Fluorocyclines. 1. 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6- deoxytetracycline: A potent, broad spectrum antibacterial agent

Article abstract of DOI:10.1021/jm201465w

This and the accompanying report (DOI: 10.1021/jm201467r) describe the design, synthesis, and evaluation of a new generation of tetracycline antibacterial agents, 7-fluoro-9-substituted-6-demethyl-6-deoxytetracyclines ("fluorocyclines"), accessible through a recently developed total synthesis approach. These fluorocyclines possess potent antibacterial activities against multidrug resistant (MDR) Gram-positive and Gram-negative pathogens. One of the fluorocyclines, 7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6- deoxytetracycline (17j, also known as TP-434, 50th Interscience Conference on Antimicrobial Agents and Chemotherapy Conference, Boston, MA, September 12-15, 2010, poster F1-2157), is currently undergoing phase 2 clinical trials in patients with complicated intra-abdominal infections (cIAI).

Full text of DOI:10.1021/jm201465w

Article
pubs.acs.org/jmc
Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6-
deoxytetracycline: A Potent, Broad Spectrum Antibacterial Agent
Xiao-Yi Xiao,*,† Diana K. Hunt,† Jingye Zhou,† Roger B. Clark,† Nick Dunwoody,§ Corey Fyfe,‡
Trudy H. Grossman,‡ William J. O’Brien,‡ Louis Plamondon,† Magnus Ronn,§ Cuixiang Sun,†
̈
Wu-Yan Zhang,§ and Joyce A. Sutcliffe‡
‡
§
†Discovery Chemistry, Microbiology, and Process Chemistry R&D, Tetraphase Pharmaceuticals, 480 Arsenal Street, Watertown,
Massachusetts 02472, United States
ABSTRACT: This and the accompanying report (DOI:
10.1021/jm201467r) describe the design, synthesis, and
evaluation of a new generation of tetracycline antibacterial
agents, 7-fluoro-9-substituted-6-demethyl-6-deoxytetracyclines
(“fluorocyclines”), accessible through a recently developed
total synthesis approach. These fluorocyclines possess potent
antibacterial activities against multidrug resistant (MDR) Gram-positive and Gram-negative pathogens. One of the fluorocyclines,
7-fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline (17j, also known as TP-434, 50th Interscience Conference on
Antimicrobial Agents and Chemotherapy Conference, Boston, MA, September 12−15, 2010, poster F1−2157), is currently
undergoing phase 2 clinical trials in patients with complicated intra-abdominal infections (cIAI).
improved antibacterial activity and led to the discovery and
development of clinically important antibiotics like minocycline
and tigecycline.16−20 It has been observed that physicochemical
properties such as the polarity and electronegativity of the C7
substituent can influence tetracycline’s intrinsic antibacterial
potency.24,25 Additionally, as demonstrated by tigecycline,
substitutions at C9 can dramatically improve both intrinsic
antibacterial potency and activity against resistant pathogens
expressing tetracycline-specific efflux and ribosomal protection
mechanisms.18−20 X-ray crystallorgraphy studies by Saenger et
al. indicated that bulky substitution groups at C9 can render the
Tet repressor protein ineffective.26 Traditionally, substituents at
C7 and C9 have been introduced through semisynthetic
approaches and are largely limited to chemical groups that can
be incorporated by direct electrophilic aromatic substitutions
(e.g., halogenation and nitration) and through further
modifications of these functional groups via a very limited set
of chemical transformations.14,16,25 The total synthesis
approach recently developed by Myers et al.27−29 and expanded
by Tetraphase,30,31 on the other hand, has the potential to
introduce a more diverse set of substituents at more positions
including C7, C8, and C9 on the D ring27 as well as the ability
to incorporate heterocyclic27,30 and polycyclic ring systems29,31
into the tetracycline scaffold. We therefore decided to explore
new 7,9-disubstituted tetracycline analogues with diverse C7
substituents (e.g., fluoro, methoxy, trifluoromethoxy, trifluor-
omethyl, and cyano) coupled with a variety of C9 substituents
(e.g., alkyl, aryl, amino, aminoalkyl, and amido). In addition to
improved antibacterial activity, these new substituents and
substitution patterns could potentially impart favorable
INTRODUCTION
■
Tetracyclines are a class of broad spectrum antibiotics first
discovered in the mid-1940s.2 They have been used to treat
many bacterial infections caused by both Gram-positive and
Gram-negative pathogens.3 However, decades of widespread
tetracycline use has resulted in significant bacterial resistance
and has drastically decreased these agents’ efficacy against a
wide range of organisms.4,5
Two main mechanisms of tetracycline resistance have been
reported to date: (1) active drug efflux (tet(A)−tet(D), and
tet(K)−tet(L)), widely found in both Gram-positive and Gram-
negative pathogens,6,7 and (2) ribosomal protection (e.g.,
tet(M)−tet(O)), more commonly seen in Gram-positive
organisms such as Staphylococcus aureus and Streptococcus
spp.8,9 Since the discovery of early generations of natural
tetracyclines (chlorotetracycline (1),10 oxytetracycline (2),11
and tetracycline (3)12,13), a number of non-natural tetracycline
antibiotics have been developed to combat tetracycline
resistance. These synthetic tetracyclines include doxycycline
(4),14,15 minocycline (5),16,17 and, more recently, tigecycline
(6),18−20 all derived from naturally occurring tetracycline
intermediates via semisynthetic approaches (Figure 1).
Structure−activity relationships (SAR) of early generations
of tetracycline analogues21,22 and the recently solved
tetracycline-30S ribosome cocrystal structure23 indicate that
the “northwest” region of the molecule is not directly involved
in interactions with the ribosome and can be modified without
substantial loss of activity. In contrast, the “southeast” portion is
directly involved in an extensive H-bond network in the A-site
of the bacterial ribosome and conservation of this H-bond
network is essential to retain tight ribosomal binding. Structural
modifications at C7 and C9 of the tetracycline D ring have
emerged as one of the most promising approaches for
Received: October 31, 2011
Published: December 9, 2011
© 2011 American Chemical Society
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Figure 1. Generations of tetracycline antibiotics.
