October 1999
`
`Chem. Pharm. Bull. 47(10) 1417--1425 (1999)
`
`1417
`
`Synthesis and Antifungal Activities of (2R,3R)-2-Aryl-l-azolyl-3-
`(substituted amino)-2-butanol Derivatives as Topical Antifungal Agents
`
`Hironobu OGURA,* Haruhito KOBAYASHI, Kiyoshi NAGAI, Zokiko NISH1DA, Takanobu NAITO,
`Yoshiyuki TATSUM1, Mamoru YOKOO, and Tadashi AR~KA
`
`Development Research Laboratories, Kaken Pharmaceutical Co., Ltd., 14, Shinomiya Minamikawara-cho, Yamashina-ku,
`Kyoto 607-8042, Japan. Received May 10, 1999; accepted July 20, 1999
`
`2-Aryl-l-azolyl-3-(substituted amino)-2-butanol derivatives I were prepared by ring-opening reaction of
`epoxides II with excess amine, and their antifungal activities were evaluated as topical agents. Azolyl-cyclic
`amine derivatives with a methylene group showed extremely strong activity with a broad spectrum in vitro. In
`general, anti-Trichophyton mentagrophytes activities of most of the topical antifungal agents are substantially re-
`duced by addition of keratin (a major constituent of the keratinized tissue). However, the triazole derivative
`(2R~3R)-2-(2~4-di~u~ropheny~)-3-(4-methy~enepiperidin~)-1-(1H-~~2~4-triaz~~-1-y~)-2-butan~~ ((-)-40, KP-103)
`showed very little deactivation by addition of keratin. This biological characteristic of triazole derivative (-)-40
`resulted in excellent therapeutic efficacy on dermatophytosis superior to that of the corresponding imidazole de-
`rivative ((-)-41).
`
`Key words KP-103; topical antifungal agents; 4-methylenepiperidine; 1,2,4-triazole; structure-activity relationship
`
`Many kinds of topical antifungal agents have been devel-
`oped and are available as commercial products. These agents
`are classified into a number of groups, i.e. allylamines, ben-
`zylamines, imidazoles, thiocarbamates and morpholines,
`which exhibit quite different biological properties. Benzy-
`lamines and allylamines show strong antifungal activities
`against dermatophytes such as Trichophyton mentagrophytes,
`but show weak activity against yeast-like fungi such as Can-
`dida albicans. Thiocarbamates and morpholines show very
`weak effects against yeast-like fungi and Aspergillus spp., re-
`spectively. Imidazoles have moderate antifungal activities,
`but their activity and spectra are wider than those of the other
`groups. These agents are used for the treatment of dermato-
`mycosis, but relapse of infection is often observed after ther-
`apy. Therefore, there it is still necessary to develop new topi-
`cal antifungal agents with broad spectrum, strong in vitro an-
`tifungal activity and excellent therapeutic efficacy.
`For this purpose, we focused our attention on triazole de-
`rivatives. Similarly to imidazoles, triazoles have been shown
`to inhibit the conversion of lanosterol to ergosterol,J) which
`is an important constituent of the fungal cell membrane. Al-
`though a large number of triazole derivatives have been
`synthesized, only two triazole drugs (fluconazole,2) itracona-
`zole3)) have been developed commercially for treatment of
`systemic fungal infection, but they can not be used topically.
`Among the triazoles currently under development, the sul-
`fonyl derivative SM-866841 (Chart l, active enantiomer has a
`
`O
`N~"X.N~Me / 70-100°C
`
`~on~y amine
`/ protie solvent
`
`[
`
`(2R,3R)-absolute configuration), showed the strongest and
`broadest spectrum antifungal activity, and was used as the
`reference drug in the present study. We replaced the sulfonyl
`group at the 3-position of SM-8668 with a substituted amino
`group.
`Here, we describe the preparation of 2-aryl-l-azolyl-3-
`(substituted amino)-2-butanol derivatives I, which we called
`"azolylamine derivatives," and the antifungal activities of I
`
`Me
`
`N~N~Am
`
`A= CH, N
`X=2,4-F2, 2,4-C12, 4-C1
`Am=substituted amino group
`
`x
`
`F
`
`OCGHll ’ 1-101
`
`SM-8668
`
`Clotrimazole
`
`Neticonazole
`
`Chart 1
`
`¯
`.
