Optically active antifungal azoles. XII. Synthesis and antifungal activity of the water-soluble prodrugs of 1-[(1R,2R)-2-(2,4difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1- yl)propyl]3-[4-(1H-1-tetrazolyl) phenyl]-2-imidazolidinone

Article abstract of DOI:10.1248/cpb.49.1102

1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1- yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone (1: TAK-456) was selected as a candidate for clinical trials, but since its water-solubility was insufficient for an inj

Full text of DOI:10.1248/cpb.49.1102

1102
Chem. Pharm. Bull. 49(9) 1102—1109 (2001)
Vol. 49, No. 9
Optically Active Antifungal Azoles. XII.1) Synthesis and Antifungal
Activity of the Water-Soluble Prodrugs of 1-[(1R,2R)-2-(2,4-
Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-
3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone
Takashi ICHIKAWA,*,a Tomoyuki KITAZAKI,b Yoshihiro MATSUSHITA,a Masami YAMADA,b
a
Ryogo HAYASHI,c Masashi YAMAGUCHI,d Yutaka KIYOTA,d Kenji OKONOGI,e and Katsumi ITOH
Medicinal Chemistry Research Laboratories I,a Medicinal Chemistry Research Laboratories II,b Pharmacology Research
Laboratories II,c Drug Analysis & Pharmacokinetics Research Laboratories,d and Research Management Department,e
Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., 17–85, Jusohonmachi 2-chome, Yodogawa-ku,
Osaka 532–8686, Japan. Received March 16, 2001; accepted June 5, 2001
1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-3-[4-(1H-1-tetra-
zolyl)phenyl]-2-imidazolidinone (1: TAK-456) was selected as a candidate for clinical trials, but since its water-
solubility was insufficient for an injectable formulation, the quaternary triazolium salts 2 were designed as water-
soluble prodrugs. Among the prodrugs prepared, 4-acetoxymethyl-1-[(2R,3R)-2-(2,4-difluorophenyl)-2-hydroxy-
3-[2-oxo-3-[4-(1H-1-terazolyl)phenyl]-1-imidazolidinyl]butyl]-1H-1,2,4-triazolium chloride (2a: TAK-457) was se-
lected as an injectable candidate for clinical trials based on the results of evaluations on solubility, stability, he-
molytic effect and in vivo antifungal activities.
Key words water-soluble prodrug; triazolium salt; antifungal azole; TAK-457; TAK-456; 1,2,3-trisubstituted-2-butanol
In the course of our study on optically active azolone- hydrolyzed enzymatically and/or non-enzymatically in vivo
based antifungal azoles, we selected 1-[(1R,2R)-2-(2,4-difluo- to generate the parent compound TAK-456. It was also ex-
rophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]- pected that the water-solubility and the liability to hydrolysis
3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone (1: TAK- would depend on the physicochemical properties of the sub-
456) as a candidate for clinical trials (Chart 1). In previous stituent R. To search for a prodrug suitable for injectable for-
reports, we described the stereocontrolled synthesis of TAK- mulation, we planned to prepare a series of compounds 2
4561) and the antifungal activities in vitro and in vivo.2) TAK- with different physicochemical properties. We chose alkyl,
456 exhibited strong growth inhibitory activity against not alkoxy and substituted alkoxy groups (see Chart 2) for the
only yeasts such as Candida albicans (C. albicans) and substituent R and prepared the triazolium salts 2 (Table 1). In
Cryptococcus neoformans but also against molds such as As- this paper, we describe the synthesis of 2 as well as the re-
pergillus fumigatus (A. fumigatus), as well as highly potent sults of evaluations on their water-solubility, stability, he-
protective effects against candidiasis and aspergillosis in molytic effect and antifungal activities.
mice via oral administration.3)
Synthesis The water-soluble prodrugs (2a—g, i: Table
Invasive aspergillosis still remains resistant to antifungal 1) were prepared by alkylation of the parent compound 1
chemotherapy, although injectable amphotericin B has been with halomethyl esters or halomethyl carbonates (4—6) in an
used for the treatment of this disease in spite of the severe aprotic polar solvent such as acetonitrile (MeCN) or acetone
toxicity.4) It is, therefore, considered that an injectable formu- within a range of temperatures between 50 and 100 °C as
lation of TAK-456 would have potential in the clinical treat- shown in Chart 3. In the case of reaction of 1 with 5 or 6, the
ment of this serious disease. We attempted to prepare an in- resulting triazolium bromides or iodides were converted to
jectable solution of TAK-456 with sufficient concentration the corresponding chlorides (2a, d, e, g, i) by anion-ex-
for experimental and clinical uses, but several trials by addi- change with Dowex 1ϫ8 ClϪ or mixing with aqueous satu-
tion of pharmacologically acceptable cosolvents and/or ex- rated sodium chloride (NaCl) solution (brine). The hydroxy-
cipients were unsuccessful because of the poor water-solubil- propoxy derivative 2h was prepared from the benzyl-
ity (0.005 mg/ml) of TAK-456. Then, we turned our efforts to oxypropoxy derivative 2j by catalytic hydrogenolysis with
preparing a water-soluble prodrug of TAK-456. Bodor et al. palladium on carbon (Pd–C). The compounds 2a—h, except
has described the general concept of “soft drugs” or “pro- 2i (amorphous), were obtained as crystals after purification
drugs”, for modifying the physicochemical properties, in-
cluding water-solubility and pharmacokinetic profiles, of
drugs containing a tertiary nitrogen atom by quarternizing
them to the corresponding ammonium salts with a enzymati-
cally and/or chemically labile group.5) Thus, we designed the
quaternary triazolium salts depicted by the general formula
2. The prodrug strategy is represented by the scheme shown
in Chart 2. Compounds 2, which could be prepared by alky-
lation of TAK-456, might have enhanced water-solubility
owing to their charged character and could be expected to be
Chart 1
To whom correspondence should be addressed. e-mail: Ichikawa_Takashi@takeda.co.jp
© 2001 Pharmaceutical Society of Japan

September 2001
1103
Chart 2
Chart 3
by chromatography [on silica gel or octadecyl silica (ODS)] 2a by NMR measurement, i.e., the nuclear Overhauser and
1
and/or by crystallization from the following solvents; ethanol exchange spectroscopy (NOESY) and H-detected multiple-
(EtOH), water, saline, ethyl acetate (AcOEt) or acetone. bond heteronuclear multiple quantum coherrence (HMBC).7)
Among the crystalline quaternary triazolium salts, the ace- Thus, in NOESY of 2a, a nuclear Overhauser effect (NOE)
toxymethyl derivative 2a was obtained in two crystalline between the methylene protons of the acetoxymethyl group
forms, i.e., the unhydrated form crystallized from EtOH–ace- and the 3- and 5-protons on the triazole ring was observed.
tone and the hydrated form crystallized from water.