a
Scheme 1. Synthesis of 7-Fluoro-9-aminoacetamido-6-demethyl-6-deoxytetracyclines
a
Reagents: (a) s-BuLi, TMEDA, THF, then CH3I; (b) (COCl)2, DMF, CH2Cl2, then PhOH, DMAP, Et3N, 52% for two steps; (c) BBr3, CH2Cl2;
(d) Boc2O, DMAP, CH2Cl2, 75% for two steps; (e) D ring precursor 12, LDA, TMEDA, THF, then enone 13, 35%; (f) aq HF, TFA, CH3CN; (g)
H2, 10% Pd−C, dioxane−CH3OH, 41% for two steps; (h) HNO3, H2SO4; (i) H2, 10% Pd−C, CH3OH, 81% for two steps; (j) RR′NCH2C(O)Cl
(HCl salt), DMF, or BrCH2C(O)Br, Na2CO3, CH3CN−DMPU, then RR′NH.
physicochemical32,33 and pharmacokinetic−pharmacodynam-
RESULTS AND DISCUSSION
■
ic34,35 properties into the new tetracycline analogues. This
Chemistry. Applying the total synthesis approach, a series
and the accompanying report (DOI: 10.1021/jm201467r)44
describe the details of our studies on 7-fluoro-9-substituted-6-
demethyl-6-deoxytetracyclines, the “fluorocyclines” (7), and the
eventual discovery and development of one of the fluorocycline
analogues, 7-fluoro-9-pyrrrolidinoacetamido-6-demethyl-6-de-
oxytetracycline (17j),1 as a potent and broad spectrum
antibacterial agent. Details of the other 7-substituted
tetracycline subclasses (such as 7-methoxy, 7-trifluoromethoxy,
7-trifluoromethyl, and 7-cyanotetracyclines) will be the subjects
of future communications.
of 7-fluoro-9-aminoacetamido-6-demethyl-6-deoxytetracyclines
(17) were synthesized via enone 1327,36 and a D ring precursor
12. As shown in Scheme 1, 2-methoxy-5-fluorobenzoic acid (8)
was methylated by regioselective deprotonation37 with s-BuLi
in the presence of TMEDA in THF at −78 °C followed by the
addition of methyl iodide to give 2-methoxy-5-fluoro-6-
methylbenzoic acid (9), which was subsequently esterified to
afford phenyl ester 10 in 52% overall yield. Demethylation of
compound 10 with BBr3 followed by protection with Boc2O
gave the desired D ring precursor 12 in 75% yield over two
steps. The tandem Michael−Dieckmann annulation27 was
carried out by the deprotonation of 12 with LDA in the
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Table 1. In Vitro Antibacterial Activity of Fluorocycline Analogues
a
Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first seven strains from the
left are Gram-positive strains. The last seven strains are Gram-negative strains. Strains with “tet(A)”, “tet(K)”, or “tet(M)” noted underneath are
tetracycline-resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Esherichia coli; AB, Acinetobacter
b
baumannii; PA, Pseudomonas aeruginosa; ECl, Enterobacter cloacae; KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI).
c
Obtained from Marilyn Roberts’ laboratory at the University of Washington.
presence of TMEDA in THF at −78 °C followed by the
addition of enone 13, yielding the pentacyclic intermediate 14
in 35% isolated yield (not optimized). Desilylation and Boc
deprotection with aqueous HF followed by catalytic hydro-
genation gave the desired 7-fluoro-6-demethyl-6-deoxytetracy-
cline (15).38 Nitration of compound 15 with nitric acid in
sulfuric acid followed by catalytic hydrogenation afforded the
aniline intermediate 16 in 81% yield over two steps. Acylation
of aniline 16 with either aminoacetyl chlorides or bromoacetyl
bromide followed by treatment with various amines afforded
the desired 7-fluoro-9-aminoacetamido-6-demethyl-6-deoxyte-
tracycline analogues 17 in moderate yields after preparative
HPLC purification.
tetracycline-resistant Gram-positive and Gram-negative bacte-
rial strains. A large variety of amines on the C9 side chain were
systematically explored. In general, a small, secondary or
tertiary alkylamine is preferred (Table 1). Among the secondary
alkylamines (17a, 17d, and 17e), potency against the tet(A)
and tet(K) strains improved with increasing size. For the
tertiary amines (17h−17l), tet(K) activity was generally good,
while tet(A) activity was significantly improved for the
pyrrolidinyl compound 17j (TP-434). Alkylamines with weak
basicity decreased potency, especially against Gram-negative
organisms (17b, 17c, and 17l). Analogues with aromatic
amines on the C9 side chain were substantially less active
against most strains in the panel (17f and 17g). Compounds
bearing a cyclic alkylamine on the C9 side chain (17i−17k) had
dramatically different potency depending on the ring size of the
Biology. The fluorocyclines were evaluated for antibacterial
activity against a panel of tetracycline-susceptible and
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Table 2. In Vitro Antibacterial Activity of Fluorocycline Analogues
a
Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first seven strains from the
left are Gram-positive strains. The last seven strains are Gram-negative strains. Strains with “tet(A)”, “tet(K)”, or “tet(M)” noted underneath are
tetracycline-resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter
b
baumannii; PA, Pseudomonas aeruginosa; ECl, Enterobacter cloacae; KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI).
c
Obtained from Marilyn Roberts’ laboratory at the University of Washington.
amine. Compound 17j, with a pyrrolidine on the C9 side chain,
was the most potent analogue in this fluorocycline series,
having MICs ≤ 1 μg/mL against Gram-negative bacteria
including those that express the Tet(A) efflux protein. In
contrast, the azetidine analogue 17i and the piperidine analogue
17k were 4- to 64-fold less potent than the pyrrolidine analogue
17j against almost all bacterial strains in the panel.
maintained potency equivalent to isolates of the same species
not harboring tet(M) (MIC of 0.0156 μg/mL). S. aureus SA158
contains tet(K) and had MICs of 32 μg/mL and 0.0156 μg/mL
for tetracycline and 17j, respectively. Both E. coli EC155 and K.
pneumoniae KP153 carry tet(A) and had MICs of >64 μg/mL
for tetracycline and MICs of 1 and 0.5 μg/mL for 17j,
respectively. As compared to tigecycline, compound 17j was 4-
to 16-fold more potent against S. aureus, E. faecalis, E. coli
EC107, and A. baumannii AB110 and was equipotent to
tigecycline against all other strains in the panel.