`~/~ @X
`~--r~ ~h--R’ 46a
`nmary amme]I
`~p
`] +
`46b
`N#"’N"~’-’~OH
`protic solvent
`Me I R I
`46c
`
`70-100°C
`
`F
`
`46a--¢
`
`* To whom correspondence should be addressed,
`
`© 1999 Pharmaceutical Society of Japan
`
`Chart 2
`
`NII-Electronic Library Service
`
`
`
`
`
`
`ARGENTUM EX1012
`
`Page 1
`
`

`

`1418
`
`against several species of fungi in comparison with those of
`clotrimazole and neticonazole5) (Chart 1). Furthermore, we
`evaluated the differences between triazole and imidazole de-
`rivatives to determine the usefulness of triazole as a topical
`agent.
`Chemistry The azolylamine derivatives I (2----43, Table
`1) were prepared by ring-opening reaction of epoxides II
`with excess amine on heating at 70---100 °C (Chart 2). This
`reaction proceeded more smoothly with the addition of a pro-
`tic solvent such as water or alcohol in comparison with that
`in the presence of only nonprotic solvents. However, when
`primary amines were used for ring-opening reaction of 2,4-
`difluorophenyl-epoxide II (X=2,4-F2), indoline derivatives6)
`(46a--c) were obtained together with the desired com-
`pounds (10, 13, 18). The epoxides II were prepared as de-
`scribed.4a’4c’7) With the exception of commercially available
`
`o
`
`50
`
`o
`
`d, oo
`
`49
`
`(Me2CH)2NLi
`M¢3SiC]
`
`OTMS
`
`Vol. 47, No. 10
`
`RICHO
`
`47
`
`1) MeNH2 ) H.N.CH2_R1
`
`2) NaBI~
`
`Me
`
`48a-e
`
`R1
`
`4~8a)
`
`~)
`48b
`
`8c) ~
`48c
`
`48d
`8~0
`~F CF3
`
`4.8eSe;’
`
`Chart 3
`
`l)
`
`AcONa I N
`
`H
`NH2OH
`2) CF3CO2H
`¯ HCI N’’’’’~ ~Or~
`51
`
`1) HN=CHNH2 . AcOH
`
`Et3N ~ 3CO2H
`
`I) N-brt)mt)succinimid¢ ~N
`"ri
`r
`thiourea
`---
`J,
`II \~-’-N H2
`H N ~v"’~k~S/
`
`2) CF3CO2H
`
`I
`Boo
`
`53
`
`54
`
`52
`
`Boe:tea-BuOCO-
`
`/(¢H2>m~
`~==O
`PhXN
`\ (CH2)n/ X=CH2, CO
`
`Wittig
`reagent
`
`Cha~ 4
`
`/(CH2)n~k
`
`~
`
`PhXN
`\ (CH2)n/
`
`?2 deprotection
`/(CH,)m\ 22
`~ HN
`_~/
`\ (CH2)n
`
`55
`
`56
`
`57 a-- f
`
`HN
`
`--t
`
`1) PhCOCI
`Py
`
`2) NaBH4
`3) SOCI2
`
`N/¢’-~ /CI
`) PhCO~
`
`1) tert-BuOK
`
`2) KOH
`
`58
`
`59
`
`HNO=
`
`57a
`
`R2
`
`H
`
`Me
`
`CH=CH2
`
`Ph
`
`H
`
`57a
`
`57b
`
`57c
`
`57d
`
`57e
`
`57f
`
`2
`
`2
`
`2
`
`2
`
`3
`
`2
`
`2
`
`2
`
`2
`
`2
`
`1
`
`1
`
`Cha~ 5
`
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`
`

`

`October 1999
`
`Table 1. MICs (/lg!ml) of Azolylamine Derivatives
`
`Compd.=)
`
`X
`
`Am
`
`Config.
`
`1c)
`
`2,4-F2
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`1419
`
`M1C (/.tg/ml)b>
`
`Ca.
`
`Cr. n.
`
`A. fu.
`
`T. m.
