Novel antifungal agents: Triazolopyridines as inhibitors of β-1,6-glucan synthesis

Article abstract of DOI:10.1016/j.bmc.2010.06.096

Preparations and in vitro antifungal activities of triazolopyridines, imidazopyridines, and a pyrazolopyridine were reported. Among those scaffolds, triazolopyridine was found to be the specific inhibitor of the synthesis of β-1,6-glucan, an essential component of the fungal cell wall, and to show potent antifungal activities against several Candida species.

Full text of DOI:10.1016/j.bmc.2010.06.096

Bioorganic & Medicinal Chemistry 18 (2010) 5845–5854
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel antifungal agents: Triazolopyridines as inhibitors
of b-1,6-glucan synthesis
a
a
a
b
Jun-ichi Kuroyanagi a, , Kazuo Kanai , Yuuichi Sugimoto , Tetsunori Fujisawa , Chikanori Morita ,
*
Takashi Suzuki a, Katsuhiro Kawakami a, Makoto Takemura a
a Lead Discovery & Optimization Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Technical Department, Daiichi Fine Chemical Co., Ltd, 530 Chokeiji, Takaoka, Toyama 933-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Preparations and in vitro antifungal activities of triazolopyridines, imidazopyridines, and a pyrazolopyri-
dine were reported. Among those scaffolds, triazolopyridine was found to be the specific inhibitor of the
synthesis of b-1,6-glucan, an essential component of the fungal cell wall, and to show potent antifungal
activities against several Candida species.
Received 26 May 2010
Revised 28 June 2010
Accepted 29 June 2010
Available online 01 July 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal activity
Triazolopyridine
b-1,6-Glucan
Candida species
4CN
1. Introduction
Pyridine core
CN
5
N
9
N
3
6
Fungal infection has dramatically increased in immunosup-
pressed patients such as those with AIDS or who have had organ
or marrow transplants over the past decades.1–3 Currently, some
antifungal agents of major drug classes: azoles, polyenes, and can-
dins, have been launched and are demonstrating remarkable suc-
cess for the treatment of fungal systemic infections.2–7 However,
these usages are often limited mainly due to their unsatisfactory
antifungal activity, narrow spectrum, and side effects, causing ra-
pid development of drug resistance and a high rate of mortal-
ity.7–10 In consideration of these facts, alternative agents for the
treatment and prevention of fungal infections are urgently re-
quired, preferably with a novel mode of action.
Pyridobenzimidazole derivatives 1, 2, and 3 have been reported
as antifungal agents inhibiting the synthesis of b-1,6-glucan, an
essential component of the fungal cell wall (Fig. 1).11,12 It was also
reported that their primary target was one of the b-1,6-glucan syn-
theses encoded by KRE6 gene, which was conserved in various fun-
gi, and that no homolog was found in mammalian cells.13–15 These
facts suggest that Kre6p could be a novel target for the treatment of
fungal infections. According to the previous structure–activity rela-
tionships study (SARs) by Takeshita et al., substituents such as cya-
no, methyl, phenyl, and (3S)-(dimethylamino)pyrrolidine on the
pyridine core of 2 were required for potent antifungal activity
N
1
N
R
2
7
N
HN
8
Benzene core
NEt2
NMe2
2 : R = Ph
1
3 : R = n-Bu
Figure 1. The structure of compounds 1–3.
against Candida species (Candida spp.).12 However, the role of the
benzene core in the tricyclic scaffold has not been elucidated so
far. Although compound 2 showed potent antifungal activity, its
physicochemical properties such as water solubility and metabolic
stability were not satisfactory for drug therapies. It is known that
water solubility and metabolic stability of compounds are often im-
proved by reducing their lipophilicity. If certain bicyclic compounds
in which the benzene core was removed from tricyclic 2 showed
antifungal activity in the same manner as compound 2, we could re-
duce lipophilicity and improve their physicochemical properties.
Herein, we would like to report the design, synthesis, and evalua-
tion of antifungal activity of novel bicyclic derivatives such as triaz-
olopyridines, imidazopyridines, and a pyrazolopyridine.
2. Design
As shown in Figure 2, three new heteroaromatic bicyclic scaf-
folds: triazolopyridines, imidazopyridines, and pyrazolopyridines,
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8609.
E-mail address: kuroyanagi.junichi.d5@daiichisankyo.co.jp (J. Kuroyanagi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.096

5846
J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
CN
8
3. Chemistry
CN
4
N1
7
Me
5
3
Me
Ph
2 R2
R2
Ph6
Triazolopyridines 9 and 13–21 were synthesized as depicted in
Scheme 1. Imidoate 5 was synthesized from cyanide 4.16 Com-
pounds 6a–6d17,18 and 5 were treated with cyanoacetohydrazide
to give triazoles 7a–7e. Compound 7a was then condensed with
ethyl 2-acetylhexanoate in the presence of ammonium acetate to
afford 8, which was chlorinated with POCl3 followed by the intro-
duction of (3S)-3-(dimethylamino)pyrrolidine to give triazolopyri-
dine 9. Triazolopyridines 13–19 and 21 were obtained from
triazoles 7a–7e, 10, and 11 following a similar manner as described
above. Compound 20 was obtained by hydrogenolysis of 19.
Imidazopyridines 29 and 30 were synthesized as shown in
Scheme 2. Imidazole 27a was prepared in five steps from (benzyl-
oxy)acetic acid (22) and 1-bromo-3,3-dimethylbutan-2-one (23).
Compounds 27a and 27b19 were converted to corresponding imi-
dazopyridines 29 and 30 by the same manner as described in
Scheme 1.
N
6
2
X
3
N
4
N
1
5N
7N
NMe2
NMe2
Pyrazolopyridines
Triazolopyridines X = N
Imidazopyridines X = CH
Figure 2. Design of novel fused-pyridine scaffolds.
were designed. Based on the results reported by Takeshita, substit-
uents such as cyano, methyl, phenyl, and (3S)-(dimethyl-
amino)pyrrolidine on the rings were considered to be essential to
show antifungal activity against Candida spp. Therefore, we
decided to fix these substituents and to focus on finding efficient
substituents on R2.
HN
a
HCl
NC
OBn
R2
MeO
OBn
4
5
HN
b
N
N
NC
HCl
R2
HN
MeO
6a : R2 = t-butyl
7a : R2 = t-butyl
7b : R2 = cyclopropyl
7c : R2 = ethyl
6b : R2= cyclopropyl
6c : R2 = ethyl
6d : R2 = n-butyl
7d : R2 = n-butyl
5 : R2 = C(Me)2CH2OBn
7e : R2 = C(Me)2CH2OBn
CN
CN
N
N
H
d, e
N
N
c
N
7a
N
N
O
NMe2
8
9
CN
CN
N
N
H
N
R2
d, e
f
N
N
NC
N
R2
R2
Ph
N
HN
N
Ph
N
O
NMe2
7a-7e
12a : R2 = t-butyl
12b : R2 = methyl
12c : R2 = n-butyl
12d : R2 = phenyl
10 : R2 = methyl
11 : R2 = phenyl
13 : R2 = t-butyl
14 : R2 = methyl
15 : R2 = n-butyl
16 : R2 = phenyl
12e : R2 = cyclopropyl
12f : R2 = ethyl
17 : R2 = cyclopropyl
18 : R2 = ethyl
12g : R2 = C(Me)2CH2OBn
19 : R2 = C(Me)2CH2OBn
CN
CN
N
N
N
N
d, h
g
12a
N
N
19
OH
Ph
Ph
HN
N
NEt2
NMe2
21
20
Scheme 1. Reagents: (a) HCl, MeOH, 59%; (b) cyanoacetohydrazide, NaOH, MeOH, 53–88%; (c) ethyl 2-acetylhexanoate, AcONH4, 63%; (d) POCl3; (e) (3S)-3-
(dimethylamino)pyrrolidine, Et3N, DMF; (f) ethyl 3-oxo-2-phenylbutanoate, AcONH4, 12–50%; (g) H2, 10% Pd–C, MeOH, 87%; (h) 2-(diethylamino)ethylamine, Et3N, DMF,
64% (two steps).

