Synthesis and evaluation of N-substituted 1,4-oxazepanyl Sordaricins as selective fungal EF-2 inhibitors.

Article abstract of DOI:10.1016/S0960-894X(02)00290-1

Sordaricin analogues possessing 6-methoxy-7-methyl-1,4-oxazepane moiety instead of the sugar part were synthesized and evaluated. It was found that N-substituents on the oxazepane ring had influence on biological activity. In particular, N-(2-methylpropenyl) derivative 12p exhibited potent in vitro antifungal activity. Furthermore, 12p maintained significant activity (MIC 0.25 microg/mL) against Candida albicans SANK51486 even in the presence of 20% horse serum.

Full text of DOI:10.1016/S0960-894X(02)00290-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1705–1708
Synthesis and Evaluation of N-Substituted 1,4-Oxazepanyl
Sordaricins as Selective Fungal EF-2 Inhibitors
Satoru Kaneko,a,* Masami Arai,a Takuya Uchida,a Tamako Harasaki,b
Takashi Fukuokab and Toshiyuki Konosua
aMedicinal Chemistry Research Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome,
Shinagawa-ku, Tokyo 140-8710, Japan
bBiological Research Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome,
Shinagawa-ku, Tokyo 140-8710, Japan
Received 13 March 2002; accepted 23 April 2002
Abstract—Sordaricin analogues possessing 6-methoxy-7-methyl-1,4-oxazepane moiety instead of the sugar part were synthesized
and evaluated. It was found that N-substituents on the oxazepane ring had influence on biological activity. In particular,
N-(2-methylpropenyl) derivative 12p exhibited potent in vitro antifungal activity. Furthermore, 12p maintained significant activity
(MIC 0.25 mg/mL) against Candida albicans SANK51486 even in the presence of 20% horse serum. # 2002 Elsevier Science Ltd.
All rights reserved.
Sordarin (1) is a diterpene glycoside isolated from Sor-
daria araneosa in the early 1970s (Fig. 1).1 Acid-cata-
lyzed hydrolysis of 1 affords sordaricin (2), which is a
caged tetracyclic aglycon in common with the congeners
of the sordarin family.2aÀe In 1987, zofimarin (3) was
isolated from Zopfiella marina SANK21274 as an anti-
fungal natural product,3 and showed moderate inhibi-
tory activity in the growth of pathogenic fungi.
Recently, a target molecule for the sordarin family has
been disclosed. Compounds of this class interfere with
fungal protein synthesis by means of selectively binding
to fungal elongation factor 2 (EF-2).4a,b,5,6 They con-
tribute to form a stable EF-2-ribosome complex and
prevent the release of EF-2 in the course of translation.7
Since the mode of action was revealed, sordarin ana-
logues have been a fascinating synthetic target for
developing novel antifungal agents.
serum.14 Nevertheless, it was demonstrated by the
Glaxo group that GW531920( 5) with a morpholine
appendage instead of the sugar component exhibited
good in vivo efficacy.9,15
Encouraged by this finding, we envisaged a new series of
sordaricin derivatives bearing an oxazepane ring,
anticipating efficient activity even in the presence of
serum. Since the hydrophobicity of the side portion was
very important to exhibit good antifungal activity,16 our
attention was focused on the introduction of different
A number of sordaricin derivatives have been known to
date,8,9 and a few structure–activity relationships were
reported during the preparation of this manuscript.10À12
We also reported 4 as one of such potent compounds.13
The only problem is that the activity of these com-
pounds is generally diminished in the presence of
*Corresponding author. Tel.: +81-3-3492-3131; fax: +81-3-5436-
8563; e-mail: skanek@shina.sankyo.co.jp
Figure 1. Chemical structures of sordarin 1 and its related compounds.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00290-1

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S. Kaneko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1705–1708
Scheme 1. Reagents and conditions: (a) NaOMe, MeOH, rt; (b) PMBCl, NaHCO3, DMF, 70 ꢀC; (c) CH(OMe)3, (CH2OH)2, TsOH, MeOH, rt; (d)
NaIO4, NaHCO3, MeOH–H2O, rt; (e) primary amine, NaBH3CN, AcOH, MeCN, rt; (f) 1 N-HCl, MeOH, rt; (g) TFA, CH2Cl2, rt.
lipophilic N-substituents. Herein, we describe the
synthesis of N-substituted 1,4-oxazepan-2-yl sordaricin
derivatives, and their in vitro antifungal activity under
conditions with or without horse serum.
ester 7 in 99% yield. At this stage, the formyl group was
protected by treatment with ethylene glycol and a cata-
lytic amount of TsOH in MeOH, to provide ethylene
acetal 8 in a quantitative yield. Oxidative cleavage of the
vicinal diol 8 by employing aq NaIO4 in MeOH gave
rise to a quantitative amount of bis-aldehyde 9, which
was observed as diastereomeric cyclic hemiacetals in
NMR experiments. Without purification, 9 was subject
to the next step. Double reductive amination of 9 using
different primary amines proceeded smoothly to afford
reclosed oxazepane derivatives 10a–j in moderate yields.