On the other hand, HMBC of 2a showed C–H long range
The position of alkylation on the 1,2,4-triazole was as- coupling between the methylene protons of the acetoxy-
sumed to be at the 4-nitrogen atom since Duffin et al. re- methyl group and the 3- and 5-carbons on the triazole.
ported that quanternization of 1-substituted 1,2,4-triazoles
Water-Solubility and Stability The prodrugs for in-
generally occurs at the 4-position to give the corresponding jectable use should have sufficient solubility and adequate
1,4-disubstituted triazolium salts.6) The assignment of the stability in water to allow production of the injectable formu-
1,4-disubstituted triazolium structure was carried out using lation. Therefore, the triazolium salts 2 were assessed for

1104
Vol. 49, No. 9
Table 1. Physicochemical Properties of Prodrugs (2)
Analysis (%)
Calcd (Found)
[a]D
{°C}
(c)
Yielda)
(%)
mp (°C)
(Solv.)b)
IR (KBr)
cmϪ1
Compd.
1H-NMR (in DMSO-d6) d
C
H
N
MeOH
2a
51f,k,i)
204
C25H26ClF2N9O4
0.98 (3H, d, Jϭ7 Hz), 2.08 (3H, s), 3.60—3.66 (1H, m), 3.95—4.08 3217, 2995, Ϫ39.7°
(dec.)
50.89 4.44 21.37 (3H, m), 4.70 (1H, q, Jϭ7 Hz), 4.86 (1H, d, Jϭ14.2 Hz), 5.07 (1H, 1760, 1706,
1527, 1421,
6.60 (1H, s), 6.96—7.06 (1H, m), 7.24—7.36 (2H, m), 7.90 (4H, s), 1261
9.07 (1H, s), 10.06 (1H, s), 10.40 (1H, s)
0.98 (3H, d, Jϭ7 Hz), 2.08 (3H, s), 3.60—3.72 (1H, m), 3.96—4.10 3226, 3120, Ϫ39.6°
{20}
(0.52)
[ET–AC] (50.61 4.38 21.24) d, Jϭ14.2 Hz), 6.10 (1H, d, Jϭ10.6 Hz), 6.19 (1H, d, Jϭ10.6 Hz),
Cl: 6.01 (5.80)
195—196
(dec.)
[W]
C25H26ClF2N9O4
·H2O
(3H, m), 4.70 (1H, q, Jϭ7 Hz), 4.87 (1H, d, Jϭ14.4 Hz), 5.13 (1H, 1754, 1706,
1525, 1415,
{20}
(0.54)
49.39 4.64 20.73 d, Jϭ14.4 Hz), 6.11 (1H, d, Jϭ10.8 Hz), 6.18 (1H, d, Jϭ10.8 Hz),
(49.56 4.64 20.85) 6.70 (1H, s), 6.94—7.03 (1H, m), 7.21—7.38 (2H, m), 7.89 (4H, s), 1261, 1230
9.06 (1H, s), 10.06 (1H, s), 10.50 (1H, s)
c)
2b
2c
2d
2e
24e,g)
217—219
(dec)
[Saline]
C27H30ClF2N9O4
·1/2H2O
0.97 (3H, d, Jϭ7 Hz), 1.07 (6H, d, Jϭ7 Hz), 2.59 (1H, quintet, Jϭ7 1753, 1694,
—
Hz), 3.61—4.08 (4H, m), 4.65—4.75 (1H, m), 4.87 (1H, d, Jϭ14
1524, 1501,
1481, 1422,
51.72 4.98 20.10 Hz), 5.11 (1H, d, Jϭ14 Hz), 6.14—6.22 (2H, m), 6.69 (1H, s),
(51.79 4.83 20.04) 6.92—7.03 (1H, m), 7.22—7.37 (2H, m), 7.90 (4H, s), 9.09 (1H, s), 1269
10.08 (1H, s), 10.48 (1H, s)
48f,h)
196—197
(dec.)
[EA]
C28H32ClF2N9O4
53.21 5.10 19.94 (3H, m), 4.71 (1H, q, Jϭ7 Hz), 4.89 (1H, d, Jϭ14.4 Hz), 5.15 (1H, 3181, 1736,
(53.17 5.15 19.76) d, Jϭ14.4 Hz), 6.17 (2H, s), 6.75 (1H, s), 6.90—7.00 (1H, m), 1481, 1420,
0.98 (3H, d, Jϭ7 Hz), 1.13 (9H, s), 3.61—3.66 (1H, m), 3.96—4.10 1707, 1522, Ϫ32.7°
{20}
(1.0)
7.23—7.34 (2H, m), 7.89 (4H, s), 9.10 (1H, s), 10.06 (1H, s), 10.55 1262, 1167
(1H, s)
42f,h,g,j,i) 203—206
C26H28ClF2N9O5
0.97 (3H, d, Jϭ7 Hz), 1.22 (3H, t, Jϭ7 Hz), 3.60—4.08 (4H, m),
3218,1752,
1709,1526,
1481, 1424,
1262
Ϫ38.7°
{20}
(0.55)
(dec)
[ET]
50.37 4.55 20.33 4.18 (2H, q, Jϭ7 Hz), 4.63—4.73 (1H, m), 4.87 (1H, d, Jϭ14 Hz),
(50.07 4.63 20.10) 5.10 (1H, d, Jϭ14 Hz), 6.13 (1H, d, Jϭ11 Hz), 6.21 (1H, d, Jϭ11
Hz), 6.66 (1H, s), 6.96—7.04 (1H, m), 7.24—7.36 (2H, m), 7.90
(4H, s), 9.09 (1H, s), 10.07 (1H, s), 10.48 (1H, s)
22f,h,g,k,i) 202—203
(dec.)
C27H30ClF2N9O5
·1/2H2O
0.88 (3H, t, Jϭ7.2 Hz), 0.98 (3H, d, Jϭ7 Hz), 1.62 (2H, tq, Jϭ7.2
Hz), 3.62—3.67 (1H, m), 3.90—4.10 (3H, m), 4.09 (2H, t, Jϭ7.2
3221, 3056, Ϫ36.7°
1752, 1709,
1524, 1422,
1264
{20}
(1.0)
[ET–AC]
50.43 4.86 19.60 Hz), 4.69 (1H, q, Jϭ7 Hz), 4.87 (1H, d, Jϭ14.2 Hz), 5.05 (1H, d,
(50.25 4.71 19.31) Jϭ14.2 Hz), 6.16 (1H, d, Jϭ11 Hz), 6.20 (1H, d, Jϭ11 Hz), 6.53
Cl: 5.51 (5.42)
(1H, s), 6.94—7.04 (1H, m), 7.23—7.35 (2H, m), 7.89 (4H, s), 9.09
(1H, s), 10.05 (1H, s), 10.38 (1H, s)
2f
17f,h,i)
220—222
(dec.)
C27H30ClF2N9O5
0.98 (3H, d, Jϭ7.4 Hz), 1.24 (6H, d, Jϭ6.2 Hz), 3.62—3.66 (1H,
3181, 1746, Ϫ37.2°
51.15 4.77 19.88 m), 3.98—4.00 (3H, m), 4.69 (1H, q, Jϭ7.4 Hz), 4.80 (1H, quintet, 1709, 1524,
{20}
(1.0)
[ET–AC] (50.79 4.60 19.62) Jϭ6.2 Hz), 4.87 (1H, d, Jϭ14.4 Hz), 5.04 (1H, d, Jϭ14.4 Hz), 6.11 1426, 1267,
(1H, d, Jϭ11 Hz), 6.19 (1H, d, Jϭ11 Hz), 6.51 (1H, s), 6.96—7.03 1098
(1H, m), 7.22—7.38 (2H, m), 7.90 (4H, s), 9.10 (1H, s), 10.06 (1H,
s), 10.36 (1H, s)
218—220
(dec.)
