A solid-phase approach to analogues of the antibiotic mureidomycin

Article abstract of DOI:10.1016/S0960-894X(00)00560-6

A library of 80 analogues of the antibacterial compound mureidomycin was prepared using solid-phase chemistry techniques. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00560-6

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2759±2763
A Solid-Phase Approach to Analogues of the Antibiotic
Mureidomycin
Andrea Bozzoli, Wieslaw Kazmierski,* Gordon Kennedy,
Alessandra Pasquarello and Angelo Pecunioso
GlaxoWellcome SpA, Medicines Research Centre, via Fleming 4, Verona 37135, Italy
Received 10 July 2000; revised 29 September 2000; accepted 29 September 2000
AbstractÐA library of 80 analogues of the antibacterial compound mureidomycin was prepared using solid-phase chemistry techniques.
# 2000 Elsevier Science Ltd. All rights reserved.
Introduction
although it lacks whole cell activity against these bacteria.
These interesting biological properties, combined with
mureidomycin's modular structure (Fig. 1) which is
potentially amenable to a combinatorial chemistry
approach, make it an attractive synthetic target. In this
communication, we wish to report our initial synthesis of
a library of analogues of mureidomycin in the search for
novel antibacterial compounds.
The increasing incidence of resistance amongst patho-
genic bacteria is becoming a serious health problem
across the world.1 This has led to attempts to improve
the activities of existing antibacterial agents in order to
extend their coverage to drug resistant bacteria and also
to discover new classes of antibacterial agents with
mechanisms of action distinct from the classical agents
such as the b-lactams, macrolides, aminoglycosides and
tetracyclines.2 The mureidomycins (Fig. 1) and the rela-
ted pacidamycins, have recently been described to be
inhibitors of bacterial translocase, an essential enzyme
involved in the synthesis of the peptidoglycan compo-
nent of the bacterial cell wall.3 One of the traits of mure-
idomycin is its unusual C- to N-peptide sequence which is
caused by the reversal of the peptide chain polarity by the
urea within the chain. Alignment of mureidomycin and
the UDP-MurNAc-pentapeptide substrate of the
enzyme along their respective deoxyribose-uridine/
aminoribosyl-uridine moieties shows that the peptide
chains have the same polarity caused by the urea and
the meso-diaminopimellic acid units.
The cornerstone of the synthetic strategy designed to
provide a library of analogues of mureidomycin was the
building up of the modular structure anchored to a suit-
able solid-phase support. Overall, the structure of mure-
idomycin presents several challenges for the development
of solid-phase chemistry including: the choice of anchor
point which dictates the chemical strategy for building up
the molecule; the introduction of variation into the frame-
work; and the availability of suitable building blocks. The
solid-phase synthesis of peptides combines robust proce-
dures with the availability of a large number of amino
acids in suitably protected form. This consideration,
together with the synthetic eciency which would be
gained by anchoring the uridyl±sugar moiety to a resin
followed by introduction of variation along the peptide
chain, implied that the best strategy would be to link a
suitably modi®ed uridyl building block to the resin and
grow the peptide chain in such a way as to be able to
deprotect and release the compounds in the ®nal step or
steps of the synthetic sequence.
Mureidomycin exhibits strong activity both in vitro and
in vivo against strains of the dicult pathogen Pseudo-
monas aeruginosa and is equipotent to cefsoludin and
ceftazidime in an in vivo model of P. aeruginosa infection
in the mouse. It is active against isolated translocases
from both Escherichia coli and Staphylococcus aureus
The most obvious point for attaching the sugar to a resin
would be via an acetal based linker which is suitable for
diols and therefore for ribose but not deoxyribose ana-
logues. The known activity of the ribose-containing
*Corresponding author. Present address: GlaxoWellcome Research, NC
27615, USA; e-mail: wmk25860@glaxowellcome.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00560-6

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A. Bozzoli et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2759±2763
uridyl peptide antibiotics tunicamycin and liposidomy-
cin against translocase3 implied that substitution of the
deoxyribose by ribose could be justi®ed in the ®rst
instance for the development of the chemistry in the
solid phase. The choice of acetal linker and acid labile
protecting groups in the residues of the peptide chain
would allow cleavage from the resin and overall depro-
tection in one step under acidic conditions. However, it
was felt that the unusual enamide moiety of mure-
idomycin would present problems at this stage and
therefore this grouping was substituted by a simple
amide in the ®rst instance. For the initial library, the
branch point residue N-b-dimethyl-a,b-diamino pro-
pionic acid was substituted by commercially available
diaminopropionic acid in order to simplify the chemistry.
