Practical Gram-Scale Synthesis of Iboxamycin, a Potent Antibiotic Candidate

Article abstract of DOI:10.1021/jacs.1c03529

A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is presented. A pivotal transformation in the route involves an intramolecular hydrosilylation-oxidation sequence to set the ring-fusion stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective alkylation of a pseudoephenamine amide, a convergent sp3-sp2 Negishi coupling, and a one-pot transacetalization-reduction reaction to form the target compound's oxepane ring. Implementation of this synthetic strategy has provided ample quantities of iboxamycin to allow for its in vivo profiling in murine models of infection.

Full text of DOI:10.1021/jacs.1c03529

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Article
Practical Gram-Scale Synthesis of Iboxamycin, a Potent Antibiotic
Candidate
Jeremy D. Mason, Daniel W. Terwilliger, Aditya R. Pote, and Andrew G. Myers*
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ABSTRACT: A gram-scale synthesis of iboxamycin, an antibiotic candidate bearing a fused bicyclic amino acid residue, is
presented. A pivotal transformation in the route involves an intramolecular hydrosilylation−oxidation sequence to set the ring-fusion
stereocenters of the bicyclic scaffold. Other notable features of the synthesis include a high-yielding, highly diastereoselective
alkylation of a pseudoephenamine amide, a convergent sp3−sp2 Negishi coupling, and a one-pot transacetalization−reduction
reaction to form the target compound’s oxepane ring. Implementation of this synthetic strategy has provided ample quantities of
iboxamycin to allow for its in vivo profiling in murine models of infection.
INTRODUCTION AND BACKGROUND
■
The lincosamide antibiotics are orally available, nontoxic,
bacteriostatic agents that act to inhibit translation by binding
to the 23S subunit of the bacterial ribosome, near the peptidyl
transferase center.1 The founding member of the class,
lincomycin (1, Figure 1), was isolated in 1963 by scientists
at The Upjohn Company from a culture of the soil bacterium
Streptomyces lincolnensis.2 Lincomycin quickly gained FDA
approval (1964) for the treatment of infections caused by
Gram-positive pathogens. The semisynthetic derivative clinda-
mycin (2), prepared by selective, stereoinvertive deoxy-
chlorination of lincomycin at C7, showed improved in vitro
and in vivo activity relative to lincomycin and was approved for
use in humans in 1970.3 In addition to its antibacterial
properties, clindamycin can also be used for the treatment of P.
falciparum malaria.4 Clindamycin is commonly used to treat
dental infections5 and osteomyelitis,6 and in topical form as an
Figure 1. Structures and key characteristics of lincomycin (1),
clindamycin (2), and the novel bicyclic analog iboxamycin (3), whose
antiacne agent,7 but its widespread oral and parenteral
gram-scale synthesis is outlined herein.
