calcd for C13H11N3O4S·
1
A pale brown solid (56%). mp 246—248 °C. H-NMR (DMSO-d6) d: 3.85 0.2EtOH·0.4H2O: C, 50.02; H, 4.07; N, 13.06. Found: C, 50.05; H, 4.01;
(3H, s), 6.09 (2H, br s), 7.04—7.08 (1H, m), 7.16 (2H, br), 7.33 (1H, d, Jꢂ N, 12.90.
2 Hz), 7.91 (1H, d, Jꢂ9 Hz). Anal. Calcd for C12H10N2O3S: C, 54.95; H,
3.84; N, 10.68. Found: C, 54.66; H, 3.82; N, 10.57.
3-[(Aminocarbonyl)amino]-6-bromo[1]benzothieno[3,2-b]furan-2-
carboxamide (13f) This compound was prepared from crude 12f by a sim-
3-Amino-8-methoxy[1]benzothieno[3,2-b]furan-2-carboxamide (12e) ilar procedure described in the synthesis of 13a as a pale brown solid (15%
1
A gray solid (68%). mp 219—221 °C. 1H-NMR (DMSO-d6) d: 3.97 (3H, s),
from 11f). mp ꢀ300 °C. H-NMR (DMSO-d6) d: 6.90 (2H, br), 7.50—7.90
6.08 (2H, br s), 6.99—7.02 (3H, m), 7.36 (1H, d, Jꢂ8 Hz), 7.57 (1H, d, (2H, br), 7.63 (1H, d, Jꢂ9 Hz), 7.76 (1H, d, Jꢂ8 Hz), 8.30 (1H, s), 9.23 (1H,
Jꢂ8 Hz). Anal. Calcd for C12H10N2O3S: C, 54.95; H, 3.84; N, 10.68. Found: s). Anal. Calcd for C12H8BrN3O3S: C, 40.69; H, 2.28; N, 11.86. Found: C,
C, 55.04; H, 4.04; N, 10.42.
40.44; H, 2.29; N, 11.69.
3-Amino-6-bromo[1]benzothieno[3,2-b]furan-2-carboxamide (12f)
A mixture of 11f (16.8 g) and sodium ethoxide (428 mg) in EtOH (20 ml)
3-[(Aminocarbonyl)amino]-7-bromo[1]benzothieno[3,2-b]furan-2-
carboxamide (13g) A pink solid (40%). mp ꢀ300 °C. H-NMR (DMSO-
1
was heated to reflux for 1 h. The mixture was diluted with H2O, and the pre- d6) d: 6.89 (2H, br s), 7.54—7.80 (3H, m), 7.94—7.98 (2H, m), 9.20 (1H, s).
cipitate was collected by filtration, washed with H2O and EtOH. The solid Anal. Calcd for C12H8BrN3O3S·1.0H2O: C, 38.72; H, 2.71; N, 11.29. Found:
was dissolved in MeOH/THF/EtOAc, filtered through the short pad of NH2- C, 38.87; H, 2.84; N, 10.99.
coated silica gel eluting with THF/EtOAc, and the filtrate was concentrated
3-[(Aminocarbonyl)amino]-6-phenyl[1]benzothieno[3,2-b]furan-2-
in vacuo. The residue was recrystallized from THF/EtOH and DMF/H2O to carboxamide (13h) A solid (87%). mp ꢀ300 °C. 1H-NMR (DMSO-d6) d:
afford a crude 12f (0.345 g) as a white solid. This material was used for the 6.87 (2H, br), 7.38—7.82 (8H, m), 7.93 (1H, d, Jꢂ8 Hz), 8.31—8.32 (1H,
next reaction without further purification.
m), 9.27 (1H, s). Anal. Calcd for C18H13N3O3S: C, 61.53; H, 3.73; N, 11.96.
3-Amino-7-bromo[1]benzothieno[3,2-b]furan-2-carboxamide (12g) Found: C, 61.23; H, 3.66; N, 11.87.
1
A pale brown solid (40%). mp 249 °C (decomposed). H-NMR (DMSO-d6)
3-[(Aminocarbonyl)amino]-7-phenyl[1]benzothieno[3,2-b]furan-2-
d: 6.12 (2H, s), 7.19 (2H, br s), 7.57 (1H, dd, Jꢂ9, 2 Hz), 7.96 (1H, d, carboxamide (13i) A pale brown solid (85%). mp ꢀ300 °C. 1H-NMR
Jꢂ2 Hz), 8.03 (1H, d, Jꢂ9 Hz). Anal. Calcd for C11H7BrN2O2S: C, 42.46; H, (DMSO-d6) d: 6.90 (2H, br), 7.18 (2H, br), 7.41 (1H, t, Jꢂ7 Hz), 7.50—
2.27; N, 9.00. Found: C, 42.52; H, 2.22; N, 8.99.
7.76 (7H, m), 8.06—8.08 (2H, m), 9.25 (1H, s). Anal. Calcd for
3-Amino-6-phenyl[1]benzothieno[3,2-b]furan-2-carboxamide (12h) C18H13N3O3S·0.3H2O: C, 60.60; H, 3.84; N, 11.78. Found: C, 60.62; H,
This compound was prepared from 11h by a similar procedure described in 3.78; N, 11.70.
the synthesis of 12a as a pale brown solid (35%). mp 266—268 °C. 1H-
NMR (DMSO-d6) d: 6.13 (2H, br s), 7.20 (2H, br), 7.40 (1H, t, Jꢂ7 Hz),
3-Amino-6-hydroxy[1]benzothieno[3,2-b]furan-2-carboxamide (14)
To a stirred solution of 12c (10.2 g) in CH2Cl2 (200 ml) was added dropwise
7.51 (2H, t, Jꢂ7 Hz), 7.77—7.83 (3H, m), 7.92 (1H, d, Jꢂ8 Hz), 8.38—8.39 BBr3 (25.0 g) at 0 °C, and the mixture was heated to reflux for 1 d. After
(1H, m). Anal. Calcd for C17H12N2O2S: C, 66.22; H, 3.92; N, 9.08. Found: cooling, the reaction was quenched with ice, and the precipitate was col-
C, 66.20; H, 4.03; N, 9.16.
lected by filtration. The solid was dissolved in DMF, and filtered through the
3-Amino-7-phenyl[1]benzothieno[3,2-b]furan-2-carboxamide (12i) short pad of NH2-coated silica gel with MeOH/EtOAc (20%). The filtrate
1
A pale brown solid (28%). mp 269—271 °C. H-NMR (DMSO-d6) d: 6.13 was concentrated in vacuo, and the residue was recrystallized from
(2H, br s), 7.18 (2H, br), 7.41—7.56 (3H, m), 7.74—7.70 (3H, m), 8.08 (1H, DMF/MeOH/EtOH to afford the title compound (7.28 g, 75%) as crystals.
s), 8.14 (1H, d, Jꢂ9 Hz). Anal. Calcd for C17H12N2O2S·0.2EtOH: C, 65.81; mp 285—290 °C (decomposed). 1H-NMR (DMSO-d6) d: 6.04 (2H, br s),
H, 4.19; N, 8.82. Found: C, 65.60; H, 3.97; N, 8.79.
