Combinatorial modification of natural products: Synthesis and in vitro analysis of derivatives of thiazole peptide antibiotic GE2270 A: A-ring modifications

Article abstract of DOI:10.1016/S0960-894X(03)00811-4

Thiazole peptide GE2270 A (1) possesses potent antimicrobial activity against many gram-positive pathogens, including methicillin resistant Staphylococcus aureus (S. aureus, MRSA; MIC₉₀=0.06 μg/mL) and vancomycin resistant Enterococcus spp. (VRE; MIC₉₀=0.03 μg/mL); however its poor aqueous solubility has prohibited its development for the clinical treatment of infections. An integrated combinatorial and medicinal chemistry program was employed to identify derivatives of 1 that retain activity but possess greatly enhanced aqueous solubility.

Full text of DOI:10.1016/S0960-894X(03)00811-4

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3409–3414
Combinatorial Modification of Natural Products: Synthesis and
In Vitro Analysis of Derivatives of Thiazole Peptide Antibiotic
GE2270 A: A-Ring Modifications
Jeffrey Clough,a,y Shaoqing Chen,a,{ Eric M. Gordon,a,x Corinne Hackbarth,a
Stuart Lam,a,x Joaquim Trias,a Richard J. White,a Gianpaolo Candiani,b
Stefano Donadio,b Gabriella Romano,b Romeo Ciabattib and Jeffrey W. Jacobsa,*,x
aVicuron, Inc., 34790 Ardentech Court, Fremont, CA 94555, USA
bVicuron, Inc., 21040 Gerenzano (VA), Italy
Received 11 March 2003; revised 23 July 2003; accepted 29 July 2003
Abstract—Thiazole peptide GE2270 A (1) possesses potent antimicrobial activity against many gram-positive pathogens, including
methicillin resistant Staphylococcus aureus (S. aureus, MRSA; MIC90=0.06 mg/mL) and vancomycin resistant Enterococcus spp.
(VRE; MIC90=0.03 mg/mL); however its poor aqueous solubility has prohibited its development for the clinical treatment of
infections. An integrated combinatorial and medicinal chemistry program was employed to identify derivatives of 1 that retain
activity but possess greatly enhanced aqueous solubility.
# 2003 Elsevier Ltd. All rights reserved.
The emergence of multi-drug resistant strains of bacterial
pathogens is a significant clinical problem.1 Of key concern
are the Gram positive pathogens, especially Staphylococcus
aureus, Staphylococcus pneumoniae, Entoerococcus faecium
and Enterococcus faecalis.1 Efforts to improve or
expand the utility of existing antibiotics have been only
moderately successful; hence, there is an urgent need for
novel classes of antibacterial agents that act on pre-
viously unexploited biochemical targets essential to the
pathogen life-cycle. One such novel class of anti-infec-
tive not currently used clinically is the thiazole peptides.
We report herein the synthesis and in vitro antibacterial
properties of a series of derivatives of thiazole peptide
GE2270 A (1, Scheme 1) that were engineered to possess
greatly enhanced aqueous solubility.2
their thiazole heterocycles.3 These natural products are
noted for their potent antibacterial activity, the pre-
dominant mechanism of which is inhibition of protein
synthesis.3 Thiazole peptide GE2270 A (1) was selected
as a starting point for an anti-infective discovery pro-
gram for the following reasons. First, 1 is a potent
inhibitor of elongation factor Tu (EF-Tu; IC50=5 nM),
the molecular chaperone responsible for translocating
aminoacylated tRNAs to the bacterial ribosome.4 EF-
Tu is essential for bacterial protein biosynthesis, and no
antibiotics currently used clinically act via this mechan-
ism. Second, EF-Tu from several bacterial species are
sensitive to 1,5a whereas the mammalian counterpart to
this protein (eukaryotic elongation factor, EF-1 alpha)
is structurally distinct from the Escherichia coli pro-
tein,5b and 1 is inactive in a mammalian cell-free protein
synthesis system.6 Third, 1 possesses exquisite anti-
microbial activity against many gram-positive patho-
gens, including MRSA (MIC90 0.06 mg/mL) and VRE
(MIC90 0.03 mg/mL),2d and has also been shown to be
highly efficacious in animal models of infection.7 The
key limitation to the development of this compound has
been its very poor solubility in water (<0.0001 mg/mL
in PBS pH 7.4 buffer), making formulation for systemic
administration to humans difficult. Therefore, the goal
of the chemistry program was identification of analogues
of 1 which retain potency yet possess greatly enhanced
Thiazole peptides comprise a family of non-ribosomally
synthesized compounds classified by the architecture of
*Corresponding author at present address: Rigel, Inc., 240 East Grand
Avenue, South San Francisco, CA 94080, USA. Tel.:+1-650-266-
3628; fax: +1-650-266-3501; e-mail: jjacobs@sunesis.com
yPresent address: Rigel, Inc., 240 East Grand Avenue, South San
Francisco, CA 94080, USA.
{Present address: Discovery Chemistry, Hoffmann-La Roche, Inc., 340
Kingsland St., Nutley, NJ 07110, USA.
xPresent address: Sunesis Pharmaceuticals, Inc., 341 Oyster Point
Boulevard, South San Francisco, CA 94080, USA.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00811-4

