1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of β-1,6-glucan synthesis inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.08.044

Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7- carbonitrile 3, 1,3-benzoxazole-4-carbonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzoxazole-4-carbonitrile was found to be a superior sca

Full text of DOI:10.1016/j.bmc.2010.08.044

Bioorganic & Medicinal Chemistry 18 (2010) 7593–7606
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,3-Benzoxazole-4-carbonitrile as a novel antifungal scaffold of b-1,6-glucan
synthesis inhibitors
⇑
Jun-ichi Kuroyanagi , Kazuo Kanai, Yuuichi Sugimoto, Takao Horiuchi, Issei Achiwa,
Hiroshi Takeshita, Katsuhiro Kawakami
Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and in vitro antifungal evaluations of 1,3-benzoxazole-7-carbonitrile 3, 1,3-benzoxazole-4-car-
bonitrile 4, benzofuran 5, benzoxazine 7, and benzimidazole 8 were reported. Among them, 1,3-benzox-
azole-4-carbonitrile was found to be a superior scaffold structure with moderate growth inhibition
against Candida species. 1,3-Benzoxazole-4-carbonitrile 6 showed potent activity against Candida species
compared to 5-desmethyl compound 4 and triazolopyridine 2. Compound 6 was efficiently prepared
from versatile intermediate 24, which possessed six different substituents on the benzene ring. Conver-
sion of benzene 24 into various 1,3-benzoxazole derivatives such as 2-aliphatic 34, 2-amino 35, and lac-
tone 38 was demonstrated.
Received 2 August 2010
Revised 21 August 2010
Accepted 24 August 2010
Available online 25 September 2010
Keywords:
Antifungal activity
Candida species
Bicyclic scaffold
Fully substituted benzene
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
derivatives was established starting from benzene derivative 24,
which has six different functional substituents as a versatile
Candida species (spp.), including Candida albicans, Candida glab-
rata, and Candida krusei are considered to be significant pathogens
of systemic fungal infections.1,2 So far, several kinds of antifungal
drugs such as polyenes, azoles, and candins have been launched
for the treatment of these opportunistic infections, which show
efficacies against Candida spp.2–6 However, the usage of these
agents is often limited due to their unsatisfactory antifungal activ-
ity, narrow spectrum, and side effects. These unresolved issues of
antifungal therapy have caused a high rate of mortality and drug
resistance.6–9 Therefore, new antifungal drugs with a novel mode
of action have been constantly required in clinical therapy.
intermediate.
CN
CN
1
8
7
N
N
2
6
N
N
N 3
Ph
Ph
4
5
N
N
NMe2
NMe2
2
1
It was reported that pyridobenzimidazole 1 and its derivatives
exhibited antifungal activities against Candida spp., based on selec-
tive inhibition of the biosynthesis of b-1,6-glucan which is con-
served in various fungi, but not in mammalian cells (Fig. 1).10–12
In the course of our program toward the discovery of new anti-
fungal scaffolds, we have found that triazolopyridine 2 inhibited
b-1,6-glucan synthesis and showed potent antifungal activities
against Candida spp. (Fig. 1).13 Herein, we would like to report
the synthesis of various bicyclic scaffolds and the discovery of
1,3-benzoxazole-4-carbonitrile as a novel bicyclic scaffold with
potent antifungal activity (Fig. 2). A practical and efficient route
for the preparation of 1,3-benzoxazole-4-carbonitrile 6 and its
Figure 1. Structure of compounds 1 and 2.
CN
O
CN
CN
N
R5
N
O
N
X
X
Ph
Ph
Ph
N
N
NMe2
NMe2
NMe2
: X = O, R5 = H
: X = O, R5 = Me
4
6
3
7
: X = N
5 : X = CH
8 : X = NH, R5 = Me
⇑
Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: kuroyanagi.junichi.d5@daiichisankyo.co.jp (J.-i. Kuroyanagi).
Figure 2. Synthesized bicyclic derivatives 3–8.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.08.044

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J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
CO2H
OH
CO2Bn
OH
CO2Bn
CO2Me
OH
CO2Me
OH
CO2Me
O
c
a, b
O
a, b, c
d, e
N
Br
Br
NH2
10
Br
Br
I
Br
F
F
F
NHAc
NHAc
NHAc
11
21
9
19
20
CO2H
CN
CN
CO2Me
O
O
N
h, i, j
f, g
j
O
g, h, i
d, e, f
O
N
Ph
Ph
Ph
F
F
Ph
12
13
Br
23
Br
22
CN
O
l
k
3-HCl salt
CN
N
Ph
O
N
Ph
NMe2
N
3
NMe2
Scheme 1. Synthesis of 1,3-benzoxazole-7-carbonitrile 3. Reagents and conditions:
(a) f.HNO3, H2SO4, 83%; (b) BnBr, K2CO3, DMF, 88%; (c) Fe, AcOH, 34%; (d) pivaloyl
chloride, pyridine, benzene, 83%; (e) p-TsOHꢀH2O, benzene, 43%; (f) PhB(OH)2,
Pd(PPh3)4, K3PO4, THF, 87%; (g) H2, 10% Pd–C, EtOAc, 96%; (h) (COCl)2, DMF, CH2Cl2;
(i) 28% NH4OH, EtOAc; (j) Tf2O, 4-DMAP, pyridine, 90% (three steps); (k) 14, Et3N,
DMSO, 78%; (l) 1 M HCl in EtOH, 91%.
5
Scheme 3. Synthesis of benzofuran-7-carbonitrile 5. Reagents and conditions: (a)
NBS, DMF, 45%; (b) BnNMe3Cl2I, NaHCO3, CH2Cl2–MeOH, 100%; (c) 3,3-dimethyl-1-
butyne, Cl2Pd(PPh3)2, CuI, Et3N, 1,4-dioxane, 54%; (d) PhB(OH)2, Pd(PPh3)4, 2 M
Na2CO3 aq, toluene–DME, 91%; (e) 4 M HCl, MeOH, 48%; (f) tert-BuONO, CuBr2,
MeCN, 99%; (g) KOH, THF–MeOH, 94%; (h) 28% NH4OH, EDCꢀHCl, HOBt, DMF, 99%;
(i) Tf2O, Et3N, CH2Cl2, 87%; (j) 14, Pd(OAc)2, (R)-BINAP, NaOtBu, toluene, 33%.
2. Chemistry
pling reaction18 followed by intramolecular cyclization to give ben-
zofuran 21. Subsequent Suzuki cross-coupling reaction,
deacetylation and Sandmeyer reaction19 afforded 22, which was
then converted to benzofuran-7-carbonitrile 5 by the same manner
as described in Schemes 1 and 2.
As in Scheme 2, our methodology for the synthesis of 4 required
multiple steps in quite low yield (1.3% total yield for 11 steps).
Moreover, functionalization at the vacant 5-position, which was
required for synthesis of 6, has not been achieved because of the
hindered position or highly functionalized benzene structure. Since
both potent antifungal analogs 1 and 2 possessed methyl groups,
methylation of the 1,3-benzoxazole-4-carbonitrile was particularly
interesting. The synthetically and biologically attractive structure
of the fully substituted 1,3-benzoxazole-4-carbonitrile 6 motivated
us to develop an efficient and versatile synthetic methodology that
would allow ready access to various analogs for further biological
As shown in Scheme 1, 1,3-benzoxazole-7-carbonitrile 3 was
synthesized. Nitrization of salicylic acid 914 followed by esterifica-
tion and reduction gave 10, which was condensed with pivaloyl
chloride to afford 1,3-benzoxazole-7-carboxylate 11 as a key
intermediate. Compound 12 was obtained from 11 using Suzuki
cross-coupling reaction15 and hydrogenolysis. (3S)-3-(Dimethyl-
amino)pyrrolidine (14) was introduced to the fluoride 13 to afford
3, which was subsequently treated with hydrochloric acid to pro-
vide HCl salt of 3.
As shown in Scheme 2, 1,3-benzoxazole-4-carbonitrile 18 was
prepared from anthranilic acid 1516 following the similar manner
described in Scheme 1. Palladium catalyzed cross-coupling reac-
tion17 of 18 with 14 gave 1,3-benzoxazole-4-carbonitrile 4, which
was converted into HCl salt of 4.
As outlined in Scheme 3, compound 20, obtained from methyl
4-acetamido-2-hydroxybenzoate (19), was subjected to cross-cou-
CO2H
NH2
CO2H
CO2H
d,e, f
N
a,b,c
N
O
OH
Br
O
Br
Ph
NO2
Br
17
15
16
CN
CN
N
j
k
g, h, i
N
O
4-HCl salt
O
Ph
Ph
N
Br
18
NMe2
4
Scheme 2. Synthesis of 1,3-benzoxazole-4-carbonitrile 4. Reagents and conditions: (a) pivaloyl chloride, pyridine, benzene, 98%; (b) f.HNO3, AcOH, 13%; (c) p-TsOHꢀH2O,
benzene, 98%; (d) PhB(OH)2, Pd(PPh3)4, 2 M Na2CO3 aq, toluene–EtOH; (e) H2, 10% Pd–C, MeOH, 66% (two steps); (f) tert-BuONO, CuBr2, MeCN, 91%; (g) (COCl)2, cat. DMF,
CH2Cl2; (h) 28% NH4OH, EtOAc; (i) Tf2O, 4-DMAP, pyridine, 62% (three steps); (j) 14, Pd(OAc)2, NaOtBu, rac-BINAP, toluene, 34%; (k) 1 M HCl in EtOH, 80%.

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7595
Heterocyclic
CN
CN
ring formation
NH2
OH
a
CN
b, c
NH2
OH
CN
NH2
OH
N
O
R2
Br
Ph
R6
F
Br
F
R7
F
28
24
24
5-Methyl-1,3-benzoxazoles
Cross-coupling
reaction
CN
N
CN
Nucleophilic aromatic
substitution
d,e
N
O
N
O
Ph
Ph
Figure 3. Design of key intermediate 24.
F
evaluation. Our strategy was shown in Figure 3. We envisioned
that the fully substituted 1,3-benzoxazole-4-carbonitirile deriva-
tives could be formed from hexasubstituted benzene 24 through
appropriate functionalization. Bromo group can be replaced to var-
ious substituents such as aromatic rings, unsaturated aliphatic
groups, and amino moieties by means of cross-coupling reactions.
Fluoro group can also be converted into amino substituents by
SNAr reactions. Amino and hydroxyl groups are used for heteroar-
omatic cyclization to afford 1,3-benzoxazole skeleton.
29
R
N
HCl
6 : R = Me
30 : R = H
Scheme 5. Synthesis of 5-methyl-1,3-benzoxazole-4-carbonitriles
6
and 30.
Reagents and conditions: (a) PhB(OH)2, Pd(PPh3)4, K3PO4, 1,4-dioxane-H2O, 91%;
(b) pivaloyl chloride, iPr2NEt, CH2Cl2; (c) p-TsOHꢀH2O, toluene, 93% (two steps); (d)
amine 14 or 31, Et3N, DMSO; (e) 4 M HCl in 1,4-dioxane.
The key intermediate benzene 24 was synthesized as follows:
2-Fluoro-4-methylphenol (25) was selectively nitrized at the
ortho-position of the hydroxyl group, followed by methylation of
the hydroxyl group and reduction of the nitro group to afford aniline
26. Subsequent regioselective halogenations gave fully substituted
benzene 27 in good yield. Finally, palladium-catalyzed cyanization
and subsequent demethylation gave 24 in seven steps and an overall
33% yield from commercially available 25 (Scheme 4).
3. Results and discussion
Synthesized bicyclic compounds 3–8 and 30 as well as pyrido-
benzimidazole 1, triazolopyridine 2, and fluconazole (FLCZ) as po-
sitive control agents were evaluated for their in vitro antifungal
activity against Saccharomyces cerevisiae YPH500 (S. cerevisiae),
Candida albicans ATCC 24433 (C. albicans), Candida glabrata ATCC
48435 (C. glabrata), Candida tropicalis IAM4965 (C. tropicalis), and
Candida krusei ATCC 44507 (C. krusei) (Table 1). Minimum inhibi-
tory concentration MIC-2 (the lowest drug concentration showing
50% growth inhibition compared to the control without drug) was
determined by using the microdilution method with alamar
blue.10–13,20–22 As shown in Table 1, 1,3-benzoxazole-7-carbonitrile
3 exhibited weak growth inhibition against S. cerevisiae. On the
other hand, 1,3-benzoxazole-4-carbonitrile 4, which possessed a
different array of nitrogen and oxygen atoms on the benzoxazole
scaffold from 3, showed moderate growth inhibitory activities
As outlined in Scheme 5, methylated derivatives 6 and 30 were
prepared from 24 and 14 or (3S)-3-(methylamino)pyrrolidine (31)
in a manner similar to that described in Scheme 1.
Benzenes 24 and 28 were converted into various 1,3-benzoxaz-
oles to demonstrate their utility as versatile intermediates. As
shown in Scheme 6, 24 was condensed with various acid chlorides
to give 32. Using this strategy, the 2-position was easily converted.
Cross-coupling reaction of 32 followed by the introduction of 14
gave 34, indicating that this route was applicable to the optimiza-
tion of the 6-position. Benzene 28, possessing a phenyl group, was
used to synthesize various 2-substituted 1,3-benzoxazoles such as
33, which was converted into 34 as described above. Benzene 28
was also useful for the preparation of 2-amino analog 35
(Scheme 7). Synthesis of lactone 38 demonstrated that the 4- and
5-positions can be cyclized easily by using a well-known procedure
(Scheme 8). To extend the utility of fully substituted benzene, ben-
zoxazine 7 was prepared from 27 (Scheme 9). Benzimidazole 8 was
also synthesized from fully substituted benzene 42 (Scheme 10).
against S. cerevisiae and C. glabrata (MIC-2 = 0.25 and 0.25 lg/mL,
respectively). Evaluation of benzofuran derivative 5 exhibited
weak antifungal activity. These results indicate that the positions
of heteroatoms such as nitrogen and oxygen on the bicyclic ring
could strongly affect the potencies of their antifungal activities.
In addition, the nitrogen atom at the ring juncture, as seen at the
4-position of 2 (Fig. 1), was unessential for antifungal activity.
The requirement of the nitrogen atom at the 3-position, as seen
in 4 (Fig. 2), also seems to be endorsed by the result obtained in
the previous study of fused pyridine derivatives.13 The 5-methyl-
ated analog 6 exhibited potent growth inhibition against various
Candida spp., which suggests that the introduction of methyl group
at the 5-position shows more than fourfold enhancement in anti-
fungal activities compared to 5-desmethyl analog. Compound 30,
possessing monomethylamino pyrrolidine moiety, decreased
inhibitory activity. Although 7 had a desired array of heteroatoms
on the benzoxazine skeleton and appropriate substituents at each
position, it showed little effects. Benzimidazole 8 was less active
than benzoxazole 6. These results implied that slight modification
of scaffolds might cause a significant loss of potency.
NH2
a, b, c
OH
O
F
F
25
26
I
CN
NH2
O
f, g
NH2
OH
d, e
Br
Br
F
27
F
24
As shown in Table 2, synthesized 1,3-benzoxazole-4-carbonitr-
iles 34, 35, and lactone 38 as well as triazolopyridine 2 and FLCZ as
positive control agents were evaluated for their in vitro antifungal
activity against FLCZ-susceptible Candida albicans ATCC 90028 (C.
albicans-s), FLCZ-resistant Candida albicans ATCC MYA-573 (C. albi-
cans-r), Candida glabrata ATCC 48435 (C. glabrata), Candida tropicalis
Scheme 4. Synthesis of hexa-substituted benzene 24. Reagents and conditions:
(a) f.HNO3, H2SO4, 98%; (b) Me2SO4, Na2CO3, toluene, 69%; (c) H2, Pd–C, MeOH, 81%;
(d) NBS, DMF, 81%; (e) NIS, AcOH, 98%; (f) Zn(CN)2, Pd(dba)2, DPPF, DMF–H2O, 80%;
(g) BBr3, CH2Cl2, 96%.