J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
5847
O
The MIC-0 (the lowest drug concentration producing an opti-
cally clear well) of the triazolopyridine derivatives 9, 13–16,
and 21 were shown in Table 1. All the derivatives showed no inhib-
itory activity against C. albicans. Although pyridobenzimidazole 3
with n-butyl group exhibited potent activity against C. glabrata
O
a
O
Br
BnO
CO2H
22
+
BnO
O
23
24
(60.125
weak antifungal activity against C. glabrata (32
the 6-phenyl analog 13 exhibited potent activity against C. glabrata
(60.125 g/mL). The substituents at the 2-position of compounds
13–16 strongly affected their antifungal activities. Among 2-
substituted analogs 13–16, 2-tert-butyl derivative 13 was proved
to exhibit the most potent inhibitory activity against Candida
spp. The methyl and n-butyl derivatives 14 and 15 showed weak
l
g/mL), the corresponding triazolopyridine 9 showed
c, d
b
N
BnO
lg/mL). However,
HN
e
l
25
N
N
NC
Cl
HN
HN
26
27a
antifungal activity against C. glabrata (2 and 1 lg/mL, respectively).
No activity was observed for 2-phenyl derivative 16. The 5-[(3S)-3-
(dimethylamino)pyrrolidin-1-yl] (cyclic amine) derivative 13
showed more potent antifungal activity than the 5-{[2-(diethyla-
mio)ethyl]amino} (acyclic amine) derivative 21, which was in
agreement with the study of pyridobenzimidazole derivatives.12
Among these derivatives, 14 showed significantly improved water
solubility and metabolic stability.
CN
CN
CN
N
H
N
f
g, h
R2
N
R2
N
R2
Ph
N
HN
Ph
N
O
27a : R2 = t-butyl
27b : R2 = methyl
NMe2
28a : R2 = t-butyl
28b : R2 = methyl
Since our product-treated cells often grew well but were se-
verely clumped and could not be uniformly mixed, it was difficult
to visually define the endpoint. To measure a reproducible and
accurate MIC, an oxidation–reduction indicator, alamar blue was
added to the medium.11 Our compounds exhibited trailing growth
phenomenon, which lead to significant differences in the MIC val-
ues among C. albicans and C. tropicalis.25 For that reason, MIC-2 (the
lowest drug concentration showing 50% growth inhibition com-
pared to the control without drug) was measured as an endpoint
for the experiments with Candida spp. (Table 2). Compared to the
results of the MIC-0s for compound 2, 3, 13, and FLCZ, the MIC-
2s showed similar tendency of SARs and thus seemed not to have
any problem to evaluate our derivatives in detail (Table 2). The
MIC-2s of various triazolopyridines 13, 17-20, imidazopyridines
29 and 30, and a pyrazolopyridine 36 were shown in Table 2. Al-
most all the bicyclic compounds showed no inhibitory activity
against C. albicans. Among several 2-aliphatic triazolopyridine ana-
logs, 2-tert-butyl derivative 13 exhibited the most potent growth
inhibition against Candida spp., followed by cyclopropyl and ethyl
29 : R2 = t-butyl
30 : R2 = methyl
Scheme 2. Reagents: (a) K2CO3, DMF, 80%; (b) AcONH4, 76%; (c) H2, 10% Pd–C,
EtOH; (d) SOCl2, 80% (two steps); (e) KCN, EtOH–H2O, 67%; (f) ethyl 3-oxo-2-
phenylbutanoate, AcONH4, 33–61%; (g) POCl3; (h) (3S)-3-(dimethylamino)pyrrol-
idine, Et3N, DMF; 24–96% (two steps).
Synthesis of pyrazolopyridine 36 was accomplished from pyra-
zole 3120 as shown in Scheme 3. Pyrazole 31 was converted to cya-
nide 32, which on treatment with but-2-enenitrile 34 to give
pyrazolopyridine 35. Sandmeyer reaction21 of 35, followed by the
introduction of (3S)-3-(dimethylamino)pyrrolidine gave pyrazolo-
pyridine 36.
4. Results and discussion
Synthesized compounds 9, 13–21, 29, 30, and 36 were evalu-
ated for their in vitro antifungal activity against Saccharomyces
cerevisiae (S. cerevisiae), Candida albicans (C. albicans), Candida
glabrata (C. glabrata), Candida tropicalis (C. tropicalis), and Candida
krusei (C. krusei). Pyridobenzimidazoles 2, 3, and Fluconazole
(FLCZ) were used as positive control agents.
derivatives 17 and 18 (0.25, 1, and 1 lg/mL against C. tropicalis,
respectively). Results obtained above suggested that the preferable
number of carbon atoms for potent antifungal activity was two to
four for a substituent at the 2-position. Moreover, a sterically bulky
substituent, such as tert-butyl and cyclopropyl, was required
for potent antifungal activity. Interestingly, 2-(benzyloxy)-1,1-
dimethylethyl derivative 19, possessing a rather bulky substituent,
also gave potent activity. In addition, the hydroxyl group, attached
to the bulky substituent as in compound 20, retained moderate
a
NC
Cl
HN
HN
N
N
32
31
activity against C. glabrata and C. tropicalis (0.25 and 0.25 lg/mL,
respectively). These results indicated that the hydrophilic func-
tional group, such as hydroxyl or alkoxy group, at the 2-position
was tolerated for showing potent growth inhibition.
Similar results were observed in the SARs of imidazopyridine
analogs 29 and 30. The 2-tert-butyl analog 29 was more potent
compared to the corresponding 2-methyl derivative 30 (0.25 and
O
OMe
CN
b
Ph
CN
Ph
33
34
CN
0.5 lg/mL against C. glabrata, respectively), as in Table 2. On the
CN
other hand, pyrazolopyridine 36 with preferable substituents ob-
served in 13 and 29 exhibited weak antifungal activities against
several Candida spp.
These results suggested that the nitrogen atoms at the 1-posi-
tion as in triazolopyridine 13 and imidazopyridine 29 were indis-
pensable for potent antifungal activity in comparison with the
non-nitrogen pyrazolopyridine 36 at the 3-position (Fig. 2).
Pyrazolopyridine 36 showed high lipophilicity and poor solubil-
ity. Triazolopyridine 13 and imidazopyridine 29 demonstrated
d, e
c
N
32 34
+
N
Ph
N
N
Ph
N
NH2
35
NMe2
36
Scheme 3. Reagents: (a) KCN, EtOH–H2O, 85%; (b) MeC(OMe)3, 57%; (c) lithium
diisopropylamide, THF, 42%; (d) t-BuONO, CuCl2; (e) (3S)-3-(dimethylamino)pyr-
rolidine, Et3N, DMF, 12% (two steps).

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J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
Table 1
In vitro activity and physicochemical property of triazolopyridine derivatives
CN
N
CN
N
N
R2
N
N
R6
Ph
N
N
HN
NEt2
NMe2
9, 13-16
21
Compd
R2
R6
MIC-0 (
l
g/mL)a
Log Dc
Sol.d
MSe
S. cerevisiae
C. albicans
C. glabrata
C. tropicalis
C. krusei
pH 6.8
pH 6.8
Human (%)
AY-14
ATCC 24433
IFO 0622
TIMM 0313
TIMM 0269
(lg/mL)
9
t-Bu
t-Bu
Me
n-Bu
Ph
n-Bu
Ph
Ph
Ph
Ph
—
64
60.125
8
2
>128
0.5
>128
>128
>128
>128
>128
>128
32
60.125
2
1
>128
0.5
>128
2
>128
>64
>128
>128
>128
16
>128
>64
>128
>128
3.2
3.1
1.7
3.2
NTf
3.3
146
16
376
33
NT
2
11
9
84
5
NT
10
13
14
15
16
21
—
2
3
—
—
—
—
—
—
60.125
0.25
4
>128
>128
0.25
60.125
60.125
2
0.5
0.5
0.25
60.125
0.5
16
3.1
3.2
0.4g
5.3
26
>2000
29
57
96
FLCZb
a
b
c
d
e
f
MIC-0s (in micrograms per milliliter) were determined by using the microdilution method.
The lowest drug concentration producing a prominent reduction in turbidity was employed as an endpoint.
Log D values were determined from the partition coefficient for 1-octanol/phosphate buffer saline (BPS) at pH 6.8.
Water solubility was measured at pH 6.8.
Metabolic stability for human liver microsome.
Not tested.
g
Log D value at pH 7.4.
Table 2
In vitro activity and physicochemical property of various bicyclic derivatives
CN
N
CN
CN
N
R2
R2
N
N
N
N
N
Ph
Ph
Ph
N
N
N
NMe2
NMe2
NMe2
13, 17-20
29, 30
36
Compd
R2
MIC-2 (
l
g/mL)a
Log Db
Sol.c
MSd
S. cerevisiae
C. albicans
C. glabrata
C. tropicalis
C. krusei
pH 6.8
pH 6.8
Human (%)
AY-14
ATCC 24433
ATCC 48435
IAM 4965
ATCC 44507
(lg/mL)
13
17
18
19
20
29
30
36
t-Bu
Cyclopropyl
Ethyl
C(Me)2CH2OBn
C(Me)2CH2OH
t-Bu
0.063
0.032
0.063
0.063
0.125
0.125
1
>4
>4
>4
>4
>4
4
0.063
0.063
0.125
0.125
0.25
0.25
0.5
0.25
1
1
0.125
0.25
4
>4
1
3.1
2.4
2.1
3.8
1.8
3.0
1.9
4.8
16
57
228
<3
140
27
356
<2
9
48
54
9
64
16
71
12
>4
>4
>4
>4
>4
4
Me
—
>4
4
>4
1
1
1
2
3
FLCZ
—
—
—
0.004
0.125
4
>4
>4
0.25
0.063
0.125
2
0.25
0.5
0.25
0.125
1
16
3.1
3.2
0.4e
5.3
26
>2000
29
57
96
a
b
c
MIC-2 was 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 6.8.
