Synthesis and antibacterial activity of desosamine-modified macrolide derivatives

Article abstract of DOI:10.1016/j.bmcl.2012.05.110

Structural factors behind erm macrolide resistance were studied through synthesis of new macrolide derivates possessing truncated desosamine sugar moieties and subsequent determination of their antibacterial activity. Synthesized compounds with 2′-deoxy a

Full text of DOI:10.1016/j.bmcl.2012.05.110

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4575–4578
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial activity of desosamine-modified
macrolide derivatives
Nicolas LeTourneau a, Pavan Vimal a, Dorota Klepacki b, Alexander Mankin b, Artem Melman a,
⇑
a Department of Chemistry & Biomolecular Science, Clarkson University, 8 Clarkson Ave., Potsdam, NY 13699-5810, United States
b Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 S. Ashland Ave., Chicago, IL 60607, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Structural factors behind erm macrolide resistance were studied through synthesis of new macrolide der-
ivates possessing truncated desosamine sugar moieties and subsequent determination of their antibacte-
rial activity. Synthesized compounds with 20-deoxy and 30-desmethyl desosamine rings demonstrated
decreased antibacterial activity on the native Staphylococcus aureus strain and were inactive against con-
stitutively resistance S. aureus. The obtained results indicate that steric repulsion between the dimethy-
lated A2058 and desosamine ring cannot be considered as a primary reason for erm-resistance.
Ó 2012 Published by Elsevier Ltd.
Received 23 April 2012
Revised 22 May 2012
Accepted 29 May 2012
Available online 6 June 2012
Keywords:
Macrolide antibiotics
Desosamine
Erm resistance
Deoxygenation
Macrolide antibiotics of the first (erythromycin 1a) and second
(clarithromycin 1b, azithromycin 1c) generations are commonly
used for the treatment of infectious diseases.1 More than 50 years
of macrolide antibiotics research have revealed both their
strengths—low toxicity, high efficacy, and the rare occurrence of
allergic reactions—as well as their drawbacks—poor bioavailability
and rapidly spread resistance.2 Ketolides such as telithromycin3 2d,
cethromycin4 2e, and solithromycin5 2f constitute the third gener-
ation of macrolide antibiotics that demonstrate improved activity
against a number of resistant isolates and represent successful
examples of chemical modifications of the macrolide platform
which results in superior antibacterial agents.
Macrolide antibiotics inhibit protein synthesis by blocking the
progression of nascent proteins through the exit tunnel in the
50S ribosomal subunit preventing the elongation of the polypep-
tide chain.6,7 All macrolide antibiotics exhibit remarkable consis-
tency in their binding modes to the binding pocket in the exit
tunnel. Their high affinities originate mainly from hydrophobic
interactions of their macrocyclic lactone rings and hydrogen bond-
ing of their desosamine sugars with rRNA. While Van der Waals
interactions between rRNA and the macrolactone ring are impor-
tant for the drug binding, a substantial part of the binding energy
comes from contacts that involve C5-desosamine that approaches
adenines residues 2057, 2058, and 2059 of 23S rRNA (Fig. 1).8
Two major components dominate the binding of desosamine: (a)
hydrogen bond between the 20 hydroxy group and the N-1 of
A2058, and (b) electrostatic interaction between the protonated
30-dimethylamino group with neighboring phosphate groups
(4.5 Å distance, Fig. 1). The placement of desosamine in the ribo-
some is essentially identical for all studied macrolide antibiotics.
Acylation of 20-OH group yields inactive but labile 20-esters that
are hydrolyzed by water or alcohols to regenerate initial macro-
lides.9 Other chemical modifications of desosamine such as
replacement of the 30-dimethylamino group with acylamino
groups results in complete loss of antibacterial activity.10
Dimethylation of N6 of A2058 residue by Erm-type methyl-
transferases dramatically decreases affinity of macrolides to ribo-
some rendering bacteria that produce these enzymes resistant to
all known types of macrolide antibiotics.11 Despite significant ef-
fort invested in structural modifications of macrolides and keto-
lides,12 there have been no reports of successful macrolide
antibiotics capable of binding to A2058 dimethylated ribosomes.
Although structural reasons for Erm-mediated resistance have
been proposed on the basis of crystallographic analysis of native
ribosome complexed with macrolide antibiotics, the Erm-modified
ribosome has never been investigated crystallographically. As a re-
sult, predictions about the interactions of macrolide antibiotics
with A2058 dimethylated ribosomes are currently based solely
on theoretical considerations.13 A graphic representation based
on the crystal structure of ketolide telithromycin 2d bound to na-
tiveEscherichia coli ribosome13b is shown inFigure 1. Three possible
factors could contribute to a sharply decreased affinity of macro-
lides to the A2058 dimethylated ribosome:
(a) Introduction of methyl groups on N-6 of A2058 can disrupt
the hydrogen bonding between 20-OH of macrolides and N-
1 of A2058;
⇑
Corresponding author.
E-mail address: amelman@clarkson.edu (A. Melman).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.110