The building blocks were chosen to most closely resem-
ble the residues present in the natural product and, as the
absolute con®guration of each residue of mureidomycin
is unknown, both enantiomers of each building block
were included in the design. The enantiomers of
diaminopropionic acid M1, methionine M3 and p-tyr-
osine in position M2 and M4 are commercially avail-
able, while the two enantiomers of suitably protected m-
tyrosine M4 were synthesised ex novo and tert-butyl 2-
phenyl malonic acid ester, M4, was prepared as a race-
mate. The monomers used are shown in Figure 2.
The array of compounds was built up by splitting the
loaded resin at each addition of a residue in such a way
as to couple each batch of resin produced with the
individual monomer residues for that stage. The library
was planned to provide a total of 80 (2(M1)Â4(M2)Â
2(M3)Â(4+1)(M4)) individuals as discrete compounds.
Results and Discussion
Prior to starting the solid-phase chemistry, the key
intermediate acetal protected (50-N-Fmoc-aminoribo-
syl)-uridine 1 was synthesised in seven steps starting
from commercially available isopropylidene uridine
(Scheme 1).4 Conversion of the 50-hydroxyl function
into the azido group occurred smoothly, however, the
attempted reduction of the azido group with PPh3 yiel-
ded exclusively the product of Michael attack of the free
amine on the uridine ring. In contrast, the amine was
successfully obtained by catalytic hydrogenation of the
azide in the presence of acetic acid. After Fmoc protec-
tion and removal of the dimethylacetonide, the sugar
Figure 1. Mureidomycin and the basic sca€old for a library of
compounds.
Scheme 1. Reagents: (a) MsCl/DMAP; yield=80% (15 g scale); (b)
NaN3/DMF/70 ꢀC; yield=88%; (c) 1 atm H2/10% Pd/C/MeOH/
AcOH; (d) FmocONSu/10% (aq) Na2CO3/dioxane; yield=64% (two
steps); (e) TFA/H2O 95/5; yield=95%; (f) pTsOH/(EtO)3CH/DMF,
rt; yield=89%; (g) Na2CO3(aq) 3%, dioxane, rt; yield=59%.
Figure 2. Monomers used in the preparation of the library.

A. Bozzoli et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2759±2763
2761
hydroxyls were protected as the benzylidene acetal
through an exchange reaction with the diethylacetal of
methyl phenoxy acetic aldehyde pretreated with ethyl
orthoformate. The ®nal hydrolysis of the methyl ester
was carried out with Na2CO3.
accomplished with 0.25 N HCl without a€ecting the
acid labile tert-butyl group.
The crude mixture of (S)-O-tert-Bu-m-tyrosine methyl
ester and (R)-valine methyl ester obtained was treated
with di-tert-butylpyrocarbonate to give a mixture of (S)-
N-Boc-O-tert-Bu±m-tyrosine methyl ester and (R)-N-
Boc-valine methyl ester which were separated by col-
umn chromatography. The enantiomeric excess of the
product was determined to be >92% by HPLC analysis
after puri®cation and con®rmed by NMR analysis of
the (+)-methyl mandelate ester.
Both enantiomers of Boc protected m-tyrosine required
for the termination of the two chains (M2 and M4) were
prepared in enantiomerically enriched form according
to the Schollkopf asymmetric amino acid synthesis
(Scheme 2).5 (R)( )-Bis-lactim ether was reacted with 3-
tert-butoxy-a-bromotoluene to give the alkylated inter-
mediate. The cleavage of the dihydropyrazine ring was
The tyrosine building blocks used in position M4 were
prepared using a less direct route which involved sub-
stituting the methyl ester for the acid labile tert-butyl
ester. Thus (S)-O-tert-Bu±m-tyrosine methyl ester (as a
crude mixture with (R)-valine methyl ester) was treated
with N-Cbz-OnSu and the resulting mixture chromato-
graphed to remove the valine contaminant. The methyl
ester was cleaved and replaced with the tert-butyl group6
and the Cbz group removed by hydrogenolysis. The
p-nitrophenylcarbamate was formed by reaction of the
amine with p-nitrophenylchloroformate.7
The solid-phase chemistry was rehearsed by synthesising
one of the planned library members which was then used
as a reference compound during the preparation of the
actual library. Thus, the uridine intermediate 1 was linked
to an aminomethyl polystyrene (manufacturer-determined
loading of 0.9 mmol g 1) through the carboxylate group
of the acetal linker (Scheme 3).8
Scheme 2. Reagents: (a) BuLi 1.6 M/ 78 ꢀC then 3-(tButoxy)-a-bro-
motoluene; yield=70%; (b) HCl 0.25M; (c) NH4OH (conc); yield=
.
