Synthesis and antifungal activity of rhodopeptin analogues. 1. Modification of the east and south amino acid moieties

Article abstract of DOI:10.1021/ol005631c

(equation presented) East Amino Acid n=8, 10 South Amino Acid Synthetic Rhodopeptin Analogs Structure - activity relationships of the east and south amino acid modified analogues of rhodopeptins, novel antifungal cyclic tetrapeptides isolated from Rhodococcus species Mer-N1033, have been investigated. It was observed that a basic amino acid moiety (lysine or ornithine) as the east amino acid and a hydrophobic and bulky neutral amino acid (i.e., γ-methylleucine) as the south amino acid were indispensable structure motifs for antifungal activity of rhodopeptin analogues.

Full text of DOI:10.1021/ol005631c

ORGANIC
LETTERS
2000
Vol. 2, No. 7
973-976
Synthesis and Antifungal Activity of
Rhodopeptin Analogues. 1. Modification
of the East and South Amino Acid
Moieties
Haruko C. Kawato, Kiyoshi Nakayama, 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
kawatlm8@daiichipharm.co.jp
Received February 7, 2000
ABSTRACT
Structure−activity relationships of the east and south amino acid modified analogues of rhodopeptins, novel antifungal cyclic tetrapeptides
isolated from Rhodococcus species Mer-N1033, have been investigated. It was observed that a basic amino acid moiety (lysine or ornithine)
as the east amino acid and a hydrophobic and bulky neutral amino acid (i.e., γ-methylleucine) as the south amino acid were indispensable
structure motifs for antifungal activity of rhodopeptin analogues.
The occurrence of opportunistic fungal infections, caused
by Candida sp., Cryptococcs neoformans, and Aspergillus
sp., has risen sharply in recent years among patients
immunocompromised by cancer chemotherapy, organ trans-
plant, hematological malignancy, and HIV infection. There
are only a few drugs available for treating systemic antifungal
infection, e.g., amphotericin B (AMPH) and the azoles such
as fluconazole (FLCZ) and itraconazole (ITCZ), and thus
the need for other effective antifungal agents has intensified.1
Rhodopeptins comprise a family of novel antifungal cyclic
tetrapeptides, isolated from Rhodococcus species Mer-
N1033.2 The rhodopeptins, which are composed of three
R-amino acids and one â-amino acid with a lipophilic side
chain (Figure 1), exhibit fungicidal activity against Candida
sp. and Cryptococcus neoformans and fungistatic activity
against Aspergillus fumigatus.
Prompted by the unique structure and antifungal activity,
we executed a structure-activity relationship (SAR) study
(1) (a) De Lucca, A. J.; Walsh, T. J. Antimicrob. Agents Chemother.
1999, 43, 1. (b) Barrett, J. F.; Klanbert, D. H. Ann. Rep. Med. Chem. 1992,
27, 149. (c) Barrett, J. F.; Hey, R. J.; Peterson, P. K. New strategies in
Fungal Disease; Churchill Livingstone: Edinburgh, 1992. (d) Yamaguchi,
H.; Kobayashi, G. S.; Takahashi, H. Recent Progress in Antifungal
Chemotherapy; Marcell Dekker Inc.: New York, 1991. (e) Balkovec, J.
M. Exp. Opin. InVest. Drugs 1994, 3, 65.
Figure 1. 1: n ) 3, R ) (CH2)6CH(CH3)CH2CH3 (rhodopeptin
C1). 2: n ) 4, R ) (CH2)8CH(CH3)2 (rhodopeptin B5). 3: n ) 4,
R ) (CH2)8CH3.
10.1021/ol005631c CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/15/2000

