Optically active antifungal azoles. XIII. Synthesis of stereoisomers and metabolites of 1-[(1Rr,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 (TAK-456)

Article abstract of DOI:10.1248/cpb.49.1110

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)phenyll-2-imidazolidinone [(1R,2R)-1: TAK-456] is a new antifungal agent selected as a candidate for clinical trials. The three stereoisomers [(1S

Full text of DOI:10.1248/cpb.49.1110

1110
Chem. Pharm. Bull. 49(9) 1110—1119 (2001)
Vol. 49, No. 9
Optically Active Antifungal Azoles. XIII.1) Synthesis of Stereoisomers and
Metabolites 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 (TAK-456)
Takashi ICHIKAWA, ,a Masami YAMADA,b Masashi YAMAGUCHI,c Tomoyuki KITAZAKI,b
*
a
Yoshihiro MATSUSHITA,a Keiko HIGASHIKAWA,a and Katsumi ITOH
Medicinal Chemistry Research Laboratories I,a Medicinal Chemistry Research Laboratories IIb and Drug Analysis &
Pharmacokinetic Research Laboratories,c 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 [(1R,2R)-1: TAK-456] is a new antifungal agent selected as a candidate for clini-
cal trials. The three stereoisomers [(1S,2R)-, (1S,2S)- and (1R,2S)-1] of this compound were prepared as authen-
tic samples to determine the enantiomeric and diastereomeric purity of TAK-456 as well as to compare their in
vitro antifungal activity. Pharmacokinetic studies of TAK-456 using rats identified the existence of metabolites in
the liver homogenate. The structures of the major metabolites were assigned as 4-hydroxy-2-imidazolidinone (3)
and/or 5-hydroxy-2-imidazolidinone (4), based on HPLC and LC/MS/MS analyses. These hydroxylated com-
pounds, 3 and 4, were prepared by reduction of the corresponding imidazolidinediones, 11 and 12, and con-
firmed to be identical to the metabolites by HPLC. In vitro antifungal activities of the three stereoisomers and the
synthesized metabolites were considerably weaker than TAK-456.
Key words TAK-456; optically active antifungal triazole; stereoisomer; metabolite; stereocontrolled synthesis; antifungal
activity
We have recently reported the synthesis and potent anti- the three stereoisomers [(1S,2R)-, (1S,2S)- and (1R,2S)-1]
fungal activity of 1-[(1R,2R)-2-(2,4-difluorophenyl)-2-hy- were needed as authentic samples to determine the enan-
droxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-3-[4-(1H-1- tiomeric and diastereomeric purity of the TAK-456 bulk syn-
tetrazolyl)phenyl]-2-imidazolidinone [(1R,2R)-1: TAK-456]2) thesis product, as well as to compare the in vitro antifungal
and the water-soluble triazolium salt 2 (TAK-457: prodrug of activities of the stereoisomers.
TAK-456)1) (Chart 1). Since these compounds exhibited po-
In addition, pharmacokinetic studies on TAK-456 using
tent therapeutic activities against various models of candidia- rats revealed the existence of some metabolites in the liver
sis and aspergillosis using mice, TAK-456 and TAK-457 homogenate. The structures of the major metabolites were
were designated as the candidates for development of two assigned as 1-[(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-
types of formulation for oral and intravenous administra- methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-4-hydroxy-3-[4-(1H-
tions, respectively, in clinical use.3)
1-tetrazolyl)phenyl]-2-imidazolidinone (3: Chart 1) and/or 1-
In the course of development of these antifungal agents, [(1R,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-methyl-3-(1H-
Chart 1
To whom correspondence should be addressed. e-mail: Ichikawa_Takashi@takeda.co.jp
© 2001 Pharmaceutical Society of Japan

September 2001
1111
Chart 2
1,2,4-triazol-1-yl)propyl]-5-hydroxy-3-[4-(1H-1-tetrazolyl)- lowed by catalytic hydrogenation of (1S,2R)-9 over palladium
phenyl]-2-imidazolidinone (4: Chart 1) based on the results on carbon (Pd–C) in acetic acid (AcOH) to afford (1S,2R)-1.
of HPLC and LC/MS/MS analyses. Definitive structural de- For synthesis of the other two stereoisomers, the oxiranes 6
termination and synthesis of the metabolites were necessary with (2S,3R)- and (2S,3S)-configurations were prepared from
in order to use these compounds as the authentic samples for the oxiranylethanols, (1S,2ЈS)-54b,e) and (1R,2ЈS)-5,4b) respec-
future metabolic studies in various animals.
tively. These oxiranes were converted to the corresponding
In this paper, we describe the stereocontrolled synthesis of imidazolidinones, (1S,2S)-1 and (1R,2S)-1, via the same se-
the three stereoisomers of TAK-456 and the structural deter- quence of reactions used for (1S,2R)-1. The physicochemical
mination of the metabolites by chemical synthesis, as well as properties for the three stereoisomers prepared are shown in
the in vitro antifungal activities of these compounds in com- Table 1 together with those for TAK-456.2a) The diastere-
parison with TAK-456.
omeric (% de) and enantiomeric (% ee) purities were deter-
Synthesis We have previously reported an improved mined by HPLC on an octadecyl silica (ODS) column and a
route for the synthesis of TAK-456 starting from (2R,3S)-2- chiral stationary column, respectively. The four stereoiso-
(2,4-difluorophenyl)-2-(1H-1,2,4-triazol-1-yl)methyl-3- mers are each of sufficient purity to enable comparison of
methyloxirane [(2R,3S)-6],2b) which was derived from methyl their in vitro antifungal activities.
(R)-lactate by stereocontrolled synthesis.4a) Preparation of the
During preliminary pharmacokinetic studies on TAK-456,
three stereoisomers was carried out using optically active HPLC analysis of rat liver homogenate was carried out after
oxiranes 64a,c,e—g) with the appropriate stereochemistry as the oral administration of TAK-456. Some peaks in the chro-
key intermediates via the same synthetic route used for TAK- matograph were observed at shorter retention times com-
456 as shown in Chart 2.
pared with that of TAK-456 as shown in Fig. 1 and were con-
Thus, the optically active oxiranylethanol (1S,2ЈR)-54b,d,e) sidered to be metabolites with increased hydrophilicity. The
was converted to the oxirane (2R,3R)-64a) in 71% yield major metabolite (peak A) was analyzed by LC/MS/MS and
by reaction with methanesulfonyl chloride (MsCl) in the the molecular weight was determined to be 497; i.e., 16 mass
presence of triethylamine (Et3N) followed by treatment units larger than that of TAK-456, suggesting that oxidation
with the sodium salt of 1H-1,2,4-triazole (Tri-H).5) Com- had occurred on the skeleton of TAK-456. Since the ethylene
pound (2R,3R)-6 was reacted with 2,2-diethoxyethylamine moiety in the imidazolidin-2-one nucleus appears to be the
[H2NCH2CH(OEt)2] in the presence of titanium tetraiso- most susceptible to metabolic oxidation in TAK-456, the
propoxide [Ti(O-isoPr)4] in propanol (PrOH) to give the structures of the major metabolites were thought to be the 4-
ring-opened product (2R,3S)-7 as an oil in 52% yield, which and/or 5-hydroxy-2-imidazolidinones, 3 and/or 4. We ex-
was then treated with phenyl 4-(1H-1-tetrazolyl)phenylcarba- pected that the hydroxy-2-imidazolidinone could be smoothly
mate (10)2) at 80 °C in N,N-dimethylformamide (DMF) to dehydrated to form the 2-imidazolone under acidic condi-
give the urea (1S,2R)-8 (79% isolated yield). Subsequent in- tions and thus the rat liver homogenate containing the
tramolecular cyclization of (1S,2R)-8 under acidic conditions metabolites was treated with 6 N HCl and then analyzed by
using 1 N hydrochloric acid in methanol (1 N HCl–MeOH) HPLC. As expected, peak A disappeared and another strong
gave the imidazolone (1S,2R)-9 in 82% yield which was fol- peak appeared at the same retention time as that of the 2-imi-

1112
Vol. 49, No. 9
Table 1. Physicochemical Properties of Stereoisomers (1)
Analysis (%)
Calcd (Found)
[a]D
{°C}
(c)
mp (°C)
IR (KBr)
cmϪ1
dec)
(%)
eed)
(%)
Compd.
Yield (%)
1H-NMR d
(Solvent)b)
C
H
N
[Solvent]
(1R,2R)-1: TAK-456a) 211—213
C22H21F2N9O2
1.08 (3H, d, Jϭ7 Hz), 3.69—4.14 (4H, m), 4.52 (1H, 3400, 3120, Ϫ60.6° Ͼ99 Ͼ99
(EtOH)
54.88 4.40 26.18 d, Jϭ14 Hz), 4.65—4.80 (1H, m), 5.12 (1H, d, Jϭ
(54.79 4.42 26.00) 14 Hz), 5.35 (1H, br), 6.74—6.84 (2H, m), 7.36—
7.49 (1H, m), 7.68 (2H, d, Jϭ9 Hz), 7.77 (1H, s),
7.82 (2H, d, Jϭ9 Hz), 7.87 (1H, s), 8.98 (1H, s) (in
CDCl3)
1680, 1610,
1520, 1498,
1480
{20}
(1.0)
[MeOH]
(1S,2R)-1
(1S,2S)-1
(1R,2S)-1
30
19
19
206—207
(EtOH)
C22H21F2N9O2
54.88 4.40 26.18 d, Jϭ14 Hz), 4.63 (1H, q, Jϭ7 Hz), 4.88 (1H, d, Jϭ 1705, 1680,
(54.92 4.30 26.12) 14 Hz), 6.16 (1H, s), 6.77—6.87 (1H, m), 7.01— 1524, 1429,
1.37 (3H, d, Jϭ7 Hz), 3,42—3.74 (4H, m), 4.56 (1H, 3241, 3135, Ϫ91.7° Ͼ99
95.3
99.9
94.3
{20}
(1.0)
[DMSO]
7.13 (1H, m), 7.32—7.44 (1H, m), 7.62 (2H, d, Jϭ 1271
9 Hz), 7.70 (1H, s), 7.79 (2H, d, Jϭ9 Hz), 8.29 (1H,
s), 10.00 (1H, s) (in DMSO-d6)
210—211
(EtOH)
C24H21F2N7O3
1.09 (3H, d, Jϭ7 Hz), 3,68—4.13 (4H, m), 4.52 (1H, 3400, 3121, ϩ60.7° Ͼ99
54.88 4.40 26.18 d, Jϭ14 Hz), 4.72 (1H, q, Jϭ7 Hz), 5.12 (1H, d, Jϭ 1682, 1617,
{20}
(1.0)
[MeOH]
(54.64 4.39 26.16) 14 Hz), 5.35 (1H, br), 6.73—6.85 (2H, m), 7.37—
7.49 (1H, m), 7.68 (2H, d, Jϭ9 Hz), 7.77 (1H, s),
7.82 (2H, d, Jϭ9 Hz), 7.87 (1H, s), 8.98 (1H, s) (in
CDCl3)
1524, 1424,
1267
207—208
(EtOH)
C24H21F2N7O3
1.37 (3H, d, Jϭ7 Hz), 3,42—3.74 (4H, m), 4.56 (1H, 3243, 3135, ϩ88.1° Ͼ99
54.88 4.40 26.18 d, Jϭ14 Hz), 4.63 (1H, q, Jϭ7 Hz), 4.88 (1H, d, Jϭ 1705, 1680,
(55.01 4.38 26.00) 14 Hz), 6.16 (1H, s), 6.78—6.86 (1H, m), 7.01—7.12 1524, 1429,
(1H, m), 7.32—7.45 (1H, m), 7.62 (2H, d, Jϭ9 Hz), 1271
7.69 (1H, s), 7.78 (2H, d, Jϭ9 Hz), 8.29 (1H, s),
{20}
(1.0)
[DMSO]
10.00 (1H, s) (in DMSO-d6)
a) Reported in ref. 2a. b) Recrystallization solvent. c) Determined by HPLC using an ODS column. d) Determined by HPLC using Chiralpak AS.
