Synthesis and biological activities of benzofuran antifungal agents targeting fungal N-myristoyltransferase

Article abstract of DOI:10.1016/S0968-0896(03)00429-2

The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro. Further

Full text of DOI:10.1016/S0968-0896(03)00429-2

Bioorganic & Medicinal Chemistry 11 (2003) 4463–4478
Synthesis and Biological Activities of Benzofuran Antifungal
Agents Targeting Fungal N-Myristoyltransferase
Miyako Masubuchi, Hirosato Ebiike, Ken-ichi Kawasaki, Satoshi Sogabe,
Kenji Morikami, Yasuhiko Shiratori, Shinji Tsujii, Toshihiko Fujii, Kiyoaki Sakata,
,
Michiko Hayase, Hidetoshi Shindoh, Yuko Aoki, Tatsuo Ohtsuka* and Nobuo Shimma
Chugai Pharmaceutical Kamakura Research Center (formerly Nippon Roche Research Center), 200 Kajiwara,
Kamakura, Kanagawa 247-8530, Japan
Received 19 February 2003; accepted 18 June 2003
Abstract—The C-4 side chain modification of lead compound 1 has resulted in the identification of a potent and selective Candida
albicans N-myristoyltransferase (CaNmt) inhibitor RO-09-4609, which exhibits antifungal activity against C. albicans in vitro.
Further modification of its C-2 substituent has led to the discovery of RO-09-4879, which exhibits antifungal activity in vivo. The
drug design is based on X-ray crystal analysis of a CaNmt complex with benzofuran derivative 4a. The optimization incorporates
various biological investigations including a quasi in vivo assay and pharmacokinetic study. The computer aided drug design,
synthesis, structure–activity relationships, and biological properties of RO-09-4879 are described in detail.
#
2003 Elsevier Ltd. All rights reserved.
Introduction
interactions with various drugs by inhibiting metabolic
1
enzymes, the CYP450 isoenzymes. The antifungal
The incidence of fungal infection has increased sig-
nificantly in the past 25 years. The growing number of
immunocompromised patients as a result of cancer
chemotherapy, organ transplantation, and HIV infec-
tion are the major factors contributing to this incidence.
Since Candida albicans (C. albicans), and Aspergillus
fumigatus (A. fumigatus) are the main causative fungi of
the systemic mycosis, antifungal drugs for treating
patients of deep mycosis should have a broad antifungal
spectrum including at least these microorganisms. Cur-
rently only four classes of antifungal drugs, polyene
macrolides (amphotericin B), azoles (fluconazole, mico-
nazole, itraconazole and voriconazole), flucytosine, and
candins (caspofungin acetate and micafungin), are
available for treatment of systemic mycoses. Unfortu-
nately, none of them is ideal in terms of efficacy, anti-
fungal spectrum or safety. Although amphotericin B is
efficacious against both candidiasis and aspergillosis, it
shows severe renal toxicity. Azoles show drug–drug
spectra of fluconazole and flucytosine are narrow
(mainly against C. albicans) and they are prone to
develop drug resistance. Although caspofungin and
micafungin are fungicidal in Candida, they are fungi-
static in Aspergillus and inactive against Cryptococcus
neoformans (C. neoformans), a clinically emerging fun-
gus. In addition, these drugs are given only by iv infu-
sion due to poor oral bioavailability. To overcome the
drawbacks of the current antifungal drugs and to obtain
more efficacious drugs, an antifungal drug having a
novel mode of action should be developed. Myristoyl
CoA:protein N-myristoyltransferase (Nmt; EC 2.1.3.97)
is a cytosolic monomeric enzyme that catalyzes the
transfer of the myristoyl group from myristoyl CoA to
the N-terminal glycine of a number of eukaryotic cel-
2ꢀ4
lular proteins and viral proteins. N-Myristoylproteins
have diverse functions and intracellular destinations. N-
Myristoylation is an irreversible protein modification
that occurs co-translationally after the removal of the
initiator methionine by cellular methionylaminopepti-
5,6
dases. N-Myristoylation results in an increase of lipophi-
licity that triggers the promotion of reversible protein–
*Corresponding author at present address: Product planning depart-
7
,8
membrane and protein–protein interactions. N-Myr-
istoylation relates to diverse biological processes including
ment, Chugai Pharmaceutical Co., LTD. 1–9 Kyobashi 2-Chome,
Chuo-Ku, Tokyo, 104-8301, Japan. Tel.: +81-3-3273-8549; fax: +81-
9,10
3-3281-6675; e-mail: ohtsukatto@chugai-pharm.co.jp
signal transduction cascades and apoptosis.
N-Myr-
0
968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00429-2

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M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
istoylation of several G-proteins, Gpa1, Arf1, Arf2, and
Vps15, which are essential for fungal growth in vitro,
has been reported to be indispensable for their function
and 3b were prepared from 4-hydroxybenzofuran deri-
vative 2 by treatment with excess (2S)- and (2R)-glycidyl
tosylate in the presence of NaH in N,N-dimethylforma-
mide (DMF), respectively. (R,S)-4-Oxiranylmethoxy
derivative 3 was prepared from 2 by treatment with
excess epichlorohydrin in the presence of Cs CO in
1
1ꢀ14
in Saccharomyces cerevisiae.
Nmt has also been
proven to be essential for viability in vitro of fungi,
including medically important pathogenic fungi such as
C. albicans and C. neoformans, which cause systemic
2
3
ꢁ
acetonitrile at 60 C. Amination of epoxides 3, 3a, and
ꢁ
1
5,16
fungal infections in immunocompromised patients.
Furthermore, we have recently reported that a defect of
Nmt causes the loss of the ability of C. albicans to infect
3b with tert-butylamine in ethanol at 60 C gave amino
compounds 4, 4a, and 4b, respectively. Treatment of 2
with excess alkylene dibromide in the presence of
potassium carbonate in DMF gave O-bromoalkyl deri-
vatives 5, 6, 7, and 8. Treatment of 5, 6, 7, and 8 with
1
7
in mice. These data suggest that fungal Nmt is a
potential target enzyme for the development of novel
fungicidal drugs having a broad antifungal spectrum. In
addition, since the mode of action is novel, the Nmt
inhibitors might overcome the drawbacks of current
antifungal drugs, such as resistance or drug–drug inter-
actions. On the other hand, since Nmt is also distributed
in mammalian cells, the inhibitors need to be fungal
Nmt selective to avoid adverse events caused by inhi-
biting human Nmt. So far, peptidomimetic
ꢁ
various amines in ethanol at 50–70 C gave the desired
amino derivatives 9, 10a–j, 11, and 12, respectively. 3-
(Pyridin-3-ylmethoxy)propoxy analogue 13 was also
prepared from 6 by treatment with 3-pyridinemethanol
in the presence of NaH in DMF. Ethers 15a–k, 16, and
17 were synthesized by Mitsunobu reaction of appro-
priate phenols and 14 that was obtained by LiAlH4
reduction of 10h. The modified Mitsunobu reaction
1
8ꢀ26
27,28
0
inhibitors,
zole inhibitors,
and benzofuran inhibitors
myristic acid analogues,
benzothia-
31
using 1,1 -(azodicarbonyl)dipiperidine (ADDP) and tri-
2
9,30
p-toluenesulfonamide inhibitors,
butylphosphine gave 15f and 15g in higher yields (40–
80%) than the conventional Mitsunobu reaction using
diethyl azodicarboxylate (DEAD) and triphenylphos-
phine (TPP).
3
2ꢀ39
have been reported to be
fungal Nmt inhibitors. Among them, the benzofuran
inhibitors that we synthesized showed high selectivity
over human Nmt and exhibited antifungal activity in
vivo. We identified lead compound 1 that competitively
inhibited CaNmt (IC : 0.98 mM) with high selectivity
5
0
Results and Discussion
over human Nmt (IC : 194 mM) by a random screening
5
0
of the Roche chemical libraries. Since antifungal activity
in vitro of 1 was only marginal (IC50 against C. albicans
CY1002: 390 mM), our first aim was to improve the in
vitro activity. The C-4 side-chain modification of 1
resulted in finding the potent and selective CaNmt inhi-
bitor 10h (RO-09-4609), which exhibited antifungal
activity against C. albicans in vitro. Further modifi-
cation of 10h led to the discovery of 15g (RO-09-4879),
which exhibited antifungal activity in vivo. The drug
design was based on X-ray crystallographic analysis of a
Modification of the C-4 substituent of 1
Lead compound 1 was originally reported to have b-
4
5
adrenoceptor blockade. Since it is well known that an
aromatic compound having a b-aminoalcohol moiety
shows b-blocking action, we removed the hydroxy
group of 1 to eliminate this activity. As expected,
removing the hydroxy group lowered the b-adreno-
ceptor blockade significantly without affecting its
CaNmt enzyme inhibitory activity (Table 1). The IC s
5
0
3
8
CaNmt complex with a benzofuran derivative 4a. The
optimization incorporated various biological investiga-
of 10a and 10b in the b-adrenoceptor blockade assay
were 38 and 25 mM, respectively, whereas those of the
corresponding hydroxy derivatives 1 and 4 were 0.098
and 0.0043 mM, respectively. To find the optimal chain
length of the C-4 position, ethylene 9, tetramethylene
11, and pentamethylene 12 derivatives were also pre-
pared and their enzyme inhibitory activity was com-
pared with that of the trimethylene derivative 10b. The
result indicated that the optimal chain length was C3
(trimethylene).
4
0,41
tions including a quasi in vivo assay
antifungal assay in 80% serum) and a cassette dosing
pharmacokinetic (PK) study.
(an in vitro
4
2ꢀ44
This paper describes
in full the drug design, synthesis, structure–activity
relationships (SARs), and lead optimization process.
The biological properties of 15g (RO-09-4879) are also
discussed in detail.
For further optimization, we used the crystal structure
of a binary complex of CaNmt and 4a that was
˚
obtained at 3.5 A resolution by a soaking experiment
3
8
(
Fig. 1). The benzofuran moiety is located at the cen-
ter of a deep pocket, surrounded by hydrophobic resi-
dues. The C-4 substituent on the benzofuran ring
extends to a C-terminal leucine (Leu 451) of the poly-
peptide chain and the tert-butyl group is surrounded by
a hydorophobic pocket consisting of aromatic amino
acid residues, Tyr 107, Tyr 119, and Phe 176. The amino
group in the C-4 side chain makes a salt bridge with the
carboxyl group of Leu 451. This interaction should be
essential for the enzyme inhibitory activity because the
Chemistry
The general synthesis of the benzofuran derivatives is
outlined in Scheme 1. 4-Oxiranylmethoxy derivatives 3a