Water solubility was measured at pH 6.8.
Metabolic stability for human liver microsome.
Log D value at pH 7.4.
d
e
similar physicochemical properties. Potent antifungal activity
and improved physicochemical properties were observed for
triazolopyridine derivatives such as 17, 18, and 20 compared to 2
and the other bicyclic compounds. These results suggested that
triazolopyridine was considered to be the most promising scaffold
with potent antifungal activities and improved physicochemical
properties.
Incorporation studies of pyridobenzimidazole analogs 1 and 2
with growing cells of S. cerevisiae or C. albicans using [14C]-glucose
revealed that they were specific inhibitors of b-1,6-glucan synthe-
sis. To determine the mode of action of our bicyclic compounds ob-
tained above, we selected two potent antifungal analogs,
triazolopyridine 13 and imidazopyridine 29, and adopted the bio-
chemical approach based on the methods described by Kitamura

J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
5849
et al.11 Triazolopyridine 13 and imidazopyridine 29 were evaluated
by incorporation studies with growing cells of C. albicans using
[
edly reduced the radioactivity in the fraction of b-1,6-glucan in a
dose-dependent manner. No reductions in the fractions of b-1,3-
glucan and chitin were observed in each case. Results obtained
above suggested that bicyclic analogs 13 and 29 were also specific
inhibitors of b-1,6-glucan synthesis.
(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, UV-lamp, iodine, or Wako
Ninhydrin Spray.
14C]-glucose. As shown in Figure 3, these bicyclic analogs mark-
6.1.1. Methyl 3-(benzyloxy)-2,2-dimethylpropanimidoate
hydrochloride (5)
Gaseous hydrogen chloride was bubbled through a stirred solu-
tion of 2,2-dimethyl-3-(benzyloxy)propionitrile (4)16 (18.9 g,
99.9 mmol) and MeOH (8.1 mL, 200 mmol) in CH2Cl2 (200 mL) at
ꢀ5 °C for 1 h. After being stirred at 0 °C for 15 h, the mixture was
concentrated to dryness. Et2O was added to the residue and the
resulting precipitate was collected by filtration and washed with
Et2O to afford the title compound (15.2 g, 59%) as a pale yellow so-
lid. 1H NMR (CDCl3) d: 1.38 (6H, s), 3.63 (2H, s), 4.33 (3H, s), 4.55
(2H, s), 7.27–7.37 (5H, m).
5. Conclusion
We prepared triazolopyridines 9 and 13–21, imidazopyridines
29 and 30, and pyrazolopyridine 36 and evaluated their antifungal
activities against Candida spp. Among them, triazolopyridine was
found to be the most promising scaffold for specific inhibitors of
b-1,6-glucan synthesis with potent antifungal growth inhibition
and improved physicochemical properties.
6.1.2. General procedure for preparation of (1H-1,2,4-triazol-5-
yl)acetonitrile 7a–7e
6. Experimental section
6.1. Chemistry
A mixture of sodium hydroxide (51.0 mmol), imidoate hydro-
chloride (50.0 mmol), and cyanoacetohydrazide (51.5 mmol) in
dry MeOH (100 mL) was refluxed for 2.5 h. The solvent was evap-
orated, and the residue was chromatographed on a silica gel col-
umn (CHCl3/MeOH = 50/1, v/v) to afford compounds 7a–7e.
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 order: number of protons, multiplicity (s, sin-
glet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad), cou-
pling 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 conditions (EI),
electron spray ionization conditions (ESI), or fast atom bombard-
ment ionization conditions (FAB). Elemental analyzes are indicated
only by the symbols of the elements; analytical results were within
0.4% of the theoretical values unless otherwise noted, which indi-
cates P95% purity of the tested compounds. Column chromatogra-
phy refers to flash column chromatography conducted on Merck
silica gel 60, 230–400 mesh ASTM. Thin-layer chromatography
6.1.2.1. (3-tert-Butyl-1H-1,2,4-triazol-5-yl)acetonitrile (7a).
Yield,
63% from methyl 2,2-dimethylpropanimidoate hydrochloride (6a),17
colorless solid. Mp: 110–112 °C. MS (FAB) m/z: 165 (M+1)+. 1H NMR
(CDCl3) d: 1.42 (9H, s), 3.87 (2H, s), 11.0 (1H, br s). 13C NMR (CDCl3)
d: 18.1, 29.0 (3C), 32.3, 115.8, 154.2, 166.6. IR (KBr): 2973, 2255,
1565, 1414, 1255, 1049, 1021, 940 cmꢀ1. Anal. Calcd for C8H12N4: C,
58.51; H, 7.37; N, 34.12. Found: C, 58.40; H, 7.30; N, 33.76.
6.1.2.2. (3-Cyclopropyl-1H-1,2,4-triazol-5-yl)acetonitrile (7b).
Yield,
53% from methyl cyclopropanecarboximidoate hydrochloride (6b),
colorless solid. Mp: 138–140 °C. HRMS (ESI) Calcd for C7H8N4+H:
149.0827. Found: 149.0849. 1H NMR (CDCl3) d: 1.06–1.14 (4H,
m), 1.93–2.00 (1H, m), 3.79 (2H, s), 10.44 (1H, br s). 13C NMR
(CDCl3) d: 7.1, 8.4 (2C), 17.7, 115.6, 153.8, 161.0. IR (KBr): 3024,
2903, 2751, 2658, 2255, 1582, 1519, 1439, 1353, 1101 cmꢀ1
.
6.1.2.3. (3-Ethyl-1H-1,2,4-triazol-5-yl)acetonitrile (7c).
Yield,
88% from 6c,18 colorless solid. MS (FAB) m/z: 137 (M+1)+. 1H
NMR (CDCl3) d: 1.37 (3H, t, J = 7.5 Hz), 2.86 (2H, q, J = 7.5 Hz),
3.88 (2H, s), 11.61 (1H, br s).
Compound 29
Compound 13
120
120
100
100
80
80
60
60
β-1,6-glucan
β-1,3-glucan
Chitin
β-1,6-glucan
β-1,3-glucan
Chitin
40
40
20
0
20
0
Mannan
Mannan
0
0.016 0.063 0.25
Concentration ( g/mL)
1
4
0
0.016 0.063 0.25
Concentration ( g/mL)
1
4
μ
μ
Figure 3. Incorporation studies of the bicyclic analogs 13 and 29 with growing cells of C. albicans ATCC MYA-573 using [14C]-glucose.

5850
J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
6.1.2.4. (3-Butyl-1H-1,2,4-triazol-5-yl)acetonitrile (7d).
Yield,
CHCl3/MeOH = 19/1, v/v to give the crude product. The crude prod-
uct was recrystallized from MeCN to yield 12a (621 mg, 33%) as a
pale yellow solid. Mp: >280 °C. MS (ESI) m/z: 307 (M+1)+. 1H NMR
(CDCl3) d: 1.43 (9H, s), 2.25 (3H, s), 7.26–7.44 (5H, m). 13C NMR
(CDCl3) d: 19.8, 28.3 (3C), 32.7, 71.8, 115.7, 119.4, 127.5, 128.4
(2C), 130.9 (2C), 135.4, 145.8, 148.2, 156.2, 162.2. IR (KBr): 2973,
2213, 1653, 1524, 1291, 989 cmꢀ1. Anal. Calcd for C18H18N4O: C,
70.57; H, 5.92; N, 18.29. Found: C, 70.26; H, 5.77; N, 18.25.
76% from methyl pentanimidoate hydrochloride 6d, colorless solid.
Mp: 70–71 °C. HRMS (ESI) Calcd for C8H12N4+H: 165.1140. Found:
165.1148. 1H NMR (CDCl3) d: 0.95 (3H, t, J = 7.4 Hz), 1.36–1.45 (2H,
m), 1.70–1.79 (2H, m), 2.80 (2H, t, J = 7.7 Hz), 3.85 (2H, s), 10.79
(1H, br s). 13C NMR (CDCl3) d: 13.6, 18.1, 22.2, 26.2, 29.8, 115.8,
154.2, 159.1. IR (KBr): 2934, 2260, 1665, 1566, 1508, 1417,
1068 cmꢀ1
.