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J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
CN
CN
CN
NH2
OH
a, b
c, d
N
O
N
O
R2
R2
Br
Ph
Br
N
F
F
32a : R2 = Me
24
32b : R2 = cyclopropyl
NMe2
34a : R2 = Me
d
: R2 = cyclopropyl
34b
34c
2
: R = cyclobutyl
CN
CN
N
34d : R2 = cyclopentyl
NH2
OH
a, b
34e : R2 = cyclopent-1-enyl
R2
O
Ph
Ph
F
F
33a : R2 = cyclopropyl
28
33b : R2 = cyclobutyl
2
33c
33d
: R = cyclopentyl
: R2 = cyclopent-1-enyl
Scheme 6. Synthesis of 2-aliphatic 1,3-benzoxazole-4-carbonitriles 34. Reagents and conditions: (a) R2COCl, iPr2NEt, EtOAc; (b) p-TsOHꢀH2O, toluene; (c) PhB(OH)2,
Pd(PPh3)4, K3PO4, 1,4-dioxane-H2O; (d) 14, Et3N, DMSO.
CN
N
CN
N
I
CN
NH2
O
N
O
NH2
O
NH2
NH2
OH
c, d
a, b
a, b
Ph
Br
Br
Ph
F
F
27
28
NMe2
NMe2
CN
39
35
CN
Scheme 7. Synthesis of 2-amino-1,3-benzoxazole-4-carbonitriles 35. Reagents and
conditions: (a) di(1H-imidazol-1-yl)methanimine, THF, 87%; (b) 14, Et3N, DMSO,
NH2
N
O
e, f
30%.
Ph
OH
Ph
N
N
CN
OAc CN
HCl
a, b
c
N
O
N
O
NMe2
NMe2
40
7
Ph
Ph
F
F
Scheme 9. Synthesis of benzoxazine 7. Reagents and conditions: (a) Zn(CN)2,
Pd(dba)2, DPPF, DMF–H2O, 80%; (b) 14, Et3N, DMSO, 44%; (c) PhB(OH)2, Pd(PPh3)4,
K3PO4, 1,4-dioxane-H2O, 39%; (d) AlCl3, CH2Cl2, 62%; (e) 1-bromo-3,3-dimethylb-
utan-2-one, Bu4NHSO4, K2CO3, CH2Cl2–H2O; (f) 4 M HCl in 1,4-dioxane, Et2O, 64%
(two steps).
36
33a
O
O
OAc CN
N
O
N
O
d
Ph
Ph
CN
CN
N
N
NH2
NH2
NO2
c, d
a, b
NMe2
NMe2
Br
37
38
F
F
42
Scheme 8. Synthesis of lactone 38. Reagents and conditions: (a) NBS, AIBN, CCl4,
82%; (b) NaOAc, AcOH, 84%; (c) 14, Et3N, DMSO, 44%; (d) K2CO3, MeOH, 33%.
41
CN
CN
CN
N
ATCC 44508 (C. tropicalis), and Candida krusei ATCC 44507 (C. krusei).
YPD (1% yeast extract, 2% peptone, and 20% glucose) was used as the
medium. MIC-1 (the lowestdrug concentrationshowing 80% growth
inhibition compared to the control without drug) was evaluated for
each compound. Among various 2-aliphatic 1,3-benzoxazole-4-car-
bonitriles, tert-butyl and cycloalkyl derivatives 6 and 34b–d showed
potent growth inhibition against Candida spp. Moreover, these
derivatives exhibitedantifungal activity against both FLCZ-suscepti-
NH2
NO2
NH2
N
e
f, g
N
H
Ph
Ph
NH2
Ph
N
N
NMe2
43
NMe2
NMe2
8
44
ble and FLCZ-resistant C. albicans (MIC-1 = 0.063 and 0.063 lg/mL
Scheme 10. Synthesis of benzimidazole 8. Reagents and conditions: (a) NBS, DMF;
(b) f.HNO3, H2SO4, 48% (two steps); (c) 14, Et3N, DMSO; (d) PhB(OH)2, Pd(PPh3)4,
K3PO4, 1,4-dioxane-H2O, 87% (two steps); (e) FeCl3ꢀ6H2O, Zn, DMF–H2O, 97%;
(f) pivaloyl chloride, CHCl3; (g) AcOH, 15% (two steps).
for 34b, respectively). Conversion of the 2-substitute to NH2 de-
creased growth inhibition. Lactone 38 maintained moderate activity
without the 5-methyl and 4-cyano groups.

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7597
Table 1
In vitro activity of bicyclic derivatives
CN
N
CN
N
CN
N
R5
Ph
N
O
O
X
N
X
Ph
Ph
R7
NMe2
N
NMe2
4 : X = O, R5 = H, R7 = Me
6 : X = O, R5 = Me, R7 = Me
7
3 : X = N
5
: X = CH
5
7
30
8
: X = O, R = Me, R = H
: X = NH, R5 = Me, R7 = Me
Compd
MIC-2 (
l
g/mL)a
Log Db
Sol.c
MSd
S. cerevisiae
YPH500
C. albicans
ATCC 24433
C. glabrata
ATCC 48435
C. tropicalis
IAM 4965
C. krusei
ATCC 44507
pH 7.4
pH 6.8 (
l
g/mL)
Human (%)
3
4
5
6
30
7
4
0.25
2
60.016
0.125
8
>4
16
>4
4
16
>16
4
>4
0.25
>4
60.016
0.125
8
>4
0.5
4
0.063
0.25
16
>16
16
>4
NTe
3.8
5.4
4.6
3.4
4.4
4.7
NT
16
<2
5
120
56
8
NT
8
20
6
54
NT
21
4
>16
>16
>16
8
0.5
0.5
2
1
2
FLCZ
0.004
0.063
4
>4
>4
0.25
0.063
0.063
2
0.25
0.25
0.25
0.125
>4
16
3.1f
3.1f
0.4
5
16
>2000
29
9
96
a
b
c
d
e
f
MIC-2s (in micrograms per milliliter) were determined by using the microdilution method with alamar blue.
Log D values were determined from the partition coefficient for 1-octanol/phosphate buffer saline (BPS) at pH 7.4.
Water solubility was measured at pH 6.8.
Metabolic stability for human liver microsomes.
Not tested.
Log D value at pH 6.8.
Physicochemical properties of the obtained compounds were
1,3-Benzoxazole-4-carbonitrile 6 demonstrated that the methyl
group at the 5-position enhanced antifungal efficacy significantly
compared to 5-desmethyl compound 4. The structurally and func-
tionally attractive key intermediate benzene 24, which possessed
six different functional groups, was synthesized efficiently. Con-
version of benzene 24 into various 1,3-benzoxazoles afforded
derivatives such as 2-aliphatic 34, 2-amino 35, and lactone 38,
indicating that benzene 24 proved to be a useful intermediate for
preparing 1,3-benzoxazoles with various substituents at the 2, 4,
5, 6, and 7-positions.
shown in Tables 1 and 2. Although compound 6 exhibited high
lipophilicity, poor water solubility, and low metabolic stability,
monomethylamino analog 30 demonstrated improved physico-
chemical properties. 2-Methyl analog 34a also showed high water
solubility. These results suggest that physicochemical properties
could be improved by reducing lipophilicity.
To confirm the mode of action of 1,3-benzoxazole-4-carbonitr-
iles such as 30 obtained above, we adopted the biochemical ap-
proach based on the methods described by Kitamura et al.10
Compound 30 was evaluated by incorporation studies with grow-
ing cells of C. albicans ATCC 90028 using [14C]-glucose. As shown in
Figure 4, 30 markedly reduced the radioactivity in the fraction of b-
1,6-glucan in a dose-dependent manner. Significant reductions in
the fractions of b-1,3-glucan, chitin, and mannan were not ob-
served in each case. The result obtained above suggested that
1,3-benzoxazole-4-carbonitrile such as 30 was also a specific
inhibitor of b-1,6-glucan synthesis as well as pyridobenzimidazole
1 and triazolopyridine 2.
5. Experimental section
5.1. Chemistry
Unless otherwise noted, materials were obtained from commer-
cial suppliers and used without further purification. Melting points
were taken on a Yanako MP-500D melting point apparatus and are
uncorrected. Optical rotations were measured in a 0.5-dm cell at
25 °C at 589 nm with a HORIBA SEPA-300 polarimeter. 1H NMR
spectra were determined on a JEOL JNM-EX400 spectrometer. 13C
NMR spectra were determined on a JEOL JNM-ECP500 spectrome-
ter. Chemical shifts are reported in parts per million relative to tet-
ramethylsilane as an internal standard. Significant 1H NMR data
are tabulated in the following order: number of protons, multiplic-
ity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br,
broad), and coupling constant(s) in hertz. Infrared (IR) spectra were
obtained on a HORIBA FT-720 spectrometer or a JASCO FT/IR-6100
type A. High-resolution mass spectra were obtained on a JEOL
JMS-700 mass spectrometer under electron impact ionization
4. Conclusion
We prepared benzoxazole, benzofuran, benzoxazine, and benz-
imidazole as scaffolds and evaluated their antifungal activities.
Among them, 1,3-benzoxazole-4-carbonitrile was found to be a
superior scaffold of b-1,6-glucan synthesis inhibitors with potent
antifungal activities. It was revealed that the nitrogen atom at
the ring juncture was considered unessential for antifungal activ-
ity. On the other hand, the nitrogen atom at the 3-position, as seen
in compound 4, was indispensable for potent growth inhibition.

