Synthesis and Antibacterial Activity of Isoindoline-containing Pentacyclines: A Novel Class of Tetracycline Analogs with Oral

Bioavailability

R. Clark, D. Hunt, M. He, C. Fyfe, W. O’Brien, T. Grossman, J. Sutcliffe, and X. Xiao*

Tetraphase Pharmaceuticals, Watertown, US

22nd ECCMID31 March – 3 April 2012

London, United Kingdom

P 1451

Contact:Leland Webster

Tetraphase Pharmaceuticalslwebster@tphase.com

Abstract

Introduction

Results

Methods

Time Kill Assays

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 DE-ring 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.

SA: S. aureus; SE: S. epidermidis; EF: E. faecalis; SP: S. pneumoniae; HI: H. influenzae; MC: M. catarrhalis; n/t: not tested.

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 community-acquired 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.

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.

SA101 SA161 SA158 SE164 EF159 SP106 SP160 HI MC205 IV PO29213 MRSA,tet (M) tet (K) 12228 tet (M) 49619 tet (M) 33929 8176 3 mg/kg 30 mg/kg

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

MIC (µg/mL) Survival (%)

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).

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).

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.

Scheme 1. Synthesis of isoindoline pentacyclines

F

OH

OPh

O

CH3

1(3)

Br2/HOAc

F

OH

OPh

O

CH3

Br

BnBrCs2CO3

F

OBn

OPh

O

CH3

Br

a) LDAb) DMF

F

OBn

OPh

O

CH3

Br

OHC

4

32

NaBH4

F

OBn

OPh

O

CH3

Br

5

HO

a) i-PrMgCl /LiClb) (CH2O)n

F

OBn

OPh

O

CH3

6

HO

HO

SOCl2t-Bu4NCl

F

OBn

OPh

O

CH3

7

Cl

Cl

9RNH2

F

OBn

OPh

O

CH3

8

N9R

HO O

NH

NO

OBn

OTBS

H3C CH3

O

HF

OBn

N9R

OH O HO O

NHH

H3C CH3

OH

NH2

OO H

F(or R7)

N9R

1011

O O

NH

NO

OBn

OTBS

H3C CH3

9(1)

+

LDATMEDA

1) aq HFb) H2/Pd-C

ABCDE

ABDE

ABCDE

Table 1. In vitro antibacterial activity of isoindoline pentacyclines with various C7-substituents

OH O HO O

NHH

H3C CH3

OH

NH2

OO H

R7

NH3C

H3C

Table 2. In vitro antibacterial activity of isoindoline pentacyclines with various N9-substituents

(a) 9R = 1,1-dimethylpropyl. (b) SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli.

OH O HO O

NHH

H3C CH3

OH

NH2

OO H

F

N9R

Table 3. In vivo activity of isoindoline pentacyclines

MIC MICSA101 IV PO SA101 IV POµg/mL 3 mg/kg 30 mg/kg µg/mL 3 mg/kg 30 mg/kg

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%

TP-9506 F 0.5 83% 0% TP-7525 (CH3)2N 0.5 100% 100%

R7 R9

Survival (%)TP

Number R9R7

Survival (%)TP

Number

N

O

F

H3C

H3C

H3C

H3CH3C

H3C

H3C

H3CH3C

H3C

H3C

H3C

H3C

OH O HO O

NHH

H3C CH3

OH

NH2

OO H

R7

N9R

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)

SA: S. aureus; EF: E. faecalis; SP: S. pneumoniae; EC: E. coli.

SA: S. aureus.

1) M.G. Charest, C.D. Lerner, J.D. Brubaker, D.R. Siegel, A.G. Myers, Science, 308, 395 (2005).2) Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 8 th Ed, 29 (2009).3) X.-Y. Xiao, D. K. Hunt, J. Zhou, R. B. Clark, N. D. Dunwoody, C. Fyfe, T. H. Grossman, W. H. O’Brien, L. Plamondon, M. Ronn, C. Sun, W.-Y. Zhang, J. A. Sutcliffe, J. Med. Chem., 55, 597 (2012).

SA101 SA161 SA158 EF159 SP106 SP160 EC107 EC155ATCC29213 MRSA,tet (M) tet (K) tet (M) ATCC49619 tet (M) ATCC25922 tet (A)

TP-2516(a) H 1 4 0.5 2 0.25 0.25 2 4

TP-5984 N(CH3)2 1 4 0.25 4 ≤0.0156 0.25 1 8

TP-6515 F 0.5 2 0.125 2 0.0156 0.25 1 4

TP-9229 OCH3 0.5 4 0.0625 4 ≤0.0156 0.125 1 8

TP-4404 Cl 2 4 0.5 4 0.25 1 4 16

TP-6017 CF3 0.5 2 0.25 2 ≤0.0156 0.25 4 8

1 32 >32 >32 0.0625 >32 1 >32

TP Number

MIC (µg/mL)(b)

R7

Tetracycline

SA101 SA161 SA158 EF159 SP106 SP160 EC107 EC155ATCC29213 MRSA,tet (M) tet (K) tet (M) ATCC49619 tet (M) ATCC25922 tet (A)

TP-1489 0.5 16 4 16 0.125 2 1 >32

TP-9131 0.5 2 0.25 2 0.0156 0.25 0.5 4

TP-1689 0.25 2 0.25 4 0.0156 1 2 32

TP-7113 0.5 8 0.5 8 0.0625 0.5 2 32

TP-317 0.5 1 1 1 4 8 8 16

TP-135 0.5 1 0.125 2 0.0625 0.25 1 4

TP-8601 0.5 8 0.25 8 0.0625 2 4 >32

TP-6861 1 1 1 0.5 8 8 4 >32

TP-9394 2 4 0.5 2 0.0625 0.25 4 16

TP-8078 1 4 0.25 2 0.0313 0.25 1 >32

TP-8500 0.5 1 0.25 1 0.125 0.5 2 32

TP-9506 0.5 2 0.125 8 0.0156 0.125 1 >32

TP Number

MIC (µg/mL)R9

H3CCH3H3C

F

H3C

H

H3CO

O

H

N

O

CH3H3C

H3C

H3CH3C

CH3H3C

N

N

O

F

CH3H3C

HO

References

OH O HO O

NHH

H3C CH3

OH

NH2

OO H

R7

N9R ABCDE