In this case, condensation and the subsequent ring-closure
proceeded smoothly, and the desired imidazolidine-2,4-dione
11 was obtained in 78% isolated yield.
The reduction of 11 with sodium borohydride (NaBH4)
was carried out in ethanol (EtOH) at 0 °C. The HPLC and
TLC analyses of the reaction mixture indicated that two
products, a less polar product and a more polar product, were
formed in a ratio of approximately 2 : 1.7) Treatment of the
products with a catalytic amount of hydrochloric acid gave
the imidazolone (1R,2R)-9, exclusively.8) Therefore, these
were considered to be the epimers of the desired 4-hydroxy-
2-imidazolidinones (3a, b) resulting from the newly formed
chiral center attached to the hydroxyl group in the imidazo-
lidinone nucleus. During separation of the two epimers by
chromatography, it was suspected that epimerization oc-
curred to some extent on the silica gel, because clear separa-
tion could not be attained. The less polar epimer (3a: major
product) was obtained as colorless prisms by recrystallization
of the residue from the eluate containing 3a as the major
component. Purification of the more polar epimer (3b: minor
product) was carried out by ODS column chromatography
under neutral conditions affording 3b as an amorphous pow-
der. In order to examine the liability of 3a, b to epimeriza-
tion, the 3a-enriched mixture (3a : 3bϭ97 : 3) was heated at
50—60 °C in EtOH. HPLC analysis of the resulting solution
indicated that the ratio of 3b increased with time and that
Fig. 1. Chromatograms of Rat Liver Homogenate
dazolone (1R,2R)-9.2a) Furthermore, LC/MS/MS analysis in-
dicated that its molecular weight was identical to that of
(1R,2R)-9. On the basis of the above analytical results, we
planned to synthesize 3 and 4 to confirm the structure of the
major metabolites, as well as to determine the stereochem-
istry of the new chiral center attached to the hydroxyl group.
Our initial retrosynthetic analysis for compounds 3 and 4
is shown in Chart 3. The final step is reduction of the imida-
zolidinediones, 11 and 12, to the corresponding hydroxyimi-
dazolidinones, 3 and 4. Compounds 11 and 12 may be de-
rived from the esters 13 and 14 which could be used as the
precursors for ring-closure and could be prepared via appro-
priate functionallization of (2R,3R)-3-amino-2-(2,4-difluo-
rophenyl)-1-(1H-1,2,4-triazol-1-yl)-2-butanol (15).6)
equilibrium was reached after 6 h with
a ratio of
The synthetic routes that were initially investigated are
shown in Chart 4. Compound 15 was allowed to react with
ethyl bromoacetate (BrCH2COOEt) in the presence of Et3N
to give the N-substituted glycinate 16 in 80% yield. Then,
compound 16 was reacted with the carbamate 10 to give 13
under similar conditions to those used for the synthesis of 8.
3a : 3bϭ44 : 56.8)
The physicochemical properties of 3a and 3b are shown in
Table 2 and support the structural assignment. However, the
stereochemistry of the newly formed chiral center at the 4-
position in the imidazolidinone nucleus could not be deter-

September 2001
1113
Chart 3
Chart 4
mined by spectroscopic methods such as NMR. Thus crys- not give the desired product and resulted in decomposition or
talline 3a was submitted to X-ray crystallographic analysis recovery of the starting materials. Another approach to pre-
and the stereochemistry of the 4-position in 3a was deter- pare 14 by reaction of compound 18a or more reactive 18b
mined to be (S)-configuration relative to the known (R)-con- with the glycinates 19 also failed to proceed successfully.
figurations of the two chiral centers in the trisubstituted bu-
We then turned our efforts to investigating a new route
tanol skeleton as shown in Fig. 2. Therefore, the configura- which involves ring closure of the carbamate 22 to the 2,5-
tion of the 4-position in the other epimer 3b was assigned to dione 12 as the key step (Chart 5). We carried out the synthe-
be (R). Compounds 3a and 3b prepared above were analyzed sis of 22 via two routes.
by HPLC using the same conditions as used for analysis of
rat liver homogenate and confirmed to be identical to the sponding acid 20 in 74% yield, which was then condensed
peaks B and C (Fig. 1), respectively. with the amine 15 in the presence of diethyl cyanophospho-
Firstly, the ethyl glycinate 19 was hydrolyzed to the corre-
Next, we attempted to prepare the 2,5-dione 12 according nate (DEPC) and Et3N in DMF to obtain the amide 21 (88%
to the synthetic plan shown in Chart 3. However, reaction of isolated yield). Compound 21 was allowed to react with ethyl
15 with the phenylcabamate 17 to obtain the precursor 14 did chlorocarbonate (ClCOOEt) to give the desired product 22 in

1114
Vol. 49, No. 9
Table 2. Physicochemical Properties of 1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-4- and 5-Hydroxy-3-[4-
(1H-1-tetrazolyl)phenyl]-2-imidazolidinones
Analysis (%)
Calcd (Found)
[a]D
{°C}
(c)
mp (°C)
IR (KBr)
cmϪ1
Compd.
1H-NMR (in DMSO-d6) d
(Solvent)a)
C
H
N
MeOH
3a
211.5—212.5
C22H21F2N9O3
0.98 (3H, d, Jϭ7.0 Hz), 3.37 (1H, d, Jϭ11 Hz), 4.17 (1H, dd, Jϭ11, 7.4 Hz), 3438, 3134, Ϫ37.9°
(Acetone–AcOEt) 53.12 4.25 25.34
4.53 (1H, d, Jϭ14 Hz), 4.78 (1H, q, Jϭ7.0 Hz), 4.87 (1H, d, Jϭ14 Hz), 5.79 1688, 1615,
{20}
(1.0)
(53.06 4.38 25.38) (1H, d, Jϭ16 Hz), 5.83 (1H, d, Jϭ16 Hz), 6.69 (1H, d, Jϭ8.8 Hz), 6.86—
1522, 1426,
1260, 1206,
1134, 1105
3123, 2984,
[SI-MS: 498 (MHϩ)] 6.96 (1H, m), 7.09—7.34 (2H, m), 7.63 (1H, s), 7.88 (2H, Jϭ9.0 Hz), 8.00
(2H, d, Jϭ9.0 Hz), 8.27 (1H, s), 10.05 (1H, s).
b)
3b
Amorphous
Amorphous
C22H21F2N9O3
·1/2AcOEt
53.23 4.65 23.28
0.92 (3H, d, Jϭ7.0 Hz), 3.72 (1H, d, Jϭ10 Hz), 3.80—3.89 (1H, m), 4.70
—
(1H, d, Jϭ14 Hz), 4.73—4.87 (1H, m), 4.85 (1H, d, Jϭ14 Hz), 5.81 (1H, d, 1701, 1617,
Jϭ15 Hz), 5.84 (1H, d, Jϭ15 Hz), 6.71 (1H, d, Jϭ8.6 Hz), 6.91—7.00 (1H, 1524, 1431,
(52.96 4.59 23.03) m), 7.20—7.36 (2H, m), 7.69 (1H, s), 7.80 (2H, d, Jϭ9.0 Hz), 8.00 (2H, d,
1267, 1207,
1140, 1094
3108, 1676,
[SI-MS: 498 (MHϩ)] Jϭ9.0 Hz), 8.27 (1H, s), 10.06 (1H, s)
4a
4b
C22H21F2N9O3
1.10 (3H, d, Jϭ7.0 Hz), 3.73 (1H, d, Jϭ10 Hz), 4.22—4.31 (1H, m), 4.52
—
[SI-MS: 498 (MHϩ)] (1H, d, Jϭ14 Hz), 4.60—4.78 (1H, m), 4.83 (1H, d, Jϭ14 Hz), 5.59 (1H, br), 1618, 1524,
6.59 (1H, br), 6.86—6.95 (1H, m), 7.13—7.24 (1H, m), 7.29—7.40 (1H, m), 1267, 1138
7.60 (1H, s), 7.90 (4H, s), 8.27 (1H, s), 10.06 (1H, s).
153—154
(AcOEt)
C22H21F2N9O3
·1/2H2O
52.17 4.38 24.89
1.03 (3H, d, Jϭ7.0 Hz), 3.84 (1H, d, Jϭ10.6 Hz), 4.26 (1H, dd, Jϭ10.6,
7.2 Hz), 4.61 (1H, d, Jϭ15 Hz), 4.65—4.96 (2H, m), 4.93 (1H, d, Jϭ15 Hz), 1709, 1620,
5.43 (1H, d, Jϭ6.2 Hz), 6.00—6.60 (1H, br), 6.86—6.94 (1H, m), 7.16—
3258, 2718, Ϫ74.8°
{20}
(0.5)
1609, 1524,
1420, 1258,
1136, 1044
(52.26 4.54 24.77) 7.36 (2H, m), 7.59 (1H, s), 7.91 (4H, s), 8.20 (1H, s), 10.07 (1H, s).
