Carboxylic acid and phosphate ester derivatives of fluconazole: Synthesis and antifungal activities

Article abstract of DOI:10.1016/j.bmc.2004.08.049

Carboxylic acid and phosphate ester derivatives of fluconazole were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters, were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic acid ester derivatives of fluconazole (1a-l), such as O-2-bromooctanoylfluconazole (1g, MIC = 111 μg/mL) and O-11-bromoundecanoylfluconazole (1j, MIC = 198 μg/mL), exhibited higher antifungal activity than fluconazole (MIC ≥ 4444 μg/mL) against C. albicans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h, exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example, 2-cyanoethyl-ω-undecylenyl fluconazole phosphate (2b) with MIC value of 122 μg/mL had at least 36 times greater antifungal activity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190 μg/mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophilicity and dermal permeability than those for fluconazole. These results demonstrate the potential of these antifungal agents for further development as sustained-release topical antifungal chemotherapeutic agents.

Full text of DOI:10.1016/j.bmc.2004.08.049

Bioorganic & Medicinal Chemistry 12 (2004) 6255–6269
Carboxylic acid and phosphate ester derivatives
of fluconazole: synthesis and antifungal activities
Nguyen-Hai Nam, Soroush Sardari, Meredith Selecky and Keykavous Parang*
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, 41 Lower College Road,
Kingston, RI 02881, USA
Received 12 July 2004; revised 27 August 2004; accepted 27 August 2004
Available online 21 September 2004
Abstract—Two classes of fluconazole derivatives, (a) carboxylic acid esters and (b) fatty alcohol and carbohydrate phosphate esters,
were synthesized and evaluated in vitro against Cryptococcus neoformans, Candida albicans, and Aspergillus niger. All carboxylic
acid ester derivatives of fluconazole (1a–l), such as O-2-bromooctanoylfluconazole (1g, MIC = 111lg/mL) and O-11-bromoundeca-
noylfluconazole (1j, MIC = 198lg/mL), exhibited higher antifungal activity than fluconazole (MIC P 4444lg/mL) against C. alb-
icans ATCC 14053 in SDB medium. Several fatty alcohol phosphate triester derivatives of fluconazole, such as 2a, 2b, 2f, 2g, and 2h,
exhibited enhanced antifungal activities against C. albicans and/or A. niger compared to fluconazole in SDB medium. For example,
2-cyanoethyl-x-undecylenyl fluconazole phosphate (2b) with MIC value of 122lg/mL had at least 36 times greater antifungal activ-
ity than fluconazole against C. albicans in SDB medium. Methyl-undecanyl fluconazole phosphate (2f) with a MIC value of 190lg/
mL was at least 3-fold more potent than fluconazole against A. niger ATCC 16404. All compounds had higher estimated lipophi-
licity and dermal permeability than those for fluoconazole. These results demonstrate the potential of these antifungal agents for
further development as sustained-release topical antifungal chemotherapeutic agents.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1.Introduction
There are effective antifungal agents in the market, but
each drug carries several drawbacks. The presently mar-
keted antifungal drugs are either highly toxic (e.g.,
amphotericin B, AMB) or are becoming ineffective due
to appearance of resistant strains (e.g., flucytosine and
azoles). AMB remains the Ôgold standardÕ drug for life-
threatening fungal infections, but its use is limited due
to its severe toxicity and the inconvenience of intrave-
nous dosing.7,8 Fluconazole (FLC) is an orally effective
azole-based antifungal drug with low toxicity,9 but it
has a limited antifungal spectrum and is not fungicidal.
Although FLC shows a very significant efficacy against
Candida albicans and C. neoformans, it is not very effec-
tive against Aspergillus niger10,11 and Aspergillus fumig-
atus.12 In addition, extensive use of FLC has increased
the number of FLC-resistant C. albicans isolates.13
Therefore, toxicity concerns, limited spectrum, and the
emergence of fungi resistant to currently available
agents has created a need for new and effective anti-
fungal agents against life-threatening systemic mycoses
caused by C. neoformans, Aspergillus species, and
Candida species more urgent. These efforts led to discov-
ery of several azole and triazole derivatives (e.g.,
itraconazole and voriconazole)14 as well as several
In recent years fungal infections have emerged as a ma-
jor cause of disease and mortality, in part as a conse-
quence of the increase in acquired immunodeficiency
syndrome (AIDS), the greater use of immunosuppres-
sive drugs in transplantation and chemotherapeutic
agents in cancer, long term use of corticosteroids, and
even the indiscriminate use of antibiotics.1–3 Fungal
infections are common complications of infection with
human immunodeficiency virus (HIV). Over 90% of
those diagnosed to be HIV-positive contract a fungal
infection during the course of their illness.4,5 Cryptococ-
cus neoformans is the major cause of meningitis in AIDS
patients, and it can also cause local organ dysfunction
and disseminated disease.6 In the case of transplanta-
tion, the mortality directly attributable to fungal infec-
tions carries exceedingly high costs mainly as loss of
the organ transplant and the patient.
Keywords: Antifungal activity; Synthesis; Carboxylic acid esters;
Phosphate esters.
*
Corresponding author. Tel.: +1 401 874 4471; fax: +1 401 874
5048; e-mail: kparang@uri.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.049

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N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
(1!3)-b-D-glucan synthase inhibitors (e.g., capsofungin,
echinocandine).15–17 Voriconazole has excellent activity
against Aspergillus and is generally well tolerated.
Caspofungin has recently been shown to offer an excel-
lent alternative to AMB (with less toxicity) or FLC (with
a broader spectrum) for therapy of systemic Candida
infections.15–17
activities. Three opportunistic fungal organisms, A.
niger, C. neoformans, and C. albicans, were used in
all antifungal tests.
Carboxylic acid-FLC or fatty alcohol phosphate-FLC
conjugates may allow a larger concentration of intact
conjugate ester to enter the infected cells, due to the
lipophilic nature of the fatty acids or the fatty alcohols.
In addition to the possibility of showing enhanced lipo-
philicity, potency, and/or broad-spectrum antifungal
activities, the carboxylic acid-FLC or fatty alcohol
phosphate-FLC conjugates may have additional advan-
tages. The development of fungal resistance to those
conjugates, which have two active components acting
on different targets, would be less likely than to either
compound alone since the two components of conjugate
ester could conceivably act by different mechanisms.
In search for new agents with improved antifungal pro-
files, we have designed two classes of FLC derivatives,
(a) carboxylic acid esters and (b) fatty alcohol and
carbohydrate phosphate diesters and triesters (Fig. 1)
for potential topical application. Drugs applied to the
skin are poorly absorbed. Drug absorption from skin
can be improved by increasing lipophilicity. A sustained
release of FLC from conjugate esters could be beneficial
in treatment of superficial fungal infections. This could
result in a requirement for less frequent dosages of the
conjugate ester analog compared to those of the conju-
gate ester components alone. The rationale for this drug
design approach was based on the expectation that the
carboxylic acid ester, phosphate diester, or phosphate
triester derivatives of FLC will enhance the lipophilicity
of FLC and will be slowly biotransformed through
hydrolysis and/or enzymatic cleavage to FLC and carb-
oxylic acid, fatty alcohol, or carbohydrate analogs.
Additionally, a number of selected carboxylic acids
and fatty alcohols possess antifungal activities. Each
individual component could exhibit antifungal activity
resulting in enhanced and/or broad-spectrum antifungal
FLC-carboxylic acid ester conjugates (1a–l) (Scheme 1)
were designed to increase the lipophilicity of FLC and
with the expectation that two components of the com-
pounds will be slowly released upon ester cleavage.
FLC prevents the synthesis of ergosterol, a major com-
ponent of fungal plasma membranes, by inhibiting the
cytochrome P-450-dependent enzyme lanosterol deme-
thylase (also referred to as 14a-sterol demethylase or
P-450DM).18,19
A number of fatty acids exhibit antifungal activity by
different mechanisms.20–24 However, it is proposed that
N
F
F
N
N
N
N
O
Carboxylic acids
C
O
Fluconazole
O
F
R
N
1
O
O
N
O
F
N
N
N
Fatty alcohols
Fluconazole
P
O
N
Synthesized
FLC Derivatives
O
R1
O
N
P
OR2
R1O
R1 = -CH2CH2CN, -Me
R2 = alkyl
2
3
Phosphate ester
R1 = -H
derivatives of FLC
R2 = alkyl
F
O
Fluconazole
CN
Carbohydrates
P
O
O
P
F
AcO
N
O
O
O
N
O
N
O
OAc
N
N
N
CN
OAc
XAc
4a X = O
4b X = NH
Figure 1. Two classes of synthesized FLC derivatives.