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J. Clough et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3409–3414
Scheme 1. Hydrolysis of GE2270 A and preparation of intermediates for high-throughput synthesis: (a) H2SO4, THF/H2O 6:4, 60 ꢁC, 4 h, 90%;
(b) NaOH, THF/H2O, 2 h, 90%; (c) 1-[3-(N,N-dimethylamino)propyl]-3-ethylcarbodiimide HCl, pentafluorophenol, dioxane, 16 h, 85%; (d) sodium
borohydride, THF/H2O 6:4, 73%; (e) 4-nitrophenylchloroformate (pNP-Cl), pyridine, THF, 80%; (f) MagtrieveTMa, THF (not isolated); (g) Sieber
amide resin:b (1) 20% piperidine/DMF; (2) product from ‘f’, 2% acetic acid/DMF, 12 h; (3) sodiumcyanoborohydride, 1% acetic acid/DMF, 16 h;
(4) 5% trifluoroacetic acid (TFA)/DCM, 1 h; (5) pNP-Cl, pyridine, DCM; 12 h, 70%. aAldrich Chemical Co., Milwaukee, WI, USA. bCalbiochem-
Novabiochem Co., San Diego, CA, USA.
aqueous solubility at neutral pH. MIC criteria were set
as ꢀ1 mg/mL, which is the MIC of vancomycin
obtained against a majority of clinical isolates of MRSA
and vancomycin-susceptible enterococci.8 The aqueous
solubility objective was set at a minimum of 0.5 mg/mL
in PBS buffer at neutral pH—a modest solubility
requirement but a 103-104 fold improvement over the
parent compound.9 This solubility was estimated to be
the lowest acceptable level that would still permit form-
ulation for parenteral administration using pharmaceu-
tically acceptable excipients.10
Selected compounds were resynthesized and purified to
homogeneity, then screened against both an expanded
panel of bacteria as well as tested for their aqueous
solubility. Representative data are shown in Table 1.
Table 1. Activity and solubility data for selected compounds from
A-ring dipeptide libraries
Degradation products of 1 have been described that
partially retain antimicrobial activity, including ester 2
and acid 3 (Scheme 1). Acid 3 became the initial focus of
the chemistry program because this functionality was
hoped to provide a facile entry into analogue synthesis.
Since the oxazolino-proline moiety on 1 is the dehy-
dration product of a Ser-Pro dipeptide, our first chemi-
cal modifications employed carboxylate 3 for the
combinatorial reassembly of variants of this dipeptide
motif. Although the chemistry results in peptidic com-
pounds, the diversity of side-chain functionality readily
accessible from commercially-available amino acids
allows a relatively facile survey of functional groups and
was hoped to establish a rough SARfor both the activ-
ity and solubility requirements. Acid 3 was prepared as
described (Scheme 1),2e and was found to cleanly acyl-
ate resin-bound mono- and dipeptides using standard
techniques (data not shown). Three 88-member libraries
of peptidic derivatives were prepared via parallel syn-
thesis, which surveyed a broad range of functionality,
including acidic, basic, and neutral side chains, l- and
d-configured amino acids, and many unnatural amino
acids (data not shown)—264 derivatives in total. In
most instances, crude library products exceeded 70%
purity while yields were approximately 50% as esti-
mated by reverse-phase (C18) HPLC and MS analysis.
Crude library products were initially screened against a
panel of four organisms, including three bacterial patho-
gens, and Candida albicans as a eucaryotic surrogate.8b
Compd
RMIC (
mg/mL)
Solubility
(mg/mL)c
MSSAa MRSAb
4
NT
0.25
0.06
0.004
5
6
NT
0.00011
>32
>32
>1.5
7
8
>32
>32
>32
>2.8
>2
>32
>32
9
>32
2.6
10
NT
4
0.19
aATTC25923.
bATTC33591.
cSolubility values are medians of three determinations (see ref 9).