[SI-MS: 498 (MHϩ)]
a) Recrystallization solvent. b) Not measured.
was only 27% with the 23% recovery of 22.
The 2,5-dione 12 was reduced to the 5-hydroxy compound
4 under conditions similar to those used for the synthesis of
3a, b. Formation of two products, 4a (less polar, minor) and
4b (more polar, major), was observed on TLC10) which were
considered to be epimers analogous to 3a, b.11) However, in
contrast with the case of 3a, b, these two epimers are stable
on silica gel and the less polar epimer 4a could be purified by
column chromatography and was obtained as an amorphous
powder in 13% isolated yield. The more polar epimer 4b was
obtained in 67% isolated yield as colorless prisms by recrys-
tallization after silica gel chromatography. The physicochem-
ical properties of 4a and 4b are shown in Table 2. The stereo-
chemical assignment of 5-position was also carried out by X-
ray crystallographic analysis using a crystal of 4b, and the
stereochemistry of the 5-carbon atom in 4b was determined
to be (S)-configuration as shown in Fig. 3. Therefore, the
configuration of the 5-position of the other epimer 4a was as-
signed to be (R).
Fig. 2. Molecular View of 3a
Compounds 4a and 4b prepared above were examined by
HPLC under the same conditions as used for analysis of the
rat liver homogenate, and confirmed to be identical to the
peak A (Fig. 1). At present, it is unclear whether peak A in-
cludes a single or both epimers 4a, b, because the epimers
were not separable on ODS under the conditions used in this
analysis.
In addition to the identification of the metabolites 3 and 4
by HPLC, the small peak D (Fig. 1) was identified as the imi-
dazolone (1R,2R)-9, which was presumed to be generated
from the metabolites 3 and 4 via dehydration.
Antifungal Activity The stereoisomers [(1S,2R)-,
(1S,2S)-, (1R,2S)-1] and the metabolites (3a, b and 4a, b)
prepared in this report were evaluated for in vitro antifungal
activity for comparison with TAK-456, and the results are
shown in Table 3. The activity is expressed as the minimum
inhibitory concentration (MIC, mg/ml). The MIC values for
75% yield. The second route starting from the tert-butyl gly-
cinate 23 also proceeded successfully. Compound 23 was re-
acted with ClCOOEt to obtain the carbamate 24 (85% yield).
The tert-butyl moiety of 24 was removed by treatment with
hydrogen chloride and the resulting acid 25 (85% yield) was
condensed with 15 in the presence of DEPC to obtain 22 in
87% isolated yield. In contrast to 13, compound 22 was sta-
ble and ring closure to 12 did not occur under neutral condi-
tions even at elevated temperature (80→100 °C). Albericio
and Barany reported that tetrabutylammonium fluoride
(TBAF) is an effective catalyst for ring-closure reactions to
obtain hydantoins.9) Thus we investigated the conversion of
22→12 using this catalyst. However, although use of TBAF
did give the desired compound 12, the conversion of 22 to 12
was low and we attempted to improve the yield of 12 by
modifying the reaction conditions. Nevertheless, even under
the optimized conditions, the isolated yield of the desired 12

September 2001
1115
yeast type fungi such as Candida and Cryptococcus species
were determined by an agar dilution method using RPMI
1640 medium under 20% CO2,12) and MIC values for As-
pergillus species were measured using the same medium
under ordinary air. As can be seen in Table 3, the antifungal
activities of the stereoisomers and the metabolites against
various fungi are considerably weaker compared to those of
TAK-456. The order of potency among the stereoisomers is
(1R,2R)ϾϾ(1S,2R)Ն(1S,2S)Ͼ(1R,2S), which is similar to our
previous results obtained in a series of optically active 1,2,3-
trisubstituted butanols.4a—c) Among the metabolites, 4a and
4b showed some activity, however the potency is about ten
times less than that of TAK-456.
In conclusion, we have carried out the synthesis of the
three stereoisomers of TAK-456 [(1S,2R)-, (1S,2S)-, (1R,2S)-
1] and established an efficient route for synthesis of the four
metabolites, 3a, b and 4a, b. The stereoisomers and the
metabolites exhibited considerably weaker antifungal activity
against various fungi compared with TAK-456. These
metabolites 3 and 4 were confirmed to exist in the rat liver
Fig. 3. Molecular View of 4b
Chart 5
Table 3. In Vitro Antifungal Activities
Fungus
MIC (mg/ml)
Strain
(1R,2R)-1 (1S,2R)-1 (1S,2S)-1 (1R,2S)-1
3a
3b
4a
4b
TAK-456
Candida albicans
IFO 0583
TA
TIMM1756
CA383
IFO 10241
IFO 1162
IFO 0619
TIMM1740
IFO 6344
437
0.016
0.016
0.03
4
0.25
2
0.25
0.25
0.5
1
1
4
2
2
4
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
16
Ͼ16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
4
4
16
4
4
8
0.25
0.25
1
0.25
0.5
0.5
Ͼ16
16
Ͼ16
8
Ͼ16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ16
Ͼ16
Ͼ16
8
Ͼ16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
Ͼ16
16
Ͼ16
Ͼ16
Candida tropicalis
Candida krusei
Candida utilis
Cryptococcus neoformans
Aspergillus fumigatus
4
Ͼ16
4
16
64
64
64
64
2
Ͼ16
4
Ͼ16
Ͼ16
Ͼ16
Ͼ64
Ͼ64
Ͼ64
Ͼ64
8
32
32
32
32
0.5
0.5
1
TIMM1728
IFO 4414
Aspergillus niger

1116
Vol. 49, No. 9
(190 ml). The mixture was stirred overnight, then 1 N HCl (23 ml) was
added. The whole was concentrated in vacuo to afford a residue, which was
submitted to chromatography on silica gel (AcOEt–hexane, 1 : 6, v/v) to give
(1R,2ЈS)-5 (2.87 g, 87%) as a colorless oil. This alcohol was identical to the
authentic sample4b) upon direct comparison.
homogenate after oral administration of TAK-456. Detailed
pharmacokinetic profiles of the candidates, TAK-456 and
TAK-457, will be reported in due course.
Experimental
(2R,3R)-2-(2,4-Difluorophenyl)-3-methyl-2-(1H-1,2,4-triazol-1-yl)-
Melting points were determined using a Yanagimoto melting point appa- methyloxirane [(2R,3R)-6] Et3N (5.43 ml) was added dropwise to an ice-
ratus and are uncorrected. IR spectra were measured with JASCO IR-810 cooled solution of (1S,2ЈR)-5 (5.2 g) and MsCl (4.46 g) in AcOEt (132 ml).
spectrometer. 1H-NMR spectra were recorded on a Varian Gemini-200 spec- The mixture was stirred for 30 min, then worked up (AcOEt; water, aqueous
trometer with tetramethylsilane as an internal standard. The following abbre- NaHCO3, brine) to give [(1S)-1-[(2R)-2-(2,4-difluorophenyl)-2-oxiranyl]-
viations are used: sϭsinglet, dϭdoublet, tϭtriplet, qϭquartet, mϭmultiplet ethyl] methanesulfonate as a colorless oil, which was used for the next step
and brϭbroad. The secondary ion mass spectra (SI-MS) were measured with without purification.
a Hitachi M-80A mass spectrometer. The optical rotations were recorded
with a Jasco DIP-181 or DIP-370 digital polarimeter.
Tri-H (6.28 g) was added to a stirred mixture of sodium hydride (NaH,
60% in oil, 3.12 g) and DMF (140 ml) at 0 °C. The whole was stirred for
Reactions were carried out at room temperature unless otherwise noted 10 min, then the mesylate prepared above was added. The resulting mixture
and followed by TLC on Silica-gel 60 F254 precoated TLC plates (E. Merck) was stirred for 5.5 h at 50 °C, and worked up (AcOEt; water, brine). The
or by HPLC using an ODS column (A-303, 4.6 mm i.d.ϫ250 mm, YMC residue was purified by chromatography on silica gel (AcOEt–hexane,
Co., Ltd.). Standard work-up procedures were as follows. The reaction mix- 55 : 45→3 : 2, v/v) to give (2R,3R)-6 [4.6 g, 71% from (1S,2ЈR)-5] as a col-
ture was partitioned between the indicated organic solvent such as ethyl ac- orless oil. The oxiranes, (2S,3R)-6 and (2S,3S)-6, were prepared according to
etate (AcOEt) and water. Organic extracts were combined and washed in the the same way as above from the corresponding oxiranylethanols, (1S,2ЈS)-5
indicated order using the following aqueous solutions: water, 5% aqueous and (1R,2ЈS)-5, in yields of 67% and 76%, respectively. These oxiranes pre-
sodium carbonate solution (aqueous NaHCO3), saturated sodium chloride pared were identical to the authentic samples,4a) respectively, upon direct
(NaCl) solution (brine), 1 N aqueous sodium hydroxide solution (1 N NaOH) comparison.
and 1 N HCl. Extracts were dried over anhydrous magnesium sulfate
(2R,3S)-3-(2,2-Diethoxyethyl)amino-2-(2,4-difluorophenyl)-1-(1H-
(MgSO4), filtered and evaporated in vacuo. Chromatographic separations 1,2,4-triazol-1-yl)-2-butanol [(2R,3S)-7] A mixture of (2R,3R)-6 (4.17 g),
were carried out on Silica-gel 60 (0.063—0.200 mm, E. Merck) or an ODS Ti(O-isoPr)4 (7.35 ml), H2NCH2CH(OEt)2 (48.3 ml) and PrOH (50 ml) was
column (CPO-273L, pre-packed column, 22 mmϫ300 mm, Kusano Kagaku stirred under reflux for 5.5 h under a nitrogen atmosphere. The solvent and
Kikai Co.) using the indicated eluents.