6.1.2.5. {3-[2-(Benzyloxy)-1,1-dimethylethyl]-1H-1,2,4-triazol-
6.1.5.2. 2,7-Dimethyl-5-oxo-6-phenyl-1,5-dihydro[1,2,4]triazol-
5-yl}acetonitrile (7e).
Yield, 68% from 5, colorless solid. MS
o[1,5-a]pyridine-8-carbonitrile (12b).
Following the proce-
(FAB) m/z: 271 (M+1)+. 1H NMR (CDCl3) d: 1.39 (6H, s), 3.53 (2H,
dures as described for 12a, the title compound (183 mg) was
prepared in 12% yield from 10 (730 mg, 6.00 mmol) as a pale yel-
low solid. MS (FAB) m/z: 265 (M+1)+. 1H NMR (DMSO-d6) d: 2.16
(3H, s), 2.51 (3H, s), 7.23 (2H, m), 7.38 (3H, m).
s), 3.82 (2H, s), 4.59 (2H, s), 7.29–7.41 (5H, m), 11.23 (1H, br s).
6.1.3. 6-Butyl-2-tert-butyl-7-methyl-5-oxo-1,5-dihydro[1,2,4]-
triazolo[1,5-a]pyridine-8-carbonitrile (8)
A mixture of 7a (1.00 g, 6.09 mmol), ethyl 2-acetylhexanoate
(1.19 g, 6.39 mmol), and ammonium acetate (986 mg, 12.8 mmol)
was heated at 150 °C for 3.5 h. The mixture was cooled to room
temperature and purified by silica gel chromatography eluting
with CHCl3/MeOH = 49/1, v/v to give the crude product. The crude
product was recrystallized from MeCN to yield 8 (1.09 g, 63%) as a
pale yellow solid. Mp: >280 °C. MS (FAB) m/z: 287 (M+1)+. 1H NMR
(CDCl3) d: 0.93 (3H, t, J = 7.2 Hz), 1.35–1.52 (4H, m), 1.46 (9H, s),
2.42 (3H, s), 2.68 (2H, t, J = 7.7 Hz). 13C NMR (CDCl3) d: 14.0, 18.2,
22.7, 26.5, 28.2 (3C), 30.9, 32.6, 71.0, 116.1, 118.5, 145.3, 146.3,
156.6, 161.9. IR (KBr): 2960, 2212, 1656, 1571, 1521, 1392, 1190,
990 cmꢀ1. Anal. Calcd for C16H22N4O: C, 67.11; H, 7.74; N, 19.56.
Found: C, 67.04; H, 7.72; N, 19.33.
6.1.5.3. 2-Butyl-7-methyl-5-oxo-6-phenyl-1,5-dihydro[1,2,4]triaz-
olo[1,5-a]pyridine-8-carbonitrile (12c).
Following the proce-
dures as described for 12a, the title compound (311 mg) was
prepared in 17% yield from 7d (1.00 g, 6.09 mmol) as a pale yellow
solid. Mp: >280 °C. HRMS (ESI) Calcd for C18H18N4O+H: 307.1559.
Found: 307.1547. 1H NMR (DMSO-d6) d: 0.92 (3H, t, J = 6.9 Hz),
1.29–1.40 (2H, m), 1.64–1.74 (2H, m), 2.11 (3H, s), 2.62–2.71
(2H, m), 6.67 (1H, br s), 7.17–7.40 (5H, m). IR (KBr): 3331, 2959,
2210, 1668, 1610, 1523, 1369, 1304, 727 cmꢀ1
.
6.1.5.4. 7-Methyl-5-oxo-2,6-diphenyl-1,5-dihydro[1,2,4]triazol-
o[1,5-a]pyridine-8-carbonitrile (12d). Following the proce-
dures as described for 12a, the title compound (885 mg) was
prepared in 50% yield from 11 (1.00 g, 5.43 mmol) as a colorless so-
lid. MS (FAB) m/z: 327 (M+1)+. 1H NMR (DMSO-d6) d: 2.13 (3H, s),
7.13 (1H, br s), 7.24 (3H, m), 7.36 (2H, m), 7.46 (3H, m), 8.15 (2H, m).
6.1.4. 6-Butyl-2-tert-butyl-5-[(3S)-3-(dimethylamino)pyrrolidin-
1-yl]-7-methyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (9)
A mixture of 8 (900 mg, 3.14 mmol) in phosphorus oxychloride
(5 mL) was refluxed for 1.5 h. The mixture was cooled to room
temperature and concentrated in vacuo, and then CHCl3 and water
were added. The separated organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo to give 6-butyl-
2-tert-butyl-5-chloro-7-methyl[1,2,4]triazolo[1,5-a]pyridine-8-car-
bonitrile (939 mg) as a pale yellow solid. The obtained compound
was used without further purification. To a solution of the
chloride (600 mg, 1.97 mmol) in DMF (6 mL) was added (3S)-3-
6.1.5.5.
2-Cyclopropyl-7-methyl-5-oxo-6-phenyl-1,5-dihydro-
Following
[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (12e).
the procedures as described for 12a, the title compound (366 mg)
was prepared in 21% yield from 7b (890 mg, 6.00 mmol) as a pale
yellow solid. Mp: >280 °C. HRMS (ESI) Calcd for C17H14N4O+H:
291.1246. Found: 291.1232. 1H NMR (DMSO-d6) d: 0.80–0.96 (4H,
m), 2.10 (3H, s), 2.14–2.23 (1H, m), 7.18–7.40 (5H, m). 13C NMR
(DMSO-d6) d: 8.1 (2C), 8.7, 19.3, 111.3, 118.7, 126.3, 127.8 (2C),
131.2 (2C), 137.3, 145.6, 151.9, 155.6, 166.5, 171.4. IR (KBr): 3469,
(dimethylamino)pyrrolidine (275
lL, 2.17 mmol) and triethyl-
amine (549 L, 3.94 mmol) and the mixture was stirred at 90 °C
l
3396, 3204, 2205, 1662, 1611, 1530, 1363, 1297 cmꢀ1
.
for 4 h. The mixture was cooled to room temperature and concen-
trated in vacuo. The residue was dissolved with CHCl3 and the mix-
ture was washed with satd NaHCO3 aq and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography eluting with CHCl3/MeOH = 99/1,
v/v to afford 9 (723 mg, 97%) as a pale yellow solid. Mp: 49–
53 °C. MS (FAB) m/z: 383 (M+1)+. 1H NMR (CDCl3) d: 0.97 (3H, t,
J = 7.2 Hz), 1.38–1.50 (13H, m), 2.00–2.09 (1H, m), 2.22–2.29 (1H,
m), 2.34 (6H, s), 2.63 (3H, s), 2.68–2.79 (2H, m), 3.09–3.19 (1H,
m), 3.40 (1H, t, J = 8.3 Hz), 3.53 (1H, t d, J = 8.6, 2.9 Hz), 3.67–3.73
(2H, m). IR (KBr): 2956, 2869, 2772, 2216, 1607 cmꢀ1. Anal. Calcd
for C22H34N6ꢁ0.25H2O: C, 68.27; H, 8.98; N, 21.71. Found: C,
6.1.5.6.
2-Ethyl-7-methyl-5-oxo-6-phenyl-1,5-dihydro[1,2,4]-
triazolo[1,5-a]pyridine-8-carbonitrile (12f).
Following the
procedures as described for 12a, the title compound (350 mg)
was prepared in 21% yield from 7c (820 mg, 6.00 mmol) as a pale
yellow solid. MS (FAB) m/z: 279 (M+1)+. 1H NMR (DMSO-d6) d:
1.26 (3H, t, J = 7.5 Hz), 2.13 (3H, s), 2.76 (2H, q, J = 7.5 Hz), 7.20
(2H, m), 7.29 (1H, m), 7.39 (2H, m).
6.1.5.7. 2-[2-(Benzyloxy)-1,1-dimethylethyl]-7-methyl-5-oxo-6-
phenyl-1,5-dihydro[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile
(12g).
compound (2.00 g) was prepared in 26% yield from 7e (5.00 g,
18.5 mmol) as a pale yellow solid. MS (FAB) m/z: 413 (M+1)+. 1H
NMR (CDCl3) d: 1.35 (6H, s), 2.21 (3H, s), 3.56 (2H, s), 4.42 (2H,
s), 7.18–7.42 (10H, m).