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4465
C-terminal carboxylate plays an important role in the
suggesting that the strong basicity of an aliphatic amino
group is essential for the inhibitory activity. The (pyr-
idin-3-ylmethyl)amino derivative 10h (RO-09-4609)
showed the most potent enzyme inhibitory activity
against CaNmt with extremely high selectivity (>5000-
fold) over human Nmt. Its increased enzyme inhibitory
activity over benzyl derivative 10f indicated the presence
of additional favorable hydrogen bonding between the
pyridine nitrogen and an amino acid residue of CaNmt
(vide infra). Since other heteroaromatic derivatives such
as (pyridin-2-ylmethyl)amino 10g, (pyridin-4-ylmethyl)-
amino 10i, and (pyridin-3-ylethyl)amino 10j derivatives
showed weaker activity than did 10h, the position of the
4
8
catalytic mechanism. This structural analysis sug-
gested that the replacement of the tert-butyl amino
group by another hydrophobic amino group or an alkyl
amino group with an aromatic ring would increase the
enzyme inhibitory activity. The weak inhibitory activity
of primary amine 10c supported this analysis (Table 2).
Therefore, a number of benzofurans having a hydro-
phobic group or an alkyl group with an aromatic ring
on the amino group were synthesized.
The aniline derivative 10e and pyridin-3-ylmethoxy
derivative 13 were devoid of the inhibitory activity,
Scheme 1. Preparation of benzofuran derivatives. Reagents and conditions: (a) (2S)- or (2R)-glycidyl tosylate, NaH, DMF, rt, 76–83%; (b) epi-
ꢁ
ꢁ
ꢁ
chlorohydrin. Cs
2
CO
3
, CH
3
CN, 60 C, 86%; (c) tert-BuNH
ꢁ
2
, EtOH, 60 C, 74–95%; (d) alkylene dibromide, K
, 10% Pd/C, EtOH, rt, 45% (g) 3-pyridinemethanol, NaH, DMF, rt, 11%; (h) LiAlH
2
CO
3
, DMF, rt or 100 C, 1–4 h, 43–
, THF,
1
2
8
0
8%; (e) R R NH, EtOH, 50–70 C, 34–98%; (f) H
ꢁ
2
4
C, 91%; (i) Mitsunobu reaction.

4466
M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
pyridine nitrogen was very important to gain stronger
binding affinity.
Modification of the C-2 substituent of 10h
The crystal structure of a binary complex of CaNmt and
4a (Fig. 1) revealed that the C-2 substituent is sur-
rounded by three phenylalanine residues, Phe 115, Phe
240, and Phe 339. To strengthen the binding between
the C-2 substituent and these phenylalanine residues
through aromatic–aromatic interaction, we introduced
a phenyl group to the C-2 position via various linkers:
–CONH–, –CH S–, –CH CH –, and –CH O– (15a)
To find the optimal substituent of the C-3 position, the
C-3 methyl group of 10h was replaced by hydrogen,
ethyl, cyclopropyl, and isopropyl; however, the methyl
3
2
was still the best among them.
Although 10h was about 10 times stronger than 1 in
terms of CaNmt inhibitory activity and showed clear
antifungal activity against C. albicans, it did not show
antifungal activity in vivo in a murine systemic candi-
diasis model. Its antifungal activity might be weak and
its metabolic stability (elimination half-life t1/2: 0.42 h in
mice) insufficient. The ester group of 10h was easily
hydrolyzed by esterases in mice to give an inactive car-
boxylic acid metabolite. To overcome these drawbacks,
we modified the C-2 substituent of 10h.
2
2
2
2
which are expected to be much more metabolically
stable than an ethyl ester group. Among them, com-
pound 15a having an ether linker showed the most
potent CaNmt inhibitory activity and antifungal activity.
Next, we introduced various substituents on the phenyl
3
3
ring of 15a. To increase the binding affinity, electron-
withdrawing groups, such as fluoro, bromo, cyano, and
nitro, were introduced to the phenyl ring of 15a to
Table 1. Enzyme inhibitory activity, antifungal activity, and b-adrenoceptor blockade of benzofuran derivatives (IC50: mM)
d
Compd
R
Enzyme inhibition
Antifungal activityc
C. albicans CY1002
390
b-Adrenoceptor blockade
CaNmta
HsNmtb
194
1
4
0.98
1.2
0.098
0.0043
0.079
0.38
38
470
190
210
39
4
4
1
1
9
1
1
a
1.1
b
150
>290
380
40
0a
0b
4.4
1.7
470
300
590
190
53
200
25
50
>630
>580
>550
NT
NTe
NT
1
2
4.4
15
NT
Alprenolol
NT
NT
0.0023
aInhibitory activity against C. albicans Nmt (CaNmt) as assessed by IC50 using substrate peptide GLTISKLFRR-NH
(0.5 mM) and myristoyl-CoA
(0.5 mM) and myristoyl-CoA at 0.5
HPO , pH 7).
2
at 0.5 mM.
Inhibitory activity against human Nmt (HsNmt) as assessed by IC50 using substrate peptide GNAASARR-NH
b
2
mM.
Antifungal activity against C. albicans CY1002 as assessed by IC50 in YNBPB medium (1% glucose, 0.25% K
c
2
4
d
46,47
Adrenergic, beta, non-selective binding assay, performed by NOVA SCREEN according to a literature method with modifications.
NT, Not tested.
e

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4467
Figure 1. Crystal structure of a binary complex of CaNmt and 4a. Only the binding site is shown.
interact with the aromatic residues of CaNmt more
methylbenzothiophene-4-yloxy]propyl}pyridin-3-ylme-
thylamine, a benzothiophene analogue of compound
15f, showed about 1000-fold lower inhibitory activity,
the hydrogen bond interaction of the benzofuran ring
with the imidazole ring of His 227 might greatly con-
tribute to the inhibitor binding. The phenyl ring of the
C-2 substituent of 15e is surrounded by three phenyl-
alanine residues, Phe 115, Phe 240, and Phe 339, that
make hydrophobic packing interactions, and is situated
exactly between Phe 240 and Phe 339. The crystal struc-
ture also suggested the important hydrogen bond between
4
9
strongly. As expected, most of the phenyl ether deri-
vatives having electron-withdrawing group(s) showed
stronger inhibitory activity than the un-substituted
phenyl ether 15a (Table 3). Among them, the 2-fluoro
1
5b, 4-fluoro 15d, 2,3-difluoro 15e, 2,4-difluoro 15f
RO-09-4746), 2,3,4-trifluoro 15g (RO-09-4879), 4-
(
cyano 15i, and 2-nitro-3-methyl 15j derivatives showed
very strong inhibitory activity against CaNmt (IC :
0
5
3
1
.7–9.4 nM). In contrast to this, the 3-fluoro derivative
5c was 10–30 times less active. This variation in inhi-
bitory activity among the ether derivatives could be
explained by the differences in the electron density on
the ether oxygen calculated by the electrostatic poten-
tials around the aromatic rings of the compounds using
a molecular orbital method. We also introduced het-
eroaromatics, 1-methyl-5-trifluoromethyl-1H-pyrazole
the amido-NH of Asn 392 and the ether oxygen of the C-
2
2 substituent. The 3-pyridyl group in the C-4 side chain is
surrounded by hydrophobic aromatic amino acid resi-
dues, Tyr 107, Tyr 119, and Phe 176. In addition, hydro-
gen bonding between the pyridine nitrogen and the Tyr
119 hydroxy group was observed. The increased enzyme
inhibitory activity of 10h over the benzyl derivative 10f
can be explained by this additional hydrogen bonding,
supported by the finding that the inhibitory activity of 10h
against a mutant CaNmt having alanine for Tyr 119 was
3
7
1
6 and 3-pyridyl 17, to the C-2 position via –CH O–.
2
The enzyme inhibitory activity of 16 was as strong as
the fluorophenyl derivatives. The binding mode of
phenyl ether derivatives was examined by solving a
crystal structure of a binary complex of CaNmt and 15e
3
2
14 times weaker than that against the wild CaNmt.
˚
that was obtained at 3.5 A resolution by a soaking
3
8
experiment. The positions of the fluorine atoms were
not well defined because of their poor electron density.
A schematic representation of the hydrogen-bonding
interactions between 15e and CaNmt are shown in Fig-
ure 2. 15e is located at a hydrophobic pocket composed
of hydrophobic amino acid residues including Tyr 107,
Phe 115, Phe 117, Tyr 119, Phe 176, His 227, Phe 240,
Tyr 225, Tyr 354, and Phe 339. The benzofuran ring of
Quasi in vivo assay and cassette dosing PK study for
evaluating benzofuran inhibitors
The results of enzyme inhibitory activity, antifungal
activity in vitro, quasi in vivo activity, pharmacokinetic
(PK) properties, and in vivo antifungal activity are
shown in Table 3. To predict in vivo efficacy, we intro-
duced an antifungal assay of 80% calf serum supple-
.
1
3
5e is stacked parallel to Tyr 225 and interacts with Tyr
54 in a perpendicular orientation in the proximity of
mented with 10 mM FeCl 6H O, 10 mM deferoxamine
3
2
and 2% dextrose (quasi in vivo assay). The quasi in vivo
assay was reported to be more predictive for in vivo
efficacy than the conventional in vitro antifungal
Phe 117 and Phe 339. The benzene ring of the benzo-
furan occupies the hydrophobic pocket with Leu 394,
Tyr 225, and Cys 393 of the polypeptide chain and little
space is left for an extra substituent at the C-6 and C-7
positions of the benzofuran. This observation is con-
sistent with the SARs that show mono-methylation at
the C-7 position lowered CaNmt inhibitory activity by a
factor of 250. The ring oxygen of the benzofuran is
located in the proximity of the His 227 imidazole ring
and makes a hydrogen bond to a ring nitrogen of
His 227. Since {3-[2-(2,4-difluoro-phenoxymethyl)-3-
4
0,41
assay.
In addition, a PK study would also be
important for predicting in vivo efficacy. To carry out
the PK study efficiently and to reduce the number of test
4
2ꢀ44
animals, we introduced cassette dosing PK.
Five
compounds (2 mg/kg each) were intravenously admi-
nistered to one rat and the plasma concentration of each
compound was measured by LC–MS. This method
allowed efficient selection of compounds for in vivo
tests. Compounds 15b, 15d, 15e, 15f, 15g, 15j, and 16

