COMPOSITIONS FOR THE TREATMENT OF TUBERCULOSIS AND

METHODS OF USING SAME

GOVERNMENT SUPPORT

This invention was made with government support under 5R01AI084411-03 NIH/NIAID, awarded by the National Institutes of Health. The government has certain rights in the invention.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Serial No.

61/594,167, filed February 2, 2012, which is herein incorporated by reference in its entirety.

BACKGROUND

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB) and is responsible for nearly two million deaths annually. Moreover, the emergence of multidrug-resistant (MDR) strains of Mtb seriously threatens TB control and prevention efforts. In addition, one-third of the 42 million people living with HIV/ AIDS worldwide are co-infected with Mtb. Recent studies have shown that infection with Mtb enhances replication of HIV and may accelerate the progression of HIV infection to AIDS. There are significant problems associated with treatment of AIDS and Mtb co-infected patients.

In addition to the necessity of drugs for the treatment of MDR- Mtb, the development of drugs that kill Mtb in any state is very important. However, no current TB drugs are effective in killing the dormant form of Mtb in vivo.

SUMMARY

This disclosure provides compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent tuberculosis.

Thus, in one aspect provided herein is a compound of Formula I. In certain embodiments, Formula I is represented as Formulae A, B, C, D or E. In another aspect, the compounds of Formula I are provided in a pharmaceutical composition with a pharmaceutically acceptable carrier. The compounds of Formulae A, B, C, D or E can also be provided in a pharmaceutical composition with a

pharmaceutically acceptable carrier.

In another aspect, provided herein is a method for the treatment or prevention of a bacterial infection in a subject in need thereof, comprising administering the compound of Formula I (e.g., Formulae A, B, C, D or E) to the subject or a pharmaceutical composition comprising Formula I (e.g., Formulae A, B, C, D or E) made thereof to the subject. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating a subject with a disease caused by Mycobacterium tuberculosis, comprising administering the compound of Formula I or Formulae A, B, C, D or E to the subject or a pharmaceutical composition made thereof. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating a subject with a mycobacterium infection, comprising administering the compound of Formula I (e.g, Formulae A, B, C, D or E) to the subject or a pharmaceutical composition made thereof. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis. In one embodiment, the mycobacterium infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating or preventing tuberculosis in a subject in need thereof, comprising administering the compound of Formula I (e.g„ Formulae A, B, C, D or E) to the subject or a pharmaceutical

composition made thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a schematic that shows the electron flow system from M.

tuberculosis.

Figure 2 is a chart describing an assay to identify selective MenA inhibitors against M. tuberculosis. Figure 3 is a chart showing bactericidal activity of selected MenA inhibitor molecules.

Figure 4 is a schematic showing the synthesis of Class A optically active MenA inhibitors. Reagents and conditions: (a) Ac20/Py. (1 : 1), room temperature; (b) BnBr, NaH, DMF/THF (4: 1). 0 ^° C; (c) TMSNCO, DMAP, CH ₂ C1 ₂ , room temperature.

Figure 5 is a schematic showing the syntheses of Class B and C optically active MenA inhibitors. Reagents and conditions: (c) TMSNCO, DMAP, CH ₂ C1 ₂ , room temperature; (d) IN NaOH, CH ₃ CN, 0 ^° C, (f) Jocobsen' s kinetic resolution with (R,R)- salene Co(III); (g) Jocobsen' s kinetic resolution with (S^-salene Co(III).

Figure 6 is a schematic showing the syntheses of Class D and E optically active

MenA inhibitors. Reagents and conditions: (c) TMSNCO, DMAP, CH ₂ C1 ₂ , room temperature; (d) IN NaOH, CH ₃ CN, 0 ^° C.

DETAILED DESCRIPTION Provided herein are compounds, intermediates thereto and derivatives thereof, as well as pharmaceutical compositions containing the compounds, for use in treatment of tuberculosis. The compounds of the invention or compositions thereof are useful for the treatment of tuberculosis, as well as patients who suffer from acquired immune deficiency syndrome (AIDS).

Rifampicin and isoniazid induce the cytochrome P450 3A4 enzyme which shows significant interactions with anti-HIV drugs such as protease inhibitors. In addition, rifampicin strongly interacts with non-nucleoside reverse transcriptase and protease inhibitors for HIV infections. Therefore, clinicians avoid starting Highly Active Antiretro viral Therapy (HAART), which consists of three or more highly potent reverse transcriptase inhibitors and protease inhibitors, until the TB infection has been cleared. M. tuberculosis is recognized to lie in a non-replicating state (dormancy), particularly in the caseous nodules of the lungs where the lesions have little access to oxygen, and can survive for many years in the host by entering a dormant state. About 10% of patients with latent Mtb are reactivated, causing the risk of fatal diseases. In certain

embodiments, the compounds and compositions described herein are effective in the treatment of tubercuslosis without inhibiting the effectiveness of protease inhibitors or other HIV therapies. In other embodiments, the compounds and compositions described herein can eliminate tuberculosis in the activated or dormant phases.

The function of ubiquinone (coenzyme Q 10) as a component of the mitochondrial respiratory chain in human is well established ("the chemiosmotic theory", Mitchell, 1978). In prokaryotes, especially in Gram-positive bacteria, menaquinone transfers two electrons in a process of either aerobic or anaerobic respiration (Figure 1). On the other hand, a majority of Gram-negative organisms utilize ubiquinone under aerobic conditions and menaquinone under anaerobic conditions in their electron transport systems.

Recently, menA knockdown mutant M. tuberculosis were generated possessing TetON (tetracycline-inducible expression system). It has been demonstrated that MenA is essential for growth of Mtb via mouse infection experiments with the menA

knockdown Mtb mutant. The electron transport system couples with ATP synthase to produce ATP through oxidative phosphorylation. Bacterial ATP synthase, FIFO-ATPase, is a viable target for treatment of MDR Mtb infections. Only a few studies have investigated the electron transport system for development of new antibacterial drugs. It was reported that inhibitors of type II NADH:menaquinone oxidoreductase effectively killed Mtb in vitro and they concluded that type II NADH dehydrogenase could be a unique and interesting antimicrobial target.

It has been shown that inhibition of MenA (l,4-dihydroxy-2-naphthoate preny transferase), which catalyzes a formal decarboxylative prenylation of 1,4- dihydroxy-2-napthoate (DHNA) to form demethylmenaquinone (DMMK) in

menaquinone biosynthesis (Figure 1), showed significant growth inhibitory activities against drug resistant Gram-positive bacteria including M. tuberculosis. In menaquinone biosynthesis, MenD (2-succinyl-5-enoylpyruvyl-6-hydroxy-3-cyclohexane- 1-carboxylic acid synthase), MenE (an acyl-CoA synthase), and MenB (1,4-dihydroxynaphtoyl-CoA synthase) have recently been studied for the development of novel drug lead for Gram- positive pathogens including M. tuberculosis.