2g
2h
27f,h,j)
C28H30ClF2N9O7
0.98 (3H, d, Jϭ7 Hz), 3.61—3.66 (1H, m), 3.87—4.02 (7H, m),
3252, 1765, Ϫ31.8°
49.60 4.46 18.59 4.52—4.60 (1H, m), 4.68—4.89 (3H, m), 4.89 (1H, d, Jϭ13.8 Hz), 1703, 1524,
(49.60 4.46 18.40) 5.14 (1H, d, Jϭ13.8 Hz), 6.18 (1H, d, Jϭ11 Hz), 6.26 (1H, d, Jϭ11 1422, 1260
Hz), 6.70 (1H, s), 6.92—7.02 (1H, m), 7.22—7.38 (2H, m), 7.89
{20}
(1.0)
[ET]
(4H, s), 9.11 (1H, s), 10.01 (1H, s), 10.56 (1H, s)
0.98 (3H, d, Jϭ7 Hz), 1.74 (2H, quintet, Jϭ6 Hz), 3.44 (2H, dt, Jϭ 3216, 3127, Ϫ36.7°
33g,i)
181—185
(dec.)
[ET]
C27H30ClF2N9O6
49.89 4.65 19.39 6, 5 Hz), 3.60—4.10 (4H, m), 4.20 (2H, t, Jϭ6 Hz), 4.59 (1H, t, Jϭ 1755, 1705,
(49.48 4.75 19.18) 5 Hz), 4.65—4.75 (1H, m), 4.87 (1H, d, Jϭ14 Hz), 5.10 (1H, d, Jϭ 1524, 1481,
14 Hz), 6.13 (1H, d, Jϭ11 Hz), 6.21 (1H, d, Jϭ11 Hz), 6.63 (1H, s), 1424, 1265
6.95—7.05 (1H, m), 7.25—7.37 (2H, m), 7.89 (4H, s), 9.09 (1H, s),
10.06 (1H, s), 10.48 (1H, s)
{20}
(1.1)
2i
36f,h,g,j)
A.P.d)
C28H31ClF2N10O6
·2H2O
0.97 (3H, d, Jϭ7 Hz), 1.83 (2H, quintet, Jϭ6 Hz), 3.20 (3H, s), 3.35 3237, 1763, Ϫ33.5°
(2H, t, Jϭ6 Hz), 3.55—3.70 (1H, m), 3.90—4.10 (3H, m), 4.18 (2H, 1690, 1661,
{20}
(1.0)
47.16 4.95 19.64 t, Jϭ6 Hz), 4.64—4.75 (1H, m), 4.87 (1H, d, Jϭ14 Hz), 5.11 (1H,
(47.09 4.92 19.51) d, Jϭ14 Hz), 6.14 (1H, d, Jϭ11 Hz), 6.22 (1H, d, Jϭ11 Hz), 6.66
(1H, s), 6.65—7.05 (1H, m), 7.25—7.39 (2H, m), 7.90 (4H, s), 9.10
(1H, s), 10.07 (1H, s), 10.48 (1H, s)
1524, 1483,
1424, 1267
a) 1a—g, i: yields based on the parent compound (2); 1h: yield of the deprotection reaction. b) Recrystallization solvent: EA, AcOEt; W, water; AC, acetone; ET, EtOH.
c) Not measured. d) A.P.: Amorphous powder. e) The reaction was carried out in acetone. f) The reaction was carried out in MeCN. g) The product was purified by chro-
matography using a pre-packed column CPO-273LR (ODS). h) The product was purified by silica gel column chromatography. i) The product was purified by crystallization.
j) Anion exchange was done with Dowex 1ϫ8 ClϪ. k) Anion exchange was done by mixing with brine.
their solubility and stability in 5% glucose as an injectable tained. The solubility (mg/ml) was calculated based on the
carrier.8) The solubilities (mg/ml) of 2b, d—i were measured amount of 5% glucose added. In the case of 2a, c, the solu-
by the following simple method: 5% glucose was added por- bilities were determined by another method: a mixture of 2
tionwise (0.1 ml aliquots) to compound 2 (1 mg) with shak- and 5% glucose was allowed to stand in a sonicator for
ing followed by sonication until a clear solution was ob- 10 min at room temperature and the remaining solid (2) was

September 2001
1105
with the parent compound 1 (TAK-456) and the non-prodrug
quaternary salt 3 (Table 3). The in vitro activities are ex-
pressed as the minimum inhibitory concentrations (MIC,
mg/ml), which were determined by an agar-dilution method
on RPMI 1640 medium under 20% CO2.10) Compounds 2a—
filtered off using a 0.2 mm membrane filter. The concentra-
tion (solubility: mg/ml) of 2 in the filtrate was determined by
HPLC based on the peak area.9) On the other hand, the stabil-
ities were measured by the following method: a 1—5 mg/ml
solution of 2 in 5% glucose was allowed to stand for 1 d at
room temperature and the relative % content of 1 formed by
hydrolysis of 2 was determined by HPLC.9) This % value was
regarded as the stability of the prodrugs 2. The solubility and
the stability results are shown in Table 2.
Table 2. Solubility and Stability of the Prodrugs
Compounds 2 generally had good water-solubility (1.5—
Ն10 mg/ml). Notably, compounds 2h, i, which have hy-
drophilic hydroxyl and acetoamino groups, respectively, re-
vealed water-solubility higher than 10 mg/ml. In the case of
2a, the solubilities of two crystalline forms were somewhat
different; i.e., 10 mg/ml for the unhydrated form and 4 mg/ml
for the hydrated form.
Solubility
Stability
Compd.
No.
R
in 5% glucose
(mg/ml)
Content (%) of 1 after
1 d in 5% glucose
The alkanoyl derivatives (2a—c) and the alkoxycarbonyl
derivatives (2d—f) were considerably resistant to hydrolysis
in 5% glucose (stability: 0.1—2.1%). Among these prodrugs
(2a—f), the compounds having secondary and tertiary alkyl
groups (2b, c, f: 0.3, 0.1, 0.3%, respectively) were found to
be more stable than the compounds having unbranched alkyl
groups (2a, d, e: 1.8, 2.1, 1.9%, respectively). On the other
hand, 2g—i with hydrophilic moieties were considerably un-
stable (6.4—25.6%). Especially, the hydroxyl compound 2h
showed the lowest stability (25.6%), which might be due to
the neighboring group participation of the hydroxyl group
accelerating the hydrolysis.
2a
2b
2c
2d
2e
2f
CH3-
(CH3)2CH-
(CH3)3C-
CH3CH2O-
CH3(CH2)2O-
(CH3)2CHO-
4,a) 10b)
1.8
0.3
0.1
2.1
1.9
0.3
3
6
3
1.5
2
2g
5—8
6.4
2h
2i
Ͼ10
Ͼ10
25.6
6.4
Biological Activity The in vitro antifungal activities of
the triazolium salts 2 against C. albicans TA were compared
a) Hydrated form of 2a. b) Unhydrated form of 2a.