N-Substituents are listed in Table 1. In some cases,
undesired deprotection of 1,3-dioxolan-2-yl moiety
occurred partially. Therefore, crude oxazepanes 10a–j
Chemistry
The synthetic route is outlined in Scheme 1. The syn-
thesis commenced with the degradation of 3 produced
by fermentation. Treatment of 3 with sodium methoxide
afforded sodium salt 6 as a colorless powder in 86%
yield. The resulting salt 6 was treated with p-methoxy-
benzyl chloride and NaHCO3 in DMF to furnish PMB
Table 1. N-Substitutents on the 1,4-oxazepane ring and the chemical yields of 11 and 12
R
Yielda (%)
of 11
Yield (%)
of 12
R
Yielda (%)
of 11
Yield (%)
of 12
R
Yielda (%)
of 11
Yield (%)
of 12
a
58
48
g
64
77
n
76c
81
e
b
c
d
e
f
44
46
86
68
49
65
81
h
i
5069
48
o
p
q
r
—
78
98
87
96
77
86
69
79
76
91d
49d
72d
72c
43
j
85
48
l
52c
64d
40b
m
s
aIsolation yield in two steps from 9 unless otherwise noted.
bCompound 11f was prepared by reductive amination of 11k employing NaBH3CN.
cN-Alkylation was performed with K2CO3 and KI in MeCN.
dN-Alkylation was performed with NaHCO3 and NaI in EtOH.
eCompound 11o was prepared by a different route as follows: (i) 11k, ethyl 2-(bromomethyl)acrylate, K2CO3, MeCN, rt, 73%; (ii) CH(OMe)3,
(CH2OH)2, TsOH, MeOH, rt, 100%; (iii) DIBAL, THF, À60 ꢀC, 61%; (iv) DAST, CH2Cl2, À60to 0 ꢀC, 50%; (v) 1 N-HCl, MeOH, rt, 84%.

S. Kaneko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1705–1708
1707
N-alkylation of 11k was performed with alkyl halides.
Substituted alkyl groups and yields of 11l–s are listed in
Table 1. At last, deprotection of 11l–s led to the desired
products 12l–s, respectively, in good yields.17,18
Biological Activity
The N-substituted oxazepane derivatives 12a–j, 12l–s
were assayed for their in vitro antifungal activity under
conditions with or without horse serum.20 The results
are summarized in Tables 2 and 3.
Scheme 2. Reagents and conditions: (a) Rh(PPh3)3Cl, aq EtOH,
reflux; (b) alkyl halide, K2CO3, KI, MeCN, rt; (c) alkyl halide,
NaHCO3, NaI, EtOH, rt; (d) TFA, CH2Cl2, rt.
All of the N-benzyl substituted compounds 12a–i,
except 12d, exhibited excellent antifungal activity (MIC
ꢁ0.125 mg/mL) against Candida albicans including
azole-low-susceptible strains. They also showed moder-
ate activity (MIC 0.25–8 mg/mL) against Candida glab-
rata and Candida tropicalis. In addition, 12g–i had
somewhat potency (MIC 2 mg/mL) against Cryptococcus
neoformans. In particular, 12i is the most effective com-
pound under the standard conditions used in our study.
However, the activity of these compounds were
decreased as well as that of 4 in the presence of 20%
horse serum in the medium. In the case of 12i, the MIC
were treated with 1 N-HCl in MeOH, to furnish alde-
hydes 11a–j. Yields in two steps from 9 are shown in
Table 1. Finally, the PMB groups of 11a–j were
removed to afford the desired products 12a–j in good
yields.17
When the primary amine was not commercially avail-
able, an alternative route using N-alkylation was adop-
ted as shown in Scheme 2. N-Allyl derivative 11j was
dealkylated by employing Wilkinson’s catalyst, to gen-
erate N-liberated derivative 11k in 91% yield.19 Then,
Table 2. In vitro antifungal activity of sordaricin derivatives 12a–i
Organism
MIC (mg/mL)
3
4
12a
12b
12c
12d
12e
12f
12g
12h
12i
Candida albicans ATCC24433
Candida albicans SANK51486
Candida albicans TIMM3164a
Candida albicans ATCC64550a
Candida parapsilosis ATCC90018
Candida glabrata ATCC90030
Candida tropicalis ATCC750
0.5
0.031 ꢁ0.063
0.063 ꢁ0.063
0.5
0.063
0.063 ꢁ0.063
0.063
0.031
0.016
0.031
0.031
0.25 ꢁ0.016 ꢁ0.063 ꢁ0.031 ꢁ0.063
0.25 ꢁ0.031 ꢁ0.031 ꢁ0.063 ꢁ0.031
0.5
0.5
>4
>4
0.5
0.063
0.125 ꢁ0.063
>8 >32
0.5
0.125
0.063 ꢁ0.063
0.063 ꢁ0.063
0.5
1
>32
8
2
>32
>32
0.125
0.125
>16
2
1
>16
>16
0.063 ꢁ0.063
0.125 ꢁ0.063
0.063
0.125
>16
8
0.5
2
>16
>16
2
0.5
8
>16
>32
>16
>32
>16
0.031
2
1
0.5
4
>32
2
0.5
>16
>16
0.25
0.25
2
0.5
0.25
2
0.25
4
Cryptococcus neoformans TIMM1855
Aspergillus fumigatus ATCC26430
0.25 >8
>8
>4
>32
>32
>16
Candida albicans ATCC24433b
Candida albicans SANK51486b
4
NTc
8
4
1
0
2
.5
8
1
32
4
8
16
4
2
1
2
0
1
.5
2
2
0
aLow susceptibility to fluconazole (MIC >4 mg/mL).
