Determination of the relative and absolute configuration of the dimethylmyristoyl side chain of pneumocandin B0 by asymmetric synthesis

Article abstract of DOI:10.1021/ol0261940

(Equation presented) The relative and absolute configuration of the pneumocandin B₀ side chain has been established as (10R,12S)-dimethylmyristoyl by the stereocontrolled synthesis of both antipodes of the side chain acid and their comparison to a sample derived from the natural product.

Full text of DOI:10.1021/ol0261940

ORGANIC
LETTERS
2002
Vol. 4, No. 24
4201-4204
Determination of the Relative and
Absolute Configuration of the
Dimethylmyristoyl Side Chain of
Pneumocandin B0 by Asymmetric
Synthesis
William R. Leonard, Jr.,* Kevin M. Belyk, Dean R. Bender, David A. Conlon,
David L. Hughes, and Paul J. Reider
Department of Process Research, Merck Research Laboratories, Merck & Co.,
P.O. Box 2000, Rahway, New Jersey 07065
bill_leonard@merck.com
Received May 15, 2002 (Revised Manuscript Received September 4, 2002)
ABSTRACT
The relative and absolute configuration of the pneumocandin B0 side chain has been established as (10R,12S)-dimethylmyristoyl by the
stereocontrolled synthesis of both antipodes of the side chain acid and their comparison to a sample derived from the natural product.
Serious and life-threatening fungal infections have increased
dramatically over the past several decades due to the
increased use of invasive medical procedures and broad-
spectrum antibiotics, as well as a burgeoning immune-
compromised patient population resulting from cancer and
organ transplantation chemotherapy, malignancies, and AIDS.
The few antifungal agents available are limited by their
toxicity, drug interactions, and growing antifungal resistance.1
â-(1,3)-D-glucan, an essential cell wall component of many
pathogenic fungi.3
One of the echinocandins, pneumocandin B0 (1), was
isolated from the fermentation of the fungus Glarea lozoy-
ensis.4 It has been employed in the semisynthesis of several
potent antifungal drug candidates,5 including CANCIDAS
(2) For reviews on the antifungal properties and SAR studies of
echinocandins, see: (a) Hossain, M. A.; Ghannoum, M. A. Exp. Opin. InVest.
Drugs 2000, 9, 1797-1813. (b) Balkovec, J. M.; Black, R. M.; Bouffard,
F. A.; Dropinski, J. F.; Hammond, M. L. Pharmacochem. Libr. (XIVth
International Symposium on Medicinal Chemistry, 1996) 1997, 28, 1-13.
(c) Debono, M. Exp. Opin. Ther. Patents 1995, 5, 771-786. (d) Hammond,
M. L. In Cutaneous Antifungal Agents; Rippon, J. W., Fromtling, R. A.,
Eds.; Marcel Dekker: New York, 1993; pp 375-393.
The echinocandin class of natural products has recently
emerged as a promising antifungal therapy.2 Their fungal-
specific mode of action is inhibition of the biosynthesis of
(1) (a) Groll, A. H.; Walsh, T. J. Curr. Opin. Infect. Dis. 1997, 10, 449.
(b) Kauffman, C. A.; Carver, P. L. Drugs 1997, 53, 539.
(3) Kurtz, M. B.; Douglas, C. M. J. Med. Vet. Mycol. 1997, 35, 79-86
10.1021/ol0261940 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/08/2002

