Total synthesis of the myxobacterial macrolide ripostatin b?

Article abstract of DOI:10.2533/chimia.2013.227

This article describes the total synthesis of ripostatin B, which is a 14-membered macrolide of myxobacterial origin that inhibits E. coli RNA polymerase by a different mechanism of action than the first-line anti-tuberculosis drug rifampicin. Structurally, ripostatin B features a labile and synthetically challenging doubly skipped triene motif embedded in the macrolactone ring. Key steps in the synthesis were a Paterson aldol reaction, a low-temperature Yamaguchi esterification and an alkene metathesis reaction to close the macrolide ring. The natural product was synthesized in a longest linear sequence of 21 steps and 3.6% overall yield. Schweizerische Chemische Gesellschaft.

Full text of DOI:10.2533/chimia.2013.227

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 227
doi:10.2533/chimia.2013.227
Chimia 67 (2013) 227–230 © Schweizerische Chemische Gesellschaft
Total Synthesis of the Myxobacterial
Macrolide Ripostatin B‡
Florian Glaus§ and Karl-Heinz Altmann*
§SCS-Metrohm Foundation Award for best oral presentation
Abstract: This article describes the total synthesis of ripostatin B, which is a 14-membered macrolide of
myxobacterial origin that inhibits E. coli RNA polymerase by a different mechanism of action than the first-line
anti-tuberculosis drug rifampicin. Structurally, ripostatin B features a labile and synthetically challenging doubly
skipped triene motif embedded in the macrolactone ring. Key steps in the synthesis were a Paterson aldol
reaction, a low-temperature Yamaguchi esterification and an alkene metathesis reaction to close the macrolide
ring. The natural product was synthesized in a longest linear sequence of 21 steps and 3.6% overall yield.
Keywords: Antibiotics · Ring-closing metathesis · Ripostatin · RNA polymerase · Total synthesis
Ripostatins A and B (1) (Fig. 1) were
pound binds to the hinge that mediates
opening and closing of the RNAP clamp,
thus trapping the enzyme in a closed con-
formation and preventing access of the
first isolated in 1995 by Reichenbach,
Höfle and co-workers from the myxobac-
terium Sorangium cellulosum (strain So ce
377) found in a soil sample from Kenya.[1]
Both compounds are 14-membered macro-
lides that differ only in the oxidation state
at C(15) (ripostatin A exists as a hemiac-
O
O
O
dsDNA to the active-site cleft.[4,11] This
O
OH
OH
binding mode is fundamentally different
from that of the rifamycin-type inhibitors,
which prevent the extension of RNA be-
Ripostatin A(hemiacetal form)
etal/ketone mixture in a 4:3 ratio). The two
yond a length of 2–3 nucleotides by bind-
ing to a site close to the active-site cleft.
Based on the location of RNAP mutations
in myxopyronin- and ripostatin A-resistant
E. coli, Ebright and co-workers have sug-
gested that ripostatin A (as well as a num-
ber of other natural products) likewise
inhibit(s) bacterial RNAP by binding to
ripostatins exhibit a similar, albeit narrow
spectrum of antibiotic activity against
gram-positive bacteria, with minimum in-
hibitory concentrations (MICs) of less than
1 µg/mL only against S. aureus and the hy-
perpermeable E. coli strain DH21tolC.[1a]
Additionally, ripostatins A[1a] and B[2] both
inhibit the DNA-dependent RNA poly-
merase (RNAP) from E. coli with sub-µM
activity, while being inactive against eu-
karyotic RNA polymerase II.[1a]
H
O
O
O
O
OH
OH
the hinge region of the enzyme.
Ripostatin B(1)
The attractive biological profile of
the ripostatins as well as their interesting
chemical structure (notably the C(2)–C(9)
Fig. 1. Ripostatins A and B.