Table 3. Antifungal Activities and Hemolytic Activities of the Prodrugs (2)
In vitro
MIC (mg/ml)
C. albicans TA
In vivo
Compd. No.
R-
Candidiasisc)
N
Hemolysis (%)
d) (Day)e)
2a
2b
2c
2d
2e
2f
CH3-
(CH3)2CH-
(CH3)3C-
CH3CH2O-
CH3(CH2)2O-
(CH3)2CHO-
0.06b)
0.016a)
0.5a)
3/5 (4.5)
—
1/5 (5.0)
4/5 (6.0)
—
1.8
89
100
7.0
64
f)
0.13b)
0.13b)
0.25b)
3/5 (5.5)
81
2g
0.06b)
—
2.2
2h
2i
0.06b)
0.06b)
—
—
1.1
1.4
3
Ͼ16a)
—
—
1 (TAK-456)
0.002,a) 0.008b)
2/5 (4.3)g)
—
a) Determined under condition A. b) Determined under condition B. c) Prodrugs were administered interavenously to the mice at a dose of 1 mmol/kg immediately after
infection. d) The number (N) of survivors per total number of mice (nϭ5) on day 7 after infection. e) The mean survival days (Day) of the mice which had died up to 7 d after
infection. Control mice died up to 0.8 d. f) Not determined. g) Administered orally at a dose of 1 mmol/kg.

1106
Vol. 49, No. 9
Table 4. In Vivo Antifungal Activity of Compound 2a
i (MICs, 0.016—0.5) were about ten times or more less po-
tent than the parent compound 1 (MIC, 0.002—0.008). On
the other hand, the N-methyl triazolium salt 3, which is stable
to hydrolysis, was substantially inactive (MIC, Ͼ16). There-
fore, it is considered that the antifungal activity of compound
2 is inherently weak and the observed activity could be due
to a small amount of the parent compound 1, which is
formed by hydrolysis in the assay medium during the experi-
ment.
ED50 (mg/kg)e)
Compd.
No.
C. albicans TA
A. fumigatus 437
2aa)
0.62
0.77
0.22—0.39
5.19
4.49
3.56
1: TAK-456b)
Fluconazolec)
Itraconazoled)
179
19
a) Administered intravenously with the solution in 5% glucose. b) Administered
orally as a 0.5% carboxymethylcellulose (CMC) suspension. c) Administered orally
as an aqueous solution. d) Administered orally as a 2-hydroxypropyl b-cyclodextrin
solution. See ref. 11. e) The dose of the test compound which allows 50% survival of
the infected mice.
Four compounds 2a, c, d, f were evaluated for in vivo anti-
fungal activity using C. albicans TA infected mice (Table 3).
The test compounds (1 mmol/kg) were administered intra-
venously once, immediately after infection. The activity is
expressed as the number (N) of survivors per total number of
mice (nϭ5) on day 7 after infection and as the mean survival
days (Day) of the mice which had died up to 7 d after infec-
tion. All of the prodrugs tested were found to exert strong
protective effects (Nϭ1/5—3/5, Dayϭ4.5—6.0) against can-
didiasis comparable to that of the parent compound 1
(Nϭ2/5, Dayϭ4.3). These results of antifungal activities in
vitro and in vivo suggested that compounds 2 are hydrolyzed
to the parent compound 1 in blood after intravenous adminis-
tration.
Table 5. In Vitro Half-Lives (T1/2) of 2a (TAK-457, 12.25 mg/ml) in
Plasma
T1/2 (min)
Mouse
5.1
Rat
6.4
Human
6.0
In general, some amphiphilic compounds such as quater-
nary salts, which contain both hydrophobic and hydrophilic
moieties, have been known to induce hemolysis. Thus, com-
pounds 2 were assessed for hemolytic activity using rat
blood. Table 3 shows the % of hemolysis caused by com-
pounds 2. Compounds 2g—i with the hydrophilic moieties
were found to have low hemolytic activity (1.1—2.2%).
Among the alkanoyl derivatives (2a—c) and the alkoxycar-
bonyl derivatives (2d—f), compounds 2b, c, e, f, possessing
lipophilic alkyl or alkoxy groups showed high hemolytic ac-
tivity (64—100%). On the other hand, the activity of com-
pounds 2a, d that have smaller alkyl or alkoxyl groups,
proved to be low. Especially, compound 2a bearing the ace-
toxymethyl group was substantially devoid of this effect
(1.8%).
Fig. 1. Plasma Concentrations (Mean Values, nϭ3) of 2a (᭹) and TAK-
456 (1: ᭿) after a 1 mg/kg Intravenous Dose of [14C]-2a in Rats
From the series of prodrugs, we selected 2a as the most
promising compound for further evaluation.
In Vivo Antifungal Activity of 2a The in vivo antifun- those of fluconazole (ED50, 179 mg/kg) and itraconazole
gal activity of 2a against C. albicans TA and A. fumigatus (ED50, 19 mg/kg).
437 in mice was evaluated and compared with the parent
Pharmacokinetics of 2a The in vitro half-lives of 2a
compound 1 (TAK-456), fluconazole and itraconazole. C. al- were measured in the plasma of various animals, including
bicans TA infected mice and A. fumigatus 437 infected neu- mouse, rat and human. The results are shown in Table 5.
tropenic mice were used for the in vivo assay. In the in vivo Compound 2a was hydrolyzed rapidly to form TAK-456 and
assay against C. albicans TA, the test compounds were ad- the T-half-lives in vitro were about 5—6 min in the plasma of
ministered once, intravenously or orally, immediately after all the animals tested (mouse 5.1 min; rat 6.4 min; human
infection. On the other hand, in the case of the in vivo assay 6.0 min). It is noteworthy that there was no species difference
against A. fumigatus 437, the test compounds were adminis- in hydrolysis.
tered intravenously or orally, once on the day of infection and
This rapid conversion of 2a to TAK-456 was also con-
twice daily on the following 2 d. The in vivo activity (Table firmed in vivo using rats. Figure 1 shows the time course of
4) is expressed in terms of ED50 (mg/kg, the dose of the test the plasma concentrations of 2a and TAK-456 after intra-
compound which allows 50% survival of the infected mice). venous administration of [14C]-2a (1 mg/kg).12) As seen in
The activity (ED50, 0.62 mg/kg) of 2a against C. albicans TA Fig. 1, 2a was rapidly hydrolyzed with a T-half-life of 6 min
infection was comparable to those of 1 (ED50, 0.77 mg/kg) and the parent compound TAK-456 appeared quickly. From
and fluconazole (ED50, 0.22—0.39 mg/kg), and superior to the basis of the above in vitro and in vivo studies, 2a is ex-
that of itraconazole (ED50, 5.19 mg/kg). Furthermore, in the pected to be converted smoothly to TAK-456 in humans.
therapeutic effect against aspergillosis in neutropenic mice, Based on the results of the evaluations described above, com-
2a showed a low ED50 value (ED50, 4.49 mg/kg), comparable pound 2a (TAK-457) was designated as the injectable candi-
to that of 1 (ED50, 3.56 mg/kg), and remarkably superior to date for clinical trials.