Compounds of Formula I (e.g., Formulae A, B, C, D and E) were evaluated in an enzymatic assay in vitro (IC50) against MenA and in bacterial growth inhibitory assays (MIC). Figure 2 illustrates the assay scheme to identify selective MenA inhibitors against M. tuberculosis. In these library molecules, 26 molecules were identified to exhibit the in vitro biological activities which met the assay criteria summarized in Figure 2. It became evident that the topology of the N atom in the inhibitor molecules plays an important role in selectivity of the MenA enzymatic and bactericidal activities (Mtb vs. S. aureus). As summarized in Figure 3, selective mycobactericidal molecules (2-6) possess the secondary or tertiary amine in the near center of the molecules, whereas the topology of the N atom of the molecules possessing antibacterial activities against both Mtb and S. aureus (7-10) locates the right half of the molecules.

Compounds of the Invention In one aspect, the invention rovides compounds of the Formula I:

I

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ³ is a Ci_6 alkyl group, a -C(H)(OR ² )-Ci_ ₆ alkyl group, or a -C(H)(CH ₂ OR ² )-Ci_ ₆ alkyl group, wherein the Ci_ ₆ alkyl, -C(H)(OR ² )-Ci_ ₆ alkyl, and -C(H)(OR ² )-Ci_ ₆ alkyl groups are independently substituted by one or more of the following moieties:

-C(H)(OR ² )-, -C(H)(CH ₂ OR ² )-, -Ph- -N(R ⁶ )-, -Ph-C(H)(OR ² )-, or

-Ph-C(H)(CH ₂ OR ² )-; wherein R ² is independently H, Ci_ ₄ alkyl, or C(0)NH ₂ ; and wherein R ⁶ is H, C _1-10 alkyl, C _1-3 alkylaryl or C _1-3 alkylheteroaryl, wherein the aryl or heteroaryl rings can be substituted with C _1-3 alkyl, C _1-3 alkoxy, halogen, hydroxyl, cyano, nitro, thiol, sulfonyl, or amino;

R ⁴ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl; R ⁵ is hydrogen, halogen, Ci_io alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl;

X is O or NOCi_ ₄ alkyl;

ring A is phenyl or a 5-, 6- or 7-membered heterocycle; and

n is 1, 2, or 3.

In one embodiment of Formula I,

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H or CONH ₂ ;

R ³ is a Ci_6 alkyl group, a -C(H)(OR ² )-Ci_ ₆ alkyl group, or a -C(H)(CH ₂ OR ² )-Ci_ ₆ alkyl group, wherein the Ci_ ₆ alkyl, -C(H)(OR ² )-Ci_ ₆ alkyl, and -C(H)(OR ² )-Ci_ ₆ alkyl groups are independently substituted by one or more of the following moieties:

-C(H)(OR ² )-, -Ph- -N(H)-, or -Ph-C(H)(OR ² )-, wherein R ² is independently H, Ci_ ₄ alkyl, or C(0)NH ₂ ;

R ⁴ is hydrogen or halogen;

R ⁵ is hydrogen or halogen;

X is O or NOCi_ ₄ alkyl;

ring A is phenyl or a 5-. 6- or 7-membered heterocycle; and

n is 1, 2, or 3.

3 2

As used herein, the expression "R ^J is a Ci_6 alkyl group, a -C(H)(OR")-Ci_ ₆ alkyl group, or a -C(H)(CH ₂ OR ² )-Ci_ ₆ alkyl group, wherein the Ci_ ₆ alkyl, -C(H)(OR ² )-Ci_ ₆ alkyl, and -C(H)(OR )-C _1-6 alkyl groups are independently substituted by one or more of the following moieties..." means that the Ci_ ₆ alkyl, -C(H)(OR ^A )-C _1-6 alkyl, and

-C(H)(OR )-Ci_6 alkyl chains are interrupted at least once by any of the listed substituent groups. By "interrupted," it is meant that the one or more of the substituents are substituted in between adjacent carbons of the R group. By "interrupted," it is also meant that the carbon chains can be terminated by one of the listed substituent groups, i.e., the substituent group can be the last substituent before the R ¹ group. In one embodiment, the Ci_ ₆ alkyl, -C(H)(OR ² )-Ci_ ₆ alkyl, and -C(H)(OR ² )-Ci_ ₆ alkyl chains are interrupted by one, two or three of any of the listed substituent groups. The chains can also be interrupted by non- identical substituent groups, e.g., both -Ph- and -N(R ⁶ )-. In one embodiment of Formula I, R 3 is a (CH ₂ ) _m , -C(H)(OR 2 )-(CH ₂ ) _m , or

2 3

-C(H)(CH ₂ OR")-(CH ₂ ) _m group, wherein m is 1, 2, 3, 4, 5 or 6, and wherein the R groups are independently substituted by one or more of the following moieties: -C(H)(OR )-, -C(H)(CH ₂ OR ² )-, -Ph- -N(R ⁶ )-, -Ph-C(H)(OR ² )-, or -Ph-C(H)(CH ₂ OR ² )-.

In other embodiments of Formula I, R ⁵ is CI or F; X is O or NOMe; R ⁴ is H, CI or F; n is 1; A is phenyl or piperidinyl; R ³ is CH ₂ C(H)(OR ² )(CH ₂ ) ₃ , CH ₂ PhC(H)(OR ² )CH ₂ , or C(H)(CH ₂ OH)N(H)(CH ₂ ) ₂ Ph; R ¹ is CH ₃ , OCH ₃ , C ₃ H ₇ , or C ₆ Hi ₃ ; or R ² is H or CONH ₂ .

In another embodiment of the invention, the compound of formula I is a compound of formula A:

A

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ;

R ⁵ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl; and

X is O or NOCi_4 alkyl.

In another embodiment of the invention, the compound of formula A has the formula A':

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ; and X is O or NOCi_ ₄ alkyl.

In certain embodiments, the compound of Formula A' is one of the following

(S)-2 X, R-i R ₂ = 0, CH ₃ , H

(R)-2 X, R-i R ₂ = 0, CH ₃ , H

(S)-11 X, R _{1 f} R ₂ = 0, C ₃ H ₇ , H

(R)-11 X, R _{1 f} R ₂ = 0, C ₃ H ₇ , H

(S)-12 X, R _{1 f} R ₂ = 0, C ₃ H ₇ , CONH ₂

(R)-12 X, R _{1 f} R ₂ = 0, C ₃ H ₇ , CONH ₂

(R)-13 X, R _{1 f} R ₂ = NOMe, C ₃ H ₇ , CONH ₂

In another embodiment of the invention, the compound of formula I is a compound of formula B:

B

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_ ₄ alkyl, or C(0)NH ₂ ;

R ⁵ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl; and

X is O or NOCi_ ₄ alkyl.

In another embodiment of the invention, the compound of formula B has the formula B':

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ; and

X is O or NOCi_4 alkyl.