administration (the latter in the form of the 2-phosphate
ester prodrug) is contraindicated due to the risk of promotion
of secondary Clostridioides difficile infections.8 In addition, as
with most antibiotics in use for decades, multiple bacterial
resistance genes have evolved and are now widespread in
bacteria within the community.9 In spite of much effort, no
new members of the lincosamide class have advanced to the
clinic in the past five decades, which has possibly promoted a
Received: April 2, 2021
Published: July 15, 2021
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self-sustaining perception that the class has little to offer in
terms of future antibiotics.10
Scheme 1. Retrosynthetic Analysis of the N-Protected
Amino Acid Component 4
Over a period of more than 5 years, our laboratory has been
involved in the continuous development of new methodology
for the chemical synthesis of novel lincosamides in a search for
next-generation antibiotic candidates that (1) are effective
against current multidrug resistant clinical isolates and (2)
have the potential to overcome the C. difficile liability of
clindamycin. In his Ph.D. research, Dr. Matthew Mitcheltree
pioneered new chemistry permitting fully synthetic construc-
tion of novel lincosamides with modified aminooctose subunits
and initiated the discovery and construction of novel bicyclic
scaffolds to replace the 4′-n-propyl hygric acid residue of
lincomycin and clindamycin.11 The Ph.D. thesis research of
Drs. Katherine Silvestre and Ioana Moga permitted an
extensive investigation of scaffold substituents, resulting in a
large expansion of our synthetic lincosamide-based library
(currently >500 members), with >80% of the analogs having
demonstrable antibacterial activity.12 Dr. Katherine Silvestre
discovered the molecule which bears the name iboxamycin (3)
whose remarkable antibacterial effects, ribosomal-binding
studies, and efficacy in murine infection models have been
detailed elsewhere.13 Relative to clindamycin (2), iboxamycin
(3) displays an expanded spectrum of activity, encompassing
Gram-negative species, enterococcal clinical isolates, and
strains expressing Erm- and Cfr-ribosome methyltransferases
that together confer resistance to canonical lincosamides,
macrolides, phenicols, oxazolidinones, pleuromutilins, and
streptogramins. These results, together with the compound’s
favorable performance in in vitro safety profiling and mouse
pharmacokinetic studies, encouraged us to evaluate iboxamycin
(3) in mouse models of infection, necessitating the preparation
of the substance on gram scale.
The original synthetic route to iboxamycin (3) provided
quantities of material that were sufficient for extensive
minimum inhibitory concentration assays against various
bacterial strains (∼50 mg, isolated as its formic acid salt after
purification by RP-HPLC), but the sequence was lengthy and
laborious as it had been designed as a discovery engine, not for
efficient synthesis of iboxamycin (3) specifically. Considerable
efforts were made to improve and substantially scale the
discovery route to provide the greater quantities of iboxamycin
needed to explore its efficacy in murine infection models.
However, this work ultimately only reinforced the sense that an
entirely new and different sequence would have to be
developed to produce iboxamycin. Here, we present that
route, its development and refinement, and implementation to
produce >1 g of iboxamycin (3). Should it be warranted, we
believe the route presented could provide the basis of a
manufacturing route.
in one operation. Disconnection of 6 by targeting the alkene
appendage using a Negishi coupling14 transform gave rise to
the known vinyl triflate 715 and the elaborated organozinc
reagent 8 as starting materials. The latter was envisioned to
arise from a straightforward sequence initiated in the synthetic
direction by an asymmetric alkylation reaction of (R,R)-
pseudoephenamide 10 toward α-substituted amide 9, as
depicted.16
Implementation of an Asymmetric Alkylation Proto-
col to Construct the Direct Precursor to the Elaborated
Organozinc Reagent 8. By modifying literature protocols,
we were able to prepare the carboxylic acid 12 in multideca-
gram quantities (Scheme 2).17 Specifically, we learned that
Scheme 2. Synthesis of Alkyl Iodide 16
RESULTS AND DISCUSSION
■
Retrosynthetic Analysis. Simplification of the novel
bicyclic amino acid component of iboxamycin (in N-Cbz
protected form, subtarget 4) was reimagined along the lines
presented in the retrosynthetic analysis shown in Scheme 1.
Our initial disconnection targeted the oxepane ring, which we
proposed might be constructed in a reductive cyclization
reaction of the dihydroxy acetal 5. This, in turn, was
considered to arise from an anti-Markovnikov, suprafacial
hydration of the alkene 6, which we anticipated would require
methodological development, but was considered strategic
because it would set the stereochemistry of positions 3′ and 4′
formation of the Grignard reagent from the commercially
available alkyl bromide 11 was best achieved by activation of
the magnesium metal turnings using 1 mol % of diisobutyla-
luminum hydride according to the procedure of Tilstam and
Weinmann.18 After sparging the solution of Grignard reagent
with carbon dioxide with care to control the exotherm, the
carboxylic acid 12 was obtained as an amorphous white solid
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after extractive isolation (35.7 g, 72%). Amide bond formation
between 12 and (R,R)-pseudoephenamine (13)19 by mixed
anhydride activation of 12 with pivaloyl chloride provided
pseudoephenamide 10 as white needles after extractive
isolation and recrystallization from 1:1 ethyl acetate−hexanes
(56.0 g, first crop, mp 111−112 °C, 8.0 g, second crop, 84%
total). An additional 3.3 g of product (4%, total yield 88%) was
obtained by flash chromatography of the residue obtained from
concentration of the mother liquors using acetone−hexanes as
eluent.