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6257
they act by a combination of mechanisms. Fatty acids,
in general, have been reported to inhibit cellular respira-
tion,25 phosphoceramide synthase of the sphingolipid
biosynthetic pathway,26 and fungal precursor protein
myristoylation or more specifically myristoyl-CoA:
protein N-myristoyl transferase (NMT).27 The partial
incorporation of fatty acids through membrane lipid
biosynthesis may account for their differential activity.28
For example, studies with 4-oxatetradecanoic acid ana-
logs have already indicated that they enter fungal cells
and are converted to their CoA thioesters by cellular
acylCoA synthetase, and are delivered to target proteins
by NMT. Many fungal species like Saccharomyces cere-
visiae, C. albicans, and C. neoformans synthesize a vari-
ety of N-myristoyl proteins including ADP ribosylation
factor (Arf1p, Arf2p) using NMT as catalyst.27,29,30 In
several cases their mechanism of antifungal activity is
not known. The 2-substituted tetradecanoic acids were
reported to be active against C. neoformans, A. niger,
C. albicans, and S. cerevisiae.21 Thus, a number of 2-
bromosubstituted fatty acyl ester analogs of FLC were
synthesized to determine their antifungal properties.
this research, two phosphate triester glucose derivatives
of FLC (4a and 4b) (Scheme 3) were synthesized and
their antifungal activities were compared with those of
the fatty alcohol phosphate triesters derivatives of
FLC (2a–h).
2.Results and discussion
2.1. Chemistry
Scheme 1 shows the procedure for the preparation of
carboxylic acid ester derivatives of FLC (1a–h) from
the reaction of acyl chlorides with FLC in the presence
of sodium hydride. Acyl chlorides were synthesized from
the corresponding carboxylic acids with thionyl chloride
in dry benzene if they were not commercially available.
Scheme 2 demonstrates the preparation of phosphate
triester derivatives of FLC (2a–h). The reaction of alco-
hol (ROH) with phosphorylating reagents, cyanoethyl
N,N-diisopropylchlorophosphoramidite, or methyl
N,N-diisopropylchlorophosphoramidite (5), in the pres-
ence of diisopropyl ethylamine in dry DCM gave N,N-
diisopropylamine phosphine derivatives (6). Coupling
reactions of FLC with 6 in the presence of 1-H-tetrazole
in DCM followed by oxidation with t-butyl hydroperox-
ide afforded phosphate triesters (2a–h). The cyanoethyl
protecting group in 2a–d was cleaved using ammonium
hydroxide to yield the corresponding phosphate diester
derivatives of FLC (3a–d).
Fatty alcohol derivatives such as pramanicin,31 lipox-
amycin,32 and neoenactin A33–35 have been shown to
have promising antifungal activities. A series of aliphatic
alkanols have been reported to exhibit significant anti-
fungal activity.36 In this work we selected several fatty
alcohols and conjugated them with FLC through a
phosphate linker. It was expected that resulting dou-
ble-barreled compounds, fatty alcohol phosphate tri-
ester (2a–h) and phosphate diester (3a–d) derivatives of
FLC (Scheme 2), to have enhanced antifungal activity
compared to those of carboxylic acid ester derivatives
of FLC (1a–l) and FLC.
Scheme 3 displays the synthesis of carbohydrate
phosphate triester derivatives of FLC (4a and 4b). The
reaction of b-D-glucosepentaacetate (7a) or 2-acetam-
ido-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranoside
(7b) with benzylamine in THF resulted in the selective
removal of the anomeric O-acetyl group to produce
the reducing carbohydrates 8a and 8b. Reaction of 8
with cyanoethyl N,N-diisopropylchlorophosphorami-
dite followed by coupling with FLC and oxidation reac-
tion in the presence of t-butyl hydroperoxide furnished
carbohydrate phosphate triesters (4a and 4b).
Several carbohydrates and carbohydrates derivatives
such as CAN-296,37,38 derivatives of 2-amino-2-deoxy-
D-glucitol-6-phosphate,39 zargozic acid and its deriva-
tives,40,41 papulacandins such as papulacandin B,42
and carbohydrates with structures similar to inositol,43
have been reported to have antifungal activities by a
variety of mechanisms. Many fungi possess specific
carbohydrates integrated in the structure of their cell
wall.44–46 The absence of some of these specific carbohy-
drates in the structure of mammalian cells may create an
opportunity to design conjugate ester analogs, which
may selectively inhibit fungal cells. For the purpose of
2.2. Antifungal activity
Biological data for FLC derivatives show that the
in vitro antifungal activity is clearly dependent upon the
_
N
N
a R = CH3
_
b R = n-C13H27
F
F
F
F
N
N
N
N
c R = CH3CH(Br)_
N
N
a, b
d R = C2H5CH(Br)_
e R = n-C3H7CH(Br)_
f R = n-C4H9CH(Br)_
g R = n-C6H13CH(Br)_
h R = n-C10H21CH(Br)_
i R = n-C12H25CH(Br)_
N
N
OH
O
R
N
N
O
Fluconazole (FLC)
1
_
j R = n-CH2(Br)C9H18
k R = ClCH2CH2O_
l R = 2-OCOCH3-C6H4
_
Scheme 1. Synthesis of carboxylic acid ester derivatives of FLC. Reagents and conditions: (a) NaH, DMF, rt, 2h; (b) RCOCl, rt, 3h.

6258
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
N
F
F
N
N
(i-C3H7)2N
(i-C3H7)2N
b, c
OR
Cl
N
a
P
P
N
O
O
OR1
OR1
N
P
5
6
OR2
2
R1O
N
a R1 = C2H4CN, R2 = n-C11H23
b R1 = C2H4CN, R2 = n-CH2=CHC9H18
c R1 = C2H4CN, R2 = n-BrCH2C10H20
d R1 = C2H4CN, R2 = n-C14H29
e R1 = C2H4CN, R2 = n-C8H17
f R1 = CH3-, R2 = n-C11H23
g R1 = CH3-, R2= n-CH2=CHC9H18
h R1 = CH3-, R2 = n-C8H17
-
-
F
F
N
N
N
-
-
N
-
d
-
O
O
N
P
-
OR
HO
-
3
a R = n-C11H23
b R = n-CH2=CHC9H18
c R = n-BrCH2C10H20
d R = n-C14H29
-
-
-
-
Scheme 2. Synthesis of alkylphosphate derivatives of FLC. Reagents and conditions: (a) ROH, DIEA, DCM, rt, 20h; (b) FLC, 1H-tetrazole, DCM,
2h, rt; (c) tBuOOH (28%), 0ꢁC, 1h; (d) NH4OH/MeOH, 24h, rt.
AcO
AcO
AcO
O(CH2)2CN
P N(i-C3H7)2
OAc
O
OH
O
O
O
OAc
OAc
a
b
OAc
OAc
OAc
OAc
7
XAc
8
XAc
XAc
9
F
AcO
O
F
N
N
c, d
a X = O
O
P
O
O
N
b X = NH
OAc
OAc
N
O
N
N
CN
XAc
4
Scheme 3. Synthesis of carbohydrate phosphate triester derivatives of FLC. Reagents and conditions: (a) BnNH2, THF; (b) 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite, DIEA, DCM, 20h; (c) 1. FLC, 1H-tetrazole, DCM, 2h, rt; (d) tBuOOH, 0ꢁC, 1h.
nature of substituent(s) attached to FLC and/or incor-
porated into the chain of carboxylic acids or fatty alcoh-
ols. These groups of compounds exhibited a broad range
of antifungal activities.
exhibited antifungal activity against C. albicans in
RPMI and were the most active antifungal agents in this
group of compounds against all fungi tested. Since these
compounds are carboxylic esters, it was expected that
they could be partially or completely hydrolyzed in vi-
tro, releasing FLC and carboxylic acids. Therefore, the
true activity of these compounds may not be determined
accurately in vitro RPMI medium with pH = 6.9. The
half-life for chemical hydrolysis of all 2-bromosubsti-
tuted ester derivatives of FLC (1a–i) was calculated to
be approximately 74days at pH6.9 using the HYDRO-
WIN (Version 1.67) estimation program. The hydrolysis
rate is slow probably due to the steric hindrance around
the ester functional group, surrounded by three aro-
matic groups and long chain alkyl chain, sometimes sub-
stituted with bulky substituents such as bromine. On the
2.2.1. Carboxylic acid ester derivatives of FLC (1). The
in vitro antifungal test results for carboxylic acid ester
derivatives of FLC (1) are presented in Table 1. FLC
was active against C. albicans in RPMI medium
(MIC = 2.3lg/mL), but showed complete resistance
against C. albicans in SDB medium (MIC P 4444lg/
mL). All carboxylic acid derivatives of FLC (1a–l)
exhibited lower antifungal activity than FLC in RPMI
1640growth medium. Two compounds, O-2-bromo-
octanoylfluconazole (1g) (MIC 6 14lg/mL) and O-11-
bromoundecanoylfluconazole (1j, MIC = 12lg/mL)