Following the procedures as described for 12a, the title
68.45; H, 8.89; N, 21.85. ½a D25
ꢂ
ꢀ49.9 (c 1.00, CHCl3).
6.1.5. General procedure for the synthesis of 7-methyl-5-oxo-6-
phenyl-1,5-dihydro[1,2,4]triazolo[1,5-a]pyridine-8-carbonitriles
12a–12g
6.1.5.1. 2-tert-Butyl-7-methyl-5-oxo-6-phenyl-1,5-dihydro[1,2,4]-
triazolo[1,5-a]pyridine-8-carbonitrile (12a). A mixture of 7a
(1.00 g, 6.09 mmol), ethyl 3-oxo-2-phenylbutanoate (1.32 g, 6.39
mmol), and ammonium acetate (986 mg, 12.8 mmol) was heated
at 150 °C for 3.5 h. The mixture was cooled to room tempera-
ture and purified by silica gel chromatography eluting with
6.1.6. 2-tert-Butyl-5-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-7-
methyl-6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (13)
Compound 13 was prepared from 12a, as described above for 9.
Yield, 99% as a pale yellow solid. Mp: 177–179 °C. MS (FAB) m/z:
403 (M+1)+. 1H NMR (CDCl3) d: 1.47 (9H, s), 1.56–1.69 (1H, m),
1.92–2.00 (1H, m), 2.16 (6H, s), 2.24 (3H, s), 2.57–2.67 (1H, m),

J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
5851
3.23–3.36 (2H, m), 3.50–3.59 (2H, m), 7.11–7.15 (1H, m), 7.22–7.26
(1H, m), 7.38–7.49 (3H, m). 13C NMR (CDCl3) d: 20.4, 29.6 (3C),
29.7, 33.4, 43.9 (2C), 51.2, 55.9, 65.2, 89.5, 114.9, 116.1, 128.2,
128.8, 129.0, 131.2, 131.4, 136.5, 144.0, 148.3, 151.6, 174.2. IR
151.7, 168.8. IR (KBr): 2771, 2210, 1606, 1542, 1508, 1477, 1360,
1270 cmꢀ1. Anal. Calcd for C23H26N6ꢁ0.5H2O: C, 69.85; H, 6.88; N,
21.25. Found: C, 69.92; H, 6.68; N, 21.19. ½a D25
ꢂ
+44.4 (c 1.01, CHCl3).
(KBr): 2964, 2772, 2210, 1606, 1508 cmꢀ1
.
Anal. Calcd for
6.1.11. 5-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2-ethyl-7-
methyl-6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (18)
Compound 18 was prepared from 12f, as described above for 9.
Yield, 98% as a pale yellow solid. Mp: 151–153 °C. MS (FAB) m/z:
375 (M+1)+. 1H NMR (CDCl3) d: 1.42 (3H, t, J = 7.5 Hz), 1.63 (1H,
m), 1.98 (1H, m), 2.15 (6H, s), 2.26 (3H, s), 2.61 (1H, m), 2.95
(2H, q, J = 7.5 Hz), 3.21 (1H, dd, J = 8.7, 9.9 Hz), 3.32 (1H, m), 3.45
(1H, dd, J = 7.2, 9.6 Hz), 3.60 (1H, d t, J = 6.9, 10.2 Hz), 7.13 (1H,
m), 7.24 (1H, m), 7.34–7.49 (3H, m). 13C NMR (CDCl3) d: 12.5,
20.4, 22.4, 29.9, 44.1 (2C), 51.1, 56.0, 65.3, 89.5, 115.4, 115.7,
128.3, 128.8, 129.1, 131.1, 131.3, 136.3, 143.9, 148.5, 151.7,
168.1. IR (KBr): 2770, 2211, 1603, 1541, 1508, 1480, 1358,
1268 cmꢀ1. Anal. Calcd for C22H26N6ꢁ0.2H2O: C, 69.89; H, 7.04; N,
C
24H30N6: C, 71.61; H, 7.51; N, 20.88. Found: C, 71.44; H, 7.49; N,
21.01. ½a 2D5
ꢂ
+24.8 (c 0.839, CHCl3).
6.1.7. 5-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2,7-dimethyl-
6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (14)
Compound 14 was prepared from 12b, as described above for 9.
Yield, 41% as a pale yellow solid. Mp: 182–184 °C. MS (FAB) m/z:
361 (M+1)+. 1H NMR (CDCl3) d: 1.55–1.68 (1H, m), 1.93–2.02 (1H,
m), 2.14 (6H, s), 2.26 (3H, s), 2.55–2.65 (1H, m), 2.60 (3H, s), 3.16
(1H, t, J = 9.5 Hz), 3.29–3.34 (1H, m), 3.42 (1H, dd, J = 10.0,
7.2 Hz), 3.58–3.65 (1H, m), 7.14 (1H, d, J = 7.4 Hz), 7.23–7.27 (1H,
m), 7.40–7.49 (3H, m). 13C NMR (CDCl3) d: 14.7, 20.4, 30.0, 44.1
(2C), 51.0, 56.0, 65.3, 89.6, 115.5, 115.7, 128.3, 128.9, 129.1,
131.0, 131.2, 136.2, 143.8, 148.6, 151.8, 163.5. IR (KBr): 2768,
22.23. Found: C, 70.15; H, 6.99; N, 21.97. ½a D25
ꢂ
+44.7 (c 1.03, CHCl3).
2216, 1607, 1541, 1510, 1485, 1351 cmꢀ1
.
Anal. Calcd for
6.1.12. 2-[2-(Benzyloxy)-1,1-dimethylethyl]-5-[(3S)-3-(dimeth-
ylamino)pyrrolidin-1-yl]-7-methyl-6-phenyl[1,2,4]triazolo[1,5-
a]pyridine-8-carbonitrile (19)
C
21H24N6: C, 69.97; H, 6.71; N, 23.32. Found: C, 69.70; H, 6.73; N,
23.02. ½a 2D5
ꢂ
+49.2 (c 0.438, CHCl3).
Compound 19 was prepared from 12g, as described above for 9.
Yield, 95% as a pale yellow solid. Mp: 131–133 °C. MS (FAB) m/z:
509 (M+1)+. 1H NMR (CDCl3) d: 1.49 (6H, s), 1.61 (1H, m), 1.93
(1H, m), 2.14 (6H, s), 2.41 (3H, s), 2.60 (1H, m), 3.23 (1H, dd,
J = 9.0, 9.9 Hz), 3.31 (1H, m), 3.48–3.59 (2H, m), 3.78 (2H, s), 4.55
(2H, m), 7.10–7.48 (10H, m). 13C NMR (CDCl3) d: 20.4, 24.6, 24.6,
29.8, 38.2, 44.0 (2C), 51.3, 56.0, 65.2, 73.3, 78.4, 89.4, 114.8,
116.1, 127.3, 127.3 (2C), 128.2, 128.2 (2C), 128.8, 129.0, 131.2,
131.4, 136.5, 138.9, 144.0, 148.4, 151.6, 171.7. IR (KBr): 2869,
2207, 1604, 1537, 1504, 1468, 1349, 1091 cmꢀ1. Anal. Calcd for
6.1.8. 2-Butyl-5-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-7-
methyl-6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (15)
Compound 15 was prepared from 12c, as described above for 9.
Yield, 89% as a pale yellow solid. Mp: 108–109 °C. MS (FAB) m/z:
403 (M+1)+. 1H NMR (CDCl3) d: 0.97 (3H, t, J = 7.4 Hz), 1.42–1.50
(2H, m), 1.58–1.67 (1H, m), 1.81–1.89 (2H, m), 1.94–2.01 (1H,
m), 2.14 (6H, s), 2.26 (3H, s), 2.57–2.64 (1H, m), 2.92 (2H, t,
J = 7.7 Hz), 3.20 (1H, t, J = 9.5 Hz), 3.29–3.34 (1H, m), 3.45 (1H,
dd, J = 9.7, 6.9 Hz), 3.55–3.62 (1H, m), 7.12–7.15 (1H, m), 7.23–
7.26 (1H, m), 7.39–7.48 (3H, m). 13C NMR (CDCl3) d: 13.9, 20.4,
22.5, 28.6, 29.9, 30.3, 44.0 (2C), 51.1, 56.0, 65.3, 89.6, 115.5,
115.7, 128.3, 128.8, 129.1, 131.1, 131.3, 136.3, 143.9, 148.5,
151.6, 167.2. IR (KBr): 2956, 2771, 2209, 1610, 1508 cmꢀ1. Anal.
Calcd for C24H30N6: C, 71.61; H, 7.51; N, 20.88. Found: C, 71.49;
C
31H36N6O: C, 73.20; H, 7.13; N, 16.52. Found: C, 73.04; H, 7.11;
N, 16.38. ½a 2D5
ꢂ
+27.4 (c 1.04, CHCl3).
