Synthesis and antifungal activity of rhodopeptin analogues. 2. Modification of the west amino acid moiety.

Article abstract of DOI:10.1021/ol005630k

Structure-activity relationships of the west amino acid modified analogues of rhodopeptins, novel antifungal tetrapeptide isolated from Rhodococcus species Mer-N1033, have been investigated. Among the analogues synthesized, 2,2-difluoro and 2-hydroxy deri

Full text of DOI:10.1021/ol005630k

ORGANIC
LETTERS
2000
Vol. 2, No. 7
977-980
Synthesis and Antifungal Activity of
Rhodopeptin Analogues. 2. Modification
of the West Amino Acid Moiety
Kiyoshi Nakayama, Haruko C. Kawato, Hiroaki Inagaki, Ryohei Nakajima,
Akihiro Kitamura, Kazuhiko Someya, and Toshiharu Ohta*
New Product Research Laboratories I, Daiichi Pharmaceutical Co. Ltd,
1-16-13 Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan
nakaygoi@daiichipharm.co.jp
Received February 7, 2000
ABSTRACT
Structure−activity relationships of the west amino acid modified analogues of rhodopeptins, novel antifungal tetrapeptide isolated from
Rhodococcus species Mer-N1033, have been investigated. Among the analogues synthesized, 2,2-difluoro and 2-hydroxy derivatives retained
the antifungal activity with better physical properties, i.e., solubility or acute toxicity.
Rhodopeptins, a new family of antifungal cyclic lipopeptides,
were isolated from Rhodococcus species Mer-N1033.1 They
are composed of three R-amino acids and one â-amino acid
with a lipophilic side chain as shown in Figure 1.
Fascinated by the attractive fungicidal activity of the
rhodopeptins along with the unique structural characteristics,
we executed a structure-activity relationship (SAR) study
on the molecule. The SAR of the south and the east amino
acid moieties has been reported in the preceding paper.2 The
result of this SAR study revealed that a hydrophobic and
bulky neutral amino acid (i.e., γ-methylleucine) as the south
amino acid and a basic amino acid moiety (lysine or
ornithine) as the east amino acid were indispensable structural
motifs for antifungal activity. We have also found that the
structure of the lipophilic side chain does not have a crucial
(1) (a) Chiba, H.; Agematu, H.; Kaneto, R.; Terasawa, T.; Sakai, K.;
Dobashi, K.; Yoshioka, T. J. Antibiot. 1999, 52, 695. (b) Chiba, H.;
Agematu, H.; Dobashi, K.; Yoshioka, T. J. Antibiot. 1999, 52, 700. (c)
Chiba, H.; Agematu, H.; Sakai, K.; Dobashi, K.; Yoshioka, T. J. Antibiot.
1999, 52, 710.
Figure 1.
(2) Kawato, H.; Nakayama, K.; Inagaki, H.; Nakajima, R.; Kitamura,
A.; Someya, K.; Ohta, T. Org. Lett. 2000, 2, 973-976.
10.1021/ol005630k CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