September 2001
1107
was stirred at 80 °C until the clear solution was obtained. After having been
cooled to room temperature (r.t.), silica gel (400 mg) was added, and the
mixture was stirred for 10 min at r.t. The silica gel was filtered off and
washed with MeCN–tetrahydrofuran (THF) (1 : 1, v/v, 2 mlϫ3). The mother
liquor and the washings were combined, and brine (10 ml) was added. The
In conclusion, we identified a new antifungal prodrug
TAK-457 for injectable use. This biologically labile quater-
nary triazolium salt had sufficient solubility and adequate
stability for an injectable formulation and exhibited excellent
therapeutic effects against candidiasis as well as aspergillosis resulting mixture was stirred for 30 min at r.t., and the organic layer was sep-
arated. The same operations as above, the addition of brine (10 ml) to the or-
ganic layer followed by the stirring for 30 min at r.t. and the subsequent sep-
aration of the organic layer, were performed further three times. The organic
layer was dried over MgSO4, and the solvent was distilled off under reduced
in mice via intravenous administration. Detailed studies on
the therapeutic effects against aspergillosis using various ex-
perimental models will be described in a separate paper.13)
pressure. The residue was dissolved in THF (3 ml), and the solution was al-
Experimental
lowed to stand for 5 h at r.t. The precipitate (179 mg) was collected by filtra-
Melting points were determined using a Yanagimoto melting point appa- tion and dissolved in methanol (MeOH)–THF (1 : 1, v/v, 4 ml). The solution
ratus and are uncorrected. IR spectra were measured with a JASCO IR-810 was concentrated in vacuo and the residue was dissolved in THF (2 ml). The
spectrometer or a Shimazu FTIR-8200PC spectrometer. NMR spectra were solution was allowed to stand for 5 h at r.t. The precipitate was collected by
recorded on Varian Gemini-200 and Bruker Avance DPX-300 spectrometers filtration and dissolved in ethanol (EtOH)–acetone (12 : 1, v/v, 6.5 ml). The
with tetramethylsilane as an internal standard. The following abbreviations resulting solution was concentrated in vacuo to a volume of ca. 2 ml and al-
are used: sϭsinglet, dϭdoublet, tϭtriplet, qϭquartet, mϭmultiplet, brϭ lowed to stand for 3 h at r.t. The precipitate was collected by filtration to give
broad. The optical rotations were recorded with a JASCO DIP-181 or DIP- the unhydrated form of 2a (126 mg, 51%) as a white crystalline powder.
370 digital polarimeter.
Compound 2a (0.63 g) obtained above was recrystallized from water (10 ml)
Reactions were followed by TLC on Silica-gel 60 F254 precoated TLC to give the hydrate form (0.61 g) as a white crystalline powder.
plates (E. Merck) or by HPLC using an ODS column (A-303, 4.6 mm
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
i.d.ϫ250 mm, YMC Co., Ltd.). Standard work-up procedures were as fol-
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-[(2-methylpropanoyloxy)methyl]-
lows. The reaction mixture was partitioned between the indicated solvent 1H-1,2,4-triazolium Chloride (2b: Table 1) A mixture of 1 (0.5 g), 4b
and water. Organic extracts were combined and washed in the indicated (1.37 g) and acetone (20 ml) was refluxed for 50 h. The resulting mixture
order using the following aqueous solutions; water, 1 N aqueous sodium hy-
was concentrated in vacuo and diisopropylether (isoPr2O, 10 ml) was added
droxide solution (1 N NaOH), 5% aqueous NaHCO3 solution (aqueous to the residue. The precipitate was collected by filtration and purified by
NaHCO3), 1 N hydrochloric acid (HCl) and brine. Extracts were dried over
ODS column chromatography (MeOH–H2O, 3 : 2, v/v) to give 2b (0.15 g,
anhydrous magnesium sulfate (MgSO4), filtered and evaporated in vacuo.
24%) as a white amorphous powder. This powder (50 mg) was crystallized
Chromatographic separations were carried out on Silica gel 60 (0.063— from saline (1 ml) to obtain 2b (41 mg) as a white crystalline powder.
0.200 mm, E. Merck) or ODS (CPO-273LR, pre-packed column, 22 mmϫ
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
300 mm, Kusano Kagaku Kikai Co.) using the indicated eluents.
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-[(2,2-dimethylpropanoyloxy)-
Chloromethyl pivalate (4c), bromomethyl acetate (5a) were purchased methyl]-1H-1,2,4-triazolium Chloride (2c: Table 1) A mixture of 1
from Aldrich Chemical Company, Inc.. Chloromethyl isobutylate (4b),14) (0.5 g, 1.04 mmol), 4c (15.7 g, 104.2 mmol) and MeCN (3 ml) was stirred at
chloromethyl propyl carbonate (4e), chloromethyl isopropyl carbonate 100 °C for 6.5 h. The resulting mixture was concentrated in vacuo and
(4f),15) ethyl iodomethyl carbonate (6d)16) were prepared according to the lit- isoPr2O was added to the residue. The precipitate was collected by filtration
erature.
Chloromethyl (1,3-Dioxan-5-yl) Carbonate (4g)
and purified by silica gel flush column chromatography (AcOEt→acetone→
solution of acetone–EtOH, 10 : 1→5 : 1, v/v) to give 2c (0.32 g, 48%) as a white amor-
A
chloromethyl chloroformate (25 g) in diethyl ether (Et2O, 50 ml) was added phous powder. This powder (0.4 g) was crystallized from AcOEt to obtain 2c
dropwise to a stirred solution of glycerol formal (14 g) and pyridine (15 g) in (0.3 g) as a white crystalline powder.
Et2O (400 ml) over a period of 10 min at 0 °C. After stirring for 20 h, the pre-
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
cipitate was removed by filtration. The filtrate was worked up (Et2O; brine). zolyl)phenyl]-1-imidazolidinyl]butyl]-4-ethoxycarbonyloxymethyl-1H-
The residue was purified by silica gel flush column chromatography 1,2,4-triazolium Chloride (2d: Table 1) A mixture of 1 (1.31 g, 2.72
(AcOEt–hexane, 1 : 5→1 : 3, v/v) to give 4g (1.7 g, 5%) as a colorless oil.
1H-NMR (CDCl3) d: 4.05 (4H, d, Jϭ3.2 Hz), 4.67 (1H, quintet, Jϭ3.2 Hz), 14 h under an argon atmosphere. The resulting mixture was concentrated in
4.82 (1H, d, Jϭ6.2 Hz), 4.95 (1H, d, Jϭ6.2 Hz), 5.75 (2H, s).
vacuo. The residue was purified by silica gel flush column chromatography
mmol), 6d (1.25 g, 5.43 mmol) and MeCN (15 ml) was stirred at 60 °C for
[2-(N-Acetylamino)ethyl] chloromethyl carbonate (4i) and 3-benzy- (AcOEt→acetone→acetone–EtOH, 4 : 1, v/v) followed by ODS column
loxypropoxy chloromethyl carbonate (4j) were prepared from the corre- chromatography (MeOH–H2O, 3 : 2, v/v) to give the iodide salt (1.1 g) as a
sponding alcohols (N-acethylethanolamine, 3-benzyloxypropanol17)) by the pale yellow amorphous powder. The iodide salt (1.1 g) was then ion-ex-
1
same method as described above. 4i (85%): H-NMR (CDCl3) d: 2.01 (3H,
s), 3.58 (2H, q, Jϭ6 Hz), 4.32 (2H, t, Jϭ6 Hz), 5.75 (2H, s), 5.87 (1H, br).