4576
N. LeTourneau et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4575–4578
Figure 1. Fragment of crystal structure of telithromycin–E. coli 50S ribosome subunit complex (PDB 3OAT) with measured distances between N6 of A2058 residue and nearby
30-NMe2 (4.3 Å) and 20-OH (3.5 Å) groups in desosamine sugar of telithromycin, and between N6 of A2058 residue and water molecules (3.4 and 3.9 Å) coordinated to
magnesium cation in 23S RNA. Two yellow methyl groups attached to N6 were manually added to the crystal structure to illustrate possible non-bonding interactions arising
in A2058 dimethylated ribosome.
X
9
O
(b) The methyl group on N-6 of A2058 may sterically clash with
20-OH and/or 30-dimethylammonium groups of the desos-
amine. Due to the rigidity of the six-membered desosamine
scaffold the clash may result in squeezing the desosamine
sugar out of its binding pocket.
(c) The N-6 methyl group(s) on A2058 may clash with neighbor-
ing water molecules (3.9 Å, 3.4 Å, Fig. 1) bound to the Mg2+
cation that is chelated by phosphate groups of G2056 and
G2057. In this case, positioning of 23S rRNA residues in
the whole macrolide binding pocket may be altered.
R1
N
O
R2
OR
11 OH
Me
Me
HO
6
C
HO
NMe2
12
O
3'
5'
O
6
5
5
HO
2'
NMe2
Me
2'
3'
O
O
Me
1
3
O
Et
O
O
O
5'
O
1
3
O
Desosamine
OMe
O
O
Y
Desosamine
2
O
3''
4''
Y=H; R1= (CH2)4
N
3-pyridyl
1
d
e
f
R2=Me
N
OH
Cladinose
Y=H; R2=
R1=H
3-quinolyl
a
b
c
R=H, X = CO
R=Me, X = CO
R=H, X=N(Me)CH2
Y=F; R1=
(CH2)4
N
N N
3-NH2Ph
Discrimination between these possibilities can be done through
synthesis of macrolide derivatives containing a modified desos-
amine cycle and examining their antibacterial activity on native
and Erm-modified strains. Particularly, the contribution of hydro-
gen bonding between N-1 of A2058 and 20-OH can be tested by
comparing the antibacterial activity of macrolide antibiotics with
their 20-deoxy derivatives in which 20-OH of desosamine is re-
placed with a hydrogen atom. In this Letter we report on synthesis
of new macrolide derivatives possessing desosamine sugar modi-
fied in positions 20 and 30, and their corresponding antibacterial
activity on macrolide susceptible and macrolide resistant strains
of Staphylococcus aureus.
Synthesis of all new macrolide compounds was done starting
from the available erythromycin, clarithromycin, telithromycin,
and cethromycin (ABT-773) antibiotics. N-Desmethyl erythromy-
cin 3a and clarithromycin 3b were prepared by reaction of corre-
sponding macrolides 1a,b with iodine and sodium acetate in
methanol.14 N-Desmethyl clarithromycin 2b was used for prepara-
tion of 30-(1-methylguanidino) clarithromycin by refluxing with
excess of cyanamide in ethanol. O-Deoxygenation15 of erythromy-
cin and clarithromycin was performed by free radical reduction
using treatment with 2 equiv of PhOC(S)Cl/triethylamine to afford
corresponding thionocarbonates that were converted into deoxy-
genated macrolides 5a,b through subsequent reaction with
Bu3SnH/AIBN (see Schemes 1 and 2).
R2=Me
Scheme 1. Macrolide antibiotics erythromycin 1a, clarithromycin 1b, azithromycin
1c, and ketolide telithromycin 2d, cethromycin 2e, solithromycin 2f.
replacement of additional groups in the desosamine ring we also
introduced n-Bu chain in 30-desmethyltelithromycin 6d by reduc-
tive alkylation with excess of sodium cyanoborohydride and butyr-
aldehyde to give ketolide 8d.
Initial attempts for deoxygenation of telithromycin 2d and
cethromycin 2e through preparation of corresponding 20-thiono-
carbonate with PhOC(S)Cl/triethylamine were not successful due
to formation of complex mixture of products during the reaction.
Negative results were also obtained by using other reagents
including PhOC(S)Cl/pyridine, PhOC(S)Cl/ dimethylaminopyridine,
and C6F5OC(S)Cl/dimethylamino-pyridine. The deoxygenation of
telithromycin was eventually achieved through its prolonged
treatment with an excess of N,N-thiocarbonyldiimidazole at room
temperature followed by one pot reaction of the reaction mixture
with Bu3SnH/AIBN to give compound 7d. The same procedure
was used for preparation of 20-deoxycethromycin 7d, and
20-deoxy-30-(n-butylmethylamino) telithromycin 9d.
All new macrolide derivatives possessing a modified desos-
amine function showed moderate antibacterial activity on native,
antibiotic-sensitive S. aureus strain ATCC29213 (Table 1). Five
new 20-deoxymacrolide derivatives 5a, 5b, 7d, 7e, and 9d showed
MIC values ranging between 4 and 64
tially higher than those of parent macrolides. These results are
Modification of the desosamine ring in ketolides 2d,e required
different set of reaction conditions (Scheme 3). N-Desmethyl teli-
thromycin 6d was prepared through treatment of telithromycin
with N-iodosuccinimide in acetonitrile.16 To study the effect of
lg/mL which were substan-

