4-Aminothiazolyl analogs of GE2270 A: Design, synthesis and evaluation of imidazole analogs

Article abstract of DOI:10.1016/j.bmcl.2011.04.048

Imidazole analogs of the antibiotic natural product GE2270 A (1) were designed, synthesized, and evaluated for Gram positive bacteria growth inhibition. A recently reported, copper-mediated synthesis was exploited to prepare 4-thiazolyl imidazole analogs

Full text of DOI:10.1016/j.bmcl.2011.04.048

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3210–3215
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Aminothiazolyl analogs of GE2270 A: Design, synthesis and evaluation
of imidazole analogs
Matthew J. LaMarche a, , Jennifer A. Leeds b, JoAnne Dzink-Fox b, Steve Mullin b, Michael A. Patane a,
⇑
Elin M. Rann a, Stacey Tiamfook b
a Global Discovery Chemistry, Novartis Institutes for Biomedical Research, 250 Massachusetts Avenue, Cambridge, MA 02139, USA
b Infectious Disease Area, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Imidazole analogs of the antibiotic natural product GE2270 A (1) were designed, synthesized, and eval-
uated for Gram positive bacteria growth inhibition. A recently reported, copper-mediated synthesis was
exploited to prepare 4-thiazolyl imidazole analogs of 1. The synthesis described represents a structurally
complex, natural product-based application of this recently reported synthetic methodology. In addition,
the biological evaluation of the imidazole-based analogs further define the SAR of the 4-aminothiazolyl-
based antibacterial template.
Received 16 March 2011
Accepted 12 April 2011
Available online 21 April 2011
Keywords:
GE2270 A
Curtius
Ó 2011 Elsevier Ltd. All rights reserved.
Elongation factor Tu
Antibiotic
Imidazole
Drug discovery
Antibacterial
Natural product
Introduction: In 1991, Selva et al. from Lepetit Research Institute
reported the structure and antibiotic activity of GE2270 A (1). This
thiopeptide-based natural product was isolated from a fermenta-
tion broth of Planobispora rosea1 and displayed exquisite in vitro
antibacterial activity against methicillin resistant staphylococci
(MRSA) and vancomycin resistant enterococci (VRE), with mini-
Recently there has been increased interest in 1 and related thio-
peptides. In 2003, Vicuron Inc. reported semisynthetic derivatives
of 1 intended for parenteral administration.5 A previously de-
scribed acid-mediated decomposition of the oxazoline sidechain
allowed for SAR investigation of carbon-based sidechain analogs,
which included terminal carboxylic acids.6 One compound was
reportedly selected for advancement to the clinic as a topical treat-
ment for acne.7 In addition, partial and total syntheses of 1 have
been achieved by several academic groups.8 Structural papers9
and solution conformation studies10 of related natural products
have also recently been reported. Our own drug discovery efforts
have included the fermentation, isolation, and characterization of
the Thiomuracins.3
More recently, we described the identification, decomposition,
and subsequent stabilization of 4-aminothiazolyl analogs of 1.11
Due to promising initial in vitro antibacterial results, the 4-
aminothiazolyl template was selected for further medicinal
chemistry optimization. In order to further extend the SAR and
mimic the oxazoline ring structure of GE2270 A while retaining
exquisite antibacterial activity, 4-thiazolyl N-linked imidazole
analogs were envisioned. Thus, the acid sensitive oxazoline
sidechain1 of 1 would be replaced by a 4-thiazolyl, N-linked
imidazole. This aromatic heterocyclic replacement offered the
mum inhibitory concentrations <1 lg/mL. Thiopeptide-based acti-
nomycetes metabolites are characterized by their highly modified,
sulfur containing macrocyclic peptide structures, which possess a
tri- or tetra-substituted nitrogen-containing heterocycle core (blue
color, Fig. 1). Nearly all of the members of the thiopeptide class in-
hibit bacterial growth by inhibiting protein synthesis, however
their cellular targets are distinct. For example, the structurally
complex polycycles of the Thiostreptons2 (2, Fig. 1) and Nocathiac-
ins2 (3) bind to the 23S rRNA component of the bacterial 50S ribo-
somal subunit at the same site as the L11 ribosomal subunit, while
1 and the Thiomuracin3 (4) monocycles target the prokaryotic
chaperone, elongation factor Tu (EF-Tu).4
Abbreviations: MIC, minimum inhibitory concentration; G+, gram positive;
MRSA, methicillin resistant staphylococci; VRE, vancomycin resistant enterococci;
S. aureus, Staphylococcus aureus; E. faecalis, Enterrococcus faecalis; E. faecium,
Enterococcus faecium; S. pyogenes, Streptococcus pyogenes; EF-Tu, elongation factor
Tu.
opportunity to stabilize the sidechain structure of
1 while
⇑
Corresponding author. Tel.: +1 617 871 7729.