In certain embodiments, the compound of Formula B' is one of the following

(S)-3 X, R ₂ = 0, H

(R)-3 X, R ₂ = 0, H

(S)-14 X, R ₂ = 0, CONH ₂

(R)-14 X, R ₂ = 0, CONH ₂

(R)-15 X, R ₂ = NOMe, OH

In another aspect, the invention provides compounds of the Formula C:

C

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or Ci_8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ;

R ⁵ is hydrogen, halogen, Ci_io alkyl, Ci_io alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, Ci_io alkylthiol, carboxy, CMO alkylcarboxy, or carbamoyl; and

X is O or NOCi_4 alkyl.

In another embodiment of the invention, the compound of formula C has the formula:

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein C-C bond a is (R) ((R)-4)) or (S) ((S)-4)). In another embodiment of the invention, the compound of formula I is a compound of formula D:

D

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ;

R ⁴ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl;

R ⁵ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-io haloalkyl, amino, Ci-io alkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl; and

X is O or NOCi_4 alkyl.

In another embodiment of the invention, the compound of formula D has the formula D':

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ; and

X is O or NOCi_4 alkyl.

In certain embodiments, the compound of Formula D' is one of the following rac-5 ^χ · ^R 1- ^R 2 ^~ 0> ^Η ₃ , H

rac-16 X, R _1f R ₂ = 0, CH ₃ , CONH ₂

(S)-5 X, R _1l R ₂ = 0, CH _3l H

(R)-5 X, R ₁ , R ₂ = 0, CH ₃ , H

(S)-16 X, R ₁ , R ₂ = 0, C ₃ H ₇ , H

(R)-16 X, R ₁ , R ₂ = 0, C ₃ H ₇ , H

(S)-17 X, R _1f R ₂ = 0, C ₃ H ₇ , C0NH ₂

(S)-18 X, R _1f R ₂ = NOMe, C ₃ H ₇ , CONH ₂

In another embodiment of the invention, the compound of formula I is a compound of formula E:

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ¹ is Ci-8 alkyl or C _1-8 alkoxy;

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ;

R ⁴ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-iohaloalkyl, amino, Ci-ioalkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl;

R ⁵ is hydrogen, halogen, C _1-10 alkyl, C _1-10 alkoxy, hydroxyl, cyano, nitro, thiol, sulfonyl, Ci-iohaloalkyl, amino, Ci-ioalkylamino, C _1-10 alkylthiol, carboxy, C _1-10 alkylcarboxy, or carbamoyl; and

XisOor NOCi_4 alkyl.

In another embodiment of the invention, the compound of formula E has the formula E':

and pharmaceutically acceptable salts, enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates thereof, wherein

R ² is H, Ci_4 alkyl, or C(0)NH ₂ ; and

X is O or NOCi_ ₄ alkyl.

In certain embodiments, the compound of Formula E' is one of the following rac-6 X, R ₂ = 0, H

(S)-6 X, R ₂ = 0, H

(R)-6 X, R ₂ = 0, H

(S)-19 X, R ₂ = 0, CONH ₂

(R)-19 X, R ₂ = 0, CONH ₂

(S)-20 X, R ₂ = NOMe, OH

(R)-20 X, R ₂ = NOMe, OH

Preferred embodiments of Formula I (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof) and are shown below in Table A and are also considered to be "compounds of the invention." The compounds of the invention are also referred to herein as "tuberculosis inhibitors" or "Mtb inhibitors."

Table A

Class A

Compound

X

Number Ri R ₂

(S)-2, (R)-2 CH ₃ 0 H

(S)-11, (R)-11 C ₃ H ₇ 0 H

(S)-12, (R)-12 C ₃ H ₇ 0 CONH ₂

(S)-13, (R)-13 C ₃ H ₇ NOMe CONH2

(S)-30, (R)-29 CH ₃ NOMe H

(S)-31, (R)-30 C ₃ H ₇ NOMe H

(S)-32, (R)-32 CH ₃ 0 Ac

(S)-33, (R)-33 CH ₃ NOMe Ac

(S)-34, (R)-34 CH ₃ 0 Bn

(S)-35, (R)-35 CH ₃ NOMe Bn

(S)-36, (R)-36 CH ₃ 0 CONH2

(S)-37, (R)-37 CH ₃ NOMe CONH2

(S)-38, (R)-38 C ₃ H ₇ 0 Ac

(S)-39, (R)-39 C ₃ H ₇ NOMe Ac

(S)-40, (R)-40 C ₃ H ₇ 0 Bn

(S)-41, (R)-41 C ₃ H ₇ NOMe Bn

Class B

OR ² Compound

X

Number R ₂

(S)-3, (R)-3 0 H

(S)-14, (R)-14 0 CONH ₂

(S)-15, (R)-15 NOMe H

(S)-46, (R)-46 0 Ac

(S)-47, (R)-47 NOMe Ac

Class C

Compound Number: (S)-4 and (R)-4

Class D

Compound i X R ₂ Number

(S)-5, (R)-5 CH ₃ 0 H

(S)-16, (R)-16 C ₃ H ₇ 0 H

(S)-17, (R)-17 C ₃ H ₇ 0 CONH2

(S)-64, (R)-64 0 Ac

(S)-65, (R)-65 NOMe Ac

Methods of Treatment

Compounds of the present invention are useful for the treatment of tuberculosis. As used herein, the term "tuberculosis" includes any disorders or states caused by

Mycobacterium tuberculosis. This includes multidrug-resistant (MDR) tuberculosis. In certain embodiments, a subject suffering from tuberculosis is also suffering from other pathologies. These pathologies include human immunodeficiency virus (HIV) infection and AIDS.

The compounds of the present invention can reduce the viability of M.

tuberculosis in vivo and in vitro. M. tuberculosis strains that can be vulnerable to the compounds of the present invention include drug resistant and non-drug resistant varieties.

The compounds of the present invention can be used to ameliorate symptoms associated with tuberculosis. These symptoms include cough, weight loss, fatigue, fever, night sweats, chills, loss of appetite, chest pain, or pain with breathing or coughing, spinal pain, joint destruction, meningitis, liver or kidney dysfunction and cardiac tamponade. The compounds of the invention can improve and/or end symptoms associated with tuberculosis.

The compounds of the present invention can be used to treat tuberculosis in mammals. Specifically, the mammal can be a human. In other embodiments, the mammal is pig, cow, dog, cat, goat, rat or mouse.

In certain embodiments, the compounds of the invention are used alone or in combination with other therapeutic agents, e.g. rifampicin, isoniazid, pyrazinamide, ethambutol, amikacin, kanamycin, capreomycin, viomycin, enviomycin, ciprofloxacin, levoflovacin, moxifloxacin, ethionamide, rifabutin, clarithomycin, linezolid,

thioacetazone, arginine, vitamin D and/or R207910.

In another embodiment, the invention provides a pharmaceutical composition of any of the compounds of the present invention. In a related embodiment, the invention provides a pharmaceutical composition of any of the compounds of the present invention and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the invention includes the compounds as novel chemical entities.

In other embodiments, the present invention provides a method for inhibiting the growth and/or replication of M. tuberculosis. The method includes contacting a cell with any of the compounds of the present invention. In a related embodiment, the method further provides that the compound is present in an amount effective to selectively inhibit the growth and/or replication of M. tuberculosis.