6.96 (1H, d, Jꢂ8 Hz), 7.06 (2H, br), 7.33 (1H, s), 7.65 (1H, d, Jꢂ9 Hz),
Typical Procedure G. 3-[(Aminocarbonyl)amino][1]benzothieno- 10.00 (1H, br). Anal. Calcd for C11H8N2O3S: C, 53.22; H, 3.25; N, 11.28.
[3,2-b]furan-2-carboxamide (13a) Method A: A mixture of 12a (220
Found: C, 53.22; H, 3.19; N, 11.20.
mg) and potassium cyanate (153 mg) in AcOH (3.0 ml) was heated to 110 °C
Typical Procedure H. tert-Butyl (2-{[3-Amino-2-(aminocarbonyl)-
for 3 d, and then concentrated in vacuo. The residue was dissolved in THF, [1]benzothieno[3,2-b]furan-6-yl]oxy}ethyl)carbamate (15a) To a stirred
washed with aq. NaHCO3 and saturated brine, dried over MgSO4, and con- solution of 14 (300 mg) in DMF (5.0 ml) was added NaH (72.5 mg, 60% in
centrated in vacuo. The residue was purified by column chromatography on oil), and the mixture was heated to 60 °C for 20 min. To this mixture was

April 2007
621
added a solution of tert-butyl (2-bromoethyl)carbamate (325 mg) in DMF
(1.0 ml), and the mixture was heated to 60 °C for 6 h. The mixture was parti-
3-Amino-6-(3-phenylpropoxy)[1]benzothieno[3,2-b]furan-2-carbox-
amide (15j) This compound was prepared from 14 and 1-bromo-3-phenyl-
tioned between H2O and EtOAc. The organic phase was separated, washed propane by a similar procedure described in the synthesis of 15a as a pale
with 1 N aq. KHSO4 and brine, dried over Na2SO4, and concentrated in
brown solid (50%). mp 185—187 °C. 1H-NMR (DMSO-d6) d: 2.03—2.09
vacuo. The residue was purified by column chromatography on NH2-coated (2H, m), 2.77 (2H, t, Jꢂ8 Hz), 4.06 (2H, t, Jꢂ6 Hz), 6.06 (2H, br s), 7.04—
silica gel with EtOAc/hexane (90—100%), and recrystallization from 7.34 (8H, m), 7.63 (1H, d, Jꢂ2 Hz), 7.72 (1H, d, Jꢂ9 Hz). Anal. Calcd for
EtOAc/hexane to afford the title compound (391 mg, 83%) as a white solid. C20H18N2O3S·0.1H2O: C, 65.23; H, 4.98; N, 7.61. Found: C, 65.00; H, 5.02;
mp 212—214 °C. 1H-NMR (DMSO-d6) d: 1.38 (9H, s), 3.31—3.34 (2H, m, N, 7.62.
overlapped with DMSO), 4.01—4.07 (2H, m), 6.07 (2H, br s), 7.05—7.11
Compounds 16a—j were prepared according to the typical procedure G
(3H, m), 7.65 (1H, d, Jꢂ2 Hz), 7.72 (1H, d, Jꢂ9 Hz). Anal. Calcd for (method B).
C18H21N3O5S: C, 55.23; H, 5.41; N, 10.73. Found: C, 55.16; H, 5.56; N,
10.49.
tert-Butyl (3-{[3-Amino-2-(aminocarbonyl)[1]benzothieno[3,2-b]furan-
6-yl]oxy}propyl)-carbamate (15b) This compound was prepared from 14
tert-Butyl [2-({2-(Aminocarbonyl)-3-[(aminocarbonyl)amino][1]ben-
zothieno[3,2-b]furan-6-yl}oxy)ethyl]carbamate (16a) white solid
(81%). mp 209—211 °C. H-NMR (DMSO-d6) d: 1.39 (9H, s), 3.31—3.33
(2H, m, overlapped with DMSO), 4.05 (2H, t, Jꢂ6 Hz), 6.65—6.95 (2H, br),
A
1
and tert-butyl (3-bromopropyl)carbamate by a similar procedure described 7.04—7.09 (2H, m), 7.40—7.70 (2H, br), 7.57 (1H, d, Jꢂ2 Hz), 7.72 (1H, d,
1
in the synthesis of 15a as a white solid (79%). mp 159—161 °C. H-NMR Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C19H22N4O6S·1.0H2O: C, 50.43; H,
(DMSO-d6) d: 1.37 (9H, s), 1.84—1.99 (2H, m), 3.10 (2H, q, Jꢂ6 Hz), 5.35; N, 12.38. Found: C, 50.38; H, 5.36; N, 12.34.
4.06 (2H, t, Jꢂ6 Hz), 6.06 (2H, br s), 6.91—6.93 (3H, m), 7.07—7.10 (3H,
tert-Butyl [3-({2-(Aminocarbonyl)-3-[(aminocarbonyl)amino][1]ben-
white solid
C19H23N3O5S·0.5EtOH: C, 56.06; H, 6.12; N, 9.81. Found: C, 55.81; H, (79%). mp 221—223 °C. H-NMR (DMSO-d6) d: 1.37 (9H, s), 1.86—1.91
5.85; N, 9.74. (2H, m), 3.11 (2H, q, Jꢂ6 Hz), 4.05 (2H, t, Jꢂ6 Hz), 6.70—7.00 (3H, br),
m), 7.63 (1H, d, Jꢂ2 Hz), 7.72 (1H, d, Jꢂ9 Hz). Anal. Calcd for zothieno[3,2-b]furan-6-yl}oxy)propyl]carbamate (16b)
A
1
3-Amino-6-[2-(dimethylamino)ethoxy][1]benzothieno[3,2-b]furan-2- 7.04 (1H, dd, Jꢂ9, 3 Hz), 7.31 (1H, d, Jꢂ3 Hz), 7.56 (1H, br), 7.76 (1H, br),
carboxamide (15c) This compound was prepared from 14, 2-dimethyl- 7.84 (1H, d, Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C20H24N4O6S·0.2H2O:
aminoethyl chloride hydrochloride and sodium iodide by a similar procedure C, 53.14; H, 5.44; N, 12.39. Found: C, 52.90; H, 5.53; N, 12.32.
described in the synthesis of 15a as a brown solid (71%). mp 184—186 °C.
