Total synthesis of the thiopeptide amythiamicin D.

Article abstract of DOI:10.1039/b401580k

The first total synthesis of the thiopeptide antibiotic amythiamicin D is described.

Full text of DOI:10.1039/b401580k

Total synthesis of the thiopeptide amythiamicin D†
Rachael A. Hughes,a Stewart P. Thompson,a Lilian Alcarazb and Christopher J. Moody*a
a Department of Chemistry, University of Exeter, Exeter, UK EX4 4QD
b Medicinal Chemistry Department, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, UK
LE11 5RH
Received (in Cambridge, UK) 3rd February 2004, Accepted 25th February 2004
First published as an Advance Article on the web 16th March 2004
The first total synthesis of the thiopeptide antibiotic amythiami-
cin D is described.
not disclosed. Therefore, we assume that they derive from natural -
L
amino acids, an assumption supported by the structure of the
closely related antibiotic GE2270A.3,22 Our synthetic strategy is
indicated in Scheme 1, and disconnections at the amide bonds
reveal the building blocks as two thiazoles 2 and 3, a simple glycine
derivative 4, and the pyridine 5. In 1978 Bycroft and Gowland, as
well as reporting the structure of micrococcin P1,23 suggested that
its pyridine ring (as well as the tetrahydropyridine in thiostrepton)
could result biogenetically “from the interaction of two dehy-
droalanine units,” themselves derived from serine residues. This
interesting proposal for the biosynthesis of the pyridine ring in
thiopeptides was subsequently supported by isotopic labeling
experiments by Floss and co-workers.24,25 Floss also viewed the
Bycroft proposal for the biosynthesis of the pyridine ring as a
cycloaddition (not necessarily concerted) followed by aromatisa-
tion. We have recently reported the realisation of Bycroft’s original
The amythiamicins are members of the thiopeptide family of
antibiotics, a class of sulfur-containing highly modified cyclic
peptides characterized by several common structural features such
as the presence of thiazole and, in some cases, oxazole rings,
unusual and dehydro amino acids, and a heterocyclic centerpiece of
a tri- or tetra-substituted pyridine all in a macrocyclic array. Most
of the thiopeptide antibiotics inhibit protein synthesis in bacteria,
and share common modes of action. They act by binding to the
complex of 23SrRNA with ribosomal protein L11, inhibiting the
action of GTP-dependent elongation factors.1,2 Alternatively, other
thiopeptides such as GE2270A, a compound structurally related to
the amythiamicins, act directly on the elongation factor proteins
inhibiting their action.3
The amythiamicins, isolated from a strain of Amycolatopsis sp.
MI481-42F4, and their structures determined by degradative and
spectroscopic techniques,4–6 were originally reported to inhibit the
growth of Gram-positive bacteria including MRSA,4 but more
recently, in common with other thiopeptides such as thiostrepton
and micrococcin,7,8 they have been shown to exhibit antimalarial
activity against Plasmodium falciparum,9 the parasite that causes
the majority of malarial infections in humans. Despite the
fascinating biological activity of the thiopeptide antibiotics,
relatively little synthetic work has been carried out to date, and the
only reported total syntheses are that of micrococcin P1,10–12 and
our own work on promothiocin A.13 However, the syntheses of
various fragments of other thiopeptides have been reported, for
example the pyridine fragments of dimethyl sulfomycinamate,14,15
nosiheptide,16 A10255,17 and GE2270A,18 and Nicolaou has
recently reported substantial progress towards the synthesis of
thiostrepton.19,20 We now report the first synthesis of one of the
amythiamicins, amythiamicin D 1, using a biosynthesis inspired
Diels–Alder route to the pyridine core of the antibiotic as the key
step.21
Scheme 2 Synthesis of thiazole 2. Reagents and conditions: (i) (a) EtO2CCl
(1 eq), Et3N (1 eq), THF, 0 °C; (b) aq. NH3 (35%), THF, 0 °C, 96%; (ii)
methyl 2-diazo-3-oxobutanoate (1.2 eq), Rh2(Oct)4 (0.025 eq), CH2Cl2,
reflux, 74%; (iii) Lawesson’s reagent (2 eq), THF, reflux, 65%; (iv) H2 (60
psi), 10% Pd/C, MeOH, reflux, 80%; (v) (a) EtO2CCl (1 eq), Et3N (1 eq),
THF, 0 °C; (b) 2 M MeNH2 in THF (3 eq), 0 °C, 73%; (vi) 1 M aq. LiOH,
MeOH, rt, 79%. Oct = octanoate.