4468
M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
were as potent as fluconazole (IC : 0.5 mM in this assay
ED s of 7.1 mg/kg (Fig. 3). The in vivo activity of 15h
50
5
0
protocol) in the quasi in vivo assay, although the addi-
tion of calf serum lowered their antifungal activity. All
the ether derivatives in Table 3 with the exception of 17
showed longer t1/2 than did 10h, as we had expected.
Thus, most of the compounds selected for further in
vivo evaluation showed reasonable PK profiles and
strong quasi in vivo activity. Among them the fluoro
derivatives 15f and 15g, which showed the strongest
quasi in vivo antifungal activity, exhibited the most
potent antifungal activity in the in vivo model with
was not as strong (ED : >30 mg/kg) in spite of its rea-
5
0
sonable PK profile. Its weak activity in the quasi in vivo
assay may explain its weak in vivo activity. The in vivo
activity of the ether derivatives was well predicted by the
results of the quasi in vivo assay and PK properties.
Biological properties of 15g
Antifungal activity in a rat systemic candidiasis model.
Fisher rats (n=5) were infected intravenously with a
Table 2. Optimization of the C-4 substituent (IC50:mM)
Compound
R
Enzyme inhibition
Antifungal activityc
C. albicans CY1002
470
CaNmta
4.4
HsNmtb
380
1
0a
0b
1
1.7
200
300
1
0c
0d
NH
2
11
>720
260
84
1
4.1
38
1
1
1
1
1
1
1
0e
>570
3.3
>570
530
>280
10
0f
0g
11
270
200
1.6
0h (RO-09-4609)
0.1
>540
>540
>520
>540
0i
0j
3
1.9
180
7.8
0.39
>540
NTd
SC-58272e
0.83
NT
>140
200
0.72
Fluconazole
NT
aInhibitory activity against C. albicans Nmt (CaNmt) as assessed by IC50 using substrate peptide GLTISKLFRR-NH
(0.5 mM) and myristoyl-CoA
2
at 0.5 mM.
Inhibitory activity against human Nmt (HsNmt) as assessed by IC50 using substrate peptide GNAASARR-NH
b
2
(0.5 M) and myristoyl-CoA at 0.5
HPO , pH 7).
mM.
Antifungal activity against C. albicans CY1002 as assessed by IC50 in YNBPB medium (1% glucose, 0.25% K
Not tested.
c
2
4
d
eA dipeptide CaNmt inhibitor.22,23,25

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4469
Figure 2. Schematic drawing of the interactions between CaNmt and 15e, generated by LIGPLOT.50 Residues forming van der Waals interactions
are indicated; those participating in hydrogen bonds are represented by a ball-and-stick figure. The inhibitor is also represented by a ball-and-stick
˚
figure. Hydrogen bonds are depicted as dotted lines with the donor-acceptor distance given in A.
lethal dose of C. albicans (CY1002) and treated intra-
venously (iv) with multiple doses (2 tid) of the test
compound. Efficacy of the compounds was calculated as
the effective dose (mg/kg) for 50% survival (ED ) on
intraperitoneally (ip). Figure 4 shows the mean log10
colony forming unit (CFU) per brain or kidney at day 2
after infection in the groups treated with antifungal
agents and that of sham-treated control groups. Com-
pound 15g reduced the counts of yeast cells recovered
from the brain and kidney significantly. The results
suggest that the survival of the rats tested was pro-
longed by inhibiting fungal growth in the rats.
5
0
day 7. The antifungal activity in vivo of the representa-
tive compound 15g is shown in Figure 3. All the animals
in the control group died within 2 days. The benzofuran
derivative produced a statistically significant increase in
survival compared with the control in a dose-dependent
manner, although its efficacy was fairly weaker than
fluconazole (ED : 0.5 mg/kg, iv).
Time–kill kinetics of 15g. To confirm the antifungal
activity of 15g, the time–kill kinetics of 15g for C. albi-
cans CY3003 in 80% calf serum was investigated at 2, 4,
15, and 30 times concentrations of the IC50 (Fig. 5).
Compound 15g reduced CFU in a dose- and time-
dependent manner at concentrations of 15ÂIC and
5
0
Efficacy in the disseminated candidiasis target organ assay
of 15g. The results of the target organ assay of 15g in a
rat systemic candidiasis model are shown in Figure 4.
The benzofuran derivative was tested for its activity in
reducing the numbers of recoverable yeast cells from the
brain and kidney of Fisher rats (n=3) challenged intra-
5
0
30ÂIC showing that it had fungicidal activity. In con-
5
0
trast, fluconazole did not reduce the CFU at a concen-
tration of 200ÂIC50 in the same experiment.
3
venously with C. albicans CY1002 (3Â10 cells/g, 0.5
mL). Compound 15g was administered intravenously in
the disseminated candidiasis models, three rats per
therapy group. Fluconazole (FCZ) was administered
Antifungal spectrum of 15g. The antifungal spectra of
15g and fluconazole are shown in Table 4. Antifungal
susceptibility assays were performed using the broth

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M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
Table 3. Enzyme inhibitory activity, antifungal activity in vitro, quasi in vivo activity, pharmacokinetic (PK) properties, and in vivo antifungal
activity
Compd
Enzyme inhibition IC50 (mM)
Antifungal activity IC50 (mM)
PK parameters in ratse
.
In vivo antifungal activityf
ED50 (mg/kg)
CaNmta
HsNmtb
Serum (ꢀ)
c
Serum (+)d
AUC (ng h/mL)
t
1/2 (h)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0h (RO-09-4609)
5a
5b
5c
5d
5e
5f (RO-09-4746)
5g (RO-09-4879)
5h
5i
5j
5k
6
0.10
>450
77
>470
>110
67
1.6
0.37
0.040
0.11
0.021
0.080
0.030
0.035
0.074
0.16
NTg
NT
0.60
3.3
0.70
0.57
0.34
0.33
1.3
1.4
0.65
5.5
0.86
5.2
330
670
560
370
340
500
420
330
360
340
320
400
260
420
0.42
1.9
1.6
1.5
1.9
1.9
1.6
2.0
3.6
1.2
1.4
1.6
1.7
0.37
NT
NT
NT
NT
NT
NT
7.1
7.1
>30
20
0.072
0.0083
0.11
0.0052
0.0037
0.0075
0.0057
0.028
0.0094
0.0039
0.19
62
>450
>430
>400
>460
>280
430
0.12
0.21
0.12
0.20
22
>15
16
0.0071
0.057
>420
120
7
>30
aInhibitory activity against C. albicans Nmt (CaNmt) as assessed by IC50 using substrate peptide GLTISKLFRR-NH
(0.5 mM) and myristoyl-CoA
2
at 0.5 mM.
Inhibitory activity against human Nmt (HsNmt) as assessed by IC50 using substrate peptide GNAASARR-NH
b
2
(0.5 mM) and myristoyl-CoA at 0.5
HPO , pH 7).
mM.
Antifungal activity against C. albicans CY1002 as assessed by IC50 in YNBPB medium (1% glucose, 0.25% K
c
2
4
d
3.
2
Antifungal activity against C. albicans CY1002 as assessed by IC50 in 80% calf serum (80% calf serum supplemented with 10 mM FeCl 6H O, 10
mM deferoxamine, 2% glucose).
Cassette dosing: Five compounds were intravenously administered to a rat and the plasma concentration of each compound was measured by LC-
e
MS.
f
Fisher rats (n=5) were infected intravenously with a lethal dose of C. albicans (CY1002) and treated intravenously (iv) with multiple doses (2 tid) of
the test compound. Efficacy of the compounds was calculated as the effective dose (mg/kg) for 50% survival (ED50) on day 7.
g
NT, Not tested.
parapsilosis. It was also active against mucorales
including Absidia corymbifera and dermatophytes such
as Trichophyton mentagrophytes. It was, however, only
marginally active against Cryptococcus neoformans and
Aspergillus fumigatus.
Antifungal activity of 15g against C. albicans mutants in
efflux transporter. Compound 15g, fluconazole, and
itraconazole were tested for their antifungal activity
against C. albicans mutants with deletions in several
multidrug efflux transporter genes including CDR1,
CDR2, CaMDR1, and FLU1 (Table 5). The assays of
the two azoles resulted in greatly enhanced activity
against all deleted mutants. Especially, of all the trans-
porter mutants tested, the quadruple deletion mutants
(
Á cdr1 Á cdr2 Á flu1 Á camdr1) were most susceptible
to both azoles. In contrast, the antifungal activity of 15g
was not affected by the deletion of the transporters. This
clearly indicates that 15g is not a substrate either for
ABC (ATP binding cassette, CDR1 and CDR2) trans-
porters or for MFS (major facilitators superfamily,
CaMDR1 and FLU1) transporters, suggesting that 15g
would show antifungal activity against azole resistant C.
albicans strains expressing multidrug efflux transporters.
Figure 3. Antifungal activity of 15g in a rat systemic candidiasis
model. Animals: normal male Fischer rats, mean B.W. 56.9 g on day
4
0. Infection: C. albicans CY1002, 2.5Â10 cells/g, 0.5 mL iv on day 0.
Treatment: 15g, 5% DMSO, 5% PEG and 10% HPCD, 0.5 mL, iv 2
tid.
dilution method according to M27-A guidelines recom-
mended by the National Committee for Clinical
Laboratory Standards (NCCLS). Compound 15g dis-
played a broad spectrum and potent inhibitory activity
against a variety of fungal species. This compound
exhibited stronger antifungal activity in vitro against all
the strains tested than did fluconazole. It exhibited
excellent antifungal activity against a wide range of
Candida species including clinically isolated C. albicans
and non-albicans Candida such as C. tropicalis and C.
Summary and Conclusion
The modification work was carried out by the combi-
nation of rational drug design based on the crystal
structures of CaNmt bound with benzofuran inhibitors,
and SAR analysis guided by various biological assays
including a quasi in vivo assay and cassette dosing PK
studies in rats. This ‘multidimensional optimization’