(caspofungin acetate)6 (2), which has recently been approved
by the U.S. FDA for treatment of invasive aspergillosis in
patients who are refractory to or intolerant of standard
therapy. Caspofungin is also in late-stage clinical testing for
other indications, including empirical antifungal therapy.7
Caspofungin is proving to be a valuable antifungal agent due
to its specific mode of action, broad spectrum, and low
toxicity.
both the antifungal potency and the toxicity due to red blood
cell hemolysis.2c,d
Herein, we report the determination of the relative and
absolute stereochemistry of the pneumocandin B0 dimeth-
ylmyristoyl side chain by comparison of the hydrolyzed side
chain acid to samples of both antipodes produced by
enantioselective synthesis.
A sample of 1 was obtained by fermentation, isolation,
and purification as previously described.9 The sample was
92% pure. The impurities were mostly compounds that
contained analogous amino acids. Impurities that differed
in the side chain were analyzed at <0.7%. The pneumocandin
was methanolized (MeOH, AcCl, reflux, 73 h), and the
pentane extract was purified by bulb-to-bulb distillation (95-
105 °C oven, 0.35 Torr) to give the methyl ester in 72%
yield. The ester was hydrolyzed (THF/H2O, LiOH, 25 °C,
16 h) to the acid in quantitative yield. The side chain acid
1
was homogeneous by H and 13C NMR analysis, and the
specific rotation was determined to be [R]25
(c 0.010, CHCl3).
+16.6°
405
To aid in the identification of the relative stereochemistry
of the side chain, a short racemic synthesis of a mixture of
the syn and anti compounds was conducted (Scheme 1).
The structures of pneumocandin B0 (1) and caspofungin
(2) consist of a polar cyclic hexapeptide core with a lipophilic
side chain containing two stereogenic methyl groups. The
structure and absolute stereochemistry of the peptide core
of 1 was determined by NMR, X-ray crystallographic, and
chromatographic amino acid analysis; however, the lipophilic
side chain was disordered in the crystallographic study, and
the stereochemistry of the two methyl groups could not be
elucidated.8 Structure-activity relationship experiments have
shown that the structure and stereochemical configuration
of the echinocandin side chain is of central importance for
Scheme 1a
a Key: (a) THF, from 0 to 25 °C; (b) 3 equiv of Br-Ph3P+CH2-
(CH2)6CO2H, THF, 0 °C, LiHMDS, 30 min; (c) Wittig, from 0 °C
to rt, 4 h. (d) H2, Pd/C
(4) (a) Schwartz, R. E.; Sesin, D. F.; Joshua, H.; Wilson, K. E.; Kempf,
A. J.; Goklen, K. A.; Kuehner, D.; Gailliot, P.; Gleason, C.; White, R.;
Inamine, E.; Bills, G.; Salmon, P.; Zitano, L. J. Antiobiotics 1992, 45, 1853-
1866. (b) Bills, G. F.; Platas, G.; Pelaez, F.; Masurekar, P. Mycol. Res.
1999, 103, 179-192.
(5) (a) Bouffard, F. A.; Zambias, R. A.; Dropinski, J. F.; Balkovec, J.
M.; Hammond, M. L.; Abruzzo, G. K.; Bartizal, K. F.; Marrinan, J. A.;
Kurtz, M. B.; McFadden, D. C.; Nollstadt, K. H.; Powles, M. A.; Schmatz,