Ribosomal inhibition by compound 17j and tetracycline was
characterized in an in vitro E. coli coupled transcription/
translation assay39 where inhibition of translation of the
reporter gene encoding firefly luciferase was detected by
luminescence. Compound titrations were tested, and 50%
inhibition of total luminescence (IC50) was determined. In a
representative assay (Figure 2), compound 17j displayed 7-fold
greater potency than tetracycline (IC50 of 0.62 μM (0.39 μg/
mL) vs 4.58 μM (2.2 μg/mL)).
The excellent in vitro activity of compound 17j extended to
promising in vivo efficacy in a number of animal infection
models, including a murine septicemia model challenged with a
SHV-family40 extended-spectrum β-lactamase producing E. coli
also encoding a tetracycline-specific tet(B) efflux gene (EC133)
and a neutropenic thigh model challenged with MRSA
containing the ribosomal protection gene tet(M) (SA191). As
shown in Table 3, compound 17j administered intravenously
had a PD50 of 1.3 mg/kg in the septicemia model, compared to
3.5 mg/kg for tigecycline in the same model. The MIC for both
A series of substituted pyrrolidines were then explored for
potential potency improvement over compound 17j. As shown
in Table 2, fluoro-substitution on the pyrrolidine ring retained
substantial Gram-positive activity (17m and 17n) with almost
no difference between the two diastereomers. Gram-negative
activity, on the other hand, was substantially reduced relative to
17j. Again, similar to compounds in Table 1, pyrrolidines with
polar substituents (17o−17q) had dramatically decreased
activity against most strains in the panel, especially Gram-
negative organisms. Analogues with a bicyclic pyrrolidine (17r
and 17s) on the C9 side chain retained activity against the
majority of Gram-positive strains in the panel. However, none
of the substituted pyrrolidine analogues displayed improved
activity over the unsubstituted pyrrolidine analogue 17j.
Compound 17j displayed broad spectrum activity against all
Gram-negative bacteria in the panel except P. aeruginosa, as well
as excellent in vitro activity against major Gram-positive
pathogens, including methicillin-resistant S. aureus (Table 1).
Further, compound 17j’s activity was minimally affected by the
presence of tet(M), tet(K), or tet(K). S. aureus SA161, E. faecalis
EF159, and S. pneumoniae SP160 all express tet(M) and had
MICs of ≥32 μg/mL for tetracycline, but compound 17j
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C9 side chain indicated that a small alkylamine is particularly
preferred for increased antibacterial activity. This led to the
discovery of the highly potent fluorocycline 17j, 7-fluoro-9-
pyrrolidinoacetamido-6-demthyl-6-deoxytetracycline. In addi-
tion to its potent, broad spectrum in vitro antibacterial
activities, compound 17j also displayed potent ribosomal
inhibition, promising in vivo efficacy in animal infection
models and desirable pharmacokinetic properties in rats.
Compound 17j was selected for further preclinical studies
and demonstrated favorable pharmacological and toxicological
profiles. Following IND filing, compound 17j has successfully
completed phase 1 single- and multiple-ascending dose studies
in man. Currently, compound 17j is being evaluated for
treatment of adult community-acquired intra-abdominal
infections in a global phase 2 trial.
Figure 2. Transcription−translation activity of compound 17j and
tetracycline.
EXPERIMENTAL SECTION
■
Chemistry. All commercially available solvents, including anhy-
drous solvents, and reagents were used without further purification. All
reactions under dry conditions were performed under nitrogen
atmospheres. 1H NMR (nuclear magnetic resonance) spectra were
recorded on a 400 MHz JEOL ECX-400 spectrometer. Thin layer
chromatography (TLC) analysis was performed on Merck silica-gel 60
F254 and visualized under UV light. Flash chromatography was
performed on Merck silica gel 60 (40−43 μm). Purity of tested
compounds was determined to be ≥95% by reverse phase analytical
HPLC/MS analysis (high performance liquid chromatography/mass
spectrometry) performed on a Waters Alliance system (column,
SunFire C18, 5 μm, 4.6 mm × 50 mm; solvent A, water with 0.1%
formic acid; solvent B, acetonitrile with 0.1% formic acid; MS detector,
Waters 3100). Reverse phase preparative HPLC was performed on a
Waters Autopurification system with mass-directed fraction collection
(for final compounds: column, Polymerx RP-1 100A, 10 μm, 150 mm
× 21.20 mm; flow rate, 20 mL/min; solvent A, water with 0.05 N HCl;
solvent B, acetonitrile. For intermediates: column, SunFire Prep C18
OBD, 5 μm, 19 mm × 50 mm; flow rate, 20 mL/min; solvent A, water
with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid).
3-Fluoro-6-methoxy-2-methylbenzoic Acid (9). To a THF (5
mL) solution of 5-fluoro-2-methoxybenzoic acid (500 mg, 2.94 mmol,
1 equiv) cooled at −78 °C was added s-BuLi (4.60 mL, 1.40 M/THF,
6.44 mmol, 2.2 equiv) and TMEDA (0.97 mL, 6.47 mmol, 2.2 equiv).
The reaction was stirred at −78 °C for 2 h. Methyl iodide (1.10 mL,
17.64 mmol, 6 equiv) was added dropwise. The reaction was allowed
to warm to 25 °C over 1 h and stirred at 25 °C for 1 h. Aqueous
NaOH (6 N, 20 mL) was added. The resulting mixture was extracted
with t-butyl methyl ether (20 mL × 2). The aqueous layer was
acidified with aqueous HCl (6 N) to pH 1 and extracted with EtOAc
(20 mL × 4). The combined EtOAc extracts were dried (Na21SO4) and
concentrated to give compound 9 (510 mg, crude, 94%). H NMR
(400 MHz, CDCl3) δ 7.06 (dd, J = 9.8, 8.5 Hz, 1 H), 6.75 (dd, J = 9.8,
3.7 Hz, 1 H), 3.86 (s, 3 H), 2.34 (d, J = 2.4 Hz, 3 H). MS (ESI) m/z
185.12 (M + H).
17j and tigecycline against the challenging strain was 0.125 μg/
mL. Table 3 also shows the comparison of compound 17j
(MIC = 0.25 μg/mL) to tigecycline (MIC = 0.25 μg/mL) and
vancomycin (MIC = 1 μg/mL) in the neutropenic thigh model
where the reduction in bacterial burden is used to measure
antibacterial activity. Compound 17j demonstrated a one-log
bacterial burden reduction at an IV dose of 0.6 mg/kg and a
three-log reduction at 3 mg/kg, while doses required for the
two comparators to reach the same levels of bacterial
reductions were 3 mg/kg and 17.3 mg/kg for tigecycline and
0.75 mg/kg and 10 mg/kg for vancomycin, respectively.
