A click chemistry approach to pleuromutilin conjugates with nucleosides or acyclic nucleoside derivatives and their binding to the bacterial ribosome

Article abstract of DOI:10.1021/jm800261u

Pleuromutilin and its derivatives are antibacterial drugs that inhibit protein synthesis in bacteria by binding to ribosomes. To promote rational design of pleuromutilin based drugs, 19 pleuromutilin conjugates with different nucleoside fragments as side

Full text of DOI:10.1021/jm800261u

J. Med. Chem. 2008, 51, 4957–4967
4957
A Click Chemistry Approach to Pleuromutilin Conjugates with Nucleosides or Acyclic
Nucleoside Derivatives and Their Binding to the Bacterial Ribosome
Line Lolk,# Jacob Pøhlsgaard,‡ Anne Sofie Jepsen,# Lykke H. Hansen,‡ Henrik Nielsen,# Signe I. Steffansen,# Laura Sparving,#
Annette B. Nielsen,# Birte Vester,*,‡ and Poul Nielsen*,#
Nucleic Acid Center, Department of Physics and Chemistry and Department of Biochemistry and Molecular Biology, UniVersity of Southern
Denmark, 5230 Odense M, Denmark
ReceiVed March 12, 2008
Pleuromutilin and its derivatives are antibacterial drugs that inhibit protein synthesis in bacteria by binding
to ribosomes. To promote rational design of pleuromutilin based drugs, 19 pleuromutilin conjugates with
different nucleoside fragments as side chain extensions were synthesized by a click chemistry protocol.
Binding was assessed by chemical footprinting of nucleotide U2506 in 23S rRNA, and all conjugates bind
to varying degree reflecting their binding affinity to the peptidyl transferase center. The side chain extensions
also show various protections at position U2585. Docking studies of the conjugates with the highest affinities
support the conclusion that despite the various conjugations, the pleuomutilin skeleton binds in the same
binding pocket. The conjugated triazole moiety is well accommodated, and the nucleobases are placed in
different pockets in the 50S ribosomal subunit. The derivative showing the highest affinity and a significantly
better binding than pleuromutilin itself contains an adenine-9-ylpropylene triazole conjugate to pleuromutilin
C-22.
Introduction
The need for new or better antibiotics increases in parallel to
the appearance and spread of bacterial resistance to many
antibiotics. Despite synthesis and screening of numerous new
compounds, very few new antibiotics have appeared on the
marked in recent years. Advances in knowledge of binding and
mechanism of action of antibiotics in connection to modeling
now allow new approaches in the field of drug development.
Pleuromutilin1 1 (Figure 1) is a natural drug with modest
antibacterial activity. Pleuromutilin and derivatives thereof bind
to bacterial ribosomes in the peptidyl transferase center and
inhibits peptide bond formation.2 The semisynthetic pleuro-
mutilin derivative retapamulin 2 has recently been developed
as a drug for human use but only for topical treatment of skin
infections.3 Other derivatives named tiamulin 3 and valnemulin
4 are used in veterinary medicine to treat diseases in pigs and
poultry. Valnemulin 4 had also in extraordinary cases been used
in humans4 but are probably too toxic for approved use.
Resistance to pleuromutilin drugs have been reported5 but are
so far not very widespread and apparently only slowly develop-
ing. All in all, pleuromutilin derivatives have a potential for
human use with improvement in pharmacology, binding, and
ability to resist development of resistance.
Figure 1. Chemical structure of pleuromutilin and derivatives thereof.
The peptidyl transferase center (PTCa) where amino acids
are joined to produce nascent peptides is composed mainly of
the central loop of domain V of 23S rRNA and contains binding
sites for various antibiotics of clinical and veterinary importance
(Figure 2). Tiamulin 3 and valnemulin 4 have been footprinted
to the PTC.2 Numerous data on X-ray structures of 50S
ribosomal subunits have revealed the three-dimensional structure
of the PTC with drugs bound,6 including pleuromutilin deriva-
tives (Figure 2).7,8 The crystal structures have also revealed that
while the binding site of the pleuromutilin core is constant, the
RNA moieties interacting with the side chains move to
accommodate the variable sizes and chemical groups of the
drugs. This combination of a consistent core binding pocket
and a permissible area for side chain binding makes the
pleuromutilin family well suited for further modification. A large
number of derivatives of pleuromutilin have been investigated,
and the most numerous and successful group is semisynthetic
analogues like 2-4 with different moieties on the C-14 ester
* To whom correspondence should be addressed. For B.V.: phone, +45
65502377; fax, +45 65502467; e-mail, b.vester@bmb.sdu.dk. For P.N.:
phone, +45 65502565; fax, +45 66158780; e-mail, pon@ifk.sdu.dk.
#
Department of Physics and Chemistry.
Department of Biochemistry and Molecular Biology.
‡
a Abbreviations: PTC, peptidyl transferase center; TIPDS, 1,1,3,3-
tetraisopropyl-1,3-disiloxanediyl; TBDMS, tert-butyldimethylsilyl; TBDPS,
tert-butyldiphenylsilyl; TBAF, tetrabutylammonium fluoride; NOE, nuclear
Overhauser effect; CMCT, N1-cyclohexyl-N3-(2-morpholino)ethylcarbo-
diimide p-toluenesulfonate; AMV, avian myeloblastosis virus; VMD, visual
molecular dynamics.
10.1021/jm800261u CCC: $40.75
2008 American Chemical Society
Published on Web 08/05/2008

4958 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lolk et al.
Figure 2. (A) Model of the 70S bacterial ribosome, based on the E. coli structure.11 The 50S subunit is shown in blue and gray, and the 30S
subunit is shown in yellow and orange. (B) Secondary structure of the central part of domain V of E. coli 23S rRNA from the 50S subunit.
Footprinted nucleotides are indicated in red and orange, while potential stacking partners for the drugs are indicated in magenta. (C) The 50S
subunit of T. thermophilus12 cut in half to reveal the peptide exit tunnel and the pleuromutilin binding site indicated by a circle. tRNA in the P site
is also shown. (D) Tiamulin from a D. radiodurans 50S-tiamulin complex7 aligned into the PTC of the 50S ribosomal subunit of E. coli. The drug
is shown in stick representation and the surrounding rRNA as gray surface with the nearby 23S rRNA residues U2585 and U2506 indicated in red
and orange, respectively, and U2586 and A2062 indicated in magenta.
of pleuromutilin.9,10 Most of the pleuromutilin derivatives have
been made without any knowledge of their interactions in the
ribosome. Isosteric replacements of the thioether of 2-4 have
in general led to less active compounds, although 14-carbamates
have shown good activity. The presence of amino groups in
the side chain, like in 2-4, in general improves the water
solubility significantly.9
paving the way for rational design of new successful pleuro-
mutilin derivatives.
Results and Discussion
Design. The design in our approach hinges on two basic
criteria: (1) the synthetic availability of the new variants and
(2) the available space and chemical environment in the drug
binding pocket. In order to obtain a convenient and simple
parallel synthetic strategy for a range of pleuromutilin derivates,
we designed a click chemistry based approach for conjugating
a range of functionalities at the easily accessible C-22 position
of pleuromutilin (Figure 1). A linker containing a triazole as a
substitute of the thioether in the established derivatives 2-4
was obtained in all cases. Simple inspection of 50S X-ray crystal
structure data reveals that ample space exists for extension of
the drug in this direction and for accommodating the triazole
(Figure 2). The tricyclic pleuromutilin core is situated in a site
pocket of the PTC, while the introduced triazole is expected to
be positioned in the narrowest (but sufficiently broad) part of
PTC leading into the main peptide exit tunnel and allowing room
for the placement of bulky substituents. Because the ribosomal
PTC and exit tunnel are rich in interaction possibilities, primarily
in the form of RNA backbone, we synthesized the modified
drugs based on the space available. A detailed modeling
approach was only used on conjugates that showed a good
affinity for the ribosome binding site based on the footprinting
results. Therefore, the conjugates to be synthesized were chosen
to represent a broad spectrum of various nucleobases as well
as various linker lengths and constitutions. With respect to
synthetic availability, simple acyclic nucleosides displayed the
variation on nucleobases, whereas uridine and thymidine deriva-
tives were chosen for the variation in attachment points of
nucleoside ribose moieties. A systematic variation of 2′,3′,5′-
O- and 2′,3′,5′-C-alkynylation of the nucleoside was employed.
Chemical Synthesis. A general click chemistry strategy based
on the usual Huisgen-type [3 + 2] cycloaddition reaction
Since launched by Sharpless in 2001,13 the concept of click
chemistry has grown in impact within drug discovery.14 In
particular, the Huisgen [3 + 2] cycloaddition reacting terminal
alkynes with azides forming 1,2,3-triazoles has been a conve-
nient reaction. The reaction is chemoselective, it accepts almost
all other functional groups, it can be performed in aqueous
mixtures, and all atoms are used without the production of side
products. By use of Cu(I) to catalyze the reaction, a complete
regioselectivity for forming only 1,4- over 1,5-disubstituted
triazoles has been achieved with a very efficient procedure.15
In the present project, we use the pleuromutilin skeleton and
its strong and selective binding in the ribosome PTC as our
starting point. By converting the primary alcohol of pleuro-
mutilin into an azide, we attach a range of nucleoside derivatives
to this position in a parallel synthetic strategy using click
chemistry. Hereby we search the binding site of pleuromutilin
for further interactions and the nucleobases can potentially bind
strong and selectively herein because of their inherent H-bonding
properties in combination to stacking properties. Like the amine
moieties of 2-4, the nucleobases are expected also to improve
the water solubility of pleuromutilin. A variation of linker length
and constitution are explored as well as the presence of the intact
ribose or 2′-deoxyribose skeleton. Hence, a combination of
structural data from the X-ray structures of pleuromutilin-50S
complexes, molecular modeling, parallel synthesis of 19 pleu-
romutilin conjugates, and biochemical information from chemi-
cal footprinting is used to find high-affinity pleuromutilin
derivatives and to improve our understanding of drug binding

A Click Chemistry Approach to Pleuromutilin Conjugates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4959
Scheme 1. General Parallel Strategy for the Synthesis of Pleuromutilin Derivatives by Click Chemistrya
a Yields: 5 (37%), 6 (92%), 26 (99%), 27 (93%), 28 (91%), 29 (55%), 30 (91%), 31 (38%), 32 (41%), 33 (68%), 34 (48%), 35 (64%), 36 (83%), 37
(81%), 38 (54%), 39 (63%), 40 (96%), 41 (73%), 42 (87%), 43 (78%), 44 (75%).
between an azide and a terminal alkyne was used (Scheme 1).
Hence, pleuromutin 1 was converted into the known p-
toluenesulfonyl ester 5,16 which by a nucleophilic substitution
was further converted into the azide 6.16 This azide was reacted
with 19 different terminal alkynes 7-25 that were all linked to
nucleosides, nucleobases, or a simple aromatic replacement
thereof, through a variation of linker lengths. For the cycload-
dition, a standard protocol15 based on the catalysis by Cu(I),
which is generated in situ by Cu(II)SO4 and sodium ascorbate,
was applied. Microwave heating was used in most cases.
Hereby, the pleuromutilin derivates 26-44 all containing a
triazol-1,4-diyl linkage were prepared in mostly very good yields
(Scheme 1).
