Synthesis and Antibacterial Activity of Isoindoline-containing
Pentacyclines: A Novel Class of Tetracycline Analogs with Oral
Bioavailability
P 1451
R. Clark, D. Hunt, M. He, C. Fyfe, W. O’Brien, T. Grossman, J. Sutcliffe, and X.
22nd ECCMID
Xiao*
31 March – 3 April 2012
Tetraphase Pharmaceuticals, Watertown, US
London, United Kingdom
Abstract
Results
Objectives: Antimicrobial resistance drives the increasing need for discovery
and development of new antibiotics. Novel pentacyclic tetracycline analogs
containing an isoindoline moiety, accessible by total synthesis, were designed
to explore their potential to achieve antibacterial potency and overcome
tetracycline resistance while retaining oral bioavailability and other desirable
physicochemical properties of the tetracycline class.
Method: Pentacyclic tetracycline analogs containing an isoindoline as the DEring moiety were synthesized from a DE-ring and an AB-ring precursor via a
tandem Michael-Dieckmann annulation. These new isoindoline analogs were
evaluated for in vitro antibacterial activity by susceptibility testing according to
CLSI guidance against a panel of bacteria strains including organisms
expressing tetracycline-resistant ribosomal protection tet(M) or efflux tet(K) or
tet(A). In vivo efficacy was assessed in a mouse septicemia model against
Staphylococcus aureus ATCC 13709.
Results: Antibacterial activity of representative isoindoline analogs are shown
in the table below. Additional studies showed that compound TP-834 had
48.3% oral bioavailability in rats and ED50 values of 1.5 mg/kg (IV) and 6.2
mg/kg (PO) in the mouse septicemia model.
Table 1. In vitro antibacterial activity of isoindoline pentacyclines with various C7-substituents
Survival (%)
IV
PO
3 mg/kg 30 mg/kg
SA101
SA161
SA158
29213 MRSA,tet(M) tet (K)
MIC (µg/mL)
SE164
EF159
SP106
12228 tet (M) 49619
SP160
tet (M)
HI
33929
MC205
8176
TP-834
0.25
2
0.25
0.0313
1
0.0156
0.0625
1
0.25
100%
83%
TP-6860
0.25
2
0.0625
≤0.0156
2
≤0.0156
0.5
1
0.125
50%
50%
TP-9215
0.25
1
0.125
n/t
1
0.0156
0.125
n/t
n/t
50%
0%
TP-5648
0.5
2
0.125
0.0625
2
≤0.0156
0.0625
0.5
0.125
100%
33%
TP-3114
1
2
0.25
0.0625
2
≤0.0156
0.25
2
0.5
100%
20%
TP-4045
0.5
2
0.125
0.0625
2
≤0.0156
0.125
1
0.25
100%
100%
TP
Number
Contact:
Leland Webster
Tetraphase Pharmaceuticals
lwebster@tphase.com
MIC (µg/mL)(b)
EF159
SP106
tet (M) ATCC49619
TP
Number
R7
TP-2516(a)
H
1
4
0.5
2
TP-5984
N(CH3)2
1
4
0.25
TP-6515
F
0.5
2
TP-9229
OCH3
0.5
TP-4404
Cl
TP-6017
CF3
SA101
SA161
ATCC29213 MRSA,tet (M)
SP160
tet (M)
EC107
ATCC25922
EC155
tet (A)
0.25
0.25
2
4
4
≤0.0156
0.25
1
8
0.125
2
0.0156
0.25
1
4
4
0.0625
4
≤0.0156
0.125
1
8
2
4
0.5
4
0.25
1
4
16
0.5
2
0.25
2
≤0.0156
0.25
4
8
1
32
>32
>32
0.0625
>32
1
>32
SP160
tet (M)
EC107
ATCC25922
EC155
tet (A)
Tetracycline
SA158
tet (K)
(a) 9R = 1,1-dimethylpropyl. (b) SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli.
Table 2. In vitro antibacterial activity of isoindoline pentacyclines with various N9-substituents
SA: S. aureus; SE: S. epidermidis; EF: E. faecalis; SP: S. pneumoniae; HI: H. influenzae; MC: M. catarrhalis; n/t: not tested.
TP
Number
Conclusion: Novel isoindoline-containing pentacyclines have potent in vitro
activities against tetracycline-resistant, Gram-positive and Gram-negative
bacterial strains, especially pathogens commonly implicated in communityacquired bacterial pneumonia (CABP). A number of the new analogs showed
excellent IV and oral in vivo efficacy in a mouse septicemia model of infection.
Compound TP-834 demonstrated promising oral bioavailability (%F = 48.3%,
rats) and IV/oral efficacy (ED50 = 1.5 mg/kg IV, 6.2 mg/kg PO, mouse
septicemia) and was selected for further pre-clinical development.