H2NCH2CH(OEt)2 were distilled off under reduced pressure. The residue
(1S)-1-[(2R)-2-(2,4-Difluorophenyl)-2-oxiranyl]ethanol [(1S,2؅R)-5] was diluted with AcOEt (50 ml). 2 N NaOH (50 ml) saturated with NaCl was
This compound was prepared according to the method described in our pre- added and the resulting mixture was stirred for 1 h. A milky precipitate was
vious report.4d)
filtered off and washed with AcOEt (50 ml). The filtrate and the washing
(1S)-1-[(2S)-2-(2,4-Difluorophenyl)-2-oxiranyl]ethanol [(1S,2؅S)-5]
A
were combined and extracted with 1 N HCl (50 mlϫ3). The aqueous extracts
mixture of 2-(2,4-difluorophenyl)-2-[(1S)-1-(3,4,5,6-tetrahydro-2H-pyran-2- were combined and neutralized with 2 N NaOH saturated with NaCl (75 ml)
yloxy)ethyl]oxirane13) (49.6 g), pyridinium p-toluenesulfonate (4.3 g) and at 0 °C. The whole was extracted with AcOEt (150 ml). The extract was
EtOH (600 ml) was stirred for 1.5 h at 55 °C, then concentrated in vacuo and washed with water (100 ml) and brine (100 ml), successively, dried over
worked up (AcOEt; water, brine). The residue was purified by flash chro- MgSO4 and evaporated in vacuo. The residue was purified by chromatogra-
matography on silica gel [hexane–AcOEt, 20 : 1→10 : 1→5 : 1, v/v] to give phy on silica gel (AcOEt–hexane, 4 : 1, v/v) and crystallized from AcOEt–
(1S)-1-[2-(2,4-difluorophenyl)-2-oxiranyl]ethanol (23.5 g, 70%) as a pale hexane to give (2R,3S)-7 (3.3 g, 52%) as a white crystalline powder. mp
1
yellow oil. H-NMR (CDCl3) d: 1.15—1.22 (3H, m), 1.78, 2.03 (1H), 2.80, 81—82 °C. Anal. Calcd for C18H26F2N4O3: C, 56.24; H, 6.82; N, 14.57.
2.92 (1H), 3.28—3.31 (1H, m), 4.02—4.18 (1H, m), 6.76—6.95 (2H, m), Found: C, 56.06; H, 6.52; N, 14.29. 1H-NMR (CDCl3) d: 1.10 (3H, d,
7.32—7.48 (1H, m).
Jϭ7 Hz), 1.06—1.13 (1H, br), 1.16 (3H, t, Jϭ7 Hz), 1.25 (3H, d, Jϭ6.6 Hz),
This oil (21.1 g) and 3,5-dinitrobenzoyl chloride (31.7 g) were dissolved 2.23 (1H, dd, Jϭ12, 5.2 Hz), 2.57 (1H, dd, Jϭ12, 5.8 Hz), 3.20 (1H, q,
in tetrahydrofuran (THF, 360 ml). Et3N (19.1 ml) was added dropwise to this Jϭ6.6 Hz), 3.33—3.68 (4H, m), 4.34 (1H, dd, Jϭ5.8, 5.2 Hz), 4.56 (1H, d,
solution at 0 °C. After having been stirred for 2 h, the mixture was washed Jϭ14 Hz), 4.81 (1H, d, Jϭ14 z), 5.03 (1H, s), 6.69—6.81 (2H, m), 7.37—
(water, aqueous NaHCO3) and concentrated in vacuo. The residue was chro- 7.50 (1H, m), 7.69 (1H, s), 8.08 (1H, s). IR (KBr): 3320, 2976, 1617, 1599,
matographed on silica gel (hexane–AcOEt, 20 : 1→10 : 1→5 : 1, v/v) and 1499, 1418, 1273, 1123, 1061, 966 cmϪ1. [a]2D0: Ϫ29.4° (cϭ0.99, MeOH).
crystallized from AcOEt–MeOH (1 : 1, v/v, 100 ml) to give [(1S)-1-[(2S)-2-
(2S,3S)-7: The oxirane (2S,3R)-6 was allowed to react with H2NCH2CH-
(2,4-difluorophenyl)-2-oxiranyl]ethyl] 3,5-dinitrobenzoate (15.5 g, 38%) as (OEt)2 to obtain (2S,3S)-7 in the same manner as described above. Yield
colorless needles. mp 108.5—109.5 °C. Anal. Calcd for C17H12F2N2O7: C, 85% (a pale yellow oil). Anal. Calcd for C18H26F2N4O3: C, 56.24; H, 6.82;
51.79; H, 3.10; N, 7.10. Found: C, 51.81; H, 3.07; N, 7.15. 1H-NMR N, 14.57. Found: C, 56.11; H, 6.70; N, 14.57. 1H-NMR (CDCl3) d: 0.91
(CDCl3) d: 1.46 (3H, dd, Jϭ6.6, 1.6 Hz), 3.01 (1H, d, Jϭ4.6 Hz), 3.23 (1H, (3H, d, Jϭ6.6 Hz), 1.00—1.20 (1H, br), 1.23 (6H, t, Jϭ7 Hz), 2.67 (1H, dd,
d, Jϭ4.6 Hz), 5.34 (1H, q, Jϭ6.6 Hz), 6.85—7.06 (2H, m), 7.48—7.59 (1H, Jϭ12, 4.6 Hz), 2.95 (1H, dd, Jϭ12, 6.4 Hz), 3.11 (1H, q, Jϭ6.6 Hz), 3.48—
m), 9.13 (2H, d, Jϭ2.2 Hz), 9.25 (1H, t, Jϭ2.2 Hz). IR (KBr): 1734, 1549, 3.80 (4H, m), 4.55 (1H, dd, Jϭ6.4, 4.6 Hz), 4.75 (1H, d, Jϭ14 Hz), 4.86
1346, 1279, 1169 cmϪ1. [a]2D0: ϩ23.2° (cϭ1.0, CHCl3).
(1H, s), 4.89 (1H, d, Jϭ14 Hz), 6.70—6.81 (2H, m), 7.33—7.45 (1H, m),
A 1 N NaOH solution (36 ml) was added dropwise to a solution of the 7.76 (1H, s), 7.93 (1H, s). IR (neat): 2976, 1617, 1501, 1273, 1136, 1100,
dinitrobenzoate prepared above (7.04 g) in MeOH (350 ml). The mixture 1063, 964 cmϪ1. [a]2D0: ϩ70.2° (cϭ1.0, MeOH).
was stirred for 1 h, then 1 N HCl (18 ml) was added. The whole was concen-
(2S,3R)-7: The oxirane (2S,3S)-6 was allowed to react with H2NCH2CH-
trated in vacuo and worked up (AcOEt; brine) to afford a residue, which was (OEt)2 to obtain (2S,3R)-7 in the same manner as described above. Yield
purified by chromatography on silica gel (AcOEt–hexane, 1 : 4→1 : 3, v/v ) 72% (a white crystalline powder). mp 82—83 °C (AcOEt–hexane). Anal.
to give (1S,2ЈS)-5 (3.5 g, 98%) as a pale yellow oil. This alcohol was identi- Calcd for C18H26F2N4O3: C, 56.24; H, 6.82; N, 14.57. Found: C, 56.49; H,
cal to the authentic sample4b) upon direct comparison.
6.87; N, 14.78. 1H-NMR (CDCl3) d: 1.10 (3H, d, Jϭ7 Hz), 1.13—1.20 (1H,
(1R)-1-[(2S)-2-(2,4-Difluorophenyl)-2-oxiranyl]ethanol [(1R,2؅S)-5] br), 1.16 (3H, t, Jϭ7 Hz), 1.26 (3H, d, Jϭ6.6 Hz), 2.23 (1H, dd, Jϭ12,
Triphenylphosphine (13 g), benzoic acid (6.05 g) and diethyl azodicarboxy- 5.2 Hz), 2.57 (1H, dd, Jϭ12, 5.8 Hz), 3.20 (1H, q, Jϭ6.6 Hz), 3.33—3.70
late (40% in toluene, 21.6 g) were added to an ice-cooled solution of (4H, m), 4.34 (1H, dd, Jϭ5.8, 5.2 Hz), 4.56 (1H, d, Jϭ14 Hz), 4.81 (1H, d,
(1S,2ЈS)-5 (4.96 g) in THF (100 ml). The mixture was stirred for 1 d, then Jϭ14 Hz), 5.03 (1H, s), 6.69—6.81 (2H, m), 7.37—7.49 (1H, m), 7.68 (1H,
worked up (AcOEt; aqueous NaHCO3, water, brine), and the residue was s), 8.07 (1H, s). IR (KBr): 3314, 2976, 1615, 1599, 1499, 1418, 1273, 1121,
chromatographed on silica gel (AcOEt–hexane, 1 : 30, v/v) to give [(1R)-1- 1063, 966 cmϪ1. [a]2D0: ϩ31.2° (cϭ1.0, MeOH).
[(2S)-2-(2,4-difluorophenyl)-2-oxiranyl]ethyl] benzoate (5.15 g, 68%) as a
1-(2,2-Diethoxyethyl)-1-[(1S,2R)-2-(2,4-difluorophenyl)-2-hydroxy-1-
colorless oil. 1H-NMR (CDCl3) d: 1.38 (3H, d, Jϭ6.6 Hz), 2.89 (1H, d, methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-
Jϭ5.2 Hz), 3.28 (1H, d, Jϭ5.2 Hz), 5.36 (1H, q, Jϭ6.6 Hz), 6.75—6.93 (2H, urea [(1S,2R)-8] A mixture of (2R,3S)-7 (2.85 g), 10 (1.88 g) and DMF
m), 7.38—7.60 (4H, m), 7.95—8.00 (2H, m). IR (neat): 1719, 1617, 1601, (29 ml) was stirred for 6 h at 80 °C under a nitrogen atmosphere. The mix-
1508, 1453, 1427, 1267, 1096 cmϪ1
.
ture was cooled and worked up (AcOEt; water, brine). The residue was puri-
A 28% sodium methoxide–MeOH solution (4.12 ml) was added dropwise fied by silica gel column chromatography (AcOEt–hexane, 8.5 : 1, v/v→
to an ice-cooled solution of the benzoate prepared above (5.0 g) in MeOH AcOEt–MeOH, 8 : 1, v/v) to give (1S,2R)-8 (3.33 g, 79%) as a white powder.