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6259
Table 1. General structure, physicochemical properties, and antifungal activity (MIC values, lg/mL)a against Candida albicans (RPMI and SDB),
Cryptococcus neoformans (SDB), and Aspergillus niger (SDB) at 35–37ꢁC after 24–48h and for carboxylic acid ester derivatives of FLC (1)
N
F
F
N
N
N
N
O
R
N
O
1
Compound
MIC (lg/mL)
LogPb
Kp (l/h)c
C. albicans
C. albicans
C. neoformans
A. niger
ATCC 14053, RPMI
ATCC 14053, SDB
ATCC 66031, SDB
ATCC 16404, SDB
1a
1b
>14201>4124020
718
656
P14201.3
P718
>656
1.1
718
656
718
656
7.2
2.01.1
2.5
1610
1c
1d
319
275
636
552
>1276
1100
492
P1276
P1100
P979
111
1.9
3.5
1e
3.0
3.5
1f
1g
61
492
111
6.5
614
1466
731
14
4.5
6.4
21.8
246
826
1h
1i
1466
731
1466
731
P1466
>731
395
7.4
1j
1k
12
977
198
6.1
6.0151
1.9
2.3
>977
>1212
P4444
1.5
>977
>1212
2.4
>977
>1212
>580
1.2
1.2
1l
P1212
2.3
0.7
FLC
AMB
DMSO
0.3
À3.8
0.4
5.1 · 10À8
<3.7
>5000
0.4
>5000
>5000
>5000
a The result is the average of three separate experiments.
b Partition coefficient of the compound calculated using the KowWin program.
c Dermal permeability coefficient of the compound calculated using the DermWin program (l/h).
other hand, all compounds showed higher antifungal
activity than FLC against C. albicans in SDB medium
(pH = 5.6). FLC was completely inactive against C. albi-
cans in SDB suggesting that the antifungal effects
of carboxylic ester derivatives of FLC (1a–l) are not
exclusively due to ester hydrolysis and the release
of FLC. Compounds 1g (MIC = 111lg/mL) and 1j
(MIC = 198lg/mL) were at least 40- and 22-fold more
active against C. albicans in SDB, respectively, than that
for FLC. Furthermore, compounds 1g (MIC = 111lg/
mL) and 1j (MIC = 395lg/mL) were at least 5.2- and
1.5-fold more potent than FLC against A. niger indicat-
ing the importance role of carboxylic acids attached to
FLC through the ester moiety in enhancing antifungal
activity. The antifungal activity of carboxylic ester 1j
against C. neoformans was comparable to FLC.
Although compounds 1g and 1j exhibited 8- and 17-fold
less antifungal activity against C. albicans in SDB than
that in RPMI, respectively, they were still significantly
more active than FLC and other analogs in SDB, indi-
cating the presence of an additional mechanism(s) other
than ester hydrolysis for antifungal activity of these
compounds.
such as bromine, affected lipophilicity dramatically (Ta-
ble 1). The differences in lipophilicity and the size of the
alkyl ester substituent in carboxylic acid ester derivatives
of FLC could provide a correlation of antifungal acti-
vity with physicochemical properties. All compounds
(1a–l) showed larger estimated partition coefficients
(logP = 1.3–7.4) than that for FLT (logP = 0.3). Simi-
larly all carboxylic ester derivatives of FLC (1a–l) had
higher estimated dermal permeability coefficients (Kp)
(1.1–1610l/h) than that for FLC (Kp = 0 .4l/h) indicat-
ing the potential for using carboxylic ester analogs for
dermal delivery of FLC. Since all carboxylic ester deriv-
atives of FLC are lipophlic, their penetration through
the lipid matrix of the stratum corneum of skin and also
their penetration into cells might be more efficient. More
research is required to confirm this postulation. There
appears to be a relationship between antifungal activity
against all fungi tested and the calculated logP for these
carboxylic ester derivatives of FLC. There is a correl-
ation between the number of methylene groups and
the antifungal activity of 2-bromosubstituted ester
derivatives of FLC (1c–i). As the number of carbon
atoms increases, the antifungal activities are increased
up to the maximum level in O-2-bromooctanoylflucon-
azole (1g). The antifungal activities are then diminished
significantly in compounds 1h and 1i, indicating the limit
of tolerance for hydrophobicity in generating maximum
antifungal activity in this group of compounds. These
data suggest that there is an optimal partition coefficient
Estimated partition coefficients (logP), which can be
used as an indicator of passive diffusion across cell mem-
branes and cellular uptake, were determined for all carb-
oxylic ester derivatives of FLC (1a–l). Increasing the
length of ester chain or attaching lipophilic substituents

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N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
range for maximum antifungal activity. The conjugate
ester O-2-bromooctanoylfluconazole (1g) has an esti-
mated logP value of 4.5 that was optimal among
2-bromosubstituted ester derivatives of FLC (1c–i) for
maximum antifungal activity. The highly lipophilic com-
pounds, such as 1h and 1i, exhibited weaker antifungal
activity than other analogs. 2-Bromosubstituted ester
analogs with shorter chains, such as 1c–f or carboxylic
acid ester derivatives without a bromine group, such
as 1k and 1l, generally exhibited weak antifungal acti-
vity. Another potent antifungal compound, O-11-bro-
moundecanoylfluconazole (1j), has a logP value of 6.0
suggesting that additional factors such as the intracellu-
lar rate of ester hydrolysis, the stability of ester prior to
membrane penetration or in culture medium, and the
nature, size, and position of the substituent attached
to the chain, may also contribute to overall antifungal
effect observed. Differences in the ester hydrolysis are
expected to be dependent upon the steric size of the
attached acyl group where more sterically hindered
esters should possess longer half-lives.
half-life for all 2-bromosubstituted carboxylic acid esters
at pH5.6 is approximately 4years. Therefore, chemical
hydrolysis does not probably contribute significantly
to release of components. It remains to be seen whether
the esters are hydrolyzed in vitro by the action of fungal
esterases. Further stability studies are underway to
determine the stability of these compounds in the pres-
ence of fungi and SDB medium and to identify the active
component(s) after incubation.
2.2.2. Carboxylic acid and fatty alcohol derivatives. The
in vitro antifungal results for several carboxylic acids
and fatty alcohols are presented in Table 2. In general,
fatty alcohols showed significantly higher antifungal
activities against C. neoformans and A. niger than those
of the corresponding fatty acids. For example, tetrade-
canol (MIC = 14lg/mL) and 11-bromoundecanol
(MIC =12lg/mL) were more potent than tetradecanoic
acid (MIC > 529lg/mL) and 11-bromoundecanoic acid
(MIC = 23lg/mL), against C. neoformans in SDB, respec-
tively. The antifungal activities of primary aliphatic alcoh-
ols from C6 to C13 have been previously reported against
S. cerevisae.36 It was proposed that the primary mecha-
nism for the antifungal action of alkanols is due to their
amphipathic characteristics and ability as nonionic surf-
actants to disrupt the native membrane-associated func-
tion of the integral proteins.36 The reason for the higher
antifungal activity of the fatty alcohols compared to the
corresponding carboxylic acids with similar chain length
is not known, but could be due to their better surfactant
effect or other mechanism(s) of antifungal action.
In general 2-bromosubstituted analogs are expected to
be much more stable than other carboxylic esters as pre-
viously shown in 50-O-myristoyl analog derivatives of
zidovudine.47 Stability of fatty acid derivatives were esti-
mated using HYRDROWIN (Version 1.67) at pH5.6 as
used in SDB medium indicating high stability of these
compounds probably due to the presence of a tertiary
ester functional group surrounded by bulky aromatic
and nonaromatic groups. For example the estimated
Table 2. Physicochemical properties and antifungal activity (MIC values, lg/mL)a against Candida albicans (RPMI and SDB), Cryptococcus
neoformans (SDB), and Aspergillus niger (SDB) at 35–37ꢁC after 24–48h and for carboxylic acid and fatty alcohols
Compound
MIC (lg/mL)
C. neoformans
ATCC 14053, RPMI ATCC 14053, SDB ATCC 66031, SDB ATCC 16404, SDB
LogPb Kp (l/h)c
C. albicans
C. albicans
A. niger
d
Acetic acid
P220
468
513
P220
468
P220
117
.1
P2200
468
6.2
2.8
2-Bromopropionyl chloride
2-Bromobutyric acid
2-Bromovaleric acid
2-Bromohexanoic acid
2-Bromooctanoic acid
2-Bromolauric acid
2-Bromomyristic acid
11-Bromoundecanoic acid
Decanoic acid
0.3
1.3f
1.8
>513
578
>513
290
NDe
18.6
29.9
P578
P710731505
865
P578
721.30
54.7
865
155
433
19
ND
78
3.3
5.3
184
2070
155
12
>54015
185
99
68
6.3
93
6970
1520
371
791
23
4.9
4.0g
1280
1360
16,800
1850
ND
<12
h
Tetradecanoic acid
1-Undecanol
>876
ND
ND
1750>529
87
>529
6.0
28
87
4.3
x-Undecylenyl alcohol
Tetradecanol
11014
172014
95
27
27
4.1
5.8
24
i
ND
ND
17,900
1070
0.4
5.1 · 10À8
11-Bromoundecanol
FLC
AMB
12
4.6
0.3
2.3
0.7
P4444
1.5
>5000
2.4
<3.7
>5000
>580
0.4
>5000
À3.8
DMSO
>5000
a The result is the average of three separate experiments.
b Partition coefficient of the compound calculated using the KowWin program.
c Dermal permeability coefficient of the compound calculated using the DermWin program (l/h).
d Experimental logP = À0.17.52
e ND = Not determined.
f Experimental logP = 1.42.52
g Experimental logP = 4.09.52
h Experimental logP = 6.11.53
i Experimental logP = 6.03.54