RNA polymerase is a suitable target for
antibacterial therapy.[3] It is an essential en-
zyme (permitting efficacy) and it is highly
conserved across gram-positive and -nega-
tive bacteria (permitting broad-spectrum
activity), while it differs significantly from
doubly skipped triene motif) prompted
us to embark on the total synthesis of ri-
mammalian RNA polymerases I, II and
III (providing therapeutic selectivity).[4–6]
RNAP is also a clinically validated target,
sinceitisthetargetoftherifamycin-classof
antibiotics(themostprominentmemberbe-
postatin B[12] with the goal to provide a
chemical basis for future SAR studies. No
total synthesis of either ripostatin had been
published prior to our initial communica-
ing rifampicin).[7,8] The rifamycins are clin-
tion. However, concurrent with our own
work total syntheses of ripostatin B were
independently developed by Christmann
ically used for the treatment of both gram-
positive and gram-negative bacteria.[9]
and co-workers, and Prusov and Tang.[13a,b]
They have proven particularly effective in
In mid-2012, Prusov and Tang also pub-
the treatment of tuberculosis, where they
are used as first-line drugs.[6,7] However,
the number of bacterial strains resis-
tant to the rifamycins is increasing.[7,10]
Consequently, there is an urgent need for
new RNAP-inhibitors that exhibit a differ-
ent mode of action.
Ebright and co-workers have recently
reported an X-ray crystal structure of a
complex between T. thermophilus RNAP
and myxopyronin, another myxobacterial
lished a total synthesis of ripostatin A.[13c]
Due to the anticipated tendency of
the skipped triene-motif (C(2)–C(9)) for
double-bond migration, we based our ret-
rosynthetic analysis on a ring-closing me-
tathesis (RCM), which, due to the absence
of base in the ring-closing step, should
minimize problems with the double-bond
framework (Scheme 1).[14] After RCM, the
primary hydroxyl group would be depro-
*Correspondence: Prof. Dr. K.-H. Altmann
Swiss Federal Institute of Technology (ETH) Zürich
Institute of Pharmaceutical Sciences
HCI H 405, Wolfgang-Pauli-Strasse 10
CH-8093 Zürich
Tel.: +41 44 633 73 90
E-mail: karl-heinz.altmann@pharma.ethz.ch
‡An original report on this work has been published
previously: F. Glaus, K.-H. Altmann, Angew. Chem.
Int. Ed. 2012, 51, 3405.
RNA polymerase inhibitor.[4] The com-
tected selectively and oxidized, thereby

228 CHIMIA 2013, 67, Nr. 4
Laureates: awards and Honors, sCs FaLL Meeting 2012
installing the vinylogous malonate-type
metathesis
C(1)-C(3)-C(28) framework. The RCM-
precursor 2 could be accessible by Stille-
1
1
coupling between an allylstannane and
vinyl iodide 3. The Stille reaction was spe-
5
H
O
O
O
TB
S
O
O
stille
OTBS
3
1
O
OH
28
O
cifically chosen because it is usually per-
formed at neutral pH, which would mini-
mize double-bond migration in the dienoic
ester. Vinyl iodide 3 should be accessible
by esterification between alcohol 4 and io-
doacrylic acid 5.[15] In principle, perform-
ing the Stille coupling with acid 5 (or a
suitably protected derivative thereof) prior
to esterification should lead to a more con-
vergent approach toward the natural prod-
1
5
O
H
OTBS
Ripostatin B(1)
2
anti 1,3-
reduction
TB
S
O
O
I
TB
aldol
SO
O
H
I
O
OTBS
O
uct. This approach, however, had failed in
H
O
OTBS
OTB
S
O
the hands of Kirschning and co-workers,
due to the pronounced tendency for dou-
ble-bond migration in the dienoic acid un-
der a variety of basic esterification condi-
tions (under neutral or acidic conditions,
yields were generally unsatisfactory).[16]
We intended to set the C(13)- and C(15)-
stereocenters by an aldol reaction between
ketone 6 and aldehyde 7 (stereocenter at
C(13)), followed by an anti 1,3-reduc-
tion (stereocenter at C(15)). Compound 7
would in turn be accessible from epoxide
8[17] by epoxide opening and subsequent
elaboration of the diene moiety.
esterification
5
3
4
O
O
+
PMBO
TBSO
6
7
8
O
Scheme 1. Retrosynthetic analysis of ripostatin B. PMB = para-methoxybenzyl,TBS = tert-butyldi-
methylsilyl.