The alkynyl building blocks were prepared in a few steps
except for 7, 8, and 9, which were commercially available. The
known N1-propargylthymine 1017 was made by alkylation of
N3-benzoylthymine18 with propargyl bromide followed by
hydrolysis (Scheme 2). The two other N1-alkylated thymine
derivatives 11 and 12 were made by Mitsunobu reactions19 with
3-butyne-1-ol and 4-pentyne-1-ol, respectively, on N3-ben-
zoylthymine followed by hydrolysis. In a similar way, the two
N1-alkylated cytosine derivatives 1317 and 14 were made from
N4-acetylcytosine by using a basic alkylation20 and a Mitsunobu
reaction, respectively, followed by hydrolysis. The N9-alkylated
adenine derivatives 15,21 16, and 17 were made from unpro-
tected adenine using again either basic alkylation with propargyl
bromide or Mitsunobu protocols (Scheme 2).
The alkynyl nucleoside derivatives were made in a few steps
from either thymidine or uridine. The 2′-C-ethynyl arabino-
configured uridine derivative 18 was made from a known
stereoselective organolithium reaction on 5′,3′-TIPDS-protected
and 2′-oxidized uridine,22 followed by global desilylation. The
known 3′-C-ethynylxylo-configured uridine derivative 19 was
made in a similar way.23 The 5′-C-ethynylthymidine derivate
20 was made as an inseparable 2.6:1 epimeric mixture from
3′-TBDPS-protected thymidine 45, which was oxidized with a
Swern protocol and alkylated by a Grignard reaction24 to give
46 (Scheme 3). Deprotection afforded 20 in a good overall yield.
The two epimeric 5′-C-propargylthymidine derivatives 21 and
22 were made from 3′-O-TBDMS-protected thymidine 47,
which was oxidized and alkylated using a Barbier-type reaction
with zink dust and propargyl bromide (Scheme 4). After
chromatographic separation, 48 and 49 were obtained in good
yields from a 2:1 epimeric mixture and deprotected separately

4960 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Scheme 2. Synthesis of Alkynylated Nucleobasesa
Lolk et al.
Scheme 4. Synthesis of 5′-C-, 5′-O- and
3′-O-Propargylthymidine as well as 2′-O-Propargyluridinea
a Reagents and conditions: (a) (i) 3-brompropyne, K2CO3, DMF (100%);
(ii) aqueous NH3 (94% 10); (b) (i) 3-butyne-1-ol, PPh3, DEAD, toluene,
DCM (74%); (ii) NaOCH3, CH3OH (74% 11); (c) (i) 4-pentyne-1-ol, PPh3,
DEAD, toluene, DCM (85%); (ii) NaOCH3, CH3OH (83% 12); (d) (i)
3-brompropyne, K2CO3, DMF (40%); (ii) aqueous NH3, CH3OH (97% 13);
(e) (i) 4-pentyne-1-ol, PPh3, DEAD, toluene, DCM (9%); (ii) aqueous NH3,
CH3OH (100% 14); (f) 3-brompropyne, K2CO3, DMF (57% 15); (g)
3-butyne-1-ol, PPh3, DEAD, toluene, DCM (8% 16); (h) 4-pentyne-1-ol,
PPh3, DEAD, toluene, DCM (31% 17).
a Reagents and conditions: (a) (i) Dess-Martin periodinane, DCM; (ii)
Zn, 3-brompropyne, toluene, THF, NH4Cl (aq) (50%); (b) TBAF, THF (92%
21, 77% 22); (c) TIPDSCl2, pyridine (52% 50, 43% 51); (d) 3-brompropyne,
NaH, THF (40% 53, 88% 55, 43% 56); (e) TBAF, THF (69% 23, 32% 24,
88% 25). TBDMS ) tert-butyldimethylsilyl.
the corresponding contacts for 50 were only of approximately
1%. This established 50 and thereby 21 to have 5′(R)-
configuration and 51 and 22 to have 5′(S)-configuration.
The known 2′-O-propargyluridine 2326 was made by alky-
lation of 5′,3′-TIPDS-protected but N3-unprotected uridine 52
to give 53 followed by deprotection (Scheme 4). 3′-O-Propar-
gylthymidine 2427 was made similarly from alkylation of 5′-
O-TBDMS-thymidine 54 to give 55 followed by desilylation.
Finally, the new 5′-O-propargylthymidine 25 was made with a
similar strategy without protecting the nucleobase. Basic alky-
lation of 47 to give 56 was followed by desilylation to give 25
(Scheme 4).
Scheme 3. Synthesis of 5′-C-Ethynylthymidinea
Chemical Footprinting of Pleuromutilin Derivatives. The
interaction of the 19 pleuromutilin derivatives 26-44 with the
peptidyl transferase cavity was examined to compare ribosomal
binding of the fungal natural product pleuromutilin 1 and the
semisynthetic derivative tiamulin 3 with binding of the synthe-
sized derivatives. The drug binding affinities on the ribosome
were studied by chemical probing, where accessible rRNA
nucleotides were modified by N1-cyclohexyl-N3-(2-morpholi-
no)ethylcarbodiimide p-toluenesulfonate (CMCT). After forma-
tion of pleuromutilin derivative-70S ribosome complexes, the
antibiotic-70S complexes and control samples of 70S ribosomes
were treated with CMCT to probe the accessibility of N3 of
uridines. Primer extension with reverse transcriptase was then
used to identify alterations induced by drug binding. Autorad-
iograms showing the effect on nucleotides U2506 and U2585
in 23S rRNA are presented in Figure 3, and the quantification
a Reagents and conditions: (a) (i) ClCOCOCl, DMSO, DCM, Et3N; (ii)
HCt CMgBr,THF(30%);(b)TBAF,THF(71%).TBDPS)tert-butyldiphenylsilyl.
to give 21 and 22. In order to establish the 5′-configuration in
the two stereoisomers, each of these were converted to the
TIPDS-protected compound, 50 and 51, respectively, in order
to use the decrease in conformational freedom of the bicyclic
1
system in combination with NOE difference H NMR spec-
troscopy. This method has been used in other studies of 5′-C-
alkylated thymidine derivatives.24,25 Thus, large mutual NOE-
contacts of 6-8% were detected between H3′ and H5′ in the
NOE difference spectra for compound 50 proving the 5′(R)-
configuration, whereas no mutual contacts between H3′and H5′
were detected for 51. On the other hand, strong mutual contacts
of 8-9% between H4′ and H5′ were detected for 51, whereas

A Click Chemistry Approach to Pleuromutilin Conjugates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4961
conserved tricyclic cores of the compounds are placed identically
in a binding pocket involving nucleotide U2506 as illustrated
in Figure 2. The differences in the footprints of the drugs at
U2506 thus reflect the binding affinity of the compounds. In
contrast, the effects at U2585 are considered correlated with
placement of the various conjugated substituents because
previous data on pleuromutilin derivatives indicated both
enhancement and protection effects depending on various side
chain extensions.28 The weakness of this particular chemical
footprinting assay for assessing affinity of the drug derivatives
for the ribosomal binding site is that the footprint at U2506 is
relatively weak. Thus, we are quantifying and comparing
delicate effects on a weak modification site implying some
inaccuracy. The strength of this assay, on the other hand, is
that the assayed protection of modification of specific nucle-
otides ensures that we are looking at specific binding.
The overall picture from the data in Table 1 is that all the
tested compounds bind to the pocket in PTC (near U2506) as
do tiamulin and pleuromutilin. The CMCT accessibility repre-
senting binding affinity varies from 0.17 to 0.73 at 5 µM, but
at 50 µM all compounds bind strongly with accessibilities from
0.06 to 0.19. In other words, the affinity of the mutilin skeleton
is strong and sufficient to allow the entire range of nucleoside
derivatives attached to it, as well the triazole linkers, to follow
it into the binding pocket. Concerning the interaction with
U2585, all the compounds show some interaction though with
larger variation. However, in contrast to pleuromutilin, all the
other compounds protect well, at least at a 50 µM concentration.
Many of the new compounds demonstrate a stronger interaction
with this position than does tiamulin, but tiamulin has the highest
measured protection in the binding pocket for the mutilin core
as seen at the U2506 footprint.
Figure 3. Gel autoradiograms showing footprints of the conjugates
on ribosomes modified with CMCT. Nucleotides exhibiting altered
reactivities in the presence of the pleuromutilin derivatives are indicated.
Dideoxy sequencing lanes are denoted by G, A, U, and C. Lanes are
labeled to denote samples from chemically unmodified 70S ribosomes
in the absence of drugs (control, Co) and 70S ribosomes modified in
the absence of drugs (CMCT, M). Ribosomes modified in the presence
of the derivatives are marked with their numbers, and wedges denote
a high (50 µM) or low (5 µM) drug concentration.
Table 1. Footprinting Data on Pleuromutilin and Derivatives Thereof
U2506c
U2585c
length of
linkera nucleobaseb 5 µM 50 µM 5 µM 50 µM
compd
tiamulin 3
0.10
0.17
0.21
0.24
0.27
0.29
0.31
0.32
0.33
0.37
0.38
0.41
0.42
0.44
0.53
0.54
0.61
0.61
0.62
0.69
0.73
0.05
0.06
0.08
0.03
0.05
0.05
0.06
0.09
0.09
0.10
0.10
0.08
0.10
0.12
0.10
0.14
0.18
0.16
0.19
0.18
0.19
0.59
0.33
0.48
0.45
0.56
0.61
0.54
0.59
0.69
0.40
0.50
0.46
0.99
0.73
0.57
0.72
0.74
0.69
0.93
0.75
0.73
0.58
0.22
0.41
0.18
0.26
0.35
0.29
0.29
0.35
0.26
0.25
0.24
0.98
0.41
0.36
0.29
0.35
0.38
0.36
0.37
0.37
The best analogues in the series appear to have acyclic linkers
of three carbon atoms between the triazole and the nucleobase.
However, also a cyclic analogue with the same length, 38, is
among the best binding compounds. The adenine derivative 36
shows the highest affinity, but in general the constitution of the
nucleobase does not seem very important, as also the phenyl
derivative 28 is binding well. Another general trend is that the
compounds with the shortest linkers of only one carbon atom
between the nucleobase and the triazol are showing the lowest
binding affinities. Then again, there are some deviations to this
observation, as compound 26 binds better than expected,
whereas the thymine derivative 31 binds with a lower affinity
than to be expected. All the remaining compounds bind with
intermediate affinities.
36
28
38
33
27
26
40
41
3
3
3
3
2
1
5
5
2
4
3
A
Ph
U
C
Ph
Ph
T
T
A
T
35
39
31
T
pleuromutilin 1
43
30
42
37
32
44
29
34
5
2
4
2
1
6
1
1
T
T
U
U
C
T
T
A
Docking Studies. The two pleuromutilin derivatives display-
ing the best binding affinities, compounds 36 and 28, were
chosen for a molecular docking study. Attempts to place the
drugs in the binding pocket by the use of a standard docking
protocol were unsuccessful. For agreement with the published
drug binding modes, we had to employ a simulated flexible site
(explained in details in the Experimental Section) consisting of
all residues containing an atom within 25 Å of 23S rRNA
residue 2504. The calculations with the flexible docking protocol
placed the drugs correctly into the binding pocket. The criterion
for successful docking was set as placement of the pleuromutilin
core in the consensus site to be in agreement with the
footprinting data on the U2506 position. The final docking
results showed similar binding modes for the two drugs, with
the conjugated groups extending into the peptide exit tunnel,
pointing toward the exit itself (Figure 4). No specific hydrogen
bonding with ribosomal bases was detected for the adenine in
36, and the terminal groups of both 28 and 36 seem to have a
a The length of the linker defined as the number of atoms separating the
triazole from the nucleobase. b A) adenin-9-yl, C ) cytosin-1-yl, U )
uracil-1-yl, T ) thymin-1-yl, Ph ) phenyl. c Positions in E. coli 23S rRNA
affected by CMCT modification: U2506 at the binding pocket of the mutilin
core and U2585 near the variations in pleuromutilin conjugations (see text).