N. LeTourneau et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4575–4578
4577
O
O
NH2
HN
6 OR
OR
O
HO
HO
6
OH NH
3'
OH 3'N
2'
a) R = H
b) R = Me
OH
OH
5
5
Me
Me
2'
O
O
O
H2NCN
5'
5'
3
3
O
O
O
O
O
O
OMe
3''
OMe
3''
I2, NaOAc,
MeOH
O
O
4''
4''
3a,b
4b
OH
OH
O
O
OR
6
OR
H
O
HO
6
OH 3' N
2'
H
N
OH
OH
3'
1. PhOC(S)Cl,
Et3N
2. Bu3SnH, AIBN,
toluene, Δ
H
5
Me
5
Me
2'
O
3
O
O
3
O
5'
5'
O
O
O
O
O
O
OMe
3''
OMe
3''
O
O
1a,b
4''
5a,b
4''
OH
OH
Scheme 2. Synthesis of N-desmethyl macrolides 3a,b, 30-(1-methylguanidino)clarithromycin 4b, and 20-deoxymacrolides 5a,b.
O
O
O
R1
N
R1
N
R1
N
O
O
O
R2
O
R2
O
R2
O
C
C
C
O
O
O
6
5
6
5
6
5
H
OH
2'
OH
2'
3'
N
3'
N
3'
NH
2'
H
O
Me
Me
Me
Et
O
Et
O
O
Et
O
O
5'
5'
5'
1
1
1
3
3
3
O
O
O
O
O
O
O
O
O
7d, e
2d, e
6d
O
O
R1
N
R1
O
O
R2
O
R2
O
N
C
C
N
O
O
d
e
R1=
R
6
5
6
OH
N n-Bu
H
(CH2)4
2 = Me
R1 = H
R2
N
N
n-Bu
2'
2'
H
O
5
N
Me
Me
3'
5'
3'
5'
Et
O
Et
O
O
1
1
3
3
O
O
=
3-quinolyl
O
O
O
O
9d
8d
Scheme 3. Preparation of 30-desmethyltelithromycin 6d, 20-deoxy-30-(n-butylmethylamino)telithromycin 9d and 20-deoxyketolides 7d,e, 9d.
comparison clearly indicates that the far more dramatic decrease
in antibacterial activity observed in erm-positive resistant bacteria
(see Table 1, ermA-positive strain ATCC33591) cannot be attrib-
uted solely to the loss of the hydrogen bond between 20-OH of
macrolide antibiotics and A2058.
Table 1
Antibacterial activity of macrolide and ketolide derivatives
Compound
MIC for S. aureus
ATCC29213
MIC for S. aureus
ATCC33591 (ermA,
constitutive), lg/mL
(wild type),
lg/mL
All new 20-deoxy macrolide derivatives were completely inac-
tive against ermA-positive strain ATCC33591. This result argues
against any considerable contribution of a steric clash between
N-6 methyl groups of A2058 and 20-OH of macrolide derivative
to erm-mediated resistance.
3a
3b
4b
5a
5b
6d
7d
7e
8
4
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
32
64
64
16
8
Similarly to 20-deoxygenated macrolide derivatives; 30-desm-
ethyl macrolide derivatives 3a,b and 6d showed lower antibacte-
rial activity in the native strain of S. aureus. A moderate decrease
in antibacterial activity was observed in ketolide 8d bearing n-
BuNMe group in 30-position instead of dimethylamino group.
Replacement of 3-methylamino group in clarithromycin analog
5b with 1-methylguanidino group resulted in a significant drop