Although the detailed structural assignment of amythiamicin D 1
was reported,5 the stereochemistry of the three chiral centres was
† Electronic supplementary information (ESI) available: Spectroscopic data
for synthetic 1. See http://www.rsc.org/suppdata/cc/b4/b401580k/
Scheme 1 Retrosynthetic analysis of amythiamicin D 1.
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T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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biosynthesis proposal in a biomimetic cycloaddition route to
2,3,6-trisubstituted pyridines, involving the Diels–Alder reaction of
serine-derived 1-alkoxy-2-azadienes with dehydroalanine deriva-
tives,26 and therefore a cycloaddition approach to the pyridine 5
remained the keystone of our overall strategy.27
converted into the bis-thiazole 19 by a standard ester to amide to
thioamide and Hantzsch reaction sequence. The carboxamide 19
was then reacted with triethyloxonium hexafluorophosphate to give
the imidate 20, reaction of which with the serine derivative 18 gave
the complex 2-azadiene 21 after elimination of acetate using DBU
as base (Scheme 5).
Our synthesis started with the construction of the thiazole 2 from
commercially available N-Boc-
L
-aspartic acid 4-benzyl ester
The stage was now set for the key ‘biomimetic’ hetero-Diels–
Alder-aromatisation sequence which was carried out under our
previously developed thermal conditions,26 rather than ‘biological’
conditions. Thus heating the two components 15 and 21 under
microwave irradiation in toluene at 120 °C for 12 hours gave the
required 2,3,6-tris(thiazolyl)pyridine 5, albeit in modest yield
(Scheme 6). With all the components 2–5 now in hand, the
assembly of the natural product could be addressed. Deprotection
(Scheme 2). Thus the corresponding amide 6 underwent an N–H
insertion reaction with the rhodium carbene derived from methyl
2-diazo-3-oxobutanoate to give the 1,4-dicarbonyl compound 7 in
good yield, a reaction we have used previously as a key step in a
route to oxazole building blocks of nostocyclamide and promothio-
cin A.13,28 However, on this occasion rather than dehydrating the
ketoamide 7 to give an oxazole, it was treated with Lawesson’s
reagent to give the corresponding thiazole 8.29 It thus remained to
install the correct side-chain and this was achieved by hydro-
genolysis of the benzyl ester and amide formation to give 9; this
was followed by alkaline hydrolysis to reveal the free thiazole-
4-carboxylic acid 2 for subsequent coupling reaction.
The synthesis of the second thiazole 3 was readily achieved from
the known (S)-thiazole 1030 as shown in Scheme 3. Hydrolysis of
the ethyl ester was followed by re-esterification using phase-
transfer catalyzed alkylation with tert-butyl bromide,31 and then
selective removal of the Boc-group.32 The resulting amine 3 was
coupled to the thiazolecarboxylic acid 2 using carbodiimide
methodology to give the bis-thiazole 11, which was prepared for a
further coupling reaction by a second selective removal of an N-
Boc-group in the presence of a tert-butyl ester to give the bis-
thiazole amine hydrochloride 12.
