Novel 3-O-carbamoyl erythromycin A derivatives (carbamolides) with activity against resistant staphylococcal and streptococcal isolates

Article abstract of DOI:10.1016/j.bmcl.2013.01.067

A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity versus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains) and streptococci are reported. An (R)-2-aryl

Full text of DOI:10.1016/j.bmcl.2013.01.067

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1727–1731
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel 3-O-carbamoyl erythromycin A derivatives (carbamolides)
with activity against resistant staphylococcal and streptococcal
isolates
c,
Thomas V. Magee a, , , Seungil Han b, , Sandra P. McCurdy , Thuy-Trinh Nguyen f,
,
⇑
⇑
Karl Granskog f, , Eric S. Marr b, Bruce A. Maguire d, Michael D. Huband e, , Jinshan Michael Chen f,
Timothy A. Subashi d, Veerabahu Shanmugasundaram g
a Pfizer Cardiovascular, Metabolic, and Endocrine Diseases (CVMED) Chemistry, 620 Memorial Dr, Cambridge, MA 02139, USA
b Pfizer Structural Biology and Biophysics, Eastern Point Rd, Groton, CT 06340, USA
c Cubist Pharmaceuticals, 65 Hayden Ave, Lexington, MA 02421, USA
d Pfizer Primary Pharmacology Group, Eastern Point Rd, Groton, CT 06340, USA
e Astra-Zeneca, 35 Gatehouse Lane, Waltham, MA 02451, USA
f Pfizer Exploratory Discovery Chemistry, Eastern Point Rd, Groton, CT 06340, USA
g Pfizer Computational Chemistry, Eastern Point Rd, Groton, CT 06340, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 3-O-carbamoyl erythromycin A derived analogs, labeled carbamolides, with activity ver-
sus resistant bacterial isolates of staphylococci (including macrolide and oxazolidinone resistant strains)
and streptococci are reported. An (R)-2-aryl substituent on a pyrrolidine carbamate appeared to be crit-
ical for achieving potency against resistant strains. Crystal structures showed a distinct aromatic interac-
tion between the (R)-2-aryl (3-pyridyl for 4d) substituent on the pyrrolidine and G2484 (G2505,
Escherichia coli) of the Deinococcus radiodurans 50S ribosome (3.2 Å resolution).
Received 16 November 2012
Revised 7 January 2013
Accepted 16 January 2013
Available online 26 January 2013
Keywords:
Bacterial resistance
cfr
Ó 2013 Elsevier Ltd. All rights reserved.
erm
MLSb
MRSA
Macrolide
Carbamolide
Clarithromycin
Oxazolidinone
Linezolid
Ribosome X-ray
Deinococcus radiodurans
Staphylococcus aureus infections, common in both hospital and
in community settings, are increasingly becoming resistant to
available therapies and current standard treatment options such
as the natural glycopeptide vancomycin are losing their effective-
ness.1 The newer and purely synthetic oxazolidinone class,
represented by linezolid (Fig. 1), is not exempt from this trend
either—particularly in light of the plasmid-carried multi-drug
resistant cfr gene and the possibility of horizontal resistance trans-
fer.2 The macrolide antibiotics such as clarithromycin (Fig. 1) have
been very successful for decades combating community-acquired
respiratory tract infections, caused by primarily Streptococcus
pneumoniae rather than S. aureus.3
However streptococcal susceptibilities have inevitably de-
creased with use of these agents as well, and multidrug-resistant
pathogens to the macrolide class are a persistent and growing
health concern.4 In order to combat this problem, a number of
semi-synthetic variants on the macrolide structure have been dis-
covered.5 The structural changes of these newer macrolides have
enabled different pharmacophoric elements to interact with the
bacterial ribosome—with the ketolides for example.6 These deriv-
atives have resulted in improved resistance profiles versus strains
with the capacity to modify the target ribosome (erm)7 and over-
come macrolide efflux (mef).8 The strategies generally rely upon
⇑
Corresponding authors.
E-mail addresses: thomas.v.magee@pfizer.com (T.V. Magee), seungil.han@pfi-
zer.com (S. Han).
Current address.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.067

