Total Synthesis and Absolute Configuration of Simpotentin, a Potentiator of Amphotericin B Activity

Article abstract of DOI:10.1021/acs.orglett.9b01945

The total synthesis of simpotentin (1), a new potentiator of amphotericin B activity against Candida albicans, was achieved. Our research results enabled the access of all stereoisomers of 1 and the elucidation of the unknown absolute configuration of 1.

Full text of DOI:10.1021/acs.orglett.9b01945

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis and Absolute Configuration of Simpotentin, a
Potentiator of Amphotericin B Activity
Masaki Ohtawa,† Eri Shimizu,† Atsushi Saito,† Sayuri Sakamoto,† Ai Waki,† Ariko Kondo,† Akiho Yagi,‡
Ryuji Uchida,‡ Hiroshi Tomoda,† and Tohru Nagamitsu*,†
†Laboratory of Synthetic Natural Products Chemistry and Medicinal Research Laboratories, School of Pharmacy, Kitasato
University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan
‡Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai-shi,
Miyagi 891-8558, Japan
S
* Supporting Information
ABSTRACT: The total synthesis of simpotentin (1), a new
potentiator of amphotericin B activity against Candida albicans, was
achieved. Our research results enabled the access of all stereoisomers
of 1 and the elucidation of the unknown absolute configuration of 1.
Furthermore, one of the stereoisomers is a better amphotericin B
potentiator than 1 and is an excellent lead compound for the
development of a novel amphotericin B potentiator.
Scheme 1. Retrosynthesis of (3R,5R,13R)-1
ver the past 30 years, the number of patients with
Omycoses has continued to increase, primarily due to
highly advanced medical treatments, such as organ trans-
plantation, the use of immunosuppressive drugs, radiotherapy,
and antitumoral chemotherapy. Among them, Candida
albicans, Aspergillus fumigatus, Cryptococcus neoformans, and
Figure 1. Structures of AMPB and its potentiators and the MIC
values of 1, AMPB, and their mixture against C. albicans.
Received: June 5, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b01945
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Letter
Scheme 2. Total Synthesis of (3R,5R,13R)-1
Scheme 3. Synthesis of All Stereoisomers of Simpotentin (1)
Rhizopus oryzae are pathogenic fungi that occur via invasive
fungal infections.1 C. albicans is especially implicated in
approximately 50% of patients with candidasis.2 Although
several antifungal antibiotics are currently available for their
treatment, amphotericin B (AMPB), a polyene macrolide
antifungal antibiotic, has been the “gold standard” since its
introduction into clinical practice in the 1950s.3 Additionally,
liposomal AMPB was developed to improve the tolerability
profile of AMPB deoxycholate in the 1990s.4 However, AMPB
has serious side effects, such as nephrotoxicity, hypokalemia,
fever, chills, and vomiting, which have led to discontinuation of
this treatment. The development of potentiators for AMPB has
reduced the dose of AMPB and the severe side effects. A few
potentiators for AMPB have been reported thus far (Figure 1).
Ogita et al. reported allicin, which enhances the membrane
permeability of AMPB, as the first report of a potentiator for
AMPB from garlic.5 Serotonin has been reported by Hellar et
al. to have a similar mechanism of action.6 Recently,
shearinenes D and E, alkaloid metabolites produced by a
Alaskan soil-derived Penicillium sp., have shown an enhance-
ment in the antifungal activity of AMPB, according to
Cichewicz et al.7 During our continuous screening for
microbial AMPB potentiators against C. albicans, a novel
compound, simpotentin (1), was isolated from the culture
broth of Simplicillium sp. FKI-4981B (Figure 1).8
Simpotentin (1) is a β-mannoside, and its aglycone is 3-
hydroxy-5-[(3-hydroxyoctanoyl)oxy]decanoic acid with three
stereogenic centers. Simpotentin (1) is a nonantibiotic with no
antifungal activity against C. albicans even at 512 μg/mL via
the liquid microdilution method. On the other hand, the
MIC90 value of AMPB against C. albicans was found to be
0.500 μg/mL. However, the MIC values decreased to 0.06 and
0.03 μg/mL in combination with 32 and 64 μg/mL of 1,
respectively. Thus, we found that 1 enhanced AMPB activity
against C. albicans dose dependently.
Simpotentin (1) is a new potent potentiator of AMPB;
however, the absolute configuration of the aglycone of 1 has
not been clarified. To determine the absolute configuration of
the natural 1, a concise total synthesis of all simpotentin
stereoisomers (total eight diastereomers) was required.
