Desmethyl macrolides: Synthesis and evaluation of 4,8,10-tridesmethyl telithromycin

Article abstract of DOI:10.1021/ml1002184

There is an urgent need to discover new drugs to address the pressing problem of antibiotic resistance. Macrolide antibiotics such as erythromycin (1) are safe, broad-spectrum antibiotics used in the clinic since 1954. Herein, we report the synthesis and

Full text of DOI:10.1021/ml1002184

pubs.acs.org/acsmedchemlett
Desmethyl Macrolides: Synthesis and Evaluation of
4,8,10-Tridesmethyl Telithromycin
Venkata Velvadapu,† Tapas Paul,† Bharat Wagh,† Dorota Klepacki,‡ Olgun Guvench,§,
Alexander MacKerell, Jr.,§ and Rodrigo B. Andrade*,†
†Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122, United States, ‡Center for Pharmaceutical
Biotechnology, University of Illinois, Chicago, Illinois 60607, United States, and §Department of Pharmaceutical Sciences,
University of Maryland, Baltimore, Maryland 21201, United States
ABSTRACT There is an urgent need to discover new drugs to address the pressing
problem of antibiotic resistance. Macrolide antibiotics such as erythromycin (1) are
safe, broad-spectrum antibiotics used in the clinic since 1954. Herein, we report the
synthesis and evaluation of 4,8,10-tridesmethyl telithromycin (3), a novel des-
methyl analogue of the third-generation drug telithromycin (2), which is a
semisynthetic derivative of 1. Analogue 3 was found to possess antibiotic activity
and was superior to telithromycin (2) when tested against resistant strains of
Staphylococcus aureus possessing an A fT mutation at position 2058 (Escherichia
coli numbering).
KEYWORDS Total synthesis, ketolide antibiotics, antibiotic resistance, telithromy-
cin, desmethyl, analogues
he rapid emergence of antibiotic-resistant bacteria
represents a serious public health threat.1 The pro-
blem is compounded by the sharp decline in drug
(Escherichia coli numbering) and do not efficiently bind
macrolides 1 and 2. However, a point mutation of guanine to
adenine at position 2058 of 23S rRNA (i.e., G2058A) rendered
mutants susceptible to the antibiotics, allowing the structure of
telithromycin (2) bound to the HmA2058 mutant at 2.6 Å
resolution to be obtained (Figure 2A). From these data, Steitz
argued that in bacteria, A2058G mutations confer resistance
due to a steric clash of the amino group of guanine 2058 with C4
methyl of the drug (Figure 2B, both in red). In turn, we hypo-
thesize that replacement of the C4 methyl group in 2 with
hydrogen should relieve this steric clash and thus offer a means
of addressing antibiotic resistance arising from mutation.
To rapidly access analogues for biological testing by facil-
itating synthesis, we first targeted 4,8,10-tridesmethyl teli-
thromycin (3). Prior to embarking on our synthetic campaign,
we examined the conformational consequences of replacing the
methyl groups in telithromycin (2) with hydrogens (Figure 3).
A priori, the removal of any methyl group from the 14-
membered macrolactone scaffold of telithromycin (2) has
two major consequences that directly impact binding: (1)
conformational changes caused by removing syn-pentane
interactions that serve to bias the classical Perun-Celmer
modified diamond lattice structure and (2) loss of potential
van der Waals contributions (ΔGvdW) to the overall binding.7
To address the former, we employed the conformationally
sampled pharmacophore (CSP) protocol that uses molecular
dynamics to look at distribution of distances (Figure 3A-D).8
T
companies with active antimicrobial research programs;
therefore, new sources of antibiotics are critical.2,3 To ad-
dress this need, we have initiated a structure-based drug
design program wherein desmethyl analogues (i.e., CH3 fH) of
the third-generation macrolide antibiotic telithromycin (2) are
prepared via chemical synthesis (Figure 1). Herein, we report
the synthesis and biological evaluation of 4,8,10-tridesmethyl
telithromycin (3) against both wild-type and macrolide-resistant
bacteria.