Selected rat pharmacokinetic (PK) parameters of compound
17j are summarized in Table 4. When dosed intravenously, 17j
had an AUC of 1.766 μg·hr/mL, lower than tetracycline but
70% greater than tigecycline. Its clearance was similar to
tetracycline but 50% slower than tigecycline. All three
compounds had similar elimination half-lives of 4−5 h. These
PK characteristics of compound 17j most likely contributed to
its higher in vivo efficacy than tigecycline as data in Table 3 has
shown. The oral bioavailability was poor in rats for compound
17j but was only moderate for tetracycline in the same species.
Of note, the oral bioavailability of tetracycline in man is 60−
80%,41 suggesting that lower mammals may not model this
parameter well. In fact, we later found compound 17j to have
better oral bioavailability in higher mammals.42
CONCLUSIONS
■
We have designed and synthesized a series of fluorocycline
analogues using the total synthesis approach. These fluorocy-
clines possess potent, broad spectrum antibacterial activities
against multidrug resistant (MDR) Gram-positive and Gram-
negative pathogens. Optimization of the amino group on the
Phenyl 3-Fluoro-6-methoxy-2-methylbenzoate (10). Oxalyl
chloride (0.95 mL, 11.10 mmol, 4 equiv) and dry DMF (0.1 mL) were
a
Table 3. In Vivo Efficacy of Compound 17j (IV)
model
strain
17j
tigecycline
vancomycin
murine septicemia
EC133 tet (B)
MIC (μg/mL)
0.125
1.3
0.125
3.5
NT
NT
PD50 (mg/kg)
neutropenic thigh
SA191 tet (M) (MRSA)
MIC (μg/mL)
0.25
0.6
3
0.25
3
1
dose at 1 log reduction (mg/kg)
dose at 3 log reduction (mg/kg)
0.75
10
17.3
a
SA, Staphylococcus aureus; EC, Esherichia coli. EC133 was obtained from Clinical Microbiology Institute, Wilsonnillw, OR, and SA191 was obtained
from Vivisource, Waltham, MA. NT, not tested.
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a
Table 4. Rat PK Parameters of Compound 17j and Comparators
compd
17j
dosage route (mg/kg)
CLs (L/h/kg)
0.564
Vz (L/kg)
T1/2 (L/kg)
Cmax (hr)
AUClast
% F (%)
IV (1)
3.2
4.0
6.9
0.812
0.045
1.766
0.295
PO (10)
1.7
tetracycline
tigecycline
IV (1)
0.542
0.929
1.2
4.6
5.2
2.664
0.791
3.083
4.536
PO (10)
14.9
1.0
IV (1)
6.12
4.6
0.428
1.052
0.107
PO (10)
3.98
0.0278
a
Measured in Sprague−Dawley rats (n = 3). Animals were fasted overnight (minimum of 12 h) and given a single oral (10 mg/kg) or IV (1 mg/kg)
dose followed by a sampling scheme for 24 h. Sterile water was used as vehicle. PK parameters were calculated by noncompartmental analysis using
WinNonlin.
added to compound 9 (510 mg, 2.77 mmol, 1 equiv) in dry CH2Cl2
(15 mL). The reaction was stirred at 25 °C for 1 h and concentrated.
The resulting solid was redissolved in dry CH2Cl2 (15 mL). Phenol
(520 mg, 5.50 mmol, 2 equiv), DMAP (670 mg, 5.60 mmol, 2 equiv),
and triethylamine (1.90 mL, 13.90 mmol, 7 equiv) were added. The
reaction was stirred at 25 °C for 12 h and concentrated. The residue
was redissolved in EtOAc (100 mL). The solution was washed with
aqueous NaOH (1 N, 100 mL × 1), water (100 mL × 1), and brine
(50 mL × 1), dried (Na2SO4), and concentrated. Flash chromatog-
raphy on silica gel (40:1 hexanes/EtOAc) yielded the desired product
(s, 9 H), 0.82 (s, 9 H), 0.26 (s, 3 H), 0.12 (s, 3 H). MS (ESI) m/z
735.45 (M + H).
(1R,3S,4S,11S)-8-(Benzyloxy)-4-(dimethylamino)-19-fluoro-
1 1 , 1 2 , 1 6 - t r i h y d r o x y - 6 - o x a - 7 - a z a p e n t a c y c l o -
[11.8.0.0.3,110.5,9015,20]henicosa-5(9),7,12,15(20),16,18-hex-
aene-10,14-dione. Aqueous HF (3 mL, 48%) and TFA (4 mL) were
added to a CH3CN solution (7 mL) of 14 (210 mg, 0.29 mmol) in a
polypropylene tube at 25 °C. The reaction was stirred at 25 °C for 18
h. The resulting mixture was poured into an aqueous solution of
K2HPO4 (21 g, dissolved in 150 mL water). The mixture was extracted
with EtOAc (50 mL × 3). The combined EtOAc extracts were dried
(Na2SO4) and concentrated to yield the title compound (180 mg,
crude). 1H NMR (400 MHz, CDCl3) δ 14.64 (s, 1 H), 11.47 (s, 1 H),
7.49−7.45 (m, 2 H), 7.39−7.32 (m, 3 H), 7.14 (dd, J = 9.2, 8.8 Hz, 1
H), 6.77 (dd, J = 9.2, 4.3 Hz, 1 H), 5.36 (s, 2 H), 3.68 (d, J = 3.7 Hz, 1
H), 3.09 (dd, J = 15.6, 4.6 Hz, 1 H), 3.02−2.92 (m, 1 H), 2.84−2.79
(m, 1 H), 2.49 (s, 6 H), 2.34−2.22 (m, 1 H), 2.09−2.02 (m, 1 H),
1.55−1.44 (m, 1 H). MS (ESI) m/z 521.30 (M + H).
1
10 (400 mg, 52% for 2 steps). H NMR (400 MHz, CDCl3) δ 7.47−
7.41 (m, 2 H), 7.31−7.24 (m, 3 H), 7.08 (dd, J = 9.2, 9.2 Hz, 1 H),
6.77 (dd, J = 9.2, 3.7 Hz, 1 H), 3.88 (s, 3 H), 2.36 (d, J = 2.3 Hz, 3 H).
