Efficient total synthesis of (+)-negamycin, a potential chemotherapeutic agent for genetic diseases

Article abstract of DOI:10.1039/b801498a

Herein, we describe an efficient strategy for the total synthesis of (+)-negamycin using commercially available achiral N-Boc-2-aminoacetaldehyde as starting material with 42% overall yield for a limited number of steps. The Royal Society of Chemistry.

Full text of DOI:10.1039/b801498a

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Efficient total synthesis of (+)-negamycin, a potential chemotherapeutic
agent for genetic diseasesw
Yoshio Hayashi,*ab Thomas Regnier,a Shigenobu Nishiguchi,a Magne O. Sydnes,a
Daisuke Hashimoto,c Junya Hasegawa,c Takahiro Katoh,c Tetsuya Kajimoto,c
Masataka Shiozuka,d Ryoichi Matsuda,d Manabu Nodec and Yoshiaki Kiso*a
Received (in Cambridge, UK) 28th January 2008, Accepted 25th February 2008
First published as an Advance Article on the web 18th March 2008
DOI: 10.1039/b801498a
Herein, we describe an efficient strategy for the total synthesis of
(+)-negamycin using commercially available achiral N-Boc-2-
aminoacetaldehyde as starting material with 42% overall yield
for a limited number of steps.
This resulting chiral amino alcohol was directly engaged
without further purification to form the target oxazolidine 4
by treatment with 2,2-dimethoxypropane (DMP) in the pre-
sence of boron trifluoride diethyl ether complex (BF3ÁEt2O) in
acetone. As a result, 4 was generated in high yield after
purification by silica gel column chromatography (90%). To
prepare the key intermediate 3, a cross-metathesis (CM)
reaction between 4 and tert-butyl acrylate 6 was investigated.
Although the efficiency of ruthenium-based catalysts for ring-
opening metathesis polymerization (ROMP) and ring-closing
metathesis (RCM) is now well established, most alkene CM
variants have fewer successful applications because of the
multiple possible side reactions that cause relatively low
synthetic yields.7 Because our substrates are categorized as
rapid and slow homodimerizable compounds according to
Grubbs et al.’s empirical model for predicting the outcome
of CM reactions,8 we screened different reaction conditions to
avoid forming unwanted dimers and selectively provide the
target compound 3 by varying catalysts (Grubbs first [Ru-I]
and second-generation [Ru-II] catalysts), amount of reactant
(1 or 5 equiv. of 6), duration of reaction as well as heating
method (conventional or microwave-assisted heating).
(+)-Negamycin (1, Scheme 1), an unusual antibiotic contain-
ing a hydrazine peptide bond was isolated for the first time by
Umezawa et al. in 1970 from culture filtrates of three strains
related to Streptomyces purperfuscus. This natural product
exhibits very low acute toxicity and strong inhibitory activity
against multiple drug-resistant enteric Gram-negative bacteria
including Pseudomonas aerginosa.1 (+)-1’s anti-microbial ac-
tivity is derived from a genetic miscoding on bacterial riboso-
mal systems, and thereby leading to a specific inhibition of
protein biosynthesis.2 Because this miscoding causes read-
through of termination signals, considerable attention is fo-
cused on (+)-1 as a potential therapeutic agent against genetic
diseases. Indeed, the aminoglycoside antibiotic gentamicin and
the less toxic negamycin both restore dystrophin expression in
skeletal and cardiac muscles of mdx mice, an animal model of
Duchenne muscular dystrophy (DMD) with a nonsense muta-
tion in the dystrophin gene.3 Therefore, an efficient shortened
synthetic route of (+)-1 and its derivatives appears significant
to develop promising new therapeutic candidates for DMD
and other diseases caused by nonsense mutations. The first
total synthesis of (+)-1 from D-galacturonic acid was reported
in 1972 and confirmed the assigned structure of the natural
product.4 Over three decades, numerous total syntheses have
been reported on both racemic and optically active (+)-1 but
with moderate overall yield.5
As detailed in the ESI,w the conversion and chemoselectivity
enhancements are definitely more pronounced for [Ru]-II than
[Ru]-I. Furthermore, we observed that microwave irradiation
drastically shortened the CM reaction time by 20-fold. As it
pertains to microwave-assisted synthesis, this acceleration is
commonly attributed to the very high local temperatures and
the ease to which microwave irradiation reaches such condi-
tions but the scientific community is still divided in opinion on
the involvement of a specific non-thermal effect induced by the
dielectric heating produced using microwaves.9 Interestingly,
although our observations provide a new example for the
thermal effect, the involvement of such ‘‘specific effect’’ is
neither confirmed nor disproved in these reaction conditions.
Bargiggia et al. arrived to similar conclusions while studying