Additionally, a method of the invention includes administering to a subject an effective amount of a compound of the invention, e.g., compounds of Formula I, (e.g., Formula A, B, C, D or E), as well as Table A (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof).

In certain embodiments, the compounds of the invention are used for the treatment of tuberculosis. These compounds include compounds of Formula I, (e.g., Formula A, B, C, D or E), as well as Table A (including pharmaceutically acceptable salts thereof, as well as enantiomers, stereoisomers, rotamers, tautomers, diastereomers, atropisomers or racemates thereof).

In other embodiments, the present invention provides a use of any of the compounds of the invention for manufacture of a medicament to treat tuberculosis.

In other embodiments, the invention provides a method of manufacture of a medicament, including formulating any of the compounds of the present invention for treatment of a subject.

In another aspect, provided herein is a method for the treatment or prevention of a bacterial infection in a subject in need thereof, comprising administering the compound (R)-12 to the subject or a pharmaceutical composition comprising (R)-12 to the subject. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating a subject with a mycobacterium infection, comprising administering the compound (R)-12 to the subject or a pharmaceutical composition made thereof. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis. In one embodiment, the

mycobacterium infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating or preventing tuberculosis in a subject in need thereof, comprising administering the compound (R)-12 to the subject or a pharmaceutical composition made thereof.

In another aspect, provided herein is a method for the treatment or prevention of a bacterial infection in a subject in need thereof, comprising administering the compound (R)-13 to the subject or a pharmaceutical composition comprising (R)-13 to the subject. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating a subject with a mycobacterium infection, comprising administering the compound (R)-13 to the subject or a pharmaceutical composition made thereof. In one embodiment, the bacterial infection is caused by Mycobacterium tuberculosis. In one embodiment, the

mycobacterium infection is caused by Mycobacterium tuberculosis.

In another aspect, provided herein is a method of treating or preventing tuberculosis in a subject in need thereof, comprising administering the compound (R)-13 to the subject or a pharmaceutical composition made thereof.

Abbreviations

Mtb, Mycobacterium tuberculosis; S. aureus, Staphylococcus aureus; MDR, multi-drug resistant; VK ₂ , vitamin K ₂ (menaquinone); MenA, l,4-dihydroxy-2- naphthoate prenyltransferase; Qio, coenzyme Qio (ubiquinone); MIC, minimum inhibitory concentration; MABA, microplate alamar blue assay; ROLA, low-oxygen recovery assay; MK, menaquinone; DMMK, demethylmenaquinone; SI, selectivity index; REF, rifampin; INH, isoniazid; EMB, ethanbutol; TMC207, (1R,2S)- l-(6-bromo- 2-methoxyquinolin-3-yl)-4-(dimethylamino)-2-(naphthalen- 1-yl)- l-phenylbutan-2-ol (a diarylquinolone TB drug lead); Tet, tetracycline; DIAD, Diisopropyl azodicarboxylate; TPP, triphenylphosphine; CBS, Corey-Bakshi-Shibata reduction; Py, pyridine; DMAP, dimethylaminopyridine. Definitions

The term "treat," "treated," "treating" or "treatment" include the diminishment or alleviation of at least one symptom associated or caused by the state, disorder or disease being treated. In certain embodiments, the treatment comprises the onset of tuberculosis, followed by the administration of the compound of the invention, which would in turn diminish or alleviate at least one symptom associated or caused by M. tuberculosis. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder.

The term "use" includes any one or more of the following embodiments of the invention, respectively: the use in the treatment of tuberculosis; the use for the

manufacture of pharmaceutical compositions for use in the treatment of tuberculosis, e.g., in the manufacture of a medicament; methods of use of compounds of the invention in the treatment of tuberculosis; pharmaceutical preparations having compounds of the invention for the treatment of tuberculosis; and compounds of the invention for use in the treatment of tuberculosis; as appropriate and expedient, if not stated otherwise.

The term "subject" is intended to include organisms, e.g., eukaryotes, which are capable of suffering from or afflicted with tuberculosis. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from tuberculosis. In certain embodiments, the subject is also suffering from HIV or AIDS. In another embodiment, the subject is a cell.

As used herein, the term "alkyl" refers to a fully saturated branched or

unbranched hydrocarbon moiety. Preferably the alkyl comprises 1 to 20 carbon atoms, more preferably 1 to 16 carbon atoms, 1 to 10 carbon atoms, 1 to 7 carbon atoms, 1 to 6 carbons, 1 to 4 carbons, or 1 to 3 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, w-propyl, wo-propyl, w-butyl, sec-butyl, iso- butyl, ie/t-butyl, w-pentyl, isopentyl, neopentyl, w-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, w-heptyl, w-octyl, w-nonyl, w-decyl and the like. Furthermore, the expression "C _x -C _y - alkyl", wherein x is 1-5 and y is 2-10 indicates a particular alkyl group (straight- or branched-chain) of a particular range of carbons. For example, the expression Ci-C4-alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

As used herein, the term "cycloalkyl" refers to saturated or unsaturated monocyclic, bicyclic or tricyclic hydrocarbon groups of 3-12 carbon atoms, preferably 3- 9, or 3-7 carbon atoms. Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclo butyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl and the like. Exemplary bicyclic hydrocarbon groups include bornyl, indyl, hexahydroindyl, tetrahydronaphthyl, decahydronaphthyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.1]heptenyl, 6,6-dimethylbicyclo[3.1.1]heptyl, 2,6,6- trimethylbicyclo[3.1.1]heptyl, bicyclo[2.2.2]octyl and the like. Exemplary tricyclic hydrocarbon groups include adamantyl and the like.

"Alkoxy" refers to those alkyl groups, having from 1 to 10 carbon atoms, attached to the remainder of the molecule via an oxygen atom. Alkoxy groups with 1-8 carbon atoms are preferred. The alkyl portion of an alkoxy may be linear, cyclic, or branched, or a combination thereof. Examples of alkoxy groups include methoxy, ethoxy, isopropoxy, butoxy, cyclopentyloxy, and the like. An alkoxy group can also be represented by the following formula: -OR ¹ , where R ¹ is the "alkyl portion" of an alkoxy group.

The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, consisting of the stated number of carbon atoms and from one to five heteroatoms, more preferably from one to three heteroatoms, selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroalkyl group is attached to the remainder of the molecule through a carbon atom or a heteroatom.

The term "alkylcarbonyl" refers to a group having the formula -C(0)-R", wherein

R" is an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. An "alkylcarbonyl" group can be attached to the remainder of the molecule via an alkyl group (i.e., -alkyl-C(0)-R").

The term "alkoxycarbonyl" refers to a group having the formula -C(0)0-R ^m , wherein R ^m is an alkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. An "alkoxycarbonyl" group can be attached to the remainder of the molecule via an alkyl group (i.e. , -alkyl-C(0)0- R'").

The term "heteroalkylcarbonyl" refers to a group having the formula -C(0)R ^1V , wherein R ^1V is a heteroalkyl group as defined above and wherein the total number of carbon atoms refers to the combined alkyl and carbonyl moieties. A

"heteroalkylcarbonyl" group can be attached to the remainder of the molecule via an alkyl or heteroalkyl group (i.e., -alkyl-C(0)0-R ^iv or -heteroalkyl-C(0)0-R ^iv ).