effect on the activity as long as the total number of carbons
ranges from 9 to 11.1 The compounds that fulfilled these
structural requirement exhibited antifungal activities with the
same magnitude as amphotericin B (AMPH) against Candida
albicans, Cryptococcus neoformans, and Aspergillus fumi-
gatus, the main fungi that cause systemic antifungal infec-
tions. However, these analogues also displayed acute toxicity
and poor water solubility. Our next concern, therefore, was
to identify new rhodopeptin analogues with decreased
toxicity and improved physicochemical properties.
To accomplish these objectives, our effort was focused
on the introduction of hydroxy and difluoro groups3 to the
west amino acid moiety. We also targeted an alkylated
analogue as a means of further exploring the SAR of the
â-amino acid moiety. In this Letter, we report the syntheses
and resultant antifungal activities of these analogues.
Our initial targets were difluoro analogues 9a,b, which
were synthesized by utilizing â-lactams as intermediates4 for
the â-amino acid moiety (Scheme 1). Reformatsky reaction
conversion accomplished by treatment with ceric(IV) am-
monium nitrate (CAN) and di-tert-butyl carbonate (Boc2O)
was necessary for purification of the intermediate. After
removal of the Boc group to provide 6, conventional methods
for peptide synthesis were used to obtain tetrapeptides 8a,b,
which were separated by flash chromatography. Cyclization
of each diastereomer with diphenylphosphoryl azide (DPPA)5
and hydrogenolysis in TFA furnished the desired difluoro
analogues 9a,b.
Introduction of a methyl group onto the â-amino acid was
next examined (Scheme 2). Condensation of commercially
Scheme 2
Scheme 1
available (S)-3-hydroxytetradecanoic acid6 with p-anisidine,
followed by Mitsunobu reaction, provided optically pure
â-lactam 12. Alternatively 12 could be obtained in 99% ee
via reduction of compound 10 with Ru-(S)-BINAP com-
plex.7 Formation of 12 was complicated by competitive
between 1 and 2 was followed by formation of the p-
methoxyphenyl (PMP) amide and intramolecular Mitsunobu
reaction to provide racemic â-lactam 4. Following ring
opening with sodium methoxide to yield 5, a protecting group
(5) Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972, 94,
6203.
(6) (S)-3-Hydroxytetradecanoic acid was kindly provided by Daiichi Pure
Chemical Co. Ltd. The enantiomeric excess was determined on compound
12 by comparison of the [R]D value. The [R]D values of compound 12 are
from BINAP reduction, -67.6°, and from the authentic sample, -67.9°,
respectively.
(7) (a) Taber, D. F.; Silverberg, L. J. Tetrahedron Lett. 1991, 32, 4227.
(b) King, S. A.; Thompson, A. S.; King, A. O.; Verhoeven, T. R. J. Org.
Chem. 1992, 57, 6689.
(3) Uoto, K.; Ohsuki, S.; Takenoshita H.; Ishiyama T.; Iimura S.; Hirota
Y.; Mitsui I.; Terasawa H.; Soga T. Chem. Pharm. Bull. 1997, 45, 1793.
(4) (a) Thaisrivongs, S.; Pals, D. T.; Kati, W. M.; Turner, S. R.;
Thomasco, L. M.; Watt, W. J. Med. Chem. 1986, 29, 2080. (b) Thaisrivongs,
S.; Schostarez, H. J.; Pals, D. T.; Turner, S. R. J. Med. Chem. 1987, 30,
1837. Typical coupling constants for trans isomer ca. 2 Hz, for cis isomer,
ca. 5 Hz.
978
Org. Lett., Vol. 2, No. 7, 2000

â-elimination, resulting in a diminished yield in comparison
to that of the corresponding difluoro analogue. Methylation
of 12 furnished a single product, whose structure was
confirmed to be the trans isomer 13 by the coupling constant
of the protons on the â-lactam ring.5 Subsequent removal of
the PMP group, protection with a Boc group, and hydrolysis
of the lactam gave the â-amino acid component 14.
Conventional methods for peptide synthesis were used to
provide linear tetrapeptide 15, which was cyclized with
DPPA, followed by removal of the Cbz group to complete
compound 16 in good yield.
provided anti-3-hydroxy-2-bromo ester 19. Subsequent ep-
oxide formation and hydrolysis gave epoxy acid 20, which
furnished anti-R-hydroxy-â-amino acid moiety 21 upon
treatment with sodium azide. The corresponding syn-R-
hydroxy-â-amino acid 23 was prepared as follows. Treatment
of diol 18 with p-nitrobenzenesulfonyl chloride (NsCl)
selectively provided the 2-O-sulfonated compound, which
was then cyclized to afford epoxide 22. Hydrolysis of 22,
followed by treatment with sodium azide, completed 23.
Compounds 21 and 23 were independently converted to the
linear tetrapeptides 26a,b by conventional methods, followed
by cyclization with DPPA and deprotection of the Cbz group
complete compounds 27a,b.
Finally, the synthesis of analogues containing a hydroxyl
group was investigated (Scheme 3). The 2-hydroxy-â-amino
The antifungal activity of our new analogues are sum-
marized in Table 1. The activity of amphotericin B (AM-
Scheme 3
Table 1. Antifungal Activity11,12 (MIC, µg/mL), Acute
Toxicity (MTD, maximum tolerance dose, mg/kg), and
Solubility in Water (mg/mL) of Rhodopeptin Analogues
antifungal activity
Candida Cryptococcus Aspergillus
acute
compd albicans12 neoformans12 fumigatus12 toxicity solubility
9a
9b
16
27a
27b
28
29
30
64
16
128
16
32
4
16
4
4
8
4
8
4
8
4
8
4
2
8
>128
16
128
32
32
>128
32
0.2
1.9
>40
20
>4
5
10
20
3
0.9
3.1
2.6
8
4
AMPH
FLCZ
>128
>128
PH),10 fluconazole (FLCZ),10 and the nonsubstituted rhodopep-
tin analogues 28-30 (Figure 2) are cited as references. The
difluoro analogue 9b showed moderate activity against all
(9) (a) Sharpless, K. B.; Amberg, W.; Bennani, Y. L.; Crispino, G. A.;
Hartung, J.; Jeong, K.-S.; Kwong, H.-L.; Morikawa, K.; Wang, Z.-M.; Xu,
D.; Zhang, X.-L. J. Org. Chem. 1992, 57, 2768. (b) Sharpless, K. B.;
Fleming, P. R. J. Org. Chem. 1991, 56, 2869. (c) Chong, J. M.; Sharpless,
K. B. J. Org. Chem. 1985, 50, 2869.
(10) Drugs used: amphotericin B (AMB; Fungizon, Bristol Myers
Squibb, New Brunswick, NJ) and fluconazole (FCZ; Diflucan, Pfizer, New
York, NY) were obtained commercially. Cyclic peptides were initially
dissolved in dimethyl sulfoxide (DMSO; Nakarai Chemicals, Kyoto, Japan)
and further diluted with distilled water. The final concentration of DMSO
contained in culture in wells was 2% at maximum.
(11) The in vitro susceptibility testings were done by a microdilution
method, using 96-well microplates (flat bottom). Synthetic amino acid
medium fungal (SAAMF; pH 7.0 at 30 °C, Nippon Bio-Supp. Center,
Tokyo, Japan) was used throughout in this study. Freshly grown yeasts on
slopes of sabouraud dextrose agar (SDA; Difco Laboratories, Detroit, MI)
were suspended with physiological saline and counted in a hemacytometer,
and cell concentration was adjusted to 1 × 106 cells per mL. When A.
fumigatus was studied, subcultured organisms were suspended with saline
containing 0.1% Tween 80 (monooleate polyoxyethylenesorbitan, Sigma
Chemicals, St. Louis, MO), and then conidia were collected by passing the
culture through a glass filter. Serial 2-fold dilutions of test compounds (256
to 0.125 µg/mL, 100 µL per well) were dispensed with an aid of an
automatic dispenser (Model, Dinatech, Kyoto, Japan) and 2× concentrated
medium was added to each well (100 µL per well). After inoculation (5 µL
per well) by use of an automatic inoculator (Model, Dinatech), plates were
gently but thoroughly shaken and were incubated at 30 °C for 48 h (C.
albicans) or 72 h (C. neoformans and A. fumigatus). The inoculum size
was 5 × 103 cells/mL. Minimal inhibitory concentration (MIC) was defined
as the lowest concentration of a compound which gave no visibly detectable
fungal growth.
acid moieties 21 and 23 were synthesized according to the
Shioiri protocol.8,9 Thus, after Sharpless asymmetric dihy-
droxylation9 of alkene 17, bromination and methanolysis
(8) Matsuura, F.; Hamada, Y.; Shioiri, T. Tetrahedron 1994, 50, 11303.
Org. Lett., Vol. 2, No. 7, 2000
979