We found that both isobutyl iodide and isobutyl triflate were
not sufficiently reactive as substrates for high-yielding
alkylation of the lithium enolate of pseudoephenamide 10,
but methallyl bromide provided excellent results. Thus,
formation of the lithium enolate using the standard protocol
(LiCl, 6 equiv, LDA, 2.1 equiv, tetrahydrofuran (THF),
−78 °C → 0 °C → 23 °C → 0 °C) followed by addition of
methallyl bromide (2 equiv) at 0 °C provided the alkylation
product 14 as a colorless gum in 92% yield after extractive
isolation and purification by flash column chromatography.16
The product was shown to have a dr of >19:1 by using the
oxazolinium triflate method.20
contrast, when we employed freshly prepared 2% copper−zinc
couple26 in lieu of zinc metal, we found that organozinc
formation proceeded smoothly at 55 °C (benzene−N,N-
dimethylformamide (DMF) as solvent) and was easily
reproduced on scales of as much as 10 g (larger scales were
not attempted). Negishi coupling between the elaborated
organozinc reagent 8 (formed in situ using 10.0 g of 16) and
the vinyl triflate 7 (17.4 g, 1.1 equiv, prepared by slight
modifications of literature procedures;15 see Supporting
Information) in the presence of lithium chloride (1.5 equiv)
27
using [1,1′-bis(diphenylphosphino)ferrocene]-
dichloropalladium(II), complex with dichloromethane
(PdCl2(dppf)·CH2Cl2) as catalyst (0.02 equiv) in benzene−
DMF as solvent at 55 °C afforded the coupled product 17 as a
colorless oil in 57% yield (9.5 g) after extractive isolation and
purification by flash-column chromatography. In separate
chromatography fractions, we also recovered the vinyl triflate
7 (38%) and a dienyl dimerization product (18, 7% yield).
Desilylation of coupling product 17 with tetra-N-butylammo-
nium fluoride (TBAF) in THF at 23 °C gave rise to the key
homoallylic alcohol intermediate 6 as a light-yellow oil (97%
yield, 7.2 g).
Stereo- and Regioselective Hydration of Homoallylic
Alcohol 6. The next and, as it transpired, greatest challenge in
the synthetic route was to affect a suprafacial, anti-
Markovnikov hydration of the alkene function within
homoallylic alcohol 6 from its more hindered facethat
which bears the hydroxymethyl group appendage (Scheme 4).
In a single experiment, we also evaluated the alkylation
conditions recently reported by Collum and co-workers21
(sodium diisopropylamide as base at −78 °C rather than
LiCl−LDA at 0 °C) and found them to be effective as well as
convenient, providing 14 in 83% yield (287 mg) with >19:1 dr.
The isobutenyl side chain of amide 14 was hydrogenated (Pt/
C catalysis) in ethyl acetate as solvent to provide the saturated
product 9 in 95% yield (30.5 g) as a thick, colorless oil.
Reduction of 9 with lithium amidotrihydroborate22 provided
the primary alcohol 15 in 91% yield (12.5 g), and (R,R)-
pseudoephenamine (13) was recovered in 92% yield by a
simple extractive isolation. Using the Mosher ester method,23
we ascertained that the transformation of 9 to 15 had
proceeded without detectable epimerization (dr >19:1).