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4471
Figure 4. Efficacy of 15g in a candidiasis target organ assay. Animals: Normal male Fischer rats, mean B.W. 62.5 g on day 0. Infection: C. albicans
3
CY1002, 3Â10 cells/g, 0.5 mL iv on day 0. Treatment: 15g: 5% DMSO, 5% PEG, 10% HPCD, 0.5 mL, iv tid (30 mg/kg) or 2 tid (5 and 15 mg/kg).
Fluconazole (FCZ): saline, 0.5 mg/kg, 0.5 mL, ip 2 tid. Control: 5% DMSO, 5% PEG, 10% HPCD and distilled water, 0.5 mL, iv 2 tid. *p<0.05,
**<0.01
Figure 5. Time–kill curve of 15g for C. albicans CY3003 in 80% calf serum. Medium: 80% calf serum (80% calf serum supplemented with 10 mM
.
ꢁ
5
FeCl 6H
fluconazole against C. albicans CY3003 were 0.5 and 1.6 mM, respectively, under the following conditions: medium: 80% calf serum (80% calf serum
3
2
O, 10 mM deferoxamine, 2% glucose). Strain: C. albicans CY3003. Incubation: 35 C. Inoculum size: 1Â10 (CFU/mL). IC50s of 15g and
.
4
supplemented with 10 mM FeCl
3
6H
2
O, 10 mM deferoxamine, 2% glucose). Inoculum size: 1Â10 (CFU/mL).
enabled us to rapidly identify compounds 15f and 15g
that were active in vivo, starting from the enzyme inhi-
bitor 1 with only marginal antifungal activity even in
vitro. The candidiasis target organ assay suggested that
the survival of the rats tested was prolonged by inhibit-
ing fungal growth. Further biological investigations on
efflux transporters that cause azole resistance. In addi-
tion, since the oral bio-availability (F value) of 15g in
dogs was determined to be 33% and its stability in
human liver microsome was better than in dog, oral
administrations of this agent might be clinically useful.
The novel mode of action, fungicidal activity, and
potent antifungal activity in vivo make the benzofurans
attractive for the development of antifungal drugs for
systemic mycoses.
1
5g revealed that (1) it had stronger and broader anti-
fungal activity in vitro than did fluconazole, (2) it had
fungicidal activity, and (3) it could not be a substrate of

4472
M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
Table 4. Antifungal spectrum of 15g
Strains
15g (mM)
Fluconazole (mM)
IC50
IC80
MIC
0.11
IC50
0.82
11
2
IC80
MIC
Candida albicans
Candida glabrata
Clinical Isolate
IFO0005
0.026
2.1
0.081
0.033
0.0054
4.6
7.2
7.2
0.64
0.044
7.5
0.13
0.046
0.01
5.9
12
19
2.9
22
2.1
1.2
22
2.4
>650
82
10
>650
20
41
>650
>650
>650
>650
330
14
Candida guilliermondii
Candida tropicalis
Candida parapsilosis
Cryptococcus neoformans
Aspergillus fumigatus
Aspergillus niger
ATCC9058
ATCC13803
ATCC22019
MTU13001
MTU06002
IFO6341
0.44
0.11
0.055
6.9
>650
>650
2.30
9.5
1.6
4.6
313
>650
>650
>650
30
55
555
55
14
55
14
>650
>650
>650
144
Absidia corymbifera
Fusarium solani
IFO8084
IFO8509
13
1.4
Trichophyton tonsurans
Trichophyton mentagrophytes
Trichophyton rubrum
IFO5928
IFO5974
IFO5807
0.44
0.64
0.7
0.83
1.7
3.4
0.98
2.22
1.2
3.6
5.1
10
Medium: YNBPB (=YNB+1% glucose+0.25%
2 4
K HPO ) (pH 7.0) for yeasts. Medium: YNBPA (=YNB+1% glucose+0.25%
ꢁ
4
K
2
HPO
4
+LMPA) (pH 7.0) for filamentous fungi. Incubation: 35 C. Inoculum size: 1Â10 (CFU/mL).
Table 5. Effect of transporter disruption on antifungal activity of 15g, fluconazole, and itraconazole
Strains
IC50 (mM)
Fluconazole
1
5g
Itraconazole
C. albicans CAF2-1 parent (wild type)
C. albicans DSY448 Á cdr1
C. albicans DSY465 Á camdr1
C. albicans DSY468 Á cdr1 Á camdr1
C. albicans DSY653 Á cdr2
C. albicans DSY654 Á cdr1 Á cdr2
C. albicans DSY1024 Á cdr1 Á cdr2 D flu1 Á camdr1
0.042
0.042
0.059
0.039
0.046
0.039
0.021
>650
1.9
>650
0.78
>280
0.0068
0.78
0.0032
0.028
0.0025
0.0017
7.8
2
0.24
ꢁ
4
Medium: YPD (1% Bacto Yeast extract+2% glucose+2% Bacto Peptone). Incubation: 35 C. Inoculum size: 1Â10 (CFU/mL).
Experimental
methane=5:1) to afford 3a (104 mg, 83%) as a white
2
2
1
powder. [a]D (c 0.5, CHCl ) +17.9. H NMR (CDCl ) d:
3
3
General methods
1.44 (3H, t, J=7 Hz), 2.77 (3H, s), 2.83 (1H, dd, J=5, 2.5
1H NMR spectra were recorded on a Jeol JNM-EX270
spectrometer; chemical shifts are reported in parts per
million (ppm) relative to TMS. Mass spectra were
recorded on a Jeol DX-303 spectrometer. Optical rota-
tion measurements were made using a JASCO DIP-140
polarimeter. All solvents and reagents were purchased
Hz), 2.96 (1H, dd, J=5, 4.5 Hz), 3.41-3.46 (1H, m), 4.08
(1H, dd, J=11, 5.5 Hz), 4.37 (1H, dd, J=11, 3 Hz), 4.44
(2H, q, J=7 Hz), 6.62 (1H, d, J=8 Hz), 7.15 (1H, d, J=8
Hz), 7.31 (1H, t, J=8 Hz). HRMS (m/z): calcd for
C H O , 276.0998; found, 276.0996.
1
5
16
5
from commercial sources and used without further pur-
1
(R,S)-3-Methyl-4-oxiranylmethoxybenzofuran-2-carb-
oxylic acid ethyl ester (3). To a mixture of 4-hydroxy-3-
methylbenzofuran-2-carboxylic acid ethyl ester 2 (104
mg, 0.47 mmol) and cesium carbonate (240 mg, 0.74
mmol) in acetonitrile (5 mL) was added epichlorohydrin
(440 mg, 4.7 mmol) at room temperature. The mixture
ification. Wakogel
C-200 (Wako Pure Chemical
Industries, Ltd., Japan) was used for chromatographic
purification unless otherwise indicated. Thin layer
chromatography (TLC) was performed on precoated
silica gel 60F254 glass plates (Merck, Germany).
ꢁ
was heated to 60 C with stirring for 24 h. The reaction
(S)-3-Methyl-4-oxiranylmethoxybenzofuran-2-carboxylic
acid ethyl ester (3a). A mixture of 4-hydroxy-3-methyl-
mixture was diluted with ethyl acetate (20 mL) and
washed with water (20 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated to dry-
ness. The residue was purified by silica gel column
chromatography (hexane/dichloromethane=4:1) to
5
1
benzofuran-2-carboxylic acid ethyl ester 2 (100 mg,
.45 mmol) and 60% NaH in mineral oil (22 mg, 0.55
0
mmol) in DMF (3 mL) was stirred at room temperature
for 15 min. To the suspension was added (2S)-(+)-gly-
cidyl tosylate (99 mg, 0.43 mmol) at room temperature.
The mixture was stirred at room temperature for 23 h.
The reaction mixture was diluted with ethyl acetate (50
mL) and washed with brine (50 mL). The organic layer
was dried over anhydrous sodium sulfate and
evaporated to dryness. The residue was purified by silica
1
afford 3 (112 mg, 86%) as a white powder. H NMR
(CDCl ) d: 1.44 (3H, t, J=7 Hz), 2.77 (3H, s), 2.82 (1H,
3
dd, J=5, 2.5 Hz), 2.95 (1H, dd, J=5, 4.5 Hz), 3.40–3.46
(1H, m), 4.08 (1H, dd, J=11, 5.5 Hz), 4.37 (1H, dd,
J=11, 3 Hz), 4.44 (2H, q, J=7 Hz), 6.62 (1H, d, J=8
Hz), 7.15 (1H, d, J=8 Hz), 7.31 (1H, t, J=8 Hz). EI–
+
1
MS: m/z 276 (M ). The H NMR data were practically
5
2
gel
column
chromatography
(hexane/dichloro-
identical with the reported values.