4j (89%): 1H-NMR (CDCl3) d: 2.01 (2H, quintet, Jϭ6.2 Hz), 3.57 (2H, t,
Jϭ6.0 Hz), 4.36 (2H, t, Jϭ6.4 Hz), 4.51 (2H, s), 5.71 (2H, s), 7.33 (5H, s).
changed using Dowex 1ϫ8 ClϪ (water) followed by crystallization from
EtOH to give 2d (0.7 g, 42%) as a white crystalline powder.
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-propoxycarbonyloxymethyl-1H-
(1,3-Dioxan-5-yl) Iodomethyl Carbonate (6g) A mixture of 4g (1.7 g), 1,2,4-triazolium Chloride (2e: Table 1) Compound 1 (1.0 g) was allowed
sodium iodide (5.1 g) and MeCN (40 ml) was stirred at 60 °C for 2 h. The re-
sulting mixture was concentrated in vacuo. The residue was worked up
(Et2O; 5% aqueous sodium thiosulfate solution, water, brine) to give 6g
(3.2 g, 100%) as a pale yellow oil. 1H-NMR (CDCl3) d: 4.04 (4H, d,
to react with 6e (0.9 g) in a manner similar to that described for the synthesis
of 2d to obtain the iodide (1.0 g) as a pale yellow amorphous powder.
This iodide (0.3 g) was dissolved in THF–AcOEt (3/1, 100 ml) and ion-
exchanged by mixing with brine (50 mlϫ4). The organic layer was dried
Jϭ3.0 Hz), 4.66 (1 H, quintet, Jϭ3.0 Hz), 4.81 (1H, d, Jϭ6.2 Hz), 4.95 (1H, over MgSO4, and concentrated in vacuo followed by crystallization from
d, Jϭ6.2 Hz), 5.97 (2H, s).
Iodomethyl propyl carbonate (6e), [2-(N-acetylamino)ethyl] iodomethyl
EtOH–acetone to give 2e (0.09 g, 22% from 1) as a white crystalline powder.
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
carbonate (6i) and 3-benzyloxypropoxy iodomethyl carbonate (6j) were pre- zolyl)phenyl]-1-imidazolidinyl]butyl]-4-isopropoxycarbonyloxymethyl-
pared from the corresponding chloromethyl carbonates 4e, i, j by the same 1H-1,2,4-triazolium Chloride (2f: Table 1) The reaction of 1 (0.5 g) with
method as described above. 6e (88%): 1H-NMR (CDCl3) d: 0.97 (3H, t,
4f (3.17 g) was carried out in a manner similar to that described for the syn-
Jϭ7.0 Hz), 1.73 (2H, m), 4.19 (2H, t, Jϭ7.0 Hz), 5.96 (2H, s). 6i (48%): 1H- thesis of 2c. Compound 2f (110 mg, 17%) was obtained as a white crys-
NMR (CDCl3) d: 2.00 (3H, s), 3.54 (2H, q, Jϭ6 Hz), 4.28 (2H, t, Jϭ6 Hz), talline powder (recrystallized from AcOEt–acetone).
1
5.93 (2H, s), 6.12 (1H, br). 6j (93%): H-NMR (CDCl3) d: 2.00 (2H, quin-
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera-
tet, Jϭ6.2 Hz), 3.57 (2H, t, Jϭ6.0 Hz), 4.36 (2H, t, Jϭ6.4 Hz), 4.51 (2H, s), zolyl)phenyl]-1-imidazolidinyl]butyl]-4-[(1,3-dioxan-5-yl)oxycarbony-
5.94 (2H, s), 7.34 (5H, s).
4-Acetoxymethyl-1-[(2R,3R)-2-(2,4-difluorophenyl)-2-hydroxy-3-[2-
oxo-3-[4-(1H-1-terazolyl)phenyl]-1-imidazolidinyl]butyl]-1H-1,2,4-tria-
loxymethyl-1H-1,2,4-triazolium Chloride (2g: Table 1) The reaction of
1 (1.0 g) with 6g (0.9 g) was carried out in a manner similar to that described
for the synthesis of 2d to obtain the iodide (4.22 g) as a pale yellow amor-
zolium Chloride (2a: Table 1) A solution of 1 (200 mg) and 5a (77.1 mg) phous powder. This iodide salt (1 g) was subjected to column chromatogra-
in MeCN (2 ml) was stirred for 7 h at 80 °C under an argon atmosphere. The phy on Dowex 1ϫ8 ClϪ (water). The eluate was lyophilized to obtain 2g
reaction mixture was diluted with MeCN (11 ml), and the resulting mixture (0.23 g, 27%) as a white amorphous powder. This powder (0.15 g) was crys-

1108
Vol. 49, No. 9
tallized from EtOH (20 ml) to give 2g (0.14 g) as a white crystalline powder.
ED50 values of the compounds against aspergillosis were determined by
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera- the following method: Six-week-old CDF1 female mice were infected intra-
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-[(3-hydroxypropyl)oxycarbony-
venously with 6ϫ104 CFU of A. fumigatus 437 per mouse 4 d after the in-
loxymethyl]-1H-1,2,4-triazolium Chloride (2h: Table 1) The reaction of traperitoneal administration of 200 mg/kg of cyclophosphamide. The test
1 (2.0 g) with 6j (2.9 g) was carried out in a manner similar to that described
compounds were administered intravenously or orally 2 h after infection and
for the synthesis of 2d. The product was ion-exchanged by Dowex 1ϫ8 ClϪ twice daily on the following 2 d. ED50 values (Table 4) were calculated by
(H2O) to obtain 4-[(3-benzyloxypropoxy)carbonyloxymethyl]-1-[(2R,3R)-2- the method of Reed and Muench from survival rates on day 10 after infec-
(2,4-difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-terazol-1-yl)phenyl]-1-
imidazolidinyl]butyl]-1H-1,2,4-triazolium chloride (2j, 0.79 g, 26%) as a
white amorphous powder. Anal. Calcd for C34H36ClF2N9O6·1/2H2O: C,
tion.
In Vitro Half-Lives of 2a (Table 5) The standard solution (10 ml) of 2a
(1.225 mg/ml) was added to the animal [SD(IGS) rat or ICR mouse] or
54.51; H, 4.98; N, 16.83. Found: C, 54.37; H, 5.10; N, 16.87. 1H-NMR human plasma (990 ml), which had been pre-incubated for 5 min at 37 °C.