The term "aryl" includes aromatic monocyclic or multicyclic e.g. , tricyclic, bicyclic, hydrocarbon ring systems consisting only of hydrogen and carbon and containing from six to nineteen carbon atoms, or six to ten carbon atoms, where the ring systems may be partially saturated. Aryl groups include, but are not limited to, groups such as phenyl, tolyl, xylyl, anthryl, naphthyl and phenanthryl. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g. , tetralin).

The term "heteroaryl," as used herein, represents a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. Heteroaryl groups within the scope of this definition include but are not limited to: acridinyl, carbazolyl, cinnolinyl, quinoxalinyl, pyrrazolyl, indolyl, benzotriazolyl, furanyl, thienyl,

benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively.

The term "heterocycle" or "heterocyclyl" refers to a five-member to ten-member, fully saturated or partially unsaturated nonaromatic heterocylic groups containing at least one heteroatom such as O, S or N. The most frequent examples are piperidinyl, morpholinyl, piperazinyl, pyrrolidinyl or pirazinyl. Attachment of a heterocyclyl substituent can occur via a carbon atom or via a heteroatom. Moreover, the alkyl, alkenyl, cycloalkyl, cycloalkenyl, alkoxy, aryl, heteroaryl, and heterocycle groups described above can be "unsubstituted" or "substituted." The term "substituted" is intended to describe moieties having substituents replacing a hydrogen on one or more atoms, e.g. C, O or N, of a molecule. Such substituents can independently include, for example, one or more of the following: straight or branched alkyl (preferably C ₁ -C ₅ ), cycloalkyl (preferably C ₃ -C ₈ ), alkoxy (preferably Ci-C ₆ ), thioalkyl (preferably Ci-C ₆ ), alkenyl (preferably C ₂ -C ₆ ), alkynyl (preferably C ₂ -C ₆ ), heterocyclic, carbocyclic, aryl (e.g. , phenyl), aryloxy (e.g. , phenoxy), aralkyl

(e.g., benzyl), aryloxyalkyl (e.g. , phenyloxy alkyl), arylacetamidoyl, alkylaryl, hetero aralkyl, alkylcarbonyl and arylcarbonyl or other such acyl group,

heteroarylcarbonyl, or heteroaryl group, (CR'R")o- ₃ NR'R" (e.g. , -NH ₂ ), (CR'R") ₀ - ₃ CN (e.g., -CN), -N0 ₂ , halogen (e.g. , -F, -CI, -Br, or -I), (CR'R") ₀ - ₃ C(halogen) ₃ (e.g., -CF ₃ ), (CR'R") ₀ - ₃ CH(halogen) ₂ , (CR'R") ₀ - ₃ CH ₂ (halogen), (CR'R") ₀ - ₃ CONR'R",

(CR'R")o- ₃ (CNH)NR'R", (CR'R") _{0 3} S(0)i_ ₂ NR'R", (CR'R") ₀ - ₃ CHO,

(CR'R")o- ₃ 0(CR'R")o- ₃ H, (CR'R") ₀ - ₃ S(O) ₀ - ₃ R' (e.g. , -S0 ₃ H, -OS0 ₃ H),

(CR'R")o- ₃ 0(CR'R")o- ₃ H (e.g., -CH ₂ OCH ₃ and -OCH ₃ ), (CR'R") ₀ - ₃ S(CR'R") ₀ - ₃ H

(e.g., -SH and -SCH ₃ ), (CR'R") ₀ - ₃ OH (e.g., -OH), (CR'R") ₀ - ₃ COR\

(CR'R") ₀ - ₃ (substituted or unsubstituted phenyl), (CR'R") ₀ - ₃ (C ₃ -C ₈ cycloalkyl),

(CR'R")o- ₃ C0 ₂ R' (e.g. , -C0 ₂ H), or (CR'R") ₀ - ₃ OR' group, or the side chain of any naturally occurring amino acid; wherein R' and R" are each independently hydrogen, a C ₁ -C ₅ alkyl, C ₂ -C ₅ alkenyl, C ₂ -C ₅ alkynyl, or aryl group.

The term "amine" or "amino" should be understood as being broadly applied to both a molecule, or a moiety or functional group, as generally understood in the art, and may be primary, secondary, or tertiary. The term "amine" or "amino" includes compounds where a nitrogen atom is covalently bonded to at least one carbon, hydrogen or heteroatom. The terms include, for example, but are not limited to, "alkyl amino," "arylamino," "diary lamino," "alkylarylamino," "alkylaminoaryl," "arylaminoalkyl," "alkaminoalkyl," "amide," "amido," and "aminocarbonyl." The term "alkyl amino" comprises groups and compounds wherein the nitrogen is bound to at least one additional alkyl group. The term "dialkyl amino" includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups. The term "arylamino" and "diarylamino" include groups wherein the nitrogen is bound to at least one or two aryl groups, respectively. The term "alkylary lamino," "alky lamino aryl" or "arylamino alkyl" refers to an amino group which is bound to at least one alkyl group and at least one aryl group. The term "alkamino alkyl" refers to an alkyl, alkenyl, or alkynyl group bound to a nitrogen atom which is also bound to an alkyl group.

The term "amide," "amido" or "aminocarbonyl" includes compounds or moieties which contain a nitrogen atom which is bound to the carbon of a carbonyl or a thiocarbonyl group. The term includes "alkaminocarbonyl" or "alkylaminocarbonyl" groups which include alkyl, alkenyl, aryl or alkynyl groups bound to an amino group bound to a carbonyl group. It includes arylaminocarbonyl and arylcarbonylamino groups which include aryl or heteroaryl moieties bound to an amino group which is bound to the carbon of a carbonyl or thiocarbonyl group. The terms "alkylaminocarbonyl,"

"alkenylaminocarbonyl," "alkynylaminocarbonyl," "arylaminocarbonyl,"

"alky lcarbony lamino," "alkenylcarbonylamino," "alkynylcarbony lamino," and

"arylcarbonylamino" are included in term "amide." Amides also include urea groups (aminocarbonylamino) and carbamates (oxycarbonylamino).

In a particular embodiment of the invention, the term "amine" or "amino" refers to substituents of the formulas N(R ⁸ )R ⁹ , CH ₂ N(R ⁸ )R ⁹ and CH(CH ₃ )N(R ⁸ )R ⁹ , wherein R ⁸ and R ⁹ are each, independently, selected from the group consisting of H and (Ci-C ₄ - alkyl)o-iG, wherein G is selected from the group consisting of COOH, H, P0 ₃ H, S0 ₃ H, Br, CI, F, 0-Ci_4-alkyl, S-Ci_ ₄ -alkyl, aryl, C(0)OCi-C ₆ -alkyl, C(0)Ci-C ₄ -alkyl-COOH, C(0)Ci-C ₄ -alkyl and C(0)-aryl.