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6261
All carboxylic acids were equally active against C. albi-
cans in SDB and RPMI media with the exception of
2-bromomyristic acid and 11-bromoundecanoic acid,
which showed higher and lower antifungal activity in
RPMI, respectively, than that in SDB. 2-Bromolauric,
2-bromomyristic, and 11-bromoundecanoic acids were
found to be the most active antifungal agents in this
group of carboxylic acids against all fungi tested. 11-
Bromoundecanoic acid showed comparable activity to
2-bromolauric acid against all three fungi tested in
SDB medium. The antifungal activity of 2-bromomyris-
tic acid and 11-bromoundecanoic acid against C. albi-
cans in RPMI medium was in agreement with our
earlier studies.21 There appears to be a relationship be-
tween antifungal activity against all fungi tested and
the calculated logP for 2-bromosubstituted analogs.
These data suggest that there is an optimal partition
coefficient range for maximum antifungal activity for
2-bromosubstituted carboxylic acids. A logP value in
the range of 5.3–6.3 is required for the broad-spectrum
antifungal activity against fungi tested in SDB medium.
2-Bromosubstituted analogs with shorter chain fatty
acids or carboxylic acids without a bromine group gen-
erally exhibited weak antifungal activity suggesting the
importance of hydrophobicity in the activity of fatty
acid derivatives. The cutoff in the observed antifungal
activity could be due to a corresponding limit in the
absorption of carboxylic acids into lipid-bilayer portions
of membranes. Shorter chain carboxylic acids probably
enter the cell by passive diffusion across plasma mem-
brane and do not incorporate into the cell membrane.
Longer chain and nonbrominated carboxylic acids are
probably soluble in the membrane phospholipids, and
therefore are incorporated into hydrophobic domain
of the membrane. It seems that only amphipathic med-
ium-chain carboxylic acids act as surfactants. All carb-
oxylic acids and fatty alcohols had higher estimated
dermal permeability coefficient (Kp = 2.8–17,900l/h)
than FLC (Kp = 0.39l/h).
2.2.3. Phosphate ester derivatives of FLC (2–4). Based on
the antifungal activity data for compounds in Table 2,
the fatty alcohols were selected for further preparation
of phosphate diester and triester derivatives of FLC.
The in vitro antifungal test results for the fatty alcohol
phosphate triester (2a–h) and diester (3a–d) derivatives
of FLC are presented in Table 3. In general, in compar-
ison to FLC, this group of compounds had a much
stronger activity against C. albicans in SDB medium in
which FLC was inactive indicating the importance of
fatty alcohol portion and phosphate linker in enhancing
antifungal activities. FLC phosphate triesters of FLC (2)
were more potent antifungal agents than phosphate
diesters (3). Compounds 2a, 2b, and 2g were the most
potent antifungal agents in this group against C. albi-
cans in SDB medium. Compounds 2a, 2c, and 2f were
found to be potent antifungal agents against C. neofor-
mans (MIC = 12–31lg/mL). Compound 2f with a MIC
Table 3. General structure, physicochemical properties, and antifungal activity (MIC values, lg/mL)a against Candida albicans, Cryptococcus
neoformans, and Aspergillus niger in SDB at 35–37ꢁC after 24–48h and for fatty alcohol phosphate diester (3) and triester (2) derivatives of FLC and
controls
N
F
F
N
N
N
N
O
O
N
P
OR2
R1O
Compound
R1
R2
MIC (lg/mL)
LogPb
Kp (l/h)c
C. albicans
ATCC 14053
C. neoformans
ATCC 66031
A. niger
ATCC 16404
2a
2b
CNCH2CH2–
CNCH2CH2–
n-C11H23
–
185
122
23
31
26
7404.7
9804.6
1658
10
8.3
5.1
n-CH2@CH–C9H18
n-BrCH2C10H20
–
2c
2d
CNCH2CH2–
CNCH2CH2–
CNCH2CH2–
–
1658
940235
680170 1360 3.2
5.8
n-C14H29
–
–
940 6.2
1.6
61.9
2e
n-C8H17
–
2f
2g
CH3–
CH3–
CH3
H
n-C11H23
762
228
12
29
1905.2
1825
6.3
38.5
5.1
n-CH2@CH–C9H18
n-C8H17
n-C11H23
n-CH2@CH–C9H18
n-BrCH2C10H20
n-C14H29
–
31.7
2h
3a
–
47059
76095
1501
4703.7
7605.1
–
42.8
3b
3c
H
–
1501
185
1501
1477
264
5.0
5.5
35.2
24.8
H
–
1477
940235
3d
H
–
940 6.6
FLC
AMB
DMSO
P4444
1.5
>5000
2.4
<3.7
5000
>580
0.4
>5000
0.3
À3.8
0.4
5.1 · 10À8
a The result is the average of three separate experiments.
b Partition coefficient of the compound calculated using the KowWin program.
c Dermal permeability coefficient of the compound calculated using the DermWin program (l/h).

6262
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
Table 4. General structure, physicochemical properties, and antifungal activity (MIC values, lg/mL)a against Candida albicans, Cryptococcus
neoformans, and Aspergillus niger in SDB at 35–37ꢁC after 24–48h and for carbohydrate phosphate triester derivatives of FLC (4) and controls
F
AcO
O
P
F
N
O
O
O
N
OAc
OAc
N
OCH2CH2CN
N
N
N
XAc
Compound
MIC (lg/mL)
LogPb
Kp (l/h)c
C. albicans ATCC 14053
C. neoformans ATCC 66031
A. niger ATCC 16404
4a
4b
1999
>3399
P4444
1.5
551
306
1999
1699
>580
0.4
À1.3
À0.8
0.3
4.4 · 10À5
1.1 · 10À4
0.4
5.1 · 10À8
FLC
AMB
DMSO
2.4
<3.7
>5000
À3.8
>5000
>5000
a The result is the average of three separate experiments.
b Partition coefficient of the compound calculated using the KowWin program.
c Dermal permeability coefficient of the compound calculated using the DermWin program (l/h).
value of 190lg/mL was the most potent compound
tested in this group against A. niger. The in vitro anti-
fungal activity for these compounds could be dependent
upon a number of factors, which include: (i) the intrinsic
activity of the intact ester derivative; (ii) the rate and ex-
tent of cellular uptake; and/or (iii) the rate and extent at
which the two substrate moieties FLC and the fatty
alcohol analog are released following hydrolysis of the
conjugate ester. The mechanism of intrinsic activity of
these derivatives could be due to their amphipathic
structure. Hydrophilic parts of the molecule such as
protonated triazoles might bind with the hydrophilic
portion of fungal cell membrane, while the hydrophobic
tail may enter into the membrane lipid bilayer of the
membrane. Furthermore, other mechanisms such as
hydrolysis of the molecule to FLC and fatty alcohols,
which have different mechanisms of antifungal action,
may be involved. All compounds in this group had
higher estimated partition coefficients (logP = 3.2–6.6)
and dermal permeability coefficient (Kp = 1.6–264l/h)
than those for FLC (logP = 0.3; Kp = 0 .4l/h). The data
suggest that there is an optimal partition coefficient
range (4.6–5.2) for maximum antifungal activity of these
compounds against C. neoformans.
2.3. Interaction studies
Although many factors may influence the clinical effi-
cacy of antifungal therapy in vivo, a great effort has
been directed toward identifying combinations that are
synergistic or antagonistic in vitro. These studies were
carried out in detecting and determining such interac-
tions in compounds 1g and 1j against C. albicans in
SDB growth medium, in which FLC was inactive
(Fig. 2). Fractional inhibitory concentration (FIC)
index [FICindex = FICA + FICB] calculation was used
4444
5000
(a)
3000
865
1000
216
111
111 111
1g (+2-BO)(+FLC)
2-BO (+FLC)
Fluconazole (FLC)
-1000
Compound name
5000
3000
1000
-1000
4444
(b)
To understand the importance of fatty alcohols in the
antifungal activity of 2, fatty alcohols were replaced
with carbohydrates. Table 4 shows the antifungal
activities for carbohydrate phosphate triester derivatives
of FLC (4). The removal of fatty alcohols in the
conjugate and substitution with carbohydrates de-
creased the antifungal activities significantly against
all fungi tested. Compounds 4a (logP = À1.3) and 4b
(logP = À0.8) showed lower estimated partition coeffi-
cients than that for FLC (logP = 0.3). The data
indicates the significant role of fatty alcohols in anti-
fungal activity of phosphate triester and diester deriva-
tives of FLC.
198
198
185
1j (+11-BU)(+FLC)
11-Bu (+FLC)
Fluconazole (FLC)
Compound name
Figure 2. The interaction study of FLC, 2-bromooctanoic acid (2-BO),
11-bromoundecanoic acid (11-Bu) with the conjugate ester antifungal
agents, 1g and 1j, respectively, against C. albicans ATCC 14053 (SDB).