E-mail address: matthew.lamarche@novartis.com (M.J. LaMarche).
also acting as a linchpin for further diversification and SAR
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.048

M. J. LaMarche et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3210–3215
3211
H N
2
O
O
O
NH
2
HN
H
O
H
N
O
N
N
NH
O
H
H
NH
O
S
N
N
N
NH HO
O
N
N
S
N
HN
O
OH
H
O
N
H
O
S
H
HN
OH
O
N
S
H
N
S
O
HN
N
HN
O
S
S
N
O
HN
N
H
S
N
NH
NH
O
H
O
HN
S
O
H
HN
OH
O
NH
S
S
OH
O
OH
HN
N
H
O
GE2270A (1)
Thiostrepton A (2)
NH
2
O
O
H
OH
HN
N
O
O
N
O
H
S
S
N
N
S
N
N
HO
HO
N
O
N
N
S
H
O
NH
S
N
N
S
O
O
N
S
O
OH
O
N
HN
O
NH
O
O
O
O
S
NH
O
H
O
HN
O
HN
OH
N
S
HN
O
O
N
H
N
S
N
N
H2N
O
HO
44
O
S
H
HO
Nocathiacin I (3)
Thiomuracin A (4)
Figure 1. Thiopeptide-based natural products.
characterization. Towards this end, N-linked imidazole analogs
were designed, synthesized, and evaluated for Gram positive
bacterial growth inhibition.
Results and discussions: In the forward synthetic sense (Scheme
2), selective removal of the Boc protecting group under acidic con-
ditions (TFA or HCl)11 provided the stable amine salt which was
used directly in a formylation reaction, furnishing formamide 10.
Treatment with phosphorous oxychloride then afforded isonitrile
7, the imidazole precursor. In the cyclization event, commercially
available isocyanoacetate, copper oxide, and phenanthroline were
stirred in the presence of the isonitrile at elevated temperature
(80 °C, THF). This process furnished the imidazole-4-carboxylic
acid ethyl ester in good overall yield (11). Base-mediated hydroly-
sis then afforded the imidazole-4-carboxylic acid analog (6). Sub-
sequent amide coupling utilizing polymer supported DCC13
afforded proline analog 5, and by the same methods the acyclic
analog (12).
In 2006, Kanazawa et al. reported the efficient, direct synthesis
of imidazoles through a copper-catalyzed cross-cycloaddition be-
tween two different isocyanides.12 Therefore, a 4-thiazolyl isocya-
nide would be required to form the N-linked imidazole analogs of
1 (Scheme 1). In the retrosynthetic sense, imidazolyl-proline 5 was
disconnected at the proline amide bond revealing imidazole-acid
6. Disconnection of the imidazole ring revealed isocyanate 7,
which was envisioned to arrive from the previously described
Boc-protected 4-aminothiazole (8).11 This strategic intermediate
results from the Curtius rearrangement of known acid 9,1 a deriv-
ative of 1.

3212
M. J. LaMarche et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3210–3215
O
O
O
OH
N
N
O
N
OH
N
N
NC
HN
N
O
S
N
N
S
N
S
N
S
N
S
6
7
8
O
N
S
O
NH
2
OH
O
N
S
O
O
N
N
N
HN
N
OH
O
O
S
N
S
HN
N
NH
NH
O
O
S
9
GE2270A (1)
NH
S
O
5
Scheme 1. Retrosynthesis of imidazole-based analogs.
Analogs 1, 6, 5, and 12 were then evaluated in MIC (minimum
inhibitory concentration) assays for Gram positive bacteria growth
inhibition (Table 1).14 Four organisms comprised our antibacterial
screen and included Enterrococcus faecalis, Enterococcus faecium,
Staphylococcus aureus, and Streptococcus pyogenes. All three imidaz-
ole-based analogs showed similar bacterial growth inhibition re-
sults. Unfortunately, overall the imidazole congeners all proved
mization efforts regarding 4-aminothiazole derivatives of 1 will be
reported in due course.