`
`(KC-03)
`
`(KC-201)
`
`(KA-01)
`
`(KD-04)
`
`25.0
`
`>100
`
`>100
`
`>100
`
`6.25
`
`12.5
`
`0.78
`
`0.78
`
`0.39
`
`O. 1
`
`O. 1
`
`50.0
`
`50.0
`
`1.56
`
`1.56
`
`1.56
`
`0.78
`
`0.39
`
`25.0
`
`25.0
`
`3.13
`
`1.56
`
`3.13
`
`1.56
`
`1.56
`
`-NH2
`
`--N(Me)2
`
`-Q
`-©
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.2
`
`0.2
`
`0.05
`
`0.1
`
`0.1
`
`_ N~).jPh
`
`(2R*,3R*)
`
`<0.025
`
`<0.025
`
`-NN.Ph
`-N’--~o
`,...%
`H
`- N....,%
`.Me
`- N ,,,.~
`
`8
`
`9
`
`10
`
`11
`
`12
`
`13
`
`14
`
`15
`
`16
`
`17
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.05
`
`0.025
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`0.025
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`0.025
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.05
`
`0.05
`
`0.78
`
`3.13
`
`0.78
`
`O. I
`
`3.13
`
`0.39
`
`0.39
`
`0.39
`
`O. 1
`
`3.13
`
`1 O0
`
`3.13
`
`1.56
`
`3.13
`
`50.0
`
`6.25
`
`1.56
`
`50.0
`
`12.5
`
`0.78
`
`3.13
`
`6.25
`
`12.5
`
`6.25
`
`0.78
`
`0.78
`
`1.56
`
`6.25
`
`6.25
`
`18
`
`19
`
`20
`
`21
`
`22
`
`23
`
`24
`
`25
`
`26
`
`27
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`N
`
`(2R*,3R*)
`
`<0.025
`
`0.78
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.025
`
`0.05
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`<0.025
`
`O. 1
`
`0.2
`
`0.2
`
`0.2
`
`0.1
`
`1.56
`
`25.0
`
`>100
`
`1.56
`
`0.78
`
`(2R*,3R*)
`
`0.2
`
`0.78
`
`12.5
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.05
`
`0.2
`
`0.2
`
`0.1
`
`6.25
`
`0.39
`
`0.39
`
`1.56
`
`0.39
`
`0.78
`
`6.25
`
`0.39
`
`1.56
`
`6.25
`
`1.56
`
`1.56
`
`3.13
`
`25.0
`
`25.0
`
`28
`
`29
`
`30
`
`31
`
`32
`
`33
`
`34
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`2,4-F2
`
`(-)-35
`
`2,4-F2
`
`(-)-36
`
`2,4-F2
`
`(-)-37
`
`2,4-F2
`
`N
`
`N
`
`N
`
`N
`
`CH
`
`N
`
`N
`
`N
`
`N
`
`N
`
`(2R*,3R*)
`
`0.78
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`0.78
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`0.05
`
`(2R*,3R*)
`
`<0.025
`
`(2R*,3R*)
`
`<0.025
`
`6.25
`
`0.39
`
`3.13
`
`0.78
`
`1.56
`
`0.1
`
`0.05
`
`1.56
`
`12.5
`
`0.78
`
`6.25
`
`0.39
`
`0.39
`
`0.78
`
`12.5
`
`1.56
`
`1.56
`
`0.78
`
`0.78
`
`(2R,3R)
`
`(2R,3R)
`
`(2R,3R)
`
`<0.025
`
`0.78
`
`>100
`
`>100
`
`<0.025
`
`<0.025
`
`0,1
`
`0.1
`
`0.2
`
`0.2
`
`0.39
`
`0.2
`
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`
`

`

`1420
`
`Table 1. (continued)
`
`Compdfl
`
`X
`
`(-)-38
`
`2,4-F2
`
`(-)-39
`
`2,4-F2
`
`40
`
`2,4-F2
`
`(-)-40
`
`2,4-F2
`
`A
`
`N
`
`N
`
`N
`
`N
`
`Vo|. 47, No. 10
`
`MIC (flg/ml)b)
`
`Am
`
`Config.
`
`C a.
`(KC-03)
`
`Cr. n.
`(KC-201)
`
`A. fu.
`(KA-01)
`
`T. m.