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6263
to determine synergism between different components of
the synthesized compounds while administered sepa-
rately, where FICA or FICB equals to MIC of com-
pounds A or B in combination divided by MIC of
drug A or B alone. If the FIC index is 60.5, the combi-
nation is synergistic. If the FIC value is equal to 1, the
combination is additive or ÔindifferentÕ and if greater
than 4.0, the combination is antagonistic.
remain speculative until further mechanistic investiga-
tions have been carried out. Bioconversion studies are
underway to determine the stability of candidate com-
pounds following incubation with fungi and SDB med-
ium. Accordingly, in vitro antifungal activity appears
to be dependent on several factors, which include intrin-
sic activity, lipophilicity, rate of cellular uptake, and cel-
lular rate of ester or phosphate ester hydrolysis. The
utility of derivatives of FLC as therapeutic agents will
be enhanced by a clear understanding of their uptake,
metabolism, and incorporation. The in vitro results
from this study provide a rationale upon which several
candidate compounds can be selected for topical anti-
fungal agents. This strategy can be used for other triaz-
ole derivatives such as voriconazole that has a better
antifungal profile.
While FLC did not show inhibition against C. albicans
at the highest tested concentration (4444lg/mL), the
mixture of FLC and 2-bromooctanoic (2-BO) showed
antifungal activity (Fig. 2a). FIC index values for a mix-
ture of 2-BO and FLC (FIC index = 0.30) indicated a
synergism effect between these two components when
mixed. There was no synergistic effect when 1g was
mixed with FLC (FIC index = 1.05). The presence of
FLC and/or 2-BO did not affect the inhibition level of
1g. A mixture of 2-BO and FLC had approximately 2-
fold less antifungal inhibition than when 1g was used
alone or mixed with any of the components (2-BO or
FLC). The inequality in the MIC values of the mixed
components and the corresponding conjugate ester 1g
could be due to differences in cell penetration and dispo-
sition of individual components compared with the ester
derivative of FLC.
4.Experimental
4.1. General
All products were homogenous by thin-layer chroma-
tography (TLC), performed on Whatmanꢂ 250lm Sil-
ica Gel GF Uniplates and visualized under UV light at
254nm. Melting points were determined with an electro-
thermal melting point apparatus and are uncorrected.
Chromatographic purification was done by the open
flash silica gel column chromatography using Merck
silica gel 60 (240–400 mesh). Nuclear magnetic reso-
nance spectra (1H NMR) were recorded using tetra-
methylsilane as an internal standard on a Bruker
400MHz spectrometer with CDCl3 as a solvent unless
otherwise indicated. Chemical shifts are reported in
parts per millions (ppm) downfield from tetramethyl-
silane as an internal standard. Electrospray ionization
(ESI) and high-resolution mass spectra were obtained
using PE Biosystems API 2000 and Marinerꢂ mass
spectrometers, respectively. Reagents and solvents were
purchased from Aldrich or Fluka Chemical Corp.
(Milwaukee, WI, USA) unless noted otherwise. Solvents
were distilled and dried before use.
Some differences were observed in the interaction studies
using 11-bromoundecanoic acid (11-Bu) and 1j (Fig.
2b). The mixture of 11-Bu with FLC showed synergistic
effect (FIC index = 0.52) and fungal inhibitory effect
that was approximately equal to that of 1j. While the
presence of FLC did not affect the antifungal activity
of 1j against C. albicans in SDB (FIC index = 1.04),
addition of 11-Bu reduced the activity of 1j. The lack
of interaction of the designed conjugate esters, 1j and
1g, with their individual components (FLC and carb-
oxylic acids), indicate that the ester analogs may have
some intrinsic antifungal activity in addition to that of
their hydrolysis products, FLC and carboxylic acids,
in the site of action.
3.Conclusion
4.2. General procedure for the synthesis of carboxylic acid
ester derivatives of FLC (1a–h)
In conclusion, two series of double-barreled carboxylic
acid and fatty alcohol derivatives of FLC were designed
and their in vitro antifungal structure–activity relation-
ships were determined. By virtue of being prodrugs, all
of the synthesized compounds were expected to be less
active in vitro in comparison to FLC. Surprisingly, a
large difference in antifungal activities was observed
for conjugate ester analogs investigated. The advantage
of carboxylic acid ester derivatives of FLC is their activ-
ity against C. albicans ATCC 14053 in SDB medium in
which FLC was inactive. The in vitro antifungal activi-
ties of several fatty alcohol phosphate diester and tri-
ester derivatives of FLC were stronger against C.
albicans and A. niger than those of FLC. Several of these
phosphate derivatives were found to be potent anti-
fungal agents against all three fungi indicating their
broad-spectrum activities. The exact mode of action of
conjugate ester analog derivatives of FLC will obviously
Dry dimethyl formamide (DMF) (2mL) was added to
sodium hydride (48mg, 40% immersion in oil, 1.2mmol,
prewashed with dry hexane) and the mixture was cooled
to 0ꢁC. A solution of FLC (300mg, 1mmol) in dry
DMF was slowly added to this mixture. After stirring
for 2h at room temperature, the reaction mixture was
cooled down again in an ice bath; thereafter a solution
of acyl chloride (1.5mmol, freshly prepared by refluxing
the corresponding carboxylic acid with thionyl chloride
if not commercially available) in dry benzene was added
dropwise during 30min. The reaction mixture was stir-
red for a further period of 3h at room temperature
and poured into a separatory funnel containing cold
aqueous sodium bicarbonate (5%, 100mL). The crude
product was extracted with ethyl acetate (EA)
(2 · 100mL). The organic layers were combined, dried

6264
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
over magnesium sulfate (MgSO4), and concentrated
in vacuo. The residue, which consisted of one major
product, was purified by a silica gel column chromatog-
raphy using hexane/acetone (3:1 v/v to 1:1 v/v) as eluting
solvents to yield final products (Scheme 1).
(MÀC2H2N3, 79Br), 402.1 (MÀC2H2N3, 81Br), 289.3
(MÀC5H8BrO2), 220.1 (MÀC7H10BrN3O2).
4.2.6. O-2-Bromohexanoylfluconazole (1f). The general
synthetic method described above afforded 1f (69.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.16 (2H,
4.2.1. O-Acetylfluconazole (1a). The general synthetic
method described above afforded 1a (78.0%) as an
amorphous powder; mp 131.5–133.1ꢁC; 1H NMR
(CDCl3, 400MHz): d 7.97 (2H, s), 7.88 (2H, s), 6.90–
6.74 (3H, m), 5.14 (2H, d, J = 14.5Hz), 5.05 (2H, d,
J = 14.5Hz), 2.15 (3H, s). HR-MS (ESI-TOF) calcu-
lated for C16H16F2N6O2 348.1146, found 349.1222
(M+H). ESI m/z 349.2 (M+H), 220.2 (MÀC4H5N3O2).
s), 7.98 (2H, s), 7.09 (1H, d, J = 8.3Hz), 6.88 (1H, d, J =
2.5Hz), 6.79 (1H, dd, J = 8.3, 2.5Hz), 5.38 (2H, d,
J = 14.2Hz), 5.11 (2H, d, J = 14.2Hz), 4.41 (1H, t,
J = 7.7Hz), 2.05–1.85 (2H, m), 1.33–1.20 (4H, m, meth-
ylene envelope), 0.99 (3H, t, J = 7.1Hz). HR-MS (ESI-
TOF) calculated for C19H21BrF2N6O2 482.0877, found
483.0711 (M+H, 79Br), 485.1021 (M+H, 81Br). ESI m/z
483.0(M+H,
79Br), 485.1 (M+H, 81Br), 414.0
(MÀC2H2N3, 79Br), 416.1 (MÀC2H2N3, 81Br), 289.3
4.2.2. O-Tetradecanoylfluconazole (1b). The general
synthetic method described above afforded 1b (67.0%)
as an amorphous powder; mp 142–143ꢁC; 1H NMR
(CDCl3, 400MHz): d 7.95 (2H, s), 7.84 (2H, s), 6.88–
6.75 (3H, m), 5.15 (2H, d, J = 15.3Hz), 5.50(2H, d,
J = 15.3Hz), 2.37 (2H, t, J = 7.5Hz), 1.54–1.22 (22H,
m, methylene envelope), 0.99 (3H, t, J = 8.2Hz). HR-
MS (ESI-TOF) calculated for C28H40F2N6O2 516.3024,
found 517.3057 (M+H). ESI m/z 517.3 (M+H), 220.1
(MÀC16H29N3O2).
(MÀC6H10BrO2), 220.1 (MÀC8H12BrN3O2).
4.2.7. O-2-Bromooctanoylfluconazole (1g). The general
synthetic method described above afforded 1g (75.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.20(2H,
s), 7.82 (2H, s), 7.05 (1H, d, J = 8.8Hz), 6.92 (1H, d, J =
2.7Hz), 6.80(1H, dd, J = 8.8, 2.7Hz), 5.39 (2H, d,
J = 15.1Hz), 5.09 (2H, d, J = 15.1Hz), 4.31 (1H, t,
J = 7.7Hz), 2.05–1.99 (2H, m), 1.59–1.32 (8H, m, meth-
ylene envelope), 0.90 (3H, m). HR-MS (ESI-TOF)
calculated for C21H25BrF2N6O2 510.1190, found
511.2011 (M+H, 79Br), 513.3101 (M+H, 81Br). ESI
m/z 511.2 (M+H, 79Br), 513.3 (M+H, 81Br), 289.3
(MÀC10H16BrO2), 220.1 (MÀC8H12BrN3O2).
4.2.3. O-2-Bromopropionylfluconazole (1c). The general
synthetic method described above afforded 1c (38.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.20(2H,
s), 7.90(2H, s), 7.08 (1H, d, J = 8.1Hz), 7.00 (1H, d, J =
2.2Hz), 6.80(1H, dd, J = 8.1, 2.2Hz), 5.39 (2H, d,
J = 15.0Hz), 5.20 (2H, d, J = 15.0Hz), 4.45 (1H, q,
J = 7.5Hz), 1.83 (3H, d, J = 7.5Hz). HR-MS (ESI-
TOF) calculated for C16H15BrF2N6O2 440.0408, found
441.0411 (M+H, 79Br), 443.1416 (M+H, 81Br). ESI m/z
441.1 (M+H, 79Br), 443.1 (M+H, 81Br), 372.0
(MÀC2H2N3, 79Br), 374.0(M ÀC2H2N3, 81Br), 289.3
(MÀC3H4BrO2), 220.1 (MÀC5H6BrN3O2).
4.2.8. O-2-Bromolauroylfluconazole (1h). The general
synthetic method described above afforded 1h (91.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.19 (2H,
s), 7.90(2H, s), 7.08 (1H, d, J = 8.1Hz), 6.88 (1H, d, J =
2.4Hz), 6.77 (1H, dd, J = 8.1, 2.4Hz), 5.32 (2H, d,
J = 13.7Hz), 5.07 (2H, d, J = 13.7Hz), 4.28 (1H, t,
J = 7.5Hz), 2.13–1.98 (2H, m), 1.59–1.32 (16H, m,
methylene envelope), 0.94 (3H, m). HR-MS (ESI-
TOF) calculated for C25H33BrF2N6O2 566.1816, found
567.1823 (M+H, 79Br), 569.1821 (M+H, 81Br). ESI m/z
567.3 (M+H, 79Br), 569.4 (M+H, 81Br), 498.3
(MÀC2H2N3, 79Br), 500.3 (MÀC2H2N3, 81Br), 289.3
(MÀC12H22BrO2), 220.1 (MÀC14H24BrN3O2).
4.2.4. O-2-Bromobutyrylfluconazole (1d). The general
synthetic method described above afforded 1d (31.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.20(2H,
s), 7.99 (2H, s), 7.10(1H, d, J = 8.5Hz), 7.05 (1H, d, J =
2.1Hz), 6.88 (1H, dd, J = 8.5, 2.1Hz), 5.39 (2H, d,
J = 13.4Hz), 5.15 (2H, d, J = 13.4Hz), 4.30(1H, t,
J = 7.5Hz), 1.49–1.40(2H, m), 1.15 (3H, t, J = 8.2Hz).
HR-MS (ESI-TOF) calculated for C17H17BrF2-
N6O2 454.0564, found 455.0531 (M+H, 79Br), 457.0542
(M+H, 81Br). ESI m/z 455.3 (M+H, 79Br), 457.3
(M+H, 81Br), 386.1 (MÀC2H2N3, 79Br), 388.1
(MÀC2H2N3, 81Br), 289.3 (MÀC4H6BrO2), 220.1
(MÀC8H8BrN3O2).
4.2.9. O-2-Bromomyristoylfluconazole (1i). The general
synthetic method described above afforded 1i (95.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.59 (2H,
s), 7.88 (2H, s), 7.02 (1H, d, J = 8.4Hz), 6.92 (1H, d, J =
2.3Hz), 6.81 (1H, dd, J = 8.4, 2.3Hz), 5.34 (2H, d,
J = 14.6Hz), 5.07 (2H, d, J = 14.6Hz), 4.31 (1H, t,
J = 7.5Hz), 2.15–1.97 (2H, m), 1.60–1.21 (20H, m,
methylene envelope), 0.95 (3H, m). HR-MS (ESI-
TOF) calculated for C27H37BrF2N6O2 594.2129, found
595.2121 (M+H, 79Br), 597.2132 (M+H, 81Br). ESI
m/z 595.3 (M+H, 79Br), 597.2 (M+H, 81Br), 526.3
(MÀC2H2N3, 79Br), 528.3 (MÀC2H2N3, 81Br), 289.4
(MÀC14H26BrO2), 220.1 (MÀC16H28BrN3O2).
4.2.5. O-2-Bromovaleroylfluconazole (1e). The general
synthetic method described above afforded 1e (55.0%)
1
as a syrup; H NMR (CDCl3, 400MHz): d 8.22 (2H,
s), 8.00 (2H, s), 7.15 (1H, d, J = 8.3Hz), 7.09–6.80
(2H, m), 5.40(2H, d,
J = 16.1Hz), 5.12 (2H, d,
J = 16.1Hz), 4.38 (1H, t, J = 7.2Hz), 2.12–2.00 (2H,
m), 1.49–1.42 (2H, m), 0.96 (3H, t, J = 7.5Hz). HR-MS
(ESI-TOF) calculated for C18H19BrF2N6O2 468.0721,
found 469.0712 (M+H, 79Br), 471.0711 (M+H, 81Br).
ESI m/z 469.0(M+H, 79Br), 471.1 (M+H, 81Br), 400.0
4.2.10. O-11-Bromoundecanoylfluconazole (1j). The
general synthetic method described above afforded 1j
(88.0%) as a syrup; 1H NMR (CDCl3, 400MHz): d
7.95 (2H, s), 7.85 (2H, s), 6.95–6.88 (1H, m), 6.87–