September 2001
1117
1
SI-MS (m/z): 572 (MHϩ). H-NMR (CDCl3) d: 1.24 (3H, t, Jϭ7 Hz), 1.32
(3H, t, Jϭ7 Hz), 1.52 (3H, d, Jϭ7 Hz), 3.21 (2H, br), 3.44—3.99 (5H, m),
4.55 (1H, d, Jϭ14 Hz), 4.52—4.61 (1H, m), 4.76 (1H, d, Jϭ14 Hz), 6.72—
6.86 (2H, m), 7.33 (2H, d, Jϭ9 Hz), 7.42—7.55 (1H, m), 7.57 (2H, d,
Jϭ9 Hz), 7.66 (1H, s), 7.83 (1H, s), 8.20 (1H, s), 8.52 (1H, s), 8.93 (1H, s).
zolones [(1S,2S)-9 and (1R,2S)-9].
The % de of the stereoisomers was determined by HPLC using an ODS
column under the following conditions: mobile phase, MeOH–water–AcOH,
7 : 3 : 0.02, v/v; flow rate, 0.8 ml/min; detection, UV at 262 nm. The % ee of
the stereoisomers was determined by HPLC using a chiral stationary phase
column (Chiralpak AS 4.6 mm i.d.ϫ250 mm, Daicel Chemical Industries,
Tokyo, Japan) under the following conditions: mobile phase, hexane–EtOH,
3 : 7, v/v; flow rate, 0.8 ml/min; detection, UV at 262 nm.
Ethyl N-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-
1,2,4-triazol-1-yl)propyl]glycinate (16) A mixture of 15 (1.04 g),6) THF
(15 ml), BrCH2COOEt (0.65 ml) and Et3N (1.08 ml) was stirred for 22 h
under a nitrogen atmosphere. An additional amount of BrCH2COOEt
(0.22 ml) and Et3N (0.27 ml) was added to the reaction mixture. The whole
was stirred for further 63 h under a nitrogen atmosphere and concentrated in
vacuo. The concentrate was dissolved in AcOEt (70 ml) and washed with
5% aqueous. NaHCO3 (35 ml) and brine (35 ml). The organic layer was
dried over MgSO4 and evaporated in vacuo. The residue was purified by sil-
ica gel column chromatography (AcOEt–hexane, 1 : 3→1 : 4, v/v) to give 16
(1.1 g, 80%) as a colorless oil. 1H-NMR (CDCl3) d: 0.93 (3H, dd, Jϭ6.6,
1.2 Hz), 1.30 (3H, t, Jϭ7 Hz), 1.46 (1H, br), 3.11 (1H, dq, Jϭ6.6, 1.8 Hz),
4.22 (2H, q, Jϭ7 Hz), 4.82 (1H, d, Jϭ14 Hz), 4.95 (1H, d, Jϭ14 Hz), 4.96
(1H, s), 6.69—6.81 (2H, m), 7.36—7.49 (1H, m), 7.76 (1H, s), 7.94 (1H, s).
IR (KBr): 3331, 2980, 1736, 1618, 1597, 1501, 1420, 1273, 1138 cmϪ1. SI-
MS (m/z): 355 (MHϩ).
1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-tri-
azol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2,4-imidazolidinedione
(11) A mixture of 16 (0.92 g), 10 (0.88 g) and DMF (10 ml) was stirred at
80 °C for 20 h under a nitrogen atmosphere. After having been cooled, the
mixture was diluted with AcOEt (100 ml) and washed with water (50 ml).
The aqueous layer was extracted with AcOEt (30 ml). The AcOEt layers
were combined and washed with 0.1 N HCl (50 ml), water (50 ml) and brine
(50 ml), successively. The organic layer was dried over MgSO4 and evapo-
rated in vacuo. The residue was purified by silica gel column chromatogra-
phy (AcOEt–hexane, 6 : 1→AcOEt–acetone, 6 : 1, v/v) and crystallized from
AcOEt–isoPr2O to give 11 (1.0 g, 78%) as white powdery crystals. mp
136—137 °C. Anal. Calcd for C22H19F2N9O3: C, 53.33; H, 3.87; N, 25.44.
Found: C, 53.10; H, 3.89; N, 25.15. 1H-NMR (CDCl3) d: 1.13 (3H, d,
Jϭ7 Hz), 4.27 (1H, d, Jϭ19 Hz), 4.47 (1H, d, Jϭ14 Hz), 4.69 (1H, d,
Jϭ19 Hz), 5.00 (1H, dq, Jϭ7, 1.8 Hz), 5.17 (1H, d, Jϭ14 Hz), 5.39 (1H, d,
Jϭ1.8 Hz), 6.75—6.87 (2H, m), 7.34—7.46 (1H, m), 7.76—7.88 (6H, m),
9.06 (1H, s). IR (KBr): 3385, 3127, 1771, 1715, 1617, 1524, 1424,
1207 cmϪ1. [a]2D0: Ϫ65.6° (cϭ1.0, MeOH).
(4S)-1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-
1,2,4-triazol-1-yl)propyl]-4-hydroxy-3-[4-(1H-1-tetrazolyl)phenyl]-2-imi-
dazolidinone (3a: Table 2) and (4R)-1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-
hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-4-hydroxy-3-[4-(1H-
1-tetrazolyl)phenyl]-2-imidazolidinone (3b: Table 2) Compound 11
(0.70 g) was added to a stirred solution of NaBH4 (0.11 g) in EtOH (60 ml)
at 0 °C under a nitrogen atmosphere. The mixture was stirred at 0 °C under a
nitrogen atmosphere. During stirring, NaBH4 (0.11 gϫ3) was added to the
mixture at 30 min intervals. After the final addition of NaBH4, the whole
was stirred for further 1.5 h at 0 °C under a nitrogen atmosphere and diluted
with AcOEt (100 ml). The solution was washed with brine (50 mlϫ3), dried
over MgSO4 and evaporated in vacuo. The residue was purified by silica gel
column chromatography (AcOEt→AcOEt–acetone, 8 : 1, v/v). The first elu-
ate containing 3a as the major component was evaporated in vacuo. The
residue was crystallized from AcOEt to give a white crystalline powder,
which was recrystallized from acetone-AcOEt to give 3a (0.22 g, 31%) as
colorless prisms. The second eluate containing 3a and 3b in a ratio of 36 : 64
was evaporated in vacuo to give a white residue (0.12 g, 17%), which was
separated using ODS column chromatography [acetonitrile (MeCN)–water,
3 : 7, v/v; flow rate 0.3 ml/min]. The fractions containing 3b was combined
and evaporated in vacuo. The residue was dissolved in AcOEt (100 ml) and
washed with water (50 ml) and brine (40 ml), successively. The AcOEt layer
was dried over MgSO4 and evaporated in vacuo to give 3b (0.042 g, 6%) as a
white amorphous powder.
IR (KBr): 3308, 3114, 2978, 1651, 1522, 1499, 1472, 1273, 1132 cmϪ1
.
[a]2D0: ϩ169.4° (cϭ1.0, MeOH).
(1S,2S)-8: Compound (2S,3S)-7 was converted to (1S,2S)-8 by the reac-
tion with 10 under the same condition as described above. Yield 93% (a
white crystalline powder). mp 179—181 °C [AcOEt–diisopropyl ether
(isoPr2O)]. Anal. Calcd for C26H31F2N9O4: C, 54.63; H, 5.74; N, 22.05.
Found: C, 54.38; H, 5.37; N, 22.07. 1H-NMR (CDCl3) d: 1.08 (1.5H, d,
Jϭ7 Hz), 1.24 (1.5H, d, Jϭ7 Hz), 1.31—1.40 (6H, m), 3.52—4.09 (7H, m),
4.39 (0.5H, d, Jϭ14 Hz), 4.56 (0.5H, d, Jϭ14 Hz), 4.76 (0.5H, m), 4.93—
5.08 (1H, m), 5.26—5.47 (1.5H, m), 6.73—6.83 (2H, m), 7.32—7.68 (5.5H,
m), 7.79 (0.5H, s), 7.82 (0.5H, s), 8.24 (0.5H, s), 8.85 (0.5H, br), 8.94 (0.5H,
s), 8.96 (0.5H, s), 9.15 (0.5H, s). IR (KBr): 3312, 3112, 2978, 1651, 1524,
1499, 1470, 1248, 1134 cmϪ1. [a]2D0: ϩ110.4° (cϭ1.0, MeOH).
(1R,2S)-8: Compound (2S,3R)-7 was converted to (1R,2S)-8 by the reac-
tion with 10 under the same condition as described above. Yield 82% (a
white powder). SI-MS (m/z): 572 (MHϩ). H-NMR (CDCl3) d: 1.24 (3H, t,
1
Jϭ7 Hz), 1.32 (3H, t, Jϭ7 Hz), 1.51 (3H, d, Jϭ7 Hz), 3.21 (1H, br), 3.44—
3.98 (6H, m), 4.55 (1H, d, Jϭ14 Hz), 4.52—4.63 (1H, m), 4.75 (1H, d,
Jϭ14 Hz), 6.71—6.86 (2H, m), 7.33 (2H, d, Jϭ9 Hz), 7.42—7.55 (1H, m),
7.57 (2H, d, Jϭ9 Hz), 7.66 (1H, s), 7.83 (1H, s), 8.20 (1H, s), 8.52 (1H, s),
8.92 (1H, s). IR (KBr): 3312, 3131, 2978, 1651, 1524, 1499, 1472, 1273,
1132 cmϪ1. [a]2D0: Ϫ172.4° (cϭ1.0, MeOH).
1-[(1S,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-tri-
azol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2-(1H,3H)-imidazolone
[(1S,2R)-9] 1 N HCl (46.5 ml) was added to a solution of (1S,2R)-8 (2.66 g)
in MeOH (47 ml). The mixture was stirred for 5 h at 55 °C and cooled. After
having been neutralized with 2 N NaOH (23.5 ml), the whole was evaporated
in vacuo to remove MeOH and extracted with AcOEt–THF (1 : 1, v/v;
120 ml). The extract was washed with water (50 ml) and brine (50 ml), suc-
cessively, dried over MgSO4 and evaporated in vacuo. The residue was puri-
fied by silica gel column chromatography (AcOEt→AcOEt/MeOH, 7 : 1,
v/v) and crystallized from AcOEt–THF to give (1S,2R)-9 (1.83 g, 82%) as a
white crystalline powder. mp 203—204 °C. Anal. Calcd for C22H119F2N9O2:
C, 55.11; H, 3.99; N, 26.29. Found: C, 55.08; H, 4.19; N, 26.10. H-NMR
(CDCl3) d: 1.61 (3H, d, Jϭ7 Hz), 4.57 (1H, d, Jϭ14 Hz), 4.82 (1H, q,
Jϭ7 Hz), 4.88 (1H, d, Jϭ14 Hz), 6.31—6.46 (3H, m), 6.63—6.78 (2H, m),
7.35—7.47 (1H, m), 7.64 (2H, d, Jϭ9 Hz), 7.73 (2H, d, Jϭ9 Hz), 7.75 (1H,
s), 8.06 (1H, s), 8.99 (1H, s). IR (KBr): 3125, 1684, 1617, 1526, 1429, 1271,
1256, 733 cmϪ1. [a]2D0: Ϫ97.1° [cϭ1.0, dimethylsulfoxide (DMSO)].