MeMgBr,
1) Mg,PhCH2CHO
O
O
C
H3NHOCH3·HCl
Br
The synthesis of ketone 6 started
OEt
2) CH3CH2COOH,
78%
with the addition of the Grignard reagent
derived from 2-bromopropene to phen-
CH3C(OEt) ,
3
9
6
49%
ylacetaldehyde (Scheme 2). The result-
ing secondary alcohol was submitted to
a Johnson-Claisen rearrangement with
triethyl orthoacetate in the presence of a
catalytic amount of propionic acid at 145
°C, giving γ,δ-unsaturated ester 9[18] in
49% yield for the two-step sequence. By
using Williams’ procedure, which involves
treatment of the ester with an excess of
the Grignard-reagent in presence of N,O-
dimethylhydroxylamine hydrochloride, 9
could be transformed into methyl ketone
Scheme 2. Synthesis of ketone 6.
Scheme 3. Synthesis
of acid 5. Red-Al® =
sodium bis(2-
methoxyethoxy)alu-
minum dihydride.
1) TBSCl,
OH
Red-Al®,
thenI
imidazole,92%
OH
I
2
OTBS
2) nBuLi,
(CH2O) ,93%
74%
n
10
1) MnO
O
I
2
HO
OTBS
2) Pinnickox.
HO
OTBS
6 in one pot.[19]
[15]
11
5
The known carboxylic acid 5
was
57%(twosteps)
synthesized from 3-butyn-1-ol, which
was TBS-protected and homologated with
sulting primary alcohol 14 was converted
into the corresponding allylic bromide us-
paraformaldehyde in 86% yield (two steps, by treatment of the diol with base (NaH)
Scheme 3). The triple bond was then re-
and PMBBr (Scheme 4). PMB-protected
duced stereoselectively with Red-Al® and epoxy alcohol 8 was obtained in 61%
the intermediate aluminate was quenched yield for the three-step sequence. Epoxide
with iodine to give vinyl iodide 11 in 74% opening with the anion derived from ethyl
ing Appel’s conditions (CBr , PPh3) in the
presence of 2,6-lutidine;[22]4this bromide
was then converted into 1,4-diene 15 by
Stille reaction with Bu SnCH=CH using
yield. A two-step oxidation involving propiolate followed by TBS-protection of
3
Pd2(dba)3 (12 mol%) as palladium2source
MnO -mediated conversion of the allylic the resulting secondary alcohol then gave
alcoh2ol into the aldehyde, followed by a TBS-ether 12 in 83% yield (two steps).
and 0.5 equiv. AsPh .[23] PMB-removal
from 15 then turned3 out to be rather
Pinnick reaction afforded then iodoacrylic The trisubstituted E-configured double
challenging. Deprotection could not be
achieved under standard DDQ conditions,
which might be attributed to the presence
of the 1,4-diene moiety.[24] After the inves-
tigation of a range of methods, we finally
acid 5 in 57% yield from 11.