J. Clough et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3409–3414
3411
Compound 4 was quite active and is equivalent to the
product derived from the hydration of the oxazoline
ring of 1, liberating a serine side chain. Unfortunately,
solubility was only marginally improved (4 mg/mL, see
Table 1). Compound 5 contains an azetidine 3-carbox-
amide derivative off the A-ring, and retains potency
equivalent to the parent compound, but its aqueous
solubility is just barely detectable at 0.1 mg/mL. In con-
trast, several compounds had excellent solubility char-
acteristics (e.g., examples 6–9) but were devoid of any
antibacterial activity in our assay (MICMRSA >32 mg/
mL). These compounds are bis-acids, and perhaps have
difficulty crossing the bacterial cell-wall. Interestingly,
no basic compounds were identified with aqueous solu-
bilities >10 mg/mL although over 50 were prepared
(data not shown). The compound which best approa-
ched key criteria in this exercise is mono-carboxylic acid
10.
mediate for urea synthesis via oxidation of 12 to an
aldehyde followed by a solid-phase mediated reductive
amination (see Scheme 1).11 Active intermediates 11, 13
and 14 were employed to acylate a broad range of
functionalized amines and the resulting amides, carba-
mates and ureas analyzed for their antimicrobial activ-
ity and solubility properties (Table 2). Most acylations
employing 11 or 13 proceeded in high yield at room
temperature in under an hour, while carbamate 14
required heating at 50 ꢁC overnight to obtain the
desired ureas (Scheme 2). All products were purified by
semi-preparative reverse-phase (C18) HPLC and con-
verted to either their sodium or hydrochloride salt prior
to testing. Over 150 diverse GE2270 A analogues were
prepared via Scheme 2, and representative derivatives
are shown in Table 2.
A diverse range of functionality was surveyed at the A-
ring, including alcohols (15–19), amines and amine
derivatives (20–27), and carboxylic acids (28–41).
Including the derivatives made on solid phase, nearly
400 compounds were prepared before the first derivative
with an acceptable MIC and solubility profile was iden-
tified (32, Table 2). An examination of the accumulated
SARsuggested that carboxylate derivatives afforded the
highest solubility at pH 7.4, and that this functional
group must be positioned at least five atoms from the A-
ring in order to retain antibacterial potency. Once these
criteria were defined, the next 25 compounds we pre-
pared yielded four analogues fulfilling the key objectives
(examples 34–37). Acceptable derivatives were identified
only in the amide and carbamate series; no urea analo-
gues satisfied the activity goals (cf 33 vs 34). N-Methyl-
ation to afford tertiary amides and carbamates resulted
in a significant improvement in aqueous solubility (e.g.,
37 vs 38, and 40 vs 41), perhaps due to conformational
changes and/or reduced A-ring conjugation with the
tertiary amide. Ultimately, eight compounds were iden-
tified that satisfied activity and solubility criteria (32,
34–39, 41), and their potency against an expanded panel
of bacteria is illustrated in Table 3. MICs against
MRSA and VRE were ꢀ1 mg/mL and all derivatives
were at least equipotent to vancomycin against suscep-
tible strains of these pathogens (Table 3). Most analo-
gues in Table 3 possess a Gram positive spectrum of
activity consistant with that of parent compound 1, but
with potency attenuated approximately 10-50-fold. One
exception is 38, which was inactive against the indicated
Streptococcal strains (MICs >16 mg/mL). Interestingly,
some derivatives were active against the Gram negative
pathogen Haemophilus influenzae (38, 39, and 41) while
1 is inactive against this strain (MIC >16 mg/mL). In
our assay, the aqueous solubility of all compounds in
Table 3 was improved approximately 10,000-fold over
that of 1 (Table 2).9
We next wished to survey a broader range of function-
ality at the A-ring to more thoroughly establish the
MIC and solubility SARs. It was reasoned that an
active ester derivative of 3 could permit the rapid
incorporation of highly functionalized amines in
unprotected form. Thus, the pentafluorophenol (PFP)
ester of 3 was prepared (11, Scheme 1), and as desired,
ester 11 could be used to cleanly acylate a broad cross-
section of functionally dense amine nucleophiles
(Scheme 2 and Table 2). Acylations were initially per-
formed in DMF in the presence of N,N-diisopropyl-
ethylamine (DIEA); however many hydrophilic amines
were insoluble under these conditions. In these instan-
ces, most amines were cleanly acylated after first treat-
ing them with chlorotrimethylsilane (TMS-Cl) and
DIEA to render them soluble in organic solvent
(Scheme 2). Desired products were typically isolated in
30–50% yield after semi-preparative reverse-phase
(C18) HPLC purification.
Carbamates and ureas could be prepared analogously to
amides using appropriate derivatives of GE2270 A. As
illustrated in Scheme 1, 2 was further employed as a
synthon for the preparation of active intermediates for
carbamate and urea synthesis. Thus, reduction of 2 with
sodium borohydride (NaBH4) produced alcohol 12, and
acylation of 12 with p-nitrophenylchloroformate (pNP-
Cl) afforded carbonate 13—an intermediate for the
facile synthesis of carbamates from amine nucleophiles
(Scheme 1). Carbamate 14 was prepared as an inter-
An integrated program of combinatorial solid-phase
synthesis and solution-phase medicinal chemistry was
employed to synthesize approximately 500 A-ring ana-
logues of thiazole peptide GE2270 A. This exercise
rapidly explored the scope and limitations of function-
ality tolerated at the A-ring and ultimately led to the
identification of eight compounds meeting activity and
Scheme 2. Acylation of amines with reactive intermediates: (i) Amines
soluble in DMF: (a) (11 or 13) DIEA, DMF, 1 h; (b) (14) DMF,
DIEA, 50 ꢁC, 16 h. (ii) Hydrophilic amines not soluble in DMF: (c)
amine, chlorotrimethylsilane (TMS-Cl), DIEA, DCM, 40 ꢁC, 1–3 h,
concentrate, then: (1) 11 or 13, DIEA, DMF, 1 h; or (2) 14, DMF,
DIEA, 50 ꢁC, 16 h.