The numbers represent the relative CMCT accesibility in the presence of
the drug in 5 or 50 µM concentrations compared to absence of the drug.
For example, 0.10 means that the intensity of the band on the gel in presence
of the drug is only 10% of the intensity of the corresponding band from
samples without the drug.
of protection against CMCT modification is tabulated in Table
1 ordered by the binding affinity for U2506 as measured at a 5
µM concentration. The footprinting data show that all derivatives
protect U2506 and U2585 when bound to the 70S ribosome.
Both footprinting data and X-ray data on binding of pleuro-
mutilin derivatives to the PTC center2,7,8,28 indicate that the

4962 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lolk et al.
Figure 4. Docking studies of pleuromutilin derivative 36 (A) and 28 (B) in the PTC of the 50S ribosomal subunit. The heavy atoms of the 10 best
scoring solutions of each drug are shown as overlaying stick representation. The cut surface is shown in green as in Figure 2. The surrounding
rRNA is shown as gray surface with the nearby 23S rRNA residues U2585 and U2506 indicated in red and orange, respectively. Nucleotides
U2586 and A2062, possibly implicated in stacking interactions, are shown in magenta.
large degree of freedom, interacting with the ribosome mainly
through stacking with either nucleobase A2062 or U2586.
A2062 is known to be highly flexible7 and might adopt a
conformation more favorable for stacking with the conjugates
than can be modeled by the flexibility in our docking assay. A
stacking interaction between the triazole and U2585 is also
indicated.
the demand of protecting groups and one or more global
deprotection steps would have rendered this study impossible.
We expect to continue the design of easily available pleuro-
mutilin derivatives with improved affinity in the PTC on the
basis of the present click chemistry protocol.
Conclusion
Discussion. The results of the present study follow the line
from the literature demonstrating that the C-14 side chain of
pleuromutilin can be voluminously derivatized and thereby
improving the binding affinity of the pleuromutilin core for the
PTC binding pocket. It is remarkable that all the 19 synthesized
derivatives actually bind in the PTC. The present data also prove
that nucleobases can be applied in the side chain of C-14
modified pleuromutilin derivatives. Thus, derivatives with simple
linkers containing the 1,4-disubstituted triazole in connection
to a three-carbon chain show stronger affinities for the PTC
than the parent pleuromutilin. The almost similar affinity of the
adenine and the phenyl derivatives (36 and 28, respectively)
indicates that aromatic rings in general can be used to further
improve the binding affinity. The study also demonstrates that
the triazole is well accepted in the PTC and can replace the
typically used thioether or carbamate linkers without compro-
mising the binding affinity significantly. In fact, a stacking
interaction with U2585 might favor the binding. Hence, small
heterocycles can be adopted near the mutilin core in the binding
pocket, and importantly, future drug optimization can be based
on the triazole and the click chemistry approach. Conformational
restriction of the three-carbon linker in 36 is an obvious target
for improvement. Compound 38 can be considered a confor-
mationally restricted derivative of 31, and the better binding of
38 indicates that a similar or maybe different conformational
restriction of the adenine derivative 36 might lead to a further
increase in affinity. With a view to further steps toward drug
development we also expect the conjugation of natural nucleo-
bases to pleuromutilin to improve water solubility and eventually
body distribution as well as being of low toxicity.
The pleuromutilin derivatives synthesized and examined in
this work were all prepared in very few steps by a semisynthesis
from pleuromutilin. They all bind to the peptidyl transferase
center in the ribosome and most of them with a higher affinity
than the natural pleuromutilin antibiotic. Chemical footprints
and docking studies in combination rationalized the binding of
the mutilin core near the U2506 in the PTC site and the
nucleobase conjugated side chains near position U2585 in 23S
rRNA. There is seemingly a potential for drug improvement
by lengthening and derivitization of the pleuromutilin side chain
that can be explored by molecular modeling of drug molecules
in the ribosome based on X-ray crystallography models of
ribosomes. A click chemistry approach demonstrated a conve-
nient approach toward a library of active pleuromutilin deriva-
tives in the search for future therapeutic antibiotics.
Experimental Section
General. Reactions were performed under an atmosphere of
nitrogen when anhydrous solvents were used. Column chromatog-
raphy was carried out on glass columns using silica gel 60
(0.040-0.063 mm). NMR spectra were recorded at 300 MHz for
1H NMR and 75 MHz for 13C NMR. The δ values are in ppm
relative to tetramethylsilane as internal standard. Assignments of
NMR spectra are based on 2D spectra and follow the standard
numbering of pleuromutilin as shown below as well as standard
carbohydrate and nucleoside style; i.e., the carbon atom next to a
nucleobase is assigned C-1′, etc. HR ESI mass spectra were
recorded in positive-ion mode.
General Procedure for Synthesizing the Pleuromutilin
Conjugates from Pleuromutilin Azide (6) and Nucleoside
Alkyne Derivatives (7-25). Compound 6 (0.20 mmol) and the
alkyne derivate (0.22 mmol) were dissolved in a mixture of t-BuOH
and water (2.5 mL, 1:1 v/v) in a microwave vial. Sodium ascorbate
(4 mg, 0.02 mmol) and CuSO4 · 5H2O (3 mg, 0.01 mmol) were
added, and the vial was sealed and heated with stirring in the
microwave reactor (Emrys Creator) at 110 °C for 30 min. The
residue was concentrated under reduced pressure and purified by
The employed click chemistry strategy has made a simple
preparation of a series of compounds directly possible in
medium to high yields and, importantly, without the need for
protecting groups. The cycloaddition of terminal alkynes and
an azide is completely chemoselective, thus allowing the
presence of unprotected functional groups. Other methods with

A Click Chemistry Approach to Pleuromutilin Conjugates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4963
121.9 (CH(triazole)), 117.5 (C20), 74.4 (C11), 70.7 (C14), 57.9
(C4), 51.4 (C22), 45.3 (C9), 44.6 (C13), 43.9 (C12), 41.8 (C5),
36.5 (C6), 36.0 (C10), 35.3 (PhCH2), 34.3 (C2), 30.9 (Ph(CH2)CH2),
30.3 (C8), 26.7 (C7), 26.3 (C18), 25.0 (PhCH2CH2), 24.7 (C1),
16.7 (C16), 14.6 (C15), 11.4 (C17); HRMS (ESI) m/z (548.3485
[M + H]+, C33H46N3O4 calcd 548.3483).
Synthesis of 22-(4-(Thymine-1-ylmethyl)-1,2,3-triazole-1-yl)-22-
deoxypleuromutilin (29). The general procedure using compound
6 (149 mg, 0.37 mmol), compound 10 (60 mg crude, 0.22 mmol),
t-BuOH/H2O (4 mL), sodium ascorbate (6 mg, 0.03 mmol), and
CuSO4 ·5H2O (4 mg, 0.02 mmol) was applied. Yield, 68 mg, 55%;
Rf ) 0.48 (15% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 9.36 (1H,
m, NH), 7.85 (1H, s, CH(triazole)), 7.32 (1H, d, J ) 1.4, H6(T)),
6.38 (1H, dd, J ) 11.1, 17.3, H19), 5.77 (1H, d, J ) 8.4, H14),
5.31 (1H, d, J ) 11.1, H20), 5.20 (1H, d, J ) 17.3, H20), 5.12-5.00
(2H, m, H22), 4.97 (2H, AB, J ) 15.0, NCH2), 3.35 (1H, dd, J )
6.5, 10.4, H11), 2.30-2.02 (5H, m, H2, H4, H10, H13), 1.90 (3H,
d, J ) 1.4, CH3(T)), 1.85-1.05 (9H, m, H1, H6, H7, H8, 11-OH,
H13), 1.27 (3H, s, H15), 1.16 (3H, s, H18), 0.86 (3H, d, J ) 7.0,
H17), 0.65 (3H, d, J ) 6.9, H16); 13C NMR (CDCl3) δ 216.5 (C3),
164.8, 164.0 (C21, C4(T)), 151.0 (C2(T)), 142.4 (C(triazole)), 140.0
(C6(T)), 138.5 (C19), 125.2 (CH(triazole)), 117.6 (C20), 111.4
(C5(T)), 74.4 (C11), 71.1 (C14), 57.9 (C4), 51.5 (C22), 45.3 (C9),
44.6 (C13), 44.0 (C12), 43.0 (NCH2), 41.8 (C5), 36.5 (C6), 36.0
(C10), 34.3 (C2), 30.2 (C8), 26.7 (C7), 26.3 (C18), 24.7 (C1), 16.8
(C16), 14.5 (C15), 12.3 (CH3(T)), 11.4 (C17); HRMS (ESI) m/z
(590.2928 [M + Na]+, C30H41N5O6Na calcd 590.2950).
Synthesis of 22-(4-(2-(Thymine-1-yl)ethyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (30). The general procedure using compound
6 (94 mg, 0.23 mmol), compound 11 (33 mg, 0.18 mmol), t-BuOH/
H2O (3 mL), sodium ascorbate (4 mg, 0.02 mmol), and
CuSO4 ·5H2O (3 mg, 0.01 mmol) was applied. Yield, 97 mg, 91%;
Rf ) 0.68 (20% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 9.39 (1H,
s, NH), 7.46 (1H, s, CH(triazole)), 6.95 (1H, d, J ) 1.1, H6(T)),
6.36 (1H, dd, J ) 10.7, 17.3, H19), 5.77 (1H, d, J ) 8.5, H14),
5.30 (1H, dd, J ) 1.1, 10.7, H20), 5.18 (1H, dd, J ) 1.1, 17.3,
H20), 5.09-4.97 (2H, m, H22), 4.07 (2H, t, J ) 6.8, NCH2), 3.34
(1H, m, H11), 3.13 (2H, t, J ) 6.8, NCH2CH2), 2.29-2.03 (5H,
m, H2, H4, H10, H13), 1.82 (3H, d, J ) 1.1, CH3(T)), 1.77-1.05
(9H, m, H1, H6, H7, H8, 11-OH, H13), 1.32 (3H, s, H15), 1.15
(3H, s, H18), 0.86 (3H, d, J ) 7.0, H17), 0.68 (3H, d, J ) 7.0,
H16); 13C NMR (CDCl3) δ 216.6 (C3), 165.0, 164.4 (C21, C4(T)),
151.0 (C2(T)), 143.8 (C(triazole)), 141.0 (C6(T)), 138.5 (C19),
123.5 (CH(triazole)), 117.5 (C20), 110.3 (C5(T)), 74.4 (C11), 70.9
(C14), 57.9 (C4), 51.4 (C22), 48.0 (NCH2), 45.3 (C9), 44.6 (C13),
43.9 (C12), 41.7 (C5), 36.4 (C6), 35.9 (C10), 34.3 (C2), 30.2 (C8),
26.7 (C7), 26.3 (C18), 24.9 (NCH2CH2), 24.7 (C1), 16.7 (C16),
14.5 (C15), 12.1 (CH3(T)), 11.4 (C17); HRMS (ESI) m/z (604.3078
[M + Na]+, C31H43N5O6Na calcd 604.3107).
column chromatography (0-10% CH3OH in CH2Cl2) to give the
product as a white foam.