moderate activity, with the R-hydroxy analogue 27a being
slightly more active.
The acute toxicity (MTD; maximum tolerance dose) and
solubility in water of the new analogues were also measured.
The nonsubstituted analogue 30 was safe up to the dose of
20 mg/kg on iv injection in mice (no lethal toxicity). The
hydroxyl analogue 27a displayed similar toxicity to that of
30, while the difluoro analogue 9b was found to be at least
2 times safer. As for the solubility in water, analogue 9b
displays solubility at 1.9 mg/mL, which is comparable to
compound 30 (2.6 mg/mL), while hydroxy analogue 27a has
an increased solubility (above 4 mg/mL).
In summary, introduction of difluoro, methyl, and hydroxyl
groups to the â-amino acid moiety of the 13-membered
rhodopeptin peptide ring has been achieved. The requsite
â-amino acid fragments were synthesized via the corre-
sponding â-lactam compounds or through the use of Sharp-
less asymmetric dihydroxylations. The difluoro and hydroxy
analogues retained the antifungal activity of the nonderiva-
tized rhodopeptins and displayed improved physical proper-
ties. The synthesis and biological evaluation of additional
derivatives are currently underway in our laboratory.
Acknowledgment. We thank Mr. Hiroshi Kuga, Ms. Chie
Makino for measuring the solubility, and Prof. William
Moser of IUPUI for critiquing the manuscript. We also thank
Mr. Chiba (Mercian Co. Ltd.) for useful discussions.
Figure 2.
fungi tested, while the C3 stereoisomer 9a was devoid of
activity against Candida albicans and Aspergillus fumigatus.
The methylated analogue 16 displayed the same loss of
activity as 9a. Both of the hydroxyl analogues 27a,b showed
Supporting Information Available: Spectroscopic and
analytical data for compounds 3-9, 11-16, and 18-27.
Protocol of the acute toxicity (MTD) assay. This material is
available free of charge via the Internet at http://pubs.acs.org.
(12) Fungal strains: C. albicans (ATCC 24433), A. fumigatus (TIMM
0063), and C. neoformans (IFO 1420) were employed.
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Org. Lett., Vol. 2, No. 7, 2000