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J. Clough et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3409–3414
Table 2. Structure–activity relationship of A-ring amide, carbamate and urea analogues of GE2270 A
Compd
RMIC (
mg/mL)
VSEc
Solubility
(mg/mL)e
MSSAa
0.03
MRSAb
0.03
VREd
0.015
1
0.015
<0.0001
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
n=1
n=3
0.06
0.125
0.125
0.06
0.06
2
0.03
0.06
0.015
0.125
2
0.03
0.125
0.015
0.06
4
<0.0001
0.0002
<0.0001
<0.0002
0.0012
0.001
0.0005
0.014
0.005
<0.01
<0.01
<0.01
<0.01
0.2
0.125
0.125
0.25
2
n=1
n=2
n=1
n=2
n=1
n=2
n=1
n=3
n=1
n=4
0.5
0.25
0.06
1
0.06
0.06
0.5
1
0.06
0.06
2
0.125
1
2
2
2
2
1
2
2
1
1
2
2
4
8
8
8
8
4
8
8
0.5
0.06
4
0.5
0.015
4
0.06
0.03
0.5
0.03
0.25
2
0.06
0.03
1
0.05
0.2
4
2
0.25
0.25
0.2
1
0.25
8
0.5
8
>16
0.66
1
0.5
0.5
1
1.2
(continued)