In order to set up the key Diels–Alder reaction we required both
diene and dienophile components to contain a thiazole-4-carbox-
ylate, the ester groups of which needed to be differentiated. Since
the target molecule 1 bears a thiazole methyl ester at the pyridine-
6-position, this fixes the corresponding substituent at the 3-position
of the proposed 2-azadiene component, and therefore a dienophile
containing an orthogonally protected carboxyl was prepared
(Scheme 4). Starting from cysteine ethyl ester hydrochloride,
benzyl 2-acetylthiazole-4-carboxylate 13 was built up by reaction
with pyruvic aldehyde, manganese(IV) oxide oxidation of the
resulting thiazoline, and ester exchange. Conversion into the oxime
14 (E/Z ~ 1 : 2) was followed by reduction to the required
‘dehydroalanine’ dienophile, the N-acetylenamine 15, using Burk’s
iron/acetic anhydride/acetic acid protocol.33
Scheme 4 Synthesis of dienophile 15. Reagents and conditions: (i) pyruvic
aldehyde (1 eq), KHCO3 (1 eq), EtOH/H2O (1 : 1), rt; (ii) MnO2 (20 eq),
MeCN, 60 °C, 55% over 2 steps; (iii) Cs2CO3, THF/H2O (1 : 1), rt, 100%;
(iv) BnBr (1 eq), Et3N (1 eq), EtOAc, reflux, 89%; (v) H2NOH·HCl (1 eq),
AcONa (1 eq), MeOH, rt, 99% (E : Z ratio ~ 1 : 2); (vi) Fe (2 eq), AcOH
(3 eq), Ac2O (3 eq), toluene, 70 °C, 57%.
The 2-azadiene component 21 which contains the remaining
three thiazole rings was constructed from the serine-derived
thiazole 18 and the valine-derived bis-thiazole 20 using the imidate-
based methodology developed in our preliminary studies.26 N-Boc-
Serine methyl ester was converted into the thiazole-4-ester 16 in
four straightforward steps, and this was followed by desilylation
and acetylation to give 17, and final deprotection of the Boc-group
to deliver the amine hydrochloride 18. The lower half of the
azadiene was formed from the valine-derived thiazole 10 that was
Scheme 5 Synthesis of diene 21. Reagents and conditions: (i) tBuPh2SiCl
(TBDPSCl) (1.1 eq), imidazole (2.5 eq), THF, rt, 100%; (ii) aq. NH3 (35%),
MeOH, rt, 100%; (iii) Lawesson’s reagent (0.5 eq), CH2Cl2, rt, 91%; (iv) (a)
methyl bromopyruvate (3.5 eq), KHCO3 (4 eq), DME, 0 °C to rt; (b) TFAA
(3 eq), 2,6-lutidine (6 eq), DME, 0 °C to rt, 88%; (v) 1 M nBu4NF in THF
(1.5 eq), THF, rt, 94%; (vi) Ac2O/py (1 : 2), CH2Cl2, rt, 93%; (vii) 4 M HCl
in dioxane (8 eq); (viii) aq. NH3 (35%), MeOH, rt, 100%; (ix) Lawesson’s
reagent (0.5 eq), CH2Cl2, rt, 89%; (x) (a) ethyl bromopyruvate (3.5 eq),
KHCO3 (4 eq), DME, 215 °C; (b) TFAA (3 eq), 2,6-lutidine (6 eq), DME,
230 °C, 94%; (xi) aq. NH3 (35%), MeOH, rt, 98%; (xii) Et3O+PF62 (1.15
eq), CH2Cl2, rt, 100%; (xiii) (a) CH2Cl2, rt; (b) DBU (2 eq), CHCl3, 63%
over 3 steps from 17.
Scheme 3 Synthesis of bis-thiazole fragment 12. Reagents and conditions:
(i) 1 M aq. LiOH, MeOH, rt, 100%; (ii) BTEAC (1 eq), BuBr (48 eq),
t
K2CO3 (24 eq), THF, reflux, 92%; (iii) 4 M HCl in dioxane (8 eq), rt, 1 h,
88%; (iv) 2, EDCI (1 eq), HOBt (1 eq), DMF, 0 °C, 1 h, rt, overnight, 48%;
(v) 4 M HCl in dioxane (8 eq), 88%. BTEAC = benzyltriethylammonium
chloride; EDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride; HOBt = 1-hydroxybenzotriazole.