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6265
6.71 (2H, m), 5.20(2H, d, J = 15.5Hz), 5.03 (2H,
d, J = 15.5Hz), 3.40(2H, –C H2Br, t, J = 7.7Hz),
2.32 (2H, COCH2–, t, J = 7.7Hz), 1.90–1.81 (2H,
–CH2CH2Br, m), 1.61–1.51 (2H, COCH2CH2–, m),
1.45–1.20(12H, m, methylene envelope). HR-MS
(ESI-TOF) calculated for C24H31BrF2N6O2 552.1660,
found 553.1647 (M+H, 79Br), 555.2111 (M+H, 81Br).
ESI m/z 553.4 (M+H, 79Br), 555.2 (M+H, 81Br),
484.2 (MÀC2H2N3, 79Br), 486.2 (MÀC2H2N3,
81Br), 289.3 (MÀC11H20BrO2), 220.1 (MÀC13H22-
BrN3O2).
4.3.1. 2-Cyanoethyl-n-undecanyl fluconazole phosphate
(2a). The general synthetic method described above af-
forded 2a (24.0%) as a wax; mp 75.4–77.0ꢁC; 1H
NMR (CDCl3, 400MHz): d 8.31 (2H, s), 7.93 (2H, s),
7.22–7.15 (1H, m), 6.94–6.83 (1H, m), 6.83–6.72 (1H,
m), 5.21 (2H, d, J = 14.2Hz), 5.03 (2H, d,
J = 14.4Hz), 4.15 (2H, –OCH2CH2CN, t, J = 7.2Hz),
3.93 (2H, –OCH2–, t, J = 7.7Hz), 2.74 (2H,
–OCH2CH2CN, t, J = 7.2Hz), 1.57 (2H, –OCH2-
CH2CH2–, t, J = 7.6Hz), 1.41–1.27 (16H, methylene
envelope), 0.92–0.83 (3H, m). HR-MS (ESI-TOF)
calculated for C27H38F2N7O4P 593.2691, found
594.2710(M+H).
4.2.11. O-2-Chloroethyloxycarbonylfluconazole (1k). The
general synthetic method described above afforded 1k
(75.0%) as a syrup; H NMR (CDCl3, 400MHz) 8.05
1
4.3.2. 2-Cyanoethyl-x-undecylenyl fluconazole phosphate
(2b). The general synthetic method described above af-
forded 2b (36.1%) as a wax; mp 72.6–73.6ꢁC; 1H
NMR (CDCl3, 400MHz): d 8.35 (2H, s), 7.95 (2H, s),
7.20–7.10 (1H, m), 6.91–6.81 (1H, m), 6.81–6.71 (1H,
m), 5.88–5.75 (1H, CH2@CH–, m), 5.23 (2H, d,
J = 15.1Hz), 5.05 (2H, d, J = 15.2Hz), 5.01–4.85
(2H, CH2@CH–, m), 4.20(2H, –OC H2CH2CN, t,
J = 7.5Hz), 3.98 (2H, –OCH2–, t, J = 7.8Hz), 2.72
(2H, –OCH2CH2CN, t, J = 7.5Hz), 2.11–2.00 (2H,
CH2=CHCH2–, m), 1.58 (2H, –OCH2CH2CH2–, t,
J = 7.2Hz), 1.40–1.15 (12H, methylene envelope). HR-
MS (ESI-TOF) calculated for C27H36F2N7O4P
591.2534, found 592.2541.
(2H, s), 7.95 (2H, s), 7.05–6.90 (2H, m), 6.88–6.80
(1H, m), 5.15 (2H, d, J = 14.3Hz), 5.05 (2H, d,
J = 14.3Hz), 4.50(2H, –OC H2–, t, J = 9.2Hz), 3.76
(2H, –OCH2CH2Cl, t, J = 9.2Hz). HR-MS (ESI-TOF)
calculated for C16H15ClF2N6O3 412.0862, found
413.0866 (M+H, 79Br), 415.0811 (M+H, 81Br). ESI m/z
413.1
(M+H),
344.1
(MÀC2H2N3),
289.3
(MÀC3H4ClO3), 220.1 (MÀC5H6ClN3O3).
4.2.12. O-Salicylfluconazole (1l). The general synthetic
method described above afforded 1l (44.0%) as an amor-
phous powder; mp 142–143ꢁC. 1H NMR (CDCl3,
400MHz): d 8.31 (2H, s), 8.05 (1H, d, J = 8.3Hz),
7.93 (2H, s), 7.56–7.50(1H, m), 7.31–7.22 (2H, m),
7.07–6.92 (2H, m), 6.87–6.78 (1H, m), 5.21
(2H, d, J = 15.1Hz), 5.08 (2H, d, J = 15.1Hz), 2.15
(3H, s). ESI m/z 469.0(M+H), 426.9 (M ÀC2H3O+H),
399.9 (MÀC2H2N3), 289.3 (MÀC9H7O4), 220.1
(MÀC11H9N3O4).
4.3.3. 11-Bromoundecanyl-2-cyanoethyl fluconazole phos-
phate (2c). The general synthetic method described
above afforded 2c (28.1%) as a wax; mp 77.2–78.9ꢁC;
1H NMR (CDCl3, 400MHz): d 8.32 (2H, s), 7.88 (2H,
s), 7.20–7.10 (1H, m), 6.92–6.82 (1H, m), 6.82–6.71
(1H, m), 5.22 (2H, d, J = 16.0Hz), 5.05 (2H, d,
J = 16.0Hz), 4.15 (2H, –OCH2CH2CN, t, J = 8.1Hz),
3.98 (2H, –OCH2–, t, J = 6.4Hz), 3.43 (2H, –CH2Br, t,
J = 8.0Hz), 2.71 (2H, –OCH2CH2CN, t, J = 8.1Hz),
1.88–1.80(2H, –C H2CH2Br, m), 1.75–1.63 (2H,
COCH2CH2–, m), 1.43–1.21 (14H, m, methylene enve-
lope). HR-MS (ESI-TOF) calculated for C27H37-
BrF2N7O4P 671.1796, found 672.1855 (M+H,
79Br), 674.1819 (M+H, 79Br).
4.3. General procedure for the synthesis of phosphate
triester derivatives of FLC (2a–h)
2-Cyanoethyl N,N-diisopropylchlorophosphoramidite
(267lL, 1.2mmol) (for 2a–e) or methyl N,N-diisoprop-
ylchlorophosphoramidite (240lL, 1.2mmol) (for 2f–h)
and diisopropyl ethylamine (209lL, 1.2mmol) were
added to a solution of alcohol (ROH) (1.0mmol) in
dry DCM (2mL) under a N2 atmosphere. After stirring
for 20h, the reaction mixture was partitioned between
EA (10mL) and distilled water (10mL). The organic
layer was then washed with brine (10mL) and dried over
anhydrous MgSO4. The solvent was removed in vacuo
and the syrup was dried under vacuum overnight.
FLC (200mg, 0.6mmol) and 1H-tetrazole (84mg,
1.2mmol) were dissolved in dry dichloromethane
(DCM, 2mL) under a N2 atmosphere. The syrup was
dissolved in dry DCM (1mL) and was added to this
reaction mixture. After stirring for 3h, the reaction
was cooled to 0ꢁC and t-butyl hydroperoxide (0.3mL)
was added. After stirring for 1h, the reaction mix-
ture was partitioned between EA (10mL) and distilled
water (10mL). The organic phase was then washed with
brine (10mL) and dried over anhydrous MgSO4. The
solvent was removed in vacuo. The syrup residue was
purified by silica gel column chromatography, eluting
with hexane and acetone from 3:1 (v/v) to 1:1 (v/v) to
yield 2a–h (Scheme 3).
4.3.4. 2-Cyanoethyl-tetradecanyl fluconazole phosphate
(2d). The general synthetic method described above
afforded 2d (27.3%) as a wax; mp 79.2–83.5ꢁC; 1H
NMR (CDCl3, 400MHz): d 8.33 (2H, s), 7.88 (2H, s),
7.20–7.11 (1H, m), 6.90–6.80 (1H, m), 6.80–6.71 (1H,
m), 5.22 (2H, d, J = 14.9Hz), 5.10(2H, d,
J = 14.8Hz), 4.18 (2H, –OCH2CH2CN, t, J = 7.8Hz),
3.96 (2H, –OCH2–, t, J = 7.4Hz), 2.78 (2H, –OCH2CH2-
CN, t, J = 7.7Hz), 1.56 (2H, –OCH2CH2CH2–, t,
J = 7.4Hz), 1.38–1.18 (22H, methylene envelope), 0.86
(3H, t, J = 7.5Hz). HR-MS (ESI-TOF) calculated for
C30H44F2N7O4P 635.3160, found 636.3221 (M+H).
ESI m/z 636.4 (M+H).
4.3.5. 2-Cyanoethyl-n-octyl fluconazole phosphate (2e).
The general synthetic method described above afforded
2e (3.9%) as a syrup; 1H NMR (CDCl3, 400MHz)
8.35 (2H, s), 7.91 (2H, s), 7.12–7.22 (1H, m), 6.95–6.85
(1H, m), 6.85–6.75 (1H, m), 5.23 (2H, d, J = 15.1Hz),