All macrolide antibiotics target the bacterial ribosome and
reversibly inhibit the 50S subunit by binding in the peptidyl
transferase center, thus blocking protein synthesis.4 Telithro-
mycin (2) is a third-generation semisynthetic drug derived
from the classic macrolide antibiotic erythromycin (1) and
has been used in the clinic since 2004.5
Macrolide antibiotic resistance mechanisms fall into three
major categories: (1) enzymatic modification of the drug, (2)
drug efflux from the cell encoded by mef genes, and (3)
ribosomal modification arising from either ribonucleotide
N-methylation of residues critical for binding (e.g., A2058) or
single point mutations (e.g., A2058G). Third-generation
ketolides have largely addressed the first two issues yet
remain susceptible to ribosomal modification.4,5
The rationale behind our desmethylation strategy is
grounded in structural data obtained by Steitz and co-work-
ers who successfully cocrystallized various macrolide drugs
with mutant large ribosomal subunits of the archaeon
Haloarcula marismortui (Hm).6 Unlike eubacteria that pos-
sess an adenine at 2058, all archea possess a guanine
Received Date: September 17, 2010
Accepted Date: October 12, 2010
Published on Web Date: October 18, 2010
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We began with the preparation of known diol 10 via known
aldehyde 8 (Scheme 1).10-12 Swern oxidation of commercially
available 3-benzyloxy-propanol (11), addition of 2-propenyl
MgBr, and subsequent Johnson-Claisen orthoester rearrange-
ment afforded enoate 12 in 48% overall yield. Reduction of
the ester and protection of the newly formed alcohol as its
tert-butyldimethylsilyl (TBS) ether provided 13 (78% yield over
two steps).13 Sharpless dihydroxylation (AD mix-β) established
the requisite stereochemistry of hydroxyls at C5 and C6 in 91%
yield (er > 20:1).14,15 Selective protection of the secondary C5
alcohol with triethylsilylchloride (TESCl) followed by methyla-
tion of the tertiary C6 alcohol with Me3OBF4 and Proton
Sponge afforded 14 in 80% over two steps.16 Hydrogenolysis
of the benzyl ether (85% yield) followed by Swern oxidation
furnished aldehyde 15, which was subjected to an Evans aldol
reaction with 16 to set the stereochemistry at C2 and C3.17 In
the event, desired aldol product 17 was obtained in 78% yield
(dr > 20:1) over two steps. Protection of the aldol with TBSOTf
and removal of the auxiliary secured acid 18 in 85% yield (two
steps). Chemoselective Yamaguchi esterification of 18 and diol
10 afforded ester 19 in 78% yield.
Figure 1. Structures of erythromycin (1), telithromycin (2), and
novel analogue 4,8,10-tridesmethyl telithromycin (3).
To prepare the 14-membered macroketolactone, we em-
ployed a ring-closing metathesis (RCM) strategy first estab-
lished by Kang.18 Toward this end, we first removed the
primary TBS ether chemoselectively with tetrabutylammo-
nium fluoride (TBAF) and AcOH to furnish an alcohol (60%
yield, 86% borsm) that was oxidized to the aldehyde with
the Dess-Martin periodinane (DMP).19 The addition of vinyl
MgBr and subsequent DMP oxidation afforded vinyl ketone
20 in 60% yield overall. Treatment of dienone 20 with
20 mol % Grubbs' second-generation catalyst effected the
desired RCM, affording macroketolactone 21 in 90% yield
(borsm).20
Figure 2. (A) Telithromycin and A2058 interactions in Hm with
select distances in Angstroms (Steitz et al., PDB = 1YIJ). (B) Steric
consequences of A2058G mutation.
To chemoselectively install desosamine at the C5 position,
it was critical to (1) reduce the C9 ketone to avoid ketalization
with the hydroxyl at C5 and (2) protect the tertiary C12
hydroxyl to avoid undesired glycosylation.21 Toward these
goals, the C9 ketone was first subjected to a Luche reduction.
Silylation of C9 and C12 hydroxyls with TESOTf and subse-
quent treatment with p-toluenesulfonic acid (PTSA) selec-
tively left only the tertiary C12 hydroxyl protected (52%
yield over three steps). Chemoselective silylation of the
allylic C9 alcohol with TESCl furnished 22, which was sub-
jected to glycosylation with known thiopyrimidine desosa-
mine donor 23 (50% yield over two steps) under the agency
of AgOTf and 2,6-di-t-Bu-4-Me-pyridine (DTBMP).22,23 Fluoride-
mediated cleavage of silyl ethers at C9, C12 and DMP oxida-
tion afforded glycosylated macroketolactone 24 (84% yield
over two steps).
Figure 3. Conformationally sampled pharmacophore (CSP) anal-
ysis of telithromycin (2, solid line) and analogue 3 (dashed line).
The end game for desmethyl analogue 3 began with a
sequence employed by Aventis24 to prepare C11-C12 ox-
azolidinones originally developed by Baker and co-workers
at Abbott.25 Activation of the C12 alcohol with NaH and
carbonyldiimidazole (CDI) and treatment with primary
amine 2526 effected a tandem carbamoylation/intramolec-
ular aza-Michael sequence to stereoselectively afford oxazo-
lidinone 26 in 35% overall yield. Removal of the C3 TBS
ether with tris(dimethylamino)sulfonium difluorotrimethyl-
silicate (TAS-F) proceeded in 70% yield.27 Corey-Kim
While the distributions for desmethyl analogue (3, dashed
lines) differ from those of telithromycin (2, solid lines),
analogue 3 does sample conformations that overlap with
2, suggesting that 3 can bind the ribosome.