C h e m . C o m m u n . , 2 0 0 4 , 9 4 6 – 9 4 8
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Scheme 6 Synthesis of amythiamicin D 1. Reagents and conditions: (i) toluene, 120 °C, microwave (CEM Discover™ Focused Synthesizer), 150 W, 12 h,
33%; (ii) TFA, CHCl3, rt; (iii) Boc-Gly-OH 4 (1.5 eq), PyBOP (1.3 eq), iPr2NEt (4 eq), CH2Cl2, rt, 95% over 2 steps; (iv) H2 (1 atm), Pd black, MeOH, rt;
(v) 12, PyBOP (1.3 eq), iPr2NEt (10 eq), DMF, 60% over 2 steps; (vi) TFA, CHCl3, rt; (vii) DPPA (2 eq), iPr2NEt (10 eq), DMF (1 mM), 0 °C, 73% over
2 steps. PyBOP = benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate; DPPA = diphenylphosphoryl azide.
Bycroft–Gowland structure is correct, and the only remaining issue is
the stereochemistry at three centers: B. Fenet, F. Pierre, E. Cundliffe and
M. A. Ciufolini, Tetrahedron Lett., 2002, 43, 2367. In view of these
results, there must be some doubt about the earlier reported synthesis of
micrococcin P1 (refs. 11 and 12).
of the terminal N-Boc-group on the bis-thiazole moiety 5 was
followed by a PyBOP-mediated coupling to N-Boc-glycine 4 to
give the complete right-hand fragment of amythiamicin D 22
(Scheme 6). After much experimentation, it was found that the
benzyl ester in compound 22 could be removed by hydrogenolysis
over palladium black to give the corresponding carboxylic acid. A
second PyBOP-mediated reaction successfully coupled the left-
hand bis(thiazole) 12 to provide the cyclization precursor 23. The
terminal N-Boc and tert-butyl ester groups in 23 were simultane-
ously cleaved using TFA, and the resulting amino acid treated with
DPPA and Hünig’s base in DMF. This resulted in macrolactamiza-
tion in a yield of 73% (from 23) to give amythiamicin D 1.
Our synthetic material had properties consistent with those
reported for the natural product,5,† suggesting that our original
assumptions about the stereochemistry of the three chiral centers
was correct. Subsequent correspondence with the original authors
revealed that unpublished X-ray crystallographic data substantiate
the (10S,19S,29S)-stereochemistry thereby providing final con-
firmation that we had completed the first synthesis of the natural
product amythiamicin D, and paving the way for syntheses of other
thiopeptide natural products.
11 K. Okumura, A. Ito, D. Yoshioka and C.-G. Shin, Heterocycles, 1998,
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14 T. R. Kelly and F. Lang, J. Org. Chem., 1996, 61, 4623.
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21 Very recently, an alternative synthesis of the amythiamicin pyridine
core has been reported: M. C. Bagley, J. W. Dale, R. L. Jenkins and J.
Bower, Chem. Commun., 2004, 102.
22 P. Tavecchia, P. Gentili, M. Kurz, C. Sottani, R. Bonfichi, E. Selva, S.
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We thank the EPSRC and AstraZeneca for financial support, Dr
I. Prokesˆ for NMR spectroscopy, the EPSRC Mass Spectrometry
Centre at Swansea for mass spectra, and Drs Yoshikazu Takahashi
and Yuzuru Akamatsu (Microbial Chemistry Research Foundation)
for helpful exchange of information.
Notes and references
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10 The structure of micrococcin P1 is in doubt since the synthetic material
is different from the natural: (a) M. A. Ciufolini and Y.-C. Shen, Org.
Lett., 1999, 1, 1843; (b) recent studies have confirmed that the overall
27 Nicolaou has also reported a biomimetic Diels–Alder approach to the
tetrahydropyridine ring of thiostrepton: K. C. Nicolaou, M. Nevalainen,
B. S. Safina, M. Zak and S. Bulat, Angew. Chem., Int. Ed., 2002, 41,
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28 C. J. Moody and M. C. Bagley, J. Chem. Soc., Perkin Trans. 1, 1998,
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29 T. D. Gordon, J. Singh, P. E. Hansen and B. A. Morgan, Tetrahedron
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