(1S,2S)-9: Compound (1S,2S)-8 was converted to (1S,2S)-9 under the
same condition as described above. Yield 64% (a white crystalline powder).
mp 196—197 °C (AcOEt–isoPr2O). Anal. Calcd for C22H19F2N9O2: C,
55.11; H, 3.99; N, 26.29. Found: C, 55.07; H, 4.05; N, 26.27. 1H-NMR
(CDCl3) d: 1.22 (3H, d, Jϭ7 Hz), 4.22 (1H, d, Jϭ14 Hz), 5.03 (1H, q,
Jϭ7 Hz), 5.15 (1H, d, Jϭ14 Hz), 5.46 (1H, br), 6.74—6.88 (4H, m), 7.42—
7.54 (1H, m), 7.76 (1H, s), 7.83 (2H, d, Jϭ9 Hz), 7.87 (1H, s), 7.97 (2H, d,
Jϭ9 Hz), 9.08 (1H, s). IR (KBr): 3400, 3127, 1682, 1615, 1524, 1429, 1254,
1140 cmϪ1. [a]2D0: ϩ15.4° (cϭ1.0, MeOH).
(1R,2S)-9: Compound (1R,2S)-8 was converted to (1R,2S)-9 under the
same condition as described above. Yield 74% (a white crystalline powder).
mp 204—205 °C (AcOEt–THF). Anal. Calcd for C22H19F2N9O2: C, 55.11;
H, 3.99; N, 26.29. Found: C, 54.84; H, 3.94; N, 25.99. 1H-NMR (CDCl3) d:
1.61 (3H, d, Jϭ7 Hz), 4.57 (1H, d, Jϭ14 Hz), 4.81 (1H, q, Jϭ7 Hz), 4.88
(1H, d, Jϭ14 Hz), 6.34—6.46 (3H, m), 6.63—6.78 (2H, m), 7.35—7.47
(1H, m), 7.63 (2H, d, Jϭ9 Hz), 7.73 (2H, d, Jϭ9 Hz), 7.75 (1H, s), 8.06 (1H,
s), 8.98 (1H, s). IR (KBr): 3127, 1682, 1617, 1526, 1429, 1271, 1256 cmϪ1
.
[a]2D0: ϩ99.0° (cϭ1.0, DMSO).
1-[(1S,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-tri-
azol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2-imidazolidinone
[(1S,2R)-1: Table 1] A solution of (1S,2R)-9 (1.03 g) in AcOH (75 ml)
was hydrogenated over 10% Pd–C (50% wet, 1.03 g) for 24 h under an at-
mospheric pressure. The catalyst was filtered off and washed with AcOH
(10 ml). The filtrate and the washing were combined and concentrated in
vacuo. The concentrate was worked up (AcOEt; water, aqueous. NaHCO3,
brine). The residue was purified by silica gel column chromatography
(AcOEt–MeOH, 7 : 1, v/v) and crystallized from EtOH to give (1S,2R)-1
(0.31 g, 30%) as white powdery crystals.
Ethyl N-[4-(1H-1-Tetrazolyl)phenyl]glycinate (19) A mixture of 1-(4-
aminophenyl)-1H-tetrazole (6.0 g),2a,b) THF (100 ml), BrCH2COOEt (4.56
ml) and Et3N (5.7 ml) was stirred for 4 h at 50 °C. An additional amount of
BrCH2COOEt (4.56 ml) and Et3N (5.7 ml) was added to the reaction mix-
ture. The whole was stirred for further 16 h at 50 °C. After having been
cooled, the mixture was worked up (AcOEt; water, brine). Diethyl ether
(Et2O, 100 ml) was added to the residue and the resulting precipitates were
collected by filtration to give 19 (8.77 g, 95%) as a white powder. Anal.
(1S,2S)-1 and (1R,2S)-1 (Table 1): These stereoisomers were prepared in
the same manner as above from the corresponding optically active imida-

1118
Vol. 49, No. 9
Calcd for C11H13N5O2: C, 53.43; H, 5.30; N, 28.32. Found: C, 53.43; H, (24) Pyridine (0.30 ml) and ClCOOEt (0.36 ml) were added dropwise to a
1
5.25; N, 28.06. H-NMR (DMSO-d6) d: 1.22 (3H, t, Jϭ7 Hz), 3.99 (2H, d, solution of 23 (0.43 g) in a mixture of DMF (2 ml) and THF (8 ml). After the
Jϭ6 Hz), 4.14 (2H, q, Jϭ7 Hz), 6.58 (1H, br), 6.75 (2H, d, Jϭ8.8 Hz), 7.56 mixture was stirred for 2 h, an additional amount of ClCOOEt (0.36 ml) was
(2H, d, Jϭ8.8 Hz), 9.85 (1H, s). IR (KBr): 3384, 3140, 1725, 1611, 1534,
added. After having been stirred for 15 h, the whole was diluted with AcOEt
(50 ml), washed with 5% aqueous phosphoric acid solution (30 ml), water
1231, 828 cmϪ1
.
Ethyl N-Phenoxycarbonyl-N-[4-(1H-1-tetrazolyl)phenyl]glycinate (17) (30 ml) and brine (20 ml), successively. The AcOEt layer was dried over
Pyridine (0.65 ml) and phenyl chlorocarbonate (PhOCOCl, 1.21 ml) were MgSO4 and concentrated in vacuo. The concentrate was purified by silica
added dropwise to a solution of 19 (1.0 g) in a mixture of DMF (10 ml) and
gel column chromatography (AcOEt–hexane, 1 : 1, v/v) and crystallized
THF (10 ml). After having been stirred for 5 h, the whole was worked up from AcOEt–isoPr2O to give 24 (0.46 mg, 85%) as white powdery crystals.
(AcOEt; water). The residue was purified by silica gel column chromatogra-
mp 113—114 °C. Anal. Calcd for C16H21N5O4: C, 55.32; H, 6.09; N, 20.16.
phy (AcOEt–hexane, 2 : 1, v/v) to give 17 (0.90 g, 61%) as a colorless oil. Found: C, 55.55; H, 5.91; N, 20.41. 1H-NMR (CDCl3) d: 1.26 (3H, t,
Anal. Calcd for C181H17N5O4: C, 58.85; H, 4.66; N, 19.06. Found: C, 58.72; Jϭ7 Hz), 1.49 (9H, s), 4.23 (2H, q, Jϭ7 Hz), 4.28 (2H, s), 7.55 (2H, d,
H, 4.65; N, 18.89. H-NMR (CDCl3) d: 1.31 (3H, t, Jϭ7 Hz), 4.28 (2H, q,
Jϭ9 Hz), 7.70 (2H, d, Jϭ9 Hz), 8.98 (1H, s). IR (KBr): 3123, 2982, 1742,
Jϭ7 Hz), 4.51 (2H, s), 7.11—7.40 (5H, m), 7.68 (1H, d, Jϭ8.8 Hz), 7.76 1709, 1522, 1231, 1155 cmϪ1
(1H, d, Jϭ8.8 Hz), 9.03 (1H, s). N-Ethoxycarbonyl-N-[4-(1H-1-tetrazolyl)phenyl]glycine (25) 4 N Hy-
Phenyl N-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H- drogen chloride–AcOEt solution (20 ml) was added to a solution of 24
1,2,4-triazol-1-yl)propyl]carbamate (18a) PhOCOCl (0.41 ml) was (0.49 g) in AcOEt (10 ml). After having been stirred for 17 h, the mixture
.
added dropwise to a solution of 15 (1.07 g) in a mixture of pyridine was diluted with AcOEt (70 ml). The AcOEt solution was washed with brine
(0.24 ml) and DMF (20 ml). After having been stirred for 1 h, the whole was (40 ml) and water (40 ml), successively, dried over MgSO4 and evaporated in
1
worked up (AcOEt; water). The residue was purified by silica gel column vacuo to give 25 (0.38 g, 85%) as a colorless oil. H-NMR (CDCl3) d: 1.26
chromatography (AcOEt–hexane, 1 : 1→5 : 2, v/v) and crystallized from (3H, t, Jϭ7 Hz), 4.2 (2H, q, Jϭ7 Hz), 4.4 (2H, s), 7.5 (2H, d, Jϭ8.8 Hz),
1
Et2O–isoPr2O to give 18a (1.0 g, 85%) as white powdery crystals. H-NMR 7.73 (2H, d, Jϭ8.8 Hz), 8.00 (1H, br), 9.07 (1H, s). IR (KBr): 3125, 2986,
(CDCl3) d: 1.01 (3H, d, Jϭ7 Hz), 4.48–4.56 (1H, m), 4.63 (1H, d, Jϭ14 Hz), 1705, 1607, 1522, 1381, 1213 cmϪ1
.
5.04 (1H, d, Jϭ14 Hz), 5.25 (1H, d, Jϭ1.4 Hz), 5.65 (1H, d, Jϭ9 Hz),
6.72—6.82 (2H, m), 7.15—7.43 (6H, m), 7.79 (1H, s), 7.83 (1H, s).