1H-NMR (DMSO-d6) d: 2.23 (6H, s), 2.66 (2H, t, Jꢂ6 Hz), 4.14 (2H, t,
Jꢂ6 Hz), 6.06 (2H, br s), 7.07—7.12 (3H, m), 7.66 (1H, d, Jꢂ2 Hz), 7.72
(1H, d, Jꢂ8 Hz). Anal. Calcd for C15H17N3O3S·0.1H2O: C, 56.09; H, 5.20; Jꢂ6 Hz), 4.13 (2H, t, Jꢂ6 Hz), 6.85 (2H, br), 7.08 (1H, dd, Jꢂ9, 2 Hz),
N, 13.08. Found: C, 56.11; H, 5.22; N, 13.15. 7.40—7.75 (2H, br), 7.58 (1H, d, Jꢂ2 Hz), 7.72 (1H, d, Jꢂ9 Hz), 9.22 (1H,
3-[(Aminocarbonyl)amino]-6-[2-(dimethylamino)ethoxy][1]benzothi-
eno[3,2-b]furan-2-carboxamide (16c) A pale green solid (77%). mp
290 °C (decomposed). 1H-NMR (DMSO-d6) d: 2.33 (6H, s), 2.65 (2H, t,
Ethyl {[3-Amino-2-(aminocarbonyl)[1]benzothieno[3,2-b]furan-6-yl]- s). Anal. Calcd for C16H18N4O4S·0.6H2O: C, 51.49; H, 5.19; N, 15.01.
oxy}acetate (15d) This compound was prepared from 14 and ethyl bromo- Found: C, 51.35; H, 5.17; N, 15.04.
acetate by a similar procedure described in the synthesis of 15a as a pale
Ethyl ({2-(Aminocarbonyl)-3-[(aminocarbonyl)amino][1]benzothieno-
brown solid (68%). mp 174—175 °C. H-NMR (DMSO-d6) d: 1.23 (3H, t, [3,2-b]furan-6-yl}oxy)acetate (16d) pale brown solid (80%). mp
Jꢂ7 Hz), 4.19 (2H, q, Jꢂ7 Hz), 4.89 (2H, s), 6.07 (2H, br s), 7.11—7.15
ꢀ300 °C. 1H-NMR (DMSO-d6) d: 1.23 (3H, t, Jꢂ7 Hz), 4.19 (2H, q,
1
A
(3H, m), 7.66 (1H, d, Jꢂ2 Hz), 7.74 (1H, d, Jꢂ8 Hz), 7.95 (1H, s). Anal. Jꢂ7 Hz), 4.88 (2H, s), 6.87 (2H, br), 7.09—7.13 (1H, m), 7.35—7.80 (2H,
Calcd for C15H14N2O5S·0.6DMF: C, 53.35; H, 4.85; N, 9.63. Found: C, br), 7.57 (1H, d, Jꢂ2 Hz), 7.74 (1H, d, Jꢂ8 Hz), 9.22 (1H, s). Anal. Calcd
53.08; H, 5.01; N, 9.76.
for C16H15N3O6S·0.5H2O: C, 49.74; H, 4.17; N, 10.88. Found: C, 49.85; H,
2-{[3-Amino-2-(aminocarbonyl)[1]benzothieno[3,2-b]furan-6-yl]- 4.03; N, 10.88.
oxy}ethyl Acetate (15e) This compound was prepared from 14 and 2-bro-
2-({2-(Aminocarbonyl)-3-[(aminocarbonyl)amino][1]benzothieno[3,2-
moethyl acetate by a similar procedure described in the synthesis of 15a as a b]furan-6-yl}oxy)ethyl Acetate (16e) A white solid (92%). mp 225—
1
1
white solid (76%). mp 173—176 °C. H-NMR (DMSO-d6) d: 2.05 (3H, s),
227 °C. H-NMR (DMSO-d6) d: 2.05 (3H, s), 4.29 (2H, br), 4.37 (2H, br),
4.27—4.29 (2H, m), 4.36—4.38 (2H, m), 6.07 (1H, br s), 7.10—7.14 (3H, 6.86 (2H, br), 7.08—7.12 (1H, m), 7.40—7.80 (2H, br), 7.61 (1H, s), 7.73
m), 7.69 (1H, d, Jꢂ2 Hz), 7.73 (1H, d, Jꢂ9 Hz). Anal. Calcd for (1H, d, Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C16H15N3O6S·0.3H2O: C,
C15H14N2O5S: C, 53.88; H, 4.22; N, 8.38. Found: C, 53.91; H, 4.25; N, 8.10.
50.21; H, 4.11; N, 10.98. Found: C, 50.10; H, 4.05; N, 10.95.
3-Amino-6-propoxy[1]benzothieno[3,2-b]furan-2-carboxamide (15f)
3-[(Aminocarbonyl)amino]-6-propoxy[1]benzothieno[3,2-b]furan-2-
This compound was prepared from 14 and 1-iodopropane by a similar proce- carboxamide (16f) A white solid (78%). mp ꢀ300 °C. 1H-NMR (DMSO-
dure described in the synthesis of 15a as a pale brown solid (71%). mp d6) d: 1.00 (3H, t, Jꢂ8 Hz), 1.71—1.85 (2H, m), 4.02 (2H, t, Jꢂ7 Hz), 6.86
171—173 °C. 1H-NMR (DMSO-d6) d: 1.00 (3H, t, Jꢂ7 Hz), 1.73—1.81 (2H, br), 7.08 (1H, dd, Jꢂ9, 2 Hz), 7.40—7.80 (2H, br), 7.56 (1H, d, Jꢂ
(2H, m), 4.02 (2H, t, Jꢂ7 Hz), 6.06 (2H, br s), 7.08—7.11 (3H, m), 7.64 2 Hz), 7.72 (1H, d, Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C15H15N3O4S·
(1H, s), 7.72 (1H, d, Jꢂ8 Hz). Anal. Calcd for C14H14N2O3S·0.6EtOH: C,
0.2H2O: C, 53.47; H, 4.61; N, 12.47. Found: C, 53.40; H, 4.50; N, 12.51.
57.83; H, 4.99; N, 9.50. Found: C, 57.60; H, 4.83; N, 9.48.
3-[(Aminocarbonyl)amino]-6-(2-morpholin-4-ylethoxy)[1]benzothi-
3-Amino-6-(2-morpholin-4-ylethoxy)[1]benzothieno[3,2-b]furan-2- eno[3,2-b]furan-2-carboxamide (16g) A pale green solid (53%). mp
carboxamide (15g) This compound was prepared from 14, N-(2- 299—300 °C. 1H-NMR (DMSO-d6) d: 2.47—2.51 (4H, m), 2.73 (2H, t,
chloroethyl)morpholine hydrochloride and sodium iodide by a similar proce- Jꢂ6 Hz), 3.58 (4H, t, Jꢂ5 Hz), 4.18 (2H, t, Jꢂ6 Hz), 6.86 (2H, br), 7.09
dure described in the synthesis of 15a as a pale green solid (55%). mp
(1H, dd, Jꢂ9, 2 Hz), 7.40—7.75 (2H, br), 7.59 (1H, d, Jꢂ2 Hz), 7.72 (1H, d,
170—172 °C. 1H-NMR (DMSO-d6) d: 2.49—2.50 (2H, m), 2.73 (2H, t, Jꢂ9 Hz), 9.22 (1H, s). Anal. Calcd for C18H20N4O5S·0.8H2O: C, 51.64; H,
Jꢂ6 Hz), 3.58 (4H, t, Jꢂ4 Hz), 4.18 (2H, t, Jꢂ6 Hz), 6.06 (2H, br s), 7.08—
7.12 (3H, m), 7.67 (1H, s), 7.70—7.73 (1H, m). Anal. Calcd for
C17H19N3O4S: C, 56.50; H, 5.30; N, 11.60. Found: C, 56.36; H, 5.44; N, eno[3,2-b]furan-2-carboxamide (16h) Crystals (88%). mp ꢀ300 °C. H-
5.53; N, 12.81. Found: C, 51.45; H, 5.26; N, 12.51.
