Synthesis, cleavage, and antifungal activity of a number of novel, water-soluble ester prodrugs of antifungal triazole CS-758

Article abstract of DOI:10.1016/j.bmcl.2009.04.135

In this study, the synthesis and evaluation of a number of esters of CS-758 as injectable prodrugs are described. Phosphoryl ester 1a was soluble in water (>30 mg/mL) and was converted to CS-758 in human liver microsome. It was also converted to CS-758 in rats after iv administration, wherein the bioavailability of CS-758 was 53%. Compound 1a (iv) reduced the viable cell counts in kidneys in a murine systemic Candida albicans infection model, wherein the effect was comparable to or slightly superior to that of CS-758 (po). The prodrug 1a proved to be a promising injectable antifungal agent whose further evaluation is warranted.

Full text of DOI:10.1016/j.bmcl.2009.04.135

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3559–3563
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, cleavage, and antifungal activity of a number of novel,
water-soluble ester prodrugs of antifungal triazole CS-758
Yoshiko Kagoshima a, Makoto Mori b, Eiko Suzuki c, Takahiro Shibayama c, Tamako Iida d,
Yasuki Kamai d, Toshiyuki Konosu a,
*
a Medicinal Chemistry Research Laboratories II, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
b Medicinal Chemistry Research Laboratories I, Daiichi Sankyo Co., Ltd, 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan
c Drug Metabolism and Pharmacokinetics Research Laboratories, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
d Biological Research Laboratories IV, Daiichi Sankyo Co., Ltd, 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, the synthesis and evaluation of a number of esters of CS-758 as injectable prodrugs are
described. Phosphoryl ester 1a was soluble in water (>30 mg/mL) and was converted to CS-758 in human
liver microsome. It was also converted to CS-758 in rats after iv administration, wherein the bioavailabil-
ity of CS-758 was 53%. Compound 1a (iv) reduced the viable cell counts in kidneys in a murine systemic
Candida albicans infection model, wherein the effect was comparable to or slightly superior to that of CS-
758 (po). The prodrug 1a proved to be a promising injectable antifungal agent whose further evaluation is
warranted.
Received 13 March 2009
Revised 27 April 2009
Accepted 29 April 2009
Available online 3 May 2009
Keywords:
Antifungal
CS-758
Ó 2009 Elsevier Ltd. All rights reserved.
Prodrug
Parenteral formulation
Phosphoryl ester
There is a great medical need for an injectable antifungal
agent with a broad spectrum for the treatment of severe deep
mycoses of hospitalized patients. Currently, fluconazole (or fos-
fluconazole) and amphotericin B are available for parenteral
use, but they have limitations in terms of antifungal spectra
and safety, respectively.1 Most of the azoles under development
have a broader spectrum but cannot be administered parenter-
ally without modification because of low water-solubility.2,3
There have been some efforts to overcome this problem by using
a prodrug approach.4
liberate an equivalent amount of formaldehyde or acetaldehyde
in vivo when cleaved. Fosfluconazole,1d,e the phosphoric acid es-
ter prodrug of fluconazole, has improved water-solubility, but
its antifungal spectrum is limited only to that of fluconazole.
Consequently, we focused our efforts on identifying a suitable
polar or charged group with which we could functionalize CS-
758 on the tertiary hydroxy group. In this paper, we describe
the synthesis and the characteristics of such prodrugs of CS-
758.
For water-soluble prodrugs, we first planned to prepare com-
pounds 1a–c in order to check their solubility in water and ability
to release CS-758. Then, compounds 2a and 2b, derivatives of 1b,
were investigated. Similarly, compounds 3a–c, derivatives of 1c,
were investigated (Scheme 1–5).
Previously, we identified CS-7585 (Fig. 1), which has a broad
antifungal spectrum covering Aspergillus spp., fluconazole-resistant
Candida spp. and has a good safety profile including low drug–drug
interaction. Since the water solubility of CS-758 was, however, too
low for parenteral formulation, we conducted a study on a new
prodrug of CS-758, which should have sufficient water-solubility
and efficient bioconversion.
CS-758 has two obvious functional groups, namely the ter-
tiary hydroxy and the triazole groups, with the possibility of
being linked to a pro-moiety. It is known that water-soluble
prodrugs can be readily accessed by alkylating a triazole ring
with halomethyl acetate derivatives to give
a quarternary
ammonium salt prodrug.4d,6 However, the prodrugs in this class
* Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5496 8344.
Figure 1. Structural formula of CS-758.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.135