bond was installed next by treatment of 12
with thiophenol and a catalytic amount of
The synthesis of aldehyde 7 departed
from d-(–)-aspartic acid; this was convert- sodium methoxide,[21] to provide thioether
ed into epoxide 8 via a known three-step 13 (85%), followed by displacement of the
sequence,[17,20] which involved displace- phenylsulfenyl moiety by a methyl group
ment of the amino group by bromide un- with full retention of configuration by
der retention of configuration, reduction treatment with dimethylcuprate at −78 °C
of both carboxylic acid groups to the al- and warming to −30 °C over 30–40 min-
cohol stage, and finally epoxide formation utes.[21] After DIBAL-H reduction, the re-
found that TMSI[25]
cleaves the PMB-ether
very efficiently, leading, after cleavage of
the ensuing TMS-ether with K CO3 in
methanol, to the desired primary2 alcohol

Laureates: awards and Honors, sCs FaLL Meeting 2012
CHIMIA 2013, 67, Nr. 4 229
1) NaNO2,KBr
H2SO4
1) ethylpropiolate
nBuLi, 86%
O
O
cat. SmI
2
H
O
O
O
H
(+)-Ipc B
2 Cl, NEt3, 7
TBSO
PMBO
2) BH3·THF
3) NaH, then PMBBr
61% (three steps)
2) T OTf,
BS
O
NH2
CH3CH2C
HO
8
2,6-lutidine,96%
OH
d.r.> 0:1, 62%
1
d.r.> 0:1, 93%
2
6
O
4
PhSH,
cat. NaOMe
O
T
B
S
O PhS
O
OT
B
S
1) TBSCl, imidazole,
94%
OEt
TBSO
P
MB
O
OEt
85%
TBSO
PMBO
2) DIBAL-H, 88%
O
O
OH
12
13
1) CBr4,PPh ,
OH
OTBS
3
1
6
17
2,6-lutidine,85%
OH
2) cat. Pd (dba) ,AsPh
1) MeMgBr,CuI, 95%
2) DIBAL-H, 87%
TBSO
P
MB
O
2
3
3
14
Bu SnCH=CH2,79%
3
5,2,4,6-trichloro
benzoylchloride,
MAP,tolu e, -78 °C to -40°C
D
en
T
B
S
O
O
I
1) TMSI, then
K2CO3,92%
TBSO
O
OTBS
TB
SO
80%
OTBS
P
MB
O
O
2) Swern, 98%
3
15
7
Scheme 4. Synthesis of aldehyde 7. dba = dibenzylideneacetone,
Scheme 5. Assembly of 5, 6 and 7. DMAP = 4-dimethylaminopyridine,
DIBAL-H = diisobutylaluminium hydride, DMF = N,N-dimethylformamide, (+)-Ipc2BCl = (+)-diisopinocamphenyl chloroborane.
DMSO = dimethylsulfoxide, nBuLi = n-butyllithium, Tf = trifluoro-
methanesulfonyl, TMS = trimethylsilyl.
in 92% yield. This alcohol was then oxi-
upon work-up (saturated aqueous Na2S2O3/
NaHCO3), thus leading to low and erratic
yields in a subsequent Pinnick-oxidation.
Fortunately, a one-pot procedure, involv-
tion of unidentified side products in the
metathesis reaction, Grubbs I catalyst in
dichloromethane afforded a much cleaner
reaction, which furnished 77% of the mac-
rocycle as a single isomer (Scheme 6).
NMR analysis revealed the newly formed
double bond to be E-configured. It proved
important to terminate the reaction by ad-
dition of DMSO[29] before full conversion
was reached, otherwise side products ap-
dized under standard Swern conditions to
aldehyde 7 in 98% yield.
With all three building blocks in hand,
the first step in their assembly was the
Paterson-aldol reaction[26] between alde-
ing DMP-mediated oxidation in THF
followed by Pinnick oxidation gave the
hyde 7 and ketone 6 (Scheme 5). The re-
desired carboxylic acid in 81% yield.[30]
action proceeded in moderate yield (62%)
but gave the desired aldol product 4 with
reasonable stereoselectivity (d.r. >10:1).
Finally, removal of the two remaining TBS
protecting groups with 5% aqueous HF in
acetonitrile afforded ripostatin B in 88%
The C(15) stereocenter was then set by
yield.
peared (TLC analysis) and cyclization
a samarium-mediated 1,3-anti reduction
In summary, an efficient and modu-
lar total synthesis of ripostatin B (1) is
yields were lower.
(Evans-Tishchenko reduction[27]
) that fur-
After cleavage of the primary TBS-
described. The longest linear sequence
included 21 steps and provided the target
molecule in 3.6% overall yield. Key steps
were a Paterson aldol reaction, a modified
nished 16 in high yield (93%) and with ex-
ether using pyridine buffered HF·pyridine,
the free hydroxyl group needed to be oxi-
dized to the carboxylic acid state, thereby
cellent stereoselectivity (d.r. >20:1). The
C(15) hydroxyl group was next protected
with TBS before the propionate ester was
installing the potentially labile vinylo-
gous malonate-type system. Attempts at
the direct oxidation of alcohol 18 to the
corresponding carboxylic acid met with
complete failure. Oxidation of 18 with
Dess-Martin periodinane gave an alde-
hyde that was found to be stable in the
reaction medium, but partly decomposed
reductively removed with DIBAL-H giv-
ing 17 in 83% yield for the two-step se-
quence.