3-[(Aminocarbonyl)amino]-6-[2-(1H-pyrrol-1-yl)ethoxy][1]benzothi-
1
11.35.
3-Amino-6-[2-(1H-pyrrol-1-yl)ethoxy][1]benzothieno[3,2-b]furan-2- Jꢂ2 Hz), 6.86 (2H, s), 7.05—7.09 (1H, m), 7.55 (1H, d, Jꢂ2 Hz), 7.40—
carboxamide (15h) This compound was prepared from 14 and 1-(2-bro- 7.70 (2H, br s). 9.21 (1H, s). Anal. Calcd for C18H16N4O4S·0.75H2O: C,
moethyl)-1H-pyrrole by a similar procedure described in the synthesis of 54.33; H, 4.43; N, 14.08. Found: C, 54.23; H, 4.29; N, 14.27.
NMR (DMSO-d6) d: 4.31 (4H, s), 6.00 (2H, t, Jꢂ2 Hz), 6.85 (2H, t,
1
15a as crystals (46%). mp 158—160 °C. H-NMR (DMSO-d6) d: 4.31 (4H,
s), 6.00 (2H, t, Jꢂ2 Hz), 6.05 (2H, s), 6.11 (2H, s), 6.85 (2H, t, Jꢂ2 Hz), eno[3,2-b]furan-2-carboxamide (16i) Crystals (67%). mp ꢀ300 °C. 1H-
7.07—7.10 (3H, m), 7.63 (1H, s), 7.72 (1H, d, Jꢂ8 Hz). NMR (DMSO-d6) d: 2.15—2.19 (2H, m), 3.98 (2H, t, Jꢂ6 Hz), 4.07 (2H, t,
3-[(Aminocarbonyl)amino]-6-[3-(1H-pyrrol-1-yl)propoxy][1]benzothi-
3-Amino-6-[3-(1H-pyrrol-1-yl)propoxy][1]benzothieno[3,2-b]furan-2- Jꢂ7 Hz), 5.98 (2H, t, Jꢂ2 Hz), 6.83 (2H, br s), 7.07—7.11 (1H, m), 7.40—
carboxamide (15i) This compound was prepared from 14 and 1-(3-bro- 7.74 (4H, m), 9.22 (1H, s). Anal. Calcd for C19H18N4O4S·0.75H2O: C,
mopropyl)-1H-pyrrole by a similar procedure described in the synthesis of 55.40; H, 4.77; N, 13.60. Found: C, 55.45; H, 4.58; N, 13.79.
15a as crystals (54%). mp 197—198 °C. 1H-NMR (DMSO-d6) d: 2.13—
3-[(Aminocarbonyl)amino]-6-(3-phenylpropoxy)[1]benzothieno[3,2-
2.20 (2H, m), 3.98 (2H, t, Jꢂ6 Hz), 4.07 (2H, t, Jꢂ7 Hz), 5.99 (2H, s), 6.06 b]furan-2-carboxamide (16j) A pale brown solid (90%). mp ꢀ300 °C.
(2H, s), 6.77 (2H, s), 7.08 (2H, s), 7.13 (1H, s), 7.61 (1H, s), 7.73 (1H, d, 1H-NMR (DMSO-d6) d: 2.01—2.10 (2H, m), 2.77 (2H, t, Jꢂ6 Hz), 4.06
Jꢂ9 Hz). Anal. Calcd for C18H17N3O3S: C, 60.83; H, 4.82; N, 11.82. Found: (2H, t, Jꢂ6 Hz), 6.84 (2H, br), 7.09 (1H, dd, Jꢂ9, 3 Hz), 7.16—7.32 (5H,
C, 60.53; H, 5.05; N, 11.57.
br m), 7.40—7.80 (2H, br), 7.55 (1H, d, Jꢂ2 Hz), 7.73 (1H, d, Jꢂ9 Hz), 9.22

622
Vol. 55, No. 4
(1H, s). Anal. Calcd for C21H19N3O4S·0.1H2O: C, 61.33; H, 4.71; N, 10.22.
Found: C, 61.28; H, 4.62; N, 10.12.
3-[(Aminocarbonyl)amino]-6-(2-aminoethoxy)[1]benzothieno[3,2-
trated in vacuo, and the residue was acidified with 1 N aq. KHSO4 (10 ml).
The mixture was extracted with EtOAc. The extract was washed with brine,
dried over Na2SO4, and concentrated in vacuo to give 3-[(tert-butoxycar-
b]furan-2-carboxamide Hydrochloride (16k) To a stirred solution of 16a bonyl)amino]-5-phenyl-2-furoic acid (590 mg). This crude acid (590 mg)
(100 mg) in MeOH (2.0 ml) was added 6 N aq. HCl (1.5 ml), and the mixture
was heated to 50 °C for 3 h. The mixture was concentrated in vacuo, and the
residue was recrystallized from MeOH to afford the title compound (80 mg,
94%) as a white crystal. mp 293—294 °C. H-NMR (DMSO-d6) d: 3.25—
3.27 (2H, m), 4.25 (2H, t, Jꢂ5 Hz), 6.86 (2H, br), 7.15 (1H, d, Jꢂ9 Hz),
7.50—7.80 (2H, br), 7.64 (1H, s), 7.77 (1H, d, Jꢂ9 Hz), 8.20 (3H, br), 9.23
was dissolved in CH3CN/DMF (8.0/2.0 ml), and then cooled to 0 °C. To this
mixture were added WSC (431 mg) and HOBt·NH3 (342 mg), and the mix-
ture was stirred at rt for 1 d. The mixture was diluted with H2O and extracted
with EtOAc. The organic phase was washed with 1 N aq. KHSO4, saturated
aq. NaHCO3, and brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel with
1
(1H, s). Anal. Calcd for C14H15ClN4O4S·0.5H2O: C, 44.27; H, 4.25; N, EtOAc/hexane (5—55%) and recrystallized from EtOAc/hexane to afford
14.75. Found: C, 44.02; H, 4.19; N, 14.61. the title compound (530 mg, 78% from 18) as a white crystal. mp 179—
6-[2-(Acetylamino)ethoxy]-3-[(aminocarbonyl)amino][1]benzothi- 181 °C. 1H-NMR (CDCl3) d: 1.53 (9H, s), 7.33—7.44 (3H, m), 7.51 (1H,
eno[3,2-b]furan-2-carboxamide (16l) To a stirred suspension of 16a br), 7.69—7.73 (2H, m), 8.66 (1H, br). Anal. Calcd for C16H18N2O4: C,
(113 mg) in CHCl3 (1.0 ml) was added trifluoroacetic acid (1.0 ml) at rt, and 63.56; H, 6.00; N, 9.27. Found: C, 63.70; H, 6.11; N, 9.07.