6.1.13. 5-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2-(2-hydroxy-
1,1-dimethylethyl)-7-methyl-6-phenyl[1,2,4]triazolo[1,5-a]-
pyridine-8-carbonitrile (20)
H, 7.48; N, 20.70. ½a D25
ꢂ
+42.3 (c 1.02, CHCl3).
To a solution of 19 (1.40 g, 2.80 mmol) in MeOH (14 mL) and
THF (14 mL) was added 4 M HCl in 1,4-dioxane and the mixture
was stirred for 10 min at room temperature. The mixture was con-
centrated in vacuo and the residue was dissolved with MeOH
(30 mL). To the solution was added 10% Pd–C catalyst (560 mg,
containing 55.6% of water), and the mixture was shaken vigorously
under 4.5 kg/cm2 of hydrogen for 30 min at ambient temperature.
The mixture was filtered and concentrated in vacuo. Purification of
the resulting residue by silica gel column chromatography (CHCl3/
MeOH = 50/1, v/v) gave 20 (1.0 g, 87%) as a pale yellow solid. Mp:
198–200 °C. MS (FAB) m/z: 419 (M+1)+. 1H NMR (CDCl3) d: 1.44
(6H, s), 1.63 (1H, m), 1.98 (1H, m), 2.15 (6H, s), 2.25 (3H, s), 2.61
(1H, m), 3.22 (1H, dd, J = 8.7, 10.2 Hz), 3.35 (1H, m), 3.44–3.60
(2H, m), 3.76 (1H, s), 3.86 (1H, br s), 7.11–7.14 (1H, m), 7.23–
7.26 (1H, m), 7.39–7.50 (3H, m). 13C NMR (CDCl3) d: 20.4, 24.6,
24.6, 29.9, 38.3, 44.0 (2C), 51.4, 56.2, 65.2, 71.3, 89.5, 115.5,
115.7, 128.4, 128.9, 129.1, 131.1, 131.3, 136.2, 144.0, 149.0,
151.3, 171.9. IR (KBr): 3155, 2965, 2214, 1603, 1536, 1503, 1469,
1347, 1065 cmꢀ1. Anal. Calcd for C24H30N6Oꢁ0.25H2O: C, 68.14; H,
6.1.9. 5-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-7-methyl-2,6-
diphenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (16)
Compound 16 was prepared from 12d, as described above for 9.
Yield, 91% as a pale yellow solid. Mp: 224–227 °C. MS (FAB) m/z:
423 (M+1)+. 1H NMR (CDCl3) d: 1.60–1.74 (1H, m), 1.95–2.08 (1H,
m), 2.18 (6H, s), 2.23 (3H, s), 2.59–2.70 (1H, m), 3.31 (1H, t,
J = 9.3 Hz), 3.40 (1H, m), 3.52 (1H, dd, J = 6.9, 9.9 Hz), 3.61–3.70
(1H, m), 7.14–7.19 (1H, m), 7.26–7.30 (1H, m), 7.40––7.53 (6H,
m), 8.30–8.37 (2H, m). 13C NMR (CDCl3) d: 20.4, 30.0, 44.1 (2C),
51.3, 56.2, 65.3, 89.8, 115.7, 115.8, 127.6 (2C), 128.3, 128.6 (2C),
128.9, 129.1, 130.2, 130.5, 131.1, 131.3, 136.3, 144.1, 148.9,
152.2, 163.5. IR (KBr): 2962, 2766, 2218, 1610, 1508, 1442 cmꢀ1
.
Anal. Calcd for C26H26N6ꢁ0.25H2O: C, 73.13; H, 6.25; N, 19.68.
Found: C, 72.99; H, 6.11; N, 19.74. ½a D25
ꢂ
+47.2 (c 1.03, CHCl3).
6.1.10. 2-Cyclopropyl-5-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
7-methyl-6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile
(17)
Compound 17 was prepared from 12e, as described above for 9.
Yield, 98% as a pale yellow solid. Mp: 167–169 °C. MS (FAB) m/z:
387 (M+1)+. 1H NMR (CDCl3) d: 1.03–1.09 (2H, m), 1.14–1.19 (2H,
m), 1.58–1.68 (1H, m), 1.96 (1H, m), 2.14 (6H, s), 2.22 (1H, m),
2.24 (3H, s), 2.60 (1H, m), 3.17 (1H, dd, J = 8.7, 9.9 Hz), 3.29 (1H,
m), 3.42 (1H, dd, J = 7.2, 9.9 Hz), 3.57 (1H, d t, J = 6.9, 10.2 Hz),
7.12 (1H, m), 7.24 (1H, m), 7.38–7.49 (3H, m). 13C NMR (CDCl3)
d: 9.0, 9.1, 9.6, 20.3, 29.9, 44.0 (2C), 51.0, 55.9, 65.3, 89.4, 115.5,
115.8, 128.2, 128.8, 129.1, 131.1, 131.3, 136.3, 143.7, 148.3,
7.27; N, 19.87. Found: C, 68.32; H, 7.27; N, 19.73. ½a D25
ꢂ
+36.5 (c
1.01, CHCl3).
6.1.14. 2-tert-Butyl-5-{[2-(diethylamino)ethyl]amino}-7-methyl-
6-phenyl[1,2,4]triazolo[1,5-a]pyridine-8-carbonitrile (21)
Compound 21 was prepared from 12a and 2-(diethylamino)eth-
ylamine, as described above for 9. Yield, 64% as a pale yellow solid.
Mp: 177–179 °C. MS (FAB) m/z: 405 (M+1)+. 1H NMR (CDCl3) d:
0.96 (6H, t, J = 7.2 Hz), 1.47 (9H, s), 2.22 (3H, s), 2.38–2.44 (6H,

5852
J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
m), 2.82–2.88 (2H, m), 7.19 (1H, br s), 7.24–7.27 (2H, m), 7.41–7.48
(3H, m). 13C NMR (CDCl3) d: 12.2 (2C), 20.0, 29.6 (3C), 33.5, 41.5,
46.3 (2C), 51.3, 85.0, 107.5, 116.6, 128.5, 128.9 (2C), 131.7 (2C),
135.4, 143.2, 148.2, 150.5, 174.7. IR (KBr): 2964, 2816, 2215,
1614, 1556 cmꢀ1. Anal. Calcd for C24H32N6: C, 71.25; H, 7.97; N,
20.77. Found: C, 71.07; H, 8.01; N, 20.68.
(ATR): 2962, 2258, 1464, 1365, 1203, 754 cmꢀ1. Anal. Calcd for
C9H13N3: C, 66.23; H, 8.03; N, 25.74. Found: C, 66.28; H, 8.00;
N, 25.81.
6.1.19. 2-tert-Butyl-7-methyl-5-oxo-6-phenyl-1,5-dihydroim-
idazo[1,2-a]pyridine-8-carbonitrile (28a)
Following the procedures as described for 12a, the title com-
pound (621 mg) was prepared in 33% yield from 27a (300 mg,
1.84 mmol) as a pale brown solid. Mp: >280 °C. MS (ESI) m/z:
306 (M+1)+. 1H NMR (CDCl3) d: 1.38 (9H, s), 2.28 (3H, s), 7.26–
7.40 (2H, m), 7.36–7.40 (2H, m), 7.44 (1H, s), 11.58 (1H, br s). 13C
NMR (CDCl3) d: 19.7, 29.1 (3C), 31.1, 70.0, 105.1, 116.3, 117.7,
127.2, 128.4 (2C), 131.0 (2C), 136.0, 142.2, 143.0, 147.3, 155.9. IR
(ATR): 2208, 1645, 1518, 1317, 748, 717 cmꢀ1. Anal. Calcd for
6.1.15. 3,3-Dimethyl-2-oxobutyl (benzyloxy)acetate (24)
(Benzyloxy)acetic acid (22) (1.00 g, 5.59 mmol) was dissolved in
DMF (5 mL). To the solution was added K2CO3 and 1-bromo-3,3-
dimethylbutan-2-one (23) (1.0 g, 5.59 mmol), and the mixture
was stirred at room temperature for 24 h. The mixture was poured
into 0.5 M HCl aq (40 mL) at 0 °C, and the resultant mixture was
extracted with AcOEt. The organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The crude product
was purified by silica gel column chromatography eluting with
n-hexane/AcOEt = 95/5, v/v to yield 24 (1.17 g, 80%) as a colorless
C19H19N3O: C, 74.73; H, 6.27; N, 13.76. Found: C, 74.62; H, 6.27;
N, 13.72.
oil. HRMS (ESI) Calcd for
C
15H20O4+H: 265.1440. Found:
6.1.20. 2,7-Dimethyl-5-oxo-6-phenyl-1,5-dihydroimidazo[1,2-
a]pyridine-8-carbonitrile (28b)
265.1446. 1H NMR (CDCl3) d: 1.20 (9H, s), 4.24 (2H, s), 4.67 (2H,
s), 4.97 (2H, s), 7.28–7.40 (5H, m). IR (ATR): 1764, 1722, 1192,
Following the procedures as described for 12a, the title com-
pound (330 mg) was prepared in 61% yield from 27b (250 mg,
2.06 mmol) as a brown solid. MS (FAB) m/z: 264 (M+1)+. 1H NMR
(DMSO-d6) d: 2.15 (3H, s), 2.31 (3H, s), 7.21–7.23 (2H, m), 7.31
(1H, m), 7.38–7.41 (2H, m), 7.54 (1H, s). IR (ATR): 3156, 2204,
1124, 1082, 987, 738, 698 cmꢀ1
.