`(KD-04)
`
`-NR
`
`-N~
`
`-N~=
`
`-N~)=~
`
`-N~
`
`(2R,3R)
`
`(2R,3R)
`
`0.1
`
`<0.025
`
`(2R*,3R*)
`
`<0.025
`
`(2R,3R)
`
`<0.025
`
`0.39
`
`0.39
`
`0.2
`
`0.05
`
`0.78
`
`0.39
`
`0.2
`
`0.2
`
`0.78
`
`0.78
`
`0.78
`
`0.39
`
`>100
`
`100
`
`(+)-40
`
`2,4-F2
`
`41
`
`2,4-F2
`
`(-)-41
`
`2,4-F2
`
`42
`
`43
`
`44
`
`2,4-C12
`
`4-CI
`
`Clotrimazole
`
`N
`
`CH
`
`CH
`
`N
`
`N
`
`-N~),~
`
`-N~
`
`-N~=~
`
`- N~:=
`
`(2S,3S)
`
`0.39
`
`(2R*,3R*)
`
`<0.025
`
`1.56
`
`0.05
`
`(2R,3R)
`
`<0.025
`
`_--<0.025
`
`(2R*,3R*)
`
`(2R*,3R*)
`
`0.025
`
`<0.025
`
`_--<0.025
`
`0.2
`
`0.2
`
`0.2
`
`0.2
`
`0.1
`
`0.39
`
`6.25
`
`0.78
`
`0.39
`
`0.39
`
`0.78
`
`3.13
`
`0.39
`
`a) All compounds except (-)-35, (-)-36, (-)-37, (->38, (-)-39, (-)-,10, (+)-40 and (-)-41 were racemic, b) Abbreviations: C a., Candida albicans; Cr. n., Cryptoeoc-
`eus neoformans; A. fu., Aspergillus fumigatus; 77 m., Trichophyton mentagrophytes, c) Compound I was prepared as described.7a!
`
`amines, most of the amines used in this study were prepared
`using the reported methods. For example, N-methyl sec-
`ondary amines such as 48a---e8) were prepared by reductive
`alkylation8~) (Chart 3). Heteroaromatic cyclic amines such
`as 51, 529) and 541°) were prepared from N-(tert-butoxycar-
`bonyl)piperidone (49) in three steps (Chart 4). Cyclic
`amines") with a methylene group or substituted methylene
`group (57a--f) were prepared from N-protected ketones (55)
`by the Wittig reaction and subsequent deprotection. 4-Meth-
`ylenepiperidine (57a) was also prepared from N-benzoyl-4-
`chloromethyl piperidine (59)12) by an elimination reaction
`and subsequent deprotection.
`Antifungal Activity The azolylamine derivatives I (1--
`43) obtained as described above were examined for their in
`vitro activities against Candida albicans, Cryptococcus neo-
`formans, Aspergillus fumigatus and Trichophyton mentagro-
`phytes in comparison with those of clotrimazole, and the
`minimum inhibitory concentrations (MICs) are shown in
`Table 1.
`Many of these compounds showed excellent in vitro anti-
`fungal activities. Their activities were generally exceptionally
`high against C. albicans and good against the others. The ter-
`tiary amine derivatives (11, 12, 14, 19) had higher activities
`than the corresponding secondary amine derivatives (10, 13,
`18). In particular, cyclic amine derivatives (3343) with a
`methylene group or a substituted methylene group showed
`the strongest activities. With the exception of (-)-35, only
`this class showed MIC values of <l.0/.tg/ml against all
`fungi. Among a series of 4-methylenepiperidine derivatives
`(40--43), (2R,3R)-(-)-40 was 16- to 32-fold more active
`than the (2S,3S)-enantiomer (+)-40 against C. albicans and
`C. neoformans, and (+)-40 showed no significant activity
`against A. fumigatus or T. mentagrophytes. The imidazole de-
`rivative ((-)-41) showed similar activity to the triazole deriv-
`ative ((-)-40) against these fimgi. The 2,4-C12 derivative
`(42) showed similar activity to the 2,4-F2 derivative (40),
`whereas the 4-C1 derivative (43) was less active than 40 and
`
`42 against A. fumigatus and T. mentagrophytes. In the in vitro
`assay, the cyclic amine derivatives with a methylene group
`showed strong activities, which were equivalent or superior
`to that of clotrimazole. However, we could not decide on one
`candidate for development based only on MIC values.