the mixture was stirred for 14 h. The mixture was concentrated in vacuo, and 3-Amino-5-phenyl-2-furamide (20) To stirred solution of 19
a
the residue was recrystallized from EtOH to afford 3-[(aminocarbonyl)- (111 mg) in CHCl3 (1.5 ml) was added TFA (1.0 ml) at room temperature,
amino]-6-(2-aminoethoxy)[1]benzothieno[3,2-b]furan-2-carboxamide triflu- and the mixture was stirred for 4 h. The mixture was concentrated in vacuo,
oroacetate (106 mg, 91%) as a pale yellow crystal. mp 275—280 °C (decom-
posed). H-NMR (DMSO-d6) d: 3.20—3.30 (2H, m), 4.26 (2H, t, Jꢂ5 Hz),
and the residue was purified by column chromatography on NH2-coated
silica gel with EtOAc/hexane (10—100%), and recrystallized from
1
6.88 (2H, br), 7.14 (1H, dd, Jꢂ9, 2 Hz), 7.50—7.70 (2H, br), 7.64 (1H, d, EtOAc/hexane to afford the title compound (55 mg, 74%) as a pale yellow
Jꢂ2 Hz), 7.77 (1H, d, Jꢂ9 Hz), 8.02 (3H, br), 9.23 (1H, s). Anal. Calcd for crystal. mp 159—160 °C. 1H-NMR (CDCl3) d: 4.71 (2H, br s), 5.52 (2H,
C16H15F3N4O6S·1.0H2O: C, 41.20; H, 3.67; N, 12.01. Found: C, 41.20; H, br), 6.34 (1H, s), 7.31—7.44 (3H, m), 7.63—7.68 (2H, m). Anal. Calcd for
3.65; N, 11.95. To a stirred solution of this TFA salt (52 mg, 0.12 mmol) in C11H10N2O2: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.24; H, 5.04; N,
DMF (1.5 ml) were added acetic anhydride (14.3 ml, 0.151 mmol) and Et3N 13.76.
(40.4 ml, 0.29 mmol) at rt, and the mixture was stirred at rt for 14 h. The
3-[(Aminocarbonyl)amino]-5-phenyl-2-furamide (21) To a stirred so-
mixture was diluted with H2O, and the precipitate was collected by filtration, lution of 20 (49 mg) in THF (1.5 ml) was added trichloroacetyl isocyanate
washed with H2O and EtOH to give the title compound (38 mg, 87%) as a (27.4 ml) at ꢃ70 °C, and the mixture was allowed to warm up to 0 °C over
white solid. mp ꢀ300 °C. 1H-NMR (DMSO-d6) d: 1.83 (3H, s), 3.40—3.46
(2H, m), 4.07 (2H, t, Jꢂ6 Hz), 6.87 (2H, br), 7.09 (1H, dd, Jꢂ9, 1 Hz),
3 h. To this mixture was added 2 N NH3 in MeOH (1.0 ml), and the mixture
was stirred at rt for 16 h. The mixture was concentrated in vacuo, and the
7.45—7.75 (2H, br), 7.59 (1H, s), 7.72 (1H, d, Jꢂ9 Hz), 8.10—8.18 (1H, residue was diluted with H2O. The precipitate was collected by filtration, and
m), 9.22 (1H, s). Anal. Calcd for C16H16N4O5S·0.1H2O: C, 50.81; H, 4.32; washed with H2O. The solid was recrystallized from EtOAc/hexane to afford
1
N, 14.81. Found: C, 50.55; H, 4.36; N, 14.79.
the title compound (32 mg, 54%) as a pale brown crystal. mp ꢀ300 °C. H-
3-[(Aminocarbonyl)amino]-6-(3-aminopropoxy)[1]benzothieno[3,2- NMR (DMSO-d6) d: 6.63 (2H, br), 7.34—7.48 (4H, m), 7.60 (1H, s), 7.75
b]furan-2-carboxamide Hydrochloride (16m) This compound was pre- (1H, br), 7.91—7.93 (2H, m), 8.78 (1H, s). Anal. Calcd for C12H11N3O3·
pared from 16b by a similar procedure described in the synthesis of 16k as 0.2EtOAc·0.2H2O: C, 58.09; H, 4.87; N, 15.88. Found: C, 58.11; H, 4.67;
a pale brown solid (99%). mp ꢀ300 °C. 1H-NMR (DMSO-d6) d: 2.04— N, 16.11.
2.11 (2H, m), 2.94—3.01 (2H, m), 4.17 (2H, t, Jꢂ6 Hz), 6.86 (2H, br),
Ethyl [(2-Cyano-3,4-dihydronaphthalen-1-yl)oxy]acetate (23a) A so-
7.11 (1H, dd, Jꢂ9, 2 Hz), 7.40—7.80 (2H, br), 7.58 (1H, d, Jꢂ2 Hz), lution of Ph3P (3.93 g) in THF (30.0 ml) was cooled to 0 °C, and then diethyl
7.74 (1H, d, Jꢂ9 Hz), 7.99 (3H, br), 9.22 (1H, s). Anal. Calcd for azodicarboxylate (6.53 ml, 40% solution in toluene) and ethyl glycolate
C15H17ClN4O4S·0.5H2O: C, 45.74; H, 4.61; N, 14.23. Found: C, 45.67; H, (1.42 ml) were added. To this mixture was added dropwise a solution of
4.60; N, 14.18.
({2-(Aminocarbonyl)-3-[(aminocarbonyl)amino][1]benzothieno[3,2- 8 h. The mixture was concentrated in vacuo, and the residue was purified by
22a16) (1.71 g) in THF (1.5 ml) at 0 °C, and the mixture was stirred at rt for
b]furan-6-yl}oxy)acetic Acid (16n) To a stirred solution of 16d (53 mg,
0.14 mmol) in MeOH was added 2 N aq. NaOH (0.5 ml) at rt, and the mix-
ture was stirred at rt for 3 d. The mixture was acidified with 1 N aq. HCl, and
the precipitate was collected by filtration, washed with H2O and EtOH to
column chromatography on silica gel with EtOAc/hexane (10—25%) and
recrystallized from EtOAc/hexane to afford the title compound (2.06 g,
80%) as a white crystal. mp 50—51 °C. H-NMR (CDCl3) d: 1.32 (3H, t,
Jꢂ7 Hz), 2.54—2.60 (2H, m), 2.84 (2H, t, Jꢂ8 Hz), 4.29 (2H, q, Jꢂ7 Hz),
1
give the title compound (41 mg, 84%) as a pale brown solid. mp ꢀ300 °C. 5.00 (2H, s), 7.16 (1H, d, Jꢂ7 Hz), 7.25—7.34 (2H, m), 7.72—7.75 (1H,
1H-NMR (DMSO-d6) d: 4.78 (2H, s), 6.86 (2H, br), 7.08—7.12 (1H, m), m). Anal. Calcd for C15H15NO3: C, 70.02; H, 5.88; N, 5.44. Found: C, 70.11;
7.40—7.80 (2H, br), 7.55 (1H, s), 7.74 (1H, d, Jꢂ9 Hz), 9.22 (1H, s), 13.00 H, 5.88; N, 5.42.