Yamaguchi-type esterification and a ring-
closing metathesis reaction. This strategy
enabled the incorporation of the sensitive
doubly skipped triene motif. The synthesis
of analogs and their biological evaluation
is currently ongoing in our laboratories.
A range of different esterification pro-
tocols was then examined for the conden-
sation between 17 and iodoacrylic acid
5, including the standard Yamaguchi[28]
protocol, none of which provided reason-
able amounts of ester 3; in general, the
major part of alcohol 17 was re-isolated
unchanged. Best results were finally ob-
tained using a modified Yamaguchi pro-
tocol that involved mixing of all com-
ponents in toluene at −78 °C, before the
reaction was allowed to warm to −40 °C
over 2–3 h (Scheme 5). Following this
approach 3 was obtained in 80% yield if
two equivalents of iodoacrylic acid 5 were
employed. Stille coupling between 3 and
Bu3SnCH2CH=CH2 then afforded diene 2
(Scheme 6),[23] thus setting the stage for the
key ring-closing metathesis reaction.
While the Grubbs II and Hoveyda-
Grubbs II catalysts both led to the forma-
Bu SnCH2CH=C 2
H
3
Pd (dba)3 (15mol%), AsPh (50mol%)
T
B
S
O
O
I
T
B
S
O
O
2
3
O
OT
B
S
O
OTBS
86%
OT
B
S
OTBS
3
2
1) Grubbs-I, 77%
1) D
MP, thenPinnick, 81%
T
B
S
O
O
HO
O
O
2) HF·pyr,
pyridine, 8
2) 5% aq.HFinacetonitrile,8
8%
1
%
O
OH
O
OH
OT
B
S
OH
18
Ripostatin B(1)
Scheme 6. Endgame toward ripostatin B. DMP = Dess–Martin periodinane, Grubbs I =
bis(tricyclohexylphosphine)benzylidene ruthenium(iv)dichloride.

230 CHIMIA 2013, 67, Nr. 4
Laureates: awards and Honors, sCs FaLL Meeting 2012
[10] a)A. Koul, E.Arnoult, N. Lounis, J. Guillemont, [18] A. E. Guthrie, J. E. Semple, M. M. Joullié, J.
Received: February 25, 2013
K. Andries, Nature 2011, 469, 483; b) C. E.
Org. Chem. 1982, 47, 2369.
Barry III, Curr. Top. Med. Chem. 2011, 11, [19] J. M. Williams, R. B. Jobson, N. Yasuda, G.
[1] a) H. Irschik, H. Augustiniak, K. Gerth, G.
Höfle, H. Reichenbach, J. Antibiot. 1995,
48, 787; b) H. Augustiniak, H. Irschik, H.
Reichenbach, G. Höfle, Liebigs Ann. 1996,
1657.
1216.
Marchesini, U.-H. Dolling, E. J. J. Grabowski,
[11] A. Chakraborty, D. Wang, Y. W. Ebright, Y.
Tetrahedron Lett. 1995, 36, 5461.
Korlann, E. Kortkhonjia, T. Kim, S. Chowdhury,
S. Wigneshweraraj, H. Irschik, R. Jansen, B. Abramson, H. Rapoport, Synthesis 1992, 621.
T. Nixon, J. Knight, S. Weiss, R. H. Ebright, [21] T. Murata, M. Sano, H. Takamura, I. Kadota, D.
Science 2012, 337, 591. Uemura, J. Org. Chem. 2009, 74, 4797.
[12] Ripostatin B was chosen due to its higher [22] D. Enders, J. L. Vicario, A. Job, M. Wolberg, M.
chemical stability compared to ripostatin A. Müller, Chem. Eur. J. 2002, 8, 4272.