`We next examined the effects of keratin (human hair) on
`anti-T, mentagrophytes activity. In general, the activities of
`most topical antifungal agents are greatly reduced by adsorp-
`tion to keratin,13) which is a major constituent of the kera-
`tinized tissue where fungi reside. MICs of some of these de-
`rivatives were determined by the microdilution method using
`Sabourand dextrose broth (SDB) and saline containing 5%
`human hair (Table 2). The anti-T, mentagrophytes activities
`of the reference drugs clotrimazole and neticonazole were
`markedly reduced (32-fold) by addition of human hair, but
`the activities of methylenepiperidine derivatives (33, 34, 40,
`41) were less affected (2--8-fold). The order of deactivation
`for the substituent adjacent to the double bond of these deriv-
`atives (33, 34, 40) was as follows: Ph (34)>Me (33)>H (40).
`This order suggested that addition of a lipophilic substituent
`on the double bond reduced the activity. Furthermore, we
`found that the imidazole derivatives (32, 41) were more
`markedly deactivated than corresponding triazole derivatives
`(31, 40). These results indicated that 4-methylenepiperidino
`triazole derivative (40) had low affinity to keratin and could
`retain a high level of activity in the keratinized tissue.~4)
`To clarify the difference between the triazole derivative
`((-)-40) and the imidazole derivative ((-)-41), we evaluated
`their therapeutic efficacy in the guinea pig model of Tinea
`corporis infection (Fig. 1). Topical treatment was started 3 or
`4d after infection and continued for 10d by once-a-day ap-
`plication. The T. mentagrophytes KD-04 strain used in this
`study has a strong virulence to invade the hair follicles15) and
`can invade the hair follicles deeply on 4 d after infection
`compared with on 3 d after infection. Triazole derivative
`((-)-411) showed high efficacy against Tinea eorporis both
`days after infection. On the other hand, imidazole derivative
`
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`October 1999
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`1421
`
`Table 2. Effect of Human Hair on anti-T, mentagrophytes Activity of Azolylamine Derivatives
`
`Compd.
`
`7
`
`20
`
`25
`
`26
`
`31
`
`32
`
`33
`
`34
`
`40
`
`41
`
`44
`
`45
`
`A
`
`N
`
`N
`
`N
`
`N
`
`N
`
`CH
`
`N
`
`N
`
`N
`
`CH
`
`Am
`
`-N~"Ph
`_~e~
`
`-N~[~
`
`-N~:~"F
`
`-N~)
`
`-N~
`
`-N~’
`
`-N~Ph
`
`-N~
`
`-N~
`
`Clotrimazole
`
`Netieonazole
`
`MIC (#g/ml)
`
`Saline containing
`
`SDB
`
`Saline containing
`5% hair
`
`~ SDB
`
`0.78
`
`1.56
`
`0.78
`
`0.39
`
`0.78
`
`3.13
`
`0.39
`
`0.39
`
`0.2
`
`0.2
`
`0.39
`
`0.2
`
`6.25
`
`50
`
`6.25
`
`12.5
`
`3.13
`
`100
`
`1.56
`
`3.13
`
`0.39
`
`1.56
`
`12.5
`
`6.25
`
`X8
`
`X32
`
`X8
`
`×32
`
`x4
`
`X32
`
`×4
`
`X 8
`
`X2
`
`×8
`
`x 32
`
`x 32
`
`(n=10) 100%
`100
`
`90%
`
`90%
`I
`
`5O
`
`<
`
`0%
`
`(-)-40
`
`rriazole
`derivative]
`
`(-)-41
`
`( lmidazole~
`
`derivative]
`
`None
`
`Fig. 1. Therapeutic Etiicacy of 1.0% Solution of Triazole Derivative (-)-
`40 or lmidazole Derivative (-)-41 in the Guinea Pig Model of Tinea cor-
`poris Infection
`Topical treatments were started 3 or 4d after infection and were continued for 10d
`by application of once-a-day.