Synthesis of 22-(4-Benzyl-1,2,3-triazole-1-yl)-22-deoxypleuro-
mutilin (26). The general procedure was slightly modified using
compound 6 (71 mg, 0.176 mmol), 3-phenylpropyne 7 (0.022 mL,
0.157 mmol), t-BuOH/H2O (1.5 mL), sodium ascorbate (5 mg,
0.025 mmol), and CuSO4 ·5H2O (2 mg, 0.008 mmol) and conven-
tional heating at 70 °C for 12 h. The mixture was extracted with
EtOAc (10 mL), washed with water (2 × 10 mL), dried (MgSO4),
and concentrated under reduced pressure. The residue was purified
by column chromatography (10-30% EtOAc in petrol ether). Yield,
81 mg, 99%; Rf ) 0.36 (EtOAc and petrol ether, 2:1 v/v); 1H NMR
(CDCl3) δ 7.33-7.20 (6H, m, CH(triazole), Ph), 6.39 (1H, dd, J
) 10.8, 16.8, H19), 5.78 (1H, d, J ) 8.2, H14), 5.32 (1H, d, J )
10.8, H20), 5.19 (1H, d, J ) 16.8, H20), 5.06-4.93 (2H, m,
PhCH2), 4.11 (2H, s, H22), 3.34 (1H, m, H11), 2.40-2.00 (5H, m,
H2, H4, H10, H13), 1.80-1.12 (9H, m, H1, H6, H7, H8, 11-OH,
H13), 1.28 (3H, s, H15), 1.16 (3H, s, H18), 0.87 (3H, d, J ) 6.9,
H17), 0.64 (3H, d, J ) 7.2, H16); 13C NMR (CDCl3) δ 216.6 (C3),
165.1 (C21), 148.2 (C(triazole)), 138.7 (C19), 138.5, 128.7, 128.6,
126.5 (Ph), 122.5 (triazol), 117.3 (C20), 74.5 (C11), 70.8 (C14),
58.0 (C4), 51.4 (C22), 45.4 (C9), 44.7 (C13), 44.0 (C12), 41.8 (C5),
36.6 (C6), 36.0 (C10), 34.4 (C2), 32.2 (PhCH2), 30.3 (C8), 26.8
(C7), 26.3 (C18), 24.8 (C1), 16.6 (C16), 14.6 (C15), 11.5 (C17);
HRMS (ESI) m/z (542.3014 [M + Na]+, C31H41N3O4Na calcd
542.3014).
Synthesis of 22-(4-(2-Phenylethyl)-1,2,3-triazole-1-yl)-22-deoxy-
pleuromutilin (27). The general procedure using compound 6 (77
mg, 0.19 mmol), 4-phenyl-1-butyne 8 (29 mg, 0.22 mmol), t-BuOH/
H2O (2.5 mL), sodium ascorbate (4 mg, 0.02 mmol), and
CuSO4 ·5H2O (3 mg, 0.01 mmol) was applied. Yield, 95 mg, 93%;
Rf ) 0.58 (10% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 7.31-7.19
(6H, m, CH(triazole), Ph), 6.41 (1H, dd, J ) 11.0, 17.4, H19),
5.79 (1H, d, J ) 8.4, H14), 5.33 (1H, dd, J ) 1.3, 11.0, H20), 5.20
(1H, dd, J ) 1.3, 17.3, H20), 5.06-4.94 (2H, m, H22), 3.35 (1H,
dd, J ) 6.4, 10.4, H11), 3.10-2.97 (4H, m, PhCH2CH2), 2.30-2.03
(5H, m, H2, H4, H10, H13), 1.78-1.07 (9H, m, H1, H6, H7, H8,
11-OH, H13), 1.33 (3H, s, H15), 1.17 (3H, s, H18), 0.87 (3H, d, J
) 6.9, H17), 0.68 (3H, d, J ) 6.9, H16); 13C NMR (CDCl3) δ
216.6 (C3), 165.2 (C21), 147.8 (C(triazole)), 141.0 (Ph), 138.6
(C19), 128.4, 126.1 (Ph), 122.1 (CH(triazole)), 117.6 (C20), 74.5
(C11), 70.7 (C14), 58.0 (C4), 51.4 (C22), 45.4 (C9), 44.6 (C13),
43.9 (C12), 41.8 (C5), 36.5 (C6), 36.0 (C10), 35.4 (PhCH2), 34.3
(C2), 30.3 (C8), 27.4 (PhCH2CH2), 26.7 (C7), 26.3 (C18), 24.8
(C1), 16.7 (C16), 14.6 (C15), 11.4 (C17); HRMS (ESI) m/z
(556.3168 [M + Na]+, C32H43N3O4Na calcd 556.3146).
Synthesis of 22-(4-(3-(Thymine-1-yl)propyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (31). The general procedure using compound
6 (129 mg, 0.32 mmol), compound 12 (containing 12% w/w Et3N,
50 mg, 0.23 mmol), t-BuOH/H2O (3 mL), sodium ascorbate (5.5
mg, 0.03 mmol), and CuSO4 ·5H2O (4 mg, 0.02 mmol) was applied.
Yield, 52 mg, 38%; Rf ) 0.47 (15% CH3OH in CH2Cl2); 1H NMR
(CDCl3) δ 9.49 (1H, s, NH), 7.58 (1H, s, CH(triazole)), 7.10 (1H,
d, J ) 1.0, H6(T)), 6.39 (1H, dd, J ) 10.6, J ) 17.2, H19), 5.78
(1H, d, J ) 8.4, H14), 5.30 (1H, dd, J ) 1.2, 10.6, H20), 5.08
(1H, dd, J ) 1.2, 17.2, H20), 5.14-5.02 (2H, m, H22), 3.77 (2H,
t, J ) 7.0, NCH2), 3.34 (1H, dd, J ) 6.5, 10.2, H11), 2.78 (2H, t,
J ) 7.0, NCH2CH2), 2.29-1.05 (14H, m, H1, H2, H4, H6, H7,
H8, H10, 11-OH, H13), 1.90 (3H, d, J ) 1.0, CH3(T)), 1.31 (3H,
s, H15), 1.15 (3H, s, H18), 0.86 (3H, d, J ) 7.0, H17), 0.68 (3H,
d, J ) 6.9, H16); HRMS (ESI) m/z (618.3257 [M + Na]+,
C32H45N5O6Na calcd 618.3262).
Synthesis of 22-(4-(3-Phenylpropyl)-1,2,3-triazole-1-yl)-22-deoxy-
pleuromutilin (28). The general procedure using compound 6 (71
mg, 0.18 mmol), 5-phenyl-1-pentyne 9 (29 mg, 0.20 mmol),
t-BuOH/H2O (2.5 mL), sodium ascorbate (4 mg, 0.02 mmol) and
CuSO4 5H2O (2 mg, 0.01 mmol) was applied. Yield, 88 mg, 91%;
Rf ) 0.29 (5% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 7.35 (1H,
s, CH(triazole)), 7.31-7.18 (5H, m, Ph), 6.41 (1H, dd, J ) 11.0,
17.3, H19), 5.81 (1H, d, J ) 8.5, H14), 5.33 (1H, d, J ) 11.0,
H20), 5.20 (1H, d, J ) 17.3, H20), 5.10-4.96 (2H, m, H22), 3.35
(1H, m, H11), 2.77 (2H, t, J ) 7.7, PhCH2), 2.70 (2H, t, J ) 7.6,
NCH2), 2.31-1.98 (7H, m, H2, H4, H10, H13, PhCH2CH2),
1.79-1.08 (9H, m, H1, H6, H7, H8, 11-OH, H13), 1.34 (3H, s,
H18), 1.17 (3H, s, H15), 0.88 (3H, d, J ) 6.7, H17), 0.70 (3H, d,
J ) 6.7, H16); 13C NMR (CDCl3) δ 216.6 (C3), 165.2 (C21), 148.4
(C(triazole)), 141.8 (Ph), 138.6 (C19), 128.4, 128.3, 125.8 (Ph),
Synthesis of 22-(4-(Cytosine-1-ylmethyl)-1,2,3-triazole-1-yl)-22-
deoxypleuromutilin (32). The general procedure was slightly
modified using compound 6 (65 mg, 0.16 mmol), compound 13
(20 mg, 0.14 mmol), t-BuOH/H2O (4 mL), sodium ascorbate (5.5
mg, 0.03 mmol), CuSO4 ·5H2O (4.5 mg, 0.02 mmol), and conven-
tional heating at 70 °C for 12 h. Yield, 31 mg, 41%; Rf ) 0.45

4964 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lolk et al.
(20% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 7.94 (1H, s,
CH(triazole)), 7.49 (1H, d, J ) 6.8, H6(C)), 6.34 (1H, dd, J )
11.3, 17.6, H19), 5.73-5.67 (2H, m, H14, H5(C)), 5.25 (1H, d, J
) 11.3, H20), 5.18-4.94 (5H, m, H20, H22, NCH2), 3.34 (1H, m,
H11), 2.65 (2H, br s, NH2), 2.23-1.87 (5H, m, H2, H4, H10, H13),
1.73-1.07 (9H, m, H1, H6, H7, H8, 11-OH, H13), 1.19 (3H, s,
H15), 1.11 (3H, s, H18), 0.84 (3H, d, J ) 6.7, H17), 0.60 (3H, d,
J ) 6.7, H16); 13C NMR (CDCl3) δ 217.0 (C3), 166.2, 165.0
(C2(C), C21), 156.5 (C4(C)), 145.4 (C(triazole)), 143.2 (C6(C)),
138.7 (C19), 125.5 (CH(triazole)), 117.3 (C20), 95.0 (C5(C)), 74.3
(C11), 71.0 (C14), 57.9 (C4), 51.6 (C22), 45.3 (C9), 44.5 (C13),
43.9 (C12), 41.7 (C5), 36.5 (C6), 35.9 (C10), 34.4 (C2), 30.2 (C8),
26.7 (C7), 26.5 (C18), 24.7 (C1), 16.68, 16.61 (NCH2, C16), 14.4
(C15), 11.4 (C17); HRMS (ESI) m/z (575.2962 [M + Na]+,
C29H40N6O5Na calcd 575.2953).