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4473
(S)-4-(3-tert-Butylamino-2-hydroxypropoxy)-3-methyl-
gel
column
chromatography
(dichloromethane/
benzofuran-2-carboxylic acid ethyl ester (4a). A mixture
of 3a (98 mg, 0.36 mmol) and tert-butylamine (0.4 mL)
was stirred in ethanol (1 mL) at 60 C for 13 h. The
reaction mixture was diluted with ethyl acetate (10 mL)
and washed with a saturated sodium hydrogen carbon-
ate solution (5 mL) and water (5 mL). The organic
phase was dried over anhydrous sodium sulfate and
evaporated to dryness. The residue was purified by silica
methanol=50:1!20:1) to afford 10a (46 mg, 98%) as
1
colorless crystals. H NMR (CDCl ) d: 1.30 (6H, d,
3
ꢁ
J=6.5 Hz), 1.43 (3H, t, J=7 Hz), 2.26–2.36 (2H, m), 2.73
(3H, s), 3.04 (2H, t, J=7.5 Hz), 3.07–3.17 (1H, m), 4.16
(2H, t, J=6 Hz), 4.43 (2H, q, J=7 Hz), 6.59 (1H, d, J=8
Hz), 7.11 (1H, d, J=8 Hz), 7.29 (1H, t, J=8 Hz). HRMS
(m/z): calcd for C H NO , 319.1784; found, 319.1789.
1
8
25
4
gel
column
chromatography
(dichloromethane/
4-(3-tert-Butylaminopropoxy)-3-methylbenzofuran-2-car-
boxylic acid ethyl ester (10b). This compound was pre-
pared by the same method as 10a. Yield: 95%. Colorless
methanol=50:1!5:1) to afford 4a (94 mg, 76%) as a
2
2
1
colorless oil. [a] (c 0.9, CHCl ) ꢀ23.7. H NMR
D
3
1
(CDCl ) d: 1.15 (9H, s), 1.44 (3H, t, J=7 Hz), 2.76 (3H, s),
crystals. H NMR (CDCl ) d: 1.41 (9H, s), 1.43 (3H, t,
3
3
2
.75–2.80 (1H, m), 2.95 (1H, dd, J=12, 3.5 Hz), 4.00–4.17
J=7 Hz), 2.45–2.51 (2H, m), 2.73 (3H, s), 3.10 (2H, t,
J=7 Hz), 4.15 (2H, t, J=6 Hz), 4.43 (2H, q, J=7 Hz),
6.57 (1H, d, J=8 Hz), 7.11 (1H, d, J=8 Hz), 7.29 (1H,
t, J=8 Hz). HRMS (m/z): calcd for C H NO ,
(3H, m), 4.44 (2H, q, J=7 Hz), 6.64 (1H, d, J=8 Hz), 7.13
1H, d, J=8 Hz), 7.31 (1H, t, J=8 Hz). HRMS (m/z):
(
calcd for C H NO , 349.1889; found, 349.1896.
1
9
27
5
19 27
4
333.1940; found, 333.1935.
(R)-4-(3-tert-Butylamino-2-hydroxypropoxy)-3-methyl-
benzofuran-2-carboxylic acid ethyl ester (4b). This com-
pound was prepared by the same method as 4a. Yield:
3-Methyl-4-(3-piperidin-1-ylpropoxy)benzofuran-2-carb-
oxylic acid ethyl ester (10d). This compound was pre-
2
2
5
+
6% (two steps). Colorless oil. [a] (c 1.0, CHCl3)
pared by the same method as 10a. Yield: 89%. Pale
1
D
1
23.8. H NMR (CDCl ) d: 1.14 (9H, s), 1.43 (3H, t,
orange powder. H NMR (CDCl ) d: 1.43 (3H, t, J=7
3
3
J=7 Hz), 2.75 (3H, s), 2.75–2.80 (1H, m), 2.94 (1H, dd,
J=12, 4 Hz), 3.98–4.07 (1H, m), 4.08–4.17 (2H, m), 4.44
(2H, q, J=7 Hz), 6.64 (1H, d, J=8 Hz), 7.13 (1H, d,
J=8 Hz), 7.31 (1H, t, J=8 Hz). HRMS (m/z): calcd for
Hz), 1.43–1.50 (2H, m), 1.62 (4H, quintet, J=6 Hz),
2.03-2.13 (2H, m), 2.43–2.45 (4H, m), 2.56 (2H, t, J=7
Hz), 2.75 (3H, s), 4.12 (2H, t, J=6 Hz), 4.44 (2H, q,
J=7 Hz), 6.62 (1H, d, J=8 Hz), 7.11 (1H, d, J=8 Hz),
7.30 (1H, t, J=8 Hz). HRMS (m/z): calcd for
C H NO , 345.1940; found, 345.1933.
C H NO , 349.1889; found, 349.1884.
5
1
9
27
2
0
27
4
(R,S)-4-(3-tert-Butylamino-2-hydroxypropoxy)-3-methyl-
benzofuran-2-carboxylic acid ethyl ester (4). Compound
3-Methyl-4-(3-phenylaminopropoxy)benzofuran-2-carb-
oxylic acid ethyl ester (10e). This compound was pre-
4
9
1
2
was prepared from 3 by the same method as 4a. Yield:
1
5%. Colorless oil. H NMR (CDCl ) d: 1.15 (9H, s),
pared by the same method as 10a. Yield: 34%. Light
1
3
.43 (3H, t, J=7 Hz), 2.76 (3H, s), 2.75–2.81 (1H, m),
.96 (1H, dd, J=12, 4 Hz), 4.01–4.18 (3H, m), 4.44 (2H,
brown powder. H NMR (CDCl ) d: 1.44 (3H, t, J=7
3
Hz), 2.15–2.24 (2H, m), 2.79 (3H, s), 3.42 (2H, t, J=7
Hz), 4.21 (2H, t, J=6 Hz), 4.45 (2H, q, J=7 Hz), 6.61-
6.66 (3H, m), 6.71 (1H, t, J=8 Hz), 7.13 (1H, d, J=8
Hz), 7.15–7.21 (2H, m), 7.31 (1H, t, J=8 Hz). HRMS
q, J=7 Hz), 6.64 (1H, d, J=8 Hz), 7.13 (1H, d, J=8
Hz), 7.31 (1H, t, J=8 Hz). HRMS (m/z): calcd for
C H NO , 349.1889; found, 349.1886.
1
9
27
5
(
m/z): calcd for C H NO , 353.1627; found, 353.1612.
21 23 4
4
-(3-Bromopropoxy)-3-methylbenzofuran-2-carboxylic
acid ethyl ester (6). Compound 2 (500 mg, 2.27 mmol),
potassium carbonate (1.64 g, 11.9 mmol), and 1,3-
dibromopropane (1.15 mL, 11.9 mmol) were suspended
in DMF (10 mL). The mixture was stirred at room
temperature for 4 h. The reaction mixture was diluted
with ethyl acetate (100 mL) and washed with water (100
mL). The organic layer was dried over anhydrous
sodium sulfate and evaporated to dryness. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexane=50:1) to give a crystalline product,
4-(3-Benzylaminopropoxy)-3-methylbenzofuran-2-carb-
oxylic acid ethyl ester (10f). This compound was pre-
pared by the same method as 10a. Yield: 85%. Colorless
1
oil. H NMR (CDCl ) d: 1.44 (3H, t, J=7 Hz), 2.02–
3
2.11 (2H, m), 2.67 (3H, s), 2.88 (2H, t, J=7 Hz), 3.83
(2H, s), 4.17 (2H, t, J=6 Hz), 4.43 (2H, q, J=7 Hz),
6.62 (1H, d, J=8 Hz), 7.11 (1H, d, J=8 Hz), 7.27 (6H,
m). HRMS (m/z): calcd for C H NO , 367.1784;
2
2
25
4
found, 367.1791.
which was recrystallized from hexane to give 6 (620 mg,
1
3-Methyl-4-{3-[(pyridin-2-ylmethyl)amino]propoxy}ben-
zofuran-2-carboxylic acid ethyl ester (10g). This com-
8
0%) as colorless needles. H NMR (CDCl ) d: 1.43
3
(
(
3H, t, J=7 Hz), 2.37–2.46 (2H, m), 2.74 (3H, s), 3.65
2H, t, J=6.5 Hz), 4.23 (2H, t, J=6.5 Hz), 4.45 (2H, q,
pound was prepared by the same method as 10a. Yield:
1
89%. Colorless oil. H NMR (CDCl ) d: 1.44 (3H, t,
3
J=7 Hz), 6.65 (1H, d, J=8 Hz), 7.13 (1H, d, J=8 Hz),
.31 (1H, t, J=8 Hz). HRMS (m/z): calcd for
C H BrO , 340.0310; found, 340.0306.
J=7 Hz), 2.06–2.13 (2H, m), 2.68 (3H, s), 2.91 (2H, t,
J=7 Hz), 3.95 (2H, s), 4.18 (2H, t, J=6 Hz), 4.44 (2H,
q, J=7 Hz), 6.62 (1H, d, J=8 Hz), 7.11 (1H, d, J=8
Hz), 7.12–7.16 (1H, m), 7.30 (2H, t, J=8 Hz), 7.60 (1H,
dt, J=8, 2 Hz), 8.53 (1H, d, J=4 Hz). HRMS (m/z):
calcd for C H N O , 368.1736; found, 368.1730.
7
1
5
17
4
4-(3-Isopropylaminopropoxy)-3-methylbenzofuran-2-carb-
oxylic acid ethyl ester (10a). Compound 6 (50 mg, 0.15
2
1
24
2
4
mmol) was stirred with isopropylamine (0.1 mL) in eth-
anol (1 mL) at 60 C for 9 h. The reaction mixture was
ꢁ
evaporated to dryness. The residue was purified by silica
3-Methyl-4-{3-[(pyridin-3-ylmethyl)amino]propoxy}benzo-
furan-2-carboxylic acid ethyl ester (10h). This compound