1728
T. V. Magee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1727–1731
O
Intermediates 1 and subsequently 2 were generated using
known methods starting from clarithromycin.11 Conversion of 2
to the versatile electrophilic carbamate precursor 3 required re-
peated treatment with excess di-(N-succinimidyl) carbonate in a
solution of acetonitrile with triethylamine over several days with
heating (85 °C) (Scheme 1). Succinimate 3 was purified chromato-
graphically in 44% overall yield as a stable dry powder. Coupling of
amines with 3 was affected in acetonitrile with triethylamine with
heating (50 °C) overnight in generally acceptable yields of 50% or
better. Amines that were not commercially available included the
4,4-dimethyl substituted pyrrolidines, the general synthesis of
which is outlined in Scheme 2 starting from 2,2-dimethylsuccinic
acid (5). Solvent-free condensation with urea yielded 6, which
was readily reduced with lithium aluminum hydride and con-
verted to the N-Boc intermediate 7. The key 2-arylation step was
carried out using a variety of aryl halides and following a variation
on Negishi coupling via enantio-biased anion formation.12
Thus enantio-enriched coupling products were obtained using
(ꢀ)-spartein complexed organozinc coupling to the corresponding
aryl halide via palladium catalysis. The coupled products were
subsequently crystallized to >98% enantiomeric excess as the
(+)-phencyphos salts.13 In this way, a series of amines were made
and coupled to the succinimate template 3 to yield final test
compounds (Fig. 2).
O
O
HO
HO
N
O
OH
N
F
O
O
O
O
N
O
O
O
O
O
NH
OH
linezolid
clarithromycin
Figure 1. Structures of linezolid and clarithromycin.
modification or removal of the cladinose sugar at the 3-position
and introduction of a tethered aryl moiety from different launch
points of the molecule, the latter being particularly important
for capturing erm activity.9 The degree of resistance in different
erm strains has been correlated with the degree of modification
(methylation of A2058, Escherichia coli) of the bacterial
ribosome.10
Researchers at Abbott first observed the potency advantages of
fusing a carbamate across the 11,12-positions of the macrolactone
ring,14 and this structural feature is present in the ketolides teli-
thromycin and cethromycin. We used the same 11,12-fused carba-
This disclosure is focused upon novel 3-O-carbamoyl deriva-
tives of 6-O-methyl-12,11-oxycarbonylimino erythromycin
A—herein labeled carbamolides—which begin to address the
problem of Gram-positive resistance, with an emphasis on staphy-
lococci and structural basis of these carbamolides binding in a
unique mode to large ribosomal subunit from Deinococcus radiodu-
rans (D50S). (Unless otherwise stated, in the text that follows,
nucleotide numbering will refer to D. radiodurans, but the equiva-
lent number in E. coli will be given in parentheses.)
mate feature, starting from the known intermediate
1 and
removing the cladinose provided the 3-hydroxy template (2) as a
launch point for picking up novel interactions with the ribosome
important for overcoming erm (MLSb) resistance. We found the
pyrrolidinyl carbamate without any substitution (4a) to have weak
O
O
O
NH
11
O
O
O
O
AcO
N
12
O
i
NH
3
AcO
O
N
O
O
O
O
O
O
O
O
O
OH
O
OAc
2
1
ii
O
O
O
O
O
O
O
NH
O
NH
3
iii, iv
AcO
O
N
HO
N
O
O
O
O
O
3
O
O
O
O
O
O
N
O
O
R
O
4a-n
3
Scheme 1. General route for the synthesis of the carbamolide analogs with varying amines R (see Fig. 2). Reagents and conditions: (i) HCl/EtOH/40 °C; SiO2 chromatography;
60–70% yld; (ii) di-(N-succinimidyl) carbonate (10 equiv over 5d)/TEA/MeCN/85 °C; SiO2 chromatography; 44% yld; (iii) coupling amine = R (1.2 equiv, generally as
(+)-phencyphos salt form)/TEA/MeCN/50 °C; (iv) MeOH/TEA/50 °C to remove acetate protecting group; SiO2 chromatography; 50–60% yld over two steps.