Synthesis of 22-(4-(3-(Cytosine-1-yl)propyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (33). The general procedure using compound
6 (46 mg, 0.11 mmol), compound 14 (15 mg, 0.09 mmol), t-BuOH/
H2O (2 mL), sodium ascorbate (2 mg, 0.01 mmol), and
CuSO4 ·5H2O (1.2 mg, 0.01 mmol) was applied. Yield, 33 mg, 68%;
Rf ) 0.66 (20% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 7.51 (1H,
s, CH(triazole)), 7.35 (1H, m, H6(C)), 6.37 (1H, dd, J ) 11.0, 15.9,
H19), 5.76 (1H, d, J ) 8.4, H14), 5.28-4.96 (5H, m, H5(C), H20,
H22), 3.85-3.76 (2H, m, NCH2), 3.36 (1H, m, H11), 2.78-2.70
(2H, m, N(CH2)2CH2), 2.52-1.97 (9H, m, H2, H4, H10, H13,
NCH2CH2, NH2), 1.86-1.09 (9H, m, H1, H6, H7, H8, 11-OH,
H13), 1.29 (3H, s, H15), 1.15 (3H, s, H18), 0.85 (3H, d, J ) 6.1,
H17), 0.67 (3H, d, J ) 6.1, H16); 13C NMR (CDCl3) δ 216.8 (C3),
166.0 (C2(C)), 165.2 (C21), 156.8 (C4(C)), 147.1 (C6(C)), 145.9
(C(triazole)), 138.7 (C19), 122.7 (CH(triazole)), 117.4 (C20), 94.4
(C5(C)), 74.4 (C11), 70.8 (C14), 57.9 (C4), 51.5 (C22), 49.1
(NCH2), 45.3 (C9), 44.6 (C13), 44.0 (C12), 41.8 (C5), 36.5 (C6),
36.0 (C10), 34.4 (C2), 30.3 (C8), 28.4 (N(CH2)2CH2), 26.7 (C7),
26.5 (C18), 24.7 (C1), 22.2 (NCH2CH2), 16.7 (C16), 14.6 (C15),
11.4 (C17); HRMS (ESI) m/z (603.3242 [M + Na]+, C31H44N6O5Na
calcd 603.3266).
Synthesis of 22-(4-(Adenine-9-ylmethyl)-1,2,3-triazole-1-yl)-22-
deoxypleuromutilin (34). The general procedure was slightly
modified using compound 6 (28 mg, 0.07 mmol), compound 15
(11 mg, 0.06 mmol), t-BuOH/H2O (8 mL), sodium ascorbate (1.5
mg, 0.01 mmol), CuSO4 ·5H2O (1.4 mg, 0.01 mmol), stirring at
room temperature for 19 h and then at 85 °C for 19 h. Yield, 17
mg, 48%; Rf ) 0.64 (20% CH3OH in CH2Cl2); 1H NMR (CDCl3)
δ 8.38 (1H, s, H2(A)), 7.99 (1H, s, H8(A)), 7.77 (1H, s,
CH(triazole)), 7.26 (2H, br s, NH2), 6.38 (1H, dd, J ) 11.1, 17.3,
H19), 5.76 (1H, d, J ) 8.6, H14), 5.51 (2H, s, NCH2), 5.32 (1H,
dd, J ) 1.5, 11.1, H20), 5.20 (1H, dd, J ) 1.5, 17.3, H20),
5.10-4.96 (2H, m, H22), 3.33 (1H, dd, J ) 6.8, 10.9, H11),
2.27-1.99 (5H, m, H2, H4, H10, H13), 1.77-1.07 (9H, m, H1,
H6, H7, H8, 11-OH, H13), 1.56 (3H, s, H15), 1.15 (3H, s, H18),
0.86 (3H, d, J ) 7.0, H17), 0.61 (3H, d, J ) 6.8, H16); 13C NMR
(CDCl3) δ 217.1 (C3), 165.6 (C21), 156.0 (C6(A)), 152.6 (C2(A)),
149.3 (C4(A)), 142.7 (C(triazole)), 140.7, 140.6 (C8(A), C19), 125.1
(CH(triazole)), 120.8 (C5(A)), 115.5 (C20), 72.6 (C11), 70.5 (C14),
57.1 (C4), 51.1 (C22), 44.9 (C9), 44.2 (C13), 43.3 (C12), 41.5 (C5),
38.0 (NCH2), 36.5 (C6), 36.2 (C10), 34.0 (C2), 30.1 (C8), 28.6
(C7), 26.6 (C18), 24.5 (C1), 16.1 (C16), 14.2 (C15), 11.6 (C17);
HRMS (ESI) m/z (599.3075 [M + Na]+, C30H40N8O4Na calcd
599.3064).
Synthesis of 22-(4-(2-(Adenine-9-yl)ethyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (35). The general procedure using compound
6 (17 mg, 0.09 mmol), compound 16 (53 mg, 0.13 mmol), t-BuOH/
H2O (3 mL), sodium ascorbate (2.2 mg, 0.01 mmol), and
CuSO4 ·5H2O (1.5 mg, 0.01 mmol) was applied. Yield, 34 mg, 64%;
Rf ) 0.73 (20% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 8.35 (1H,
s, H2(A)), 7.68 (1H, s, H8(A)), 7.32 (1H, s, CH(triazole)), 6.38
(1H, dd, J ) 11.1, 17.4, H19), 6.12 (2H, br s, NH2), 5.78 (1H, d,
J ) 8.4, H14), 5.32 (1H, d, J ) 11.1, H20), 5.19 (1H, d, J ) 17.4,
H20), 5.09-4.93 (2H, m, H22), 4.62 (2H, t, J ) 6.8, NCH2),
3.48-3.33 (3H, m, NCH2CH2, H11), 2.30-2.20 (3H, m, H2, H10),
2.08-2.06 (2H, m, H4, H13), 1.78-1.05 (9H, m, H1, H6, H7, H8,
11-OH, H13), 1.30 (3H, s, H15), 1.17 (3H, s, H18), 0.88 (3H, d, J
) 7.1, H17), 0.66 (3H, d, J ) 7.0, H16); 13C NMR (CDCl3) δ
216.6 (C3), 164.9 (C21), 155.6 (C6(A)), 152.9 (C2(A)), 149.8
(C4(A)), 143.8 (C(triazole)), 140.8 (C8(A)), 138.6 (C19), 123.2
(CH(triazole)), 119.5 (C5(A)), 117.4 (C20), 74.4 (C11), 71.0 (C14),
57.9 (C4), 51.4 (C22), 45.3 (C9), 44.6 (C13), 43.9 (C12), 43.1
(NCH2), 41.7 (C5), 36.4 (C6), 35.9 (C10), 34.3 (C2), 30.2 (C8),
26.7 (C7), 26.4 (C18), 26.0 (NCH2CH2), 24.7 (C1), 16.7 (C16),
14.5 (C15), 11.4 (C17), 36.2 (C10), 34.0 (C2), 30.1 (C8), 28.6 (C7),
26.6 (C18), 24.5 (C1), 16.1 (C16), 14.2 (C15), 11.6 (C17); HRMS
(ESI) m/z (613.3210 [M + Na]+, C31H42N8O4Na calcd 613.3221).
Synthesis of 22-(4-(3-(Adenine-9-yl)propyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (36). The general procedure using compound
6 (43 mg, 0.11 mmol), compound 17 (15 mg, 0.08 mmol), t-BuOH/
H2O (2 mL), sodium ascorbate (2.4 mg, 0.01 mmol), and
CuSO4 ·5H2O (1.8 mg, 0.01 mmol) was applied. Yield, 38 mg, 83%;
Rf ) 0.30 (10% CH3OH in CH2Cl2); 1H NMR (CDCl3) δ 8.32 (1H,
s, H2(A)), 7.87 (1H, s, H8(A)), 7.45 (1H, s, CH(triazole)), 6.37
(1H, dd, J ) 10.7, 17.4, H19), 6.07 (2H, m, NH2), 5.78 (1H, d, J
) 8.5, H14), 5.29 (1H, d, J ) 10.7, H20), 5.18 (1H, d, J ) 17.4,
H20), 5.12-4.96 (2H, m, H22), 4.28 (2H, t, J ) 6.9, NCH2), 3.35
(1H, m, H11), 2.74 (2H, t, J ) 7.2, N(CH2)2CH2), 2.34-2.02 (7H,
m, H2, H4, H10, H13, NCH2CH2), 1.76-1.04 (9H, m, H1, H6,
H7, H8, 11-OH, H13), 1.31 (3H, s, H15), 1.15 (3H, s, H18), 0.86
(3H, d, J ) 7.0, H17), 0.68 (3H, d, J ) 7.0, H16); 13C NMR
(CDCl3) δ 216.6 (C3), 165.1 (C21), 155.5 (C6(A)), 152.9 (C2(A)),
150.0 (C4(A)), 146.6 (C(triazole)), 140.7 (C8(A)), 138.6 (C19),
122.5 (CH(triazole)), 119.5 (C5(A)), 117.4 (C20), 74.4 (C11), 70.9
(C14), 57.9 (C4), 51.4 (C22), 45.3 (C9), 44.7 (C13), 43.9 (C12),
42.9 (NCH2), 41.8 (C5), 36.5 (C6), 36.0 (C10), 34.3 (C2), 30.2
(C8), 29.3 (N(CH2)2CH2), 26.7 (C7), 26.5 (C18), 24.7 (C1), 22.2
(NCH2CH2), 16.7 (C16), 14.6 (C15), 11.4 (C17); HRMS (ESI) m/z
(627.3266 [M + Na]+, C32H44N8O4Na calcd 627.3378).
Synthesis of 22-(4-((2(R)-(Uracil-1-yl)-3(S),4(R)-dihydroxy-5(R)-
hydroxymethyl)tetrahydrofuran-3-yl)-1,2,3-triazole-1-yl)-22-deoxy-
pleuromutilin (37). The general procedure using compound 6 (62
mg, 0.15 mmol), compound 18 (33 mg, 0.12 mmol), t-BuOH/H2O
(2 mL), sodium ascorbate (3.1 mg, 0.02 mmol), and CuSO4 ·5H2O
(3.0 mg, 0.01 mmol) was applied. Yield, 66 mg, 81%; Rf ) 0.49
(15% CH3OH in CH2Cl2); 1H NMR (CH3OH-d4) δ 8.04 (1H, d, J
) 8.3, H6(U)), 8.02 (1H, s, CH(triazole)), 6.61 (1H, s, H1′), 6.27
(1H, dd, J ) 10.9, 18.0, H19), 5.77 (1H, d, J ) 8.4, H14), 5.67
(1H, d, J ) 8.3, H5(U)), 5.27-5.13 (4H, m, H20, H22), 4.24 (1H,
d, J ) 3.9, H3′), 4.07 (1H, m, H4′), 3.97-3.83 (2H, m, H5′), 3.48
(1H, d, J ) 6.1, H11), 2.36-2.10 (5H, m, H2, H4, H10, H13),
1.82-1.19 (8H, m, H1, H6, H7, H8, H13), 1.36 (3H, s, H15), 1.15
(3H, s, H18), 0.92 (3H, d, J ) 7.0, H17), 0.73 (3H, d, J ) 6.6,
H16); 13C NMR (CH3OH-d4) δ 219.5 (C3), 167.2 (C21), 166.1
(C4(U)), 152.4 (C2(U)), 147.2 (C(triazole)), 144.7 (C6(U)), 141.0
(C19), 126.8 (CH(triazole)), 116.8 (C20), 101.5 (C5(U)), 88.3 (C1′),
86.0 (C4′), 80.2 (C2′), 79.0 (C3′), 75.4 (C11), 72.6 (C14), 62.4
(C5′), 59.2 (C4), 52.6 (C22), 46.8 (C9), 45.7 (C13), 45.4 (C12),
43.2 (C5), 38.0 (C6), 37.8 (C10), 35.3 (C2), 31.4 (C8), 28.3 (C7),
28.0 (C18), 25.8 (C1), 17.0 (C16), 15.2 (C15), 11.8 (C17); 36.2
(C10), 34.0 (C2), 30.1 (C8), 28.6 (C7), 26.6 (C18), 24.5 (C1), 16.1
(C16), 14.2 (C15), 11.6 (C17); HRMS (ESI) m/z (694.3029 [M +
Na]+, C33H45N5O10Na calcd 694.3059).