6266
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
5.07 (2H, d, J = 15.1Hz), 4.18 (2H, –OCH2CH2CN, t,
J = 6.8Hz), 3.95 (2H, –OCH2–, t, J = 8.3Hz), 2.70
(2H, –OCH2CH2CN, t, J = 6.8Hz), 1.61 (2H,
–OCH2CH2CH2–, t, J = 8.3Hz), 1.38–1.25 (10H,
methylene envelope), 0.86 (3H, t, J = 7.7Hz). HR-MS
(ESI-TOF) calculated for C24H32F2N7O4P 551.2211,
found 552.2219 (M+H).
4.4.2. x-Undecylenyl fluconazole phosphate (3b). The
general synthetic method described above afforded 3b
(65.2%) as a syrup; 1H NMR (CDCl3, 400MHz): d
8.28 (2H, s), 7.88 (2H, s), 7.15–7.00 (2H, m), 6.89–
6.75 (1H, m), 5.89–5.76 (1H, CH2@CH–, m), 5.25
(2H, d, J = 14.2Hz), 5.03 (2H, d, J = 14.2Hz), 5.00–
4.86 (2H, CH2@CH–, m), 3.99 (2H, –OCH2–, t,
J = 8.1Hz), 2.10–1.99 (2H, CH2=CHCH2–, m), 1.60
(2H, –OCH2CH2CH2–, t, J = 7.5Hz), 1.44–1.18
(12H, methylene envelope). HR-MS (ESI-TOF) calcu-
lated for C24H33F2N6O4P 538.2269, found 539.2270
(M+H).
4.3.6. Methyl-undecanyl fluconazole phosphate (2f). The
general synthetic method described above afforded 2f
(28.0%) as a syrup; 1H NMR (CDCl3, 400MHz): d
8.33 (2H, s), 7.95 (2H, s), 7.21–7.14 (1H, m), 6.93–6.82
(1H, m), 6.82–6.73 (1H, m), 5.25 (2H, d, J = 14.7Hz),
5.05 (2H, d, J = 14.7Hz), 3.94 (2H, –OCH2–, t,
J = 7.8Hz), 3.41 (3H, –OCH3, s), 1.59 (2H,
–OCH2CH2CH2–, t, J = 7.6Hz), 1.45–1.22 (16H, meth-
ylene envelope), 0.89 (3H, t, J = 7.2Hz). ESI m/z 555.2
(M+H).
4.4.3. 11-Bromoundecanyl fluconazole phosphate (3c).
The general synthetic method described above afforded
1
3c (100.0%) as a syrup; H NMR (CDCl3, 400MHz):
d 8.26 (2H, s), 7.89 (2H, s), 7.12–7.00 (1H, m), 6.93–
6.84 (1H, m), 6.83–6.72 (1H, m), 5.20(2H, d,
J = 14.9Hz), 5.02 (2H, d, J = 14.9Hz), 3.91 (2H,
–OCH2–, t, J = 8.4Hz), 3.47 (2H, –CH2Br, t,
J = 7.8Hz), 1.87–1.81 (2H, –CH2CH2Br, m), 1.76–
1.65 (2H, COCH2CH2–, m), 1.45–1.23 (14H, m, methyl-
ene envelope). ESI m/z 619.2 (M+H, 79Br), 621.2 (M+H,
81Br).
4.3.7. Methyl-x-undecylenyl fluconazole phosphate (2g).
The general synthetic method described above afforded
1
2g (36.7%) as a syrup; H NMR (CDCl3, 400MHz): d
8.31 (2H, s), 7.92 (2H, s), 7.21–7.11 (1H, m), 6.93–6.83
(1H, m), 6.82–6.72 (1H, m), 5.87–5.71 (1H, CH2@CH–,
m), 5.25 (2H, d, J = 15.4Hz), 5.05 (2H, d,
J = 15.5Hz), 5.06–4.89 (2H, CH2@CH–, m), 3.96
(2H, –OCH2–, t, J = 7.8Hz), 3.40(3H, –OCH 3, s),
2.15–2.03 (2H, CH2=CHCH2–, m), 1.58 (2H,
–OCH2CH2CH2–, t, J = 7.2Hz), 1.43–1.17 (12H, meth-
ylene envelope). ESI m/z 553.4 (M+H).
4.4.4. n-Tetradecanyl fluconazole phosphate (3d). The
general synthetic method described above afforded 3d
(100.0%) as a syrup; H NMR (CDCl3, 400MHz) 8.25
1
(2H, s), 7.86 (2H, s), 7.18–7.09 (1H, m), 6.92–6.83
(1H, m), 6.82–6.73 (1H, m), 5.20(2H, d, J = 15.1Hz),
4.99 (2H, d, J = 14.9Hz), 3.97 (2H, –OCH2–, t,
J = 7.7Hz), 1.57 (2H, –OCH2CH2CH2–, t, J = 7.7Hz),
1.41–1.98 (22H, methylene envelope), 0.88 (3H, t,
J = 7.9Hz). ESI m/z 583.3 (M+H)+.
4.3.8. Methyl-n-octyl fluconazole phosphate (2h). The
general synthetic method described above afforded 2h
(38%) as a syrup; 1H NMR (CDCl3, 400MHz): d
8.23 (2H, s), 7.99 (2H, s), 7.25–7.16 (1H, m),
7.01–6.91 (1H, m), 6.87–6.71 (1H, m), 5.25 (2H, d,
J = 14.7Hz), 5.11 (2H, d, J = 14.7Hz), 3.99 (2H,
–OCH2–, t, J = 7.8Hz), 3.57 (3H, OCH3, s), 1.69 (2H,
–OCH2CH2CH2–, t, J = 8.1Hz), 1.43–1.29 (10H,
methylene envelope), 0.91 (3H, t, J = 8.3Hz). MS
(ESI) calculated for C22H31F2N6O4P 512.2, found
513.2 (M+H).
4.5. General procedure for the synthesis of carbohydrate
phosphate triester derivatives of FLC (4a and 4b)
Benzylamine (BnNH2) (163lL, 1.5mmol) was added to
a solution of b-D-glucosepentaacetate (7a) or 2-acetam-
ido-2-deoxy-1,3,4,6-tetra-O-acetyl-b-D-glucopyranoside
(7b) (1mmol) in THF (3mL). After stirring overnight,
the reaction mixture was diluted with cold, distilled
water and extracted with DCM twice (2 · 5mL). The
combined organic phase was, respectively, washed with
ice-cold diluted hydrochloric acid, saturated sodium
bicarbonate (5mL), brine (5mL), and distilled water
(5mL). The combined extracts were dried (MgSO4)
and the solvent was removed in vacuo. The syrup was
dried in a vacuum overnight to afford 8a and 8b (Scheme
3). 2-Cyanoethyl N,N-diisopropylchlorophosphorami-
dite (236.7lL, 1mmol) and diisopropyl ethylamine
(174.3lL, 1mmol) were added to a solution of 8a or
8b (0.5mmol) in dry DCM (2mL). After stirring for
20h, the reaction mixture was extracted with EA
(10mL) and distilled water (10mL). The organic phase
was then washed with brine (10mL) and dried (MgSO4).
The solvent was removed in vacuo and the syrup was
dried under vacuum overnight to yield 9a and 9b
(Scheme 3). FLC (100mg, 0.3mmol) and 1H-tetrazole
(42mg, 0.6mmol) were dissolved in dry DCM (1mL)
under a N2 atmosphere. The syrup (9a or 9b) was dis-
solved in dry DCM (0.5mL) and added to the reaction
4.4. General procedure for the synthesis of phosphate
diester derivatives of FLC (3a–d)
Ammonium hydroxide (57lL, 0.48mmol) was added to
a solution of compound 2 (114.8mg, 0.19mmol) in
methanol (1mL) and DCM (1mL). After stirring for
24h, the solvent was evaporated in vacuo. The syrup
residue was centrifuged and lyophilized to afford 3
(Scheme 2).
4.4.1. n-Undecanyl fluconazole phosphate (3a). The gen-
eral synthetic method described above afforded 3a
(65.6%) as a syrup; 1H NMR (CDCl3, 400MHz): d
8.28 (2H, s), 7.91 (2H, s), 7.21–7.13 (1H, m), 6.92–6.82
(1H, m), 6.82–6.71 (1H, m), 5.20(2H, d, J = 15.3Hz),
5.01 (2H, d, J = 15.4Hz), 3.95 (2H, –OCH2–, t,
J = 8.1Hz), 1.59 (2H, –OCH2CH2CH2–, t, J = 8.1Hz),
1.41–1.27 (16H, methylene envelope), 0.95–0.86 (3H,
m). HR-MS (ESI-TOF) calculated for C24H35F2N6O4P
540.2425, found 541.2431 (M+H).

