Zwittermicin A: Synthesis of analogs and structure-activity studies

Article abstract of DOI:10.1016/j.bmcl.2010.02.032

Analogs and diastereomers of the natural product zwittermicin A were prepared. SAR studies of these compounds reveal the antifungal activity to be dependent singularly upon the natural constitution and configuration.

Full text of DOI:10.1016/j.bmcl.2010.02.032

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2183–2185
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Zwittermicin A: Synthesis of analogs and structure–activity studies
Evan W. Rogers a, , Doralyn S. Dalisay a,à, Tadeusz F. Molinski a,b,
*
a Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States
b Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs and diastereomers of the natural product zwittermicin A were prepared. SAR studies of these
compounds reveal the antifungal activity to be dependent singularly upon the natural constitution and
configuration.
Received 8 January 2010
Revised 4 February 2010
Accepted 8 February 2010
Available online 11 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antifungal
Amino alcohol
Synthesis
(+)-Zwittermicin A (1) (ZwA) is a highly polar aminopolyol anti-
biotic isolated from the soil-borne bacterium Bacillus cereus.1 Com-
pound 1 was first reported in 1994 and shows significant activity
against human pathogenic yeast and phytopathogenic fungi.1,2
More importantly 1 has been shown to work synergistically with
endo-toxin produced by Bacillus thuringensis for control of gypsy
moth.3 Research has shown strains of B. cereus producing 1 to be
ubiquitous in the soil1c and may be more benign to the environ-
ment than some synthetic pesticides.4 Biosynthesis of 1 arises from
a non-ribosomal peptide synthetase/polyketide synthase pathway
(NRPS/PKS) involving two new type 1 PKS extender units: hydrox-
ymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP.5
Previously, we synthesized (À)-1, the enantiomer of ZwA, its
diastereomer 2, and analogs 5 through 10 (Fig. 1).7 This Letter de-
scribes the synthesis of the additional diastereomers 3 and 4 as
well as analogs ent-6, 11 and 12 together with a comprehensive
SAR study of all compounds.
During the course of our synthesis of (À)-1 a number of models
were made to evaluate synthetic strategies and some of these were
later used to make analogs 11 and 12 as shown in Scheme 1. Ana-
log ent-6 was synthesized by reduction of diazide 13 (Scheme 1).8
The known alcohol 149 was converted to aldehyde 15 in three
high-yielding steps; MOM protection of the primary OH group,10
desilylation of the primary OTBS group,11 followed by Swern12 oxi-
dation. Evan’s aldol addition of the boron enolate of chiral glycolate
equivalent 1613 to 15 (dr 47:1) followed by separation and removal
of the chiral auxiliary under standard conditions afforded carbox-
ylic acid 17 (57% over two steps).14 Coupling of 17 to known N-ure-
O
OH NH2
NH2
H
N
7
4
15
H2N
N
H
H2N
O
OH OH OH OH
O
ido-
L
-1,3-diaminopropionamide (À)-187 gave 19 in 83% yield;
(+)-Zwittermicin A (1)
global deprotection of the latter provided the truncated ZwA ana-
log 11 in 76% yield. Similarly, analog 12 was prepared from (+)-18.
Preliminary studies show that 1 appears to exhibit a unique
mechanism of action.6 Investigations of ZwA-resistant mutants
found that the resistance mapped to rpoB and rpoC, genes that en-
code subunits of RNA polymerase.6 However, 1 showed no effect
on total RNA or DNA synthesis implying a mechanism of action that
differs from that of other antibiotics that target RNA polymerase.6
Azidodiol 20, prepared from L
-serine as described earlier,7 was
refunctionalized by TBDPS protection15 of the terminal alcohol,
MOM protection of the secondary alcohol and removal of the
TBDPS group to give 21 in high yield (86% three steps, Scheme
2). Transformation of the azido group in 21 to an N,N-dibenzyl
group by hydrogenolysis (Lindlar’s catalyst16) followed by N-ben-
zylation17 gave a primary alcohol that was easily oxidized to the
stable aldehyde 22 (74% three steps).
* Corresponding author. Tel.: +1 858 534 7115; fax: +1 858 822 0386.
E-mail address: tmolinski@ucsd.edu (T.F. Molinski).
Present address: CalciMedica, 505 Coast Boulevard South, La Jolla, CA 92037,
United States.
Aldol addition of the glycolate equivalent 2318 to aldehyde 22
(9:1 dr) followed by hydrolysis with lithium hydroxide under stan-
dard conditions19 afforded carboxylic acid 24 in (41% over two
steps).
