Novel 8-Heterocyle Substituted Tetracyclines are Potent and Broad Spectrum
Antibacterial Agents with Oral Bioavailability
Poster
F-1526
52nd Annual ICAAC
9-12 September, 2012
San Francisco, CA
X. XIAO,* Y. DENG, C. SUN, C. CHEN, D. HUNT, R. CLARK, C. FYFE, W. O’BRIEN, T. GROSSMAN, J. SUTCLIFFE
Tetraphase Pharmaceuticals, Inc., Watertown, MA
Abstract
Methods
Results
Background: Fully synthetic tetracyclines expand the chemical diversity of the tetracycline class and have the potential to overcome
tetracycline resistance. A range of heterocyclic substituents was incorporated at the C-8 position, which has been one of the least
accessed positions on the tetracycline scaffold. The resulting new tetracycline analogs displayed potent antibacterial activities against
a broad range of pathogens including those with various tetracycline resistance mechanisms. A number of these 8-heterocycle
substituted tetracyclines also demonstrated promising oral bioavailability in rodents.
Method: Novel 8-heterocycle substituted tetracycline analogs were synthesized from a properly substituted D-ring precursor and an
AB-ring intermediate via a tandem Michael-Dieckmann annulation. These new analogs’ in vitro antibacterial activities were evaluated
by susceptibility testing according to CLSI guidance. Compound evaluation included susceptibility of strains expressing tetracyclineresistant genes confering 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 8-heterocycle substituted tetracyclines
Compund
ID
A1
A2
A3
A4
SA101
29213
0.25
M RSA ,tet (M )
4
SA158
tet (K)
≤0.0156
0.125
1
≤0.0156
0.25
0.25
16
2
0.125
0.0625
EF159
tet (M)
4
1
16
2
SP160
tet (M)
1
0.5
1
0.125
EC107
25922
0.0313
≤0.0156
0.25
0.0625
Results
Results
Table 1. In vitro antibacterial activity of 7-chloro-8-heterocyclyltetracycline analogs
Table 3. In vitro antibacterial activity of 7-R-8-(N-methyl-2-pyrrolidinyl)tetracycline analogs
MIC (µg/mL)
Compund ID
8
R
A2
SA101
29213
0.125
A5
0.25
A6
0.25
SA161
MRSA,tet (M)
1
4
2
SA158
tet (K)
≤0.0156
0.03125
0.03125
EF159
tet (M)
1
2
2
SP160
tet (M)
0.5
0.25
0.5
MIC (µg/mL)
EC107
25922
≤0.0156
0.125
0.0625
EC155
tet (A)
1
1
2
KP153
tet (A)
0.5
1
1
AB250
0.5
2
2
SM256
7
R
SA101
29213
SA161
MRSA,tet (M)
SA158
tet (K)
EF159
tet (M)
SP160
tet (M)
EC107
25922
EC155
tet (A)
KP153
tet (A)
AB250
SM256
A2
Cl
0.125
1
≤0.0156
1
0.5
≤0.0156
1
0.5
0.5
0.125
A17
F
0.25
4
≤0.0156
4
1
0.03125
0.5
0.5
1
0.5
A18
OCH3
0.25
8
0.0625
8
0.5
0.0625
1
1
4
2
A19-A
OCF3
2
4
0.5
N/D
4
0.5
2
N/D
2
2
A19-B
OCF3
0.125
4
≤0.0156
N/D
≤0.0156
≤0.0156
0.25
N/D
0.125
0.125
A20
N(CH3)2
0.5
>32
0.125
32
2
0.125
2
1
16
2
A21-A
CF3
0.5
4
0.125
4
2
0.5
4
2
2
1
A21-B
CF3
0.125
2
≤0.0156
2
≤0.0156
0.03125
0.5
0.25
0.25
0.125
A22
CN
1
32
0.25
N/D
1
0.5
16
N/D
4
16
Compund ID
0.125
1
0.5
Survival (%)
MIC (µg/mL)
SA161
Contact:
Leland Webster
Tetraphase Pharmaceuticals, Inc.
lwebster@tphase.com
EC155
tet (A)
0.5
1
0.5
0.25
KP153
tet (A)
0.5
0.5
0.5
0.25
AB250
SM256
1
0.5
8
4
0.5
0.125
2
1
IV
PO
3 mg/kg
30 mg/kg
100%
100%
100%
100%
100%
100%
50%
100%
SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli; AB: A. baumannii; PA; KP: K. pneumoniae; SM, S. maltophilia.
Conclusions: Novel tetracycline analogs with heterocyclic substituents at the C-8 position displayed potent antibacterial activities
against a broad range of Gram-positive and Gram-negative pathogens including those with various tetracycline resistance
mechanisms. When administered by the oral or intravenous route, the potent in vitro activity translated into promising in vivo efficacy
in a murine septicemia model. Selected lead compounds from this novel tetracycline series are undergoing further development.
Methods
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) or Clinical Microbiology Institute (Wilsonville, OR).
In vitro Susceptibility. Compounds were dissolved in water and assayed in microtiter plates according to CLSI methodology.1
Mouse Systemic Infection Studies. Mice (n = 6) received treatment via oral gavage (PO) or intravenous (IV) injection 1 hour post
intraperitoneal (IP) infection. Percent survival was calculated at termination of study (48 hrs post-dose).
Materials. 8-Heterocycle substituted tetracycline analogs were synthesized from appropriately substituted and protected D-ring
precursors (such as 6) and the bicyclic enone 72 via a Michael-Dieckmann annulation. A typical synthesis of the 7-chloro-8-(2pyrrolidinyl)tetracycline analogs is shown in Scheme 1.
A7
0.5
A8
0.5
A9
0.125
A10
0.125
tigecycline
0.125
1
2
1
0.5
0.25
0.25
2
0.125
0.125
0.0624
1
4
1
0.5
0.0625
4
1
2
2
8
2
4
2
≤0.0156
0.03125
4
>32
>32
>32
1
4
>32
>32
>32
1
8
N/D
>32
2
8
2
N/D
Table 4. In vivo antibacterial activity of 7-R-8-(2-pyrrolidinyl)tetracycline analogs
Compound ID
2
2
MIC (µg/mL)
A2
8
R
R
R
IV
3 mg/kg
PO
30 mg/kg
8'
A2
Cl
methyl
0.125
100%
100%
A12
Cl
isopropyl
0.25
83%
83%
A13
Cl
cyclopropyl
0.5
0%
0%
A17
F
methyl
0.25
100%
100%
A23
(CH3 )2N
isopropyl
0.25
100%
50%
A24
CH3 O
isopropyl
0.25
100%
100%
1
Table 2. In vitro antibacterial activity of 7-chloro-8-(2-pyrrolidinyl)tetracycline analogs
Compund ID
7
Survival (%)
MIC (µg/mL)
SA101
29213
SA101
29213
SA161
MRSA,tet (M)
SA158
tet (K)
EF159
tet (M)
SP160
tet (M)
EC107
25922
EC155
tet (A)
KP153
tet (A)
AB250
SM256
0.125
1
≤0.0156
1
0.5
≤0.0156
1
0.5
0.5
0.125
Conclusions