7598
J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
Table 2
In vitro activity of 1,3-benzoxazole derivatives
O
CN
O
N
N
R2
O
Ph
O
Ph
N
N
NMe2
NMe2
6, 34, and 35
38
Compd
R2
MIC-1 (
l
g/mL)a
Log Db
Sol.c
C. albicans-s
C. albicans-r
C. glabrata
C. tropicalis
C. krusei
pH 7.4
pH 6.8 (lg/mL)
ATCC 90028
ATCC MYA-573
ATCC 48435
ATCC 44508
ATCC 44507
6
30
t-Bu
t-Bu
Me
0.063
0.25
2
0.063
0.063
0.125
4
0.125
0.25
2
0.063
0.125
0.125
4
0.063
0.125
0.25
0.032
60.016
0.032
0.5
0.032
0.063
0.25
0.032
60.016
0.063
1
2
>4
4
0.125
0.5
2
16
2
4.6
3.4
3.3
3.9
4.5
4.9
5.2
3.3
2.6
5
120
120
10
7
<2
<2
18
310
34a
34b
34c
34d
34e
35
Cyclopropyl
Cyclobutyl
Cyclopentyl
Cyclopenten-1-yl
NH2
1
1
2
1
0.125
0.25
0.5
0.25
38
-
2
2
FLCZ
-
-
0.25
0.25
0.5
>128
0.125
16
0.125
0.5
8
32
3.1d
0.4
16
>2000
a
b
c
MIC-1s (in micrograms per milliliter) were determined by using the microdilution method with YPD (1% yeast extract, 2% peptone, and 20% glucose) as the medium.
Log D values were determined from the partition coefficient for 1-octanol/phosphate buffer saline (BPS) at pH 7.4.
Water solubility was measured at pH 6.8.
Log D value at pH 6.8.
d
5.1.1. Benzyl 3-amino-5-bromo-4-fluoro-2-hydroxybenzoate
(10)
Compound 30
120
To a stirred solution of 5-bromo-4-fluoro-2-hydroxybenzoic
acid 914 (500 mg, 2.13 mmol) in H2SO4 (5 mL) was added fuming
HNO3 (97 lL, 2.34 mmol) at 0 °C, and the resultant mixture was
100
80
60
40
20
0
stirred at 0 °C for 1.5 h. The reaction mixture was poured into ice
water and the mixture was extracted with AcOEt. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo to afford 5-bromo-4-fluoro-2-hydroxy-3-nitroben-
zoic acid (495 mg, 83%) as a brown solid. HRMS (EI) m/z: 278.9169
(Calcd for C7H379BrNO5 278.9170). 1H NMR (CDCl3) d: 8.21 (1H, d,
J = 7.6 Hz). IR (ATR): 3076, 2860, 1668, 1541, 1431, 1228, 1213,
1167, 1072, 685, 654 cmꢁ1
Benzyl bromide (456
.
l
L, 3.83 mmol) and K2CO3 (481 mg,
3.48 mmol) were added to a solution of the obtained compound
(195 mg, 0.70 mmol) in DMF (4 mL), and the mixture was stirred
at 80 °C for 14 h. The mixture was poured into 1 M HCl aq at
0 °C, and then the mixture was extracted with AcOEt. The obtained
organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with n-hexane/AcOEt = 2:1, v/v
to afford benzyl 5-bromo-4-fluoro-2-hydroxy-3-nitrobenzoate
(226 mg, 88%) as a yellow oil. 1H NMR (CDCl3) d: 5.43 (2H, s),
7.30–7.63 (5H, m), 8.20 (1H, d, J = 7.1 Hz), 11.63 (1H, br s).
A mixture of the obtained compound (100 mg, 2.69 mmol) and
iron powder (45 mg, 8.08 mmol) in AcOH (4 mL) was stirred at
120 °C for 3 h. AcOEt was added and the resultant mixture was fil-
tered. The filtrate was concentrated in vacuo and the residue was
diluted with CHCl3/MeOH = 10:1, v/v. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluting with n-hexane/AcOEt = 9:1, v/v to give 10 (31 mg,
34%) as a colorless solid. Mp: 86–88 °C. MS (ESI) m/z: 340, 342
(M+1)+. 1H NMR (CDCl3) d: 3.95 (2H, br s), 5.36 (2H, s), 7.30–7.48
(6H, m), 10.98 (1H, d, J = 2.0 Hz).
β-1,6-glucan
β-1,3-glucan
chitin
mannan
0
0.016
0.063
0.25
1
4
Concentration (µg/mL)
Figure 4. Incorporation studies of 30 with growing cells of C. albicans ATCC 90028
using [14C]-glucose.
conditions (EI), electron spray ionization conditions (ESI), or fast
atom bombardment ionization conditions (FAB). Column chroma-
tography refers to flash column chromatography conducted on
Merck silica gel 60, 230–400 mesh ASTM. Thin-layer chromatogra-
phy (TLC) was performed with Merck silica gel 60 F254 TLC plates,
and compound visualization was effected with a 5% solution of
molybdophosphoric acid in ethanol, a UV-lamp, iodine, or Wako
Ninhydrin Spray.

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7599
5.1.2. Benzyl 5-bromo-2-tert-butyl-4-fluoro-1,3-benzoxazole-7-
carboxylate (11)
To a solution of 10 (52 mg, 0.15 mmol) in benzene (3 mL) were
added pyridine (27 lL, 0.34 mmol) and pivaloyl chloride (42 lL,
3 h. The reaction mixture was combined with AcOEt and 1 M HCl
aq, and the organic layer was washed with satd NaHCO3 aq and
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography eluting
with n-hexane/AcOEt = 5:1, v/v to give 13 (18.7 mg, 90%) as a white
solid. Mp: 119–121 °C. HRMS (EI) m/z: 294.1161 (Calcd for
0.34 mmol), and then the mixture was stirred at 85 °C for 4 h.
The reaction mixture was cooled to room temperature and diluted
with AcOEt and 1 M HCl aq. The combined organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluting with n-hexane/AcOEt = 3:1, v/v to give benzyl 5-bro-
mo-3-[(2,2-dimethylpropanoyl)amino]-4-fluoro-2-hydroxybenzo-
ate (85 mg, 83%) as a colorless oil. HRMS (EI) m/z: 423.0471 (Calcd
for C19H1979BrFNO4 423.0482). 1H NMR (CDCl3) d: 1.34 (9H, s), 5.38
(2H, s), 7.03 (1H, br s), 7.35–7.43 (5H, m), 7.95 (1H, d, J = 7.4 Hz),
11.19 (1H, d, J = 1.7 Hz).
To a solution of the obtained compound (82 mg, 0.19 mmol) in
benzene (3 mL) was added p-toluenesulfonic acid monohydrate
(37 mg, 0.19 mmol), and the mixture was stirred at 85 °C for
20 h. The reaction mixture was combined with AcOEt and satd
NaHCO3 aq. The organic layer was washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The residue was puri-
fied by silica gel column chromatography eluting with n-hexane/
AcOEt = 5:1, v/v to give 11 (38 mg, 43%) as a colorless solid. Mp:
99–101 °C. MS (ESI) m/z: 406, 408 (M+1)+. 1H NMR (CDCl3) d:
1.50 (9H, s), 5.44 (2H, s), 7.35–7.45 (3H, m), 7.50–7.56 (2H, m),
8.17 (1H, d, J = 6.4 Hz).
C
18H15FN2O 294.1169). 1H NMR (CDCl3) d: 1.56 (9H, s), 7.41–7.53
(5H, m), 7.69 (1H, d, J = 6.4 Hz). 13C NMR (CDCl3) d: 28.4 (3C),
34.8, 92.1 (d, J = 4.8 Hz), 113.9, 126.6 (d, J = 12.5 Hz), 128.6, 128.9
(2C), 129.2, 129.2, 130.8 (d, J = 3.8 Hz), 131.4 (d, J = 18.2 Hz),
133.5, 152.6 (d, J = 8.6 Hz), 153.1 (d, J = 266.8 Hz), 175.8. IR
(ATR): 2231, 1572, 1485, 1402, 1248, 1171, 1117, 1093, 918, 878,
773, 702 cmꢁ1
.
5.1.5. 2-tert-Butyl-4-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
phenyl-1,3-benzoxazole-7-carbonitrile (3)
To a stirred solution of 13 (17 mg, 0.06 mmol) in DMSO (3 mL)
were added triethylamine (24
lL, 0.17 mmol) and (3S)-3-(dimeth-
ylamino)pyrrolidine 14 (15 L, 0.12 mmol). The mixture was stir-
l
red at 90 °C for 18 h, cooled to room temperature, and then
combined with AcOEt and satd NaHCO3 aq. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by silica gel column chro-
matography eluting with CHCl3/MeOH = 9:1, v/v to give
3
(17.5 mg, 78%) as a brown oil. HRMS (EI) m/z: 388.2225 (Calcd
for C24H28N4O 388.2263). 1H NMR (CDCl3) d: 1.47 (9H, s), 1.65
(1H, dq, J = 8.8, 10.5 Hz), 1.96–2.03 (1H, m), 2.17 (6H, s), 2.52–
2.61 (1H, m), 3.40 (1H, dd, J = 8.8, 10.5 Hz), 3.50–3.65 (3H, m),
7.26–7.38 (6H, m). IR (ATR): 2216, 1618, 1471, 1338, 1119,
5.1.3. 2-tert-Butyl-4-fluoro-5-phenyl-1,3-benzoxazole-7-carbox-
ylic acid (12)
To a stirred solution of 11 (37 mg, 0.09 mmol) in THF (4 mL)
were added phenylboronic acid (22 mg, 0.18 mmol), potassium
phosphate (39 mg, 0.18 mmol), and tetrakis(triphenylphosphine)
palladium(0) (11 mg, 0.09 mmol) at room temperature. The mix-
ture was stirred at 70 °C for 16 h. The reaction mixture was com-
bined with AcOEt and satd NH4Cl aq. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluting with n-hexane/AcOEt = 5:1, v/v to give benzyl 2-tert-
butyl-4-fluoro-5-phenyl-1,3-benzoxazole-7-carboxylate (32 mg,
87%) as a white solid. MS (ESI) m/z: 404 (M+1)+. 1H NMR (CDCl3)
d: 1.53 (9H, s), 5.47 (2H, s), 7.33–7.43 (4H, m), 7.45–7.51 (2H, m),
7.52–7.61 (4H, m), 8.10 (1H, d, J = 6.9 Hz).
A mixture of the obtained compound (31 mg, 0.08 mmol) and
10% palladium on carbon (6.2 mg) in AcOEt (3 mL) was stirred un-
der a hydrogen atmosphere at room temperature for 1 h. The reac-
tion mixture was filtered, and the filtrate was concentrated under
reduced pressure to afford 12 (23 mg, 96%) as a white solid. Mp:
219–222 °C. MS (ESI) m/z: 314 (M+1)+. 1H NMR (DMSO-d6) d:
1.46 (9H, s), 7.44 (1H, t, J = 7.3 Hz), 7.51 (2H, t, J = 7.3 Hz), 7.57
(2H, t, J = 7.3 Hz), 7.89 (1H, d, J = 7.3 Hz).
710 cmꢁ1
.
5.1.6. 2-tert-Butyl-4-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
phenyl-1,3-benzoxazole-7-carbonitrile hydrochloride (3-HCl
salt)
To a stirred solution of 3 (17 mg, 0.044 mmol) in Et2O (3 mL)
was added 1 M HCl in EtOH (48 lL, 0.048 mmol), and the mixture
was stirred at room temperature for 4 h. The precipitate was col-
lected by filtration and washed with Et2O to afford the title com-
pound (17 mg, 91%) as a white solid. Mp: 201–202 °C. HRMS (EI)
m/z: 388.2225 (Calcd for C24H28N4O 388.2263). 1H NMR (DMSO-
d6) d: 1.46 (9H, s), 1.96–2.06 (1H, m), 2.10–2.20 (1H, m), 3.18–
3.28 (1H, m), 3.31 (6H, s), 3.75–4.00 (3H, m), 7.34–7.48 (6H, m),
10.74 (1H, br). 13C NMR (DMSO-d6) d: 26.0, 27.9 (3C), 33.7, 40.2,
40.9, 50.8, 52.4, 63.5, 82.4, 115.5, 125.4, 127.1, 128.3 (2C), 129.2
(2C), 130.8, 132.9, 140.2, 142.3, 150.9, 170.0. IR (ATR): 2295,
2220, 1618, 1477, 1466, 1454, 1354, 1122, 710 cmꢁ1. Anal. Calcd
for C24H28N4Oꢀ1.0HClꢀ0.2H2O: C, 67.26; H, 6.91; N, 13.07; Cl,
8.27. Found: C, 67.62; H, 6.86; N, 12.67; Cl, 8.12. ½a D25
ꢂ
+13.1
(c 1.00, MeOH).
5.1.7. 6-Bromo-2-tert-butyl-7-nitro-1,3-benzoxazole-4-
5.1.4. 2-tert-Butyl-4-fluoro-5-phenyl-1,3-benzoxazole-7-
carbonitrile (13)
To a stirred solution of 12 (22 mg, 0.07 mmol) in CH2Cl2 (3 mL)
were added DMF (one drop) and oxalyl chloride (18 lL, 0.21 mmol)
and the resultant mixture was stirred at room temperature for
13 h. The reaction mixture was concentrated in vacuo and azeotr-
oped twice with toluene, and then the residue was dissolved with
AcOEt (2 mL). Then, 28% NH3 in water (1 mL) was added and the
mixture was stirred at room temperature for 3 h. The reaction mix-
ture was combined with CHCl3/MeOH = 10:1, v/v and brine. The or-
ganic layer was washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was dissolved with pyridine
carboxylic acid (16)
Following the procedures as described for 11, 5-bromo-2-[(2,2-
dimethylpropanoyl)amino]-3-hydroxybenzoic acid was prepared
in 98% yield from 1516 and pivaloyl chloride as a brown solid.
HRMS (FAB) m/z: 316.0232 (Calcd for C12H1579BrNO4 315.9752).
1H NMR (CDCl3) d: 1.41 (9H, s), 7.04 (1H, s), 7.45 (1H, d,
J = 2.2 Hz), 7.80 (1H, d, J = 2.2 Hz). IR (ATR): 3360, 1749, 1583,
1466, 1302, 1088, 858, 789, 754 cmꢁ1
To a stirred solution of the obtained compound (149 mg,
0.47 mmol) in AcOH (3 mL) was added fuming HNO3 (22 L,
0.52 mmol) and the resultant mixture was stirred at room temper-
ature for 1 h. The reaction mixture was concentrated in vacuo and
the residue was combined with AcOEt and brine. The organic layer
was washed with brine, dried over Na2SO4, filtered, and concen-
trated in vacuo. The residue was purified by silica gel column
.
l
(3 mL). Trifluoromethanesulfonic anhydride (59 lL, 0.35 mmol)
and 4-dimethylaminopyridine (1.7 mg, 0.014 mmol) were added,
and the resultant mixture was stirred at room temperature for