`
`((-)-41) showed high efficacy on day 3 postinfection but the
`efficacy was extremely low on day 4 postinfection. These re-
`suits suggested that triazole derivative ((-)-40) showed bet-
`ter penetration into the hair follicles than imidazole deriva-
`tive ((-)-41) and the triazole group in (-)-40 was necessary
`for its property. We confirmed that (-)-40 penetrates easily
`into the skin by the transfollicular route in addition to
`transepidermal route from the distribution pattern in the skin
`of guinea pigs 24h after application of the 1% ~4C-labelled
`drug (data not shown). Lipophilic compotmds generally pen-
`etrate well into the skin through both the epidermis and hair
`folliclesJ6) The 1-octanol/water partition coefficients of (-)-
`40 and (-)-41 at pH 5 were 287.6 and 103.7, respectively,
`and (-)-40 was more lipophilic than (-)-41. This data sup-
`ported the better penetration of triazole derivative ((-)-40)
`into the skin than the imidazole derivative ((-)-41).
`
`In conclusion, many of the azolylamine derivatives pre-
`pared in this study exhibited excellent antifungal activities.
`Among these, triazole derivative ((-)-40, KP-103), which
`had a 4-methylenepiperidine moiety, was found to have a
`broad antifungal spectrum and showed excellent therapeutic
`efficacy. The excellent efficacy may be attributable to good
`penetration and low reduction of the activity in the skin in
`addition to its antifungal activity.
`
`Experimental
`Chemistry Melting points were determined on a Yanagimoto melting
`point apparatus without correction. IR spectra were measured on a Shi-
`madzu FT1R4200 spectrometer. IH-NMR and t9F-NMR spectra were
`recorded on a JEOL JNM-EX270 spectrometer using tetramethylsilane and
`trifluoroacetic acid as respective internal standards. The following abbrevia-
`tions were used: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet,
`br=broad. Mass spectra (MS) were obtained on a JEOL JMS-HX110A mass
`spectrometer. The optical rotations were determined on a Perkin-Elmer 241
`spectrometer. Elemental analysis was performed with a Yanaco MT-5 CHN
`CORDER. Thin-layer chromatography (TLC) was performed on Silica gel
`60 Fz54 precoated TLC plates, with a thickness of 0.25 mm (E. Merck). Col-
`umn chromatography was performed on Silica gel 60 (0.063~.200 mm, E.
`Merck).
`(2R,3R)-2-Aryl-3-(snbstituted amlno)-l-(lH-1,2,4-triazol-l-yl)-2-bu-
`tanols As a typical example, the preparation of (2R,3R)-2-(2,4-difluo-
`rophenyl)-3-(4-methylenepiperidino)-l-(1H-l,2,4-triazol-l-yl)-2-butanol
`((-)-40) is described. A mixture of (2R,3S)-2-(2,4-difluorophenyl)-3-
`methyl-2-[(lH-1,2,4-triazol-l-yl)methyl]oxirane (II, A=N, X=2,4-F2) (0.25
`g, l mmol), 4-methylenepiperidine (0.97g, 10mmol), EtOH (3ml) and
`water (3ml) was stirred at 85 °C for 24h. After cooling, the mixture was
`partitioned between AcOEt (20 ml) and water (20 ml). The organic layer was
`washed with brine, dried over MgSO4 and concentrated in vacuo. The
`residue (ca. 0.4g) was chromatographed on silica gel (8g, AcOEt:n-
`hexane= 1 : 5--->1 : 3, v/v). The product was recrystallized from hexane to ob-
`tain (-)-40 (0.19g, 54% yield from epoxide II) as colorless crystals. Other
`azolylamine derivatives were prepared in the same manner by ring-opening
`reaction of the epoxides II with the corresponding amine. Spectroscopic data
`and results of elementary analysis of these derivatives are shown in Table 3.
`Susceptibility Testing Procedures Candida albicans: MICs were de-
`termined by the microdilution method using synthetic amino acid medium
`fungal (SAAMF). The ftmgal cells were suspended in saline with 0.05%
`
`NII-Electronic Library Service
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`

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`NII-Eleclronic Librazy Sezvice
`Nil-Electronic Library Service
`
`Page 6
`
`

`

`October 1999
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`1424
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`Page 8
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`October 1999
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`1425
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`Tween 80 to provide a cell concentration of l×103cells/ml. Aliquots of
`300/zl of the fungal suspension were dispensed into the wells of 96-well mi-
`crodilution plates containing 3/zl of twofold serial drug dilutions (final in-
`oculumn size; 1 x 103 cells/ml) followed by incubation at 35 °C for 2 d.
`Other Fungi: MICs were determined by the agar dilution meth