bIn the presence of horse serum (20%).
cNot tested.
Table 3. In vitro antifungal activity of sordaricin derivatives 12j, l–s
Organism
MIC (mg/mL)
5
12j
12l
12m
0.25
0.125
0.25
0.5
>16
16
2
12n
12o
12p
12q
12r
12s
Candida albicans ATCC24433
Candida albicans SANK51486
Candida albicans TIMM3164a
Candida albicans ATCC64550a
Candida parapsilosis ATCC90018
Candida glabrata ATCC90030
Candida tropicalis ATCC750
0.031
0.016
0.031
0.031
8
0.063
0.031
0.063
0.063
0.125
0.063
0.125
0.25
ꢁ0.031
ꢁ0.031
ꢁ0.031
0.063
>16
1
0.5
8
>16
ꢁ0.031
ꢁ0.031
ꢁ0.031
ꢁ0.031
0.031
0.031
0.016
0.031
0.031
16
0.125
0.25
0.125
0.031
0.016
0.031
0.031
0.125
0.063
0.125
0.125
0.016
0.031
0.031
>16
>16
>16
>16
>16
>16
0.125
0.125
0.25
0.5
>16
>16
4
1
>16
>16
0.5
0.25
0.25
0.25
0.125
0.25
0.25
0.125
0.25
4
1
8
>16
Cryptococcus neoformans TIMM1855
Aspergillus fumigatus ATCC26430
16
>16
>16
>16
>16
>16
>16
>16
Candida albicans ATCC24433b
Candida albicans SANK51486b
0.5
0.25
0.25
0.25
1
0.5
2
1
2
1
0.5
0.5
0.5
0.25
0.5
0.5
1
0.5
8
4
aLow susceptibility to fluconazole (MIC >4 mg/mL).

1708
S. Kaneko et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1705–1708
values were 1 and 0.5 mg/mL against C. albicans
ATCC24433 and SANK51486, respectively.
9. Odds, F. C. Exp. Opin. Ther. Pat. 2001, 11, 283, and
references therein.
10. Arribas, E. M.; Castro, J.; Clemens, I. R.; Cuevas, J. C.;
Chicharro, J.; Fraile, M. T.; Garcıa-Ochoa, S.; Heras, F. G.;
Ruiz, J. R. Bioorg. Med. Chem. Lett. 2002, 12, 117.
11. Bueno, J. M.; Cuevas, J. C.; Fiandor, J. M.; Garcıa-Ochoa,
S.; Heras, F. G. Bioorg. Med. Chem. Lett. 2002, 12, 121.
12. Serrano-Wu, M. H.; Laurent, D. R.; Mazzucco, C. E.;
Stickle, T. M.; Barrett, J. F.; Vyas, D. M.; Balasubramanian,
B. N. Bioorg. Med. Chem. Lett. 2002, 12, 943.
13. Kaneko, S.; Uchida, T.; Shibuya, S.; Honda, T.; Kawa-
moto, I.; Harasaki, T.; Fukuoka, T.; Konosu, T. Bioorg. Med.
Chem. Lett. 2002, 12, 803.
14. It has been presumed that the sordarin family had a high
binding affinity for serum protein. See: Aviles, P.; Falcoz, C.;
Roman, R. S.; Gargallo-Viola, D. Antimicrob. Agents Che-
mother. 2000, 44, 2333.