To test our hypothesis and probe the consequences of a
desmethylation strategy, we employed chemical synthesis to
access material for biological evaluation (i.e., minimum inhibi-
tory concentrations or MICs).9 The total synthesis of 4,8,10-
tridesmethyl telithromycin (3) is shown in Scheme 1.
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Scheme 1. Synthesis of 4,8,10-Tridesmethyl Telithromycin (3)a
a Reagents and conditions: (a) Ph3P=CH2, THF. (b) 1 N HCl(aq), MeOH, 53% from 7 (see Supporting Information). (c) (COCl)2, DMSO, Et3N. (d)
2-Propenyl MgBr, THF. (e) (MeO)3CCH3, EtCO2H, 48% from 11. (f) LAH, Et2O. (g) TBSCl, imidazole, 78% from 12. (h) AD mix-β, 91%, er >20:1. (i)
TESCl, imidazole. (j) Me3OBF4, Proton Sponge, 80% (two steps). (k) H2, Pd/C, 85%. (l) (COCl)2, DMSO, Et3N. (m) Bu2BOTf, Et3N, 78%, dr > 20:1 (two
steps). (n) TBSOTf, 2,6-lutidine. (o) LiOOH, THF, H2O, 85% (two steps). (p) Cl3PhCOCl, Et3N, DMAP, 10, 78%. (q) TBAF, AcOH, 86% (borsm). (r) DMP,
NaHCO3. (s) vinyl MgBr, THF. (t) DMP, 60% (three steps). (u) 20 mol % Grubbs II cat., 90% (borsm). (v) NaBH4, CeCl3 7H2O, dr = 7:1. (w) TESOTf, 2,6-
3
lutidine. (x) PTSA, 52% (three steps). (y) TESCl, imidazole. (z) Compound 23, AgOTf, DTBMP, 50% (two steps). (aa) 1 M TBAF, THF, 95%. (ab) DMP,
CH2Cl2, 88%. (ac) NaH, CDI, THF/DMF. (ad) Compound 25, 35% over two steps. (ae) TASF, DMF/H2O, 70%. (af) NCS, DMS, Et3N, 75%. (ag) MeOH, 60%.
oxidation furnished the C3 ketone; methanolysis of the
methyl carbonate on the C20-position of desosamine
delivered 4,8,10-tridesmethyl telithromycin (3) in 45%
yield over two steps.28 With desmethyl analogue 3 in hand,
we tested it against several bacterial strains of E. coli and
Staphylococcus aureus using telithromycin (2) as comparator
(Tabl e 1 ).
While both resistant strains (entries 1 and 5) were not
susceptible to either macrolide, both E. coli wild-type and
A2058G mutant (entries 2 and 3) were inhibited by both
analogue 3 and telithromycin (2). Moreover, desmethyl
analogue 3 was less potent than telithromycin (2) by a factor
of 64 (entries 2 and 3). As described, this may be due to the
Table 1. Minimum Inhibitory Concentration (MIC) Values in μg/mL
for 4,8,10-Tridesmethyl Analogue (3) and Telithromycin (2)
entry
strain
bacteria
wt/mutant MIC (3) MIC (2)
1
2
3
4
5
SQ171/2058G E. coli
A2058G
wt
>512
32
>512
0.5
DK/pKK3535
DK/2058G
UCN14
E. coli
E. coli
A2058G
A2058T
ermA
64
1
>256
>128
S. aureus
S. aureus
32
ATCC33591
>128
desmethyl analogue 3 was found to be more potent than
telithromycin (2) against S. aureus clinical strain UCN14 with
an A2058T mutation (entry 4).29 These results demonstrate that
structural simplification (i.e., function-oriented synthesis)30 and/
or molecular editing31 of established antibiotics can result in
ꢀ
conformational flexibility of 3 vis-a-vis 2, in addition to loss
of van der Waals contacts at C4, C8, and C10. Curiously,
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analogues with improved activity against A2058T ribosomal
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SUPPORTING INFORMATION AVAILABLE General experi-
mental protocols, including the preparation of diol 10, and char-
acterization of all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author: *E-mail: randrade@temple.edu.
Present Addresses: Department of Pharmaceutical Sciences,
University of New England, Portland, Maine 04103.
Funding Sources: This work was supported by the NIH
(AI080968 and GM070855).
ACKNOWLEDGMENT We thank Dr. Alexander Mankin
(University of Illinois at Chicago) for helpful suggestions and
Dr. Charles DeBrosse (Temple University, Director of NMR Facilities)
for kind assistance with 2D NMR experiments. We also thank
Dr. Richard Pederson (Materia, Inc.) for catalyst support.
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