Exposure of alcohol 15 to a combination of iodine,
triphenylphosphine, and imidazole under the usual condi-
tions24 provided the desired primary iodide 16 in 92% yield
(18.0 g) as a colorless oil.
Scheme 4. An Attempt to Achieve a Directed,
Intramolecular Hydroboration Reaction
Formation of Elaborated Zinc Reagent 8 and Negishi
Coupling. In exploring methods to transform the primary
iodide 16 into the elaborated organozinc reagent 8, we found
that the use of chlorotrimethylsilane and/or 1,2-dibromo-
ethane as metallic zinc activators25 was not easily reproduced
on scales above a half-gram with this substrate (Scheme 3). In
The notion of using the homoallylic alcohol function to direct
the addition was obvious but challenging to implement.
Initially, we hoped that borane or another boron hydride
reagent might first engage the hydroxyl group, evolving
dihydrogen, and that the putative alkoxyboron hydride
intermediate 19 might then undergo intramolecular hydro-
boration of the adjacent alkene.28 We recognized that the
resulting [3.3.0]-bicyclooctane-type intermediate 20 would
likely suffer from a degree of strain, but we hoped that this
might be mitigated by proximity. In the event, it was not, for
only the diol arising from addition to the less hindered face of
the alkene (i.e., 21) was obtained after treatment with alkaline
peroxide. Addition of a solution of borane THF complex to
alcohol 6 led to instantaneous evolution of gas, certainly
dihydrogen, suggesting that the alkoxyboron hydride inter-
mediate 19 had likely formed, but intermolecular hydro-
boration of the less hindered face of the alkene was apparently
faster than the desired course of reaction.29 Attempts to induce
the desired transformation using equimolar quantities of
Scheme 3. Organozinc Formation and Negishi Coupling
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borane THF complex at elevated temperatures, as well as
exploration of a number of other boron hydride reagents
(thexylborane, chloroborane, to name two) were not
successful, and led to the recovery of starting material or to
the formation of the undesired diol 21 (see Supporting
Information). When exposed to the species arising from the
combination of borane dimethyl sulfide complex and
trifluoromethanesulfonic acid, 6 was observed to undergo
reductive opening of the acetal protective group.30 After much
additional experimentation not detailed here, we redirected our
focus to an alternative strategy to accomplish the desired
transformation.
We turned instead to the strategy of intramolecular
hydrosilylation followed by C−Si bond oxidation, pioneered
by Tamao and Ito.31 In an early experiment, we exposed
homoallylic alcohol 6 to an excess of 1,1,3,3,-tetramethyldisi-
lazane (4 equiv) in toluene at 23 °C, and concentration to
dryness under a vacuum afforded the crude dimethylsilyl ether
22 (Scheme 5). This intermediate was then treated directly
solvent with THF improved the yield and provided a process
that could be used to generate multigram quantities of 5.
Thus, the diphenylsilyl ether derived from alcohol 6 was
prepared by its reaction with commercially available chlor-
odiphenylsilane in ethyl ether with triethylamine as a base.
After removal of the resulting triethylammonium chloride
precipitate by filtration, the solution was thoroughly dried
under vacuum to provide the diphenylsilyl ether as a thick
colorless oil. In the optimized intramolecular hydrosilylation
process, a dilute (0.025 M) solution of the crude diphenylsilyl
ether in THF at reflux was treated with a catalytic amount of
Karstedt’s catalyst (ca. 0.01 equiv). After consumption of the
starting silyl ether (3.5 h), the crude mixture was cooled and
directly subjected to Tamao−Fleming oxidation conditions,
providing, the diol 5 (4.3 g, 58%) as a colorless oil that
solidified upon standing after purification by flash-column
chromatography.
