Synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition

Article abstract of DOI:10.1016/j.bmcl.2013.12.116

The synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal growth inhibition and significantly improved MIC against Candida parapsilosis and echinocandin resistant-Candida is described.

Full text of DOI:10.1016/j.bmcl.2013.12.116

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1172–1175
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antifungal activity of ASP9726, a novel echinocandin
with potent Aspergillus hyphal growth inhibition
a
a
a
a
Hiroshi Morikawa a, , Masaki Tomishima , Natsuko Kayakiri , Takanobu Araki , David Barrett ,
Souichirou Akamatsu b, Satoru Matsumoto b, Satoko Uchida b, Toru Nakai b, Shinobu Takeda b,
Katsuyuki Maki b
⇑
a Chemistry Research Laboratories, Astellas Pharma. Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
b Pharmacology Research Laboratories, Astellas Pharma. Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and antifungal activity of ASP9726, a novel echinocandin with potent Aspergillus hyphal
growth inhibition and significantly improved MIC against Candida parapsilosis and echinocandin resis-
tant-Candida is described.
Received 5 September 2013
Revised 24 December 2013
Accepted 28 December 2013
Available online 6 January 2014
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
ASP9726
Echinocandin
Candida spp.
Aspergillus fumigatus
Natural product
In recent years, with the growing number of immunosup-
pressed patients (transplants, cancer, AIDS, etc.), invasive mycoses
have become an increasingly serious problem.1 Especially, deep
seated mycosis caused by Aspergillus spp. and Candida spp. is highly
lethal and immediate treatment with antifungal agents is
required.2 Only a limited number of antifungal substances are cur-
rently available at the market as azoles, polyenes, and candins.
However, the treatment of invasive fungal diseases still remains
unsatisfied as mortality rate, even under treatment, is still unac-
ceptable high.3 For example, azoles and polyenes often have side
effects and/or drug–drug interactions, and some organisms are
resistant to these antifungals. Furthermore, effective treatment of
aspergillosis is still a major challenge.4 In addition, mycoses due
to non-albicans Candida and development of fungi resistant to
newer drugs are also of concern.5
Our research in this area started with a search for antifungal
natural products.6,7 In particular, we focused on 1,3-b-glucan
synthesis as an attractive target, since 1,3-b-glucan is a primary
component of the fungal cell wall with no counterpart in mamma-
lian cells. In earlier publications from our laboratories, we de-
scribed the chemical modification of the side chain of a natural
echinocandin-type 1,3-b-glucan synthase inhibitor FR901379,7,8
and we identified a number of echinocandin derivatives with im-
proved MIC/MEC against Aspergillus spp. and Candida spp., efforts
that culminated in the discovery of micafungin (2) (Fig. 1).9 Subse-
quently, our efforts were directed towards the identification of
next generation echinocandins with superior antifungal properties.
As part of these efforts, we found that modification of the lipophilic
side chain, the primary amide group of the glutamate side chain,
and the homotyrosine sulfate ester group, lead to enhancement
of the antifungal potency. Additionally, to our surprise, we also
found that the maximum inhibitory effect on Aspergillus spp. hy-
phae (Emax) differs between echinocandin structural types, and that
agents with a strong Emax potentially have significantly improved
in vivo antifungal activity against Aspergillus spp. as compared with
existing echinocandins. Emax was assessed by a scoring system (1–
6; 6 is the strongest effect) based on microcolony size and growth
of hyphal tip in human serum at 4-fold MEC by microscopy.10
Extensive synthetic modification and screening by Emax led to
the discovery of ASP9726 (1), as a novel echinocandin with potent
Aspergillus hyphal growth inhibition and significantly improved
MIC against Candida parapsilosis (C. parapsilosis) and echinocan-
din-resistant-Candida glabrata (echinocandin-resistant-C. glabrata),
as compared with caspofungin. In this communication, we wish to
report the synthesis and antifungal activity of this new agent.
ASP9726 (1) was synthesized as shown in Figure 2. The
de-acylated hexapeptide nucleus 3 was prepared by enzymatic
⇑
Corresponding author.
E-mail address: hiroshi.morikawa@astellas.com (H. Morikawa).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.116

H. Morikawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1172–1175
1173
OH
N
OH
O
O
OH
O
O
OH
NH
S
OH
N
N
O
N
H
N
N N
H
NH
N O
O
HO
HO
O
HO
HO
H
N
H2N
O
O
OH
O
OH
HN
N
HN
NH
NH
O
O
O
O
H
H
HO
N
N
N
ASP9726 (1)
2HCl
OH
Micafungin (2)
OH
O
O
OH
OH
HO
O
HO
OSO3Na
Figure 1. Structure of ASP9276 (1) and Micafungin (2).
OH
O
OH
N
O
OH
N
OH
OH
O
OH
OH
N
H
N
H
N
H
O
N
O
N
H
N
H
NH2
O
O
HO
HO
O
O
O
HO
HO
H2N
O
O
OH
O
HN
N
HN
H2N
NH
NH
O
OH
N
OH
HN
g, h, i
e, f
NH
O
O
O
O
H
H
O
O
O
N
N
H
a, b, c, d
N
N
N
OH
O
O
OH
O
OH
OH
OH
OH
O
OSO3Na
HO
OSO3Na
HO
OSO3Na
4
5
3
OH
OH
O
O
OH
OH
Fmoc
N
H
N
H
O
H
N
O
N
S
O
NH2
N
H
N
O
HO
HO
HO
O
HO
N N
(8)
O
N
O
OH
HN
O
OH
NH
HN
N
NH
N
O
O
O
ASP9726 (1)
H
O
O
O
H
N
N
N
n
j, k, l, m
O
OH
O
OH
OH
OH
HO
HO
7
6
Figure 2. Synthesis of the modified hexapeptide core nucleus 7 and APS9726 (1). Reagents and conditions:13 (a) benzyl chloroformate, THF, pH6.86 standard buffer solution,
68%; (b) Et3SiH, TFA, CH2Cl2, 45%; (c) H2, Pd/C, H2O, 73%; (d) (Boc)2O, NaOH, H2O, 1,4-dioxane, 88%; (e) BnBr, LiOH–H2O, DMF, 85%; (f) MsCl, NaHCO3, i-Pr2NEt, zeolite, DMF,
41%; (g) 10% HCl–MeOH, MeOH; 86%; (h) MeI, LiOHÁH2O, DMF, 76%; (i) H2, Pd/C, MeOH, quant.; (j) NaBH4, CoCl2–6H2O, MeOH, H2O, 85%; (k) NaBH3CN, dihydroxyacetone,
AcOH, MeOH, 73%; (l) Fmoc-Cl, i-Pr2NEt, DMF 46%; (m) TFA, Et3SiH, CH2Cl2, 83%; (n) 8, NaBH3CN, AcOH, MeOH, DMF, CHCl3, then piperidine; 67%.
a, b
c, d, e
O
O
O
O
O
O
O
N
N
O
N
H2N N
H
11
10
9
f, g
O
O
h, i, j
O
S
N
8
N N
12
Figure 3. Synthesis of the echinocandin side chain 8. Reagents and conditions: (a) chloro(cyclohexyl)magnesium, CeCl3, THF, 95%; (b) MeI, NaH, DMF, 89%; (c) TFA, anisole,
CH2Cl2; (d) ethyl 4-fluorobenzoate, K2CO3, DMSO, 76% (2 steps); (e) hydrazine monohydrate, EtOH, THF, 97%; (f) trans-4-(methoxycarbonyl)cyclohexanecarboxylic acid, EDC,
HOBt, Et3N, DMF, quant.; (g) P2S5, THF, 81%; (h) KOH, THF, EtOH, 86%; (i) N,O-dimethylhydroxylamine hydrochloride, HBTU, i-Pr2NEt, DMF, 88%; (j) LiAlH4, THF, 96%.
deacylation of the natural product FR901379.11 The primary amino
group of 3 was protected with a carboxybenzyloxy moiety, the
aminal hydroxy group and homotyrosine benzylic hydroxyl group
were reduced with TFA-Et3SiH, followed by hydrogenolysis to re-
move the carbobenzoxy group (cbz), followed by reprotection as
a tert-butoxycarbonyl group (t-Boc) moiety, to afford compound
4 in 68%, 45%, 73%, and 88% yields, respectively. Protection of the
homotyrosine phenol group as a benzyl ether, followed by dehy-
dration of the primary amide group of the glutamate side chain
with MsCl led to the nitrile 5 in 85%, and 41% yields, respectively.