Therefore, we performed the total synthesis of (3R,5R,13R)-
1 (Simpotentin numbering), which was selected as a primary
target molecule among the eight diastereomers. The
retrosynthetic analysis is shown in Scheme 1. Accordingly,
(3R,5R,13R)-1 could be synthesized via a stereoselective β-
mannosylation of the key aglycone, (3R,5R,13R)-3, with ulosyl
bromide 2,9 followed by the reduction of a carbonyl group and
deprotection.10 Aglycone (3R,5R,13R)-3 could be produced
from the acylation of (3R,5R)-5 with acyl chloride (13R)-4.
Then (3R,5R)-5 could be derived from a commercially
available n-hexanal via two sequential SmI2-mediated stereo-
B
DOI: 10.1021/acs.orglett.9b01945
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
protection16 of (3R,5R)-9 followed by removal of the chiral
auxiliary with LiOOH17 afforded the carboxylic acid, (3R,5R)-
10, in 71% yield in two steps. Subsequent TBS deprotection of
(3R,5R)-10 with TBAF afforded the desired 5-hydroxycarbox-
ylic acid but was accompanied by formation of a six-membered
lactone during workup after reaction and silica gel chromatog-
raphy. Thus, after completing the TBS deprotection of
(3R,5R)-10 with TBAF, benzyl bromide was added to the
reaction mixture in a one-pot manner. Esterification proceeded
smoothly to yield the benzyl ester, (3R,5R)-5, in 81% yield
without lactonization. Acylation of (3R,5R)-5 with acyl
chloride (13R)-4, which was easily derived from (R)-8 in
three steps,18 in the presence of Et3N gave (3R,5R,13R)-11 in
78% yield. PMB deprotection via treatment with DDQ
afforded the desired aglycone, (3R,5R,13R)-3, in 93% yield.
With the desired aglycone (3R,5R,13R)-3 synthesized, β-
mannosylation by PMB ether-mediated intramolecular aglycon
delivery19 was first investigated; however, it was unfruitful.
Next, the β-glycosylation of (3R,5R,13R)-3 with ulosyl
bromide 29 was attempted as a key reaction. β-Glycosylation
in the presence of AgOTf furnished the desired β-mannoside
(3R,5R,13R)-12 in 72% yield in two steps as a single isomer
after the reduction of the corresponding ketone with NaBH4.
Finally, deprotection of all benzyl groups via hydrogenolysis
gave the (3R,5R,13R)-1 in 86% yield. Because the total
synthesis of (3R,5R,13R)-1 was established, the other stereo-
isomers of simpotentin (1) were also synthesized in the same
manner (Scheme 3).18 This means that our total synthesis was
very concise and practical.
Figure 2. HPLC analyses of natural simpotentin (1), its synthetic
stereoisomers, and the absolute structure of simpotentin (1). HPLC
conditions: column, CHRALCEL OJ-RH (DAICEL) (ϕ 4.6 mm ×
150 mm); mobile phase, 20−25% MeCN−0.1% HCO2H (30 min
gradient); flow rate, 1 mL/min; column temperature, 10 °C;
detection, ELSD (temp 40 °C, gas flow; 1.5 L/min); injection, 5 μg.
For the determination of the absolute configuration of
natural simpotentin (1), its physicochemical properties were
compared with those of all the synthetic stereoisomers of 1.
Consequently, (3R,5R,13R)-1 was completely identical to
1
natural 1 in all respects ([α]D, H, and 13C NMR, IR, and
ESIMS).17 Furthermore, HPLC analyses of natural 1 and all
synthetic stereoisomers were performed (Figure 2). Most of
the stereoisomers, except for (3S,5R,13R)-1 and (3S,5S,13S)-1,
were separated. Then we found that the retention time of
(3R,5R,13R)-1 was completely identical to that of natural 1.
Additional HPLC analysis of the mixture of natural 1 with all
synthetic stereoisomers showed that the only peak for
(3R,5R,13R)-1 was clearly amplified. These data also revealed
that the absolute configuration of natural simpotentin (1) is
3R,5R,13R.
selective Reformatsky reactions11 with the chiral N-bromoace-
tyl oxazolidinone (R)-612 [(5R)-7 → (3R,5R)-5 and n-hexanal
→ (R)-8]. Then (13R)-4 could be synthesized from a common
intermediate, (R)-8. This synthetic strategy would also allow
the synthesis of the other simpotentin stereoisomers by using
the chiral oxazolidinone (S)-6 instead of (R)-6.