7600
J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
chromatography eluting with n-hexane/AcOEt = 3:1, v/v to give
5-bromo-2-[(2,2-dimethylpropanoyl)amino]-3-hydroxy-4-nitro-
benzoic acid (22.0 mg, 13%). 1H NMR (CDCl3) d: 1.43 (9H, s), 7.95
(1H, s).
was stirred at 80 °C for 14 h. The mixture was cooled, and the pre-
cipitate was removed by filtration and washed with AcOEt. The
filtrate was diluted with AcOEt and brine. The obtained organic
layer was washed with brine, dried over Na2SO4, filtered, and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography eluting with CHCl3/MeOH = 19:1, v/v to give the
title compound (18.5 mg, 34%) as a brown oil. HRMS (EI) m/z:
388.2251 (Calcd for C24H28N4O 388.2263). 1H NMR (CDCl3) d:
1.50 (9H, s), 1.63–1.74 (1H, m), 1.98–2.08 (1H, m), 2.17 (6H, s),
2.57–2.66 (1H, m), 3.26 (1H, t, J = 9.5 Hz), 3.40–3.50 (3H, m),
7.30–7.40 (6H, m). IR (ATR): 2218, 1610, 1475, 1410, 1192, 1138,
Following the procedures as described for 11, 16 was prepared
in 98% yield from the obtained compound and p-toluenesulfonic
acid monohydrate as a brown solid. Mp: 206–209 °C. HRMS (EI)
m/z: 341.9821 (Calcd for C12H1179BrN2O5 341.9852), 343.9803
(Calcd for C12H1181BrN2O5 343.9831). 1H NMR (CD3OD) d: 1.52
(9H, s), 8.20 (1H, s). 13C NMR (CD3OD) d: 28.6 (3C), 35.7, 109.7,
131.2, 132.5, 136.7, 142.9, 145.3, 168.0, 177.5. IR (ATR): 1705,
1533, 1190, 1136, 808 cmꢁ1
.
750, 698 cmꢁ1
.
5.1.8. 7-Bromo-2-tert-butyl-6-phenyl-1,3-benzoxazole-4-carbox-
ylic acid (17)
5.1.11. 2-tert-Butyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
6-phenyl-1,3-benzoxazole-4-carbonitrile hydrochloride (4-HCl
salt)
Following the procedures as described for 12, 2-tert-butyl-7-ni-
tro-6-phenyl-1,3-benzoxazole-4-carboxylic acid was prepared
from 16 and phenylboronic acid as a brown oil. The obtained
compound was used as such without further purification. HRMS
(EI) m/z: 340.1064 (Calcd for C18H16N2O5 340.1059). 1H NMR
(CDCl3) d: 1.41 (9H, s), 7.26–7.32 (2H, m), 7.38–7.45 (3H, m),
7.94 (1H, s). IR (ATR): 2976, 1711, 1533, 1358, 1236, 1115, 812,
Following the procedures as described for 3-HCl salt, 4-HCl salt
was prepared in 80% yield from 4 as a white solid. Mp: 180–183 °C.
MS (ESI) m/z: 389 (M+1)+. 1H NMR (CD3OD) d: 1.53 (9H, s), 2.05
(1H, dq, J = 12.7, 8.3 Hz), 2.33–2.40 (1H, m), 2.70–2.91 (6H, br m),
3.47–3.61 (3H, m), 3.71 (1H, dd, J = 10.9, 7.4 Hz), 3.87 (1H, dq,
J = 7.3, 7.3 Hz), 7.37–7.61 (6H, m). 13C NMR (DMSO-d6) d: 26.1,
28.0 (3C), 33.9, 40.0, 41.2, 50.2, 51.7, 63.6, 90.0, 116.9, 126.3,
127.4, 128.3 (2C), 129.2 (2C), 133.1, 136.0, 139.7, 141.1, 142.7,
174.6. IR (ATR): 2216, 1610, 1483, 1406, 1140, 714 cmꢁ1. Anal.
Calcd for C24H28N4Oꢀ0.25H2Oꢀ1.0HCl: C, 67.12; H, 6.92; N, 13.05;
698 cmꢁ1
.
A mixture of the obtained compound (3.00 g) and 10% palla-
dium on carbon (430 mg) in MeOH (30 mL) was stirred under a
hydrogen atmosphere at room temperature for 16 h. The reaction
mixture was filtered and the filtrate was concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with CHCl3/MeOH = 49:1, v/v to give 7-amino-2-tert-bu-
tyl-6-phenyl-1,3-benzoxazole-4-carboxylic acid (1.27 g, 66% in
two steps) as a pale brown solid. HRMS (EI) m/z: 310.1295 (Calcd
for C18H18N2O3 310.1318). 1H NMR (CDCl3) d: 1.52 (9H, s), 4.62
(2H, br), 7.35–7.50 (5H, m) 7.89 (1H, s). IR (ATR): 3323, 3209,
Cl, 8.25. Found: C, 67.23; H, 6.71; N, 12.82; Cl, 8.17. ½a D25
ꢂ
+79.6 (c
0.697, CHCl3/MeOH = 1:1, v/v).
5.1.12. Methyl 4-acetamido-5-bromo-2-hydroxy-3-
iodobenzoate (20)
To a solution of 19 (1.1 g, 5.0 mmol) in DMF (8 mL) was added
N-bromosuccinimide (0.94 g, 5.3 mmol) and the mixture was re-
fluxed for 2 h. AcOEt was added and the mixture was washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The
residue was purified by silica gel column chromatography eluting
with CHCl3/MeOH = 100:1, v/v to give methyl 4-acetamido-5-bro-
mo-2-hydroxybenzoate (640 g, 45%) as a pale red solid. Mp:
171–173 °C. MS (FAB) m/z: 288 (M+1 for 79Br)+, 290 (M+1 for
81Br)+. 1H NMR (CDCl3) d: 2.26 (3H, s), 3.94 (3H, s), 7.73 (1H, br
s), 8.00 (1H, s), 8.16 (1H, s), 10.73 (1H, s).
To a solution of the obtained compound (576 mg, 2.0 mmol) in
CH2Cl2 (20 mL) and MeOH (10 mL) were added BnNMe3Cl2I
(835 mg, 2.4 mmol) and NaHCO3 (1.1 g, 13.0 mmol). The mixture
was stirred at room temperature for 30 h and then filtered. The fil-
trate was concentrated in vacuo and the residue was diluted with
CHCl3 and satd NH4Cl aq. The combined organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with CHCl3/MeOH = 50:1, v/v to give the title compound
(889 mg, 100%) as a pale yellow solid. Mp: 222–224 °C. MS (FAB)
m/z: 414 (M+1 for 79Br)+, 416 (M+1 for 81Br)+. 1H NMR (CDCl3) d:
2.26 (3H, s), 4.00 (3H, s), 7.13 (1H, br s), 8.12 (1H, s), 11.73 (1H,
s). 13C NMR (DMSO-d6) d: 22.7, 53.2, 94.9, 111.7, 112.5, 132.3,
145.6, 159.3, 167.5, 167.6. IR (KBr): 3219, 3178, 1672, 1436,
1232, 794 cmꢁ1. Anal. Calcd for C10H9BrINO4ꢀ0.2H2O: C, 28.76; H,
2.27; N, 3.35; Br, 19.13; I, 30.39. Found: C, 28.86; H, 2.20; N,
3.43; Br, 19.27; I, 30.14.
1724, 1635, 1556, 1363, 1329, 1227, 1153, 1109, 775, 702 cmꢁ1
.
A mixture of copper(II) bromide (1.98 g, 8.86 mmol) and tert-
butyl nitrite (1.07 mL, 8.06 mmol) in MeCN (20 mL) was stirred
at 60 °C for 5 min. A solution of the obtained compound (1.25 g,
4.03 mmol) in MeCN (110 mL) was added, and the resultant mix-
ture was stirred at the same temperature for 50 min. The mixture
was combined with AcOEt and 0.5 M HCl aq. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy eluting with CHCl3/MeOH = 49:1, v/v to give 17 (1.37 g, 91%) as
a brown solid. Mp: 211–213 °C. HRMS (EI) m/z: 373.0312 (Calcd for
C
18H1679BrNO3 373.0314), 375.0291 (Calcd for C18H1681BrNO3
375.0295). 1H NMR (CDCl3) d: 1.55 (9H, s), 7.38–7.52 (5H, m),
8.04 (1H, br s). IR (ATR): 2974, 1689, 1545, 1236, 1117, 931, 742,
698 cmꢁ1
.
5.1.9. 7-Bromo-2-tert-butyl-6-phenyl-1,3-benzoxazole-4-car-
bonitrile (18)
Following the procedures as described for 13, 18 was prepared
in 62% yield from 17 as a colorless solid. Mp: 160–163 °C. HRMS
(EI) m/z: 354.0368 (Calcd for C18H1579BrN2O 354.0368). 1H NMR
(CDCl3) d: 1.56 (9H, s), 7.38–7.51 (5H, m), 7.60 (1H, s). IR (ATR):
2231, 1726, 1556, 1460, 1385, 1250, 1124, 1043, 771, 708 cmꢁ1
.
5.1.10. 2-tert-Butyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
6-phenyl-1,3-benzoxazole-4-carbonitrile (4)
A suspension of ( )-2,20-bis(diphenylphosphino)-1,10-binaph-
thyl (13.2 mg, 0.02 mmol) in toluene (2 mL) was heated at 80 °C
until the reagent was dissolved completely. Palladium acetate
(3.2 mg, 0.014 mmol) was added to the solution and the mixture
was stirred at room temperature for 5 min. To the stirred mixture
5.1.13. Methyl 4-acetamido-5-bromo-2-tert-butyl-1-
benzofuran-7-carboxylate (21)
To a stirred solution of triethylamine (4 mL) in 1,4-dioxane
(6 mL) were added 20 (414 mg, 1.0 mmol), bis(triphenylphos-
phine)palladium(II) dichloride (49 mg, 0.07 mmol), copper(I)
iodide (19 mg, 0.1 mmol), and 3,3-dimethyl-1-butyne (0.13 mL,
1.1 mmol) and the mixture was stirred at 30 °C for 2 h and then
were added 18 (50 mg, 0.14 mmol), 14 (22
lL, 0.17 mmol), and so-
dium tert-butoxide (19 mg, 0.20 mmol) and the resultant mixture