Materials and methods: NMR: proton NMR spectra were re-
corded on a Bruker 400 MHz ultrashield spectrometer. Chemical
shifts are reported relative to methanol (d 3.31), dimethyl sulfoxide
(d 2.50), or chloroform (d 7.26).
LCMS: compounds were analyzed on an Inertsil ODS-3 column
significantly less potent (MICs 4 to >32
l
g/mL) than the natural
(C18, 50 Â 4.6 mm, 3
lM) with a 2 min gradient elution (25% ace-
tonitrile/H2O/5 mM ammonium formate) and a flow rate of 4 mL/
product (MICs <1 g/mL). Moderate activity (MICs 4–8
l
lg/mL)
was observed against E. faecalis and S. aureus assays, while no
activity was detected against E. faecium and S. pyogenes. However,
using an in vitro bacterial cell extract assay measuring inhibition of
protein synthesis in the absence of the cell membrane,15 imida-
zoles 5 and 12 both showed activities commensurate to the natural
min.
HPLC: purification utilizes a C8 or C18 column (30 Â 100 mm,
5 lM, brand: Sunfire or XTerra) and is performed according to
two methods. Method 1 consists of 0.1% TFA in 10–95% ACN in
H2O. Method 2 consists of 10 mM NH4OH in 40–95% ACN in H2O.
LCUV: analysis utilizes an Atlantis brand C18 column (150 mm)
with a 20 min gradient elution (0–95% acetonitrile in water +0.1%
TFA). All compounds tested in MIC assays were >95% purity by
LCUV analysis, LCMS, and 1H NMR.
Preparation of Imidazole (6): Steps 1 and 2: To a suspension of
Boc–amine 8 (480 mg, 0.647 mmol) in DCM (150 mL) was added
HCl (g, stream) for 20 min. The reaction was capped and stirred
for 30 min. The reaction was concentrated to dryness and taken
to the next step with no further purification. LCMS: Rt = 1.45 min,
[M+H]+ 1138. The residue was suspended in DCM (150 mL) and a
solution of formic acid in acetic anhydride (1 mL, 3:2) is added fol-
lowed by stirring at rt for 2 h. The crude formamide (10, 500 mg
crude) is concentrated to dryness and stored in vacuo for 12 h.
LCMS: Rt 1.46 min, [M+H]+ 1166.
Step 3: To a suspension of the intermediate crude formamide
(10, 90 mg, 0.771 mmol) in DCM (100 mL) was added DIPEA
(1 mL, 5.74 mmol) and POCl3 (100
was stirred at rt for 30 min and a second addition of DIPEA
(1 mL, 5.74 mmol) and POCl3 (100 L, 1.27 mmol) was added. The
reaction was stirred an additional 30 min and then concentrated
onto silica gel. Purification by flash chromatography (gradient elu-
tion: 60–100% EtOAc in heptane) affords 55 mg (63%, 3 steps) of
isonitrile (7). LCMS: Rt 1.72 min, [M] 1148.
product (50–64% inhibition @ 2 lM). We speculate that the moder-
ate MIC of compounds 6, 5, and 12 results from weaker cell pene-
tration or permeability rather than insufficient protein synthesis
inhibition. Furthermore, the ionizable nature of the imidazolyl-car-
boxylic acid derivatives may contribute to this effect.