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Y. Kagoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3559–3563
Scheme 1. Synthesis of 1a. Reagents and conditions: (a) i-Pr2NP(OAllyl)2, 1H-tetrazole, CH2Cl2, 89%; (b) t-BuOOH, CH2Cl2, 82%; (c) PdCl2(PPh3)2, n-Bu3SnH, CH2Cl2; (d)
NaHCO3 aq, then C-18 reverse-phase column chromatography, 45%, two steps.
Scheme 2. Synthesis of 1b. Reagents and conditions: (a) 1-NaHCO3, H2O, 2-4-(MeO)BnCl, 60%; (b) allyl chloroformate, DMAP, CH2Cl2, 86%; (c) anisole, TFA, quant.; (d) (COCl)2,
cat. DMF, THF, 90%; (e) 1-NaH, THF; 2–10, 16%; (f) PdCl2(PPh3)2, n-Bu3SnH, CH2Cl2, quant.
Scheme 3. Synthesis of 1c. Reagents and conditions: (a) (COCl)2, cat. DMF, CH2Cl2; (b) NaH, 13, 26%; (c) CH3NHNH2, CH2Cl2, 85%.
Scheme 4. Synthesis of compound 1b’s derivatives 2a and 2b. Reagents and conditions: (a) monoallyl succinic acid chloride, NEt3, DMAP, CH2Cl2, 77%; (b) 1-PdCl2(PPh3)2, n-
Bu3SnH, CH2Cl2, 2-NaHCO3 aq, then C-18 reverse phase column, 34%; (c) 1-i-Pr2NP(OAllyl)2, 1H-tetrazole, CH2Cl2; 2-t-BuOOH, CH2Cl2, 84%; (d) PdCl2(PPh3)2, n-Bu3SnH, CH2Cl2
then NaHCO3 aq, 52%, two steps.

Y. Kagoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3559–3563
3561
Scheme 5. Synthesis of compound 1c’s derivatives 3a–c. (a) (COCl)2, cat. DMF, THF; (b) NaH, 18, THF, 37%; (c) 1-MeOTf, i-Pr2NEt, CH2Cl2, 2-DOWEX1 Â 4 (Cl form), 32%; (d)
(COCl)2, cat. DMF, CH2Cl2; (e) NaH, 20, THF, 10%; (f) MeNHNH2, CH2Cl2, 61%.
Table 1
100
1a
Solubility in water and conversion to CS-758 (in human plasma and liver microsome)
CS-758
80
60
40
20
0
Compd
Solubility in water (mg/mL)
In vitro (human)a
Plasma Liver ms
30 min 120 min 30 min 120 min
1a
1b
2a
>30
<1
>10
–
–
+
–
–
+
–
–
b
b
b
b
–
–
–
2b
1c
3a
3b
3c
>10
<1
<1
>10
<1
–
–
–
+
–
–
–
–
–
–
–
–
–
–
–
0
60
120
Time (min)
180
240
++
–
a
–; no detection of CS-758 (<10%), ; low formation of CS-758 (10–30%), +;
middle formation of CS-758 (30–60%), ++; high formation of CS-758 (>60%).
Figure 2. Conversion of 1a (s) to CS-758 (d) in human liver microsome.
b
Compound 1b was detected.
temperature, provided a modest yield (8–10%) of the desired ester
11. The allyloxycarbonyl group of 11 was removed by bis(triphen-
ylphosphine)dichloropalladium and tri(n-buthyl)hydride to give
compound 1b.
In a similar manner, 1c was synthesized from CS-758 and acid
chloride 13 which was prepared from N-phthaloyl glycine 12.
The synthesis of 1a is shown in Scheme 1. Despite the steric hin-
drance of the tertiary hydroxy group of CS-758, compound 1a
could smoothly be synthesized from CS-758 using the phosphory-
lation procedure developed by Fraser-Reid.7 Thus, CS-758 was
treated with diallyl diisopropylphosphoramidite in the presence
of 1H-tetrazole in dichloromethane to afford the diallyl phosphite
4. Phosphite 4 was then oxidized by t-butyl hydroperoxide to give
the corresponding phosphate 5. Removal of the two allyl groups
was accomplished by use of bis(triphenylphosphine)dichloropalla-
dium and tri(n-butyl)tin hydride in dichloromethane. The sodium
salt 1a was prepared by treatment with sodium hydrogen carbon-
ate and purified by C-18 reverse-phase column chromatography.
The syntheses of 1b and 1c are shown in Schemes 2 and 3. The
OH group of glycolic acid 6 was protected by the allyloxycarbonyl
group in three steps to give 9. Treatment of 9 with oxalyl chloride
in dichloromethane afforded corresponding acid chloride 10. The
usual methods reported for the preparation of esters from alcohols
were not successful with CS-758 because of its steric hinderance.
After many attempts to esterify this hydroxyl moiety, a direct
esterification reaction of oxide anion, which was generated
from CS-758 and NaH in THF, with acid chloride 10 at room
3b
100
CS-758
80
60
40
20
0
0
60
120
Time (min)
180
240
Figure 3. Conversion of 3b (s) to CS-758 (d) in human plasma.