D. M. J. Med. Chem. 1994, 37, 222-225. (b) Abruzzo, G. K.; Flattery, A.
M.; Gill, C. J.; Kong, L.; Smith, J. G.; Krupa, D.; Pikounis, V. B.; Kropp,
H.; Bartizal, K. Antimicrob. Agents Chemother. 1995, 39, 1077-1081. (c)
Bouffard, F. A.; Dropinski, J. F.; Balkovec, J. M.; Black, R. M.; Hammond,
M. L.; Nollstadt, K. H.; Dreikorn, S. L-743,872, A Novel Antifungal
Lipopeptide: Synthesis and Structure-Activity Relationships of New Aza-
Substituted Pneumocandins. Abstracts, 36th Interscience Conference on
Antimicrobial Agents and Chemotherapy, New Orleans, LA, 1996; Ameri-
can Society for Microbiology: Washington, DC, 1997; F27.
Diastereomeric 2,4-dimethylhexanal (3) was produced by
reaction of methacrolein with s-Bu3B.10 The product was
purified by distillation to provide a 55:45 ratio of aldehyde
diastereomers. The major isomer was determined to be syn
on the basis of comparison to literature 13C NMR data for
the known syn isomer.11 The mixture was subjected to a
Wittig reaction with (7-carboxyheptyl)triphenylphosphonium
bromide. The resultant olefin was hydrogenated to provide
4 as a 54:46 ratio of diastereomers. The ratio was determined
by quantitative 13C NMR measurements in which 9 of the
16 carbon resonances of the diastereomers are distinguish-
able. The major isomer (presumed to be syn on the basis of
the major aldehyde isomer being carried forward in a similar
ratio) matched the 13C NMR spectrum of the side chain acid
(6) Formerly known as MK-0991 and L-743,872.
(7) (a) Villanueva, A.; Arathoon, E. G.; Gotuzzo, E.; Berman, R. S.;
DiNubile, M. J.; Sable, C. A. Clin. Infect. Dis. 2001, 33, 1529-1535. (b)
Arathoon, E. G.; Gotuzzo, E.; Noriega, L. M.; Berman, R. S.; DiNubile,
M. J.; Sable, C. A. Antimicrob. Agents Chemother. 2002, 46, 451-457. (c)
Maertens, J.; Raad, I.; Sable, C. A.; Ngai, A.; Berman, R.; Patterson, T. F.;
Denning, D.; Walsh, T. Multicenter, Noncomparative Study to Evaluate
Safety and Efficacy of Caspofungin in Adults with Invasive Aspergillosis
Refractory or Intolerant to Amphotericin B (AMB), AMB Lipid Formula-
tions, or Azoles. Abstracts, 40th Interscience Conference on Antimicrobial
Agents and Chemotherapy, Toronto, 2000; American Society for Micro-
biology: Washington, DC, 2000; J1103.
(8) (a) Hensens, O. D.; Liesch, J. M.; Zink, D. L.; Smith, J. L.;
Wichmann, C. F.; Schwartz, R. E. J. Antibiotics 1992, 45, 1875-1885. (b)
Schwartz, R. E., Masurekar, P. S.; White, R. F. In Cutaneous Antifungal
Agents; Rippon, J. W., Fromtling, R. A., Eds.; Marcel Dekker: New York,
1993; pp 375-393.
(9) (a) Roush, D. J.; Antia, F. D.; Goklen, K. E. J. Chromatogr. A 1998,
827, 373-389. (b) Conners, N.; Petersen, L.; Hughes, R.; Saini, K.;
Olewinski, R.; Salmon, P. Appl. Microbiol. Biotechnol. 2000, 54, 814-
818 and references therein.
(10) Brown, H. C.; Kabalka, G. W.; Rathke, M. W.; Rogic, M. M. J.
Am. Chem. Soc. 1968, 90, 4165-4166.
(11) White, J. D.; Johnson, A. T. J. Org. Chem. 1994, 59, 3347-3358.
4202
Org. Lett., Vol. 4, No. 24, 2002