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7601
refluxed for 2 h. The reaction mixture was concentrated and the
residue was purified by silica gel column chromatography eluting
with n-hexane/AcOEt = 2:1, v/v to give the title compound
(165 mg, 54%) as a white solid. Mp: 186–188 °C. MS (FAB) m/z:
368 (M+1 for 79Br)+, 370 (M+1 for 81Br)+. 1H NMR (CDCl3) d: 1.40
(9H, s), 2.30 (3H, s), 3.98 (3H, s), 6.38 (1H, s), 7.45 (1H, br s), 8.05
(1H, s). 13C NMR (DMSO-d6) d: 24.0, 28.7 (3C), 33.2, 52.3, 99.8,
109.9, 113.1, 128.8, 128.9, 131.4, 153.1, 164.1, 167.9, 168.5. IR
(KBr): 2965, 1658, 1539, 1391, 1285, 1269, 1160, 1071, 775 cm
ꢁ1. Anal. Calcd for C16H18BrNO4: C, 52.19; H, 4.93; N, 3.80; Br,
21.70. Found: C, 52.11; H, 4.86; N, 3.86; Br, 21.98.
tert-butyl-5-phenyl-1-benzofuran-7-carboxamide (2.59 g, 99%) as
a yellow solid. MS (FAB) m/z: 372 (M+1 for 79Br)+, 374 (M+1 for
81Br)+. 1H NMR (CDCl3) d: 1.45 (9H, s), 6.32 (1H, br s), 6.59 (1H,
s), 7.26 (1H, br s), 7.35–7.47 (5H, m), 8.02 (1H, s).
To a solution of the obtained compound (2.56 g, 6.88 mmol) in
CH2Cl2 (100 mL) were added triethylamine (2.88 mL, 20.7 mmol)
and trifluoromethanesulfonic anhydride (1.86 mL, 11.0 mmol).
The resultant mixture was stirred at room temperature for 2 h.
Water was added and the mixture was extracted with CHCl3. The
organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with n-hexane/AcOEt = 10:1, v/v
to give 23 (2.11 g, 87%) as a yellow solid. Mp: 126–128 °C. MS
(FAB) m/z: 354 (M+1 for 79Br)+, 356 (M+1 for 81Br)+. 1H NMR
(CDCl3) d: 1.44 (9H, s), 6.54 (1H, s), 7.35–7.50 (6H, m). 13C NMR
(CDCl3) d: 28.6 (3C), 33.5, 94.8, 101.0, 114.8, 119.6, 128.1, 128.2
(2C), 129.0, 129.6 (2C), 132.5, 137.8, 139.3, 152.2, 170.8. IR (KBr):
3116, 2961, 2236, 1594, 1452, 1439, 1394, 1087, 921 cmꢁ1. Anal.
Calcd for C19H16BrNO: C, 64.42; H, 4.55; N, 3.95; Br, 22.56. Found:
C, 64.50; H, 4.46; N, 3.97; Br, 22.87.
5.1.14. Methyl 4-bromo-2-tert-butyl-5-phenyl-1-benzofuran-7-
carboxylate (22)
Following the procedures as described for 12, methyl 4-acetam-
ido-2-tert-butyl-5-phenyl-1-benzofuran-7-carboxylate was pre-
pared in 91% yield from 21 and phenylboronic acid as a brown
solid. MS (FAB) m/z: 366 (M+1)+. 1H NMR (CDCl3) d: 1.43 (9H, s),
2.11 (3H, br s), 3.98 (3H, s), 6.40 (1H, s), 7.03 (1H, br s), 7.34–
7.50 (5H, m), 7.83 (1H, s).
To a solution of the obtained compound (2.28 g, 6.24 mmol) in
MeOH (60 mL) was added 4 M HCl in 1,4-dioxane (15.6 mL,
62.4 mmol), and the mixture was stirred at room temperature for
25 h and then stirred at 50 °C for 5 h. The reaction mixture was
poured into satd NaHCO3 aq, and the mixture was extracted with
AcOEt. The obtained organic layers were washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography eluting with n-hex-
ane/AcOEt = 3:1–1:1, v/v to give methyl 4-amino-2-tert-butyl-5-
phenyl-1-benzofuran-7-carboxylate (972 mg, 48%) as a pale yellow
solid. MS (FAB) m/z: 324 (M+1)+. 1H NMR (CDCl3) d: 1.43 (9H, s),
3.93 (3H, s), 4.35 (2H, br s), 6.31 (1H, s), 7.31–7.50 (5H, m), 7.74
(1H, s).
5.1.16. 2-tert-Butyl-4-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
5-phenyl-1-benzofuran-7-carbonitrile (5)
Following the procedures as described for 4, 5 was prepared in
33% yield from 23 as
C
a yellow oil. HRMS (ESI) Calcd. for
25H29N3O + H: 388.2389. Found: 388.2382. 1H NMR (CDCl3) d:
1.39 (9H, s), 1.60–1.74 (1H, m), 1.92–2.04 (1H, m), 2.15 (6H, s),
2.50–2.61 (1H, m), 3.13 (1H, t, J = 9.0 Hz), 3.31–3.42 (3H, m), 6.65
(1H, s), 7.24–7.38 (6H, m). 13C NMR (CDCl3) d: 28.8 (3C), 30.4, 33.0,
44.2 (2C), 51.7, 57.4, 65.5, 84.8, 100.4, 117.0, 118.7, 124.1, 126.7,
128.3 (2C), 129.4 (2C), 132.8, 142.0, 145.1, 155.3, 165.7. IR (ATR):
2969, 2869, 2774, 2219, 1602, 1465 cmꢁ1. ½a D25
ꢂ ꢁ4.16 (c 1.07, CHCl3).
Following the procedures as described for 17, 22 was prepared
in 99% yield from the obtained compound (2.50 g, 7.73 mmol), cop-
per(II) bromide, and tert-butyl nitrite as a brown oil. HRMS (ESI)
Calcd. for C20H1979BrO3 + H: 387.0596. Found: 387.0584. 1H NMR
(CDCl3) d: 1.45 (9H, s), 3.99 (3H, s), 6.52 (1H, s), 7.35–7.47 (5H,
m), 7.84 (1H, s). 13C NMR (CDCl3) d: 28.8 (3C), 33.3, 52.2, 100.3,
113.5, 118.9, 127.6, 127.9, 128.0 (2C), 129.8 (2C), 132.8, 136.8,
140.4, 151.3, 165.0, 169.8. IR (KBr): 2966, 1722, 1594, 1435,
5.1.17. 3-Fluoro-2-methoxy-5-methylaniline (26)
Following the procedures as described for 10, 2-fluoro-4-
methyl-6-nitrophenol was prepared in 98% yield from 25 and fum-
ing HNO3 in H2SO4 as a brown solid. Mp: 63–64 °C. HRMS (EI) m/z:
171.0326 (Calcd for C7H6FNO3 171.0332). 1H NMR (CDCl3) d: 2.35
(3H, s), 7.26 (1H, dd, J = 2.2, 10.5 Hz), 7.71 (1H, s), 10.29 (1H, s).
13C NMR (CDCl3) d: 20.5, 119.6 (d, J = 3.8 Hz), 124.3 (d,
J = 17.3 Hz), 129.1 (d, J = 5.8 Hz), 134.4, 142.7 (d, J = 15.4 Hz),
152.0 (d, J = 250.5 Hz). IR (ATR): 3223, 1638, 1549, 1381, 1237,
1393, 1241, 1146, 769 cmꢁ1
.
1125, 770 cmꢁ1
.
5.1.15. 4-Bromo-2-tert-butyl-5-phenyl-1-benzofuran-7-carbo-
nitrile (23)
To a solution of the obtained compound (8.79 g, 51.4 mmol) in
toluene (51 mL) was added Na2CO3 (25.6 g, 154 mmol), followed
by the dropwise addition of dimethyl sulfate (13.4 mL, 141 mol)
at room temperature. After being stirred under reflux for 6 h, the
reaction mixture was cooled to room temperature. 10% Na2CO3
aq was added, and the resulting mixture was extracted with AcOEt,
washed with water and brine, dried over Mg2SO4, filtered, and con-
centrated. Column chromatography on silica gel (elution with n-
hexane/AcOEt = 9:1, v/v) provided 6.58 g (69%) of 2-fluoro-4-
methyl-6-nitroanisole as a pale yellow oil. 1H NMR (CDCl3) d:
2.37 (3H, s), 4.02 (3H, d, J = 1.5 Hz), 7.13–7.17 (1H, m), 7.37–7.39
(1H, m). 13C NMR (CDCl3) d: 20.7, 62.6 (d, J = 5.8 Hz), 120.3 (d,
J = 2.9 Hz), 121.7 (d, J = 19.2 Hz), 134.1 (d, J = 7.7 Hz), 139.8 (d,
J = 14.4 Hz), 144.3, 155.8 (d, J = 250.5). IR (ATR): 2950, 1532,
1498, 1355, 1302, 1245, 1130, 992, 858, 771 cmꢁ1. Anal. Calcd
for C8H8FNO3: C, 51.90; H, 4.36; N, 7.56; F, 10.26. Found: C,
52.06; H, 4.55; N, 7.47; F, 10.24.
To a solution of 22 (2.90 g, 7.49 mmol) in THF (50 mL) was
added a solution of KOH (1.26 g, 22.5 mmol) in MeOH (12 mL)
and the mixture was stirred at room temperature for 3.5 h. The
mixture was concentrated and then 1 M HCl aq and water was
added to the residue. The mixture was extracted with AcOEt. The
organic layer was washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo to afford 4-bromo-2-tert-butyl-5-phe-
nyl-1-benzofuran-7-carboxylic acid (2.64 g, 94%) as a yellow solid.
MS (FAB) m/z: 373 (M+1 for 79Br)+, 375 (M+1 for 81Br)+. 1H NMR
(CDCl3) d: 1.46 (9H, s), 6.55 (1H, s), 7.37–7.50 (5H, m), 7.94 (1H, s).
To a solution of the obtained compound (2.62 g, 7.02 mmol) and
1-hydroxybenzotriazole monohydrate (1.14 g, 8.42 mmol) in DMF
(80 mL) was added EDCꢀHCl (1.61 g, 8.42 mmol) and the mixture
was stirred at the room temperature for 1 h. To the reaction mix-
ture was added 28% NH4OH aq (1.42 mL, 21.1 mmol) at 0 °C, and
then the mixture was stirred at room temperature for 1 h. The
reaction mixture was concentrated, diluted with satd NaHCO3 aq
and AcOEt, and then extracted with AcOEt. The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluting with n-hexane/AcOEt = 3:2, v/v to give 4-bromo-2-
To a solution of the obtained compound (250 mg, 1.35 mmol) in
MeOH (5 mL) was added 5% palladium on carbon (100 mg), and the
mixture was stirred under a hydrogen atmosphere at room
temperature for 30 min. The reaction mixture was filtered, and
the filtrate was concentrated under reduced pressure. Column
chromatography on silica gel (elution with n-hexane/AcOEt = 9:1,