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Y. Kagoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3559–3563
smooth conversion to the diallyl phosphite was achieved. This phos-
100
10
phite was oxidized successively with t-butyl hydroperoxide to give
the corresponding phosphate 16. Removal of the allyl groups was
accomplished by use of bis(triphenylphosphine)dichloropalladium
and tri(n-butyl)tin hydride in dichloromethane to afford 1c.
The syntheses of 3a–c are shown in Scheme 5. N,N-Dimethyla-
minoglycine ester 3a was synthesized from CS-758 and 18. 3a was
allowed to react with methyl trifluoromethanesulfonate in CH2Cl2
followed by anion exchange chromatography with DOWEX-1 to af-
ford quaternary ammonium compound 3b. b-Alanine ester 3c was
synthesized in a similar manner to that shown in Scheme 3.
The solubility in water and the conversion rate to CS-758 in hu-
man plasma and in human liver microsome (ms) are shown in
Table 1. Compound 1a has good water solubility (>30 mg/mL).
1a was converted to CS-758 in human liver ms, but it was not con-
verted to CS-758 in human plasma. The solubility of 1b and 1c
was not sufficient. Though these compounds were not converted
to CS-758 in human liver ms, they released CS-758 in human plas-
ma moderately. Compounds 2a and 2b were sufficiently improved
in water-solubility. These compounds were not converted to
CS-758 in human plasma. When 2a and 2b were incubated with
human liver ms, they were smoothly converted to intermediate
1b. However, 1b was not converted to CS-758 in human liver
ms. Compounds 3a and 3c did not show any improvement in
either water-solubility or conversion rates to CS-758, unfortu-
nately. However, betaine ester 3b has good water solubility.
Furthermore, compound 3b was quickly converted to CS-758 in
human plasma.
1a
CS-758
1
0.1
0.01
0
4
8
12
16
20
24
Time (hrs)
Figure 4. Plasma level of CS-758 after iv administration of 1a to rats at a dose of
2 mg/kg (average of three rats).
100
3b
CS-758
10
1
0.1
0.01
The disappearance of 1a and the formation of CS-758 after incu-
bation of 1a with human liver ms are shown in Figure 2, and the
disappearance of 3b and formation of CS-758 after incubation of
3b with human plasma are shown in Figure 3.
The in vivo conversion of 1a and 3b to CS-758 upon iv adminis-
tration to rats is shown in Figures 4 and 5, respectively. When 1a
was administrated to rats, CS-758 was gently formed and slowly
eliminated with t1/2 of 5.7 h. The bioavailability8 of CS-758 was
53%. When 3b was administrated to rats, CS-758 was quickly
0.001
0
2
4
6
8
Time (hrs)
Figure 5. Plasma level of CS-758 after iv administration of 3b to rats at a dose of
2 mg/kg (average of three rats).
formed and a high concentration of CS-758 was observed (Cmax
,
Removal of the phthaloyl group in 14 was achieved by the use of
methylhydrazine to afford the desired amine 1c.
1.3 g/mL). But CS-758 was quickly eliminated and the bioavail-
l
ability of CS-758 was only 3.3%. The reason for the low conversion
rate of 3b was unclear.
The syntheses of 2a and 2b are shown in Scheme 4. We at-
tempted to modify the hydroxy group of 1b with a hydrophilic
function such as succinic acid or phosphoric acid groups. 1b was
allowed to react with mono allyl succinic acid chloride in dichloro-
methane to give allyl ester 15. The allyl group in 15 was removed
by the use of Pd(0) and tri(n-butyl)tin hydride. The sodium salt 2a
was prepared by treatment with sodium hydrogen carbonate and
purified by C-18 reverse-phase column chromatography.
The in vivo efficacies of CS-758 (oral administration) and pro-
drug 1a (iv administration) were evaluated in a systemic C. albicans
infection model in mice (Fig. 6). The efficacy of iv-administered
prodrug 1a was comparable or slightly superior to the efficacy of
orally administered CS-758.
Consequently, the phosphoryl ester prodrug 1a proved to be a
promising agent as an antifungal agent for parenteral use and its
further preclinical evaluation is warranted.
When compound 1b was treated with diallyl diisopropylphosph-
oramidite in the presence of 1H-tetrazole in dichloromethane, a
Schedule
6
5
4
3
6
5
4
3
Challenge
Assay
24h
(4.2 × 105 CFU/mouse)
Administration
*
***
*** ***
2h
0.2
2
20
0.2
2
20
Control
(Vehicle)
Control
(Vehicle)
Mean+S.E. ( =8)
n
CS-758 (po
Dose (mg/kg)
)
1a (iv)
Dose (mg/kg)
*
p
<0.05, *** p<0.001
Figure 6. In vivo efficacy of compound 1a (iv) against murine intravenous infection with C. albicans SANK 51486 compared with that of CS-758 (po).

Y. Kagoshima et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3559–3563
3563
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