T. V. Magee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1727–1731
1729
O
O
Boc
i
HN
ii, iii
iv, v, vi
HO
HO
HN
N
Ar
O
O
5
6
7
8
Scheme 2. General route for the synthesis of 2-aryl substituted (R)-4,4-dimethylpyrrolidines. Reagents and conditions: (i) urea/160 °C; 51% yld; (ii) LAH/THF/reflux; (iii) Boc
anhydride/THF; 95% yld over two steps; (iv) (ꢀ)-spartein/MTBE/ꢀ78 °C/sec-BuLi; ZnCl2/THF/ꢀ78 °C to rt; aryl-halide (Ar-X; X = Br or I)/Pd(OAc)2/t-Bu3PHBF4; SiO2
chromatography; (v) TFA; (vi) (+)-phencyphos/i-PrOH/recrystallization to enhance enantiomeric excess. 10–30% yld over three steps.
R =
N
N
N
N
N
HN
N
O
4a
4b
4c
4d
4e
N
N
N
N
N
O
N
O
Cl
O
Cl
O
4f
4g
4h
4j
4i
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4k
4l
4n
4m
Figure 2. Pyrrolidine attachments (R in Scheme 1) representing key test carbamolide analogs 4a–n.
Table 1
Minimum inhibitory concentrations (MICs, mg/L) of carbamolide analogs against resistant isolates
Compound
ID
Strep.
Strep.
Staph.
epidermidis
1105-00
Staph. aureus Staph. aureus
Staph.
aureus
1281-07
MRSA, cfr
Staph.
aureus
1279-07
MRSA, cfr
Staph. aureus
1609-09
MRSA, cfr,
MLSb
Staph.
Staph. aureus
29213 ATCC
[control
pneumoniae
1243-00
MLSb
pyogenes
1304-00
MLSb
1279-07
MRSA,
2111-10
MRSA,
aureus
1015-00
MRSA,
MLSb
MRSE, MLSb
USA300
USA300 cfr
strain]
Linezolid
0.5
0.5
1
2
16
32
16
16
4
4
Clarithro
4a
4b
4c
4d
4f
4g
4h
4i
4j
>64
>64
0.5
64
0.5
0.03
0.125
0.03
0.25
0.125
0.015
1
>64
>64
0.5
32
2
0.03
0.125
0.03
0.25
0.125
0.03
1
>64
>64
>64
>64
>64
16
64
64
64
64
8
>64
>64
16
>64
4
0.5
0.5
0.25
2
2
0.5
2
1
1
>64
0.5
0.25
2
0.25
0.125
0.25
0.25
0.25
0.25
0.25
0.5
0.25
0.5
0.125
1
>64
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
64
64
64
64
16
>64
>64
>64
>64
>64
64
>64
>64
64
64
32
32
16
0.5
2
0.25
4
0.25
0.125
0.25
0.25
0.25
0.25
0.25
0.5
>64
0.25
0.125
0.25
0.25
0.25
0.25
0.25
0.5
0.25
0.125
0.25
0.125
0.25
0.25
0.25
0.25
0.25
0.5
4k
4l
4m
4n
16
8
8
0.125
0.125
0.25
0.125
0.5
0.5
0.5
0.5
0.5
0.5
8
8