(CDCl3) d: 0.97 (3H, d, Jϭ7.2 Hz), 1.89 (2H, quintet, Jϭ6.4 Hz), 3.47 (2H, The mixtures were incubated at 37 °C. An aliquot (100 ml) was taken at 5,
t, Jϭ6.2 Hz), 3.60—4.09 (4H, m), 4.22 (2H, t, Jϭ6.6 Hz), 4.44 (2H, s), 10, 15, 30, 60 and 120 min and deproteinized with MeCN–acetic acid
4.65—4.75 (1H, m), 4.86 (1H, d, Jϭ14 Hz), 5.09 (1H, d, Jϭ14 Hz), 6.12 (AcOH) (4 : 1, v/v, 100 ml). After centrifugation at 19000 g for 10 min, the
(1H, d, Jϭ11 Hz), 6.20 (1H, d, Jϭ11 Hz), 6.62 (1H, s), 6.94—7.04 (1H, m),
supernatant (150 ml) was added to 150 ml of 0.01 mol/l aqueous ammonium
7.24—7.36 (7H, m), 7.90 (4H, s), 9.09 (1H, s), 10.07 (1H, s), 10.45 (1H, s) acetate solution adjusted to pH 6.0 with AcOH. An aliquot (20 ml) of the so-
IR (KBr): 3125, 1759, 1696, 1524, 1481, 1422, 1267 cmϪ1
A solution of 2j (0.66 g) and 1 N HCl (0.89 ml) in MeOH (25 ml) was hy-
.
lution was injected to an HPLC. HPLC conditions: The HPLC system con-
sisted of an LC-10AD pump, a SIL-10A autosampler, a CTO-10AC column
drogenated over 10% Pd–C (50% wet, 0.33 g) at r.t. under atmospheric pres- oven, a SPD10AV UV-detector, and a CLASS-LC10 workstation (all from
sure for 1.5 h. The catalyst was removed by filtration, and the filtrate was Shimadzu Co., Kyoto, Japan). Column: L-Column ODS (5 mm particle size,
concentrated in vacuo. The residue was purified by ODS column chromatog- 4.6 mm i.d.ϫ150 mm; Chemicals Evaluation and Research Institute, Tokyo,
raphy (MeOH–H2O, 3 : 2, v/v) followed by crystallization from EtOH to give Japan). Mobile phase: A mixture of 0.01 mol/l aqueous ammonium acetate
2h (0.19 g, 33%) as a colorless crystalline powder.
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera- temperature: 40 °C. Flow rate: 1.0 ml/min. UV detection: 274 nm.
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-[(2-N-acetylaminoethoxy)car- Concentration of 2a and TAK-456 in Rat Plasma (Fig. 1) Animals
bonyloxymethyl]-1H-1,2,4-triazolium Chloride (2i: Table 1) The reac- used in this study were male Crj:CD(SD)IGS rats (weight, 281 to 283 g;
tion of 1 (2.0 g) with 6i (2.9 g) was carried out in a manner similar to that Charles River Japan Inc., Yokohama, Japan). They were fed laboratory chow
described for the synthesis of 2d. The iodide was ion-exchanged by Dowex (CR-LPF; Oriental Yeast Co., Ltd., Tokyo, Japan), had free access to water,
1ϫ8 ClϪ (water) to obtain 2i (0.5 g, 36%) as a white amorphous powder.
and were housed in temperature- and humidity-controlled rooms (20 to
solution adjusted to pH 6.0 with AcOH and CH3CN (67 : 33, v/v). Column
1-[(2R,3R)-2-(2,4-Difluorophenyl)-2-hydroxy-3-[2-oxo-3-[4-(1H-1-tera- 26 °C, 40 to 75%) with 12-h light–dark cycles for more than a week before
zolyl)phenyl]-1-imidazolidinyl]butyl]-4-methyl-1H-1,2,4-triazolium Io- use. [14C]-2a was dissolved in 5 w/v % glucose solution for intravenous in-
dide (3) A mixture of methyliodide (0.36 ml, 6 mmol), 1 (48 mg, 0.1 jection at a dose of 1 mg/ml/kg. The test compound was injected to fed ani-
mmol) and acetone (2 ml) was refluxed for 64 h. The whole was concen-
mals. After dosing, blood samples were taken from the tail vein, and cen-
trated in vacuo. The residue was purified by ODS column chromatography trifuged at 1870 g for 10 min at 4 °C to obtain plasma samples. The sam-
(MeOH–H2O, 3 : 2, v/v) to give 3 (47 mg, 75%) as a white amorphous pow- pling times were as follows: 5, 10, 15, 30, 45, 60 min. The samples, after de-
der. 1H-NMR (DMSO-d6) d: 0.98 (3H, d, Jϭ7 Hz), 3.50—4.05 (4H, m), termination of the radioactivity, were kept frozen at Ϫ20 °C until analyses.
3.84 (3H, s), 4.60—4.80 (1H, m), 4.86 (2H, br), 6.32 (1H, br), 7.00—7.10
Compound 2a and TAK-456 (1) in rat plasma were quantified by HPLC.
(1H, m), 7.20—7.50 (2H, m), 7.90 (4H, s), 8.83 (1H, s), 9.96 (1H, s), 10.06 Prior to the HPLC analysis, to a portion of plasma was added a half volume
(1H, s). IR (KBr): 1688, 1524 1499, 1483, 1424, 1269 cmϪ1. Anal. Calcd for of phosphate buffered saline in the ice-cooled bath, and to the supernatant
C23H24F6IN9O2·H2O: C, 43.07; H, 4.09; N, 19.65. Found: C, 43.20; H, 4.22; obtained by the centrifugation at 1870 g and 4 °C for 10 min was added 2
N, 19.17.
volumes of a mixture of MeCN and AcOH (1 : 9, v/v). The supernatant ob-
Hemolytic Activity Distilled water and 5% glucose (Otsuka Co.) was tained by the same manner described above was subjected to HPLC. The
used for positive control and blank, respectively. Compounds were dissolved HPLC separations were achieved at 40 °C with a Shield RP8 column (4.6
in 5% glucose at a concentration of 2 mg/ml and pre-warmed at 37 °C. The mm i.d.ϫ150 mm, Waters Assoc., Milford Massachusetts, U.S.A.). A mix-
solution (1 ml) and rat heparinaized whole blood (0.1 ml) were mixed and in- ture of 0.01 mol/l ammonium acetate, MeCN and AcOH (75 : 25 : 0.2, v/v/v)
cubated at 37 °C for 30 min. The mixture was centrifuged at 1870 g for and a mixture of 0.01 mol/l aqueous ammonium acetate solution, MeCN and
10 min and the hemoglobin level in the supernatant was determined using
AcOH (60 : 40 : 0.2, v/v/v) were used for the mobile phase A and B [MP(A)
HEMOGLOBIN TEST WAKO (Wako Pure Chemical Co.), according to and MP(B)], respectively. The flow rate was 1 ml/min. The time program for
manufacturer’s manual. The % of hemolysis was calculated by the following the gradient elution was as follows: the concentration of MP(B) was held at
formula: hemolysis (%)ϭOD540 of sample supernatant/OD540 of control su- 0 vol% for 15 min, linearly increased from 0 to 100 vol% over a period of
pernatant ϫ100.