The synthesis of (3R,5R,13R)-1 is shown in Scheme 2. The
first SmI2-mediated Reformatsky reaction of n-hexanal with
(R)-612 gave (R)-8 in 93% yield (diastereomeric ratio (dr) of
9.3:1). The Conversion of (R)-8 to the Weinreb amide13
(95%), TBS protection (97%), and reduction with DIBAL
afforded the aldehyde (5R)-7.14 The second Reformatsky
reaction of (5R)-7 and oxazolidinone (R)-6 gave (3R,5R)-915
in 96% yield with a high diastereoselectivity (dr of 47:1). PMB
Next, the potentiating effects of all of the simpotentin
stereoisomers on the AMPB activity against C. albicans were
evaluated. The activities of most of the stereoisomers were
similar to natural 1; however, (3R,5S,13S)-1 was found to be
better than 1 with over 16-fold potentiation of AMPB activity
a b
,
Table 1. MIC Values of Amphotericin B against C. albicans in the Presence of the Stereoisomers of 1
MIC (μg/mL) of AMPB
conc of the
stereoisomer
(μg/mL)
+
natural
1
+ (3R,5R,13R)-1 =
synthetic
simpotentin (1)
+
+
+
+
+
+
(3R,5S,13R)- (3S,5R,13R)- (3S,5S,13R)- (3R,5R,13S)-
(3S,5R,13S)- (3S,5S,13S)-
1
1
1
1
+ (3R,SS,13S)- 1
1
1
c
0
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
8
32
0.25
0.25
0.125
0.25
0.25
0.125 (x4)
0.5
0.25
0.125
0.25
0.25
0.125
0.25
0.25
0.125
0.25
0.25
0.125
0.125
0.0625
<0.0313 (>×16)
0.25
0.125
0.0625
0.25
0.25
0.0625
a
b
A smaller MIC value means that it has a more potent enhancing effect for AMPB activity against C. albicans. The stereoisomers of 1 showed no
c
antifungal activity against C. albicans even at 64 μg/mL. The MIC90 value of AMPB alone for C. albicans.
C
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(7) You, J.; Du, L.; King, J. B.; Hall, B. E.; Cichewicz, R. H. ACS
Chem. Biol. 2013, 8, 840−848.
at 32 μg/mL (Table 1). The new stereoisomer, (3R,5S,13S)-1,
has the potential to be an excellent lead compound for the
development of a novel AMPB potentiator.
(8) Uchida, R.; Kondo, A.; Yagi, A.; Nonaka, K.; Masuma, R.;
Kobayashi, K.; Tomoda, H. J. Antibiot. 2019, 72, 134−140.
(9) Lichtenthaler, F.; Schneider-Adams, T. J. Org. Chem. 1994, 59,
6728−6734.
In summary, we achieved the concise total synthesis of
simpotentin (1). Extension of this chemistry enabled the
synthesis of all stereoisomers of 1 and the determination of the
absolute configuration of natural 1. Furthermore, we
discovered a new stereoisomer, (3R,5S,13S)-1, which has a
more potent activity as an AMPB potentiator than 1.
Application of our total synthesis would allow the synthesis
of a variety of simpotentin analogues. The structure−activity
relationship study of simpotentin (1) is currently underway
and will be reported in due course.
(10) Lichtenthaler, F.; Lergenmuller, M.; Peters, S.; Varga, Z.
̈
Tetrahedron Asymmetry 2003, 14, 727−736.
(11) Fukuzawa, S.; Matsuzawa, H.; Yoshimitsu, S. J. Org. Chem.
2000, 65, 1702−1706.
(12) Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1987, 109, 7151−
7157.
(13) Venkatesham, A.; Srinvasa, R. R.; Nagaiah, K. Tetrahedron
Asymmetry 2012, 23, 381−387.
(14) Fournier, L.; Kocienski, P.; Pons, J. Tetrahedron 2004, 60,
1659−1663.
ASSOCIATED CONTENT
* Supporting Information
■
(15) The stereochemistry of (3R,5R)-9 was determined by the
ROESY of (3R,5R)-13 derived from (3R,5R)-9 via TBS deprotection
and acetonization.
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.9b01945.
Experimental procedures and characterization of com-
pounds including NMR spectra (PDF)
AUTHOR INFORMATION
■
(16) Lister, T.; Perkins, M. Org. Lett. 2006, 8, 1827−1830.
(17) Evans, D. A.; Britton, T. C.; Ellman, J. A. Tetrahedron Lett.
1987, 28, 6141−6144.
Corresponding Author
*E-mail: nagamitsut@pharm.kitasato-u.ac.jp.
ORCID
(18) See the Supporting Information.
(19) Ito, Y.; Ogawa, T. Angew. Chem., Int. Ed. Engl. 1994, 33, 1765−
1767.
Hiroshi Tomoda: 0000-0002-1241-8901
Tohru Nagamitsu: 0000-0002-4278-4507
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was partially supported by a Grant-in-Aid for
Scientific Research (C) from the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan
(M.O.: 25460152). We thank Ms. N. Sato and Dr. K. Nagai
(Kitasato University) for kindly obtaining the NMR and MS
spectra.
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DOI: 10.1021/acs.orglett.9b01945
Org. Lett. XXXX, XXX, XXX−XXX