MS (ESI) m/z 261.12 (M + H).
Phenyl 3-Fluoro-6-hydroxy-2-methylbenzoate (11). BBr3
(1.85 mL, 1 M/CH2Cl2, 1.85 mmol, 1.2 equiv) was added to
compound 10 (400 mg, 1.54 mmol, 1 equiv) in CH2Cl2 (10 mL) at
−78 °C. The reaction was warmed from −78 to 25 °C over 1.5 h and
quenched with saturated aqueous NaHCO3 (100 mL). The reaction
mixture was extracted with EtOAc (100 mL × 1, 50 mL × 2). The
combined EtOAc extracts were dried (Na2SO4) and concentrated to
yield compound 11 (360 mg, crude, 95%). 1H NMR (400 MHz,
CDCl3) δ 10.66 (s, 1 H), 7.50−7.44 (m, 2 H), 7.36−7.31 (m, 1 H),
7.26−7.18 (m, 3 H), 6.86 (dd, J = 9.3, 4.9 Hz, 1 H), 2.60 (d, J = 2.4
Hz, 3 H). MS (ESI) m/z 245.11 (M − H).
Phenyl 6-{[(tert-Butoxy)carbonyl]oxy}-3-fluoro-2-methyl-
benzoate (12). Boc2O (350 mg, 1.60 mmol, 1.1 equiv) and DMAP
(20 mg, 0.16 mmol, 0.1 equiv) were added to compound 11 (360 mg,
1.46 mmol, 1 equiv) in CH2Cl2 (8 mL). The reaction was stirred at 25
°C for 1.5 h and concentrated. Flash chromatography on silica gel
(35:1 hexanes/EtOAc) yielded the desired compound 12 (400 mg,
75% for 2 steps). 1H NMR (400 MHz, CDCl3) δ 7.46−7.41 (m, 2 H),
7.31−7.23 (m, 3 H) 7.18 (dd, J = 8.8, 8.7 Hz, 1 H), 7.10 (dd, J = 8.8,
4.4 Hz, 1 H), 2.41 (d, J = 2.3 Hz, 3 H), 1.44 (s, 9 H). MS (ESI) m/z
345.18 (M − H).
(1R,3S,4S,11S)-8-(Benzyloxy)-11-[(tert-butyldimethylsilyl)-
oxy]-4-(dimethylamino)-19-fluoro-12-hydroxy-10,14-dioxo-6-
oxa-7-azapentacyclo[11.8.0.0.3,110.5,9015,20]henicosa-5-
(9),7,12,15(20),16,18-hexaen-16-yl tert-Butyl Carbonate (14). A
THF solution (6 mL) of compound 12 (487 mg, 1.40 mmol, 2 equiv)
was added to a THF solution (5 mL) of LDA (6.30 mL, 10 wt % in
hexane, 4.20 mmol, 6 equiv) and TMEDA (1.70 mL, 11.20 mmol, 16
equiv) at −78 °C. The reaction was stirred at −78 °C for 5 min. A
THF solution of enone 13 (339 mg, 0.70 mmol, 1 equiv) was added to
the reaction mixture dropwise. The reaction was warmed from −78 to
25 °C over 1 h and quenched with saturated NH4Cl (100 mL). The
reaction mixture was extracted with EtOAc (50 mL × 3). The
combined EtOAc extracts were dried (Na2SO4) and concentrated to
yield the crude product. Preparative reverse phase HPLC purification
yielded compound 14 as a yellow solid (185 mg, 35%). 1H NMR (400
MHz, CDCl3) δ 15.67 (s, 1 H), 7.51−7.46 (m, 2 H), 7.39−7.29 (m, 3
H), 7.21 (dd, J = 8.9, 8.9 Hz, 1 H), 7.03 (dd, J = 8.9, 4.0 Hz, 1 H), 5.34
(s, 2 H), 3.93 (d, J = 10.4 Hz, 1 H), 3.30−3.21 (m, 1 H), 3.10−3.00
(m, 1 H), 2.57−2.41 (m, 3 H), 2.48 (s, 6 H), 2.17−2.12 (m, 1 H), 1.53
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-2-carboxamide (15). Palladium on carbon (35 mg, 10 wt %)
was added to a CH3OH/dioxane solution (4 mL/4 mL) of the above
crude product (180 mg). The reaction was purged with hydrogen and
stirred under a hydrogen atmosphere (1 atm, balloon) at 25 °C for 1 h.
The reaction mixture was filtered through a small Celite pad. The
Celite pad was washed with methanol (5 mL × 3). The combined
filtrates were concentrated to yield the crude product, which was
purified by preparative reverse phase HPLC to yield compound 15 (51
1
mg, yellow solid, 41% for 2 steps). H NMR (400 MHz, CD3OD) δ
7.26 (dd, J = 9.2, 9.2 Hz, 1 H), 6.80 (dd, J = 9.2, 4.3 Hz, 1 H), 4.09 (br
s, 1 H), 3.14 (dd, J = 15.0, 4.6 Hz, 1 H), 3.04 (s, 3 H), 2.96 (s, 3 H),
3.09−2.91 (m, 2 H), 2.31−2.18 (m, 2 H), 1.68−1.56 (m, 1 H). MS
(ESI) m/z 433.28 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-nitro-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahy-
drotetracene-2-carboxamide. A mixture of HNO3 (8.5 μL, 69%)
and H2SO4 (0.5 mL) was added to a H2SO4 solution (1 mL) of
compound 15 (51 mg, 0.12 mmol) at 0 °C. The reaction was stirred at
0 °C for 30 min. The reaction mixture was added dropwise to
vigorously stirred diethyl ether (60 mL). The suspension was filtered
through a small Celite pad. The Celite pad was washed with more
diethyl ether (5 mL × 4). The Celite pad was then eluted with
CH3OH until the eluent became colorless. The yellow CH3OH eluent
was collected and concentrated under reduced pressure to afford the
crude product of the title compound. 1H NMR (400 MHz, CD3OD) δ
8.03 (d, J = 8.5 Hz, 1 H), 4.09 (br s, 1 H), 3.50−2.97 (m, 3 H), 3.04
(s, 3 H), 2.96 (s, 3 H), 2.46−2.36 (m, 1 H), 2.29−2.20 (m, 1 H),
1.71−1.59 (m, 1 H). MS (ESI) m/z 478.20 (M + H).