(1H, br). Anal. Calcd for C14H11N3O6S·0.2EtOH·1.5H2O: C, 44.86; H,
Ethyl [(8-Cyano-6,7-dihydro-5H-benzo[7]annulen-9-yl)oxy]acetate
3.97; N, 10.90. Found: C, 44.69; H, 3.88; N, 10.74.
(23b) This compound was prepared from 22b17) as described in the synthe-
3-[(Aminocarbonyl)amino]-6-(2-hydroxyethoxy)[1]benzothieno[3,2- sis of 23a as a yellow oil (79%). 1H-NMR (CDCl3) d: 1.28 (3H, t, Jꢂ7 Hz),
b]furan-2-carboxamide (16o) This compound was prepared from 16e by
a similar procedure described in the synthesis of 16n as a white solid (91%). q, Jꢂ7 Hz), 4.57 (2H, s), 7.26—7.42 (4H, m).
mp ꢀ300 °C. 1H-NMR (DMSO-d6) d: 3.75 (2H, q, Jꢂ5 Hz), 4.08 (2H, t,
2-[(2-Cyano-3,4-dihydronaphthalen-1-yl)oxy]acetamide (24a) To
Jꢂ5 Hz), 4.91 (1H, t, Jꢂ5 Hz), 6.85 (2H, br), 7.07—7.10 (1H, m), 7.35— stirred solution of 23a (2.90 g) in EtOH (38.0 ml) was added 28% aq. NH3
7.80 (2H, br), 7.57 (1H, d, Jꢂ2 Hz), 7.72 (1H, d, Jꢂ8 Hz), 9.22 (1H, s). (14.0 ml) at 0 °C, and the mixture was stirred at rt for 16 h. The mixture was
Anal. Calcd for C14H13N3O5S·0.2H2O: C, 49.61; H, 3.98; N, 12.40. Found: concentrated in vacuo, and the residue was purified by column chromatogra-
2.04 (2H, t, Jꢂ7 Hz), 2.17—2.26 (2H, m), 2.70 (2H, t, Jꢂ8 Hz), 4.21 (2H,
a
C, 49.52; H, 3.91; N, 12.30.
phy on silica gel with EtOAc/hexane (25—50%) and on NH2-coated silica
gel with EtOAc/hexane (10—90%). The obtained solid was recrystallized
from EtOAc/hexane to afford the title compound (1.48 g, 57%) as a white
crystal. mp 139—141 °C. 1H-NMR (CDCl3) d: 2.54—2.60 (2H, m), 2.87
(2H, t, Jꢂ9 Hz), 4.63 (2H, s), 5.74 (1H, br), 6.83 (2H, br), 7.21—7.43 (4H,
Ethyl 3-[(tert-Butoxycarbonyl)amino]-5-phenyl-2-furoate (18) To a
stirred solution of 1711) (647 mg) and DMAP (68.0 mg) in THF (12.0 ml)
was added a solution of di-tert-butyl dicarbonate (1.83 g) in THF (2.0 ml) at
0 °C, and the mixture was stirred at rt for 16 h. The mixture was concen-
trated in vacuo, and the residue was purified by column chromatography on m). Anal. Calcd for C13H12N2O2: C, 68.41; H, 5.30; N, 12.27. Found: C,
silica gel with EtOAc/hexane (5—25%). The obtained solid was recrystal- 68.36; H, 5.30; N, 12.23.
lized from EtOAc/hexane to afford the title compound (704 mg, 76%) as a
2-[(8-Cyano-6,7-dihydro-5H-benzo[7]annulen-9-yl)oxy]acetamide
(24b) This compound was prepared from 23b by a similar procedure de-
1
white crystal. mp 109—110 °C. H-NMR (CDCl3) d: 1.37 (3H, t, Jꢂ7 Hz),
1.45 (9H, s), 4.35 (2H, q, Jꢂ7 Hz), 6.64 (1H, s), 7.33—7.45 (5H, m), 7.76— scribed in the synthesis of 24a as a white solid (74%). mp 108—109 °C. 1H-
7.79 (2H, m). Anal. Calcd for C18H21NO5·0.5EtOAc: C, 63.99; H, 6.71; N, NMR (CDCl3) d: 2.06 (2H, t, Jꢂ7 Hz), 2.18—2.28 (2H, m), 2.68 (2H, t,
3.73. Found: C, 63.92; H, 6.69; N, 3.19.
Jꢂ8 Hz), 4.30 (2H, s), 5.69 (1H, br), 6.70 (1H, br), 7.29—7.44 (4H, m).
Anal. Calcd for C14H14N2O2: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.17;
H, 5.76; N, 11.55.
tert-Butyl [2-(Aminocarbonyl)-5-phenyl-3-furyl]carbamate (19) To a
stirred solution of 18 (746 mg) in EtOH (6.0 ml) was added 8 N aq. NaOH
(1.0 ml) at rt, and the mixture was stirred for 3 h. The mixture was concen-
3-Amino-4,5-dihydronaphtho[1,2-b]furan-2-carboxamide (25a) This

April 2007
623
compound was prepared from 24a according to the typical procedure F. A (3.72 g) in EtOH (40.0 ml) was added NaSH (3.36 g) at rt, and the mixture
pale yellow solid (85%). mp 133—134 °C. 1H-NMR (CDCl3) d: 2.64 (2H, t, was heated to reflux for 16 h. NaSH (2.24 g) was added to this mixture, be-
Jꢂ8 Hz), 3.01 (2H, t, Jꢂ8 Hz), 4.65 (2H, br s), 5.53 (2H, br), 7.20—7.28 cause TLC showed the 33 was remained. After further refluxing for 1 d, the
(3H, m), 7.51 (1H, d, Jꢂ7 Hz). Anal. Calcd for C13H12N2O2: C, 68.41; H,
5.30; N, 12.27. Found: C, 68.62; H, 5.29; N, 12.38.
mixture was acidified with 1 N aq. KHSO4 (100 ml) at 0 °C. The mixture was
extracted with EtOAc. The extract was washed with brine (50.0 ml), dried
3-Amino-5,6-dihydro-4H-benzo[6,7]cyclohepta[1,2-b]furan-2-carbox- over Na2SO4, and concentrated in vacuo to give 34 (3.86 g) as a yellow oil.
amide (25b) This compound was prepared from 24b according to the typi- This was dissolved in DMF/THF (20.0/20.0 ml), and chloroacetonitrile
1
cal procedure F. A white solid (83%). mp 157—159 °C. H-NMR (CDCl3) (1.52 ml) and NaI (3.60 g) were added to this solution. The mixture was
d: 2.01—2.09 (2H, m), 2.59 (2H, t, Jꢂ7 Hz), 2.88—2.91 (2H, m), 4.56 (2H, stirred at 50 °C for 14 h, and then partitioned between H2O and EtOAc. The
br s), 5.54 (2H, br), 7.13—7.29 (3H, m), 7.83 (1H, dd, Jꢂ8, 2 Hz). Anal.