6.1.16. 2-[(Benzyloxy)methyl]-4-tert-butyl-1H-imidazole (25)
A mixture of 24 (6.46 g, 24.4 mmol) and ammonium acetate
(18.8 g, 244 mmol) was heated at 140 °C for 12 h. The mixture
was cooled to room temperature and then dissolved with CHCl3
and 1 M NaOH aq. The organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The crude product was
purified by silica gel column chromatography eluting with CHCl3/
MeOH = 49/1, v/v to yield 25 (4.52 g, 76%) as a pale yellow oil.
HRMS (EI) Calcd for C15H20N2O: 244.1576. Found: 244.1580. 1H
NMR (CDCl3) d: 1.27 (9H, s), 4.55 (2H, s), 4.61 (2H, s), 6.66 (1H,
s), 7.28–7.37 (5H, m). IR (ATR): 2960, 1456, 1363, 1092, 1072,
1650, 1616, 1523, 1317 cmꢀ1
.
6.1.21. 2-tert-Butyl-5-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
7-methyl-6-phenylimidazo[1,2-a]pyridine-8-carbonitrile (29)
Compound 29 was prepared from 28a, as described above for 9.
Yield, 96% as a pale red solid. Mp: 179–180 °C. MS (EI) m/z: 401
(M+). 1H NMR (CDCl3) d: 1.42 (9H, s), 1.60–1.72 (1H, m), 1.91–
2.01 (1H, m), 2.14 (6H, s), 2.26 (3H, s), 2.51–2.59 (1H, m), 2.79–
2.85 (1H, m), 2.95–3.05 (2H, m), 3.11 (1H, dd, J = 8.9, 7.2 Hz),
7.11–7.14 (1H, m), 7.16–7.20 (1H, m), 7.25 (1H, s), 7.40–7.49
(3H, m). 13C NMR (CDCl3) d: 19.7, 30.1, 30.2 (3C), 32.6, 44.1 (2C),
49.0, 54.1, 65.6, 96.0, 105.5, 116.2, 120.3, 128.2, 128.9, 129.0,
130.4, 130.5, 135.9, 141.3, 143.5, 143.7, 158.0. IR (ATR): 2222,
1606, 1471, 1227, 1201, 1153, 912, 704 cmꢀ1. Anal. Calcd for
735, 698 cmꢀ1
.
6.1.17. 4-tert-Butyl-2-(chloromethyl)-1H-imidazole hydrochloride
(26)
To a solution of 25 (4.48 g, 18.3 mmol) in EtOH (45 mL) was
added 10% Pd–C catalyst (896 mg, containing 55.6% of water) and
1 M HCl aq (19.3 mL, 19.3 mmol), and the mixture was shaken vig-
orously under 4 kg/cm2 of hydrogen atmosphere for 12.5 h at
ambient temperature. The mixture was filtered and concentrated
in vacuo to give a crude of (4-tert-butyl-1H-imidazol-2-yl)metha-
nol (3.34 g). Thionyl chloride (6 mL) was added to the crude mate-
rial and the mixture was stirred at ambient temperature for 9 h.
The mixture was concentrated in vacuo and the residue was dis-
solved with Et2O. The precipitate formed was collected by filtra-
tion, washed with Et2O, and dried under a vacuum to afford the
title compound (2.91 g, 80%) as a brown powder. HRMS (EI) Calcd
for C8H13ClN2: 172.0767. Found: 172.0755. 1H NMR (CDCl3) d: 1.37
(9H, s), 4.91 (2H, s), 7.32 (1H, s). IR (ATR): 2686, 1682, 1630, 1282,
C
25H31N5: C, 74.78; H, 7.78; N, 17.44. Found: C, 74.55; H, 7.79; N,
17.40. ½a 2D5
ꢀ15.8 (c 1.01, CHCl3).
ꢂ
6.1.22. 5-[(3S)-3-(Dimethylamino)pyrrolidin-1-yl]-2,7-dimethyl-
6-phenylimidazo[1,2-a]pyridine-8-carbonitrile (30)
Compound 30 was prepared from 28b, as described above for 9.
Yield, 24% as a pale red solid. Mp: 194–196 °C. MS (ESI) m/z: 360
(M+1)+. 1H NMR (CDCl3) d: 1.67 (1H, m), 1.95 (1H, m), 2.14 (6H,
s), 2.27 (3H, s), 2.52 (3H, s), 2.56 (1H, m), 2.81 (1H, m), 2.94–3.03
(2H, m), 3.12 (1H, dd, J = 7.1, 9.1 Hz), 7.15 (1H, m), 7.19 (1H, m),
7.29 (1H, br s), 7.43–7.50 (3H, m). 13C NMR (CDCl3) d: 14.7, 19.8,
30.1, 44.1 (2C), 49.0, 54.0, 65.6, 95.7, 108.6, 115.7, 120.6, 128.3,
129.0, 129.0, 130.3, 130.5, 135.7, 141.0, 143.7, 144.0, 144.2. IR
901, 823, 727 cmꢀ1
.
(ATR): 2817, 2767, 2221, 1604, 1471 cmꢀ1
. Anal. Calcd for
C
22H25N5: C, 73.51; H, 7.01; N, 19.48. Found: C, 73.24; H, 6.93; N,
6.1.18. (4-tert-Butyl-1H-imidazol-2-yl)acetonitrile (27a)
19.24. ½a 2D5
ꢀ17.6 (c 1.01, CHCl3).
ꢂ
To a stirred solution of potassium cyanide (3.57 g, 54.9 mmol)
in water (15 mL) was dropwised
a
solution of 26 (2.87 g,
6.1.23. (3-tert-Butyl-1H-pyrazol-5-yl)acetonitrile (32)
To a solution of potassium cyanide (19.2 g, 0.295 mol) in
water (23 mL) was dropwised a solution of 3-tert-butyl-5-(chlo-
romethyl)-1H-pyrazole hydrochloride (31)20 (8.80 g, 42.1 mmol)
in EtOH (80 mL) at 0 °C over 1 h. The mixture was stirred at
room temperature for 4.5 h. The reaction mixture was filtered
and concentrated in vacuo, and the residue was purified by sil-
ica gel column chromatography eluting with CHCl3/MeOH = 100/
1, v/v to yield 32 (5.87 g, 85%) as a yellow solid. MS (FAB) m/z:
164 (M+1)+. 1H NMR (CDCl3) d: 1.33 (9H, s), 3.74 (2H, s), 6.10
(1H, s).
13.7 mmol) in EtOH (61 mL) at 0 °C over 1 h. The mixture was
stirred at room temperature for 2 h. The reaction mixture was fil-
tered and concentrated in vacuo, and the residue was dissolved
with AcOEt and 1 M NaOH aq. The organic layer was washed with
brine, dried over Na2SO4, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography eluting
with n-hexane/AcOEt = 1/2, v/v to yield 27a (1.51 g, 67%) as an or-
ange solid. Mp: 134–136 °C. MS (ESI) m/z: 164 (M+1)+. 1H NMR
(CDCl3) d: 1.28 (9H, s), 3.90 (2H, s), 6.72 (1H, s). 13C NMR
(DMSO-d6) d: 17.6, 30.2 (3C), 30.9, 116.0, 117.2, 136.2, 146.5. IR

J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
5853
6.1.24. (Z)-3-Methoxy-2-phenylbut-2-enenitrile (34)
The MIC-2 (the lowest drug concentration showing 50%
growth inhibition compared to the control without drug) was
evaluated by the CLSI standard microdilution method with some
modification using RPMI 1640 media with 0.165 M MOPS buffer
as the medium. Briefly, in the experiments using RPMI 1640 med-
ia, alamar blue (Biosource, Camarillo, CA) was used to accurately
define its MIC-2.23–25 Test organisms were purchased from the
American Type Culture Collection (Rockville, MD), the Institute
for Fermentation Osaka (Osaka, Japan), or Teikyo Institute of Med-
ical Mycology (Tokyo, Japan). Initial cell densities (1 ꢃ 103 to
1 ꢃ 104 cells/mL) and incubation time (18–72 h) were decided
for each strain in consideration of its growth speed. The fungi
were incubated at 30 °C. Following incubation, OD570 was mea-
sured with a Wallac 1420 ARVOsx multi-label counter (Wallac,
Tokyo, Japan) and MIC-2s were calculated as a 50% reduction
compared with the drug-free control in absorbance at 570 nm.