4-Nitrophenyl N-[(1R,2R)-2-(2,4-Difluorophenyl)]-2-hydroxy-1-methyl-
Ethyl N-[2-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-
(1H-1,2,4-triazol-1-yl)propyl]amino-2-oxoethyl]-N-[4-(1H-1-tetrazolyl)-
phenyl]carbamate (22) 1) Pyridine (0.49 ml) and ClCOOEt (0.59 ml)
3-(1H-1,2,4-triazol-1-yl)propyl]carbamate (18b) 4-Nitrophenyl chloro- were added successively to a solution of 21 (1.2 g) in DMF (20 ml) at 0 °C.
carbonate (0.89 ml) was added dropwise to a solution of 15 (1.07 g) in a After having been stirred for 17 h, the mixture was worked up (AcOEt;
mixture of pyridine (0.32 ml) and THF (20 ml). After having been stirred for water). The residue was purified by silica gel column chromatography
1 h, the whole was worked up (AcOEt; water). The residue was purified by (AcOEt→AcOEt–THF, 5 : 1, v/v) and crystallized from isoPr2O to give 22
silica gel column chromatography (AcOEt–hexane, 1 : 1→3 : 1, v/v) to give (1.02 g, 75%) as white powdery crystals. mp 183.5—184.5 °C. Anal. Calcd
1
18b (0.34 g, 20%) as a white powder. H-NMR (CDCl3) d: 1.03 (3H, d, Jϭ for C24H25F2N9O4: C, 53.23; H, 4.65; N, 23.28. Found: C, 53.12; H, 4.79; N,
7 Hz), 4.48—4.57 (1H, m), 4.63 (1H, d, Jϭ14 Hz), 5.02 (1H, d, Jϭ14 Hz), 23.06. 1H-NMR (CDCl3) d: 0.93 (3H, d, Jϭ6.6 Hz), 1.26 (3H, t, Jϭ7.2 Hz),
5.30 (1H, d, Jϭ1.4 Hz), 5.76 (1H, d, Jϭ14 Hz), 6.74—6.87 (2H, m), 7.31—
4.11—4.92 (5H, m), 4.90 (1H, q, Jϭ6.6 Hz), 4.93 (1H, d, Jϭ14 Hz), 5.26
7.44 (3H, m), 7.81 (1H, s), 7.85 (1H, s), 8.24—8.32 (2H, m).
(1H, s), 6.53—6.82 (3H, m), 7.35—7.50 (1H, m), 7.67—7.79 (4H, m), 8.99
N-[4-(1H-1-tetrazolyl)phenyl]glycine (20) 1 N NaOH (25.4 ml) and (1H, s). IR (KBr): 3316, 1699, 1617, 1522, 1273, 1140 cmϪ1. [a]2D0: Ϫ17.4°
water (100 ml) were added to a solution of 19 (5.23 g) in a mixture of THF
(cϭ1.0, MeOH).
(150 ml) and MeOH (100 ml). After having been stirred for 30 min, the mix-
2) DEPC (0.21 ml) was added dropwise over a period of 5 min to a mix-
ture was neutralized with AcOH (4 ml) and concentrated in vacuo. The con- ture of 25 (0.38 g), 15 (0.35 g), Et3N (0.2 ml) and DMF (11.5 ml) at 0 °C
centrate was dissolved in a mixture of AcOEt (100 ml) and THF (50 ml). under a nitrogen atmosphere. After having been stirred for 1.5 h, the mixture
The solution was washed with water (100 ml), dried over MgSO4 and con-
was worked up (AcOEt; water, brine). The residue was purified by silica gel
centrated in vacuo. The precipitated crystals were collected by filtration and column chromatography (AcOEt–THF, 5 : 1→1 : 1, v/v) and crystallized
dried over phosphorous pentoxide (P2O5) at 40 °C in vacuo to obtain 20
(3.84 g, 74%) as pale brown powdery crystals. mp 202—203 °C. H-NMR
from isoPr2O–AcOEt to give 22 (0.61 g, 87%) as white powdery crystals.
1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-tri-
1
(DMSO-d6) d: 3.89 (2H, s), 6.48 (1H, br), 6.74 (2H, d, Jϭ8.8 Hz), 7.55 (2H, azol-1-yl)propyl]-3-[4-(1H-1-tetrazolyl)phenyl]-2,5-imidazolidinedione
d, Jϭ8.8 Hz), 9.84 (1H, s). IR (KBr): 3393, 2892, 1725, 1613, 1534, 1217,
828 cmϪ1
N-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-1,2,4-
(12) TBAF (9.23 ml) was added dropwise over a period of 5 min to a solu-
tion of 22 (5.0 g) in THF (200 ml). After having been stirred for 1 h, the mix-
ture was stirred for further 8 h at 30 °C. AcOH (0.52 ml) was added to the
.
triazol-1-yl)propyl]-2-[4-(1H-1-tetrazolyl)phenyl]amimoacetamide (21) mixture, and the resulting mixture was worked up (AcOEt; brine). The
DEPC (0.72 ml) and Et3N (0.6 ml) were added successively to a mixture of residue was purified by silica gel column chromatography (AcOEt–Et2O,
15 (1.07 g), 20 (0.87 g) and DMF (30 ml) at 0 °C. After having been stirred
4 : 1, v/v) to give 12 (1.23 g, 27%) as a white powder with recovery of 22
for 1 h, the mixture was worked up (AcOEt; water). The residue was purified (1.15 g, 23%). Anal. Calcd for C22H19F2N9O3·1/2AcOEt: C, 53.43; H, 4.30;
1
by chromatography on silica gel (AcOEt–THF, 2 : 5, v/v) and the eluate was N, 23.37. Found: C, 53.40; H, 4.36; N, 23.31. H-NMR (DMSO-d6) d: 1.27
concentrated in vacuo. The precipitated crystals were collected by filtration (3H, d, Jϭ7 Hz), 4.58—5.02 (5H, m), 5.83 (4/7H, s), 5.91 (3/7H, s), 6.89—
and dried over P2O5 in vacuo. Compound 21 (1.65 g, 88%) was obtained as 6.97 (1H, m), 7.16—7.26 (1H, m), 7.32—7.48 (1H, m), 7.61 (1H, s), 7.92—
1
white powdery crystals. mp 89.5—90.5 °C. H-NMR (CDCl3) d: 0.89 (3H, 8.03 (4H, m), 8.27 (1H, d, Jϭ4 Hz), 10.10 (1H, s). IR (KBr): 3345, 3129,
d, Jϭ7 Hz), 3.97 (2H, s), 4.37 (1H, d, Jϭ14 Hz), 4.81 (2H, q, Jϭ7 Hz), 4.95 1771, 1705, 1618, 1524, 1208, 1138 cmϪ1. [a]2D0: Ϫ54.9° (cϭ1.0, MeOH).
(1H, d, Jϭ14 Hz), 6.68—6.82 (3H, m), 7.02 (1H, br), 7.21—7.38 (1H, m),
7.51 (2H, d, Jϭ8.8 Hz), 7.79 (2H, d, Jϭ8.8 z), 8.89 (1H, s). IR (KBr): 3333,
1665, 1613, 1526, 1501, 1273, 1140 cmϪ1. [a]2D0: Ϫ53.1° (cϭ1.0, MeOH).
(5R)-1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-hydroxy-1-methyl-3-(1H-
1,2,4-triazol-1-yl)propyl]-5-hydroxy-3-[4-(1H-1-tetrazolyl)phenyl]-2-imi-
dazolidinone (4a: Table 2) and (5S)-1-[(1R,2R)-2-(2,4-Difluorophenyl)-2-
tert-Butyl N-[4-(1H-1-Tetrazolyl)phenyl]glycinate (23) A mixture hydroxy-1-methyl-3-(1H-1,2,4-triazol-1-yl)propyl]-5-hydroxy-3-[4-(1H-
of 1-(4-aminophenyl)-1H-tetrazole (1.02 g),2a,b) DMF (30 ml), tert-butyl 1-tetrazolyl)phenyl]-2-imidazolidinone (4b: Table 2) NaBH4 (0.55 g)
chloroacetate (1.36 ml), Et3N (1.32 ml) and potassium iodide (KI, 10.5 g) was added to a solution of 12 (2.56 g) in EtOH (170 ml) at 0 °C under a ni-
was stirred for 15 h at 50 °C under a nitrogen atmosphere. An additional trogen atmosphere. After having been stirred for 2.5 h at 0 °C, an additional
amount of tert-butyl chloroacetate (20.4 ml), Et3N (15.8 ml) and KI (31.5 g) amount of NaBH4 (0.1 g) was added to the mixture. The whole was stirred
was added to the mixture. The whole was stirred for further 5 d at 50 °C, for further 2.5 h at 0 °C and diluted with AcOEt (350 ml), water (170 ml) and
cooled and worked up (AcOEt; water, brine). The residue was purified by brine (170 ml). AcOEt layer was separated and the aqueous layer was ex-
silica gel column chromatography (AcOEt–hexane, 1 : 1, v/v) and crystal- tracted with AcOEt (100 ml). The AcOEt layers were combined, washed
lized from AcOEt to give 23 (0.49 mg, 28%) as white powdery crystals. mp
with water (10 mlϫ2), dried over MgSO4 and evaporated in vacuo. The
104—105 °C. Anal. Calcd for C13H17N5O2: C, 56.71; H, 6.22; N, 25.44. residue was submitted silica gel column chromatography (AcOEt–acetone,
1
Found: C, 56.75; H, 6.19; N, 25.55. H-NMR (CDCl3) d: 1.51 (9H, s), 3.86
(2H, d, Jϭ5.2 Hz), 4.68 (1H, br), 6.67—6.73 (2H, m), 7.44—7.50 (2H, m),
12 : 1, v/v). The eluate containing 4a and 4b was evaporated in vacuo and
the residue was crystallized from acetone–MeOH–AcOEt to give the more
polar product 4b (1.71 g, 67%) as colorless prisms. The mother liquor of this
8.85 (1H, s). IR (KBr): 3368, 1723, 1611, 1534, 1235, 1161 cmϪ1
.
tert-Butyl N-Ethoxycarbonyl-N-[4-(1H-1-tetrazolyl)phenyl]glycinate crystallization was evaporated in vacuo and the residue was submitted to sil-

September 2001
1119
were solved by direct methods using the program SIR9214) for 3a, and using
the program SYSTEM9015) for 4b. The structures were then refined by the
full-matrix least-squares refinement (SHELXL-9716)) with anisotropic tem-
perature factors for the non-hydrogen atoms and isotropic temperature fac-
tors for the hydrogen atoms. Crystal data for 3a; C22H21F2N9O3; Mϭ497.46;
orthorhombic, space group P212121 (#19), aϭ15.750 (2), bϭ23.339 (4),
cϭ6.214 (4) Å, Vϭ2284 (1) Å3, Zϭ4, Dcϭ1.446 g/cm3, R1ϭ0.046, wR2ϭ
0.128 for 1194 observed reflections with IϾ2s(I). Crystal data for 4b;
C22H21F2N9O3·1/2CH3COOC2H5; Mϭ541.52; orthorhombic, space group
P212121 (#19), aϭ18.166 (4), bϭ32.932 (5), cϭ8.519(5) Å, Vϭ5096 (2) Å3,
Dcϭ1.411 g/cm3, Zϭ8, R1ϭ0.073, wR2ϭ0.252 for 1779 observed reflec-
tions with IϾ2s(I).
ica gel column chromatography (AcOEt–acetone–hexane, 1 : 1 : 1, v/v) to
obtain the less polar product 4a (0.34 g, 13%) as a white amorphous powder.