Synthesis of 22-(4-((2(R)-(Uracil-1-yl)-3(R),4(S)-dihydroxy-5(R)-
hydroxymethyl)tetrahydrofuran-4-yl)-1,2,3-triazole-1-yl)-22-deoxy-
pleuromutilin (38). The general procedure using compound 6 (102
mg, 0.25 mmol), compound 19 (53 mg, 0.20 mmol), t-BuOH/H2O
(2 mL), sodium ascorbate (4 mg, 0.02 mmol), and CuSO4 ·5H2O
(3 mg, 0.01 mmol) was applied. Yield, 72 mg, 54%; Rf ) 0.45
1
(15% CH3OH in CH2Cl2); H NMR (CH3OH-d4) δ 8.17 (1H, m,
H6(U)), 8.03 (1H, s, CH(triazole)), 6.31 (1H, dd, J ) 11.1, 17.4,
H19), 5.91 (1H, m, H1′), 5.77 (1H, d, J ) 8.1, H14), 5.73 (1H, m,
H5(U)), 5.31-5.17 (4H, m, H20, H22), 4.66 (1H, m, H4′), 4.21
(1H, m, H2′), 4.06-3.96 (2H, m, H5′), 3.52 (1H, d, J ) 5.1, H11),
2.40-2.14 (5H, m, H2, H4, H10, H13), 1.86-1.16 (8H, m, H1,
H6, H7, H8, H13), 1.38 (3H, s, H15), 1.19 (3H, s, H18), 0.95 (3H,
d, J ) 6.7, H17), 0.77 (3H, d, J ) 6.1, H16); 13C NMR (CH3OH-

A Click Chemistry Approach to Pleuromutilin Conjugates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4965
d4) δ 219.4 (C3), 167.1 (C21), 159.1 (C4(U)), 152.3 (C2(U)), 147.8
(C(triazole)), 143.4 (C6(U)), 141.0 (C19), 126.2 (CH(triazole)),
116.7 (C20), 101.4 (C5(U)), 93.9 (C1′), 87.0 (C4′), 83.7 (C2′), 79.6
(C3′), 75.3 (C11), 72.6 (C14), 61.4 (C5′), 59.1 (C4), 52.6 (C22),
46.8 (C9), 45.7 (C13), 45.4 (C12), 43.2 (C5), 38.0 (C6), 37.7 (C10),
35.3 (C2), 31.4 (C8), 28.3 (C7), 28.0 (C18), 25.8 (C1), 17.1 (C16),
15.2 (C15), 11.8 (C17); HRMS (ESI) m/z (694.3069 [M + Na]+,
C33H45N5O10Na calcd 694.3059).
Synthesis of 22-(4-(Thymidine-5′(R/S)-C-yl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (39). The general procedure was slightly
modified using compound 6 (19 mg, 0.05 mmol), compound 20
(37 mg, 0.14 mmol), CH3OH/H2O (2 mL, 1:1 v/v), sodium
ascorbate (2 mg, 0.01 mmol), CuSO4 ·5H2O (1.5 mg, 0.01 mmol),
and only 10 min of heating. Yield, 20 mg, 63% (epimeric mixture);
Rf ) 0.46 (15% CH3OH in CH2Cl2); 1H NMR (CH3OH-d4) δ 7.95
(1H, br s, H6(T)), 7.87 (1H, br s, CH(triazole)), 6.38-6.18 (2H,
m, H19, H1′), 5.73-5.67 (1H, m, H14), 5.22-5.07 (4H, m, H20,
H22), 4.56-4.52 (2H, m, H3′, H5′), 4.17-4.13 (1H, m, H4′),
3.45-3.39 (1H, m, H11), 2.28-2.09 (7H, m, H2, H2′, H4, H10,
H13), 1.84 (3H, s, CH3(T)), 1.78-1.04 (8H, m, H1, H6, H7, H8,
H13), 1.23 (3H, s, H15), 1.10 (3H, s, H18), 0.87 (3H, d, J ) 6.8,
H17), 0.64 (3H, d, J ) 6.5, H16); HRMS (ESI) m/z (692.3238 [M
+ Na]+, C34H47N5O9Na calcd 692.3266).
under reduced pressure. The crude product was purified by column
chromatography (60-0% petrol ether and then 0-5% CH3OH in
EtOAc). Yield, 26 mg, 87%; Rf ) 0.04 (EtOAc and petrol ether,
2:1 v/v)); 1H NMR (DMSO-d6) δ 11.32 (1H, br s, NH), 8.03 (1H,
s, CH(triazole)), 7.88 (1H, d, J ) 8.1, H6(U)), 6.12 (1H, dd, J )
11.2, 17.7, H19), 5.89 (1H, d, J ) 4.8, H14), 5.63-5.56 (2H, m,
H20), 5.38-5.03 (4H, m, H5(U), H1′, 2 × OH), 4.77-4.64 (2H,
m, H22), 4.54 (1H, m, H4′), 4.10-3.83 (2H, m, H2′, H3′), 3.88
(1H, m, H11), 3.65-3.40 (2H, m, H5′), 2.40-2.00 (5H, m, H2,
H4, H10, H13), 1.80-1.09 (9H, m, H1, H6, H7, H8, 11-OH, H13),
1.26 (3H, s, H15), 1.06 (3H, s, H18), 0.81 (3H, d, J ) 6.6, H17),
0.63 (3H, d, J ) 6.6, H16); 13C NMR (DMSO-d6) δ 218.3 (C3),
167.0 (C21), 164.3 (C4(U)), 151.8 (C2(U)), 145.1 (C(triazole)),
142.0, 141.6 (C19, C6(U)), 126.7 (CH(triazole)), 116.7 (C20), 103.0
(C5(U)), 87.5 (C1′), 86.2 (C4′), 81.9 (C2′), 73.8 (C3′), 71.8 (C11),
69.5 (C14), 64.0 (C22), 61.6 (C4′) 58.0 (C5′), 52.2 (OCH2), 46.2
(C9), 45.4 (C13), 44.5 (C12), 42.8 (C5), 37.7 (C6), 37.5 (C10),
35.2 (C2), 31.3 (C8), 29.8 (C7), 27.8 (C18), 25.7 (C1), 17.4 (C16),
15.5 (C15), 12.8 (C17); HRMS (ESI) m/z (708.3184 [M + Na]+,
C34H47N5O10Na calcd 708.3215).
Synthesis of 22-(4-(Thymidine-3′-O-methyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (43). The general procedure was slightly
modified using compound 24 (94 mg, 0.23 mmol), compound 6
(67 mg, 0.24 mmol), t-BuOH/H2O (1.4 mL), sodium ascorbate (5
mg, 0.025 mmol), CuSO4 ·5H2O (4 mg, 0.016 mmol), and stirring
at 75 °C for 24 h. The mixture was extracted with EtOAc (10 mL),
and the organic phase was washed with water (2 × 10 mL), dried
(MgSO4), and concentrated under reduced pressure. The crude
product was purified by column chromatography (50-0% petrol
ether and then 0-5% CH3OH in EtOAc). Yield, 111 mg, 78%; Rf
) 0.22 (EtOAc); 1H NMR (CH3OH-d4) δ 8.04 (1H, s, H6(T)), 7.81
(1H, s, CH(triazole)), 6.32-6.28 (2H, m, H1′, H19), 5.78 (1H, d,
J ) 7.8, H14), 5.29-5.15 (4H, m, H20, OCH2), 4.71 (2H, s, H22),
4.32 (1H, m, H3′), 4.14 (1H, m, H4′), 3.84-3.71 (2H, m, H5′),
3.49 (1H, d, J ) 5.7, H11), 2.40-2.10 (7H, m, H2, H2′, H4, H10,
H13), 1.90 (3H, s, CH3(T)), 1.84-1.07 (8H, m, H1, H6, H7, H8,
H13), 1.33 (3H, s, H15), 1.17, (1H, s, H18), 0.94 (3H, d, J ) 6.9,
H17), 0.73 (3H, d, J ) 6.6, H16); 13C NMR (CH3OH-d4) δ 219.2
(C3), 166.8 (C21), 165.1 (C2(T)), 152.3 (C4(T)), 146.0 (C(triazole)),
140.9 (C19), 138.1 (C6(T)), 126.7 (CH(triazole)), 116.7 (C20),
111.7 (C5(T)), 86.6, 86.4 (C1′, C4′), 80.6 (C3′), 75.4 (C11), 72.5
(C14), 63.2, 63.1 (C5′, OCH2), 59.1 (C4), 52.6 (C22), 46.7 (C9),
45.7 (C13), 45.4 (C12), 43.1 (C2′), 38.4, 38.0, 37.7 (C5, C6, C10),
35.2 (C2), 31.4 (C8), 28.3 (C7), 28.0 (C18), 25.8 (C1), 17.0 (C16),
15.2 (C15), 12.5 (CH3(T)), 11.8 (C17); HRMS (ESI) m/z (706.3445
[M + Na]+, C35H49N5O9Na calcd 706.3423).