à
Present address: Department of Chemistry, University of British Columbia,
Vancouver, BC, Canada V6T 1Z1.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.032

2184
E. W. Rogers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2183–2185
O
OH NH2
NH2
NBn2
O
N3
NBn2
O
N3
NH2
NH2
H
N
d, e, f
OH
OMe
a, b, c
H2N
N
H
O
OH OH OH OH
O
O
OH OH
O
O
21
OH OH OH
OH
OH
O
NH2
20
5
ent-Zwittermicin A (−)-1
NBn2
NBn
2OBn
NBn2
O
NBn2
O
OH NH2
NH2
NH2
NH2
H
N
OH
i
O
g, h
H2N
N
H
O
O
O
OH
OMe
O
O
OMe
O
OH OH OH OH OH OH OH
2
O
NH2
6
24
22
NH2
NH2
O
O
OH NH2
NH2
H
N
O
NBn2
O
NBn2OBn
H
N
H2N
H2N
N
H
j
N
H
NH2
NH2
3
O
OH OH OH OH OH OH OH
OH
O
O
NH2
O
O
OH
OMe
O
7
3
O
O
OH NH2
NH2
NH2
NH2
H
N
25
k, l
OMe
N
H
OBn
O
OH OH OH OH OH OH OH
OH
24
4
NH2
23
4
8
NH2
NH2
NH2
NH2
NH2
NH2
Scheme 2. Synthesis of ZwA diastereomers 3 and 4. Reagents and conditions: (a)
TBDPSCl, imidazole, DMF, 0 °C to rt, 3.5 h, 91%; (b) MeOCH2Cl, Hünig’s base, CH2Cl2,
0 °C to rt, 48 h, 96%; (c) TBAF, THF, À10 °C, 4 h, 98%; (d) Lindlar’s cat., H2, (1 atm),
EtOH, 15 h, 89%; (e) BnBr, K2CO3, CH3CN, 83 h, 92%; (f) (i) (COCl)2, DMSO, CH2Cl2,
À78 °C, (ii) Et3N, 90%; (g) (i) 23, n-Bu2BOTf, Hünig’s base, Et2O, À78 °C, 1.5 h, (ii) 22,
À78 to 0 °C, 2 h, 49%; (h) LiOH, H2O:MeOH:THF, 0 °C, 4 h, 84%; (i) (i) EDCI, HOBt,
DMF, 0 °C, 10 min, (ii) (À)-18, Et3N, 0 °C to rt, 2.5 h, 86%; (j) (i) HCl, MeOH, H2
(5 atm), Pd/C, 1 h, (ii) HCl, H2O, H2 (5 atm), Pd/C, 1.3 h, 57%; (k) (i) 24, EDCI, HOBt,
DMF, 0 °C, 10 min, (ii) (+)-18, Et3N, 0 °C to rt, 1.5 h, 86%; (l) (i) HCl, MeOH, H2
(5 atm), Pd/C, 1 h, (ii) HCl, H2O, H2 (5 atm), Pd/C, 1 h, 73%. TBDPSCl = tert-
butyldiphenylsilyl chloride.
OH OH OH
OH OH OH
OH
OH OH OH OH
OH
9
10
ent-6
O
OH NH2
O
OH NH2
H
N
H
N
H2N
N
H
H2N
OH OH
N
H
O
O
OH OH
O
NH2
O
NH2
11
12
Figure 1. ZwA diastereomers and analogs for SAR studies.
EDCI coupling of 24 to (À)-18 gave 25 (86% yield) and global
deprotection provided zwittermicin A diastereomer 3 in 57% yield.
In a similar manner, diastereomer 4 was synthesized from (+)-18
and 24.
Antifungal assay of natural (+)-1 and the 13 synthetic com-
pounds was conducted against the fungal strains Candida albi-
cans 96-489, Candida glabrata, C. albicans UCDFR1, C. albicans
ATCC 144503, Candida krusei, and the phytopathogenic bacteria
Erwinia carotovora, and Erwinia amylovora and oomycete Phy-
Bn
O
N3
N3
BnO
a
ent-6
N
O
OH OH OH OH
O
13
16
tophthora infestans (Table 1). Natural zwittermicin A [(+)-1]
showed activity against C. albicans, C. glabrata, E. carotovora,
and E. amylovora. Despite structural similarities of the synthetic
compounds to natural ZwA, particularly the enantiomer (À)-1
and stereoisomeric 3 and 4, only the natural product showed
detectable activity.
