OH O HO O N H H H 3 C CH 3 OH NH 2 O O H 7 R N 8' R M IC (µg/m L) SA101 IV PO 29213 3 m g/kg 30 m g/kg A2 Cl m ethyl 0.125 100% 100% A12 Cl isopropyl 0.25 83% 83% A13 Cl cyclopropyl 0.5 0% 0% A17 F m ethyl 0.25 100% 100% A23 (CH 3 ) 2 N isopropyl 0.25 100% 50% A24 CH 3 O isopropyl 0.25 100% 100% Survival(% ) C om pound ID 7 R 8' R OH O HO O N H H H 3 C CH 3 OH NH 2 O O H 7 R N H 3 C SA101 SA161 SA158 EF159 SP160 EC 107 EC 155 K P153 AB 250 SM 256 29213 MRSA, tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A) 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 OCH 3 0.25 8 0.0625 8 0.5 0.0625 1 1 4 2 A19-A OCF 3 2 4 0.5 N/D 4 0.5 2 N/D 2 2 A19-B OCF 3 0.125 4 ≤0.0156 N/D ≤0.0156 ≤0.0156 0.25 N/D 0.125 0.125 A20 N(CH 3 ) 2 0.5 >32 0.125 32 2 0.125 2 1 16 2 A21-A CF 3 0.5 4 0.125 4 2 0.5 4 2 2 1 A21-B CF 3 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 M IC (µg/m L) 7 R Com pund ID OH O HO O N H H H 3 C CH 3 OH NH 2 O O H Cl 8 R Methods Abstract Methods Results Printed by Novel 8-Heterocyle Substituted Tetracyclines are Potent and Broad Spectrum Antibacterial Agents with Oral Bioavailability 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 52 nd Annual ICAAC 9-12 September, 2012 San Francisco, CA References & Notes Conclusions Contact: Leland Webster Tetraphase Pharmaceuticals, Inc. [email protected] Poster F-1526 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 tetracycline-resistant 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 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. 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 7 2 via a Michael- Dieckmann annulation. A typical synthesis of the 7-chloro-8-(2-pyrrolidinyl)tetracycline analogs is shown in Scheme 1. Scheme 1. Synthesis of 7-chloro-8-(2-pyrrolidinyl)tetracycline analogs Results Results Table 3. In vitro antibacterial activity of 7-R-8-(N-methyl-2- pyrrolidinyl)tetracycline analogs Table 1. In vitro antibacterial activity of 7-chloro-8- heterocyclyltetracycline analogs 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). 3) We thank Dr. C-H Chen and his colleagues at WuXi Apptec for medicinal chemistry support to this program. 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 7,8-disubstituted 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. Table 4. In vivo antibacterial activity of 7-R-8-(2- pyrrolidinyl)tetracycline analogs SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli; AB: A. baumannii; PA; KP: K. pneumoniae; SM, S. maltophilia. SA101 SA161 SA158 EF159 SP160 EC107 EC155 KP153 AB250 SM 256 IV PO 29213 M RSA, tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A) 3 m g/kg 30 m g/kg A1 0.25 4 ≤0.0156 4 1 0.0313 0.5 0.5 1 0.5 100% 100% A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125 100% 100% A3 0.25 16 0.125 16 1 0.25 0.5 0.5 8 2 100% 50% A4 0.25 2 0.0625 2 0.125 0.0625 0.25 0.25 4 1 100% 100% M IC (µg/m L) S urvival(% ) Com pund ID Br CH 3 CO 2 Ph OCH 3 1 Br CH 3 CO 2 Ph OCH 3 2 Cl Br CH 3 CO 2 Ph OBn 3 Cl CH 3 CO 2 Ph OBn 4 Cl O BocH N CH 3 CO 2 Ph OBn 5 Cl N H CH 3 CO 2 Ph OBn 6 Cl N R HO O N H N O OBn OTBS H 3 C CH 3 O H Cl OBn N R 8 O O N H N O OBn OTBS H 3 C CH 3 OH O HO O N H H H 3 C CH 3 OH NH 2 O O H Cl N R 9 7 + NCS 1)BBr 3 2)BnBr K 2 CO 3 a) n-BuLi b) N Boc O 1)TFA 2)N aBH 4 alkylation a)LD A/TM ED A b)enone 7 1)aq H F 2)H 2 /Pd-C A D B A B C D A B C D 8 7 1 4 Table 2. In vitro antibacterial activity of 7-chloro-8-(2- pyrrolidinyl)tetracycline analogs SA101 SA161 SA158 EF159 SP160 EC 107 EC 155 K P153 AB 250 SM 256 29213 MRSA, tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A) A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125 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 M IC (µg/m L) 8 R Com pund ID N H 3 C N F N H 3 C CH 3 N N H 3 C H 3 C N H 3 C H 3 C N H 3 C CH 3 SA101 SA161 SA158 EF159 SP160 EC 107 EC 155 K P153 AB 250 SM 256 29213 MRSA, tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A) A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125 A5 0.25 4 0.03125 2 0.25 0.125 1 1 2 1 A6 0.25 2 0.03125 2 0.5 0.0625 2 1 2 0.5 A7 0.5 1 0.25 1 4 1 4 4 8 2 A8 0.5 2 2 4 2 2 >32 >32 N/D N/D A9 0.125 1 0.125 1 8 2 >32 >32 >32 2 A10 0.125 0.5 0.125 0.5 4 2 >32 >32 2 2 tigecycline 0.125 0.25 0.0624 0.0625 ≤0.0156 0.03125 1 1 8 1 M IC (µg/m L) Com pund ID 8 R N H 3 C N CH 3 N CH 3 * diastereom erA HN N N CH 3 * diastereomerB N N