(4S,4aS,5aR,12aS)-9-Amino-4-(dimethylamino)-7-fluoro-
3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-oc-
tahydrotetracene-2-carboxamide (16). Palladium on carbon (12
mg, 10 wt %) was added to a CH3OH solution (4 mL) of the above
crude product. The reaction was purged with hydrogen and stirred
under a hydrogen atmosphere (1 atm, balloon) at 25 °C for 2 h. The
602
dx.doi.org/10.1021/jm201465w | J. Med. Chem. 2012, 55, 597−605

Journal of Medicinal Chemistry
Article
catalyst was filtered off with a small Celite pad. The Celite pad was
washed with methanol (5 mL × 3). The combined filtrates were
concentrated to yield the crude product, which was purified by
preparative reverse phase HPLC to yield the desired compound 16
(43 mg, yellow solid, 81% for 2 steps). 1H NMR (400 MHz, CD3OD)
δ 7.43 (d, J = 8.5 Hz, 1 H), 4.11 (br s, 1 H), 3.22−3.16 (m, 1 H),
3.15−3.08 (m, 1 H), 3.06−2.95 (m, 1 H), 3.04 (s, 3 H), 2.96 (s, 3 H),
2.40−2.31 (m, 1 H), 2.28−2.21 (m, 1 H), 1.71−1.59 (m, 1 H). MS
(ESI) m/z 448.24 (M + H).
(4S,4aS,5aR,12aS)-9-[2-(tert-Butylamino)acetamido]-4-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17e).
2-t-Butylaminoacetylchloride hydrochloride (4.2 mg, 0.022 mmol, 2
equiv) was added to a DMF solution (0.1 mL) of compound 16 (5
mg, 0.011 mmol, 1 equiv) at 25 °C. The reaction was stirred at 25 °C
for 30 min. The reaction mixture was diluted with 0.05 N aqueous HCl
(2 mL) and purified by preparative reverse phase HPLC to yield
compound 17e as a yellow solid (3.9 mg, bis HCl salt, 56%). 1H NMR
(400 MHz, CD3OD) δ 8.25 (d, J = 11.0 Hz, 1 H), 4.11 (br s, 1 H),
4.09 (s, 2 H), 3.22−2.86 (m, 3 H), 3.05 (s, 3 H), 2.97 (s, 3 H), 2.33−
2.20 (m, 2 H), 1.69−1.57 (m, 1 H), 1.42 (s, 9 H). MS (ESI) m/z
561.39 (M + H).
Hz, 1 H), 4.08 (s, 2 H), 4.01−3.89 (m, 1 H), 3.50−3.42 (m, 1 H),
3.20−2.84 (m, 9 H), 2.30 (at, J = 14.7 Hz, 1 H), 2.23−2.15 (m, 1 H),
1.70−1.58 (m, 1 H), 1.37 (d, J = 6.7 Hz, 6 H). MS (ESI) m/z 547.25
(M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-[2-(phenylamino)acetamido]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17f).
1H NMR (400 MHz, CD3OD) δ 8.32 (d, J = 10.4 Hz, 1 H), 7.38−7.34
(m, 2 H), 7.10−7.06 (m, 3 H), 4.17 (s, 2 H), 4.10 (s, 1 H), 3.18−2.99
(m, 11 H), 2.29 (dd, J = 15.6, 15.6 Hz, 1 H), 2.25 (ddd, J = 14.8, 5.2,
2.8 Hz, 1 H), 1.66 (ddd, J = 14.8, 14.8, 14.8 Hz, 1 H). MS (ESI) m/z
581.1 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-{2-[(pyridin-3-yl)amino]-
acetamido}-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
1
boxamide (17g). H NMR (400 MHz, CD3OD) δ 8.20 (d, J = 11.2
Hz, 1 H), 8.16 (d, J = 2.4 Hz, 1 H), 8.06 (d, J = 5.2 Hz, 1 H), 7.85−
7.78 (m, 2 H), 4.27 (s, 2 H), 4.11 (s, 1 H), 3.18−2.98 (m, 9 H), 2.32−
2.21 (m, 2 H), 1.70−1.60 (m, 1 H). MS (ESI) m/z 582.2 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-9-[2-(dimethylamino)-
acetamido]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17h).
1H NMR (400 MHz, CD3OD) δ 8.24 (d, J = 11.0 Hz, 1 H), 4.24 (s, 2
(4S,4aS,5aR,12aS)-9-[2-(Azetidin-1-yl)acetamido]-4-(dime-
thylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17i).
Anhydrous Na2CO3 (16 mg, 0.15 mmol, 5.5 equiv) was added to an
anhydrous DMPU/acetonitrile (0.2 mL, 3:1, v/v) solution of
compound 16 (12 mg, 0.027 mmol, 1 equiv). Bromoacetyl bromide
(2.8 μL, 0.032 mmol, 1.2 equiv) was added. The reaction was stirred at
25 °C for 10 min, followed by the addition of azetidine (36 μL, 0.54
mmol, 20 equiv). The reaction mixture was stirred at 25 °C for 2 h,
concentrated under reduced pressure, acidified with HCl (0.5 N in
methanol, 0.7 mL), and added dropwise to vigorously stirred diethyl
ether (10 mL). The resulting suspension was filtered through a small
Celite pad. The Celite pad was washed with more diethyl ether (5 mL
× 4) and eluted with methanol until the eluent became colorless. The
yellow CH3OH eluent was collected and concentrated under reduced
pressure to afford the crude product, which was purified by preparative
reverse phase HPLC to yield compound 17i as a yellow solid (2.0 mg,
bis HCl salt, 12%). 1H NMR (400 MHz, CD3OD) δ 8.18 (d, J = 11.0
Hz, 1 H), 4.41−4.31 (m, 2 H), 4.32 (s, 2 H), 4.24−4.13 (m, 2), 4.08
(br s, 1 H), 3.18−2.86 (m, 3 H), 3.03 (s, 3 H), 2.95 (s, 3 H), 2.71−
2.57 (m, 1 H), 2.54−2.42 (m, 1 H), 2.33−2.16 (m, 2 H), 1.69−1.57
(m, 1 H). MS (ESI) m/z 545.20 (M + H).
H), 4.09 (s, 1 H), 3.14−2.93 (m, 1 5 H), 2.24−2.18 (m, 2 H), 1.65
(dt, J = 13.4, 11.6 Hz, 1 H). MS (ESI) m/z 533.17 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-[2-(pyrrolidin-1-yl)acetamido]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17j).
