The synthesis of pseudomycin C′ via a novel acid promoted side-chain deacylation of pseudomycin A

Article abstract of DOI:10.1016/S0960-894X(00)00613-2

The γ hydroxyl present in the aliphatic side chain of the natural products pseudomycin A and C′ provided a unique handle for the pH dependent side-chain deacylation. Low pH reaction conditions were used to cleave the side chain with minimal degradation of the peptide core. The pseudomycin nucleus intermediate obtained from the deacylation of pseudomycin A was pivotal in the synthesis of novel side-chain analogues. A practical synthesis of a minor fermentation factor pseudomycin C′ and related analogues is reported.

Full text of DOI:10.1016/S0960-894X(00)00613-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 161±164
The Synthesis of Pseudomycin C0 via a Novel Acid Promoted
Side-Chain Deacylation of Pseudomycin A
Michael J. Rodriguez,Ã Matthew Belvo, Robert Morris, Douglas J. Zeckner,
William L. Current, Roberta K. Sachs and Mark J. Zweifel
Lilly Research Laboratories, A Division of Eli Lilly & Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 18 September 2000; accepted 27 October 2000
AbstractÐThe g hydroxyl present in the aliphatic side chain of the natural products pseudomycin A and C0 provided a unique
handle for the pH dependent side-chain deacylation. Low pH reaction conditions were used to cleave the side chain with minimal
degradation of the peptide core. The pseudomycin nucleus intermediate obtained from the deacylation of pseudomycin A was
pivotal in the synthesis of novel side-chain analogues. A practical synthesis of a minor fermentation factor pseudomycin C0 and
related analogues is reported. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
among the factors reside on the length and degree of the
hydroxylation state of the side chain (Fig. 1). The side
chain is thought to be partly responsible for the
observed antifungal activity. PSC0, a minor component
of the fermentation with the C-16 side chain having the
highest degree of lipophilicity exhibits the best overall in
vitro and in vivo activity against the three major fungal
pathogens of interest: Candida albicans, Cryptococcus
neoformans, and Aspergillus fumigatus. As a potential
clinical candidate, a semisynthetic approach was devel-
oped to obtain suitable quantities of this minor compo-
nent for extended biological testing by converting PSA,
the major component of the fermentation, into PSC0.
Fungal infections are a signi®cant cause of disease,
degradation of quality of life, and mortality among
humans, particularly for immune compromised
patients. The increased incidence in fungal infections is
in part due to increased numbers of people with immune
systems weakened or devastated by organ transplants,
cancer chemotherapy, AIDS, age, and other similar
disorders. Such patients are prone to attack by fungal
pathogens that are prevalent throughout the population
but are kept in check by a functioning immune system.1
The pseudomycins A±C0(PSA±C0) are part of a new
class of promising fungicidal agents derived from iso-
lates of Pseudomonas syringae. Isolates of P. syringae
that have been ®rst associated with plant diseases are
part of a large family of plant bacteria that have been
the source of several interesting bioactive metabolites
with potent antifungal activity against human patho-
gens.2 The pseudomycins belong to the nonadepsipep-
tide family of compounds, which consists of a cyclic
peptide including one or more uncommon amino acids
with a long, hydroxylated, aliphatic side chain. The
amino acid residues on the cyclic peptide are identical
for each factor. The signi®cant structural di€erences
Results and Discussion
The synthesis of PSC0 from PSA took place in three
distinct stages: (1) conversion of PSA to CBZ-protected
pseudomycin A (ZPSA), (2) deacylation of ZPSA to Z
protected nucleus (ZPSN), synthesis of pseudomycin
side chain, and (3) coupling of the side chain to the
ZPSN followed by Z deprotection. As shown in Scheme 1,
pseudomycin A was converted to ZPSN by ®rst pro-
tecting the free amine groups with benzyl carbamate by
treating ZPSA with excess (6 equiv) of N-(benzyloxy-
carbonyloxyl)-succinimide. The use of Actinoplanes uta-
hensis, a common deacylase for side-chain cleavage was
not practical under optimum catalytic conditions.3,4
Under the biocatalytic conditions (pH >7), the desired
ZPSN rapidly degraded. Several attempts to lower the
ÃCorresponding author. Tel.: +1-317-276-0305; fax: +1-317-276-
5431; e-mail: mjrodriguez@lilly.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00613-2