Synthesis of 22-(4-(Thymidine-5′-O-methyl)-1,2,3-triazole-1-yl)-
22-deoxypleuromutilin (44). The general procedure was slightly
modified using compound 25 (61 mg, 0.23 mmol), compound 6
(92 mg, 0.23 mmol), t-BuOH/H2O (1.4 mL), sodium ascorbate (4.5
mg, 0.023 mmol), CuSO4 ·5H2O (2.8 mg, 0.011 mmol), and stirring
at 75 °C for 40 h. The mixture was extracted with EtOAc (10 mL),
and the organic phase was washed with water (2 × 10 mL), dried
(MgSO4), and concentrated under reduced pressure. The crude
product was purified by column chromatography (40-0% petrol
ether and then 0-5% CH3OH in EtOAc). Yield, 115 mg, 75%; Rf
) 0.05 (EtOAc); 1H NMR (CH3OH-d4) δ 7.92 (1H, s, H6(T)), 7.57
(1H, s, CH(triazole)), 6.24-6.15 (2H, m, H1′, H19), 5.64 (1H, d,
J ) 7.8, H14), 5.19-5.03 (4H, m, H20, OCH2), 4.65 (2H, s, H22),
4.31 (1H, m, H3′), 3.91 (1H, m, H4′), 3.71-3.64 (2H, m, H5′),
3.41-3.31 (1H, m, H11), 2.24-1.96 (7H, m, H2, H2′, H4, H10,
H13), 1.68 (3H, s, CH3(T)), 1.79-1.05 (8H, m, H1, H6, H7, H8,
H13), 1.19 (3H, s, H18), 1.35, (1H, s, H15), 0.81 (3H, d, J ) 7,
H17), 0.61 (3H, d, J ) 6, H16); 13C NMR (CDCl3) δ 219.3 (C3),
166.8 (C21), 165.1 (C2(T)), 152 (C4(T)), 145.7 (C(triazole)), 140.9
(C19), 137.8 (C6(T)), 126.6 (CH(triazole)), 116.7 (C20), 111.7
(C5(T)), 87.4, 86.3 (C1′, C4′), 75.3, 72.9, 72.5, 71.4 (C3′, C5′,
C11, C14), 65.1 (OCH2), 59.1 (C4), 52.6 (C22), 46.7 (C9), 45.7
(C13), 45.4 (C12), 43.2 (C5), 41.3 (C2′), 37.9 (C6), 37.7 (C10),
35.2 (C2), 31.4 (C8), 28.2 (C7), 28.0 (C18), 25.9 (C1), 17.1 (C16),
Synthesis of 22-(4-(Thymidine-5′(R)-C-ylmethyl)-1,2,3-triazole-
1-yl)-22-deoxypleuromutilin (40). The general procedure using
compound 6 (75 mg, 0.19 mmol), compound 21 (37 mg, 0.13
mmol), t-BuOH/H2O (3 mL), sodium ascorbate (4 mg, 0.02 mmol),
and CuSO4 ·5H2O (3 mg, 0.01 mmol) was applied. Yield, 88 mg,
1
96%; Rf ) 0.47 (15% CH3OH in CH2Cl2); H NMR (CDCl3) δ
8.91 (1H, s, NH), 7.56 (1H, s, H6(T)), 7.33 (1H, s, CH(triazole)),
6.37 (1H, dd, J )11.3, 17.5, H19), 6.16 (1H, t, J ) 6.9, H1′), 5.79
(1H, d, J ) 8.7, H14), 5.31 (1H, d, J ) 11.3, H20), 5.19 (1H, d,
J ) 17.5, H20), 5.13-5.01 (2H, m, H22), 4.61 (1H, m, H3′), 4.27
(1H, m, H5′), 4.13 (1H, m, 5′-OH), 3.83 (1H, m, H4′), 3.36 (1H,
m, H11), 3.11-2.92 (2H, m, H6′), 2.35-2.04 (7H, m, H2, H2′,
H4, H10, H13), 1.90 (3H, s, CH3(T)), 1.78-1.12 (9H, m, H1, H6,
H7, H8, 11-OH, H13), 1.33 (3H, s, H15), 1.16 (3H, s, H18), 0.87
(3H, d, J ) 6.9, H17), 0.69 (3H, d, J ) 6.8, H16); HRMS (ESI)
m/z (706.3391 [M + Na]+, C35H49N5O9Na calcd 706.3423).
Synthesis of 22-(4-(Thymidine-5′(S)-C-ylmethyl)-1,2,3-triazole-
1-yl)-22-deoxypleuromutilin (41). The general procedure using
compound 6 (50 mg, 0.12 mmol), compound 22 (29 mg, 0.11
mmol), t-BuOH/H2O (1.5 mL), sodium ascorbate (4 mg, 0.02
mmol), and CuSO4 ·5H2O (3 mg, 0.01 mmol) was applied. Yield,
53 mg, 73%; Rf ) 0.48 (15% CH3OH in CH2Cl2); 1H NMR (CDCl3)
δ 8.01 (1H, s, NH), 7.62 (1H, s, H6(T)), 7.52 (1H, s, CH(triazole)),
6.40 (1H, dd, J ) 11.0, 17.3, H19), 6.29 (1H, t, J ) 7.7, H1′),
5.81 (1H, d, J ) 8.4, H14), 5.34 (1H, d, J ) 11.0, H20), 5.21 (1H,
d, J ) 17.3, H20), 5.12-5.00 (2H, m, H22), 4.60 (1H, m, H3′),
4.19 (1H, m, H5′), 3.92 (1H, m, H4′), 3.77 (1H, m, 5′-OH), 3.35
(1H, dd, J ) 5.8, J ) 9.9, H11), 3.16-2.95 (2H, m, H6′), 2.53-2.05
(7H, m, H2, H2′, H4, H10, H13), 1.93 (3H, s, CH3(T)), 1.79-1.10
(9H, m, H1, H6, H7, H8, 11-OH, H13), 1.35 (3H, s, H15), 1.18
(3H, s, H18), 0.88 (3H, d, J ) 6.7, H17), 0.70 (3H, d, J ) 6.8,
H16); 13C NMR (CH3OH-d4) δ 219.4 (C3), 167.0 (C21), 166.4
(C4(T)), 152.4 (C2(T)), 145.0 (C(triazole)), 141.0, 138.6 (C19,
C6(T)), 126.0 (CH(triazole)), 116.7 (C20), 111.6 (C5(T)), 89.5
(C1′), 86.2 (C4′), 75.4 (C11), 73.2 (C3′), 72.5 (C14), 71.5 (C5′),
59.1 (C4), 52.6 (C22), 46.7 (C9), 45.7 (C13), 45.4 (C12), 43.2 (C5),
41.1 (C2′), 38.0 (C6), 37.7 (C10), 35.3 (C2), 31.4 (C8, C6′), 28.3
(C7), 28.0 (C18), 25.8 (C1), 17.0 (C16), 15.2 (C15), 12.6 (CH3(T)),
11.8 (C17); HRMS (ESI) m/z (706.3418 [M + Na]+, C35H49N5O9Na
calcd 706.3423).
Synthesis of 22-(4-(Uridine-2′-O-methyl)-1,2,3-triazole-1-yl)-22-
deoxypleuromutilin (42). The general procedure was slightly
modified using compound 6 (28 mg, 0.071 mmol), compound 23
(20 mg, 0.071 mmol), t-BuOH/H2O (2 mL), sodium ascorbate (1.4
mg, 0.007 mmol), CuSO4 ·5H2O (0.9 mg, 0.004 mmol), and stirring
at room temperature for 4 days and at 75 °C for 24 h. The mixture
was extracted with EtOAc (10 mL), and the organic phase was
washed with water (2 × 10 mL), dried (MgSO4), and concentrated

4966 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16
Lolk et al.
15.2 (C15), 12.6 (CH3(T)), 11.8 (C17); HRMS (ESI) m/z (706.3445
[M + Na]+, C35H49N5O9Na calcd 706.3423).
Acknowledgment. We thank Novartis for providing pleu-
romutilin. We thank The Danish National Research Founda-
tion, The Danish Research Agency’s Programme for Young
Researchers, The Danish Medical Research Council, and
Møllerens Fond for financial support of the project. We thank
Birthe Haack, Dr. Mogens T. Jensen, Dr. Kenneth B. Jensen,
and the NMR laboratory and Brian Gregersen for technical
assistance. The Nucleic Acid Center is funded by the Danish
National Research Foundation for studies on nucleic acid
chemical biology.
Footprinting Experiments. E. coli strain MRE600 was used to
prepare ribosomes for the chemical footprinting experiments. The
cells were grown in LB to an optical density of A450 ) 0.4 and
harvested by cooling on ice followed by centrifugation. Cells were
washed with TMN (20 mM Tris-HCl (pH 7.8), 10 mM MgCl2,
100 mM NH4Cl), centrifuged, and resuspended in 1 mL of TMN.
Cells were opened by sconification for 4 × 15 s. The lysate was
recovered by centrifugation and layered on a 38 mL 10-40%
sucrose gradient prepared in TMN buffer and centrifuged in
ultracentrifuge SE90, AH 629 rotor, 19 000 rpm, 18 h. The gradient
was analyzed on a Amersham Fraction Collector system. The
fractions containing 70S ribosomes were collected, and the ribo-
somes were recovered by dialysis against TMN and pelleted in
ultracentrifuge SE90, Ti50 rotor, 40.000 rpm, 22 h. Ribosomes were
resuspended in 200 µL of TMN and stored at -80 °C.
Supporting Information Available: Experimental procedures
that are not covered in the main text, i.e., for the compounds 5, 6,
10-18, 20-25, 46, 48-51, 53, 55, and 56; 1H NMR spectra, 13
C
NMR spectra, and HPLC profiles for the pleuromutilin conjugates.
This material is available free of charge via the Internet at http://
pubs.acs.org.
The antibiotic binding and chemical modification were performed
as follows: Ribosomes (5 pmol) were incubated with either 5 or
50 µM drug or drug derivative in modification buffer (50 mM Tris-
HCl (pH 8.0), 10 mM MgCl2, 100 mM KCl, and 5 mM dithiotretiol)
for 30 min at 37 °C. The modification procedure is described by
Ehresmann et al.29 Aliquots (50 µL) of the antibiotic-ribosome
complexes were modified with 50 µL of CMCT (42 mg/mL in
modification buffer) for 20 min at 37 °C. The CMCT reactions
were stopped by precipitating the ribosomes. After centrifugation
the ribosomes were resuspended in 0.25 M sodium acetate, pH 6.5,
and extracted with phenol and chloroform. The rRNA was
precipitated and resuspended in water.
The chemical modifications were monitored by primer extension
analysis on 23S RNA using reverse transcriptase (AMV, Life
Sciences) and 5′-[32P]-labeled deoxyoligonucleotide primers.30 The
DNA oligonucleotide 5′-TCCGGTCCTCTCGTACT-3′, comple-
mentary to nucleotides 2654-2670 of 23S rRNA, was used as
primer in the extension reactions. The cDNA products of the primer
extension reactions were separated on 6% polyacrylamide sequenc-
ing gels. The positions of the stops in cDNA synthesis were
identified by reference to dideoxy sequencing reactions on 23S RNA
that were run in parallel. The bands were visualized, and the
intensities of the modifications were assessed using a Typhoon trio
laser scanner. Experiments were performed at least two times, and
the numbers in Table 1 are an average of protections against CMCT
modification calculated from samples in the presence of drug
relative to control samples without drug and with subtraction of
the background value from unmodified samples.
Docking Experiments. Visualization of the original X-ray crystal
structure and the docking results was performed in VMD.31 The
docking experiment was performed using the Shro¨dinger 2007
package. A flexible drug binding site containing RNA was simulated
by creating a truncated version of PTC, consisting of 25 Å of the
E.coli ribosome11 surrounding the tiamulin binding site. The drugs
were then docked using a multistep approach: The first round of
docking was performed with all atoms of the drugs scaled to 80%
of their WDV radii. The resulting complex with the highest binding
score for each drug had the ribosomal residues with atoms within
3 Å of the drug minimized with a 1 kcal/(mol ·Å2) restraint, while
the drug was unrestrained. Each drug was then redocked into this
accommodated site, using the QM polarized ligand docking
workflow, assigning the charges in context of the receptor at the
B3LYP/3-21G level of theory and a 0.9 scaling of atoms with a
partial charge less than (0.25, using XP docking precision.
Minimizations were done using the OPLS2005 forcefield in the
MacroModel module, using implicit water, vdW cutoff of 7 Å,
electrostatic cutoff of 12 Å, and H-bond cutoff of 4 Å. Initial
docking was performed using the Glide module, while the
final docking was done using the quantum polarized workflow. The
docking procedure was validated by docking tiamulin into the site
(Figure 2) showing an identical conformation to the D. radiodurans7
X-ray crystal structure of tiamulin in PTC (data not shown),
References
(1) Kavanagh, F.; Hervey, A.; Robbins, W. J. Antibiotic substances from
basidiomycetes: VIII. Pleurotus Multilus (Fr.) Sacc. and Pleurotus
Passeckerianus Pilat. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 570–
574.