1174
H. Morikawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1172–1175
Table 1
Compound
Emax (score) A. fumigatus #20024
MIC/MEC [lg/ml] (in human serum)
A. fumigatus #20024
C. albicans #20015
C. parapsilosis #20009
Echinocandin-resistant-C. glabrata FP2307
ASP9726 (1)
Caspofungin
5.5
1
0.25
0.5
0.25
0.25
4
64
2
>64
Table 2
Survival efficacy of ASP9726 (1) against mouse invasive pulmonary aspergillosis (IPA) model infected with A. fumigatus #20030, day 1117
Compound
Survival rate, day 11
1.5 mg/kg
3 mg/kg
ASP9726 (1)
Caspofungin
40%*
10%
70%*
20%
*
Significant survival advantage was shown with log-rank test.
Figure 4. Microphotograph of A. fumigatus #20024 microcolonies treated with ASP9726 (1) and caspofungin in human serum.18
Subsequently, the sulfate ester group of 5 was removed, even in the
presence of the t-Boc amine protecting group, by treatment with
hydrogen chloride gas in MeOH and the resulting phenol group
was methylated and the benzyl ether group was removed by
hydrogenation, to afford nitrile 6 in 86%, 76%, and quantitative
yields, respectively. The nitrile group of 6 was reduced with NaBH4
and CoCl2–6H2O to the primary amine, followed by reductive alkyl-
ation with dihydroxyacetone using NaBH3CN, protection of the
secondary amino group as a 9-fluorenylmethyloxycarbonyl group
(Fmoc) and removal of the t-Boc group, to afford key skeleton 7
in 85%, 73%, 46%, and 83% yields, respectively. Reductive amination
of 7 with aldehydye 8 using NaBH3CN, followed by removal of the
Fmoc protecting group with piperidine afforded ASP9726 dihydro-
chloride as an amorphous powder after lyophylization in 67%
yield.12
Key aldehyde 8, the side chain of ASP9726 (1), was synthesized
as shown in Figure 3. Commercially available piperidinone 9 was
treated with the organocerium reagent derived from cyclohexyl-
magesium chloride and CeCl3 in THF, followed by methylation of
the hydroxy group with MeI to yield 10 in 95%, and 89% yields,
respectively. Removal of the t-Boc moiety of 10 with TFA, followed
by reaction of the resulting amine with ethyl 4-fluorobenzoate and
conversion of the ester moiety to acyl hydrazide with hydrazine,
led to 11 in 76% in two steps, and 97% yields, respectively. Coupling
of 11 with trans-4-(methoxycarbonyl)cyclohexanecarboxylic acid,
formation of the thiadiazole ring system by reaction with P2S5, pro-
vided methyl ester 12 in quantitative yield, and 81% yields, respec-
tively. Hydrolytic cleavage of 12, conversion to the Weinreb amide,
and reduction with LiAlH4 led to key aldehyde 8 in 86%, 88%, and
96% yields, respectively.
shown in Table 1.14 ASP9726 (1) displayed greatly superior Emax
against A. fumigatus as compared with caspofungin, independent
of MEC. In vivo efficacy of ASP9726 (1) and caspofungin in an
aspergillosis animal model are shown in Table 2. This model is very
severe, and as is clearly shown in Table 2, survival with caspofun-
gin is very low (20% at 3 mpk), whereas for ASP9726 it was 70%.
ASP9726 (1) displayed superior in vivo efficacy by inhibition of
hyphal growth as compared with caspofungin.15 ASP9726 (1)
exhibited potent MIC/MEC in human serum against A. fumigatus
and C. albicans, and was also effective against C. parapsilosis and
echinocandin-resistant-C. glabrata.16
The potent inhibitory effort of ASP9726 (1) on hyphal elonga-
tion of A. fumigatus in comparison to caspofungin is shown in
Figure 4. Microcolonies produced by exposure to ASP9726 (1)
had severely stunted hypha, and were small, rounded and compact,
whereas those under caspofungin exposure were large and had
hyphal elongation in all directions.
In this communication, we have reported the discovery of
ASP9726 (1), a novel potent echinocandin, discovered by extensive
synthetic modification of a natural product FR901379, using a no-
vel screening efficacy endpoint, Emax. Potent Aspergillus hyphal
growth inhibition and significantly improved MIC against
C. parapsilosis and echinocandin-resistant-C. glabrata19 make this
compound a suitable candidate for further development as a novel
echinocandin. Future publications will report the detailed struc-
ture activity relationships of this novel class of echinocandin anti-
fungal agents, as well as detailed investigations of the in vivo
antifungal efficacy of ASP9726 (1).
Acknowledgement
In vitro antifungal activity of ASP9726 (1) and caspofungin
against Aspergillus fumigatus (A. fumigatus), Candida albicans (C.
albicans), C. parapsilosis and echinocandin-resistant-C. glabrata are
We thank Dr. Masashi Imanishi, Takuya Makino and all the
scientists involved in this work.