162
M. J. Rodriguez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 161±164
pH below 6 failed to produce ZPSN. It was noted dur-
ing the large production scale work up of PSA that the
percent yield recoveries varied with low pH. It was
determined that pseudomycin A was not stable under
strongly acidic work up conditions (pH ꢀ2). From the
complex decomposition mixture, one of the by-products
was identi®ed as the desired pseudomycin nucleus. By
varying the acid and organic solvent concentrations, the
production of the CBZ-protected pseudomycin nucleus
was optimized.
Typically, the deacylation was promoted with an 8% aq
TFA solution at room temperature, 24±48 h. An organic
solvent such as acetonitrile was used to enhance the
Figure 1. Pseudomycin natural product factors and broad spectrum activity.
Scheme 1. Synthesis of Z-protected pseudomycin nucleus (ZPSN).

M. J. Rodriguez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 161±164
163
solubility of ZPSA in the reaction mixture. Under these
conditions, the deacylation occurred readily (75±90%)
and the degradation of the cyclic peptide core was kept
to a minimum. The acid promoted deacylation occurs
with the participation of the g hydroxy alcohol. Without
the g hydroxy as present in PSB and PSC, no side-chain
cleavage was observed. The synthesis of the PSC0 side
chain used a multistep approach that started with tetra-
decylaldehyde and featured a Sharpless epoxidation step
(1±2, 70%) to introduce the R b-OH stereochemistry
(Scheme 2).
The regioselective epoxide ring opening (3±4, 80±90%)
using DIBAL followed by potassium permanganate
Scheme 2. Pseudomycin C0 side chain, R b-OH palmitic acid. (1) (>-O2POCH2COOEt, tBuOK, THF; (2) Dibal, CH2Cl2/hexanes; (3) Ti(O-<)4,
DET ( ), CH2Cl2; (4) Dibal, CH2Cl2/hexanes; (5) pCH3OC6H5CH(OCH3)2, pTsOH, DMF; (6) Red-Al; (7) KMnO4, KH2PO4, tBuOH; (8) H2/Pd±
C, tBuOH/H2O.
Scheme 3. Side-chain coupling and ®nal deprotection. (1) TES-Cl, imidazole, DMAP, CH2Cl2; (2) HOBT-Ms, TEA, THF; (3) ZPSN, DMF/THF,
rt; (4) H2 10%Pd/C, MeOH/AcOH.
Table 1. Antifungal activity of PSC0 and related analogues
MIC (mg/mL)
Side chain
Candida
albicans
Cryptococcus
neoformans
Aspergillus
fumigatus
Candida
parapsilosis
Histoplasma
capsulatum
0.039
0.039
5.0
<0.01
<0.01
5.0
0.312
0.625
20
0.039
0.02
0.156
0.156
5.0
5.0
0.156
10.0
<0.01
1.25
1.25
NT
0.156
10.0
0.312
10.0