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T. V. Magee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1727–1731
Figure 3. The X-ray crystal structure of 4d bound to D50S at 3.2 Å resolution (PDB access code: 4IO9). (A) The (Fo–Fc) omit map at the inhibitor binding site, contoured at 3
(B) The pose in the nascent peptide tunnel. (C) The -stacking interaction of the pyridyl moiety with G2484 (G2505).
r.
p
minimum inhibitory concentrations (MICs, Table 1) for bacterial
growth although it did show improvement versus a few of the lin-
ezolid and clarithromycin resistant strains. We were pleased how-
ever to discover that the (R)-2-phenyl substituent (4b) on the
pyrrolidine ring demonstrated a significant boost in potency
against the S. pneumoniae and Streptococcus pyogenes MLSb
isolates. The stereochemistry at the 2-position was found to be
important, as illustrated by the MIC data for the corresponding
(S)-2-phenyl diastereomer (4c). Modification of the phenyl ring
to heterocycles led to only modest potency changes as illustrated
by the 3-pyridinyl analog 4d. Extended substitutions such as the
N-acetyl at the 4-position of the phenyl ring (4e) demonstrated
no potency against resistant MLSb strains.15 We subsequently
introduced a gem-dimethyl function at the 4-position of the pyrrol-
idine with the goal of probing the effect on potency incurred by
conformational locking of this ring system. The resulting (R)-3-
(4,4-dimethylpyrrolidin-2-yl)pyridinyl analog 4f was the first
carbamolide analog to demonstrate any activity against a staphylo-
coccal MLSb strain (S. epidermidis 1105-00) albeit moderate
(MIC = 16 mg/L) and did not inhibit the growth of the extremely
resistant MLSb S. aureus strains (1609-09 and 1015-00). Both ana-
log 4d and the gem-dimethyl analog 4f gave excellent potency
against the linezolid resistant (cfr) strains (MIC 6 0.25 mg/L vs S.
aureus 2111-10, 1281-07, and 1279-07). However, the majority
of analogs showed little or no activity against the staphylococcal
MLSb strains. The robust and constitutive nature of the these
strains, owing to the methylation of A2041 (A2058) and the conse-
quent steric clash with the dimethylamino function of the desos-
amine sugar present in the macrolide antibiotics, including
clarithromycin, makes it very challenging for these templates to re-
gain any potency against them.16
We were able to obtain an X-ray structure of 4d soaked into
Deinococcus radiodurans 50S (D50S) crystals at a resolution of
3.2 Å. This structure (Fig. 3) showed that the compound bound to
the large subunit in the nascent peptide tunnel below the peptidyl
transferase center. The lactone ring was oriented almost the same
as the lactone ring of erythromycin17 and inserted into the hydro-
phobic pocket mainly formed by residues A2041 (A2058), A2042
(A2059) and U2590 (C2611) upon binding. The 20-OH group of
the desosamine sugar formed a hydrogen bond with the N1 of
A2041 (A2058). Importantly, the attachment of (R)-2-aryl
(3-pyridyl for 4d) substituent on the pyrrolidine placed the aryl
moiety into a narrow pocket composed of G2484 (G2505), C2589
(C2610) and C2590 (C2611) and confirmed our observation of ste-
reo-sensitivity in this region. The aryl ring formed additional inter-
actions with G2484 (G2505), a universally conserved nucleotide in
the peptidyl transferase loop, which was further stabilized by
Figure 4. X-ray crystal structure of 4e bound to D50S at 3.2 Å resolution (PDB
access code: 4IOA). (A) The (Fo–Fc) omit map at the inhibitor binding site,
contoured at 3r. (B) Interaction of 4e in the nascent peptide tunnel showing the
rotated pyrrolidinyl side chain (cf. Figs. 3 and 5).
forming a H-bond with C2589 (C2610) and interaction with
G2555 (G2576) (Fig. 3). Interestingly, the less active N-acetyl ana-
log (4e, Fig. 4) had the pyrrolidine rotated by almost 180° relative
to 4d such that the whole pyrrolidinyl sidechain rotated out to
interact with domain V of the 23S rRNA—presumably due to the
limited pocket size defined by G2484 (G2505), C2589 (C2610)
and C2590 (C2611) and its inability to accommodate the N-acetyl
function. As a result, the pyrrolidine made a van der Waals contact
with G2484 (G2505) and the N-acetyl formed additional stacking
interactions with C2589 (C2610). As noted, this rotation was not
beneficial for antibacterial activity. Looking to improve the potency
of our analogs, we speculated that the addition of the 4,4-dimethyl
substitution pattern on the pyrrolidine would limit the conforma-
tional flexibility on this ring system, thereby enhancing the aryl-
ribosome interaction and consequently the potency. Consistent
with this hypothesis, the crystal structure of the 4,4-dimethyl ana-
log 4f at 3.6 Å resolution showed the same aromatic stacking inter-
action with G2484 (G2505) with the gem-dimethyl function
solvent exposed rather than making a direct interaction with the
ribosome (Fig. 5). The two benzo-[1,3]-dioxole analogs 4g and 4h
however failed to show any improvement in potency. Chloro sub-
stitution in the 4- (4i) and 3- (4j) positions also did not improve
antibacterial activity, however the 2-chloro (4k) had reasonable
potency against the S. epidermidis 1105-00 (MIC = 8 mg/L). The
3,4-dichloro (4l) analog was less potent than the 2,4- (4m) and
in particular the 2,3-dichloro substitution (4n)—the latter being

T. V. Magee et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1727–1731
1731
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Figure 5. X-ray crystal structure of 4f in complex with D50S at 3.6 Å resolution
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r. (B) Details of interaction showing the same p-stacking interaction
with G2484 (G2504).
the only analog to achieve MICs 6 8 mg/L versus all of the strains
tested, including the most recalcitrant staphylococcal MLSb and
cfr isolates (S. aureus 1609-09 and 1015-00). While the aromatic
stacking interaction is clearly important for potency, the precise
nature of why this particular substitution pattern has a potency
advantage is not clear and may also be a function of bacterial cell
penetration.
We have disclosed a novel series of 3-O-carbamate erythrono-
lide A analogs, herein dubbed carbamolides, which demonstrate
antibacterial activity against macrolide and oxazolidinone resis-
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gree of activity versus MLSb S. aureus isolates. We have further
provided crystallographic evidence for a unique aromatic stacking
interaction within a tight pocket of the bacterial ribosome that ap-
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.01.
067.
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