1H NMR (400 MHz, CD3OD) δ 8.22 (d, J = 11.0 Hz, 1 H), 4.33 (s, 2
H), 4.10 (s, 1 H), 3.83−3.72 (m, 2 H), 3.25−2.89 (m, 1 2 H), 2.32−
2.00 (m, 6 H), 1.69−1.56 (m, 1 H). MS (ESI) m/z 559.39 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-[2-(piperidin-1-yl)acetamido]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17k).
1H NMR (400 MHz, CD3OD) δ 8.22 (d, J = 11.0 Hz, 1 H), 4.19 (s, 2
H), 4.09 (s, 1 H), 3.65−3.58 (m, 2 H), 3.19−2.92 (m, 1 0 H), 2.34−
2.18 (m, 2 H), 2.02−1.79 (m, 6 H), 1.69−1.50 (m, 2 H). MS (ESI)
m/z 573.35 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-[2-(morpholin-4-yl)acetamido]-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17l).
1H NMR (400 MHz, CD3OD) δ 8.24 (d, J = 11.0 Hz, 1 H), 4.28 (s, 2
H), 4.03−4.00 (m, 2 H), 3.94−3.81 (m, 2 H), 3.68-.3.55 (m, 2 H),
3.20−2.88 (m, 1 2 H), 2.36−2.18 (m, 2 H), 1.71−1.57 (m, 1 H). MS
(ESI) m/z 575.37 (M + H).
The following compounds were prepared similarly to compound
17e or 17i.
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-{2-[(3R)-3-
fluoropyrrolidin-1-yl]acetamido}-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide (17m). 1H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 11.0 Hz,
1 H), 5.54−5.31 (m, 1 H), 4.39−4.20 (m, 2 H), 4.09−4.01 (m, 1 H),
3.40−3.30 (m, 2 H), 3.09−2.89 (m, 1 2 H), 2.50−2.34 (m, 2 H),
2.34−2.25 (m, 1 H), 2.24−2.16 (m, 1 H), 1.71−1.58 (m, 1 H). MS
(ESI) m/z 577.32 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-{2-[(3S)-3-
fluoropyrrolidin-1-yl]acetamido}-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide (17n). 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 10.4 Hz, 1
H), 5.57−5.37 (m, 1 H), 4.47−4.33 (m, 2 H), 4.15−3.87 (m, 2 H),
3.72−3.40 (m, 1 H), 3.17−2.83 (m, 1 2 H), 2.55−2.34 (m, 2 H),
2.33−2.18 (m, 2 H), 1.69−1.57 (m, 1 H). MS (ESI) m/z 577.37 (M +
H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-9-[2-(ethylamino)-
acetamido]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (17a).
1H NMR (400 MHz, CD3OD) δ 8.15 (d, J = 10.8 Hz, 1 H), 4.00 (s, 1
H), 3.99 (s, 2 H), 3.10−2.87 (m, 11 H), 2.32−2.12 (m, 2 H), 1.59−
1.51 (m, 1 H), 1.26 (t, J = 7.2 Hz, 3 H). MS (ESI) m/z 533.1 (M +
H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-{2-[(2-methoxyethyl)amino]acetamido}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide (17b). 1H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 11.0 Hz, 1
H), 4.12 (s, 2 H), 4.09 (s, 1 H), 3.72−3.67 (m, 2 H), 3.43 (s, 3 H),
3.19−2.92 (m, 1 1 H), 2.35−2.18 (m, 2 H) 1.71−1.58 (m, 1 H). MS
(ESI) m/z 563.23 (M + H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-9-{2-[(3R)-3-
(dimethylamino)pyrrolidin-1-yl]acetamido}-7-fluoro-
3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-oc-
tahydrotetracene-2-carboxamide (17o). 1H NMR (400 MHz,
CD3OD) δ 8.28 (d, J = 10.7 Hz, 1 H), 4.08 (s, 1 H), 4.00−3.91 (m, 2
H), 3.09−2.57 (m, 1 8 H), 3.26−3.18 (m, 3 H), 2.49−2.34 (m, 2 H),
2.35−2.06 (m, 2 H), 1.72−1.59 (m, 1 H). MS (ESI) m/z 602.37 (M +
H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-{2-[(2,2,2-trifluoroethyl)amino]-
acetamido}-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
1
boxamide (17c). H NMR (400 MHz, CD3OD) δ 8.25 (d, J = 11.0
Hz, 1 H), 4.22 (s, 2 H), 4.14−4.05 (m, 3 H), 3.18−2.84 (m, 9 H),
2.34−2.17 (m, 2 H), 1.70−1.57 (m, 1 H). MS (ESI) m/z 587.28 (M +
H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-9-{2-[(propan-2-yl)amino]-
acetamido}-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-{2-[(3R)-3-hydroxypyrrolidin-1-yl]acetamido}-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
1
boxamide (17d). H NMR (400 MHz, CD3OD) δ 8.24 (d, J = 11.0
603
dx.doi.org/10.1021/jm201465w | J. Med. Chem. 2012, 55, 597−605

Journal of Medicinal Chemistry
Article
amide (17p). 1H NMR (400 MHz, CD3OD) δ 8.23 (d, J = 11.0 Hz, 1
H), 4.62−4.54 (m, 1 H), 4.48−4.24 (m, 2 H), 4.08 (s, 1 H), 3.99−3.69
(m, 3 H), 3.50−3.40 (m, 1 H), 3.17−2.90 (m, 9 H), 2.44−2.11 (m, 4
H), 2.10−2.00 (m, 1 H), 1.69−1.56 (m, 1 H). MS (ESI) m/z 575.27
(M + H).
infected with approximately 5 × 105 CFU/mL of bacteria (SA191
tet(M)) in a 0.1 mL volume into the right thigh. At 1.5 h postinfection,
mice received treatment via IV injection. One group of infected mice
were euthanized and thighs were excised and processed for bacterial
CFU and serves as T = 0 controls. Twenty-four hours post-treatment,
the remaining mice were euthanized, thighs aseptically removed,
weighed, homogenized, serially diluted, and CFUs per gram of thigh
tissue were calculated. The amount of test article required to achieve 1
and 3 log10 reductions from 24 h control thighs was determined.