organic phase was washed with saturated brine (30.0 ml), dried over Na2SO4,
Calcd for C14H14N2O2·0.3H2O: C, 67.89; H, 5.94; N, 11.31. Found: C, and concentrated in vacuo. The residue was purified by column chromatog-
68.06; H, 5.81; N, 11.07. raphy on silica gel with EtOAc/hexane (5—50%) and recrystallized from
3-[(Aminocarbonyl)amino]-4,5-dihydronaphtho[1,2-b]furan-2-car- EtOAc/hexane to afford the title compound (2.35 g, 53% from 33) as a white
boxamide (26a) This compound was prepared from 25a according to the
crystal. mp 72—73 °C. 1H-NMR (CDCl3) d: 1.42 (3H, t, Jꢂ7 Hz), 3.93 (2H,
s), 4.42 (2H, q, Jꢂ7 Hz), 7.19 (1H, dd, Jꢂ8, 5 Hz), 8.30 (1H, dd, Jꢂ8,
typical procedure G (method B). A white solid (52%). mp ꢀ300 °C. 1H-
NMR (DMSO-d6) d: 2.79—2.89 (4H, m), 6.46 (2H, br s), 7.20—7.31 (3H, 2 Hz), 8.66 (1H, dd, Jꢂ5, 2 Hz). Anal. Calcd for C10H10N2O2: C, 54.04; H,
m), 7.39 (1H, br), 7.62—7.66 (2H, m), 8.52 (1H, s). Anal. Calcd for
C14H13N3O3·0.3H2O: C, 60.78; H, 4.95; N, 15.19. Found: C, 60.97; H, 4.85;
N, 14.94.
4.53; N, 12.60. Found: C, 54.18; H, 4.46; N, 12.76.
3-Hydroxythieno[2,3-b]pyridine-2-carbonitrile (36) This compound
was prepared from 35 accordingyo to the typical procedure D. A white solid
3-[(Aminocarbonyl)amino]-5,6-dihydro-4H-benzo[6,7]cyclohepta[1,2- (96%). mp 196—198 °C. 1H-NMR (DMSO-d6) d: 7.54—7.59 (1H, m), 8.43
b]furan-2-carboxamide (26b) This compound was prepared from 25b (1H, dd, Jꢂ8, 1 Hz), 8.75—7.78 (1H, m), 12.68 (1H, br s). Anal. Calcd for
according to the typical procedure G (method B). A white solid (79%). C8H4N2OS: C, 54.53; H, 2.29; N, 15.90. Found: C, 54.71; H, 2.47; N, 15.83.
mp ꢀ300 °C. 1H-NMR (DMSO-d6) d: 1.84—1.88 (2H, m), 2.71 (2H, t,
2-[(2-Cyanothieno[2,3-b]pyridin-3-yl)oxy]acetamide (37) This com-
Jꢂ6 Hz), 2.86—2.90 (2H, m), 6.31 (2H, br s), 7.20—7.32 (3H, m), 7.44 pound was prepared from 36 according to the typical procedure E. A white
(1H, br), 7.84 (1H, br), 8.03 (1H, s), 8.16 (1H, d, Jꢂ7 Hz). Anal. Calcd for solid (78%). mp 249—252 °C. 1H-NMR (DMSO-d6) d: 5.06 (2H, s), 7.59—
C15H15N3O3·0.2H2O: C, 62.36; H, 5.37; N, 14.54. Found: C, 62.35; H, 5.35; 7.63 (2H, m), 7.78 (1H, br s), 8.53—8.56 (1H, m), 8.81—8.82(1H, m). Anal.
N, 14.41.
1-Hydroxy-2-naphthamide (28) A mixture of 27 (1.0 g) and WSC
Calcd for C10H7N3O2S: C, 51.49; H, 3.02; N, 18.02. Found: C, 51.29; H,
3.03; N, 17.90.
(1.22 g), and HOBt·NH3 (0.97 g) in DMF (30.0 ml) was stirred at 80 °C for
1 h. The reaction mixture was poured into 1 N aq. HCl and extracted with
EtOAc. The extract was washed with brine, dried over MgSO4, and concen-
3-Aminofuro[2ꢀ,3ꢀ:4,5]thieno[2,3-b]pyridine-2-carboxamide (38)
This compound was prepared from 37 according to the typical procedure F.
A brown solid (15%). mp 265 °C (decomposed). 1H-NMR (DMSO-d6) d:
trated in vacuo. The residue was purified by column chromatography on sil- 6.16 (2H, br s), 7.25 (2H, br s), 7.55 (1H, dd, Jꢂ8, 5 Hz), 8.24 (1H, dd, Jꢂ8,
ica gel with EtOAc/hexane (2/1) to afford the title compound (720 mg, 73%) 2 Hz), 8.59 (1H, dd, Jꢂ5, 2 Hz). Anal. Calcd for C10H7N3O2S: C, 51.49; H,
1
as crystals. mp 181—182 °C. H-NMR (CDCl3) d: 6.00 (2H, br s), 7.28—
7.32 (2H, m), 7.50—7.62 (2H, m), 7.65 (1H, d, Jꢂ7 Hz), 8.41—8.44 (1H,
m).
3.02; N, 18.02. Found: C, 51.53; H, 3.18; N, 17.90.
3-[(Aminocarbonyl)amino]furo[2ꢀ,3ꢀ:4,5]thieno[2,3-b]pyridine-2-car-
boxamide (39) To a stirred solution of 38 (26.0 mg) in THF/DMF
(3.0/1.0 ml) was added dropwise trichloroacetyl isocyanate (19.0 ml) at 0 °C,
1-(Cyanomethoxy)-2-naphthamide (29) A mixture of 28 (3.0 g), bro-
moacetonitrile (3.71 g) and K2CO3 (4.43 g) in THF (50 ml) was refluxed for and the mixture was allowed to warm up to rt over 3 h. After the mixture was
8 h. The reaction mixture was poured into H2O and extracted with EtOAc. cooled to 0 °C, trichloroacetyl isocyanate (10.0 ml) was added, and then the
The extract was washed with brine, dried over MgSO4, and concentrated in
mixture was stirred at rt for 2 h. To this mixture was added 2 N NH3–MeOH
vacuo. The residue was purified by column chromatography on silica gel (2 ml), and the mixture was stirred at rt for 2 h. The mixture was diluted with
with EtOAc to afford the title compound (1.74 g, 48%) as crystals. mp
H2O, and extracted with THF/EtOAc. The extract was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was recrystal-
151—152 °C. 1H-NMR (CDCl3) d: 4.90 (2H, s), 5.99 (2H, s), 7.00 (1H, m),
7.62—7.69 (2H, m), 7.79 (1H, d, Jꢂ8 Hz), 7.90—7.98 (2H, m), 8.18—8.21 lized from THF/EtOH to afford the title compound (5.0 mg, 16%) as a pale
(1H, m).