Oxepane Formation and Completion of the Syn-
thesis. With multigram quantities of diol 5 in hand, we turned
to formation of the oxepane ring of 4 by a transacetalization−
reduction sequence. In an initial experiment, the diol 5 was
transformed into a 9:1 mixture of C9′-epimeric methoxy
oxepane acetals 24 (stereochemistry of the major isomer not
determined) upon exposure to methanol in the presence of p-
toluenesulfonic acid monohydrate (p-TSA·H2O) at reflux
(74% yield after isolation and purification by flash-column
chromatography) (Scheme 6). When a solution of this mixture
Scheme 5. Intramolecular Hydrosilylation−Oxidation
Sequence
Scheme 6. Synthesis of Oxepane 25 by Transacetalization
Followed by Reduction
with Karstedt’s catalyst (platinum-1,3-divinyl-1,1,3,3-tetrame-
thyldisiloxane complex, Pt2(dvtms)3) in toluene at 23 °C to
form the putative oxasilacycle 23, in addition to other
unidentified byproducts, and the crude mixture was subjected
to alkaline peroxide in the workup. After purification, we
obtained the desired diol 5 in 36% yield. We also recovered
39% of the homoallylic alcohol 6, which we believe arises from
a competing dimerization of the dimethylsilyl ether 22, which
hydrolyzes to 6 upon workup. Attempts to improve the yield of
this transformation by using different transition-metal catalysts
(e.g., [Rh(cod)Cl]2, Pt(PPh3)4, and a platinum−N-hetero-
cyclic carbene catalyst32) or by slow addition of the silyl ether
to a solution of the catalyst were not successful (see
Supporting Information).
We then explored modification of the substituents on the
silicon atom. Di-tert-butylsilyl, dimesitylsilyl, and di-ortho-
tolylsilyl ethers proved to be unreactive under a variety of
conditions, and the diisopropylsilyl ether proved similar to the
dimethylsilyl ether in terms of the final yield of 5. Among the
silyl ethers investigated, the diphenylsilyl ether was found to be
optimal. In addition, intermediates and byproducts of the
diphenylsilyl ether were found to be more stable than those of
the dimethylsilyl ether, permitting their isolation and character-
ization (see Supporting Information). After additional
experimentation, we found that replacement of toluene as
was treated with triethylsilane and boron trifluoride etherate at
0 °C and warmed to 23 °C, the desired oxepane 25 was
obtained in 50% yield. Isolated from separate chromatography
fractions was the methyl ether byproduct 26 (30%), which
must certainly arise from the alternative oxocarbenium ion
formation that leads to ring-opening.
By replacing methanol with 1,1,1,3,3,3-hexafluoroisopropa-
nol (HFIP) to form mixed acetal 27 (single diastereomer,
stereochemistry not determined), ring opening during the
reduction step was suppressed, and oxepane 25 was isolated in
51% yield by a parallel two-step process. The triol 28 was also
observed as a side product in this reaction, suggesting that
oxocarbenium capture by adventitious moisture may have been
an issue.
An improved, single-operation process was developed, one
in which the diol 5 was held at reflux in a mixed solvent of
dichloromethane and HFIP in the presence of p-TSA·H2O
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(1.7 equiv) and freshly activated 3 Å molecular sieves (MS)
(Scheme 7). Subsequent addition of triethylsilane (8 equiv)
Confirmation of all stereochemistry and structural details of
iboxamycin was revealed by single-crystal X-ray analysis
(CCDC # 1949345) of a sample obtained after two
consecutive recrystallizations from absolute ethanol.
We prepared several salts of iboxamycin (3) as well as a 2-
phosphate ester prodrug for galenic evaluation. The prepara-
tion and characterization of these derivatives is described in the
Supporting Information.