In summary, 4-aminothiazolyl imidazole analogs of the antibi-
otic natural product GE2270 A were designed, synthesized, and
evaluated for Gram positive bacterial growth and protein synthesis
inhibition. A recently reported, copper-mediated isonitrile cycliza-
tion provided efficient synthetic access to the imidazole deriva-
tives. The in vitro activities of these novel congeners were
moderate in MIC assays in three of four target Gram positive
organisms and equipotent to the natural product in a cell extract
assay measuring protein synthesis inhibition. The imidazole ana-
logs described provide further understanding of the aminothiazole
sidechain SAR, and demonstrate a complex application of a useful
and efficient method of constructing imidazole heterocycles. Final-
ly, the 4-aminothiazolyl-imidazole template described herein rep-
resents a unique, semi-synthetically derived scaffold for further
antibacterial drug discovery. Certainly, the discovery of innovative
chemical scaffolds that address underexploited antibacterial mech-
anisms of action remains a pressing need in infectious disease care
in order to combat increasing clinical resistance. Further lead opti-
lL, 1.27 mmol). The reaction
l

M. J. LaMarche et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3210–3215
3213
O
H
HN
N
HN
O
O
S
N
S
N
N
N
N
S
O
N
N
S
S
S
O
O
O
O
O
N
N
S
S
HN
HN
O
O
i. HCl (g), DCM
S
S
N
HN
HN
H
N
H
N
NH
NH
ii. HOOCH,
Ac2O
O
O
N
N
S
N
S
O
O
O
O
NH
NH
8
10
O
O
NC
N
S
N
N
Cu2O, THF
POCl3, DIPEA
N
S
O
O
O
O
DCM
(63%, 3 steps)
N
S
S
N
HN
O
O
S
N
HN
H
N
NH
N
N
O
N
S
O
(80%)
O
NH
O
N
7
O
O
N
S
N
N
N
O
O
N
OH
O
6
5
N
N
S
1. NaOH
OH
OH
N
O
2. PS-DCC
O
N
S
N
S
N
N
HN
O
3. NaOH
O
O
O
S
N
HN
N
H
N
NH
O
N
12
N
S
H
O
O
NH
11
S
N
N
N
O
N
S
S
OH
O
N
S
HN
O
S
N
HN
H
N
NH
O
O
N
S
O
NH
O
Scheme 2. Synthesis of 4-thiazolyl-imidazole analogs.

3214
M. J. LaMarche et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3210–3215
Table 1
Minimum inhibitory concentrations (MIC) and inhibition of prokaryotic protein synthesis
N
N
S
OH
O
N
S
N
S
HN
N
O
O
HN
N
NH
NH
O
O
S
NH
S
O
O
O
O
O
O
O
O
NH2
OH
OH
N
N
N
O
N
OH
N
N
N
H
N
N
N
N
S
N
S
S
N
S
N
6
1
5
12
E. faecaelis
0.5
4
8
8
E. faecium
S. aureus
S. pyogenes
Inhibition of protein expression
0.25
0.25
1
>32
4
>32
0%
>32
8
>32
53%
32
4
>32
64%
61%
Step 4: To a solution of isonitrile (7, 155 mg, 0.135 mmol) in
THF (8 mL) was added Cu2O (cat.), ethyl isocyanoacetate
(13.8 mg, 0.122 mmol) and phenanthroline (3.1 mg, 0.017 mmol).
The reaction was placed in a sealed tube and heated to 80 °C for
2 h, then cooled to rt. The reaction was concentrated and purified
by flash chromatography (gradient elution: 50–100% EtOAc in hep-
tane; second column: gradient elution: 0–10% MeOH in DCM) to
furnish 136 mg (80%) imidazole-ester 11. LCMS: Rt 1.62 min,
[M+2H]+ 1262.
(gradient elution: 0–10% MeOH in DCM + 0.1% acetic acid) afforded
4.2 mg of imidazole acid 12. LCMS: Rt 1.18 min, [M+2H]+ 1249.
HRMS: (M/2) 624.6121, calculated: 624.6129. 1H NMR (DMSO-d6)
d ppm 9.12 (d, 1H), 8.68 (d, 2H), 8.55–8.63 (m, 2H), 8.36–8.48
(m, 4H), 8.27 (d, 2H), 8.02 (s, 1H), 7.19–7.41 (m, 8H), 6.17 (s,
1H), 5.16–5.32 (m, 3H), 4.95–5.03 (m, 3H), 4.23–4.33 (m, 1H),
3.74–3.84 (m, 1H), 3.61–3.69 (m, 2H), 3.38 (s, 3H), 2.67–2.76 (m,
1H), 2.58 (s, 3H), 2.45–2.48 (m, 3H), 2.12–2.21 (m, 1H), 1.27–
1.38 (m, 1H), 0.81–0.90 (m, 6H).