J. Clough et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3409–3414
3413
Table 2 (continued)
Compd
RMIC (
mg/mL)
Solubility
(mg/mL)e
MSSAa
0.5
MRSAb
0.5
VSEc
VREd
1.0
35
R
0.25
0.7
36
37
38
39
40
41
S
1
0.5
0.25
0.06
0.5
0.5
0.9
R0=H
R0=Me
0.25
0.5
0.5
0.25
0.5
0.125
0.25
0.25
0.125
0.25
0.125
0.5
0.5
0.73
0.6
0.125
0.015
0.125
0.5
R0=H
0.03
0.25
0.28
0.91
R0=Me
aATTC25923.
bATTC33591.
cBM4147.1.
dATTC51299.
eValues are medians of three determinations (see ref 9).
Table 3. MICs of selected compounds against an expanded panel of Gram-positive and Gram-negative pathogens
Bacterial strain
MIC (mg/mL)
Vanb
1
32
1
0.25
2
0.25
0.25
2
1
>16
2
>16
34
1
0.5
2
0.5
1
4
8
>16
2
>16
35
36
37
38
0.5
0.25
0.5
0.5
0.5
>16
>16
8
39
41
S. aureus (ATTC 25923)
1
1
1
2
0.03
0.03
0.03
0.015
0.015
0.06
0.5
0.5
2
0.25
1
2
2
16
1
0.5
2
0.25
0.5
4
8
16
0.25
0.125
0.25
0.06
0.125
2
0.5
0.25
0.5
0.125
0.5
2
0.5
0.25
1
0.125
0.25
2
4
2
1
>16
S. aureus (MRSA; ATTC33591)
S. epidermis (ATTC 12228)
E. faecium (BM4147.1)
E. faecalis (VRE, ATTC51299)
S. pneumoniae (ATTC49619)
S. pyogenes (ATTC19615)
>16
0.125
0.25
0.125
8
16
4
>16
4
8
Haemophilus influenzae (ATTC31517) >16
>16
>16
>16
H. influenzae (acr, LS-2)a
Eschericia coli (A G100B)
>16
>16
1
>16
1
>16
2
>16
2
>16
aEfflux pump mutant.
bVancomycin.
solubility criteria. Surprisingly, no amine-containing
compounds met the solubility requirements although
many active derivatives were identified. Acceptable
compounds shared several structural features, including
a carboxylate moiety to facilitate aqueous solubility,
and the placement of this moiety at least five atoms
removed from the A-ring thiazole. Selected compounds
will next be examined in vivo in animal models of
infection, the results from which will be reported else-
where.
References and Notes
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Lociuro, S.; Restelli, E.; Ciabatti, R. Tetrahedron 1995, 51,
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Acknowledgements
We thank S. Lopez and C. Wu for their assistance in
evaluating the compounds.

3414
J. Clough et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3409–3414
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the chromophore comprised by the A-, B-, C- and F-rings
interconnected to the central pyridyl nucleus (see Scheme 1;
l
max=340 nm, e=34,000).2b Compounds (1–2 mg; sodium
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Performance Standards for Antimicrobial Susceptibility Test-
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p90. (b) MICs were determined according to National Com-
mittee for Clinical Laboratory Standards (NCCLS) guidelines,
but with 0.02% bovine serum albumin (BSA) added to the
growth medium.
or hydrochloride salts) were vigorously shaken in 1 mL
PBS pH 7.4 buffer for 3 h, clarified by centrifugation at
10,000g for 5 min, filtered (0.2 mm Millipore Millex-LG;
PTFE membrane), and then optical densities determined.
All compounds were tested in triplicate. The limit of detec-
tion of this method was on the order of 0.1 mg/mL. Acid 3
was always included as
GE2270 A (1) was undetectable under these conditions
a
control (0.2ꢂ0.06 mg/mL);
(<0.1 mg/mL).
10. (a) Estimate based on an approximate clinical dose of 1–2
mg/kg, 80 kg average patient weight, 100 mL maximum
volume of diluent for a parenteral antibiotic, and a 2- to 4-fold
increase in formulated drug concentration via the addition of
excipients. See: Sweetana, S.; Akers, M. J. PDA J. Pharm. Sci.
Tech. 1996, 50, 330. (b) Nema, S.; Washkuhn, R. J.; Brendel,
R . J.PDA J. Pharm. Sci. Tech. 1997, 51, 166.
11. Although this solid-phase route requires several steps, it
was superior to repeated attempts at solution-phase reductive
amination using ammonia and ammonia equivalents.
9. An equilibrium solubility assay was adapted that exploits