NBn2
NBn2
b, c, d
NBn2OBn
e, f
OH
O
OH
OTBS
MOMO
H
O
OH
MOMO
NH2
14
15
17
O
NBn2OBn
H
N
Table 1
g
h
Biological testing of zwittermicin A and synthetic compounds
17
N
H
NH2
11
Pathogenic microbes
(+)-1 MICa,b
(l
g/mL)
(À)-1, 2–10, ent-6, 11
O
MOMO
OH
O
and 12 MICa,b
(lg/mL)
NH2
19
H
N
Candida albicans 96–489
C. glabrata
C. albicans UCDFR1
C. albicans ATCC 14503
C. krusei
55.7
59.5
>128
>128
>128
22.2
18.8
>32
>128
>128
>128
>128
>128
>128c
>128c
>32c
H2N
O
NH2
i, j
O
17
12
(–)-18
Erwinia carotovora
E. amylovora
Scheme 1. Synthesis of ent-6, 11, and 12. Reagents and conditions: (a) H2O, H2
(1 atm), Pd/C, 2 h, 100%; (b) MeOCH2Cl, EtN(i-Pr)2, CH2Cl2, 0 °C to rt, 14 h, 91%; (c)
TBAF, THF, À10 °C, 16 h, 93%; (d) (i) (COCl)2, DMSO, CH2Cl2, À78 °C, (ii) Et3N, 94%;
(e) (i) 16, n-Bu2BOTf, Et3N, CH2Cl2, À78 to 0 °C, 3 h, (ii) 15, À78 to 0 °C, 2.5 h, 85%, dr
47:1; (f) H2O2, LiOH, 0 °C, 30 min, 67%; (g) (i) 17, EDCI, HOBt, DMF, 0 °C, 10 min, (ii)
(À)-18, Et3N, 0 °C to rt, 2.5 h, 83%; (h) (i) HCl, MeOH, H2 (5 atm), Pd/C, 1 h, (ii) HCl,
H2O, H2 (5 atm), Pd/C, 1 h, 76%; (i) (i) 17, EDCI, HOBt, DMF, 0 °C, 10 min, (ii) (+)-18,
Et3N, 0 °C to rt, 1.5 h, 67%; (j) (i) HCl, MeOH, H2 (5 atm), Pd/C, 1 h, (ii) HCl, H2O, H2
(5 atm), Pd/C, 1 h, 73%. EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride, HOBt = 1-hydroxybenzotriazole.
Phytophthora infestansd
a
The MIC endpoint is defined as the lowest concentration (
growth inhibition.
(+)-1, (À)-1, 2–4, 11 and 12 were converted to the free amine before testing.
Compounds 5–10 were used as their HCl salts.
5–10 not tested.
The maximum dose used was 32
well diffusion assay.
lg/mL) with 90%
b
c
d
lg/disk due to limitations of the nutrient agar

E. W. Rogers et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2183–2185
2185
The biological data indicate that the mechanism of action is
References and notes
highly stereospecific. The enantiomer (À)-1 and all of the
diastereomers were inactive.7 Changing the configuration of 1 at
only two stereocenters (C-13 and C-14 in 3) resulted in complete
loss of activity, even in the most susceptible strain, E. amylovora.
None of the truncated analogs (Fig. 1) showed activity. In fact,
the activity of (+)-1 is quite singular; it not mimicked by simple
synthetic stereoisomers or analogs.
Antifungal activity in simple vicinal aminoalkanols has been ob-
served for other natural products, such as oceanapiside,20 oceana-
lin,21 even sphingosine and its synthetic short-chain analogs (C6),
irrespective of the relative configurations.22 Consequently, the
stringent stereochemical requirements observed for antifungal
activity properties in 1 were unexpected and surprising. The mech-
anism of action of 1 is presently unknown, but clearly differs from
that of the former compounds which appear to interdict sphingo-
lipid metabolism.23
In summary, two zwittermicin A diastereomers and three
analogs were synthesized and compared with natural zwittermi-
cin A and eight other previously synthesized analogs and stereo-
isomers in antifungal assays. The microbial susceptibility profile
of 1–12 indicates that the mechanism of action is highly stereo-
specific, and the complete complement of functionality found in
the natural enantiomer zwittermicin A [(+)-1] is required for
efficacy. Further investigations to identify the mechanism of ac-
tion may benefit from selective affinity tagging of 1 and deploy-
ment in cell-free pull-down experiments to identify the cellular
target.24
1. (a) He, H.; Silo-Suh, L. A.; Handelsman, J.; Clardy, J. Tetrahedron Lett. 1994, 35,
2499; (b) Silo-Suh, L. A.; Lethbridge, B. J.; Raffel, S. J.; He, H.; Clardy, J.;
Handelsman, J. Appl. Environ. Microbiol. 1994, 60, 2023; (c) Athukorala, S. N. P.;
Fernando, W. G. D.; Rashid, K. Y. Can. J. Microbiol. 2009, 55, 1021.
2. (a) Silo-Suh, L. A.; Stabb, E. V.; Raffel, S. J.; Handelsman, J. Curr. Microbiol. 1998,
37, 6; (b) Milner, L.; Stohl, E. A.; Handelsman, J. J. Bacteriol. 1996, 178, 4266; (c)
Stohl, E. A.; Brady, S. F.; Clardy, J.; Handelsman, J. J. Bacteriol. 1999, 181, 5455.