7602
J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
v/v) provided 170 mg (81%) of 26 as a yellow oil. HRMS (ESI) Calcd
for C8H10FNO + H: 156.0825. Found: 156.0813. 1H NMR (CDCl3) d:
2.19 (3H, s), 3.84 (2H, br s), 3.87 (3H, d, J = 1.2 Hz), 6.26–6.32
(2H, m). 13C NMR (CDCl3) d: 21.0 (d, J = 1.9 Hz), 60.8 (d, J =
4.8 Hz), 106.7 (d, J = 19.2 Hz), 111.5 (d, J = 1.9 Hz), 132.6 (d,
J = 13.4 Hz), 134.0 (d, J = 8.6 Hz), 140.7 (d, J = 4.8 Hz), 155.7 (d,
J = 242.8). IR (ATR): 3373, 2936, 1629, 1584, 1516, 1352, 1227,
Tribromoborane (100 mL, 100.00 mmol) (1 M in CH2Cl2) was
added dropwise to a solution of the obtained compound (8.63 g,
33.30 mmol) in CH2Cl2 (270 mL) at ꢁ20 °C, and the mixture was
stirred at room temperature for 15 h. Ice water was added and
the resulting mixture was neutralized by satd NaHCO3 aq. AcOEt
was added and the organic phase was washed with brine, dried
over Na2SO4, filtered, and concentrated. The residue was recrystal-
lized from n-hexane/CH2Cl2 = 1:1, v/v to afford 7.86 g (96%) of 24
as a pale brown solid. Mp: 214–215 °C. MS (ESI) m/z: 245 (M+1)+.
1H NMR (DMSO-d6) d: 2.36 (3H, s), 5.88 (2H, s). 13C NMR (DMSO-
d6) d: 20.2 (d, J = 1.9 Hz), 91.6, 96.7 (d, J = 20.2 Hz), 116.3, 129.8
(d, J = 17.3 Hz), 131.0, 143.8 (d, J = 7.7 Hz), 150.3 (d, J = 240.9 Hz).
IR (ATR): 3436, 3357, 3198, 2227, 1641, 1588, 1491, 1234, 1088,
888 cmꢁ1. Anal. Calcd for C8H6BrFN2O: C, 39.21; H, 2.47; N,
11.43; F, 7.75; Br, 32.61. Found: C, 39.15; H, 2.38; N, 11.47; F,
7.59; Br, 32.33.
999, 815 cmꢁ1
.
5.1.18. 4-Bromo-3-fluoro-6-iodo-2-methoxy-5-methylphenyl-
amine (27)
To a solution of 26 (1.55 g, 10.0 mmol) in DMF (20 mL) was
added N-bromosuccinimide (1.84 g, 10.2 mmol) in DMF (10 mL) at
0 °C over a period of 25 min. The mixture was heated at room tem-
perature and stirred for 18 h. Brine was added and the resulting
mixture was extracted with AcOEt. The combined organic phase
was washed with brine, dried over Na2SO4, filtered, and concen-
trated. Column chromatography on silica gel (elution with
n-hexane/AcOEt = 4:1, v/v) provided 1.91 g (81%) of 4-bromo-3-flu-
oro-2-methoxy-5-methylphenylamine as a brown solid. HRMS (ESI)
m/z: 233.9925, 235.9903 (Calcd for C8H9BrFNO + H 233.9930,
235.9909). 1H NMR (CDCl3) d: 2.27 (3H, s), 3.80–3.94 (5H, m), 6.43
(1H, s). 13C NMR (CDCl3) d: 22.2 (d, J = 2.9 Hz), 61.0 (d, J = 4.8 Hz),
99.4 (d, J = 18.2 Hz), 111.8 (d, J = 2.9 Hz), 133.6 (d, J = 15.4 Hz),
133.7, 139.6 (d, J = 4.8 Hz), 152.7 (d, J = 243.8). IR (ATR): 3433,
3312, 2951, 1632, 1575, 1499, 1363, 1248, 989, 976, 816 cmꢁ1. Anal.
Calcd for C8H9BrFNO: C, 41.05; H, 3.88; N, 5.98; F, 8.12; Br, 34.14.
Found: C, 40.84; H, 3.80; N, 6.02; F, 8.25; Br, 34.05.
To a solution of the obtained compound (1.90 g, 8.1 mmol) in
AcOH (40 mL) was added N-iodosuccinimide (1.91 g, 8.5 mmol),
and the mixture was stirred at room temperature for 67 h. Brine
was added and the resulting mixture was extracted with AcOEt.
The combined organic phases were washed with brine, dried over
Na2SO4, filtered, and concentrated. Column chromatography on
silica gel (elution with n-hexane/AcOEt = 19:1, v/v) afforded 2.86 g
(98%) of 27 as a pale brown solid. Mp: 89–90 °C. HRMS (ESI)
m/z: 359.8931, 361.8900 (Calcd for C8H8BrFINO + H 359.8896,
361.8876). 1H NMR (CDCl3) d: 2.62 (3H, s), 3.91 (3H, d, J = 1.5 Hz),
4.50 (2H, br s). 13C NMR (CDCl3) d: 29.2 (d, J = 1.9 Hz), 61.1 (d, J =
4.8 Hz), 83.4 (d, J = 2.9 Hz), 98.2 (d, J = 19.2 Hz), 131.9 (d,
J = 15.4 Hz), 135.5, 141.2 (d, J = 5.8 Hz), 152.2 (d, J = 242.8). IR
(ATR): 3363, 1606, 1464, 1433, 983, 823 cmꢁ1. Anal. Calcd for
C8H8BrFINO: C, 26.69; H, 2.24; N, 3.89; F, 5.28; Br, 22.20; I, 35.25.
Found: C, 26.59; H, 2.27; N, 3.90; F, 5.66; Br, 22.38; I, 35.30.
5.1.20. 2-Amino-4-fluoro-3-hydroxy-6-methyl-5-phenylbenzo-
nitrile (28)
A solution of 24 (30.0 g, 122 mmol), phenylboronic acid (29.8 g,
245 mmol), K3PO4 (52.0 g, 245 mmol) and tetrakis(triphenylphos-
phine)palladium(0) (13.7 g, 12.2 mmol) in 1,4-dioxane (1000 mL)
and water (100 mL) was stirred at 95 °C for 2 days. AcOEt was
added to the mixture, and the organic layer was washed with
brine, dried over Na2SO4, filtered, and concentrated. Column chro-
matography on silica gel (elution with CHCl3/AcOEt = 3:1, v/v) pro-
vided 27.1 g (91%) of 28 as a colorless solid. Mp: 197–199 °C. 1H
NMR (CDCl3) d: 2.22 (3H, s), 4.61 (2H, br s), 5.09 (1H, d,
J = 4.2 Hz), 7.19–7.46 (5H, m). 13C NMR (DMSO-d6) d: 17.8 (d,
J = 2.9 Hz), 91.5, 116.9, 118.5 (d, J = 17.3 Hz), 127.4, 128.3 (2C),
129.0 (d, J = 18.2 Hz), 130.0 (d, J = 3.8 Hz), 130.3 (2C), 133.7,
143.3 (d, J = 7.7 Hz), 151.3 (d, J = 240.9 Hz). IR (KBr): 3406, 3277,
2220, 1647, 1493, 1267, 1190 cmꢁ1. Anal. Calcd for C14H11FN2O:
C, 69.41; H, 4.58; N, 11.56; F, 7.84. Found: C, 69.50; H, 4.57; N,
11.46; F, 7.61.
5.1.21. 2-tert-Butyl-7-fluoro-5-methyl-6-phenyl-1,3-benzox-
azole-4-carbonitrile (29)
Following the procedures as described for 11, 29 was prepared
in 93% yield from the obtained compound (9.18 g, 37.46 mmol) as a
white solid. Mp: 192–194 °C. MS (ESI) m/z: 309 (M+1)+. 1H NMR
(CDCl3) d: 1.54 (9H, s), 2.41 (3H, s), 7.22–7.28 (2H, m), 7.42–7.55
(3H, m). 13C NMR (CDCl3) d: 19.2 (d, J = 1.9 Hz), 28.4 (3C), 34.8,
99.9 (d, J = 3.8 Hz), 115.0, 127.2 (d, J = 13.4 Hz), 128.5, 128.8 (2C),
130.0 (2C), 132.7, 136.6 (d, J = 12.5 Hz), 139.3 (d, J = 1.9 Hz),
145.9 (d, J = 3.8 Hz), 146.6 (d, J = 258.2 Hz), 176.6. IR (KBr): 2972,
2229, 1559, 1116, 725 cmꢁ1. Anal. Calcd for C19H17FN2Oꢀ0.2H2O:
C, 73.15; H, 5.62; N, 8.98; F, 6.09. Found: C, 73.52; H, 5.49; N,
9.07; F, 6.16.
5.1.19. 2-Amino-5-bromo-4-fluoro-3-hydroxy-6-
methylbenzonitrile (24)
A mixture of 27 (359 mg, 1.0 mmol), zinc cyanide (59 mg,
0.5 mmol), 1,10-bis(diphenylphosphino)ferrocene (67 mg, 0.12
mmol), and bis(dibenzylideneacetone)palladium(0) (58 mg, 0.1
mmol) in DMF (10 mL)-water (0.1 mL) was heated at 120–130 °C
for 3 h under a nitrogen atmosphere. Brine was added and the
resulting mixture was extracted with AcOEt. The combined organic
phase was washed with brine, dried over Na2SO4, filtered, and con-
centrated. Column chromatography on silica gel (elution with n-
hexane/AcOEt = 9:1, v/v) provided 208 g (80%) of 2-amino-5-bro-
mo-4-fluoro-3-methoxy-6-methylbenzonitrile as a white solid.
Mp: 151–152 °C. MS (FAB) m/z: 258 (M+1 for 79Br)+, 260 (M+1
for 81Br)+. 1H NMR (CDCl3) d: 2.51 (3H, s), 3.93 (3H, d, J = 1.8 Hz),
4.70 (2H, br s). 13C NMR (CDCl3) d: 21.2 (d, J = 1.9 Hz), 61.3 (d,
J = 5.8 Hz), 93.8 (d, J = 1.9 Hz), 99.9 (d, J = 20.2 Hz), 115.8 (d,
J = 1.9 Hz), 132.8 (d, J = 15.4 Hz), 136.9 (d, J = 1.9 Hz), 144.4 (d,
J = 7.7 Hz), 153.8 (d, J = 250.5). IR (ATR): 3466, 3345, 2223, 1629,
1486, 1257, 987, 781 cmꢁ1. Anal. Calcd for C9H8BrFN2O: C, 41.72;
H, 3.11; N, 10.81; F, 7.33; Br, 30.84. Found: C, 42.02; H, 3.07; N,
10.88; F, 7.52; Br, 30.56.
5.1.22. 2-tert-Butyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile
hydrochloride (6-HCl salt)
Following the procedures as described for 3-HCl salt, 6-HCl salt
was prepared in 88% yield from 29 as a pale yellow solid. Mp: 165–
168 °C. MS (FAB) m/z: 403 (M+1ꢁHCl)+. 1H NMR (DMSO-d6) d: 1.47
(9H, s), 1.87–1.97 (1H, m), 2.09–2.17 (1H, m), 2.12 (3H, s), 2.57–
2.61 (3H, m), 2.65–2.69 (3H, m), 3.03–3.10 (1H, m), 3.17–3.24
(1H, m), 3.43–3.51 (1H, m), 3.62–3.68 (1H, m), 3.71–3.80 (1H,
m), 7.23 (1H, d, J = 6.9 Hz), 7.30 (1H, d, J = 8.6 Hz), 7.41–7.51 (3H,
m). 13C NMR (DMSO-d6) d: 19.7, 26.0, 28.0 (3C), 33.9, 40.0, 40.9,
49.8, 51.7, 63.3, 91.5, 116.7, 126.8, 127.7, 128.4, 128.6, 130.6,
130.9, 135.9, 138.4, 138.5, 139.7, 142.9, 174.4. IR (KBr): 2969,
2581, 2464, 2208, 1608, 1471, 1367 cmꢁ1
. Anal. Calcd for
C
25H30N4OꢀHClꢀH2O: C, 65.70; H, 7.28; N, 12.26; Cl, 7.76. Found:
C, 65.85; H, 7.31; N, 12.14; Cl, 7.72. ½a D25
ꢂ
+36.7 (c 1.02, MeOH).

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7603
5.1.23. 2-tert-Butyl-5-methyl-7-[(3S)-3-(methylamino)pyrr-
olidin-1-yl]-6-phenyl-1,3-benzoxazole-4-carbonitrile hydro-
chloride (30)
2.45–2.68 (4H, m), 3.83–3.93 (1H, m), 7.23–7.27 (2H, m), 7.44–
7.53 (3H, m).
Following the procedures as described for 3-HCl salt, 30 was pre-
pared in 89% yield from 29 and (3S)-3-(methylamino)pyrrolidine
(31) as a pale yellow solid. Mp: 253–255 °C. MS (FAB) m/z: 389
(M+1)+. 1H NMR (DMSO-d6) d: 1.47 (9H, s), 1.86–1.94 (1H, m),
2.01–2.09 (1H, m), 2.10 (3H, s), 2.46 (3H, s), 3.18–3.38 (2H, m),
3.38–3.48 (2H, m), 3.55–3.63 (1H, m), 7.23–7.30 (2H, m), 7.41–
7.49 (3H, m). 13C NMR (DMSO-d6) d: 19.8, 27.1, 28.0 (3C), 30.6,
33.8, 49.1, 52.9, 56.6, 90.7, 116.8, 125.7, 127.6, 128.4, 128.4, 130.8,
131.0, 136.1, 138.6, 138.6, 139.2, 142.8, 174.2. IR (ATR): 3512,
2971, 2218, 1606, 1465, 1365, 1304, 709 cmꢁ1. Anal. Calcd for
C24H28N4OꢀHClꢀ0.5H2O: C, 66.42; H, 6.97; N, 12.91; Cl, 8.17. Found:
5.1.29. 2-Cyclopentyl-7-fluoro-5-methyl-6-phenyl-1,3-benzox-
azole-4-carbonitrile (33c)
Yield, 42% from 28 with cyclopentanecarbonyl chloride, as a col-
orless amorphous solid. 1H NMR (CDCl3) d: 1.72–1.89 (4H, m),
1.98–2.22 (4H, m), 2.41 (3H, s), 3.41–3.49 (1H, m), 7.25–7.29
(2H, m), 7.43–7.54 (3H, m).
5.1.30. 2-Cyclopent-1-enyl-7-fluoro-5-methyl-6-phenyl-1,3-
benzoxazole-4-carbonitrile (33d)
Yield, 56% from 28 with 1-cyclopentene-1-carbonyl chloride, as
a colorless solid. 1H NMR (CDCl3) d: 2.08–2.17 (2H, m), 2.42 (3H, s),
2.66–2.73 (2H, m), 2.92–2.99 (2H, m), 7.07–7.11 (1H, m), 7.25–7.29
(2H, m), 7.44–7.54 (3H, m).
C, 66.41; H, 6.96; N, 12.57; Cl, 8.13. ½a D25
ꢂ
+30.6 (c 1.01, MeOH).
5.1.24. 6-Bromo-7-fluoro-2,5-dimethyl-1,3-benzoxazole-4-car-
bonitrile (32a)
5.1.31. 7-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2,5-
Acetyl chloride (3.85 mL, 54.2 mmol) was added dropwise to a
solution of 24 (8.85 g, 36.1 mmol) and N,N-diisopropylethylamine
(22.0 mL, 126 mmol) in AcOEt (400 mL) at 0 °C, and then the mix-
ture was stirred at room temperature for 14 h. AcOEt was added
and the organic layer was washed with brine, dried over Na2SO4,
filtered, and concentrated. A solution of the residue and p-toluene-
sulfonic acid monohydrate (200 mg) in toluene (500 mL) was stir-
red at reflux for 3 h. The mixture was concentrated and the residue
was diluted with AcOEt. The organic layer was washed with brine,
dried over Na2SO4, filtered, and concentrated. The residue was
recrystallized from AcOEt to afford 7.07 g (73%) of 32a as a color-
less solid. MS (EI) m/z: 269 (M+). 1H NMR (CDCl3) d: 2.73 (3H, s),
2.75 (3H, s). IR (ATR): 2229, 1574, 1475, 1406, 1396, 1384, 1333,
dimethyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (34a)
Following the procedures as described for 28, 7-fluoro-2,5-di-
methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile was prepared in
45% yield from 32a as a colorless solid. MS (ESI) m/z: 267 (M+1)+.
1H NMR (CDCl3) d: 2.42 (3H, s), 2.76 (3H, s), 7.23–7.27 (2H, m),
7.40–7.53 (3H, m). To the solid (266 mg, 1.00 mmol) in DMSO
(5 mL) were added 14 (190
lL, 1.50 mmol) and triethylamine
(300 L, 2.15 mmol), and then the mixture was heated at 80 °C
l
for 14 h. The reaction mixture was diluted with CHCl3 and the or-
ganic layer was washed with water and brine, dried over Na2SO4,
filtered, and concentrated. Column chromatography on silica gel
(elution with CHCl3/MeOH = 19:1, v/v) provided 110 mg (31%) of
34a as a colorless solid. HRMS (ESI) m/z: 361.2000 (Calcd for
1315, 1273, 1198, 1134 cmꢁ1
.
C
22H24N4O + H 361.2028). 1H NMR (CDCl3) d: 1.53–1.67 (1H, m),
1.90–1.98 (1H, m), 2.19 (6H, s), 2.28 (3H, s), 2.45–2.58 (1H, m),
2.66 (3H, s), 2.90–2.98 (1H, m), 3.25–3.43 (3H, m), 7.10–7.14
(1H, m), 7.20–7.26 (1H, m), 7.32–7.50 (3H, m). 13C NMR (DMSO-
d6) d: 14.0, 19.5, 29.0, 43.1 (2C), 50.6, 55.2, 64.7, 90.6, 116.7,
126.2, 127.4, 128.4, 129.6, 130.8, 131.2, 136.8, 138.4, 139.2,
139.9, 143.5, 165.1. IR (ATR): 2210, 1670, 1608, 1585, 1442,
5.1.25. 6-Bromo-2-cyclopropyl-7-fluoro-5-methyl-1,3-benzox-
azole-4-carbonitrile (32b)
Following the procedures as described for 32a, 32b was pre-
pared in 80% yield from 24 as a pale red solid. Mp: 164–168 °C.
MS (ESI) m/z: 295, 297 (M+1)+. 1H NMR (CDCl3) d: 1.27–1.34 (2H,
m), 1.38–1.42 (2H, m), 2.25–2.32 (1H, m), 2.73 (3H, s). 13C NMR
(DMSO-d6) d: 8.9, 10.5 (2C), 21.3, 98.3, 106.9 (d, J = 17.3 Hz),
114.2, 135.5 (d, J = 10.6 Hz), 139.3, 145.1 (d, J = 196.7 Hz), 146.3,
1248, 1153 cmꢁ1. ½a D25
ꢂ
+69.2 (c 1.03, CHCl3/MeOH = 1:1, v/v).
5.1.32. 2-Cyclopropyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-
yl]-5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (34b)
Following the procedures as described for 34a, 34b was pre-
pared in 63% yield from 32b as a white solid. MS (ESI) m/z: 387
(M+1)+. 1H NMR (CDCl3) d: 1.17–2.30 (4H, m), 1.55–1.67 (1H, m),
1.90–1.98 (1H, m), 2.14 (6H, s), 2.17 (3H, s), 2.24 (1H, ddd,
J = 5.1, 8.5, 13.0 Hz), 2.48–2.57 (1H, m), 2.94 (1H, t, J = 9.3 Hz),
3.22–3.39 (3H, m), 7.10 (1H, d, J = 7.6 Hz), 7.23 (1H, d, J = 7.6 Hz),
7.31–7.42 (3H, m). 13C NMR (CDCl3) d: 9.4 (2C), 9.6, 20.1, 30.3,
44.1 (2C), 50.9, 56.3, 65.4, 91.6, 117.2, 125.9, 127.4, 128.2, 128.6,
131.0, 131.5, 136.5, 139.2, 139.5, 139.6, 144.2, 169.4. IR (ATR):
2206, 1585, 1560, 1466, 1152, 712, 702 cmꢁ1. Anal. Calcd for
172.5. IR (ATR): 3057, 2230, 1625, 1567, 1317, 1131, 1034 cmꢁ1
Anal. Calcd for C12H8BrFN2O: C, 48.84; H, 2.73; N, 9.49; F, 6.44.
Found: C, 49.02; H, 2.75; N, 9.57; F, 6.55.
.
5.1.26. General procedure for preparation of 7-fluoro-5-methyl-
6-phenyl-1,3-benzoxazole-4-carbonitrile (33)
Following the procedures as described for 32, 33 was prepared
from 28.
5.1.27. 2-Cyclopropyl-7-fluoro-5-methyl-6-phenyl-1,3-benzox-
azole-4-carbonitrile (33a)
Yield, 85% from 28 with cyclopropanecarbonyl chloride, as a
colorless solid. MS (ESI) m/z: 293 (M+1)+. 1H NMR (CDCl3) d:
1.26–2.01 (2H, m), 2.38–2.42 (2H, m), 2.28–2.34 (1H, m), 2.40
(3H, s), 7.21–7.26 (2H, m), 7.42–7.51 (3H, m). 13C NMR (CDCl3) d:
9.5, 10.4 (2C), 19.1 (d, J = 1.9 Hz), 99.1 (d, J = 3.8 Hz), 115.0, 126.8
(d, J = 14.4 Hz), 128.5, 128.8 (2C), 130.0 (2C), 132.7, 136.1 (d,
J = 13.4 Hz), 139.3, 146.3 (d, J = 3.8 Hz), 146.3 (d, J = 258.2 Hz),
C
24H26N4Oꢀ0.25H2O: C, 73.72; H, 6.83; N, 14.33. Found: C, 74.08;
H, 6.76; N, 14.35. ½a D25
ꢂ
+97.2 (c 0.746, CHCl3).
5.1.33. 2-Cyclobutyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (34c)
Following the procedures as described for 3, 34c was prepared
in 47% yield from 33b as a white amorphous. MS (ESI) m/z: 401
(M+1)+. 1H NMR (CDCl3) d: 1.56–1.68 (1H, m), 2.14 (6H, s), 2.19
(3H, s), 2.06–2.20 (3H, m), 2.41–2.56 (5H, m), 2.99 (1H, t,
J = 9.3 Hz), 3.27–3.43 (3H, m), 3.77–3.86 (1H, m), 7.09–7.13 (1H,
m), 7.23–7.27 (1H, m), 7.32–7.44 (3H, m). 13C NMR (DMSO-d6) d:
18.3, 19.6, 26.5, 26.6, 29.2, 32.8, 43.2 (2C), 50.6, 55.3, 64.7, 90.6,
116.7, 126.1, 127.4, 128.1, 128.3, 130.8, 131.3, 136.9, 138.5,
139.3, 139.6, 143.3, 170.2. .IR (ATR): 2210, 1604, 1583, 1468,
172.0. IR (ATR): 2221, 1562, 1412, 1321, 1122, 1026, 722 cmꢁ1
Anal. Calcd for C18H13FN2O: C, 73.96; H, 4.48; N, 9.58; F, 6.50.
Found: C, 73.65; H, 4.47; N, 9.54; F, 6.23.
.
5.1.28. 2-Cyclobutyl-7-fluoro-5-methyl-6-phenyl-1,3-benzoxaz-
ole-4-carbonitrile (33b)
Yield, 77% from 28 with cyclobutanecarbonyl chloride, as a col-
orless solid. 1H NMR (CDCl3) d: 2.06–2.25 (2H, m), 2.42 (3H, s),