Step 5: To a solution of imidazole 11 in MeOH (8 mL) and H2O
(2 mL) was added NaOH (s, 10 mg, 0.25 mmol) and reaction was
heated to 40 °C for 12 h. The reaction was cooled to rt and concen-
trated to dryness. Final purification by HPLC (gradient elution: 45–
55% acetonitrile in H2O with 0.1% TFA) generates 3 mg of 6 as a TFA
salt. LCMS: Rt 1.13 min, [M+H]+ 1191. HRMS: 1191.1949; calcu-
lated: 1191.1975. 1H NMR (DMSO-d6) d ppm 9.02 (d, 1H), 8.68
(d, 2H), 8.60 (s, 1H), 8.55–8.58 (m, 2H), 8.41–8.47 (m, 2H), 8.38
(d, 1H), 8.25–8.30 (m, 2H), 7.20–7.39 (m, 8H), 5.99–6.06 (m, 1H),
5.17–5.32 (m, 3H), 4.95–5.03 (m, 3H), 4.22–4.32 (m, 1H), 3.74–
3.83 (m, 1H), 3.38 (s, 3H), 2.67–2.75 (m, 1H), 2.58 (s, 3H), 2.45–
2.48 (m, 3H), 2.12–2.21 (m, 1H), 1.25–1.35 (m, 1H), 0.81–0.91
(m, 6H).
Preparation of Imidazole (5): Step 1: To a suspension of crude
imidazole acid 6 (276 mg, 0.232 mmol) in DCM (20 mL) and pyri-
dine (1.0 mL) was added PS-DCC (0.695 mmol) and (R)-pyrroli-
dine-2-carboxylic acid methyl ester (60 mg, 0.463 mmol). The
reaction was stirred and heated to 40 °C for 24 h. The reaction was
concentrated onto silica gel and purified (twice) by flash chromatog-
raphy (gradient elution: 0–5% MeOH in DCM) to generate 170 mg of
methyl ester intermediate. LCMS: Rt 1.45 min, [M+2H]+ 1303.
Step 2: The methyl ester (170 mg, 1.305 mmol) was suspended
in MeOH (10 mL) and H2O (2 mL) and NaOH (s, 40 mg 1.00 mmol)
were added. The reaction was stirred at rt for 18 h then heated to
40 °C for 12 h. The reaction was mounted onto silica gel for purifi-
cation by flash chromatography (gradient elution: 0–10% MeOH in
DCM) followed by HPLC purification (gradient elution: 40–60% ace-
tonitrile in H2O with 0.1% TFA) which afforded 5 mg of imidazole
acid 5. LCMS: Rt 1.11 min, [M+H]+ 1288. HRMS: (M/2) 644.6292;
calculated: 644.6285. 1H NMR (DMSO-d6) d ppm 9.07 (d, 1H),
8.68 (d, 2H), 8.61 (s, 1H) 8.49–8.55 (m, 1H) 8.45 (d, 3H), 8.37 (d,
1H), 8.25–8.32 (m, 2H), 7.21–7.41 (m, 8H), 6.05 (s, 1H), 5.17–
5.33 (m, 3H), 4.95–5.04 (m, 3H), 4.24–4.33 (m, 1H), 4.06–4.18
(m, 1H), 3.75–3.85 (m, 1H), 3.38 (s, 3H), 2.67–2.76 (m, 1H), 2.58
(s, 3H), 2.44–2.48 (m, 3H), 2.07–2.34 (m, 4H), 1.83–2.03 (m, 3H),
1.25–1.36 (m, 1H), 0.81–0.91 (m, 6H).
Preparation of Imidazole (12): Step 1: To a suspension of crude
imidazole 6 (180 mg, 0.151 mmol) in DCM (10 mL) and pyridine
(500 lL) is added PS-DCC (0.453 mmol) and amino-acetic acid
methyl ester (20 mg, 0.227 mmol). The reaction is stirred at rt for
48 h and then heated to 40 °C for 2 h. The residue is concentrated
onto silica gel and purified by flash chromatography (gradient elu-
tion: 50–100% EtOAc in heptanes) to generate 77 mg of methyl es-
ter intermediate. LCMS: Rt 1.35 min, [M+H]+ 1262.