The description of the disclosure herein should be construed in congruity with the laws and principals of chemical bonding. For example, it may be necessary to remove a hydrogen atom in order accommodate a substitutent at any given location. Furthermore, it is to be understood that definitions of the variables (i.e., "R groups"), as well as the bond locations of the generic formulae of the invention (e.g. , Formula I(e.g, Formulae A, B, C, D or E)), will be consistent with the laws of chemical bonding known in the art. It is also to be understood that all of the compounds of the invention described above will further include bonds between adjacent atoms and/or hydrogens as required to satisfy the valence of each atom. That is, bonds and/or hydrogen atoms are added to provide the following number of total bonds to each of the following types of atoms: carbon: four bonds; nitrogen: three bonds; oxygen: two bonds; and sulfur: two-six bonds.

The compounds of this invention may include asymmetric carbon atoms. It is to be understood accordingly that the isomers arising from such asymmetry (e.g. , all enantiomers, stereoisomers, rotamers, tautomers, diastereomers, or racemates) are included within the scope of this invention. Such isomers can be obtained in

substantially pure form by classical separation techniques and by stereochemically controlled synthesis. Furthermore, the structures and other compounds and moieties discussed in this application also include all tautomers thereof. Compounds described herein may be obtained through art recognized synthesis strategies.

It will also be noted that the substituents of some of the compounds of this invention include isomeric cyclic structures. It is to be understood accordingly that constitutional isomers of particular substituents are included within the scope of this invention, unless indicated otherwise. For example, the term "tetrazole" includes tetrazole, 2H-tetrazole, 3H-tetrazole, 4H-tetrazole and 5H-tetrazole.

Combinations

The compounds of this invention are also useful in combination with known antituberculosis agents. Such known anti- tuberculosis agents include the following:

rifampicin; isoniazid; pyrazinamide; ethambutol; aminoglycosides such as amikacin and kanamycin; polypeptides such as capreomycin, viomycin and enviomycin;

fluoroquinolones such as ciprofloxacin, levoflovacin and moxifloxacin; thiomaides such as ethionamide; rifabutin; macrolides such as clarithomycin; linezolid, thioacetazone, arginine, vitamin D and R207910.

The compounds of this invention are also useful in combination with known HIV protease inhibitors. Examples of HIV protease inhibitors include amprenavir, abacavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir, tipranavir, ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632. Examples of reverse transcriptase inhibitors include delaviridine, efavirenz, GS-840, HB Y097, lamivudine, nevirapine, AZT, 3TC, ddC, and ddl. Assays

The inhibition of the growth and viability of M. tuberculosis as well as the modulation of symptoms associated with tuberculosis by the compounds of the invention may be measured using a number of assays available in the art. Examples of such assays are described in the Exemplification section below.

Pharmaceutical Compositions

The compounds of the present invention are suitable as active agents in pharmaceutical compositions that are efficacious particularly for treating tuberculosis. The pharmaceutical composition in various embodiments has a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable excipients, carriers, fillers, diluents and the like.

The language "pharmaceutically effective amount" or "pharmaceutically acceptable amount" of the compound is that amount necessary or sufficient to treat or prevent a protein kinase-associated disorder, e.g. prevent the various morphological and somatic symptoms of tuberculosis. In an example, an effective amount of a compound of the invention is the amount sufficient to treat tuberculosis in a subject. The effective amount can vary depending on such factors as the size and weight of the subject, the strain of M. tuberculosis, or the particular compound of the invention. For example, the choice of the compound of the invention can affect what constitutes an "effective amount. "One of ordinary skill in the art would be able to study the factors contained herein and make the determination regarding the effective amount of the compounds of the invention without undue experimentation.

The regimen of administration can affect what constitutes a pharmaceutically effective amount. A compound of the invention can be administered to the subject either prior to or after the onset of tuberculosis. Further, several divided dosages, as well as staggered dosages can be administered daily or sequentially, or the dose can be continuously infused, or can be a bolus injection. Further, the dosages of the

compound(s) of the invention can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation. In one non-limiting embodiment, the phrase "pharmaceutically effective amount" refers to the amount of a compound of the present invention that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and/or ameliorate tuberculosis. In still another non-limiting embodiment, the term "pharmaceutically effective amount" refers to the amount of a compound of the present invention that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reduce or inhibit the growth and/or viability of M.

tuberculosis.

Compounds of the invention may be used in the treatment of states, disorders or diseases as described herein, or for the manufacture of pharmaceutical compositions for use in the treatment of these diseases. The invention includes methods of use of compounds of the present invention in the treatment of these diseases, or pharmaceutical preparations having compounds of the present invention for the treatment of these diseases.

The language "pharmaceutical composition" includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The phrase "pharmaceutically acceptable carrier" is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administering compounds of the present invention to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, a- tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical, buccal, sublingual, rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil- in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium

phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example,

carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol and glycerol

monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydro xypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydro xypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These

compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluent commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar- agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.

Formulations of the present invention which are suitable for vaginal

administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active compound in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral

administration comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacterio stats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally-administered drug form is

accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide.

Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly( anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral and/or IV administration is preferred.

The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal,

intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually. Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

Generally, intravenous and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day, and still more preferably from about 1.0 to about 100 mg per kg per day. An effective amount is that amount treats a protein kinase-associated disorder. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical composition.

Synthetic Procedure

Compounds of the present invention are prepared from commonly available compounds using procedures known to those skilled in the art, including any one or more of the following conditions without limitation:

Acid addition salts of the compounds of the invention are most suitably formed from pharmaceutically acceptable acids, and include for example those formed with inorganic acids e.g. hydrochloric, hydrobromic, sulphuric or phosphoric acids and organic acids e.g. succinic, malaeic, acetic or fumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalates can be used for example in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt. Also included within the scope of the invention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt is achieved by applying standard techniques, in which an aqueous solution of the given salt is treated with a solution of base e.g. sodium carbonate or potassium hydroxide, to liberate the free base which is then extracted into an appropriate solvent, such as ether. The free base is then separated from the aqueous portion, dried, and treated with the requisite acid to give the desired salt.

Examples of pharmaceutically acceptable addition salts include, without limitation, the non-toxic inorganic and organic acid addition salts such as the

hydrochloride derived from hydrochloric acid, the hydrobromide derived from

hydrobromic acid, the nitrate derived from nitric acid, the perchlorate derived from perchloric acid, the phosphate derived from phosphoric acid, the sulphate derived from sulphuric acid, the formate derived from formic acid, the acetate derived from acetic acid, the aconate derived from aconitic acid, the ascorbate derived from ascorbic acid, the benzenesulphonate derived from benzensulphonic acid, the benzoate derived from benzoic acid, the cinnamate derived from cinnamic acid, the citrate derived from citric acid, the embonate derived from embonic acid, the enantate derived from enanthic acid, the fumarate derived from fumaric acid, the glutamate derived from glutamic acid, the glycolate derived from glycolic acid, the lactate derived from lactic acid, the maleate derived from maleic acid, the malonate derived from malonic acid, the mandelate derived from mandelic acid, the methanesulphonate derived from methane sulphonic acid, the naphthalene-2-sulphonate derived from naphtalene-2- sulphonic acid, the phthalate derived from phthalic acid, the salicylate derived from salicylic acid, the sorbate derived from sorbic acid, the stearate derived from stearic acid, the succinate derived from succinic acid, the tartrate derived from tartaric acid, the toluene-p-sulphonate derived from p-toluene sulphonic acid, and the like. Particularly preferred salts are sodium, lysine and arginine salts of the compounds of the invention. Such salts can be formed by procedures well known and described in the art.