produced by hydrolysis of 1. Thus, this relative stereochem-
ical assignment, while equivocal, assisted in the selection
of the initial asymmetric route and was corroborated during
the enantioselective synthesis of the syn isomer.
Scheme 3a
With the tentative syn assignment, an enantioselective
synthesis of the (10S,12R)-acid enantiomer (4S,R) was con-
ducted using Enders’ chiral hydrazone chemistry12 to deter-
mine the absolute stereochemistry (Scheme 2). Condensation
Scheme 2a
a Key: (a) LDA, THF, 5 °C; (b) HMPA, -50 °C, 1-iodo-(2S)-
methyl-butane; (c) 1 M HCl, reflux; (d) 1 equiv of cinchonidine,
1:1 v/v H2O/acetone, from 70 °C to rt, two times; (e) Et2O/THF,
LiAlH4, 5 °C; (f) CH2Cl2, Swern, from -70 to 0 °C; (g) 3 equiv
Br-Ph3P+CH2(CH2)6CO2H, THF, 0 °C, LiHMDS, 30 min; (h)
Wittig, from 0 °C to rt, 4 h; (i) H2, Pd/C.
nol (7) was alkylated with commercially available, optically
pure 1-iodo-(2S)-methylbutane1616 and hydrolyzed to yield
the acid 9. The acid was reduced to the alcohol and oxidized
to give the aldehyde 3S,S as an 83:17 (syn/anti) mixture.
Wittig reaction, followed by hydrogenation, gave the side
chain 4R,S in 61% yield with an 80:20 (syn/anti) ratio. The
a Key: (a) LDA, THF, 0 °C, 7 h; (b) n-BuLi, -25 °C, 1 h; (c)
1-iodo-(2R)-methylbutane, from -95 to 0 °C, 10 h; (d) O3, MTBE,
-78 °C, 10 min, then 3 Å mol sieves, rt; (e) 5 equiv of
Br-Ph3P+CH2(CH2)6CO2H, THF, 0 °C, LiHMDS, 30 min; (f)
Wittig, from 0 °C to rt, 5 h; (g) H2, Pt/C, i-PrOAc, rt.
specific rotation was determined to be [R]25
(c 0.010, CHCl3).
+17.1°
405
of RAMP with propionaldehyde gave the hydrazone 5 in
96% purified yield.13 The hydrazone was alkylated with
optically pure 1-iodo-(2R)-methylbutane11 to give 6 in 46%
yield as a 95:5 (syn/anti) diastereomeric ratio.14 The alkylated
hydrazone was ozonolyzed, and the resultant aldehyde 3R,R
was immediately subjected to the Wittig reaction to provide
the olefin in 32% purified yield.15 Hydrogenation of the olefin
gave the saturated acid 4S,R in 93% yield as a 92:8 (syn:
anti) mixture of diastereomers. The 13C NMR analysis of
the major syn (10S,12R)-isomer matched that of the natural
hydrolyzed acid. The specific rotation of the synthetic side
A sample of the 4R,S side chain with an improved
diastereomeric ratio was produced by purifying a sample of
acid 9 having a 91:9 (syn/anti) ratio to >97:3 by twice
crystallizing its cinchonidine salt from acetone/water. The
resulting acid was carried through the remainder of the
synthesis to provide 4R,S as a 90:10 (syn/anti) ratio of
diastereomers having a specific rotation of [R]25 +17.0°
405
(c 0.010, CHCl3). The small difference in the specific
rotations of the samples containing 90:10 vs 80:20 (syn/anti)
ratios of the diastereomers indicates that the anti impurity
does not significantly contribute to the measured specific
rotation.17
chain sample, [R]25
-18.0° (c 0.010, CHCl3), was
405
comparable in magnitude to the natural side chain but
opposite in sign, indicating the synthesis of the antipode.
A stereoselective synthesis of the other enantiomer was
also conducted using a different and complementary asym-
metric route to provide additional support for the absolute
configuration (Scheme 3). The (2S,4S)-dimethylhexanal
intermediate was prepared according to the method of White
and Johnson as follows.11 (R)-Propanoyl-2-pyrrolidinemetha-
In summary, the pneumocandin B0 side chain has been
determined to have the syn methyl relative configuration and
the absolute stereochemistry of (10R,12S)-dimethylmyristoyl.
This conclusion was based on enantioselective syntheses of
the pneumocandin B0 side chain acid and its antipode and
the comparison of the synthetic products’ NMR spectra and
optical rotation with that of the acid derived from the natural
product.
(12) Enders, D. In Asymmetric Synthesis; Morrison, J. D., Ed.; Academic
Press: Orlando, 1984; Vol. 3, pp 275-339.
(16) Aldrich Chemical Co., Milwaukee, WI.
(13) Enders, D.; Rendenbach, B. E. M. Tetrahedron 1986, 42, 2235-
(17) As a further indication that the anti diastereomer impurity does not
confound the absolute stereochemical assignment, the specific rotation (c
0.010, CHCl3) was measured at a variety of wavelengths for the natural
side chain acid and the 80:20 syn/anti mixture: (wavelength, [R]23 natural
side chain, [R]23 80:20 syn/anti) 365, 21.3°, 22.9°; 436, 13.9°, 14.7°; 546,
8.1°, 8.7°; 578, 7.1°, 7.5°; 589, 6.9°, 7.4°.
2242.
(14) Nicolaou, K. C.; Nadin, A.; Leresche, J. E.; La Greca, S.; Tsuri,
T.; Yue, E. W.; Yang, Z. Angew. Chem., Int Ed. Engl. 1994, 33, 2184-
2187.
(15) Yields of reactions were not optimized.
Org. Lett., Vol. 4, No. 24, 2002
4203

Supporting Information Available: Experimental pro-
cedures and spectral data for 4R,S, 4S,R, and 6, the degradation
of 1, and isolation of the natural side chain ester and acid,
as well as a table of 13C NMR assignments for the natural
side chain acid and synthetic 4R,S (syn) and 4S,S (anti) side
chain acids. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL0261940
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Org. Lett., Vol. 4, No. 24, 2002