Step 2: The methyl ester (77 mg, 0.061 mmol) was suspended in
MeOH (5 mL) and H2O (1 mL) and NaOH (s, 40 mg, 1.00 mmol) was
added. The reaction was stirred at rt for 12 h and then concen-
trated onto silica gel for purification by flash chromatography
MIC assays: MIC assays were conducted according to the Clinical
and Laboratory Standards Institute (CLSI). Wikler, Cockerill, Bush et

M. J. LaMarche et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3210–3215
3215
verified: Tavecchia, P.; Gentili, P.; Kurz, M.; Sottani, C.; Bonfichi, R.; Selva, E.;
Lociuro, S.; Restelli, E.; Ciabatti, R. Tetrahedron 1995, 16, 4867. For acidic
decomposition see above.
al. (2009); Methods for Dilution Antimicrobial Susceptibility Tests
for Bacteria that Grow Aerobically; Approved Standard-Eighth
Edition. CLSI M07-A8, Vol. 29, No. 2. Bacterial strains included
E. faecalis (ATCC 29212), E. faecium (Professor Chopra, Univ. Leeds
UK, strain 7130724), S. aureus (MRSA from Professor Willinger, iso-
lated from a pharyngeal smear, AKH Vienna A4.018), S. pyogenes
(ATCC BAA-595). Wikler, Cockerill, Bush et al. (2009); Performance
Standards for Antimicrobial Susceptibility Testing; Nineteenth
informational supplement. CLSI M100-S19, Vol. 29, No. 3.
2. For a review of thiopeptide natural products see: (a) Bagley, M. C.; Dale, J. W.;
Merritt, E. A.; Xiong, X. Chem. Rev. 2005, 105, 685; (b) Rezanka, T.; Sobotka, M.;
Spizek, J.; Sigler, K. Anti-Infect. Agents Med. Chem. 2006, 5, 187; (c) Hughs, R. A.;
Moody, C. J. Angew. Chem., Int. Ed. 2007, 46, 2; Thiostrepton: (d) Mocek, U.;
Zeng, Z.; O’Hagan, D.; Zhou, P.; Fan, L. D. G.; Beale, J. M.; Floss, H. G. J. Am. Chem.
Soc. 1993, 115, 7992; Nocathiocins: (e) Pucci, M. A.; Bronson, J. J.; Barret, J. F.;
DenBleyker, K. L.; Driscotto, L. F.; Fung-Tomc, J. C.; Ueda, Y. Antimicrob. Agents
Chemother. 2004, 48, 3697; (f) Naidu, B. N.; Sorensen, M. E.; Bronson, J. J.; Pucci,
M. J.; Clark, J. M.; Ueda, Y. Bioorg. Med. Chem. Lett. 2005, 15, 2069. and
references cited therein; Amythiomicins (g) Shiminaka, K.; Kinoshita, N.;
Iinuba, H.; Hamada, M.; Takeuchi, T. J. Antibiot. 1994, 6, 668.
3. Thiomuracins: Morris, R. P.; Leeds, J. A.; Naegeli, H. U.; Oberer, L.; Memmert, K.;
Weber, E.; LaMarche, M. J.; Parker, C. N.; Burrer, N.; Esterow, S.; Hein, A. E.;
Schmitt, E. K.; Krastel, P. J. Am. Chem. Soc. 2009, 131, 5946.
4. For review of the elongation factors see: Lim, V. I.; Curran, J. F.; Garber, M. B. J.
Mol. Biol. 2005, 351, 470; Nilsson, J.; Nissen, P. Curr. Opin. Struct. Biol. 2005, 15,
349.
In vitro transcription/translation assays: The E. coli S30 extract
system for circular DNA (Promega Cat # L1020) was used per rec-
ommendation of the manufacturer with slight modifications.
Briefly, 3.5
with 1.0 L each of 1 mM ‘methionine minus’ and ‘cysteine minus’
amino acid mixes, 8 L of S30 premix, 6 L of S30 extract and
0.5
L of the test agents at 40Â final concentration, in a total vol-
ume of 20 L. The reaction mixtures were incubated for 2 h at
lL of 286 ng/ll template DNA (pBESTluc™) was mixed
l
l
l
l
5. Clough, J.; Chen, S.; Gordon, E. M.; Hackbarth, C.; Lam, S.; Trias, J.; White, R. J.;
Candiani, G.; Donadio, S.; Romano, G.; Ciabatti, R.; Jacobs, J. W. Bioorg. Med.
Chem. Lett. 2003, 13, 3409; Malabarba, M.; Cavaleri, M.; Mosconi, G.; Jabes, D.;
Romano, G.; WO03105881.