The N-aliphatic substituted compounds 12j, 12l–s
showed excellent activity (MIC ꢁ0.125 mg/mL) against
C. albicans including azole-low-susceptible strains. They
also showed moderate activity (MIC 0.25–4 mg/mL)
against C. glabrata and C. tropicalis. Remarkably,
compounds 12o–r exhibited good activity (MIC 0.125–
0.25 mg/mL) against Cr. neoformans unlike 5.21 In parti-
cular, 12p–r showed broad spectrum and excellent
activity among the sordarin family. Moreover, 12p
exhibited good activity (MIC 0.25–0.5 mg/mL) in the
medium supplemented with 20% horse serum. Unfor-
tunately, no compounds tested had any activity (MIC
ꢂ16 mg/mL) against Candida parapsilosis or Aspergillus
fumigatus.
15. Herreros, E.; Almela, M. J.; Lozano, S.; Heras, F. G.; Gar-
gallo-Viola, D. Antimicrob. Agents Chemother. 2001, 45, 3132.
16. Tse, B.; Balkovec, J. M.; Blazey, C. M.; Hsu, M.-J.; Niel-
sen, J.; Schmatz, D. Bioorg. Med. Chem. Lett. 1998, 8, 2269.
17. All new compounds gave satisfactory spectroscopic and
analytical data. The representative data are shown as follows.
In conclusion, new sordarin derivatives which possess a
1,4-oxazepane ring moiety were synthesized by cycli-
zation featuring successive reductive amination, and
were evaluated as antifungal agents. The compound 12p
proved to exhibit excellent antifungal activity in the
presence of serum. Thus, some N-substituted 1,4-oxa-
zepanyl sordaricins, 12p in particular, have been proved
to be useful for treatment for systemic mycosis including
infections with azole-resistant fungal strains. Further
studies toward this therapeutic utility are under way.
1
12i: H NMR (400 MHz, CDCl3) d: 9.85 (1H, s), 6.91 (1H, s),
6.78 (1H, d, J=8.1 Hz), 6.74 (1H, d, J=8.1 Hz), 6.04 (1H, d,
J=2.9 Hz), 5.94 (2H, s), 4.49 (1H, dd, J=8.8, 2.2 Hz), 4.29
(1H, d, J=9.5 Hz), 3.65 (1H, d, J=13.1 Hz), 3.59 (1H, t,
J=6.6 Hz), 3.53 (1H, d, J=13.1 Hz), 3.30(1H, d, J=9.5 Hz),
3.08 (3H, s), 2.99 (1H, d, J=13.9 Hz), 2.98 (1H, m), 2.96 (1H,
d, J=11.7 Hz), 2.59 (1H, dd, J=13.9, 2.2 Hz), 2.47 (1H, t,
J=3.7 Hz), 2.38 (1H, dd, J=11.7, 8.8 Hz), 2.33 (1H, quint.,
J=6.6 Hz), 2.12–0.96 (m), 1.25 (3H, d, J=6.6 Hz), 1.02 (3H,
d, J=6.6 Hz), 0.97 (3H, d, J=6.6 Hz), 0.80 (3H, d, J=6.6 Hz);
FABHRMS (m/z): calcd for C35H48NO8 ([M+H]+):
Acknowledgements
1
610.3380. Found: 610.3383. 12p: H NMR (400 MHz, CDCl3)
d: 9.85 (1H, s), 6.05 (1H, m), 4.89 (1H, s), 4.87 (1H, s), 4.47
(1H, dd, J=8.8, 2.2 Hz), 4.26 (1H, d, J=9.1 Hz), 3.35 (1H,
quint., J=6.6 Hz), 3.33 (1H, d, J=9.1 Hz), 3.27 (3H, s), 3.06
(1H, d, J=13.2 Hz), 3.05 (1H, m), 2.98 (1H, d, J=13.2 Hz),
2.95 (1H, brd), 2.95 (1H, brd), 2.55 (1H, dd, J=14.6, 2.9 Hz),
2.49 (1H, t, J=4.0Hz), 2.34 (1H, dd, J=12.5, 8.8 Hz), 2.32
(1H, quint., J=6.6 Hz), 2.12–0.96 (m), 1.76 (3H, s), 1.25 (3H,
d, J=6.6 Hz), 1.02 (3H, d, J=6.6 Hz), 0.99 (3H, d, J=6.6 Hz),
0.81 (3H, d, J=6.6 Hz); FABHRMS (m/z): calcd for
C31H48NO6 ([M+H]+): 530.3481. Found: 530.3459.
The authors thank Dr. T. Honda (Sankyo Co., Ltd.) for
helpful discussions.
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18. In NOE experiments, the stereochemistry of 12p was
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medium (for Cr. neoformans: yeast nitrogen base) buffered at
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30 ꢀC). Minimum inhibitory concentration (MIC) was defined
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presence of horse serum, the medium was supplemented with
20% horse serum during incubation.
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all azasordarins such as GW531920(MIC ꢂ16 mg/mL).11.