H. Morikawa et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1172–1175
1175
12. Characterization data for compound 1. NMR (DMSO-d6 + D2O, d): 0.89–1.27 (14H,
m), 1.30–2.01 (20H, m), 2.07–2.18 (3H, m), 2.20–2.94 (1H, m), 2.32–2.49 (3H, m),
2.54–2.63 (1H, m), 2.66–2.18 (1H, m), 0.89–1.27 (14H, m), 2.90–3.07 (5H, m), 3.09
(3H, s), 3.10–3.15 (1H, m), 3.22 (1H, t, J = 8.0 Hz), 3.52–3.71 (8H, m), 3.74 (3H, s),
3.82–4.05 (5H, m), 4.16–4.22 (3H, m), 4.36–4.44 (3H, m), 4.78–4.82 (2H, m), 6.55–
6.59 (1H, m), 6.67 (2H, d, J = 8Hz), 7.02 (2H, d, J = 8.9Hz), 7.73 (2H, d, J = 8.8Hz),
HRMS (ESI) calcd for C66H99N11O17S (M+H)+ 1350.7019, found 1350.7029.
13. Step (a)–(h), (j)–(n) were purified by ODS column chromatography.
14. Susceptibility testing was conducted based on CLSI M27-A3 or M38-A2
standard by human serum as growth medium. Inoculum concentration was
5 Â 103 cells/ml. Fungi were treated with ASP9726 (1) and caspofungin and
incubated at 37 °C under 5% CO2 for 48 h. Emax were scored (1–6) according to
the degree of hyphal growth inhibition by microscopy after A. fumigatus
#20024 was treated with ASP9726 (1) and caspofungin at 4-fold MEC and
incubated at 37 °C under 5% CO2 for 48 h.
References and notes
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17.
A mouse IPA model was established by intratracheal inoculation of A.
fumigatus #20030 conidia into 4-week-old ICR mice (n = 10)
immunosuppressed by cyclophosphamide + hydrocortisone. Intravenous
treatment of ASP9726 (1), caspofungin was initiated 1 day after infection and
continued for 10 days QD, and survivals was monitored for 11 days. Statistical
significance in survival at day 11 was calculated using log-rank test.
18. Original magnification of the microphotograph is 100-fold. A. fumigatus
#20024 was treated with ASP9726 (1) and caspofungin at 4-fold MEC and
incubated at 37 °C under 5% CO2 for 48 h.
19. Akamatsu, S.; Matsumoto, S.; Takeda, S.; Uchida, S.; Nakai, T.; Maki, K.;
Morikawa, H.; Tomishima, M.; Barrett, D. 52nd ICAAC abstr.; 2012, F-820.
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