1-(Cyanomethoxy)-2-naphthonitrile (30) To a stirred solution of 29 (1H, dd, Jꢂ8, 5 Hz), 7.62 (1H, br s), 7.87 (1H, br s), 8.21 (1H, d, Jꢂ8 Hz),
(1.65 g) in DMF (10 ml) was added SOCl2 (1.5 ml) at 0 °C. After stirring at 8.59 (1H, d, Jꢂ5 Hz), 9.26 (1H, s). Anal. Calcd for C11H8N4O3S·0.25H2O:
60 °C for 30 min, the reaction mixture was poured into H2O and extracted C, 47.06; H, 3.05; N, 19.95. Found: C, 47.24; H, 3.30; N, 19.70.
with EtOAc. The extract was washed with brine, dried over MgSO4, and Preparation of Recombinant IKKb cDNA encoding human IKKb
brown solid. mp 262—264 °C. 1H-NMR (DMSO-d6) d: 6.91 (2H, br s), 7.52
concentrated in vacuo. The residue was purified by column chromatography was isolated by PCR with primers containing sequences encoding a FLAG-
on silica gel with EtOAc/hexane (33%) to afford the title compound tag in the carboxy-terminal region and subcloned into an insect cell expres-
(820 mg, 54%) as crystals. mp 120—121 °C. 1H-NMR (CDCl3) d: 5.19 (2H, sion vector, pFASTBAC1 (Invitrogen, U.S.A.). Sf21 cells were infected with
s), 7.55 (1H, d, Jꢂ8 Hz), 7.69—7.78 (3H, m), 7.90—7.93 (1H, m), 8.26— IKKb recombinant baculovirus and cultured at 28 °C for 72 h. Cells were
8.30 (1H, m).
lysed and FLAG-tagged IKKb protein was purified by affinity chromatogra-
phy using anti-FLAG M2 affinity gel (Sigma, U.S.A.).
In Vitro Phosphorylation Assay Kinase reaction of purified human
3-Aminonaphtho[1,2-b]furan-2-carboxamide (31) A mixture of 30
(0.79 g) and sodium ethoxide (0.39 g) in EtOH (20 ml) was refluxed 3 h, and
the solvent was evaporated off. The residue was diluted with EtOAc/THF, IKKb was performed at room temperature for 1 h in kinase reaction buffer
washed with brine, dried over MgSO4, and concentrated in vacuo to give (25 mmol/l HEPES, pH 7.5, 10 mmol/l magnesium acetate, 1 mM dithiothre-
crystals. This crude crystals were purified by column chromatography on sil- itol, 0.01% bovine serum albumin, 0.01% Tween20) containing 500 nM ATP,
ica gel with EtOAc/hexane (20%) to afford the title compound (0.54 g, 63%) and bacterially expressed GST-IkBa (1—54), and then terminated by
as crystals. mp 205—207 °C. 1H-NMR (DMSO-d6) d: 6.06 (2H, s), 7.33 adding the same volume of Kinase-GloTM reagent (Promega, U.S.A.). After
(2H, s), 7.59—7.72 (3H, m), 7.89 (1H, d, Jꢂ9 Hz), 8.03 (1H, d, Jꢂ8 Hz),
8.36 (1H, d, Jꢂ8 Hz).
incubating at rt for 10 min the luminescent signal correlated with the amount
of ATP remaining in solution following the reaction was measured by Wal-
3-[(Aminocarbonyl)amino]naphtho[1,2-b]furan-2-carboxamide (32) lac Arvo HTS multilabel counter (Perkin Elmer, U.S.A.).
A mixture of 31 (0.20 g) and potassium cyanate (0.22 g) in AcOH (10 ml)
was stirred at 100 °C for 15 min, and the solvent was evaporated off. The pended in RPMI1640 medium (GIBCO, U.S.A.) containing 1% fetal bovine
residue was diluted with THF, washed with aq. NaHCO3 and brine, dried
over MgSO4, and concentrated in vacuo. The residue was purified by column well plates (Corning Coster, U.S.A.), and then incubated with test com-
Quantitation of TNF-a Human monocytic THP-1 cells were sus-
serum and 50 mg/ml gentamicin. Aliquots of 1ꢈ105 cells were seeded in 96
chromatography on silica gel with EtOAc to afford the title compound pounds for 1 h, subsequently stimulated with 5 mg/ml Lipopolysaccharide
1
(18 mg, 8%) as crystals. mp >300 °C. H-NMR (DMSO-d6) d: 6.70 (2H, s),
7.62—7.71 (4H, m), 8.01—8.21 (3H, m), 8.45 (1H, d, Jꢂ7 Hz), 9.01 (1H,
s). Anal. Calcd for C14H11N3O3·0.3H2O: C, 61.22; H, 4.26; N, 15.30. Found:
C, 61.15; H, 4.33; N, 14.92.
(Wako, Japan) for 4 h. After centrifugation, the quantity of TNF-a in the
medium was measured by a human TNF-a ELISA kit (Diaclone, France).
X-Ray Crystallography JNK3 was purified following the published
protocol.23) Crystals of the JNK3:AMPPNP complex were obtained by vapor
Ethyl 2-[(Cyanomethyl)thio]nicotinate (35) To a stirred solution of 33 diffusion in hanging drops with micro-seeding method. The complex was

624
Vol. 55, No. 4
equilibrated at 20 °C against a reservoir containing 20% Pentaerythritol
ethoxylate (15/4 EO/OH), 0.1 M ammonium sulfate in 0.1 M Bis-Tris, pH 6.5.
Crystals of the 16k compolex were obtained by soaking the AMPPNP-con-
taining crystals overnight in 1 mM compound in mother liquor. They belong
to the orthorhombic space group P212121, with unit cell parameters
aꢂ49.2 Å, bꢂ71.5 Å, cꢂ107.4 Å and one molecule per asymmetric unit.
Diffraction data set was collected on single crystals, vitrified at 100 K, using
synchrotron radiation at beamline BL32B2 in the facilities of Pharmaceuti-
cal Consortium at SPring-8. The structure was initially solved by molecular
replacement with CNX (Accelrys, U.S.A.) using the coordinates of the
JNK3:AMPPNP complex (PDB code: 1JNK) as a starting model. The struc-
ture was further refined and rebuilt to the crystallographic R-factor/free-R of
0.278/0.328, with rmsd bond length/angle of 0.008 Å/1.3°, at 2.0 Å resolu-
tion using CNX and QUANTA (Accelrys, U.S.A.).
1141—1147 (2004).
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Acknowledgements We thank Mr. Masashi Yamaguchi and Dr. Teruaki
Okuda for ADMETox studies, and Dr. Nicholas Hird and Dr. David Cork for 15) Redman A. M., Dumas J., Scott W. J., Org. Lett., 2, 2061—2063
reviewing the manuscript.
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