PK Analyses. Pharmacokinetic (PK) parameters were determined
in Sprague−Dawley rats (n = 3). Animals were fasted overnight
(minimum of 12 h) and given a single oral (10 mg/kg) or IV dose (1
mg/kg) followed by a sampling scheme for 24 h. Plasma and dosing
solution concentrations were determined by TurboIonspray LC/MS-
MS analysis using appropriate standard curves. PK parameters were
calculated by noncompartmental analysis using WinNonlin.
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-{2-[(2S)-2-(methoxymethyl)pyrrolidin-1-yl]-
acetamido}-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
1
cene-2-carboxamide (17q). H NMR (400 MHz, CD3OD) δ 8.25
(d, J = 11.0 Hz, 1 H), 4.54 (d, J = 16.5 Hz, 1 H), 4.26 (d, J = 15.9 Hz,
1 H), 4.09 (s, 1 H), 3.95−3.81 (m, 2 H), 3.81−3.75 (m, 1 H), 3.69−
3.62 (m, 1 H), 3.35 (s, 3 H), 3.23−2.92 (m, 9 H), 2.35−2.04 (m, 6 H),
1.91−1.80 (m, 1 H), 1.71−1.59 (m, 1 H). MS (ESI) m/z 603.35 (M +
H).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-9-(2-{octahydrocyclopenta[c]pyrrol-2-yl}-
acetamido)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-2-carboxamide (17r). 1H NMR (400 MHz, CD3OD, 2:1
mixture of diastereomers) δ 8.25 (d + d, J = 11.0 Hz, 1 H), 4.29, 4.24
(s + s, 2 H), 4.08 (s + s, 1 H), 4.01−3.92 (m + m, 3 H), 3.20−2.62 (m
+ m, 1 3 H), 2.35−2.16 (m + m, 3 H), 1.83−1.46 (m + m, 5 H). MS
(ESI) m/z 599.36 (M + H).
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 617-715-3553. E-mail: xyxiao@tphase.com.
(4S,4aS,5aR,12aS)-9-[2-(2,3-Dihydro-1H-isoindol-2-yl)-
acetamido]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahy-
droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
ACKNOWLEDGMENTS
■
We thank Drs. Andrew Myers, Eric Gordon, Joaquim Trias, and
Robert Zahler for valuable discussions during the course of this
work. We also thank Dr. Shu-hui Chen and his colleagues at
WuXi Apptec for external chemistry support.
1
carboxamide (17s). H NMR (400 MHz, CD3OD) δ 8.29 (d, J =
11.0 Hz, 1 H), 7.41 (s, 5 H), 4.50−4.37 (m, 2 H), 4.05 (s, 1 H), 3.95−
3.81 (m, 2 H), 3.40−3.37 (m, 1 H), 3.24−3.15 (m, 3 H), 3.10−2.70
(m, 9 H), 2.36−2.25 (m, 1 H), 2.25−2.16 (m, 1 H), 1.72−1.59 (m, 1
H). MS (ESI) m/z 607.34 (M + H).
ABBREVIATIONS USED
■
In Vitro Coupled E. coli Transcription/Translation Assay.
Antitranslational activity (IC50 values) was assessed in an E. coli in vitro
coupled transcription/translation assay (TnT)39 with a firefly
luciferase readout (cat. no. L1020, Promega, Madison, WI). Reactions
were run at a volume of 20 μL in Costar black 96-well assay plates
(Costar cat. no. 3915) for 60 min at 37 °C. The reaction was stopped
by placing on ice for 5 min, followed by addition of 25 μL of luciferase
assay substrate (cat. no. E1500, Promega, Madison, WI). Plates were
read on a LUMIStar Optima (BMG Labtech, Ortenberg, Germany)
with gain set to 3600, 0.2 s read, 0 s between wells. Percent
luminescence was plotted against inhibitor concentration with 50%
inhibition versus untreated controls marked as the IC50 value.
Susceptibility Testing. Compound stocks were prepared and
serially diluted in sterile deionized water. Minimal inhibitory
concentration (MIC) determinations were performed in liquid
medium in 96-well microtiter plates according to the methods
described by the Clinical and Laboratory Standards Institute (CLSI).43
Cation-adjusted Mueller Hinton broth was obtained from BBL (cat.
no. 212322, Becton Dickinson, Sparks, MD), prepared fresh and kept
at 4 °C prior to testing. Defibrinated horse blood (cat. no. A0432,
PML Microbiologicals, Wilsonville, OR) was used to supplement
medium, as appropriate. All test methods met acceptable standards
based on recommended quality control ranges for all comparator
antibiotics and the appropriate ATCC quality control strains.
Mouse Systemic Infection Studies. E. coli EC133 was grown
overnight with shaking in brain heart infusion (BHI) broth at 37 °C.
Five hours prior to initiation of infection, a 1:10 dilution was
performed into fresh BHI and the culture was allowed to grow for 5 h.
The 5 h culture was subsequently diluted into 5% hog gastric mucin to
achieve a bacterial inoculum that would result in 0% survival in
infected mice by 48 h postinfection (generally (1−3) × 106 colony-
forming units (CFU)/mouse in a volume of 0.5 mL). CD-1 mice
(group size of six) were inoculated intraperitoneally (IP) and received
treatment via intravenous (IV) injection 1 h postinfection. After 24 h,
percent survival was calculated and the dose (mg/kg) affecting 50%
survival, the protective dose 50% (PD50), was reported along with 95%
confidence intervals as calculated by Probit analysis using GraphPad
Prism version 4.03 (GraphPad Software).
ATCC, American Type Culture Collection; AUC, area under
curve; aq, aqueous; BHI, brain heart infusion; CFU, colony-
forming units; cIAI, complicated intra-abdominal infection; Cl,
clearance; Cmax, maximum plasma concentration; CLSI, Clinical
and Laboratory Standards Institute; DMAP, 4-
(dimethylamino)pyridine; ESI, electrospray ionization; %F,
percent oral bioavailability; HPLC, high performance liquid
chromatography; IP, intraperitoneal; IV, intravenous; LDA,
lithium diisopropylamide; MDR, multidrug resistant; MIC,
minimum inhibitory concentration; MRSA, methicillin-resistant
Staphylococcus aureus; PD50, dose at which 50% protection was
observed; PK, pharmacokinetic; PO, oral; T1/2, elimination half-
life; TBS, tert-butyldimethylsilyl; TFA, trifluoroacetic acid;
TMEDA, N,N,N′,N′-tetramethylethylenediamine; Vz, volume of
distribution
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