4
Erythromycin (1a)
Telithromycin (2d)
Cethromycin (2e)
8d
0.5
0.08
0.015
0.5
64
9d
in antibacterial activity with MIC rising from 4 to 32 lg/mL. Never-
theless, the compounds retained the general ability to inhibit bac-
terial growth thereby demonstrating for the first time, the
antibacterial activity of macrolide derivatives possessing an sp2
nitrogen function at the position 30. These results provide incentive
for further investigation of possible substitutions at this position.
Despite the removal of one methyl group at the 30 position of des-
osamine ring which was expected to decrease the possible steric
clash between a N-6 methyl group of dimethylated A2058 and des-
osamine residue, all synthesized derivatives were completely inac-
consistent with the important role of hydrogen bond between
20-OH in macrolides and N-1 in A2058. However, these macrolides
retained moderate antibacterial activities, and even the biggest dif-
ference (ꢀ500-fold) observed for 20-deoxycethromycin 7e (MIC50
4 lg/mL) versus cethromycin 2e (0.0075 lg/mL) was still much
smaller than the >104 increase in MIC for 2e in ATCC33591 strain
of S. aureus that carries the constitutively-expressed ermA gene
to macrolides in comparison to the native ATCC29213 strain. This

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N. LeTourneau et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4575–4578
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Based on our results, it can be concluded that the loss of the
hydrogen bond between 20-OH and N-1 A2058 residue as well as
the possible steric clash between 20-OH of desosamine sugar and
N-6 methyl groups in A2058-dimethylated bacterial ribosome are
not major factors that account for resistance of A2058-dimethylat-
ed strains. It can be further hypothesized that the possible struc-
tural factors for the dramatically decreased binding of macrolides
to A2058 dimethylated ribosome involve non-bonding interactions
between N-6 methyl group(s) of A2058 and water molecules coor-
dinated to the Mg2+ cation bound to phosphate groups of G2056
and G2057 residues of 23S RNA. If this hypothesis is correct, the
conformation of rRNA in A2058 dimethylated ribosome may be dif-
ferent from that seen in the native ribosome.
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