(2) Poulsen, S. M.; Karlsson, M.; Johansson, L. B.; Vester, B. The
pleuromutilin drugs tiamulin and valnemulin bind to the RNA at the
peptidyl transferase centre on the ribosome. Mol. Microbiol. 2001,
41, 1091–1099.
(3) Daum, R. S.; Kar, S.; Kirkpatrick, P. Retapamulin. Nat. ReV. Drug.
DiscoVery 2007, 6, 865–866.
(4) Heilman, C.; Jensen, L.; Jensen, J. S.; Lundstrom, K.; Windsor, D.;
Windsor, H.; Webster, D. Treatment of resistant mycoplasma infection
in immunocompromised patients with a new pleuromutilin antibiotic.
J. Infect. 2001, 43, 234–238.
(5) (a) Bosling, J.; Poulsen, S. M.; Vester, B.; Long, K. S. Resistance to
the peptidyl transferase inhibitor tiamulin caused by mutation of
ribosomal protein L3. Antimicrob. Agents Chemother. 2003, 47, 2892–
2896. (b) Pringle, M.; Pøhlsgaard, J.; Vester, B.; Long, K. S. Mutations
in ribosomal protein L3 and 23S ribosomal RNA at the peptidyl
transferase centre are associated with reduced susceptibility to tiamulin
in Brachyspira spp. isolates. Mol. Microbiol. 2004, 54, 1295–1306.
(c) Kehrenberg, C.; Schwarz, S.; Jacobsen, L.; Hansen, L. H.; Vester,
B. A new mechanism for chloramphenicol, florfenicol and clindamycin
resistance: methylation of 23S ribosomal RNA at A2503. Mol.
Microbiol. 2005, 57, 1064–1073. (d) Gentry, D. R.; Rittenhouse, S. F.;
McCloskey, L.; Holmes, D. J. Stepwise exposure of Staphylococcus
aureus to pleuromutilins is associated with stepwise acquisition of
mutations in rplC and minimally affects susceptibility to retapamulin.
Antimicrob. Agents Chemother. 2007, 51, 2048–2052.
(6) (a) Poehlsgaard, J.; Douthwaite, S. The bacterial ribosome as a target
for antibiotics. Nat. ReV. Microbiol. 2005, 3, 870–81. (b) Yonath, A.
Antibiotics targeting ribosomes: resistance, selectivity, synergism and
cellular regulation. Annu. ReV. Biochem. 2005, 74, 649–679.
(7) Schlunzen, F.; Pyetan, E.; Fucini, P.; Yonath, A.; Harms, J. M.
Inhibition of peptide bond formation by pleuromutilins: the structure
of the 50S ribosomal subunit from Deinococcus radiodurans in
complex with tiamulin. Mol. Microbiol. 2004, 54, 1287–1294.
(8) Davidovich, C.; Bashan, A.; Auerbach-Nevo, T.; Yaggie, R. D.;
Gontarek, R. R.; Yonath, A. Induced-fit tightens pleuromutilins binding
to ribosomes and remote interactions enable their selectivity. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 4291–4296.
(9) Hunt, E. Pleuromutilin antibiotics. Drugs Future 2000, 25, 1163–1168.
(10) Philips, O. A.; Sharaf, L. H. Pleuromutilin antibacterial agents: patent
review 2001-2006. Expert Opin. Ther. Pat. 2007, 17, 429–435.
(11) Schuwirth, B. S.; Borovinskaya, M. A.; Hau, C. W.; Zhang, W.; Vila-
Sanjurjo, A.; Holton, J. M.; Cate, J. H. Structures of the bacterial
ribosome at 3.5 Å resolution. Science 2005, 310, 827–834.
(12) Selmer, M.; Dunham, C. M.; Murphy, F. V.;.; Weixlbaumer, A.; Petry,
S.; Kelley, A. C.; Weir, J. R.; Ramakrishnan, V. Structure of the 70S
ribosome complexed with mRNA and tRNA. Science 2006, 313, 1935–
1942.
(13) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Click chemistry: diverse
function from a few good reactions. Angew. Chem., Int. Ed. 2001,
40, 2004–2021.
(14) (a) Kolb, H. C.; Sharpless, K. B. The growing impact of click chemistry
on drug discovery. Drug DiscoVery Today 2003, 8, 1128–1137. (b)
Sharpless, K. B.; Manetsch, R. In situ click chemistry: a powerful
means for lead discovery. Expert Opin. Drug DiscoVery 2006, 1, 525–
538.

A Click Chemistry Approach to Pleuromutilin Conjugates
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 16 4967
(15) (a) Tornøe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-
dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem.
2002, 67, 3057–3064. (b) Rostovtsev, V. V.; Green, L. G.; Fokin,
V. V.; Sharpless, K. B. A stepwise Huisgen cycloaddition process:
copper(I)-catalyzed regioselective “ligation” of azides and terminal
alkynes. Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(16) Riedl, K. Studies on pleuromutilin and some of its derivatives. J.
Antibiot. 1976, 29, 132–139.
(17) Lindsell, W. E.; Murray, C.; Preston, P. N.; Woodman, T. A. Synthesis
of 1,3-diynes in the purine, pyrimidine, 1,3,5-triazine and acridine
series. J. Tetrahedron 2000, 56, 1233–1245.
(18) (a) Cruickshank, K. A.; Jiricny, J.; Reese, C. B. The benzoylation of
uracil and thymine. Tetrahedron Lett. 1984, 25, 681–684. (b) Ludek,
O. R.; Meier, C. Influence of the N3-protection group on N1- vs. O2-
alkylation in the Mitsunobu reaction. Eur. J. Org. Chem. 2006, 94,
1–946.
restricted cyclic phosphotriester linkages. Tetrahedron 2001, 57,
10191–10201.
(25) (a) Fensholdt, J.; Wengel, J. Synthesis of diastereomeric mixtures of
5′-C-hydroxymethylthymidine and introduction of a novel class of
functionalised oligodeoxynucleotides. Acta Chem. Scand. 1996, 50,
1157–1163. (b) Wang, G.; Middleton, P. J. 5′-C-Branched thymidines:
synthesis, stereochemistry, and incorporation into oligodeoxynucle-
otides. Tetrahedron Lett. 1996, 37, 2739–2742. (c) Trafelet, H.; Stulz,
E.; Leumann, C. Synthesis of (5′S)-5′-C-alkyl-2′-deoxynucleosides.
HelV. Chim. Acta 2001, 84, 87–105. (d) Sharma, P. K.; Mikkelsen,
B. H.; Christensen, M. S.; Nielsen, K. E.; Kirchhoff, C.; Pedersen,
S. L.; Sørensen, A. M.; Østergaard, K.; Petersen, M.; Nielsen, P. A
cyclic dinucleotide with a four-carbon 5′-C-to-5′-C connection;
synthesis by RCM, NMR-examination and incorporation into second-
ary nucleic acid structures. Org. Biomol. Chem. 2006, 4, 2433–2445.
(26) (a) Soloway, A. H.; Anisuzzaman, A. K. M.; Alarn, F.; Barth, R. F.;
Liu, L. The development of carboranyl nucleic acid precursors for
use in neutron capture therapy of tumors. Pure Appl. Chem. 1991,
63, 411–413. (b) Grøtli, M.; Douglas, M.; Eritja, R.; Sproat, B. S.
2′-O-Propargyl oligoribonucleotides: synthesis and hybridisation.
Tetrahedron 1998, 54, 5899–5914.
(27) (a) Rosowsky, A.; Ruprecht, R. M.; Solan, V. C. Synthesis of 3′-O-
propargylthymidine as a candidate antiretroviral agent. Nucleosides
Nucleotides 1989, 8, 491–497. (b) Zhou, L.; Amer, A.; Korn, M.;
Burda, R.; Balzarini, J.; De Clercq, E.; Kern, E. R.; Torrence, P. A.
Synthesis and antiviral activities of 1,2,3-triazole functionalized
thymidines: 1,3-dipolar cycloaddition for efficient regioselective
diversity generation. AntiViral Chem. Chemother. 2005, 16, 385–383.
(28) Long, K. S.; Hansen, L. H.; Jakobsen, L.; Vester, B. Interaction of
pleuromutilin derivatives with the ribosomal peptidyl transferase center.
Antimicrob. Agents Chemother. 2006, 50, 1458–1462.
(19) Lee, Y.-S.; Kim, B. H. Heterocyclic nucleoside analogues: design and
synthesis of antiviral, modified nucleosides containing isoxazole
heterocycles. Bioorg. Med. Chem. Lett. 2002, 12, 1395–1397.
(20) Pelissier, H.; Rodriquez, J.; Vollhardt, K. P. C. Cobalt-mediated [2 +
2
+ 2] cycloadditions of pyrimidine derivatives to alkynes.
Chem.sEur. J. 1999, 5, 3549–3561.
(21) (a) Joshi, R. V.; Zemlicka, J. Alkylation of adenine with t-propargyl
chlorides: acetylene/allene ratio and N9/N7 regioselectivity. Tetrahe-
dron 1993, 49, 2353–2360. (b) Lazrek, H. B.; Taourirte, M.; Oulih,
T.; Barascut, J. L.; Imbach, J. L.; Pannecouque, C.; Witrouw, M.; De
Clerck, E. Synthesis and anti-HIV activity of new modified 1,2,3-
triazole acyclonucleosides. Nucleosides, Nucleotides Nucleic Acids
2001, 20, 1949–1960.
(22) Iino, T.; Yoshimura, Y.; Matsuda, A. Nucleosides and nucleotides.
134. Synthesis of 2′-C-alkynyl-2′-deoxy-1-ꢀ-D-arabinofuranosylpyri-
midines via radical deoxygenation of tert-propargyl alcohols in the
sugar moiety. Tetrahedron 1994, 50, 10397–10406.
(23) Hattori, H.; Nozawa, E.; Iino, T.; Yoshimura, Y.; Shuto, S.; Shima-
moto, Y.; Nomura, M.; Fukushima, M.; Tanaka, M.; Sasaki, T.;
Matsuda, A. Nucleosides and nucleotides. 175. Structural requirements
of the sugar moiety for the antitumor activities of new nucleoside
antimetabolites, 1-(3-C-ethynyl-ꢀ-D-ribo-pentofuranosyl)cytosine and
-uracil. J. Med. Chem. 1998, 41, 2892–2902.
(29) Ehresmann, C.; Baudin, F.; Mougel, M.; Romby, P.; Ebel, J.-P.;
Ehresmann, B. Probing the structure of RNAs in solution. Nucleic
Acids Res. 1987, 15, 9109–9128.
(30) Stern, S.; Moazed, D.; Noller, H. F. Structural analysis of RNA using
chemical and enzymatic probing monitored by primer extension.
Methods Enzymol. 1988, 164, 481–489.
(31) Humphrey, W.; Dalke, A.; Schulten, K. VMD: visual molecular
dynamics. J. Mol. Graphics 1996, 14, 27-28, 33-38.
(24) Sørensen, A. M.; Nielsen, K. E.; Vogg, B.; Jacobsen, J. P.; Nielsen,
P. Synthesis and NMR-studies of dinucleotides with conformationally
JM800261U