79%; (d) Boc2O/CH2, Cl2, rt; yield for b, c, d=79%; (e) LiOH H2O/
EtOH; yield=94%; (f) N-CbzONSu/2M Na2CO3/THF, rt; yield for b, c,
.
f=61% after chromatography; (g) LiOH H2O/EtOH; yield=95%; (h)
The best compromise in using the minimum amount of
precious sca€old and having maximum incorporation
into the resin was found with 1.1 equiv of sca€old and
PyBop as coupling reagent in the presence of Hunig's
CCl3C(ˆNH)OtBu/BF3ÐEt2O; yield=52%; (i) H2/10% Pd/C, rt;
yield=93%; (g) p-nitrophenylchloroformate/Hunig's base/CH2Cl2, rt:
yield=56%; this sequence was repeated with the other enantiomer of the
chiral auxiliary to obtain the antipodal m-tyrosine.
Scheme 3. Reagents: see Refs 8 and 11 (a) sca€old-linker (1.1 equiv), PyBop (1.1 equiv), Hunig's base (1.1 equiv), DMF, 18 h, rt; (b) Fmoc-depro-
tection: 20% piperidine in DMF, rt, 1 h; (c) coupling reaction: PyBop (5 equiv), Hunig's base (5 equiv), M (1±4) (5 equiv), DMF, rt, 18 h; (d) N-Boc-
deprotection TMSCl/PhOH 1/1 20 M, rt, 4 h; (e) urea in solid phase: p-NO2-carbamate species (5 equiv), Hunig's base (10 equiv), CH2Cl2, rt, 18 h;
(f) cleavage from the resin: TFA/H2O 95/5, 2 h, rt.

2762
A. Bozzoli et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2759±2763
1
base. The best loading obtained was 0.534 mmol g
(equivalent to 92% yield). Unreacted amino groups
were capped with 10% acetic anhydride in NMP.
Table 1. Analyses of the 10 best ®nal compounds
M1
M2
M3
M4
a/a %a
(l)-DAPA
(l)-DAPA
(d)-DAPA
(l)-DAPA
(l)-DAPA
(l)-DAPA
(d)-DAPA
(l)-DAPA
(l)-DAPA
(d)-DAPA
(R)-mTyr
(l)-Tyr
(l)-Tyr
(R)-mTyr
(l)-Tyr
(R)-mTyr
(l)-Tyr
(l)-Tyr
(l)-Tyr
(l)-Tyr
(l)-met
(l)-met
(d)-met
(d)-met
(l)-met
(d)-met
(l)-met
(d)-met
(d)-met
(d)-met
(S)-mTyr
(d)-Tyr
46
43
42
40
38
36
36
36
35
35
a-Fmoc-b-Boc-d-Diaminopropionic acid (d-M1) was
coupled to the resin-linked 50-aminoribose under standard
peptide coupling conditions after Fmoc deprotection and
an aliquot of the product cleaved from the resin by treat-
ment with TFA (step 2). The structure of the compound
was con®rmed by NMR and LC±MS and the yield of
this step estimated as 75% by the Fmoc method.9
Interestingly, no formation of the undesired Michael
addition product was detected upon release of the Fmoc
group from the bound ribose unit. Selective removal of
the Boc-protecting group in the presence of the acetal
was rehearsed in solution and the resulting procedure
(TMS-Cl/phenol 20 M) was then applied to the reaction
in the solid phase.10 In order to verify that these clea-
vage conditions were general for the resin bound case,
the Boc group was removed, the free amine capped with
acetic anhydride in NMP and the N-acetyl derivative
con®rmed after cleavage from the resin. The coupling
with d-phenylalanine as a terminal residue in the M2
position was con®rmed by HPLC, MS and NMR
analysis on a small aliquot cleaved from the support
(step 3). After removal of the Fmoc group from M1,
the d-methionine residue was coupled to the growing
chain and a small quantity of compound cleaved from
the resin for analysis (step 4). The overall yield up to this
step was estimated to be 55±62% by the Fmoc method.