6. Tavecchia, P.; Kurz, M.; Colombo, L.; Bonofichi, R.; Selva, E.; Lociuro, S.;
Marzorati, E.; Ciabatti, R. Tetrahedron 1996, 26, 8763.
l
37 °C in 384 well flat bottom white plate (Corning, catalog
# 3704). The formation of luciferase was measured by adding equal
volume (20 lL) of Steady-Glo luciferase reagent (Promega Cat
# 27104) and emitted light was detected with a luminometer
(Molecular Devices, LMaxll plate reader). Kirromycin and puromy-
cin, which are known to be protein synthesis inhibitors, were used
as positive controls. Ampicillin was used as a negative control. The
Rabbit Reticulocyte TnTÒ Quick coupled Transcription/Translation
system (Promega catalog # L1170) was used as recommended by
the manufacturer except that the assay volumes were scaled down
7. Butler, M. S. Nat. Prod. Rep. 2005, 22, 162.
8. (a) Heckmann, G.; Bach, T. Angew. Chem., Int. Ed. 2005, 44, 1199; (b) Nicolaou, K.
C.; Zou, B.; Dethe, D. H.; Li, D. B.; Chen, D. Y. K. Angew. Chem., Int. Ed. 2006, 45,
7786.
9. (a) Heffron, S. E.; Jurnak, F. Biochemistry 2000, 39, 37; (b) Heffron, S. E.; Moeller,
R.; Jurnak, F. Acta Cryst. 2006, D62, 433; (c) Parmeggiani, A.; Nissen, P. FEBS Lett.
2006, 580, 4576.
10. Lewis, R. J.; Hughs, R. A.; Alcaraz, L.; Thompson, S. P.; Moody, C. J. Chem.
Commun. 2006, 4215.
11. (a) LaMarche, M.J.; Leeds, J.A.; Dzink-Fox, J.; Gunderson, K.; Krastel, P.;
Memmert, K.; Patane, M.A.; Rann, E.M.; Schmitt, E.; Tiamfook, S.; Wang, B. J.
Med. Chem. ASAP Article March 15, 2011.; (b) LaMarche, Matthew J.; Bushell,
Simon,; Patane, Michael, Whitehead, Lewis; Aminothiazoles and Their Uses.
WO2007142986.
12. Kanazawa, C.; Kamijo, S.; Yamamoto, Y. J. Am. Chem. Soc. 2006, 128, 10662.
13. Weinshenker, G. M. et al Org. Synth., Coll. 1988, 6, 951; Guan, Y. et al J. Comb.
Chem. 2000, 2, 297; Senton, K. et al J. Comb. Chem. 2000, 5, 336; Cano, M. et al J.
Org. Chem. 2002, 67, 129.
from 50 to 20
of TnTÒ Quick master mix, 3
methionine and 0.5
L of the test compound at 40Â final concen-
tration in a final volume of 20 L. The reacation mixtures were
lL. Briefly the assay components consisted of 16
lL
l
L of (pT7luc) at 167 ng/ l, 0.5 L of
l
l
l
l
incubated at 37 °C for 75 min. Luminescence was detected as de-
scribed above.
14. MIC assays were conducted according to the Clinical and Laboratory Standards
Institute (CLSI). Wikler, M.A., Cockerill, F.R., Bush, et al (2009); Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically;
Approved Standard- Eighth Edition. CLSI M07-A8, Vol. 29, No. 2. Bacterial
strains included E. faecalis (ATCC 29212), E. faecium (Professor Chopra, Univ.
Leeds UK, strain 7130724), S. aureus (MRSA from Professor Willinger, isolated
from a pharyngeal smear, AKH Vienna A4.018), S. pyogenes (ATCC BAA-595).
Wikler, M.A., Cockerill, F.R., Bush, et al (2009); Performance Standards for
Antimicrobial Susceptibility Testing; Nineteenth informational supplement.
CLSI M100-S19, Vol. 29, No. 3.
Acknowledgments
The authors thank the associates at the Novartis Natural Prod-
ucts Unit for their collaborative efforts.
References and notes
1. Selva, E.; Beretta, G.; Montanini, N.; Saddler, G. S.; Gastaldo, L.; Ferrari, P.;
Lorenzetti, R.; Landini, P.; Ripamonti, F.; Goldstein, B. P.; Berti, M.; Montanaro,
I.; Denaro, M. J. Antibiot. 1991, 7, 693; The structure was later corrected and
15. (a) Zubay, G. Annu. Rev. Genet. 1973, 7, 267; (b) Zubay, G. Methods Enzymol.
1980, 65, 856.