N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
6267
mixture. After stirring for 3h, the reaction mixture was
cooled to 0ꢁC and t-butyl hydroperoxide (0.15mL)
was added. After stirring for 1h, the reaction mixture
was partitioned between EA (10mL) and distilled water
(10mL). The organic layer was then washed with brine
(10mL) and dried (MgSO4). The solvent was removed
in vacuo. The syrup residue was purified using silica
gel column chromatography, eluting with hexane/ace-
tone from 2:1 (v/v) to 1:1 (v/v) to afford 4a or 4b
(Scheme 3).
are estimated partition coefficient and molecular weight
for each compound.
The HYDROWIN (Version 1.67) (Mill, T., Haag, W., Pen-
well, P., Pettit, T., Johnson, H. EPZ Contract no 68-02-
4254. Menlo Park, CA: SRI International, 1987) was
used to estimate the half-lives based on the total acid-
catalyzed rate constant and the prediction methodology
developed for the US Environment Protection Agency.
The half-lives for acid-catalyzed rate constants were
calculated at pH5.6 from the general equation,
Half-life = 0.6931/(Kb)(1.0EÀ8.4), where 1.0EÀ8.4 is
the OHÀ concentration in water at pH5.6.
4.5.1. 2-Cyanoethyl-[1-(b-D-2,3,4,6-glucosyltetraacetate)]-
fluconazole phosphate (4a). The general synthetic method
described above afforded 4a (13.9%) as a syrup; 1H
NMR (CDCl3, 400MHz): d 7.73 (2H, s), 7.56 (2H, s),
7.55–7.21 (3H, m), 5.95–5.82 (1H, anomeric proton,
m), 5.60–5.42 (2H, m), 5.10 (2H, d, J = 15.1Hz), 4.90
(2H, d, J = 15.1Hz), 4.41–4.08 (6H, m), 2.78 (2H,
–OCH2CH2CN, t, J = 7.8Hz), 2.15–2.10(12H, overlap).
HR-MS (ESI-TOF) calculated for C30H34F2N7O13P
769.1920, found 770.1927 (M+H).
4.6. In vitro antifungal activity
4.6.1. Microorganisms. C. albicans ATCC 14053, A.
niger ATCC 16404, and C. neoformans ATCC 66031
were obtained from American Type Culture Collection,
Manassas, Virginia. Stock cultures were kept on Sabou-
raud dextrose agar (SDA; Becton-Dickinson and Co.,
Sparks, Maryland). Subcultures were prepared on
SDA at 35–37ꢁC. Suspension cultures were prepared
by inoculation of single colonies in 7mL of normal sal-
ine solution. Prior to preparation of susceptibility as-
says, yeast cells were resuspended in normal saline to
make a transmittance of 73–75% at 530nm that provide
equivalent concentration of 106 cells/mL and spores of
A. niger in saline medium to produce a similar transmit-
tance at 530nm compared to the control tube. The med-
ia were RPMI (RPMI 1640; ICN Biomedical, Aurora,
Ohio) adjusted to pH6.9 and Sabouraud dextrose broth
(SDB; Becton Dickinson and Co., Sparks, Maryland).
4.5.2. 2-Cyanoethyl-[1-(2-acetamido-2-deoxy-3,4,6-tri-O-
acetyl-b-D-glucopyranosidyl)]fluconazole phosphate (4b).
The general synthetic method described above afforded
3b (8.3%) as a wax; 1H NMR (CDCl3, 400MHz): d
7.81 (2H, s), 7.59 (2H, s), 7.52–7.19 (3H, m), 5.91–5.80
(1H, anomeric proton, m), 5.59–5.43 (2H, m), 5.05
(2H, d, J = 14.7Hz), 4.92 (2H, d, J = 14.7Hz), 4.42–
4.33 (2H, m), 4.39–4.05 (4H, m), 2.75 (2H,
–OCH2CH2CN, t, J = 7.3Hz), 2.12–2.06 (12H, overlap).
HR-MS (ESI-TOF) calculated for C30H35F2N8O12P
768.2080, found 769.2068 (M+H).
4.5.3. Physicochemical properties. Physicochemical prop-
erties were estimated using the KowWin program
(Version 1.67) and estimation methodology developed
at Syracuse Research Corporation (Environmental
Chemistry Center, KOWWIN—The Octanol–Water
Partition Coefficient Program, PC-Computer software,
Running Ridge Road, North Syracuse, NY, 1999).
The LogKow (KowWin) program estimates the log
octanol/water partition coefficient (logP) of organic
chemicals using an atom/fragment contribution
method.48 KowWin uses a Ôfragment constantÕ method-
ology to predict logP. In a Ôfragment constantÕ method,
a structure is divided into fragments (atom or larger
functional groups) and coefficient values of each
fragment or group are summed together to yield the
logP estimate. To confirm the validity of the logP
calculation, the reported experimental data were
compared with acquired data for some of the com-
pounds. The experimental data were in agreement with
the calculated logP values.
4.6.2. Chemicals and antifungal agents. AMB was pur-
chased from Acros, New Jersey, USA, and was kept
as a 5lM stock in DMSO at 0ꢁC and used during one
week of preparation. FLC was purchased from Medisa
Inc., New York, USA, or was provided from Vera Lab-
oratories Ltd, Hyderabad, India, and was kept as a
20lM stock solution at 0ꢁC. Test compounds were dis-
solved in DMSO (0.56mg/mL) and stored at 0ꢁC.
Working dilutions were made in RPMI or SDB med-
ium. Higher concentrations of compound were used
for compounds with weak antifungal activities. The final
maximum concentration of DMSO in the assays was 5%
(v/v). DMSO was not inhibitory to the organisms in the
concentrations tested.
4.6.3. Susceptibility testing. Microdilutions for control
experiments with C. albicans, A. niger, and C. neofor-
mans were the modified method of National Committee
for Clinical Laboratory Standards (NCCLS) method
as described by Galgiani49 and by the more recent
NCCLS M27-A microdilution methods.50 as described
previously.51 Dilutions were prepared in 0.1mL of
RPMI or SDB; the inocula were either 104 C. albicans
or C. neoformans cells or A. niger spores. The tubes
were incubated for 24–48h at 36 1ꢁC, and turbidity
was read visually. MICs were calculated in comparison
to growth control as the lowest concentration that
shows inhibition for AMB, FLC, and the test
compounds.
The Dermal Permeability Coefficient Program (Derm-
Win Version 1.43) was used to estimate the dermal per-
meability coefficient (Kp) and the dermally absorbed
dose per event (DAevent) of organic compounds. Derm-
Win estimates a LogKow for every SMILES notation by
using the estimation engine from the KowWin Program
based on the general equation, LogKp = À2.72 +
0.71LogKow À 0.0061 MW, where LogKow and MW

6268
N.-H. Nam et al. / Bioorg. Med. Chem. 12 (2004) 6255–6269
27. Langner, C. A.; Travis, J. K.; Caldwell, S. J.; Tianbao, J.
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We are grateful to the Slater Center for Biomedical
Technology for a grant to Keykavous Parang for carry-
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