(d)-Tyr
(S)-mTyr
(S)-mTyr
(l)-Tyr
(S)-mTyr
(R)-mTyr
(S)-mTyr
(l)-Tyr
aValues measured as a/a by HPLC/MS: DAPA=diaminopropionic
acid; mTyr=meta-tyrosine; Tyr=tyrosine; met=methionine.
water to give two solutions in which the ®nal concentra-
tion of the desired compound was estimated to be 30 and
60 mg mL 1. These solutions were tested for inhibition of
growth of S. aureus 853, E. coli 1852, Saccharomyces
cerevisiae NCY 81, P. aeruginosa and P. aeruginosa 2033,
however, no activity was detected for any of the pro-
ducts in the library.
While it could be argued that the analogues prepared do
not suciently resemble the parent compound in their
overall structural features, the main thrust of this work
was to develop a ¯exible solid-phase approach to this
type of complex molecule. A number of publications
containing information relevant to understanding the
mechanism of action and structure±activity relation-
ships of the mureidomycins13 and the related pacidimy-
cins14 appeared after completion of this work.
A number of attempts were made to carry out the for-
mation of the p-nitrophenylcarbamate on the resin;
however, after removal of Fmoc group, the free amine
of the methionine residue reacted with p-nitrophenyl-
chloroformate to form a cyclic product which did not
react further. The reaction of an excess of p-nitrophe-
nylcarbamate of d-phenylalanine t-butyl ester (M4) with
the resin bound amine in the presence of excess base was
more successful and gave the desired product in ca. 50%
yield after cleavage from the support (step 5).
It is apparent that the enamide group is not essential for
activity against translocase and that its replacement by
an amide makes the target analogues synthetically more
accessible but does not reduce anity for the enzyme. The
importance of the absolute stereochemistries of the various
amino acid residues present in the structure of pacidamy-
cin has been noted by Lee14 and this issue was addressed in
the current work by including both (R)- and (S)-enantio-
mers of the monomers in the library described.
The library was prepared with both enantiomers of the
available building blocks where possible as shown in
Figure 2.11 The yields of the steps during the synthesis
were calculated using the Fmoc reading where appro-
priate and the Kaiser test12 was used to check for
unreacted bound amine in all steps. The quality of each
intermediate in the synthesis was checked by LC±MS
analysis of the cleaved compound which showed that up
to the formation of the urea the chemistry proceeded
without diculties. All of the compounds in the ®nal
library were con®rmed by LC±MS although the purities
were variable (15±46% a/a). The presence of a peak in
the mass spectrum with m/z 624, corresponding to the
compound obtained from step 4, showed that the for-
mation of the urea did not go to completion and that
this step is not optimised. A selection of the ®nal com-
pounds are collected in Table 1.
The N-methyl diaminopimellic acid residue has also
been found to be essential for biological activity in the
pacidamycin series, and it seems likely that the sub-
stitution of this residue by a simpler diamino acid is an
important factor in the lack of biological activity
observed here. N-Methylation is known to a€ect the cis/
trans geometry of an amide bond and it seems likely
that the use of a simple amide in these analogues puts
the M2 side chain in a di€erent spatial orientation with
respect to that in the natural product. Furthermore, the
ribose sugar may not be an optimal substitution for the
deoxyribose sugar, however, this may be less important
considering the activity observed in the ribose contain-
ing tunicamycins and liposidomycins.
Conclusions
Stock solutions in 500 mL DMSO of 2 mg of each of the
®nal compounds were prepared and, assuming an aver-
age purity of 35% for the whole library, diluted with
Notwithstanding the shortcomings of the compounds
made during this work, the original synthetic scheme for

A. Bozzoli et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2759±2763
2763
the solid-phase synthesis of analogues of uridyl-peptide
antibiotics similar to mureidomycin was successfully
validated by the production of an 80-membered library
in which all ®nal compounds were con®rmed by LC±
MS analysis. The biological results obtained may be
attributable either to the structural approximations that
were introduced to simplify the synthesis in solid phase
or a lack of penetration into the bacterial cell. In the
light of recently published data, a further iteration of
this chemistry incorporating the crucial diaminobutyric
acid residue and addressing the ribose/deoxyribose issue
should furnish closer analogues of the mureidomycins.