Compounds were tested at different concentrations ranging from
A mixture of 3-oxo-2-phenylbutanenitrile (33) (25.0 g, 0.157
mol) and trimethyl orthoacetate (140 mL, 1.21 mol) was refluxed
for 6 h. The reaction mixture was cooled to room temperature
and concentrated in vacuo. The residue was purified by silica gel
column chromatography eluting with n-hexane/AcOEt = 10/1, v/v
to 5/1, v/v to give 34 (15.4 g, 57%) as a pale yellow oil. MS (FAB)
m/z: 174 (M+1)+. 1H NMR (CDCl3) d: 2.45 (3H, s), 3.86 (3H, s),
7.18–7.26 (1H, m), 7.26–7.37 (2H, m), 7.58–7.64 (2H, m).
6.1.25. 7-Amino-2-tert-butyl-5-methyl-6-phenylpyrazolo[1,5-
a]pyridine-4-carbonitrile (35)
Under a nitrogen atmosphere, lithium diisopropylamide solu-
tion (2.0 M in heptanes/THF/ethylbenzene, 2.02 mL, 4.05 mmol)
was added dropwise to a solution of 32 (300 mg, 1.84 mmol) in
THF (30 mL) at ꢀ30 °C. The mixture was stirred at the same tem-
perature for 30 min and then a solution of 34 (318 mg, 1.84 mmol)
in THF (5 mL) was added dropwise at ꢀ30 °C. After being stirred for
4 h at ꢀ30 °C, the mixture was stirred for 15 h at 0 °C. The reaction
mixture was quenched with satd NH4Cl aq and extracted with
AcOEt. The organic layer was washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified by sil-
ica gel column chromatography eluting with n-hexane/AcOEt = 6/
1, v/v to yield 35 (235 mg, 42%) as a pale yellow solid. MS (FAB)
m/z: 305 (M+1)+. 1H NMR (CDCl3) d: 1.42 (9H, s), 2.25 (3H, s),
5.48 (2H, br s), 6.44 (1H, s), 7.23–7.30 (2H, m), 7.42–7.56 (3H, m).
0.004 to 128 lg/mL.
6.3. Incorporation study with growing cells using [14C]-glucose
Incorporation study with growing cells was conducted based on
the methods described by Kitamura et al.11 Exponentially growing
cells of C. albicans were suspended in an 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 incubation,
samples were taken and crude fractions of b-1,3-glucan, b-1,6-glu-
can, chitin, and mannan were prepared as follows. The harvested
cells were extracted with 3% NaOH at 80 °C in 1 h. Mannan frac-
tions were prepared from the supernatant using Fehling’s reaction.
Insoluble material was washed and digested with Zymolyase 100T
(Seikagaku-kougyou) overnight. After digestion, insoluble material
was harvested as a chitin fraction. The supernatants were taken as
glucan fractions (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 scintillator. The radioactivities of the b-1,3-glucan
fraction were calculated by subtracting that b-1,6-glucan fraction
from the glucan fraction.
6.1.26. 2-tert-Butyl-7-[(3S)-3-(dimethylamino)pyrrolidin-1-yl]-
5-methyl-6-phenylpyrazolo[1,5-a]pyridine-4-carbonitrile (36)
A mixture of tert-butyl nitrite (88 lL, 0.74 mmol) and copper (II)
chloride (80 mg, 0.59 mmol) in MeCN (3 mL) was stirred at 75 °C
for 5 min. To the reaction mixture was added 35 (150 mg,
0.49 mmol) at the same temperature. After being stirred for 1 h
at 75 °C, the mixture was cooled to room temperature and diluted
with CHCl3. The resultant mixture was washed with 1 M HCl aq
and brine, dried over Na2SO4, and concentrated in vacuo. The res-
idue was purified by silica gel column chromatography eluting
with n-hexane/AcOEt = 8/1, v/v to afford a crude product of 2-
tert-butyl-7-chloro-5-methyl-6-phenylpyrazolo[1,5-a]pyridine-4-
carbonitrile. The obtained crude product was diluted with DMF
(2 mL). (3S)-3-(Dimethylamino)pyrrolidine (21 mg, 0.185 mmol)
and triethylamine (41
l
L, 0.308 mmol) was added and the result-
6.4. Distribution coefficient (Log D)
ing mixture was stirred at 90 °C for 4 h. The mixture was cooled
to room temperature and concentrated in vacuo. The residue was
dissolved with AcOEt and the mixture was washed with satd NaH-
CO3 aq and brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluting with CHCl3/MeOH = 100/1, v/v to afford 36 (23 mg, 12%)
as a yellow solid. Mp: 125–127 °C. MS (FAB) m/z: 402 (M+1)+. 1H
NMR (CDCl3) d: 1.42 (9H, s), 1.50–1.70 (1H, m), 1.88–1.97 (1H,
m), 2.17 (6H, s), 2.20 (3H, s), 2.63–2.75 (1H, m), 3.16–3.26 (2H,
m), 3.38–3.59 (2H, m), 6.45 (1H, s), 7.09–7.23 (2H, m), 7.35–7.47
(3H, m). 13C NMR (CDCl3) d: 20.0, 29.3, 30.6 (3C), 32.7, 43.6 (2C),
50.3, 54.7, 65.2, 91.8, 92.2, 115.0, 117.5, 127.8, 128.7, 128.8,
131.1, 131.2, 137.1, 140.9, 143.3, 143.9, 165.2. IR (KBr): 2960,
2865, 2817, 2768, 2209, 1603, 1515, 1468, 1443 cmꢀ1. Anal. Calcd
for C25H31N5ꢁ0.75H2O: C, 72.34; H, 7.89; N, 16.87. Found: C, 72.59;
The distribution coefficients (Log D) were determined by the
shake-flask method. Four hundred micromolar of compound solu-
tion of each compound in a 2 mL n-octanol–2 mL BPS solution was
placed on a shaker for 30 min at pH 6.8. After centrifuging each
solution separately at 3000 rpm for 10 min, an LC/MS method
was used to assay each layer. The LC/MS system consisted of an
1100 Series LC/MSD (Agilent) and an X TerraÒ MSC18 3.5
lm,
3.0 ꢃ 30 mm column (Waters). The mobile phase was a 10 mM
ammonium acetate buffer (pH 4.5)/0.05% (v/v) acetic acid mixture
in acetonitrile; the gradient condition (95/5-10/90). Analyst soft-
ware program (version 1.4, Applied BioSystems) was used to calcu-
late the Log D.
6.5. Metabolic stability
H, 7.63; N, 16.73. ½a D25
ꢀ1.14 (c 1.02, CHCl3).
ꢂ
Compounds (final 1 lM) were incubated with human liver
6.2. In vitro antifungal activity
microsome in sodium phosphate buffer (pH 7.4) for 20 min at
37 °C. The microsomal protein concentration in the assay was
0.5 mg/mL. Reaction was started by the addition of NADPH at
37 °C and stopped by addition of MeOH after 30 min. After centri-
fuging each solution separately at 3500 rpm for 10 min at 4 °C, the
corresponding loss of parent compound was determined by LC/MS/
MS.
The MIC-0 against each fungal strain was measured by the mic-
rodilution method described by the Clinical and Laboratory Stan-
dard Institute (CLSI), except that the incubation temperature was
30 °C.22 The MIC-0 was defined as the lowest drug concentration
producing an optically clear well.

5854
J. Kuroyanagi et al. / Bioorg. Med. Chem. 18 (2010) 5845–5854
9. Singh, N. Clin. Infect. Dis. 2001, 33, 1692.
Acknowledgments
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We thank the members of Biological Research Laboratories IV
for the biological evaluation. We also thank the members of Drug
Metabolism & Pharmacokinetics Research Laboratories for the
physicochemical tests.
Supplementary data
15. Umeyama, T.; Kaneko, A.; Watanabe, H.; Hirai, A.; Uehara, Y.; Niimi, M.;
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Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.096. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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