7604
J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
1362, 1153, 701 cmꢁ1. Anal. Calcd for C25H28N4Oꢀ0.25H2O: C,
mixture was concentrated and the resulting residue was purified
by silica gel column chromatography eluting with n-hexane/
AcOEt = 5:1, v/v to give 5-(bromomethyl)-2-cyclopropyl-7-fluoro-
6-phenyl-1,3-benzoxazole-4-carbonitrile (1.04 g, 82%) as a white
solid. MS (ESI) m/z: 371, 373 (M+1)+. 1H NMR (CDCl3) d: 1.29–
1.35 (2H, m), 1.39–1.45 (2H, m), 2.28–2.36 (1H, m), 4.50 (2H, s),
7.36–7.42 (2H, m), 7.50–7.55 (3H, m). IR (ATR): 2227, 1566,
74.14; H, 7.09; N, 13.83. Found: C, 74.50; H, 7.09; N, 13.72. ½a D25
ꢂ
+92.6 (c 1.05, CHCl3).
5.1.34. 2-Cyclopentyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-
yl]-5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (34d)
Following the procedures as described for 3, 34d was prepared in
33% yield from 33c as a white solid. Mp: 120–122 °C. MS (ESI) m/z:
415 (M+1)+. 1H NMR (CDCl3) d: 1.53–2.09 (8H, m), 2.13 (6H, s),
2.14–2.18 (2H, m), 2.19 (3H, s), 2.45–2.56 (1H, m), 2.98 (1H, t,
J = 9.2 Hz), 3.22–3.46 (4H, m), 7.09–7.13 (1H, m), 7.22–7.26 (1H,
m), 7.31–7.43 (3H, m). 13C NMR (DMSO-d6) d: 19.6, 25.2 (2C), 29.2,
30.9 (2C), 38.2, 43.2 (2C), 50.6, 55.4, 64.7, 90.5, 116.8, 125.9, 127.4,
128.1, 128.3, 130.8, 131.3, 136.9, 138.4, 139.3, 139.6, 143.2, 171.3.
IR (ATR): 2952, 2867, 2208, 1604, 1558, 1468, 1363, 1299, 1191,
704 cmꢁ1. Anal. Calcd for C26H30N4Oꢀ0.25H2O: C, 74.52; H, 7.34; N,
1469, 1415, 1325, 1265, 1219, 1122, 1036 cmꢁ1
.
To a solution of the solid (834 mg, 2.25 mmol) in AcOH (23 mL)
was added sodium acetate (922 mg, 11.2 mmol) and the mixture
was stirred at 100 °C for 21 h. The reaction mixture was concen-
trated and the residue was diluted with AcOEt and water. The mix-
ture was extracted with AcOEt and the organic layer was dried
(Na2SO4) and evaporated to give the crude product, which was
purified by silica gel column chromatography eluting with n-hex-
ane/AcOEt = 3:1, v/v to afford 36 (664 mg, 84%) as a white solid.
MS (ESI) m/z: 351 (M+1)+. 1H NMR (CDCl3) d: 1.29–1.35 (2H, m),
1.39–1.45 (2H, m), 2.01 (3H, s), 2.28–2.37 (1H, m), 5.08 (2H, s),
7.24–7.29 (2H, m), 7.45–7.50 (3H, m). IR (ATR): 2229, 1732,
13.37. Found: C, 74.57; H, 7.31; N, 13.31. ½a D25
ꢂ
+90.4 (c 1.04, CHCl3).
5.1.35. 2-Cyclopent-1-enyl-7-[(3S)-3-(dimethylamino)pyrroli-
din-1-yl]-5-methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile
(34e)
1556, 1417, 1365, 1308, 1236, 1227, 1124, 1026 cmꢁ1
.
5.1.38. {4-Cyano-2-cyclopropyl-7-[(3S)-3-(dimethylamino)pyrr-
olidin-1-yl]-6-phenyl-1,3-benzoxazol-5-yl}methyl acetate (37)
Following the procedures as described for 3, 37 was prepared in
44% yield from 36 as a brown oil. MS (ESI) m/z: 445 (M+1)+. 1H
NMR (CDCl3) d: 1.18–1.32 (4H, m), 1.53–1.65 (1H, m), 1.90–1.98
(1H, m), 1.97 (3H, s), 2.12 (6H, s), 2.21–2.29 (1H, m), 2.44–2.54
(1H, m), 2.88–2.96 (1H, m), 3.22–3.41 (3H, m), 4.82 (1H, d,
J = 12.2 Hz), 4.98 (1H, d, J = 12.2 Hz), 7.11–7.17 (1H, m), 7.23–
7.28 (1H, m), 7.32–7.40 (3H, m). IR (ATR): 2212, 1739, 1587,
Following the procedures as described for 3, 34e was prepared
in 18% yield from 33d as a pale yellow solid. Mp: 98–101 °C. MS
(ESI) m/z: 413 (M+1)+. 1H NMR (CDCl3) d: 1.57–1.60 (1H, m),
1.89–1.99 (1H, m), 2.13 (6H, s), 2.05–2.16 (2H, m), 2.20 (3H, s),
2.44–2.55 (1H, m), 2.60–2.68 (2H, m), 2.88–3.03 (3H, m), 3.25–
3.42 (3H, m), 6.87 (1H, br s), 7.12 (1H, d, J = 7.6 Hz), 7.25–7.27
(1H, m), 7.31–7.44 (3H, m). IR (ATR): 2202, 1603, 1466, 1442,
1362, 1302, 706 cmꢁ1
.
Anal. Calcd for C26H28N4Oꢀ1.0H2O: C,
72.53; H, 7.02; N, 13.01. Found: C, 72.62; H, 6.99; N, 12.70. ½a D25
ꢂ
1468, 1367, 1221, 1155 cmꢁ1
.
+97.8 (c 0.225, CHCl3).
5.1.39. 2-Cyclopropyl-4-[(3S)-3-(dimethylamino)pyrrolidin-1-
yl]-5-phenylfuro[3,4-e][1,3]benzoxazol-8(6H)-one (38)
5.1.36. 2-Amino-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
methyl-6-phenyl-1,3-benzoxazole-4-carbonitrile (35)
To a solution of 37 (236 mg, 531 lmol) in MeOH (5 mL) was
A suspension of 28 (1.66 g, 6.86 mmol) and di(1H-imidazol-1-
yl)methanimine (2.21 g, 13.7 mmol) in THF (33 mL) was refluxed
under nitrogen for 5 days. After the mixture was concentrated, the
residue was diluted with CHCl3/MeOH (9:1, v/v) and water, and ex-
tracted with CHCl3. The organic layer was separated, dried (Na2SO4),
and evaporated to give the crude product, which was recrystallized
from MeOH to yield 2-amino-7-fluoro-5-methyl-6-phenyl-1,3-
benzoxazole-4-carbonitrile (1.60 g, 87%) as a pale yellow solid. A
solution of the solid obtained above (200 mg, 0.75 mmol), 14
added K2CO3 (220 mg, 1.59 mmol) and the mixture was stirred at
room temperature for 24 h. The mixture was diluted with CHCl3
and the solution was washed with brine, dried over Na2SO4, and
evaporated to give the crude product, which was purified by pre-
parative TLC plates (CH2Cl2/MeOH = 10:1, v/v) to afford 38 (71 mg,
33%) as a pale brown solid. Mp: 192–194 °C. MS (ESI) m/z: 404
(M+1)+. 1H NMR (CDCl3) d: 1.17–1.24 (2H, m), 1.29–1.35 (2H, m),
1.58–1.73 (1H, m), 1.96–2.04 (1H, m), 2.15 (6H, s), 2.27–2.35 (1H,
m), 2.51–2.62 (1H, m), 3.16 (1H, t, J = 9.3 Hz), 3.39–3.54 (3H, m),
4.69 (1H, d, J = 15.4 Hz), 4.99 (1H, d, J = 15.4 Hz), 7.22–7.29 (2H,
m), 7.33–7.43 (3H, m). 13C NMR (CDCl3) d: 9.4, 9.6 (2C), 30.5, 44.3
(2C), 51.2, 56.7, 65.4, 69.5, 104.0, 117.2, 127.8 (2C), 128.6 (2C),
129.9, 137.2, 137.5, 139.1, 141.8, 147.4, 169.5, 170.3. IR (ATR):
1755, 1603, 1560, 1481, 1367, 1315, 1192, 1153, 1099 cmꢁ1. Anal.
Calcd for C24H25N3O3ꢀ0.25H2O: C, 70.66; H, 6.30; N, 10.30. Found:
(190 lL, 1.50 mmol) and triethylamine (209 lL, 1.50 mmol) in
DMSO (4 mL) was heated at 150 °C in a sealed tube. After cooling,
the mixture was diluted with AcOEt and brine and then extracted
with AcOEt. The combined organic layer was washed with brine,
dried (Na2SO4), and evaporated to give the crude product, which
was purified by preparative TLC plates (CHCl3/7 M ammonia in
MeOH = 10:1, v/v) to afford 35 (81 mg, 30%) as a brown solid: Mp
C, 71.01; H, 6.22; N, 10.59. ½a D25
ꢂ
+0.234 (c 1.02, CHCl3).
249–251 °C. HRMS (EI) m/z: 361.1904 (Calcd for
C21H23N5O
361.1903). 1H NMR (CDCl3) d: 1.38–1.49 (1H, m), 1.79–1.86 (1H,
m), 1.96 (6H, s), 2.01 (3H, s), 2.38–2.47 (1H, m), 2.67 (1H, t,
J = 8.3 Hz), 3.07–3.27 (3H, m), 7.09–7.12 (1H, m), 7.27–730 (1H,
m), 7.32–7.45 (3H, m), 7.73 (2H, s). 13C NMR (DMSO-d6) d: 19.3,
29.4, 43.5 (2C), 50.3, 55.4, 64.7, 87.9, 117.5, 123.7, 127.0, 128.0,
128.3, 130.7, 131.4, 135.1, 135.6, 135.8, 139.5, 147.1, 163.6. IR
(ATR): 3228, 2210, 1672, 1560, 1466, 1408, 1286, 739 cmꢁ1. Anal.
Calcd for C21H23N5Oꢀ0.25H2O: C, 68.92; H, 6.47; N, 19.14. Found: C,
5.1.40. 2-Amino-5-bromo-4-[(3S)-3-(dimethylamino)pyrr-
olidin-1-yl]-3-methoxy-6-methylbenzonitrile (39)
Following the procedures as described for 3, 39 was prepared in
44% yield from 2-amino-5-bromo-4-fluoro-3-methoxy-6-meth-
ylbenzonitrile as a pale brown oil. MS (FAB) m/z: 353 (M+1 for
79Br)+, 355 (M+1 for 81Br)+. 1H NMR (CDCl3) d: 1.81–1.94 (1H, m),
2.14–2.25 (1H, m), 2.30 (6H, s), 2.49 (3H, s), 2.77–2.88 (1H, m),
3.35–3.44 (2H, m), 3.47–3.54 (1H, m), 3.58–3.70 (1H, m), 3.65
(3H, s), 4.52 (2H, br s).
68.68; H, 6.41; N, 18.79. ½a D25
ꢂ
+110 (c 0.202, CHCl3).
5.1.41. 4-Amino-6-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-5-
hydroxy-2-methylbiphenyl-3-carbonitrile (40)
5.1.37. (4-Cyano-2-cyclopropyl-7-fluoro-6-phenyl-1,3-benzoxa-
zol-5-yl)methyl acetate (36)
Following the procedures as described for 28, 4-amino-6-[(3S)-
3-(dimethylamino)pyrrolidin-1-yl]-5-methoxy-2-methylbiphenyl-
3-carbonitrile was prepared in 39% yield from 39 as a pale brown
A solution of 33a (1.00 g, 3.42 mmol), N-bromosuccinimide
(913 mg, 5.13 mmol) and 2,20-azobis(2-methylpropionitrile) (56
mg, 342 lmol) in CCl4 (12 mL) was stirred at 80 °C for 13 h. The