A range of heterocyclic substituents was incorporated into the tetracycline scaffold at the C8 position
using the Tetraphase total synthesis approach. Coupled with substitutions at C7, a series of novel 7,8disubstituted tetracycline analogs was prepared with increased structural diversity and the potential to
overcome tetracycline resistance.

Among the C8 heterocyclic substituents investigated, the pyrrolidine ring was found to be preferred for
optimal antibacterial activity.

A number of the new analogs, especially A2, A17, and A24, are highly potent against a broad range of
Gram-positive and Gram-negative pathogens in vitro. These compounds have also displayed promising in
vivo activity when dosed IV and PO.

Several lead compounds from this novel chemical series have demonstrated promising oral bioavailability
in PK studies and have been advanced into further preclinical and clinical development.
Scheme 1. Synthesis of 7-chloro-8-(2-pyrrolidinyl)tetracycline analogs
A11
0.5
2
0.25
0.5
8
4
32
>32
32
4
A12
0.25
1
≤0.0156
2
0.0625
0.0625
1
1
4
0.5
A13
0.5
1
0.25
2
1
1
4
8
8
4
A14
1
2
0.5
2
2
4
>32
>32
>32
8
A15
0.25
2
0.0313
2
0.25
0.0625
1
1
2
0.5
A16
1
4
0.125
4
1
0.25
2
2
4
2
References & Notes
1) Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—
Ninth Edition. CLSI document M07-A9. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2012
2) M.G. Charest, C.D. Lerner, J.D. Brubaker, D.R. Siegel, A.G. Myers, Science, 308, 395 (2005).
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3) We thank Dr. C-H Chen and his colleagues at WuXi Apptec for medicinal chemistry support to this program.