Table 1. Antifungal Activity of the East Amino Acid Analogues of Rhodopeptina
MIC (mg/mL)b
C. albicans
C. tropicalis
C. krusei
C. glabrata
A. fumigatus
compound
east amino acid
Lys
Orn
Arg
ATCC24433
TIMM0313
TIMM0269
ATCC2001
TIMM0063
3
7
14
4
4
2
4
1
1
2
2
2
4
2
2
>128
>32
64
15
16
17
18
19
20
21
AMPH
FLCZ
His
Glu
>32
>64
4
>32
>64
2
>32
>64
8
>32
>64
4
>32
>64
>128
>128
>128
>128
>16
4
N6-Me-Lys
N6-iPr-Lys
N6,N6-di-Me-Lys
N6-Me-N6-Bn-Lys
N6-Ac-Lys
8
8
8
4
8
8
8
4
>128
>16
4
>128
>16
4
>128
>16
4
>128
>16
2
>128
>128
128
128
>128
a Boc-Lys(Me, Bn) was synthesized from Boc-Lys(Cbz).8 Reductive amination of Cbz-Lys-OMe with formaldehyde, followed by deprotection, furnished
Cbz-Lys(Me2). The other protected amino acids were commercially available. b Minimum inhibitory concentration. Medium: synthetic amino acid medium
fungal (SAAMF).
of rhodopeptin derivatives. For convenience, the cyclic
tetrapeptide structure was divided into four parts, the north,
west, south, and east amino acids as illustrated in Figure 1.
Herein we report the synthesis and antifungal activity of east
and south amino acid modified analogues of the rhodopep-
tins.
The east amino acid analogues were constructed on the
basis of the previously reported protocol for the synthesis
of rhodopeptin analogue 3.3 The synthesis of ornithine
analogue 7 is depicted as an example (Scheme 1). Starting
with dipeptide 4,3 deprotection and successive condensations
with glycine and ornithine residues provided tetrapeptide 6.
Deprotection and cyclization using diphenylphosphoryl azide
(DPPA),4 followed by removal of the Cbz group, furnished
compound 7. Analogous chemistry was utilized to prepare
the remainder of the analogues listed in Table 1.
from 9 to 11 carbons, we prepared both nonyl and undecyl
derivatives of these compounds.
Wittig reaction of n-dodecanal 8 with (carbomethoxy-
methylene)triphenylphosphorane provided the R,â-unsatur-
ated ester.3 Michael addition of (R)-1-phenylethylamine to
the ester furnished a mixture of diastereomers.5 After
chromatographic separation, hydrogenolysis of the desired
isomer 9 provided (R)-3-undecyl-â-alanine methyl ester 10.
Condensation of 10 with Boc-γ-methylleucine, followed by
removal of the Boc group and another condensation with
Boc-Gly-Lys(Cbz),3 provided tetrapeptide 12. Deprotection
with trifluoroacetic acid and cyclization with DPPA, followed
by hydrogenolysis, furnished the desired compound 13.
Scheme 1
The synthesis of γ-methylleucine (γ-MeLeu) derivative
13 exemplifies the preparation of the compounds listed in
Table 2 (Scheme 2). Anticipated steric problems dictated that
tetrapeptide cyclization for this series of compounds would
rely upon ring closure between the C-terminus of the west
amino acid and the north amino acid N-terminus. From the
results of our preliminary study which determined that
antifungal activity required lipophilic side chains ranging
(2) (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.
(3) Chiba, H.; Agematu, H.; Sakai, K.; Dobashi, K.; Yoshioka, T. J.
Antibiot. 1999, 52, 710.
(4) Shioiri, T.; Ninomiya, K.; Yamada, S. J. Am. Chem. Soc. 1972, 94,
6203.
(5) Furukawa, M.; Okawara, T.; Terawaki, Y. Chem. Pharm. Bull. 1977,
25, 1319.
974
Org. Lett., Vol. 2, No. 7, 2000