Methods
Feb 24, 2016
Novel 8-Heterocyle Substituted Tetracyclines are Potent and Broad Spectrum Antibacterial Agents with Oral Bioavailability 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 . Poster F-1526. - PowerPoint PPT Presentation
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OH O HO O
NHH
H3C CH3
OH
NH2
OO H
7R
N8'R
MIC (µg/mL)SA101 IV PO29213 3 mg/kg 30 mg/kg
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 CH3O isopropyl 0.25 100% 100%
Survival (%)Compound ID 7R 8'R
OH O HO O
NHH
H3C CH3
OH
NH2
OO H
7R
NH3C
SA101 SA161 SA158 EF159 SP160 EC107 EC155 KP153 AB250 SM25629213 MRSA,tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A)
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
MIC (µg/mL)7RCompund ID
HNN
HNN
OH O HO O
NHH
H3C CH3
OH
NH2
OO H
Cl8R
MethodsAbstract
Methods
Results
Printed by
Novel 8-Heterocyle Substituted Tetracyclines are Potent and Broad SpectrumAntibacterial Agents with Oral Bioavailability
X. XIAO,* Y. DENG, C. SUN, C. CHEN, D. HUNT, R. CLARK, C. FYFE, W. O’BRIEN, T. GROSSMAN, J. SUTCLIFFETetraphase Pharmaceuticals, Inc., Watertown, MA
52nd Annual ICAAC9-12 September,
2012San Francisco, CA
References & Notes
Conclusions
Contact:Leland Webster
Tetraphase Pharmaceuticals, Inc.
PosterF-1526
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 tetracycline-resistant 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
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.
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-(2-pyrrolidinyl)tetracycline analogs is shown in Scheme 1.
Scheme 1. Synthesis of 7-chloro-8-(2-pyrrolidinyl)tetracycline analogs
Results ResultsTable 3. In vitro antibacterial activity of 7-R-8-(N-methyl-2-pyrrolidinyl)tetracycline analogsTable 1. In vitro antibacterial activity of 7-chloro-8-heterocyclyltetracycline analogs
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).3) We thank Dr. C-H Chen and his colleagues at WuXi Apptec for medicinal chemistry support to this program.
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 7,8-disubstituted 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.
Table 4. In vivo antibacterial activity of 7-R-8-(2-pyrrolidinyl)tetracycline analogsSA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli; AB: A. baumannii; PA; KP: K. pneumoniae; SM, S. maltophilia.
SA101 SA161 SA158 EF159 SP160 EC107 EC155 KP153 AB250 SM256 IV PO29213 MRSA,tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A) 3 mg/kg 30 mg/kg
A1 0.25 4 ≤0.0156 4 1 0.0313 0.5 0.5 1 0.5 100% 100%A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125 100% 100%A3 0.25 16 0.125 16 1 0.25 0.5 0.5 8 2 100% 50%A4 0.25 2 0.0625 2 0.125 0.0625 0.25 0.25 4 1 100% 100%
MIC (µg/mL) Survival (%)Compund
ID
Br CH3
CO2PhOCH3
1
Br CH3
CO2PhOCH3
2
ClBr CH3
CO2PhOBn
3
ClCH3
CO2PhOBn4
ClO
BocHN
CH3
CO2PhOBn5
Cl
NH
CH3
CO2PhOBn6
Cl
NR
HO O
NH
NO
OBnOTBS
H3C CH3
O
HCl
OBn
NR
8O O
NH
NO
OBnOTBS
H3C CH3
OH O HO O
NHH
H3C CH3
OH
NH2
OO H
Cl
NR
9 7
+
NCS
1) BBr32) BnBr K2CO3
a) n-BuLib)
NBoc
O 1) TFA2) NaBH4
alkylation
a) LDA/TMEDAb) enone 7
1) aq HF2) H2/Pd-C
A DBABCDABCD8
7
1
4
Table 2. In vitro antibacterial activity of 7-chloro-8-(2-pyrrolidinyl)tetracycline analogsSA101 SA161 SA158 EF159 SP160 EC107 EC155 KP153 AB250 SM25629213 MRSA,tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A)
A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125
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
MIC (µg/mL)8RCompund ID
HNN
NH3C
HNN
NF
N
H3C CH3
N
NH3C
H3C
NH3C
H3C
NH3C
CH3
SA101 SA161 SA158 EF159 SP160 EC107 EC155 KP153 AB250 SM25629213 MRSA,tet (M) tet (K) tet (M) tet (M) 25922 tet (A) tet (A)
A2 0.125 1 ≤0.0156 1 0.5 ≤0.0156 1 0.5 0.5 0.125
A5 0.25 4 0.03125 2 0.25 0.125 1 1 2 1
A6 0.25 2 0.03125 2 0.5 0.0625 2 1 2 0.5
A7 0.5 1 0.25 1 4 1 4 4 8 2
A8 0.5 2 2 4 2 2 >32 >32 N/D N/D
A9 0.125 1 0.125 1 8 2 >32 >32 >32 2
A10 0.125 0.5 0.125 0.5 4 2 >32 >32 2 2
tigecycline 0.125 0.25 0.0624 0.0625 ≤0.0156 0.03125 1 1 8 1
MIC (µg/mL)Compund ID 8R
HN N
NH3C
NCH3
NCH3
*diastereomer A
HN N
HNN
NCH3
*diastereomer B
N
N
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