4474
M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
was prepared by the same method as 10a. Yield: 77%.
1
practically the same preparation method for 6. Com-
pound 11 was obtained from 7 by the same prepara-
tion method as 10a. Yield: 43% (two steps). Colorless
Colorless oil. H NMR (CDCl ) d: 1.44 (3H, t, J=7
3
Hz), 2.02–2.12 (2H, m), 2.67 (3H, s), 2.89 (2H, t, J=7
Hz), 3.84 (2H, s), 4.17 (2H, t, J=6 Hz), 4.44 (2H, q,
J=7 Hz), 6.62 (1H, d, J=8 Hz), 7.12 (1H, d, J=8 Hz),
1
crystals. H NMR (CDCl ) d: 1.43 (3H, t, J=7 Hz),
3
1.46 (9H, s), 1.89–1.97 (2H, m), 2.23–2.34 (2H, m),
2.74 (3H, s), 3.02 (2H, br t, J=8 Hz), 4.02 (2H, t,
J=6 Hz), 4.43 (2H, q, J=7 Hz), 6.51 (1H, d, J=8
Hz), 7.09 (1H, d, J=8 Hz), 7.25 (1H, t, J=8 Hz).
HRMS (m/z): calcd for C H NO , 347.2097; found,
7
.21 (1H, dd, J=8, 5 Hz), 7.30 (1H, t, J=8 Hz), 7.66
(
(
1H, dd, J=8, 2 Hz), 8.49 (1H, dd, J=5, 2 Hz), 8.57
1H, d, J=2 Hz). HRMS (m/z): calcd for C H N O ,
2
1
24
2
4
3
68.1736; found, 368.1742.
2
0
29
4
347.2095.
3-Methyl-4-{3-[(pyridin-4-ylmethyl)amino]propoxy}benzo-
furan-2-carboxylic acid ethyl ester (10i). This compound
4-(5- tert-Butylaminopentyloxy)-3-methylbenzofuran-2-
carboxylic acid ethyl ester (12). Bromopentyloxy deri-
vative 8 was synthesized from 2 and 1,5-dibromo-
pentane by practically the same preparation method for
6. Compound 12 was obtained from 8 by the same pre-
was prepared by the same method as 10a. Yield: 45%.
1
Pale yellow oil. H NMR (CDCl ) d: 1.44 (3H, t, J=7
3
Hz), 2.03–2.13 (2H, m), 2.69 (3H, s), 2.89 (2H, t, J=7
Hz), 3.85 (2H, s), 4.19 (2H, t, J=6 Hz), 4.44 (2H, q,
J=7 Hz), 6.63 (1H, d, J=8 Hz), 7.12 (1H, d, J=8 Hz),
paration method as 10a. Yield: 74% (2 steps). White
1
7
J=5, 2 Hz). HRMS (m/z): calcd for C H N O ,
.24–7.27 (2H, m), 7.31 (1H, t, J=8 Hz), 8.52 (2H, dd,
powder. H NMR (CDCl ) d: 1.43 (3H, t, J=7 Hz),
3
1.50–1.65 (2H, m), 1.51 (9H, s), 1.83–1.93 (2H, m),
2.12–2.24 (2H, m), 2.71 (3H, s), 2.95 (2H, br t, J=8 Hz),
2
1
24
2
4
3
68.1736; found, 368.1742.
4.02 (2H, t, J=6 Hz), 4.42 (2H, q, J=7 Hz), 6.57 (1H,
3-Methyl-4-{3-[2-(pyridin-3-ylethyl)amino]propoxy}benzo-
furan-2-carboxylic acid ethyl ester (10j). This compound
d, J=8 Hz), 7.09 (1H, d, J=8 Hz), 7.28 (1H, t, J=8
Hz). HRMS (m/z): calcd for C H NO , 361.2253;
found, 361.2254.
2
1
31
4
was prepared by the same method as 10a. Yield: 75%.
1
Colorless oil. H NMR (CDCl ) d: 1.43 (3H, t, J=7 Hz),
3
2
.00–2.09 (2H, m), 2.73 (3H, s), 2.87 (6H, m), 4.14 (2H, t,
J=6 Hz), 4.44 (2H, q, J=7 Hz), 6.61 (1H, d, J=8 Hz),
.12 (1H, d, J=8 Hz), 7.18 (1H, dd, J=8, 5 Hz ), 7.30
1H, t, J=8 Hz), 7.52 (1H, dd, J=8, 2 Hz), 8.44 (1H, dd,
3-Methyl-4-[3-(pyridin-3-ylmethoxy)propoxy]benzofuran-
2-carboxylic acid ethyl ester (13). A suspension of 3-
pyridinemethanol (43 mL, 0.44 mmol) and 60% NaH in
mineral oil (19 mg, 0.44 mmol) in DMF (1 mL) was
stirred at room temperature for 15 min. To the mixture
was added 6 (100 mg, 0.29 mmol) and the reaction
mixture was stirred at room temperature for 2 weeks.
The mixture was diluted with ethyl acetate (30 mL) and
washed with water (30 mL). The organic layer was dried
over anhydrous sodium sulfate and evaporated to dry-
ness. The residue was purified by preparative TLC
7
(
J=5, 2 Hz), 8.48 (1H, d, J=2 Hz). HRMS (m/z): calcd
for C H N O , 382.1893; found, 382.1899.
2
2
26
2
4
4
-(3-Aminopropoxy)-3-methylbenzofuran-2-carboxylic
acid ethyl ester (10c). Compound 10f (80 mg, 0.22
mmol) was hydrogenated over 10% Pd/C (20 mg) in
ethanol (4 mL) at room temperature for 14 h. Pd/C was
filtered through a Celite pad. The filtrate was evapo-
rated to dryness and the residue was purified by silica
(ethyl acetate/hexane=1:1) to give 13 (11.6 mg, 11%) as
1
a colorless oil. H NMR (CDCl ) d: 1.44 (3H, t, J=7
3
gel
column
chromatography
(dichloromethane/
Hz), 2.14–2.22 (2H, m), 2.68 (3H, s), 3.73 (2H, t, J=6
Hz), 4.20 (2H, t, J=6 Hz), 4.44 (2H, q, J=7 Hz), 4.55
(2H, s), 6.63 (1H, d, J=8 Hz), 7.12 (1H, d, J=8 Hz),
7.20 (1H, dd, J=8, 5 Hz), 7.31 (1H, t, J=8 Hz), 7.64
(1H, d, J=8 Hz), 8.51 (1H, dd, J=5, 1.5 Hz), 8.57 (1H,
s). HRMS (m/z): calcd for C H NO , 369.1576; found,
methanol=50:1!5:1) to give 10c (27 mg, 45%) as a
1
colorless powder. H NMR (CDCl ) d: 1.44 (3H, t, J=7
3
Hz), 1.98–2.08 (2H, m), 2.75 (3H, s), 2.99 (2H, t, J=7
Hz), 4.17 (2H, t, J=6 Hz), 4.44 (2H, q, J=7 Hz), 6.63
(1H, d, J=8 Hz), 7.12 (1H, d, J=8 Hz), 7.31 (1H, t,
J=8 Hz). HRMS (m/z): calcd for C H NO ,
2
1
23
5
369.1578.
1
5
19
4
2
77.1314; found, 277.1316.
(3-Methyl-4-{3-[(pyridin-3-ylmethyl)amino]propoxy}ben-
zofuran-2-yl)methanol (14). To a solution of 10h (7.9 g,
4
-(2-tert-Butylaminoethoxy)-3-methylbenzofuran-2-carb-
oxylic acid ethyl ester (9). Bromoethoxy derivative 5
was synthesized from 2 and 1,2-dibromoethane by
practically the same preparation method for 6 except for
21 mmol) in dry tetrahydrofuran (THF) was added
lithium aluminum hydride (3.2 g, 85 mmol) at 0 C. The
ꢁ
mixture was stirred at 0 C for 30 min. To the reaction
ꢁ
ꢁ
the reaction temperature (100 C). Compound 9 was
obtained from 5 by the same preparation method as
mixture were added a little water and then anhydrous
sodium sulfate with stirring. Inorganic salt was removed
by filtration and the filtrate was evaporated to dryness.
The residue was purified by silica gel column chroma-
tography (dichloromethane/methanol=50:1!10:1) to
1
1
0a. Yield: 63% (two steps). Colorless oil. H NMR
(
CDCl ) d: 1.20 (9H, s), 1.44 (3H, t, J=7 Hz), 2.75 (3H,
3
s), 3.08 (2H, t, J=5 Hz), 4.23 (2H, t, J=5 Hz), 4.44
2H, q, J=7 Hz), 6.62 (1H, d, J=8 Hz), 7.11 (1H, d,
J=8 Hz), 7.29 (1H, t, J=8 Hz). HRMS (m/z): calcd for
1
(
give 14 (6.31 g, 91%) as a colorless solid. H NMR
(CDCl ) d: 1.97–2.07 (2H, m), 2.22 (3H, s), 2.83 (2H, t,
3
C H NO , 319.1784; found, 319.1788.
1
J=7 Hz), 3.77 (2H, s), 4.10 (2H, t, J=6 Hz), 4.66 (2H,
s), 6.55 (1H, d, J=8 Hz), 7.00 (1H, d, J=8 Hz), 7.12
(1H, t, J=8 Hz), 7.17 (1H, m), 7.63 (1H, br d, J=5 Hz),
8.42 (2H, m). HRMS (m/z): calcd for C H N O ,
8
25
4
4
-(4-tert-Butylaminobutoxy)-3-methylbenzofuran-2-carb-
oxylic acid ethyl ester (11). Bromobutoxy derivative 7
was synthesized from 2 and 1,4-dibromobutane by
1
9
22
2
3
326.1630; found, 326.1636.