3. (a) Broderick, N. A.; Goodman, R. M.; Handelsman, J.; Raffa, K. F. Environ.
Entomol. 2003, 32, 387; (b) Broderick, N. A.; Goodman, R. M.; Raffa, K. F.;
Handelsman, J. Environ. Entomol. 2000, 29, 101.
4. (a) Stabb, E. V.; Jacoboson, L. M.; Handelsman, J. Appl. Environ. Microbiol. 1994,
60, 4404; (b) Raffel, S. J.; Stabb, E. V.; Milner, J. L.; Handelsman, J. Microbiology
1996, 142, 3425.
5. (a) Stohl, E. A.; Milner, J. L.; Handelsman, J. Gene 1999, 237, 403; (b) Emmert, E.
A.; Kilmowicz, A. K.; Thomas, M. G.; Handelsman, J. Appl. Environ. Microbiol.
2004, 70, 104; (c) Chan, Y. A.; Boyne, M. T., II; Podevels, A. M.; Klimowicz, A. K.;
Handelsman, J.; Kelleher, N. L.; Thomas, M. G. Proc. Natl. Acad. Sci. U.S.A. 2006,
103, 14349; (d) Zhao, C.; Luo, Y.; Song, C.; Liu, Z.; Chen, S.; Yu, Z.; Sun, M. Arch.
Microbiol. 2007, 187, 313.
6. Stabb, E. V.; Handelsman, J. Mol. Microbiol. 1998, 27, 311.
7. (a) Rogers, E. W.; Molinski, T. F. Org. Lett. 2007, 9, 437; (b) Rogers, E. W.; Dalisay,
D. S.; Molinski, T. F. Angew. Chem., Int. Ed. 2008, 47, 8086.
8. (a) Rogers, E. W.; Molinski, T. F. J. Org. Chem. 2009, 74, 7660; (b) Rogers, E. W.,
PhD Thesis, UC San Diego, 2008.
9. Laïb, T.; Chastanet, J.; Zhu, J. J. Org. Chem. 1998, 63, 1709.
10. Stork, G.; Takahashi, T. J. Am. Chem. Soc. 1977, 99, 1275.
11. Novachek, K. A.; Meyers, A. I. Tetrahedron Lett. 1996, 37, 1743.
12. Yakelis, N. A.; Roush, W. R. J. Org. Chem. 2003, 68, 3838.
13. Evans, D. A.; Gage, J. R. Org. Synth. 1990, 68, 83.
14. Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron Lett. 1987, 28, 6141.
15. Hulme, A. N.; Montgomery, C. H.; Henderson, D. K. J. Chem. Soc., Perkin. Trans. 1
2000, 1837.
16. Corey, E. J.; Nicolaou, K. C.; Balanson, R. D.; Machida, Y. Synthesis 1975, 590.
17. Reetz, M. T. Chem. Rev. 1999, 99, 1121.
18. Bertrand, M. B.; Wolfe, J. P. Org. Lett. 2006, 8, 4661.
19. Vicario, J. L.; Rodriguez, M.; Badía, D.; Carrillo, L.; Reyes, E. Org. Lett. 2004, 6,
3171.
Acknowledgments
20. (a) Nicholas, G. M.; Hong, T. W.; Molinski, T. F.; Lerch, M. L.; Cancilla, M. T.;
Lebrilla, C. B. J. Nat. Prod. 1999, 62, 1678; (b) Nicholas, G. M.; Molinski, T. F. J.
Am. Chem. Soc. 2000, 122, 4011.
21. Makarieva, T. N.; Denisenko, V. A.; Dmitrenok, P. S.; Guzii, A. G.; Santalova, E.
A.; Stonik, V. A.; MacMillan, J. B.; Molinski, T. F. Org. Lett. 2005, 7, 2897.
22. Nicholas, G. N.; Li, R.; MacMillan, J. B.; Molinski, T. F. Bioorg. Med. Chem. Lett.
2002, 12, 2159.
23. Dalisay, D. S.; Molinski, T. F., unpublished.
24. Hughes, C. C.; MacMillan, J. B.; Gaudencio, S. P.; Fenical, W.; La Clair, J. J. Angew.
Chem., Int. Ed. 2009, 48, 728.
HRMS measurements were made by R. New (UC Riverside) and
Y. X. Su (UC San Diego). We thank AgraQuest, Inc. for providing
Erwinia and Phytophthora strains. The NSF CRIF program
(CHE0741968) is acknowledged for acquisition of the 500 MHz
NMR spectrometers. We are grateful for generous funding for this
research from the NIH, National Institute of Allergies and Infectious
Disease (AI 039987).