164
M. J. Rodriguez et al. / Bioorg. Med. Chem. Lett. 11 (2001) 161±164
oxidation provided the pCH3O benzyl ether protected
palmitic acid (86%). The reduction step performed
under aqueous conditions provided the desired R
b-hydroxypalmitic acid (5±6, 70±80%) in gram quan-
tities (by NMR>99% ee).5 The direct coupling of the b
hydroxyl side chain using HOBT-Ms resulted in the
formation of the dehydration product 9 (Scheme 3).
3. Debono, M.; Abbott, B. J.; Fukuda, D. S.; Barnhart, M.;
Willard, K. E.; Molloy, R. M.; Michel, K. H.; Turner, J. R.;
Butler, T. F.; Hunt, A. H. J. Antibiotics 1989, 42, 389.
4. Debono, M.; Turner, W. W.; LaGrandeur, L.; Burkjardt,
F. J.; Nissen, J. S.; Nichols, K. K.; Rodriguez, M. J.; Zweifel,
M. J.; Zeckner, D. J.; Gordee, R. S.; Tang, J.; Parr, T. R., Jr.
J. Med. Chem. 1995, 38, 3271.
5. The R-OH palmitic acid was alternatively obtained in
mg quantitites via the Ru-(+)-BINAP catalyzed reduction of
b-ketopalmitic acid methyl ester which was commercially
available (note: b-keto acids readily decarboxylate and could
not be used as starting materials). The reduction produced the
desired alcohol in 42% yield with >99% ee as determined by
NMR studies doped with (S)-(+) 2,2,2-tri¯uoro-1-(9-
anthryl)ethanol chiral shift reagent.
6. In vitro susceptibility studies. A robotic microdilution
microtiter testing procedure was used to determine MICs.
Pseudomycin analogues were dissolved in 100% dimethyl
sulfoxide. Compound solutions were diluted in broth to yield a
starting concentration of 20 mg/mL in the ®rst wells following
the addition of inoculum. Serial 2-fold dilutions in 100 mL
aliquots were made in 96-well microtiter plates using a
QuadFlex automatic pipetting, liquid delivery instrument
(Titertek Instruments Inc., Huntsville, AL). Fungal yeast
isolates were grown on Sabouraud dextrose agar slants at
35 ꢁC for 24 h. C. albicans, C. parapsilosis, C. neoformans, and
H. capsulatum conidia were suspended in saline and adjusted
to 1Â105 conidia/mL in Sabouraud dextrose broth (DIFCO,
Detroit, MI). A. fumigatus spores were gently teased from
mycelia grown on potato dextrose agar (DIFCO, Detroit, MI)
in 0.1% Tween 80 and saline. Spores were adjusted to 1Â105
spores/mL in Sabouraud broth. Cell suspensions were counted
using a hemacytometer under phase contrast microscopy.
Aliquots containing 100 mL of the above were added to
each well of a 96-well microtiter plate with a Titertex Multi-
drop Dispenser (Titertek Instruments Inc., Huntsville, AL).
Plates were incubated for 48 h at 35 ꢁC in ambient air. MICs
were de®ned as the lowest concentrations of drugs which
inhibited 90±100% of visible growth compared to untreated
controls.
By protecting the b-hydroxy with TES, the HOBT acti-
vated ester was prepared without decomposition and
used without further puri®cation (6±8). The ®nal cou-
pling of the activated ester to ZPSN proceeded by using
a slight excess of the acid in a 1:1 mixture of DMF/THF
at rt for 48 h (30%). The removal of the CBZ groups to
obtain ®nal product PSC0 required acetic acid. A 10%
acetic acid/methanol solution with 10% Pd/C was used
for complete removal of the Z groups (65%). There was
no reduction of the double bond (residue 7) under these
conditions. Additional analogues were prepared with S
b-hydroxy palmitic acid (obtained from DET(+)) and
with commercially available racemic hydroxyl palmitic
acid and palmitic acid. All analogues were submitted for
broad spectrum antifungal analysis.6 The minimum
inhibitory concentration data (MICs) reported in Table 1
indicate that the synthetic side chain with the b hydroxy
in the R con®guration had identical broad spectrum
activity as shown for authentic PSC0 obtained by bio-
synthesis. The analogue with the S b-hydroxy side-chain
con®guration was 7-fold (100Â) less active against C.
albicans than PSC0. The non-hydroxylated C16 side
chain signi®cantly reduced the antifungal spectrum and
activity. In summary, we have shown a practical syn-
thesis to obtain novel pseudomycin analogues from a
relatively abundant source of ZPSN. From this study,
the b hydroxyl group appears to be essential to retain
antifungal activity. Further elaboration of the side-
chain SAR is currently in progress.
Survival studies. Mice were infected by an intravenous (iv)
injection of 0.1 mL in the lateral tail vein. Mice received 2Â106
blastoconidia per mouse. Untreated controls were moribund
within 3±4 days post-infection. Mice were treated at 0, 4, 24,
and 48 h post-infection. Mice were treated intraperitoneally
(ip) with 0.2 mL. Experimental compounds were tested at
titrated concentrations (serial 2-fold dilutions) of 20, 10, and
5.0 mg/kg. Six mice were tested at each level. Compounds were
formulated in 4.0% hydroxypropyl b cyclodextrin and sodium
acetate, pH 7.0 and 1.75% dextrose. Infected shamtreated
mice (10 animals) were dosed with the vehicle alone. Morbid-
ity and mortality were recorded for 7 days. The 50% e€ective
doses (ED500's) were determined using the method of Reed
and Muench. Statistical di€erences in treated groups com-
pared to untreated infection controls were determined using
the Student's t-test.
Acknowledgements
We thank Patrick Baker of the Natural Product Divi-
sion for his dedication and commitment of supporting
this project.
References and Notes
1. Ganguly, D.; Rodriguez, M. J. Exp. Opin. Ther. Pat. 1999,
9, 6771.
2. Harrision, L.; Teplow, D. B.; Rinaldi, M.; Strobel, G. J.
Gen. Microbiol. 1991, 137, 2857.