15 min and cycled back to the initial condition (0 vol%). The effluent was
Antifungal Activity in Vitro MICs against C. albicans TA were deter- collected into 1 ml size of fractions, followed by the counting of radioactiv-
mined by an agar dilution method using RPMI-1640 medium (Gibco BRL, ity. Data Processing: Data were expressed as the mean values or the mean
Grand Island, N.Y.). A double concentration of RPMI-1640 medium was values with standard deviations (S.D.) for the results from three to four ani-
prepared with 0.3 M morpholinepropanesulfonic acid (MOPS; Dojindo, mals, unless otherwise indicated. Half-life (T1/2) in plasma was calculated by
Tokyo, Japan) buffer (pH 7.0), sterilized by filtration through a membrane
filter (pore size, 0.45 mm), and mixed with an equal volume of 2.0% agar
(condition A) or 3.0% agar (condition B) (Wako, Osaka, Japan) which had
the linear regression analysis and trapezoidal rule.
Acknowledgments We thank Dr. S. Kishimoto and Dr. A. Miyake for
been autoclaved at 121 °C for 15 min and kept at 55 °C. The agar medium their encouragement throughout this work. We also thank Ms. N. Kitamoto
(9.9 ml) was then poured into petri dishes containing 0.1 ml of serial dilu- and Mr. T. Yoshida for the biological assay. Thanks are also due to Ms. M.
tions of antifungal agents dissolved in dimethylsulfoxide (DMSO; Wako) Murabayashi for NMR measurement.
and allowed to solidify. About 103 CFU of fungal cells suspended in saline
was inoculated with a multiple inoculator (Sakuma, Tokyo, Japan) onto the References and Notes
agar plates prepared as described above. The plates were then incubated in a
CO2 incubator at 35 °C for 20 h. The MIC was defined as the lowest concen-
tration of antifungal agent giving no visible growth or causing almost com-
plete inhibition of growth.
Antifungal Activity in Vivo In vivo antifungal activity against C. albi-
cans TA was measured by the following method: Six-week-old CDF1 female
mice were infected intravenously with 2ϫ106 CFU of C. albicans TA per
mouse. The compounds were administered intravenously or orally immedi-
ately after infection. The efficacy of the compound is expressed as N and
Day (Table 3) or ED50 values (Table 4). ED50 values were calculated by the
method of Reed and Muench18) from survival rates on day 7 after infection.
1) Part XI: Ichikawa T., Kitazaki T., Matsushita Y., Hosono H., Yamada
M., Mizuno M., Itoh K., Chem. Pharm. Bull., 48, 1947—1953 (2000).
2) Kitazaki T., Ichikawa T., Tasaka A., Hosono H., Matsushita Y.,
Hayashi R., Okonogi K., Itoh K., Chem. Pharm. Bull., 48, 1935—
1946 (2000).
3) Tsuchimori N., Hayashi R., Kitamoto N., Asai K., Kitazaki T., Iizawa
Y., Itoh K., Okonogi K., Antimicrob. Agents Chemother., submitted.
4) a) Graybill J. R., Eur. J. Clin. Infect. Dis., 8, 402—412 (1989); b) Petit
C., Cheron M., Joly V., Rodrigues J. M., Bolard J., Gaboriau F., J. An-
timicrob. Chemother., 42, 779—785 (1998); c) Sandeep S., Khan, J.
A., Ghosh. P. C., ibid., 42, 635—642 (1998); d) Tumbarello M., Tac-

September 2001
conelli E., Pagano L., La Barbera E. O., Morace G., Cauda R., Leone
1109
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9) HPLC conditions: colum, ODS (A-303, 4.6 mm i.d.ϫ250 mm, YMC
Co., Ltd.); mobile phase, MeOH–water–AcOH, 7 : 3 : 0.02, v/v; flow
rate, 0.8 ml/min; detection UV at 262 nm.
10) The agar dilution method for in vitro susceptibility testing of antifun-
gal agents under 20% CO2 was developed in our laboratories: Yoshida
T., Jono K., Okonogi K., Antimicrob. Agents Chemother., 41, 1349—
1351 (1997).
5) a) Bodor N., Kaminski J. J., Selk S., J. Med. Chem., 23, 469—474
(1980); b) Bodor N., Kaminski J. J., ibid., 23, 566—569 (1980); c)
Bodor N., Woods R., Raper C., Kearney P., Kaminski, J. J., ibid., 23,
474—480 (1980); d) Vinogradova N. D., Kuznetsov S. G., Chigareva
S. M., Khim.-Farm. Zh., 14, 41—47 (1980); e) Darvas B., Kulcsa P.,
Belai I., Matolcsy G., Entomol. Exo. Appl., 44, 295—301 (1987); f)
Bwdford C. D., Harris R. N., III, Howd R. A., Goff D. A., Koolpe G.
A., Petesch M., Miller A., Nolen H. W., II, Musallam H. A., Pick R.
O., Jones D. E., Koplovitz I., Sultan W. E., J. Med. Chem., 32, 493—
503 (1989); g) Druzgala P., Winwood D., Drewniak-Deyrup M., Smith
S., Bodr N., Kaminski J. J., Pharm. Res., 9, 372—377 (1992); h)
11) Hosteler J. S., Hanson L. H., Stevens D. A., Antimicrob. Agents
Chemother., 36, 477—480 (1992).
12) The compound [14C]-2a was synthesized by alkylation of [14C]-TAK-
456, which was prepared according to the method described in ref. 2
starting from [14C]-1H-1,2,4-triazole.
Davidson S. K., Summers J. B., Albert D. H., Holms J. H., Heyman H. 13) Hayashi R., Kitamoto N., Iizawa Y., Ichikawa T., Itoh K., Kitazaki T.,
R., Magcoc T. J., Conway R. G., Rhein D. A., Carter G. W., J. Med. Okonogi K., Antimicrob. Agents Chemother., submitted.
Chem., 37, 4423—4429 (1994); i) Krise J. P., Zygmunt J., Georg G., 14) Iyer R. P, Yu D, Ho N., Agrawal S., Synth. Commun., 25, 2739—2749
Stella V. J., ibid., 42, 3094—3100 (1999); j) Krise J. P., Narisawa S., (1995).
Stella V. J., J. Pharm. Sci., 88, 922—927 (1999); k) Krise J. P., Char- 15) Bohme H., Budde J., Synthesis, 1971, 588—590.
man W. N., Charman S. A., Stella V. J., ibid., 88, 928—932 (1999).
6) Duffin G. F., Kendall J. D., Waddington H. R. J., J. Chem. Soc., 1959,
3799—3808.
7) The structure of 2a was determined by X-ray crystallographic analysis
of the bromide of 2a. The data are not shown.
16) Li Z., Bitha P., Lang S. A., Jr., Lin Y., Bioorg. Med. Chem. Lett., 7,
2909—2912, (1997).
17) Wei Y., Wang D., Li J. S., Chan T. H., J. Org. Chem., 54, 5768—5774
(1989).
18) Reed L. J., Muench H., Am. J. Hyg., 27, 493 (1938).
8) The solubility of 2 in saline was considerably low, probably due to