Table 2. Antifungal Activity of the South Amino Acid Analogues of Rhodopeptin
MIC (mg/mL)a
C. albicans
C. tropicalis
C. krusei
C. glabrata
compound
n
south amino acid
ATCC24433
TIMM0313
TIMM0269
ATCC2001
A. fumigatus TIMM0063
3
22
23
24
25
26
27
28
8
8
8
10
10
10
10
10
10
Val
Lys
Glu
Val
Ile
Leu
Cha
tert-Leu
γ-MeLeu
4
64
64
128
16
8
64
16
4
4
4
128
>128
2
>128
>128
128
64
128
16
16
4
128
32
8
8
8
8
8
4
2
>128
>128
>128
128
16
8
8
4
32
8
4
4
>128
64
32
8
4
13
AMPH
FLCZ
8
4
128
>128
>128
128
>128
a Minimum inhibitory concentration. Medium: synthetic amino acid medium fungal (SAAMF).
The minimum inhibitory concentration values (MIC, µg/
mL) of the east amino acid analogues against four strains of
Candida sp. and Aspergillus fumigatus TIMM0063 are
presented in Table 1.6 The activities of AMPH7 and FLCZ7
are shown as a reference.
comparison to the lysine analogue 3. The arginine analogue
14 possessed higher activity against Candida sp. and was
also slightly active against Aspergillus fumigatus. In contrast,
the less basic histidine derivative 15 and the glutamic acid
derivative 16 containing an acidic functional group were
inactive at the maximum soluble concentration. A glycine
analogue was completely insoluble in the medium, precluding
measurement of its activity. The results with N6-substituted
lysine derivatives (17-20) demonstrated that mono- and bis-
substitution with a small alkyl group could be possible,
whereas substitution with a larger group, e.g., benzyl, resulted
in loss of activity. The acylated analogue 21 showed
decreased solubility in the medium and was inactive at the
maximum soluble concentration.
The ornithine analogue 7 displayed increased potency in
Scheme 2
(6) 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. Test compounds were initially dissolved in
dimethyl sulfoxide (DMSO; Nakarai Chemicals, Kyoto, Japan) and further
diluted with distilled water. Serial 2-fold dilution (256 to 0.125 µg/mL,
100 µL per well) were dispensed with the aid of an automatic dispenser
(Model, Dinatech, Kyoto, Japan) and 2× concentrated medium was added
to each well (100 µL per well). The final concentration of DMSO contained
in culture in wells was 2% at maximum. After inoculation (5 µL per well)
was done by use of an automatic inoculator (Model, Dinatech), plates were
gently but thoroughly shaken and were incubated at 30 °C for 48 h (Candida
sp.) or 72 h (A. fumigatus). The inoculum size was 5 × 103 cells per mL.
Minimum inhibitory concentration (MIC) was defined as the lowest
concentration of a compound which gave no visibly detectable fungal
growth.
(7) Drug used: amphotericin B (AMPH; Fungizone, Bristol Myers
Squibb, New Brunswick, NJ) and fluconazole (FLCZ; Diflucan, Pfizer, New
York, ) were obtained commercially.
Org. Lett., Vol. 2, No. 7, 2000
975

The antifungal activity of the south amino acid analogues
is presented in Table 2.6 The activity of lysine derivative 22
containing a basic functional group was significantly de-
creased. The glutamic acid derivative 23 with an acidic
functional group was also less active. Both isoleucine
analogue 25 and leucine analogue 26 displayed moderate
activity against Candida species, with 25 showing activity
against Aspergillus species as well. The more bulky lipophilic
amino acid derivatives, cyclohexylalanine (Cha, 27) and tert-
leucine 28, did not display significantly improved antifungal
activities. However, γ-methyllecine (γ-MeLeu) analogue 13
exhibited activity comparable to that of AMPH against both
Candida and Aspergillus species. Furthermore 13 showed
lower toxicity9 than AMPH, with GI50 values of 13.6 and
1.0 µg/mL, respectively.
In conclusion, we have found that a basic amino acid
moiety (lysine or ornithine) as the east amino acid and a
hydrophobic and bulky neutral amino acid (i.e. γ-methylleu-
cine) as the south amino acid were indispensable structural
motifs for antifungal activity of rhodopeptin analogues.
Studies on the mechanism of action of these compounds are
currently in progress in our laboratories.
Acknowledgment. We thank Mr. Chiba (Mercian Co.
Ltd.) for useful discussions. We also thank Prof. William
Moser of IUPUI for critiquing the manuscript.
Supporting Information Available: Spectroscopic and
analytical data for compounds 5-7 and 11-28. This material
is available free of charge via the Internet at http://pubs.acs.org.
(8) Andruszkiewicz, R. Pol. J. Chem. 1988, 62, 257.
(9) MTT assay against P388.
OL005631C
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Org. Lett., Vol. 2, No. 7, 2000