Other acids such as oxalic acid, which cannot be considered pharmaceutically acceptable, can be useful in the preparation of salts useful as intermediates in obtaining a chemical compound of the invention and its pharmaceutically acceptable acid addition salt.

Metal salts of a chemical compound of the invention include alkali metal salts, such as the sodium salt of a chemical compound of the invention containing a carboxy group.

In vivo hydrolyzable esters or amides of certain compounds of the invention can be formed by treating those compounds having a free hydroxy or amino functionality with the acid chloride of the desired ester in the presence of a base in an inert solvent such as methylene chloride or chloroform. Suitable bases include triethylamine or pyridine. Conversely, compounds of the invention having a free carboxy group can be esterified using standard conditions which can include activation followed by treatment with the desired alcohol in the presence of a suitable base.

Mixtures of isomers obtainable according to the invention can be separated in a manner known per se into the individual isomers; diastereoisomers can be separated, for example, by partitioning between polyphasic solvent mixtures, recrystallisation and/or chromatographic separation, for example over silica gel or by, e.g. , medium pressure liquid chromatography over a reversed phase column, and racemates can be separated, for example, by the formation of salts with optically pure salt-forming reagents and separation of the mixture of diastereoisomers so obtainable, for example by means of fractional crystallisation, or by chromatography over optically active column materials.

Intermediates and final products can be worked up and/or purified according to standard methods, e.g. , using chromatographic methods, distribution methods, (re-) crystallization, and the like.

At all stages of the reactions, mixtures of isomers that are formed can be separated into the individual isomers, for example diastereoisomers or enantiomers, or into any desired mixtures of isomers, for example racemates or mixtures of

diastereoisomers, for example analogously to the methods described in Science of Synthesis: Houben-Weyl Methods of Molecular Transformation. Georg Thieme Verlag, Stuttgart, Germany, 2005.

The solvents from which those solvents that are suitable for any particular reaction may be selected include those mentioned specifically or, for example, water, esters, such as lower alkyl-lower alkanoates, for example ethyl acetate, ethers, such as aliphatic ethers, for example diethyl ether, or cyclic ethers, for example tetrahydrofuran or dioxane, liquid aromatic hydrocarbons, such as benzene or toluene, alcohols, such as methanol, ethanol or 1- or 2-propanol, nitriles, such as acetonitrile, halogenated hydrocarbons, such as methylene chloride or chloroform, acid amides, such as dimethylformamide or dimethyl acetamide, bases, such as heterocyclic nitrogen bases, for example pyridine or N-methylpyrrolidin-2-one, carboxylic acid anhydrides, such as lower alkanoic acid anhydrides, for example acetic anhydride, cyclic, linear or branched hydrocarbons, such as cyclohexane, hexane or isopentane, or mixtures of those solvents, for example aqueous solutions, unless otherwise indicated in the description of the processes. Such solvent mixtures may also be used in working up, for example by chromatography or partitioning.

The compounds, including their salts, may also be obtained in the form of hydrates, or their crystals can, for example, include the solvent used for crystallization. Different crystalline forms may be present. The invention relates also to those forms of the process in which a compound obtainable as an intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example in a protected form or in the form of a salt, or a compound obtainable by the process according to the invention is produced under the process conditions and processed further in situ.

Prodrugs

This invention also encompasses pharmaceutical compositions containing, and methods of treating tuberculosis through administering, pharmaceutically acceptable prodrugs of compounds of the compounds of the invention. For example, compounds of the invention having free amino, amido, hydroxy or carboxylic groups can be converted into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g. , two, three or four) amino acid residues is covalently joined through an amide or ester bond to a free amino, hydroxy or carboxylic acid group of compounds of the invention. The amino acid residues include but are not limited to the 20 naturally occurring amino acids commonly designated by three letter symbols and also includes 4-hydroxyproline, hydroxylysine, demosine, isodemosine, 3- methylhistidine, norvalin, beta-alanine, gamma-aminobutyric acid, citrulline

homocysteine, homoserine, ornithine and methionine sulfone. Additional types of prodrugs are also encompassed. For instance, free carboxyl groups can be derivatized as amides or alkyl esters. Free hydroxy groups may be derivatized using groups including but not limited to hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, as outlined in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, as are carbonate prodrugs, sulfonate esters and sulfate esters of hydroxy groups.

Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with groups including but not limited to ether, amine and carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, are also encompassed. Prodrugs of this type are described in J. Med. Chem. 1996, 39, 10. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties may incorporate groups including but not limited to ether, amine and carboxylic acid functionalities.

Any reference to a compound of the present invention is therefore to be understood as referring also to the corresponding pro-drugs of the compound of the present invention, as appropriate and expedient.

Kits

Advantageously, the present invention also provides kits for use by a consumer for treating tuberculosis. The kits comprise a) a pharmaceutical composition comprising one or more compounds of the invention and a pharmaceutically acceptable carrier, vehicle or diluent; and, optionally, b) instructions describing a method of using the pharmaceutical composition for treating the specific disease. The instructions may also indicate that the kit is for treating tuberculosis.

A "kit" as used in the instant application includes a container for containing the separate unit dosage forms such as a divided bottle or a divided foil packet. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material, for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle which is in turn contained within a box.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process, recesses are formed in the plastic foil. The recesses have the size and shape of individual tablets or capsules to be packed or may have the size and shape to accommodate multiple tablets and/or capsules to be packed. Next, the tablets or capsules are placed in the recesses accordingly and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are individually sealed or collectively sealed, as desired, in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a written memory aid, where the written memory aid is of the type containing information and/or instructions for the physician, pharmacist or subject, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the tablets or capsules so specified should be ingested or a card which contains the same type of information.

Another example of such a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday," . . . etc. . . . "Second Week, Monday, Tuesday, . . . " etc. Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several tablets or capsules to be taken on a given day.

Another specific embodiment of a kit is a dispenser designed to dispense the daily doses one at a time. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter, which indicates the number of daily doses that, has been dispensed. Another example of such a memory-aid is a battery-powered micro-chip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

EXAMPLES

The invention is further illustrated by the following examples, which should not be construed as further limiting. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology and immunology, which are within the skill of the art.

Example 1. Selective M. tuberculosis MenA Inhibitors- Assay. Antimicrobial spectrum focused against Mtb (selective antimycobacterial agent) is preferable for TB chemotherapy. The peptide sequences of the Mtb menA and S.

aureus menA gene products have only 32% identity and 50% similarity according to BLAST. Many of the molecules identified herein exhibit selective MenA enzyme and bacterial growth inhibitory activities against Mtb; more than a 10-fold higher inhibitory activity against Mtb than S. aureus.