J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
7605
oil. MS (FAB) m/z: 351 (M+1)+. 1H NMR (CDCl3) d: 1.42–1.56 (1H,
m), 1.78–1.90 (1H, m), 2.09 (3H, s), 2.11 (6H, s), 2.30–2.42 (1H,
m), 2.80–2.98 (3H, m), 3.10 (1H, dd, J = 6.9, 9.0 Hz), 3.63 (3H, s),
4.51 (2H, br s), 7.04–7.09 (1H, m), 7.11–7.17 (1H, m), 7.26–7.42
(3H, m). To a suspension of AlCl3 (399 mg, 3.00 mmol) in CH2Cl2
(M+1)+. 1H NMR (CDCl3) d: 1.64 (1H, m), 1.80 (1H, m), 2.06 (6H,
s), 2.16 (3H, s), 2.35 (1H, m), 2.76 (1H, m), 2.890–2.96 (3H, m),
4.09 (2H, br s), 7.10–7.14 (2H, m), 7.34–7.49 (3H, m). IR (ATR):
3357, 3284, 2944, 2821, 2775, 2200, 1666, 1583, 1459, 1436 cmꢁ1
.
(2.0 mL) was added the obtained compound (210 mg, 599
l
mol)
5.1.46. 2-tert-Butyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
5-methyl-6-phenyl-1H-benzimidazole-4-carbonitrile (8)
in CH2Cl2 (10 mL), and the mixture was refluxed for 15 min. The
reaction mixture was cooled to room temperature. Satd NaHCO3
aq and brine were added, and the mixture was extracted with
CHCl3. The organic layer was dried over Na2SO4, filtered, and con-
centrated. The residue was purified by silica gel column chroma-
tography eluting with CHCl3/MeOH = 100:1, v/v to afford 40
(125 mg, 62%) as a brown solid. MS (FAB) m/z: 337 (M+1)+. 1H
NMR (CDCl3) d: 1.57–1.70 (1H, m), 2.05–2.15 (1H, m), 2.08 (3H,
s), 2.34 (6H, s), 2.48–2.54 (1H, m), 2.73 (1H, dt, J = 1.2, 8.7 Hz),
2.89–2.95 (1H, m), 3.01–3.18 (2H, m), 4.51 (2H, br s), 7.05–7.16
(2H, m), 7.24–7.42 (3H, m).
To a solution of 44 (1.25 g, 3.73 mmol) in CHCl3 (40 mL) was
added pivaloyl chloride (0.46 mL, 3.73 mmol), and the mixture
was stirred at room temperature for 3 h. Water was added, and
the mixture was extracted with CHCl3. The organic layer was dried
over Na2SO4, filtered, and concentrated. The residue was purified
by silica gel column chromatography eluting with CHCl3/
MeOH = 19:1, v/v to afford a brown amorphous solid. A solution
of the solid in AcOH (5 mL) was stirred at 80 °C for 2.5 h. The mix-
ture was cooled to room temperature, and then poured into aque-
ous NaOH solution (6.7 M, 15 mL). The resultant solid was
collected and purified by silica gel column chromatography eluting
with AcOEt/acetone = 1:1, v/v to afford 8 (228 mg, 15%) as a color-
less solid. MS (EI) m/z: 401 (M+). 1H NMR (DMSO-d6) d: 1.35–1.52
(1H, m), 1.42 (9H, s), 1.75–1.85 (1H, m), 2.04, (6H, s), 2.09, (3H, s),
2.50–2.55 (1H, m), 3.12–3.22 (1H, m), 3.35–3.55 (3H, m), 7.05–7.55
(5H, m), 12.16 (1H, br s). IR (ATR): 3241, 2969, 2813, 2765, 2204,
1602, 1565, 1455 cmꢁ1. Anal. Calcd for C25H31N5: C, 74.78; H,
5.1.42. 3-tert-Butyl-8-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
6-methyl-7-phenyl-2H-1,4-benzoxazine-5-carbonitrile
hydrochloride (7)
To a mixture of 40 (113 mg, 336 lmol) and 20% K2CO3 aq
(1.8 mL) in CH2Cl2 (3.6 mL) were added tetrabutylammonium
hydrogen sulfate (37.5 mg, 0.111 mmol) and 1-bromo-3,3-dimeth-
ylbutan-2-one (54.2
lL, 0.403 mmol), and the reaction mixture
7.78; N, 17.44. Found: C, 74.76; H, 7.85; N, 17.28. ½a D25
ꢂ
+152 (c
was stirred for 15 h at room temperature. Brine was added, and
the mixture was extracted with CHCl3. The organic layer was dried
over Na2SO4, filtered, and concentrated. The residue was purified
by silica gel column chromatography eluting with CHCl3/
MeOH = 100:1, v/v to afford a brown oil. To a solution of the oil
0.169, CHCl3).
5.2. In vitro antifungal activity
In vitro antifungal activity was evaluated by the serial dilution
methods in 96-well microtest plates using RPMI 1640 with
0.165 M MOPS buffer or YPD [1% yeast extract (Difco, Detroit,
MI), 2% peptone (Difco, Detroit, MI), and 20% glucose] as the med-
ium.10–13,20–22 In the experiments using RPMI 1640 media, alamar
blue (Biosource, Camarillo, CA) was added to accurately define its
MIC-2 (the lowest drug concentration showing 50% growth inhibi-
tion compared to the control without drug). Test organisms were
purchased from the American Type Culture Collection (Rockville,
MD), the Institute for Fermentation Osaka (Osaka, Japan), or Teikyo
Institute of Medical Mycology (Tokyo, Japan). Initial cell densities
(1 ꢃ 103–1 ꢃ 104 cells/mL) and incubation times (18–72 h) were
decided for each strain in consideration of their growth speed.
The fungi were incubated at 30 °C (RPMI-1640) or 37 °C (YPD). Fol-
lowing incubation, OD570 was measured with a Wallac 1420 AR-
VOsx multi-label counter (Wallac, Tokyo, Japan), and MIC-2s
(50% growth inhibition) were calculated. OD600 was measured
and MIC-1s (80% growth inhibition) were calculated. Compounds
were tested at different concentrations ranging from 0.004 to
in Et2O (15 mL) was added 4 M HCl in 1,4-dioxane (75.6 lL,
0.302 mmol), and the resultant precipitate was collected and dried
to afford 7 (97.2 mg, 64%) as a yellow solid. Mp: 130–132 °C. HRMS
(ESI) m/z: 417.2617 (Calcd for C26H32N4O + H 417.2654). 1H NMR
(CDCl3) d: 1.30 (9H, s), 1.94–2.08 (1H, m), 2.09–2.26 (1H, m),
2.16 (3H, s), 2.30–2.40 (6H, m), 3.10 (2H, t, J = 7.1 Hz), 3.25–3.33
(1H, m), 3.37–3.48 (1H, m), 3.54–3.67 (1H, m), 4.63 (2H, dd,
J = 14.7, 19.5 Hz), 7.05–7.15 (2H, m), 7.30–7.45 (3H, m). IR (KBr):
2965, 2563, 2220, 1623, 1458, 1004 cmꢁ1. ½a D25
ꢂ
+104 (c 1.02,
CHCl3/MeOH = 1:1, v/v).
5.1.43. 2-Amino-5-bromo-4-fluoro-6-methyl-3-nitrobenzo-
nitrile (42)
Following the procedures as described for 20 and 26, 42 was
prepared in 48% yield from 41 as yellow solid. MS (EI) m/z: 273,
275 (M+). 1H NMR (CDCl3) d: 2.66 (3H, s), 6.53 (2H, br s). IR
(ATR): 3455, 3324, 2227, 1625, 1589, 1560, 1508 cmꢁ1
.
5.1.44. 4-Amino-6-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-2-
methyl-5-nitrobiphenyl-3-carbonitrile (43)
16 lg/mL.
Following the procedures as described for 39 and 40, 43 was
prepared in 87% yield from 42 as a brown solid. MS (FAB) m/z:
366 (M+1)+. 1H NMR (CDCl3) d: 1.54 (1H, m), 1.83 (1H, m), 2.06
(6H, s), 2.12 (3H, s), 2.50 (1H, m), 2.65 (1H, m), 2.90–2.99 (3H,
m), 5.73 (2H, br s), 7.12–7.16 (2H, m), 7.34–7.50 (3H, m). IR
5.3. Incorporation study with growing cells using [14C]-glucose
An incorporation study with growing cells was conducted based
on the methods described by Kitamura et al.10 Exponentially grow-
ing cells of C. albicans ATCC 90028 were suspended in RPMI 1640
medium to give ꢄ0.7 of absorbance at 595 nm. After the drug
solution and radioactive [14C]-glucose were added, the reaction
tubes were incubated at 30 °C with occasional shaking. After 3 h
of incubation, samples were taken and crude fractions of b-1,3-
glucan, b-1,6-glucan, chitin, and mannan were prepared as follows.
The harvested cells were suspended in 3% NaOH aq and heated at
80 °C for 1 h. Mannan fractions were prepared from the superna-
tant using Fehling’s reaction. Insoluble material was washed and
digested with Zymolyase 100T (Seikagaku-kougyou) overnight.
(ATR): 3326, 3210, 2827, 2776, 2208, 1623, 1571 cmꢁ1
.
5.1.45. 4,5-Diamino-6-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
2-methylbiphenyl-3-carbonitrile (44)
To a solution of 43 (1.40 g, 3.83 mmol) in DMF (35 mL) and
water (14 mL) were added FeCl3ꢀ6H2O (830 mg, 3.06 mmol) and
zinc (5.01 g, 76.6 mmol), and the mixture was stirred at room tem-
perature for 1 h. The reaction mixture was filtered, and the filtrate
was diluted with AcOEt. The organic layer was washed with satd
NaHCO3 aq and brine, dried over Na2SO4, filtered, and concentrated
to afford 44 (1.25 g, 97%) as a brown solid. MS (FAB) m/z: 336
After digestion, the insoluble material was harvested as
chitin fraction. The supernatants were taken as glucan fractions
a

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J.-i. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 7593–7606
(b-1,3-glucan + b-1,6-glucan fraction) and were dialyzed overnight.
After dialysis, samples were taken as a b-1,6-glucan fraction. The
radioactivity of each fraction was counted with a toluene scintilla-
tor. The radioactivities of the b-1,3-glucan fraction were calculated
by subtracting that b-1,6-glucan fraction from the glucan fraction.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.08.044. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
5.4. Distribution coefficient (Log D)
References and notes
The distribution coefficients (Log D) were determined by the
shake-flask method. Four hundred
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Acknowledgments
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We thank Ms. Naoe Haga, Dr. Masato Hata and Dr. Akihiro
Kitamura for the biological evaluation. We also thank the members
of Drug Metabolism & Pharmacokinetics Research Laboratories for
the physicochemical tests.