Scheme 7. Completion of the Synthesis
CONCLUSION
■
In summary, we developed a convergent, stereoselective, and
potentially scalable synthesis of iboxamycin (3), proceeding in
6.7% yield over 15 steps from the commercially available alkyl
bromide 11. Key steps include a Negishi coupling involving the
elaborated organozinc reagent 8 and vinyl triflate 7, a regio-
and stereoselective olefin hydration of homoallylic alcohol 6 by
an intramolecular hydrosilylation reaction followed by
carbon−silicon bond oxidation, and a single-operation
construction of the oxepane ring by transacetalization−
reduction of diol 5. The component-based nature of the
route (7, 8, 29) makes fragment substitution quite feasible, and
thus the route provides a means to prepare a large number of
additional analogs. The material furnished in these studies has
permitted extensive in vivo analysis of iboxamycin (3) in
murine infection models as well as other biological studies that
would have been infeasible otherwise.13
ASSOCIATED CONTENT
■
and a second portion of p-TSA·H2O (1.7 equiv) to the
refluxing mixture with brief further reflux gave rise to oxepane
25 in 76% yield (2.4 g), which was purified by extractive
workup and flash-column chromatography. The undesired triol
28 was isolated separately as a side product (8%).
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c03529.
Detailed experimental procedures, supplementary tables,
and all NMR characterization data (PDF)
Oxidation of the primary alcohol function of oxepane 25 to a
carboxyl group was achieved by a conventional two-step
sequence of Dess−Martin periodinane oxidation to form the
corresponding aldehyde, followed immediately by oxidation
with sodium chlorite.33 The key carboxylic acid subtarget 4
was thus obtained in 92% yield over the two-step sequence
(2.2 g). It should be noted that other oxidants were not
evaluated for the transformation of 25 to 4. Coupling of
carboxylic acid 4 with (7S)-7-deoxychloro-methylthiolincos-
amine (7-Cl-MTL, 29)34 was best achieved using 3-
(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
(DEPBT)35 as the coupling reagent and N,N-diisopropylethyl-
amine as base with DMF as solvent at 23 °C. While other
reagents such as HATU were also effective for this coupling,
DEPBT allowed for the most facile purification protocol. After
isolation by flash-column chromatography, the amide coupling
product 30 was obtained as a white powder in 82% yield
(2.8 g). No diastereomer resulting from epimerization at C2’
was observed after this coupling. To complete the synthesis of
iboxamycin (3), all that remained was to cleave the N-
benzyloxycarbonyl protective group. For this, we employed a
full equivalent of 10% palladium on carbon (catalyst poisoning
by the methylthio function was believed to be an issue) under
1 atm of dihydrogen in THF as solvent at 23 °C (5 h). Flash-
column chromatographic purification using 1% ammonium
hydroxide in the eluent afforded the pure, free-base form of
iboxamycin (3) in 81% yield (1.5 g) as a white powder.
Prior to biological evaluation in vivo, this material was
recrystallized from absolute ethanol to provide iboxamycin as
fine white needles (first crop, 78% recovery, mp 205−206 °C).
Accession Codes
CCDC 1949345 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk, or by contacting The Cam-
bridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
■
Corresponding Author
Andrew G. Myers − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States; orcid.org/0000-0001-9602-6915;
Email: myers@chem.harvard.edu
Authors
Jeremy D. Mason − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States; orcid.org/0000-0002-1039-0773
Daniel W. Terwilliger − Department of Chemistry and
Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, United States
Aditya R. Pote − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c03529
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Notes
The authors declare the following competing financial
interest(s): The authors have filed international patent
applications WO/2019/032936 and WO/2019/032956 Lin-
cosamide Antibiotics and Uses Thereof.
ACKNOWLEDGMENTS
■
We gratefully acknowledge support from the LEO Foundation
(grant LF18006). We thank Dr. Shao-Liang Zheng for the X-
ray data collection and structure determination for iboxamycin
(3). Dr. Amarnath Pisipati is acknowledged for the in vitro and
in vivo profiling of iboxamycin (3). We also acknowledge Drs.
Matthew J. Mitcheltree, Ioana Moga, and Katherine J. Silvestre
for their work on the development of an improved synthesis of
7-Cl-MTL (29) from lincomycin.
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