The isolation group (see ref. [1b]) reported [23] V. Farina, B. Krishnan, J. Am. Chem. Soc. 1991,
[20] J. A. Frick, J. B. Klassen, A. Bathe, J. M.
[2] Unpublished data.
[3] S. A. Darst, Trends Biochem. Sci. 2004, 29, 159.
[4] J. Mukhopadhyay, K. Das, S. Ismail, D.
Koppstein, M. Jang, B. Hudson, S. Sarafianos,
S. Tuske, J. Patel, R. Jansen, H. Irschik, E.
eliminative lactone opening for ripostatin A 113, 9585.
even in a slightly basic environment (methanolic [24] See T. W. Greene, P. B. Wuts, ‘Protective
Arnold, R. H. Ebright, Cell 2008, 135, 295.
[5] A. Srivastava, M. Talaue, S. Liu, D. Degen,
buffer solution at pH 8), whereas ripostatin B
was found to be stable up to pH 11.
[13] a) P. Winter, W. Hiller, M. Christmann, Angew.
Groups in Organic Synthesis’, John Wiley
& Sons, Hoboken, New Jersey, 2007; and
references therein.
R. Y. Ebright, E. Sineva, A. Chakraborty, S. Y.
Druzhinin, S. Chatterjee, J. Mukhopadhyay, Y.
W. Ebright, A. Zozula, J. Shen, S. Sengupta, R.
Chem. Int. Ed. 2012, 51, 3396; b) W. Tang, E. V.
Prusov, Angew. Chem. Int. Ed. 2012, 51, 3401;
[25] I. Kadota,Y. Yamagami, N. Fujita, H. Takamura,
Tetrahedron Lett. 2009, 50, 4552.
c) W. Tang, E. V. Prusov, Org. Lett. 2012, 14, [26] I. Paterson, J. M. Goodman, Tetrahedron Lett.
4690. 1989, 30, 997.
[14] For a recent review on the metathesis reaction, [27] D. A. Evans, A. H. Hoveyda, J. Am. Chem. Soc.
R. Niedfeldt, C. Xin, T. Kaneko, H. Irschik, R.
Jansen, S. Donadio, N. Connell, R. H. Ebright,
Curr. Opin. Microbiol. 2011, 14, 532.
[6] E. A. Campbell, N. Korzheva, A. Mustaev, K.
Murakami, S. Nair, A. Goldfarb, S. A. Darst,
Cell 2001, 104, 901.
see: A. H. Hoveyda, A. R. Zhugralin, Nature
2007, 450, 243.
[15] M. Kanematsu, M. Shindo, M. Yoshida, K.
Shishido, Synthesis 2009, 17, 2893.
1990, 112, 6447 .
[28] J. Inanaga, K. Hirata, H. Saeki, T. Katsuki, M.
[7] P. Sensi, Ref. Inf. Dis., 1983, 5 (Suppl. 3), 402.
[8] Another RNA polymerase inhibitor, the
natural product lipiarmycin (fidaxomicin), was
approved by the FDA and the EMEA in 2011
for the treatment of C. difficile infections.
[9] A. Bryskier, in ‘Antimicrobial Agents:
Antibacterials and Antifungals’, Ed. A.
Bryskier, ASM Press, 2005, p. 922–923.
Yamaguchi, Bull. Chem. Soc. Jpn. 1979, 52,
1989.
[16] a) C. Kujat, M. Bock, A. Kirschning, Synlett [29] Y. M. Ahn, K. Yang, G. I. Georg, Org. Lett.
2006, 419; b) C. Kujat, Dissertation, Wilhelm
Leibniz Universität Hannover, 2007.
2001, 3, 1411.
[30] For another example of
a one-pot DMP/
[17] C. W. Wullschleger, J. Gertsch, K.-H. Altmann,
Org. Lett. 2010, 12, 1120.
Pinnick double oxidation, see: A. Yajima, M.
Yamamoto, T. Nukada, G. Yabuta, Biosci.
Biotechnol. Biochem. 2007, 71, 2720.