CM reactions10 and Garbaccia et al. have described similar
observations for RCM reactions in 2003.11 As a result, the
desired product 3 was isolated with 83% yield. NOE experi-
ments revealed that the stereochemistry of the olefin moiety in
3 was an E configuration (Jvinylic protons = 15.7 Hz). Thus, the
desired chiral intermediate 3 was obtained with 75% yield
after two steps from achiral N-Boc-glycinal 5.
Our fast and efficient route consists first on an asymmetric
allylboration of N-Boc-glycinal
5 using the established
Brown’s procedure for preparation of chiral allylic alcohols6
that led to a corresponding chiral intermediate (Scheme 1).
a Department of Medicinal Chemistry, Kyoto Pharmaceutical
University, Kyoto 607-8412, Japan. E-mail:
yhayashi@ps.toyaku.ac.jp, kiso@mb.kyoto-phu.ac.jp; Fax: +81-75-
595-4787; Tel: +81-75-595-4636
b Department of Medicinal Chemistry, School of Pharmacy, Tokyo
University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan
c Department of Pharmaceutical Manufacturing Chemistry, Kyoto
Pharmaceutical University, Kyoto 607-8412, Japan
d Department of Life Science, University of Tokyo, Tokyo 153-8902,
Japan
With intermediate 3 in hand, our focus shifted toward the
asymmetric Michael addition reaction. Recently, Node et al.
w Electronic supplementary information (ESI) available: Analytical
data. See DOI: 10.1039/b801498a
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Scheme 1 Total synthesis of (+)-negamycin.
reported a highly stereoselective asymmetric Michael addition
toward tert-butyl a,b-unsaturated carbonyl compounds using
chiral amine 7.12 This approach, when applied to the a,b-
unsaturated tert-butyl ester 3, allowed the introduction of the
amine moiety with an excellent enantiomeric excess.13 The
chiral reagent 7 was prepared from the corresponding keto-
pinic acid12 and reacted with 3 in the presence of n-BuLi in
THF at À78 1C to afford compound 8 as a single diastereomer
(de 499%) in 80% yield after purification. Removal of both
benzyl and 2-methoxybornyl protecting groups located on the
same amine moiety could be achieved efficiently using 4 equiv.
of N-iodosuccinimide (NIS) in dichloromethane to obtain free
amino compound 9 in 81% yield. This deprotection proceeded
by oxidation with NIS to imine and subsequent spontaneous
hydrolysis to afford tert-butyl esters of b-amino acids and 2-
methoxy-D-bornylaldehyde.12 No epimerization was observed
during this reaction. Furthermore, one of the advantages of
the protocol is that the initial chiral inducer 7 can be easily
regenerated from 2-methoxy-D-bornaldehyde, generated dur-
ing the cleavage by reductive amination, using benzylamine in
the presence of sodium cyanoborohydride (data not shown).
The last part of the synthesis of (+)-1 consisted of introdu-
cing a hydrazine unit, prior to a final deprotection. A Boc-
protection of 9 using standard procedures was first quantita-
tively performed to afford N-protected tert-butyl ester 11, that
was then efficiently converted to acid 2 by a microwave-
assisted saponification with 2M KOH in MeOH, and coupling
with hydrazine unit 12 was then performed using the classical
EDCÁHCl–HOBt method. The synthesis of hydrazine 12 was
achieved by reacting N-methyl hydrazine with tert-butyl bro-
moacetate with 40% yield after purification. Deprotection of
compound 13 and purification by ion exchange chromatogra-
derivatization of the 1 structure using the above synthetic
methodology will contribute to a better understanding of the
structure–activity relationship of 1 and the development of
more potent compounds with efficient read-through activity.
Studies in this regard are currently in progress and details
pertaining to the biological activity will soon be published
elsewhere.
In conclusion, the proposed synthetic route for the total
synthesis of optically active (+)-negamycin starting from
N-Boc-glycinal 5, led to the desired product with a total yield
of 42% over only eight steps. To our knowledge, this study
represents the most efficient strategy to prepare (+)-1. Current
efforts with this new synthetic approach are now expanding
into medicinal chemistry to discover new drug candidates with
potent read-through activity for Duchenne muscular dystro-
phy. The chemical biology of negamycin is also now being
investigated to better understand its read-through mechanism.
This research was supported by the Research Grant
(17A-10) for Nervous and Mental Disorders from the Ministry
of Health, Labour and Welfare. T. R. is grateful for the
Postdoctoral Fellowship of JSPS. We thank Dr J.-T. Nguyen
for valuable help during manuscript preparation.
Notes and references
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phy on Amberlite CG50 (NH4 form) afforded the target
25.2
compound (+)-1 in 98% yield, [a]D
+2.41 (c 0.36, H2O),
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