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4475
[
3-(3-Methyl-2-phenoxymethylbenzofuran-4-yloxy)pro-
pyl]pyridin-3-ylmethylamine (15a). To a suspension of
4 (33 mg, 0.1 mmol), phenol (10 mg, 0.11 mmol), and
(1H, d, J=7 Hz), 6.80 (1H, dq, J=8, 1.5 Hz), 6.88 (1H, dt,
J=7, 1.5 Hz), 6.90–7.00 (1H, m), 7.04 (1H, d, J=8 Hz),
7.16 (1H, d, J=8 Hz), 7.19–7.22 (1H, m), 7.65 (1H, d, J=8
Hz), 8.47 (1H, d, J=4 Hz), 8.56 (1H, s). HRMS (m/z):
calcd for C H F N O , 438.1755; found, 438.1751.
1
triphenylphosphine polymer bound (3 mmol/g resin, 50
mg, Fulka, Switzerland) in THF (1 mL) was added di-
ethyl azodicarboxylate (40% toluene solution) at
2
5
24
2
2 3
ꢁ
ꢀ
45 C under argon, and the mixture was slowly
{3-[2-(2,4-Difluorophenoxymethyl)-3-methylbenzofuran-
4-yloxy]propyl}pyridin-3-ylmethylamine (15f). To
warmed to room temperature over a period of 18 h. The
mixture was filtered off, and the filtrate was poured onto
a saturated sodium hydrogen carbonate solution (10
mL). The mixture was extracted with ethyl acetate (20
mL). The organic layer was washed with brine (10 mL),
dried over anhydrous sodium sulfate, and the solvent
was evaporated under reduced pressure. The residue
was purified on preparative TLC (dichloromethane/
a
solution of the compound 14 (65 mg, 0.20 mmol), tri-
butylphosphine (74 mL, 0.30 mmol), and 2,4-difluoro-
phenol (26 mg, 0.20 mmol) in THF (1 mL) was added
0
1,1 -(azodicarbonyl)dipiperidine (ADDP) (76 mg, 0.30
ꢁ
mmol) in THF (0.5 mL) at ꢀ45 C under argon atmos-
phere, and the mixture was slowly warmed to room
temperature over a period of 18 h. The mixture was
poured onto a saturated sodium hydrogen carbonate
solution (20 mL) and extracted with ethyl acetate (20
mL). The organic layer was washed with brine, dried
over anhydrous sodium sulfate, and evaporated to dry-
ness. The residue was purified by silica gel column
chromatography (dichloromethane/methanol=20:1) to
methanol=10:1) to afford 15a (8 mg, 20%) as a yellow
1
oil. H NMR (CDCl ) d: 2.00–2.10 (2H, m), 2.34 (3H,
3
s,), 2.88 (2H, t, J=7 Hz), 3.84 (2H, s), 4.15 (2H, t, J=6
Hz), 5.08 (2H, s), 6.60 (1H, d, J=8 Hz), 6.96–7.07 (2H,
m), 7.15 (1H, d, J=8 Hz), 7.18–7.23 (1H, m), 7.28–7.34
(
2H, m), 7.43–7.55 (2H, m), 7.66 (1H, d, J=8 Hz), 8.49
1H, d, J=4 Hz), 8.57 (1H, s). HRMS (m/z): calcd for
1
(
C H N O , 402.1943; found, 402.1939.
afford 15f (35 mg, 40%) as a yellow oil. H NMR
(CDCl ) d: 2.00–2.09 (2H, m), 2.27 (3H, s), 2.89 (2H, t,
2
5
26
2
3
3
J=7 Hz), 3.83 (2H, s), 4.13 (2H, t, J=6 Hz), 5.10 (2H,
s), 6.59 (1H, d, J=8 Hz), 6.71–7.23 (6H, m), 7.66 (1H,
dd, J=8, 2 Hz), 8.49 (1H, d, J=5 Hz), 8.56 (1H, s).
HRMS (m/z): calcd for C H F N O , 438.1755;
{
3-[2-(2-Fluorophenoxymethyl)-3-methylbenzofuran-4-
yloxy]propyl}pyridin-3-ylmethylamine (15b). This com-
pound was prepared by the same method as 15a. Yield:
1
2
5
24
2
2
3
2
1%. Pale yellow oil. H NMR (CDCl ) d: 2.00–2.09
found, 438.1749.
3
(
4
2H, m), 2.30 (3H, s), 2.87 (2H, t, J=7 Hz), 3.83 (2H, s),
.13 (2H, t, J=6 Hz), 5.15 (2H, s), 6.59 (1H, d, J=8
{3 - [2 - (2,3,4 - Trifluorophenoxymethyl) - 3 - methylbenzo-
furan-4-yloxy]propyl}pyridin-3-ylmethylamine (15g). This
compound was synthesized from 14 by practically the
same preparation method as 15f. Toluene was used as a
Hz), 6.91–6.96 (1H, m), 7.03–7.10 (4H, m), 7.16 (1H, t,
J=8 Hz), 7.20 (1H, dd, J=8, 7 Hz), 7.66 (1H, d, J=7
Hz), 8.47 (1H, d, J=4 Hz), 8.56 (1H, s). HRMS (m/z):
calcd for C H FN O , 420.1849; found, 420.1855.
reaction solvent instead of THF. Yield: 80%. Yellow
1
2
5
25
2
3
oil. H NMR (CDCl ) d: 2.00–2.10 (2H, m), 2.29 (3H,
3
{3-[2-(3-Fluorophenoxymethyl)-3-methylbenzofuran-4-
yloxy]propyl}pyridin-3-ylmethylamine (15c). This com-
s), 2.88 (2H, t, J=7 Hz), 3.84 (2H, s), 4.14 (2H, t, J=6
Hz), 5.13 (2H, s), 6.60 (1H, d, J=8 Hz), 6.75–6.91 (2H,
m), 7.04 (1H, d, J=8 Hz), 7.15–7.27 (2H, m,), 7.68 (1H,
d, J=8 Hz), 8.49 (1H, d, J=4 Hz), 8.57 (1H, s). HRMS
(m/z): calcd for C H F N O , 456.1661; found,
pound was prepared by the same method as 15a. Yield:
1
1
8%. Pale yellow oil. H NMR (CDCl ) d: 2.00–2.10
3
(
4
2H, m), 2.35 (3H, s), 2.88 (2H, t, J=7 Hz), 3.83 (2H, s),
.15 (2H, t, J=6 Hz), 5.06 (2H, s), 6.60 (1H, d, J=8
Hz), 6.67-6.81 (3H, m), 7.05 (1H, d, J=8 Hz), 7.15–7.24
3H, m), 7.66 (1H, d, J=7 Hz), 8.49 (1H, d, J=4 Hz,),
2
5
23
3
2
3
456.1653.
(
{3-[2-(4-Bromo-2-fluorophenoxymethyl)-3-methylbenzo-
furan-4-yloxy]propyl}pyridin-3-ylmethylamine (15h). To
8
4
.56 (1H, s). HRMS (m/z): calcd for C H FN O ,
2
20.1849; found, 420.1844.
5
25
2
3
0
dimethylformamide) (41 mg, 0.24 mmol), and 4-bromo-
a solution of 14 (37 mg, 0.12 mmol), 1,1 -azobis(N,N-
{3-[2-(4-Fluorophenoxymethyl)-3-methylbenzofuran-4-
yloxy]propyl}pyridin-3-ylmethylamine (15d). This com-
2-fluorophenol (20 mL, 0.18 mmol) in THF (2 mL) was
ꢁ
added tributylphosphine (89 mL, 0.36 mmol) at ꢀ30 C
pound was prepared by the same method as 15a. Yield:
1
under argon atmosphere, and the mixture was slowly
warmed to room temperature over a period of 15 h. The
precipitates were filtered off, and the filtrate was poured
onto water (30 mL) and extracted with ethyl acetate (30
mL). The organic layer was washed with brine (30 mL),
dried over anhydrous sodium sulfate, and evaporated to
dryness. The residue was purified by silica gel column
chromatography (dichloromethane/methanol=20:1) to
2
5%. Yellow oil. H NMR (CDCl ) d: 2.01–2.11 (2H,
3
m), 2.31 (3H, s), 2.88 (2H, t, J=6 Hz), 3.85 (2H, s), 4.14
2H, t, J=7 Hz), 5.03 (2H, s), 6.58 (1H, d, J=8 Hz),
.95–7.00 (4H, m), 7.04 (1H, d, J=9 Hz), 7.13 (1H, d,
J=8 Hz), 7.19–7.22 (1H, m), 7.68 (1H, d, J=8 Hz), 8.48
1H, d, J=4 Hz), 8.57 (1H, s). HRMS (m/z): calcd for
C H FN O , 420.1849; found, 420.1841.
(
6
(
2
5
25
2
3
1
afford 15h (24 mg, 40%) as a colorless oil. H NMR
{
4
3-[2-(2,3-Difluorophenoxymethyl)-3-methylbenzofuran-
-yloxy]propyl}pyridin-3-ylmethylamine (15e). This
compound was prepared by the same method as 15a.
(CDCl ) d: 2.00–2.10 (2H, m), 2.30 (3H, s), 2.87 (2H, t,
3
J=7 Hz), 3.84 (2H, s), 4.14 (2H, t, J=6 Hz), 5.13 (2H,
s), 6.59 (1H, d, J=8 Hz), 6.95–7.09 (2H, m), 7.12–7.27
(4H, m), 7.66 (1H, dt, J=8, 2 Hz), 8.49 (1H, dd, J=5, 2
Hz), 8.56 (1H, d, J=2 Hz). HRMS (m/z): calcd for
C H BrFN O , 498.0954; found, 498.0958.
1
Yield: 35%. Pale yellow solid. H NMR (CDCl ) d:
2
3
3
.00–2.09 (2H, m), 2.32 (3H, s), 2.87 (2H, t, J=7 Hz),
.83 (2H, s), 4.14 (2H, t, J=6 Hz), 5.16 (2H, s), 6.59
2
5
24
2
3