(6.2 mg, 0.0099 mmol, 1.1 equiv), PyBop (5.15 mg. 0.0099
mmol, 1.1 equiv) and DIPEA (1.7 mL, 0.0099 mmol, 1.1 equiv).
The suspension was mixed with a rotator at room temperature
for 15 h, the resin ®ltered and washed with DMF, CH2Cl2
(three times), MeOH (three times) and again CH2Cl2 (three
times). Unreacted amino groups on the resin were capped by
treatment with a solution of acetic anhydride 10% in 1-methyl-2-
pyrrolidinone (1mL). The suspension was mixed with a rotator
at room temperature for 3 h, the resin ®ltered and washed with
DMF, CH2Cl2 (three times), MeOH (three times) and again
CH2Cl2 (three times). Completeness of the reaction was checked
by the Kaiser test. The loading (42%) was determined by the
Fmoc number reading.
9. Chang, C. D. Int. J. Pept. Protein Res. 1980, 15, 59.
10. Merri®eld, R. B. J. Org. Chem. 1993, 58, 5167.
11. Preparation of the library: From the results obtained in the
rehearsal stage, it was possible to estimate the amount of resin
bound 1 required for the synthesis of at least 2.5 mg of ®nal
compound of average MW of ca. 830 as at least 2Â1600 mg.
The synthesis of the library was carried out in syringes and after
each coupling with the monomers, the resin was split into a
number of aliquots equal to the number of monomers of the next
Acknowledgements
We wish to thank our colleagues of the Combinatorial
Technology, Lead Generation, Mass Spectrometry and
NMR Spectroscopy Laboratories for the support given
to this work.
step. The uridine intermediate 1 was anchored to the aminopo-
1
lystyrene resin (see note 8) with a yield of 92% (0.534 mmol g
)
for one batch and 81% (0.475 mmol g 1) for the other. Resin
bound 1 (2.3g and 1.9 g) were coupled with l-M1 and d-M1,
respectively after Fmoc deprotection. The reactions were com-
pared with the reference compound prepared during rehearsal
(yield=80±85%; LC analysis 100% a/a). Both batches were split
into four equal parts and the Boc group removed for coupling
with M2 (yield=80±85%; LC analysis 100% a/a). In this case,
one product was cleaved for comparison to the reference pro-
duct. Each of the eight batches were divided into two parts prior
to Fmoc deprotection and subsequent coupling with l- and d-
M3 (yield=85%; LC analysis 100% a/a). For the last step, each
of the 16 batches was split into ®ve aliquots for coupling with 5
equivalents of p-nitrophenylcarbamate of M4 in the presence of
10 equivalents of Hunig's base after Fmoc deprotection
(yield=ca. 50%). Each syringe contained ca. 35 mg of resin
and gave 6±7 mg of product after cleavage with aqueous TFA.
LC-MS analysis of the ®nal products con®rmed the presence
of all desired products with purities in the range 15±45% a/a.
LC analysis was carried out using a Supelcosil ABZ+ Plus
®tted with a diode array detector and MS output: eluents:
A=water+0.01% acetic acid; B=acetonitrile; ¯ow rate
0.8 mL min 1; gradient de®ned as: 90% A, 10% B for 2 min;
gradient to 40% A, 60% B over 6 min; gradient to 20% A,
80% B over 2 min; isocratic elution 20% A, 80% B for 1 min.
12. Kaiser, E. Anal. Biochem. 1970, 34, 595.
References and Notes
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8. Example procedure for anchoring the uridine sca€old to solid
phase: PS-aminomethyl resin (60 mg, 0.9 mmol g 1) was swol-
len with dry DMF (500 mL) then reacted with uridine sca€old
13. Bugg, T. D. H.; Gentle, C. A. J. Chem. Soc., Perkin Trans.
1 1999, 1279. Bugg, T. D. H.; Gentle, C. A.; Harrison, S. A.;
Masatoshi, I. J. Chem. Soc., Perkin Trans. 1 1999, 1285.
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