R9
SA101
SA161
ATCC29213 MRSA,tet (M)
TP-1489
H
TP-9131
H3 C
TP-1689
Introduction
F
TP-7113
Tetracyclines are a class of broad spectrum antibiotics first discovered in the
mid-1940s. However, decades of widespread use of tetracyclines have led to
significant bacterial resistance and have drastically decreased these agents’
efficacy. Besides natural tetracyclines, a number of non-natural tetracycline
antibiotics have been developed to combat tetracycline resistance. These
semisynthetic tetracyclines include doxycycline, minocycline, and tigecycline,
all derived from natural tetracycline intermediates by semisynthetic
approaches. The recently developed total synthesis of tetracyclines(1) can
access novel tetracycline analogs that are inaccessible by traditional
semisynthetic methods and has the potential to uncover new tetracycline
analogs to overcome bacterial resistance. Novel pentacyclic tetracycline
analogs (pentacyclines) containing an isoindoline moiety as the D-E ring,
accessible by this total synthetic approach, were designed to explore their
potential to achieve antibacterial potency and overcome tetracycline resistance
while retaining oral bioavailability and other desirable physicochemical
properties of the tetracycline class.
H3CO
TP-317
H 3C
H 3C CH3
TP-135
H
TP-8601
O
H C
H 3C 3
H3 C
H 3C
TP-6861
H3 C
TP-9394
Methods
0.5
16
4
16
0.125
2
1
>32
0.5
2
0.25
2
0.0156
0.25
0.5
4
0.25
2
0.25
4
0.0156
1
2
32
0.5
8
0.5
8
0.0625
0.5
2
32
0.5
1
1
1
4
8
8
16
0.5
1
0.125
2
0.0625
0.25
1
4
0.5
8
0.25
8
0.0625
2
4
>32
1
1
1
0.5
8
8
4
>32
2
4
0.5
2
0.0625
0.25
4
16
1
4
0.25
2
0.0313
0.25
1
>32
0.5
1
0.25
1
0.125
0.5
2
32
0.5
2
0.125
8
0.0156
0.125
1
>32
CH 3
CH 3
N
H3 C
TP-8078
CH 3
HO
Bacterial Strains. Strains with defined tetracycline resistance mechanisms
were obtained from M. Roberts (University of Washington, Seattle, WA). Other
strains were from the American Type Culture Collection (ATCC) , Micromyx
(Kalamazoo, MI; S. aureus SA161), or Clinical Microbiology Institute
(Wilsonville, OR).
SA158
tet (K)
MIC (µg/mL)
EF159
SP106
tet (M) ATCC49619
O
TP-8500
N
O
TP-9506
N
F
In vitro Susceptibility. Compounds were dissolved in water and assayed in
microtiter plates according to CLSI methodologies.(2)
Mouse Systemic Infection Studies. Preliminary assessment of in vivo
efficacy was performed in a mouse septicemia model against Staphylococcus
aureus ATCC 13709. Mice (6 per group) received treatment via intravenous
(IV) injection (3 mg/kg) or oral gavage (30 mg/kg) 1 hour post-intraperitoneal
(IP) infection. Percent survival was calculated at the termination of study (48 h
post-dose).
SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli.
Table 3. In vivo activity of isoindoline pentacyclines
Time Kill Assays
Materials. Isoindoline pentacyclines 11 were synthesized from the bicyclic D-E
ring precursor 8 and the enone 9(1) via a Michael-Dieckmann annulation
according to Scheme 1.
Survival (%)
MIC
TP
SA101
IV
PO
Number
µg/mL 3 mg/kg 30 mg/kg
Survival (%)
MIC
SA101
IV
PO
µg/mL 3 mg/kg 30 mg/kg
TP
Number
R7
TP-9206
F
0.5
83%
100%
TP-9571
Cl
0.25
17%
0%
TP-6860
F
0.25
50%
50%
TP-4609
CH3O
0.5
83%
75%
TP-6472
F
0.5
50%
0%
TP-5754
CF3
0.25
83%
100%
0.5
83%
0%
TP-7525 (CH3)2N
0.5
100%
100%
R9
R7
R9
Scheme 1. Synthesis of isoindoline pentacyclines
O
TP-9506
F
N
F
SA: S. aureus.
Conclusions





A series of novel pentacycline analogs with an isoindoline moiety as the D-E ring were prepared using Tetraphase’s total synthesis
approach
These new analogs displayed potent activity against a range of tetracycline–resistant pathogens especially Gram-positive organisms
A variety of substituents are tolerated at the C7 position, while small alkyl groups are preferred at the N9 position
A number of the pentacyclines also showed promising oral activity in a mouse septicemia infection model
A lead compound from this series has been profiled in additional in vitro and in vivo studies (TP-834, see posters P 1427, 1428, and
1452)
References
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2)
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