4476
M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4-(3-Methyl-4-{3-[(pyridin-3-ylmethyl)amino]propoxy}-
benzofuran-2-ylmethoxy)benzonitrile (15i). This com-
pound was prepared from 14 by the same method as
Determination of enzyme inhibitory activity
Inhibitory activities against CaNmt and HsNmt were
assessed by IC . Assays (100 mL) for CaNmt contained
1
1
2
3
5h. Yield: 46%. White powder. H NMR (CDCl ) d:
.00–2.10 (2H, m), 2.35 (3H, s), 2.87 (2H, t, J=7 Hz),
.84 (2H, s), 4.15 (2H, t, J=6 Hz), 5.13 (2H, s), 6.61
3
50
3
variable amounts of an inhibitor, 0.5 mM of [ H]myr-
istoyl-CoA (250 mCi, 62.0 Ci/mmol), 0.5 mM of sub-
(
1H, d, J=8 Hz), 7.05 (1H, d, J=8 Hz), 7.07 (2H, d,
J=9 Hz), 7.19 (1H, t, J=8 Hz), 7.20–7.22 (1H, m),
.61 (2H, d, J=9 Hz), 7.67 (1H, dd, J=8, 2 Hz), 8.49
strate peptide GLTISKLFRR-NH , and 25 ng of
2
purified C. albicans Nmt in a reaction buffer [30 mM
Tris–HCl (Nacalai Tesque, Japan), pH 7.5, 0.45 mM
EDTA (Dojindo, Japan), 0.5 mM EGTA (Dojindo,
Japan), 4.5 mM 2-mercaptoethanol (Sigma, USA),
1.0% Triton-X-100 (Sigma, USA)]. Assays (100 mL) for
HsNmt contained variable amounts of an inhibitor, 0.5
7
(
(
1H, dd, J=5, 2 Hz), 8.56 (1H, d, J=2 Hz). HRMS
m/z): calcd for C H N O , 427.1896; found,
2
6
25
3
3
4
27.1894.
3
{
3-[3-Methyl-2-(3-methyl-2-nitrophenoxymethyl)benzo-
mM of [ H]myristoyl-CoA (250 mCi, 62.0 Ci/mmol), 2.5
furan-4-yloxy]propyl}pyridin-3-ylmethylamine (15j). This
compound was prepared from 14 by the same method
mM of substrate peptide peptide GNAASARR-NH2,
and 1 mg of purified human Nmt in a reaction buffer [30
mM Tris–HCl (Nacalai Tesque, Japan), pH 7.5, 0.45
mM EDTA, 0.5 mM EGTA, 4.5 mM 2-mercaptoethanol
(Sigma, USA), 1.0% Triton-X-100 (Sigma, USA)].
1
as 15h. Yield: 68%. Pale yellow powder. H NMR
(
CDCl ) d: 2.00–2.09 (2H, m), 2.29 (3H, s), 2.31 (3H, s),
3
2
.87 (2H, t, J=7 Hz), 3.84 (2H, s), 4.14 (2H, t, J=6
ꢁ
Hz), 5.17 (2H, s), 6.59 (1H, d, J=8 Hz), 6.87 (1H, d,
J=8 Hz), 7.03 (1H, d, J=8 Hz), 7.05 (1H, d, J=8 Hz),
Incubations were allowed to proceed for 60 min at 37 C.
7
.16 (1H, d, J=8 Hz), 7.19–7.24 (1H, m), 7.30 (1H, d,
J=8 Hz), 7.67 (1H, dt, J=8, 2 Hz), 8.48 (1H, dd, J=5,
Hz), 8.56 (1H, d, J=2 Hz). HRMS (m/z): calcd for
C H N O , 461.1951; found, 461.1943.
Determination of antifungal activity in vitro
2
Antifungal activity in vitro of the benzofurans was
determined using the C. albicans cells (strain CY1002)
according to the broth microdilution procedure in the
M27-A guidelines recommended by the National Com-
mittee for Clinical Laboratory Standards (NCCLS).
Ten to the fourth power of cells in 100 mL of YNBPB
medium [YNB (Difco, USA), 1% (w/v) glucose (Wako,
Japan), 0.25% K HPO (Wako, Japan), pH 7.0] con-
2
6
27
3
5
{3-[2-(4-Imidazol-1-ylphenoxymethyl)-3-methylbenzo-
furan-4-yloxy]propyl}pyridin-3-ylmethylamine (15k). This
compound was prepared by the same method as 15h.
1
Yield: 67%. Colorless oil. H NMR (CDCl ) d: 2.03–
3
2
(
.09 (2H, m), 2.35 (3H, s), 2.88 (2H, t, J=6.5 Hz), 3.84
2H, s), 4.15 (2H, t, J=6 Hz), 5.12 (2H, s), 6.61 (1H, d,
2
4
taining various concentrations of compounds were dis-
ꢁ
J=8 Hz), 7.06 (1H, d, J=8 Hz), 7.10 (2H, dd, J=6.5, 2
Hz), 7.16–7.22 (4H, m), 7.31 (2H, dd, J=6.5, 2 Hz),
7.67 (1H, d, J=7.5 Hz), 7.77 (1H, s), 8.49 (1H, dd,
J=4.5, 2 Hz), 8.56 (1H, d, J=2 Hz). HRMS (m/z):
pensed in 96-well plates and incubated at 35 C for 24 h.
4
For quasi in vivo assay, the C. albicans CY1002 (1Â10
CFU/mL) was incubated in a 100 mL of 80% calf serum
(Gibco, USA) containing 2% glucose, 10 mM
.
calcd for C H N O , 468.2161; found, 468.2166.
8
FeCl 6H O (Wako, Japan), and 10 mM deferoxamine
2
28
4
3
3
2
(Sigma, USA) with various concentrations of test com-
pound using a 96-well plate. The plates were incubated
at 35 C for 24 h. The YNBPB medium was used for
{
3
3-[3-Methyl-2-(1-methyl-5-trifluoromethyl-1H-pyrazol-
-yloxymethyl)benzofuran-4-yloxy]propyl}pyridin-3-yl-
methylamine (16). This compound was prepared by the
same method as 15h. Yield: 86%. Colorless oil. 1
NMR (CDCl ) d: 2.01–2.11 (2H, m), 2.36 (3H, s), 2.88
ꢁ
yeasts, while YNBPA medium [YNB (Difco, USA), 1%
(w/v) glucose, 0.25% K HPO , 0.2% LMPA (Low
H
2
4
Melting Point Agar, Gibco, USA), pH 7.0] was used for
filamentous fungi. The turbidity of the cell suspension was
measured using a microplate reader (WL320, Bio-Tek
Instrument, USA) at 600 nm. Antifungal activity of each
compound was indicated as 50% inhibition concentration
(IC ) values that were determined by calculation of the
3
(
2H, t, J=7 Hz), 3.83 (2H, s), 3.84 (3H, s), 4.15 (2H,
t, J=6 Hz), 5.22 (2H, s), 6.03 (1H, s), 6.59 (1H, d,
J=8 Hz), 7.05 (1H, dd, J=8, 1 Hz), 7.17 (1H, t, J=8
Hz), 7.19–7.23 (1H, m), 7.67 (1H, dt, J=8, 2 Hz),
8.49 (1H, dd, J=5, 2 Hz), 8.57 (1H, d, J=2 Hz).
HRMS (m/z): calcd for C H F N O , 474.1879;
found, 474.1884.
5
0
minimum concentration of the compound required for
50% reduction of the turbidity (OD600) of cells compared
to untreated control cells. Minimum inhibitory concentra-
tions (MICs), which were defined as the lowest concentra-
tions of compound that completely inhibited visible
growth (absence of detectable turbidity), were also deter-
mined by the microdilution format described above.
2
4
25
3
4
3
{3-[3-Methyl-2-(pyridin-3-yloxymethyl)benzofuran-4-
yloxy]propyl}pyridin-3-ylmethylamine (17). This com-
pound was prepared by the same method as 15h. Yield:
1
4
4%. Pale yellow oil. H NMR (CDCl ) d: 2.01–2.10
3
(
4
2H, m), 2.35 (3H, s), 2.88 (2H, t, J=7 Hz), 3.84 (2H, s),
.15 (2H, t, J=6 Hz), 5.13 (2H, s), 6.61 (1H, d, J=8
Hz), 7.05 (1H, d, J=8 Hz), 7.18 (1H, t, J=8 Hz), 7.25
2H, m), 7.33 (1H, ddd, J=8.5, 3, 1.5 Hz), 7.67 (1H, dd,
Antifungal activity against disrupted mutants of C. albicans
(
C. albicans DSY448(Ácdr1: :hisG-URA3-hisG/Ácdr1: :hisG),
DSY465(Ácamdr1::hisG-URA3-hisG/Ácamdr1::hisG),
DSY468 (Ácdr1::hisG/Ácdr1::hisG Ácamdr1::hisG-
URA3-hisG/Ácamdr1::hisG), DSY653 (Ácdr2::hisG-
URA3-hisG/Ácdr2::hisG), DSY654 (Ácdr1::hisG/Á-
J=8, 2 Hz), 8.25 (1H, dd, J=5, 1.5 Hz), 8.42 (1H, d,
J=3 Hz), 8.49 (1H, dd, J=5, 2 Hz), 8.56 (1H, d, J=2
Hz). HRMS (m/z): calcd for C H N O , 403.1896;
2
4
25
3
3
found, 403.1891.

M. Masubuchi et al. / Bioorg. Med. Chem. 11 (2003) 4463–4478
4477
cdr1::hisG Ácdr2::hisG-URA3-hisG/Ácdr2::hisG), and
DSY1024 (Ácdr1::hisG/Ácdr1::hisG Ácdr2::hisG/
Ácdr2::hisG Ácamdr1::hisG/Ácamdr1::hisG Áflu1::hisG/
after infection (target organ brain assay or target organ
kidney assay). The organs were put in sterile tubes con-
taining 5 mL of sterile saline and then homogenized.
The homogenates were serially diluted in saline, and
aliquots were plated onto 1ÂGYPA agar [1% Peptone
(Difco, USA), 0.5% Yeast extract, 1% glucose (Wako,
Japan), 2% Agar (Difco, USA)] plates. The plates were
incubated at room temperature for 48 h, and the colo-
nies were counted. The mean number of CFU per
organs of the treated groups was compared with those
of the organs from sham-treated control animals.
Áflu1::hisG-URA3-hisG) were constructed by targeted
gene deletions in the parent strain CAF2-1.5
3ꢀ57
In 100
mL of YPD medium [1% (w/v) BactoYeast extract
Difco, USA), 2% (w/v) glucose, 2% (w/v) Bacto Pep-
(
4
tone (Difco, USA)], the C. albicans strains (1Â10 CFU/
mL) were incubated with various concentrations of test
compound using a 96-well plate. The plates were incu-
bated at 35 C for 24 h and absorbance of wells mea-
sured with 600 nm.
ꢁ
Cassette dosing pharmacokinetics
Time–kill curve procedure
Male F344 rats (n=6), weighing 55–75 g were used in
each cassette dosing PK experiment. Five compounds
were dissolved individually in DMSO at a concentration
of 20 mg/mL. The final concentration of each com-
pound of the cocktail solution was 0.2 mg/mL in 5%
DMSO, 5% PEG300 (Wako, Japan), and 10% HPCD
(Nihon Shokuhin Kako, Japan). A 10 mL/kg cocktail
solution was administered to rats intravenously at 2 mg/
kg for each compound. Blood samples were obtained
serially at selected time points after drug administration.
After centrifugation, the resultant plasma was stored
The fungistatic or fungicidal effect of the compound was
evaluated by time–kill curve studies. In a 100 mL of 80%
calf serum containing 2% glucose, 10 mM FeCl 6H O,
.
3
2
and 10 mM deferoxamine, the C. albicans CY3003
5
(
centrations of a test compound using a 96-well plate.
1Â10 CFU/mL) was incubated with various con-
ꢁ
The plates were incubated at 35 C under 10% CO
2
condition. At predetermined times, 30 mL samples were
obtained from each well, serially diluted 10-fold, and
then 100 mL of sample was plated onto YPG agar [2%
Peptone (Becton Dickinson, USA), 1% Yeast extract
ꢁ
frozen at ꢀ20 C until analysis. Concentrations of ben-
(
Becton Dickinson, USA), 2% glucose] for colony
counting. Colony counts were performed following
zofurans in plasma were determined by the LC–MS/MS
method following a protein precipitation. An aliquot of
the extract was analyzed by LC–MS/MS in the positive
ion mode using an API-300 triple quadrupole mass
spectrometer (PE SCIEX, Concord, ON, Canada). The
chromatographic column was a Develosil C8 column
(4.6Â50 mm, 5-mm particle size; Nomura Chemical Co.
Ltd., Japan). The samples were centrifuged (3000
rpmÂ10 min), evaporated, redissolved in 65% aqueous
acetonitrile and the supernatant was injected to the
HPLC system. The HPLC mobile phase was 0.1% for-
mic acid in methanol to 0.1% formic acid in water, 60:40
to 75:25 (v/v), or methanol to 0.1% formic acid and 5
mM ammonium formate in water (approximately pH 3),
60:40 to 75:25 (v/v). The flow rate was set at 1.0 mL/min.
ꢁ
incubation at 35 C for 24 h.
Antifungal tests in vivo (rat systemic candidiasis model)
An in vivo rat candidiasis model was used to monitor
the antifungal activity of the benzofuran derivatives.
Groups of 5 male Fischer rats (mean body weight 56.9
g) were inoculated with 0.5 mL of a suspension con-
4
taining 2.5Â10 cells/g of C. albicans CY1002 by iv on
day 0. The benzofuran derivatives were administered in
.5 mL of solution of 5% DMSO (Wako, Japan), 5%
0
PEG (Wako, Japan), and 10% HPCD (Nihon Shoku-
hin Kako, Japan) in distilled water by iv after infection
(
from day 0), three times daily for 2 days (2 tid). Flu-
conazole was administered in 0.5 mL of saline iv 2 tid.
The control group received only the vehicle. The effi-
cacies of the antifungal agents were assessed as the 50%
effective dose (ED ), calculated by probit analysis at
Acknowledgements
The authors thank Professor Dominique Sanglard (Insti-
tute of Microbiology, University Hospital Lausanne) for
providing the disrupted mutants of C. albicans.
5
0
day 7 after infection.
Target organ study
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