Antifungal Activity A double concentration of RPMI-1640 medium
(Gibco BRL, Grand Island, N.Y.) was prepared with 0.3 M morpholine-
propanesulfonic acid (MOPS; Dojindo, Tokyo, Japan) buffer (pH 7.0), steril-
ized by filtration through a membrane filter (pore size, 0.45 mm) and mixed
with an equal volume of 3.0% agar (Wako, Osaka, Japan) which had been
autoclaved at 121 °C for 15 min and kept at 55 °C. The agar medium (9.9 ml)
was then poured into petri dishes containing 0.1 ml of serial dilutions of an-
tifungal agents dissolved in DMSO and allowed to solidify. About 103 CFU
of fungal cells suspended in saline was inoculated with a multiple inoculator
(Sakuma, Tokyo, Japan) onto the agar plates prepared above. For the mea-
surement of in vitro antifungal activities against Candida albicans, Candida
tropicalis, Candida krusei, Candida utilis and Cryptococcus neoformans, the
plates were incubated in a CO2 incubator at 35 °C for 20 h. After MICs for
Candida species were determined, the plates were incubated in a CO2 incu-
bator for an additional 48 h to determine MICs for Cryptococcus neofor-
mans. The MIC was defined as the lowest concentration of an antifungal
agent giving no visible growth or causing almost complete inhibition of
growth. In the other hand, the plates inoculated with Aspergillus fumigatus
and Aspergillus niger were incubated in an ordinary incubator at 35 °C for
20 h. The MIC was defined as the lowest concentration of an antifungal
agent giving no visible growth.
Acknowledgements We thank Drs. S. Kishimoto and A. Miyake for
their encouragement throughout this work. We also thank Mr. T. Yoshida for
the biological assay.
References and Notes
1) Part XII: Ichikawa T., Kitazaki T., Matsushita Y., Yamada M., Hayashi
R., Yamaguchi M., Kiyota Y., Okonogi K., Itoh K., Chem. Pharm.
Bull., 49, 1102—1109 (2001).
2) a) Kitazaki T., Ichikawa T., Tasaka A., Hosono H., Matsushita Y.,
Hayashi R., Okonogi K., Itoh K., Chem. Pharm. Bull., 48, 1935—
1946 (2000); b) Ichikawa T., Kitazaki T., Matsushita Y., Hosono H.,
Yamada M., Mizuno M., Itoh K., ibid., 48, 1947—1953 (2000).
3) a) Tsuchimori N., Hayashi R., Kitamoto N., Asai K., Kitazaki T.,
Iizawa Y., Itoh K., Okonogi K., Antimicrob. Agents Chemother., sub-
mitted; b) Hayashi R., Kitamoto N., Iizawa Y., Ichikawa T., Itoh K.,
Kitazaki T., Okonogi K., ibid., submitted.
Analysis of Rat Liver Homogenate The animals used in this study
were male CD (SD) IGS rats (8-weeks old; Charles River Japan Inc., Kana-
gawa, Japan). They were fed laboratory chow (CE-2, CLEA Japan Inc.,
Tokyo, Japan), had free access to water and were housed in temperature- and
humidity-controlled rooms (20 to 26 °C, 40 to 75% R.H.) with 12 h light-
dark cycles for more than a week before use.
TAK-456 was suspended in aqueous 0.5% methylcellulose solution at a
dose of 30 mg/5 ml/kg and administered orally to fed animals. The liver of
rats was excised under ether anesthesia at 4 h after dosing. The liver was im-
mediately homogenized with a 4-fold amount (w/v) of ice-cold saline. The
homogenate was kept frozen at Ϫ80 °C until analysis.
The rat liver homogenate (2 ml) was deproteinized with MeCN (3 ml).
After centrifugation at 19000 g for 10 min, to the supernatant (4 ml) was
added 0.01 N aqueous ammonium acetate solution (0.01 N aqueous NH4OAc,
4 ml) adjusted to pH 6.0 with AcOH. The solution (100 ml) was injected to
an HPLC and analyzed. To the supernatant (1 ml) was added 6 N HCl
(0.2 ml) and the resulting mixture was incubated at 37 °C for 20 min. After
addition of 1 N aqueous NaOH (0.12 ml), the mixture (100 ml) was injected
to an HPLC and analyzed. The eluates corresponding to the peak A and the
new strong peak appeared after HCl-treatment were individually collected,
and the aliquot (50 ml) was injected to an LC/MS/MS to determine the mole-
cular weight.
4) a) Tasaka A., Tamura N., Matsushita Y., Teranishi K., Hayashi R.,
Okonogi K., Itoh K., Chem. Pharm. Bull., 41, 1035—1042 (1993); b)
Tasaka A., Kitazaki T., Tsuchimori N., Matsushita Y., Hayashi R.,
Okonogi K., Itoh K., ibid., 45, 321—326 (1997); c) Tasaka A., Tsuchi-
mori N., Kitazaki T., Hiroe K., Hayashi R., Okonogi K., Itoh K., ibid.,
43, 441—449 (1995); d) Kitazaki T., Tamura N., Tasaka A., Ma-
tsushita Y., Hayashi R., Okonogi K., Itoh K., ibid., 44, 314—327
(1996); e) Konosu T., Miyaoka T., Tajima Y., Oida S., ibid., 39,
2241—2246 (1991); f) Konosu T., Tajima Y., Miyaoka T., Oida S.,
Tetrahedron Lett., 32, 7545—7548 (1991); g) Konosu T., Miyaoka T.,
Tajima Y., Oida S., Chem. Pharm. Bull., 40, 562—564 (1992).
5) This method has been already reported in ref. 4e.
6) a) Konosu T., Tajima Y., Takeda N., Miyaoka T., Kashihara M., Ya-
suda H., Oida S., Chem. Pharm. Bull., 39, 2581—2589 (1991); b)
Bartroli J., Turmo E., Belloc J., Form J., J. Org. Chem., 60, 3000—
3012 (1995).
HPLC conditions: The HPLC system consisted of an LC-10AD pump, an
SIL-10A autosampler, a CTO-10AC column oven, an SPD10AV UV-detec-
tor, and a CLASS-LC10 workstation (all from Shimadzu Co., Kyoto, Japan).
The analytical column was an L-Column ODS (5 mm particle size, 4.6 mm
i.d.ϫ150 mm, Chemicals Evaluation and Research Institute, Tokyo, Japan).
The mobile phase was a mixture of 0.01 N aqueous NH4OAc (pH 6.0, ad-
justed with AcOH) and MeCN (68 : 32, v/v). The column temperature and
the flow-rate were 40 °C and 1.0 ml/min, respectively. UV detection was car-
ried out at 274 nm.
LC/MS/MS conditions: A 717 puls autosampler (Nihon Waters K. K.,
Tokyo, Japan), an LC-10AD pump (Shimadzu Co., Kyoto, Japan) and a
Perkin-Elmer Sciex API 3000 triple quadrupole mass spectrometer (Perkin-
Elmer Sciex, Canada) with a turbo ionspray source for atomospheric pres-
sure ionization were used for the analysis. Data processing was achieved
with the PE software (Sample Control version 1.4 and Multivew version
1.4). The analytical column was an L-column ODS (2.1 mm i.d.ϫ150 mm).
The mobile phase was a mixture of 0.01 N aqueous ammonium formate solu-
tion (pH 3.0, adjusted with formic acid) and MeCN (1 : 1, v/v). The column
temperature and the flow-rate were 40 °C and 0.2 ml/min, respectively. The
ionspray and orifice voltage, and temperature were set at 5400 V, 50 V, and
425 °C, respectively. Data acquisition was in the positive ionization Q1 scan
mode using 1 ms dwell time per transition.
7) The ratio was determined by HPLC on an ODS column under the fol-
lowing conditions: mobile phase, MeOH–H2O–AcOH, 7 : 3 : 0.02, v/v;
flow rate, 0.8 ml/min; detection UV at 262 nm.
8) This conversion was monitored by HPLC using an ODS column and
the conditions shown in ref. 7.
9) Albericio F., Barany G., Int. J. Protein Res., 30, 177—205 (1987).
10) These epimers were not separable on an ODS column under the condi-
tions shown in ref. 7.
11) It was confirmed that the treatment of 4a, b with hydrochloric acid
gave (1R,2R)-9 similarly to the case of 3a, b.
12) 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).
13) Synthesis of this compound was carried out starting from ethyl (S)-lac-
tate according to the method described in ref. 4a. This oxirane is a
mixture of (2S)-2-(2,4-difluorophenyl)-2-[(1S)-1-(3,4,5,6-tetrahydro-
2H-pyran-2-yloxy)ethyl]oxirane and (2R)-2-(2,4-difluorophenyl)-2-
[(1S)-1-(3,4,5,6-tetrahydro-2H-pyran-2-yloxy)ethyl]oxirane in a ratio
of ca 4 : 1 and was used without separation.
14) Altomare A., Burla M. C., Camalli M., Cascarano M., Giacovazzo C.,
Guagliardi A., Polidori G., J. Appl. Cryst., 27, 435 (1994).
15) Yonggeng H., Min G., Lipu L., Peixin H., Acta Cryst., A50, 748—753
(1994).
16) SHELXL-97: Sheldrick G. M. (1997). Program for the refinement of
crystal structures. University of Göttingen, Germany.
X-Ray Crystallographic Analysis A single crystal (0.40ϫ0.20ϫ0.06
mm) of 3a was obtained by recrystallization from acetone. A single crystal
(0.80ϫ0.12ϫ0.05 mm) of 4b was obtained by recrystallization from mixture
of aceton and AcOEt. The reflection data collected on a Rigaku AFC5R dif-
fractometer with graphite monochromated CuKa radiation. The structure