In order to develop MenA inhibitors which are selective against Mtb, molecules were first evaluated in MenA enzyme inhibitory assays. Only molecules exhibited Mtb MenA activity over S. aureus MenA (IC ₅₀ < 20 μΜ, >60 μΜ against Mtb MenA and S. aureus MenA, respectively) were evaluated in bacterial growth inhibitory assays (MICs) using Mtb, S. aureus, and E. coli (See Figure 2). The molecules exhibited good activity only against Mtb (MICs for Mtb, S. aureus, and E. coli are <12.5, >60, and >125 μg/mL, respectively) were evaluated in E. coli growth inhibitory assays under anaerobic conditions followed by menaquinone supplementation assays (E. coli utilize only menaquinone in their electron transport system under anaerobic conditions). This assay confirmed that the molecules killed bacteria by targeting MenA enzyme or electron transport systems. Oxygen consumption assay using Mtb identifies electron transport system inhibitors; in this assay, selective MenA inhibitors should not show activity at concentrations below the MIC against Mtb. Selective antimycobacterial MenA inhibitors confirmed via assays with E. coli and oxygen consumption assays were evaluated for their activity against non-replicating Mtb via the low oxygen recovery assays (LORA).

Several antimycobacterial MenA inhibitors in their racemic forms were formulated. In order to obtain insight into the effect of chirality of 2-6 (Figure 3) and functional groups around the chiral center on biological activity, both enantiomers of 2-6 and their analogs were synthesized in which the secondary alcohols were functionalized with acyl, carbamate, and benzyl groups. In this study benzophenone O-methyl oxime derivatives were also introduced to obtain preliminary SAR and their cytotoxicity data. Syntheses of optically active molecules of 2-6 and their analogs are summarized in the schemes shown in Figures 4, 5 and 6. The CI- substituted benzophenone

derivatives, 21 and 23, were synthesized according to the previously reported procedures. O-Methyl oxime derivatives 22 and 24 were prepared by the treatment of 21 and 23 with MeONH ₂ *HCl in pyridine at 105 °C. The phenolic alcohol of 21 was subjected to the Mitsunobu reaction with Boc-protected piperidin-4-ylmethanol (25) to provide the piperidinyl ethers 26 in over 90% yield, after deprotection of the Boc group. Optically active (2R)- or (l^-alkyloxiranes, 28 and 29, were synthesized via Jacobsen's kinetic resolutions of the racemic epoxides. Opening of the epoxides (S)-29 with the piperidine derivatives 26 was achieved by using a stoichiometric amount of AlMe ₃ at room temperature to yield the amino -alcohols (S)-ll. The generated optically active alcohol (S)-ll was functionalized with Ac ₂ 0, BnBr, and TMSNCO to afford the corresponding acetate (S)-38, benzyl ether (S)-40, and carbamate (S)-12 (class A molecules). In order to synthesize optically active piperidinyl-benzyl alcohol derivatives 3 (class B molecules), (S)- and (R)-benzaldehyde derivatives 45 were synthesized in 4 steps from 44 via CBS reductions. The starting material 44 was readily synthesized via a Grignard reaction of 42 with a Weinreb amide 43. Reductive aminations of 26 with (S)-45 was achieved with NaBH(OAc)3 in the absence of acid to provide the desired tertiary amine (S)-46 without contamination of the diphenylmethanol by-product. Deacetylation of (S)-46 afforded the secondary alcohol (S)-3 whose alcohol was functionalized with TMSNCO to afford the corresponding carbamate (S)-14. Optically active amino-alcohol 4 (class C molecules) was synthesized via the resolution of rac-epoxide 49 followed by Zn(C10 ₄ ) ₂ -catalyzed selective opening of epoxide with the primary amine. The other optically active

secondary alcohols (class D and E molecules) were successfully synthesized via the same synthetic procedures developed for class A and B molecules. Syntheses of the molecules having (R)-configuration were also achieved by using the antipodes of the building blocks utilized for the synthesis for the (^-configuration molecules. Similarly, optically active benzophenone O-methyl oxime derivatives 13, 15, 18, 19, 59, 63, and 65 were synthesized. Thus, both (S)- and (R)- forms of 2-7 and their derivatives have been synthesized in short number of steps. Optical purity and purity of each molecule were established via HPLC analyses of Mosher esters of secondary alcohols or alcohols using a chiral column.

MenA Enzyme Inhibitory Assays

MenA enzymatic assays of generated molecules were originally performed with [ H]rated farnesyl diphosphate and MenA containing membrane fraction. This assay requires a significant effort to separate demethylmenaquinone (DMMK) from the reaction mixtures and cost for [ H]farnesyl diphosphate. In order to simplify the procedure and to reduce the cost of MenA enzyme inhibitory assays, a MenA assay has been developed using HPLC. In new MenA assay, the MenA product,

demethylmenaquinone can readily be quantitated via UV absorbance (DMMK; λ ₂ 325 nm).

A preliminary screening of the activity of compounds synthesized in the schemes shown in Figures 4, 5 and 6 at the single concentrations of 100 μΜ was performed. In these MenA enzyme inhibitory assays, the optically active molecules were assayed against M. tuberculosis and S. aureus MenA. IC50 values of the molecules which exhibited activity against Mtb MenA and inactivity against S. aureus MenA were calculated. Of 86 optically active molecules synthesized in Scheme 1, 26 molecules exhibited Mtb MenA inhibitory activity and were inactive against S. aureus MenA at 100 μΜ concentrations. Dose-response plots (DMMK formation vs. concentrations of inhibitor) were obtained for 26 molecules to determine IC ₅₀ values. Significant effect of chirality in the MenA enzyme inhibitory activity was observed in the molecules in classes A and D. On the contrary, an obvious effect of chirality in MenA enzyme inhibitory activity of the molecules in classes B, C, and E was not observed; the racemic forms and each enantiomer did not show noticeable difference in MenA enzyme inhibitory activity. It is noteworthy that the carbamate analog (R)-12 group showed 6-fold better MenA enzyme inhibitory activity compared to its alcohol from (R)-ll. Similar improvement of enzymatic inhibition by modification of the alcohol with the carbamate group is observed in the molecules in class D. Example 2. Bacterial Growth Inhibitory Assays.

All molecules that showed activity against MenA in a preliminary assay at 100 μΜ concentrations were evaluated in mycobacterial growth assay via the microplate alamar blue assay (MABA) and low oxygen recovery assay (LORA). Briefly, the MABA assay is a colorimetric assay that uses the color change of rezasurin to evaluate M. tuberculosis (Mtb) inhibitory activity under aerobic conditions. On the other hand, the LORA assay evaluates potency against non-replicating Mtb cells under low oxygen conditions using a luminescent stain. Significantly, in all cases the MIC values obtained from the LORA assays were lower than those from the MABA assays. These were the first observations of molecules that killed non-replicating Mtb at a concentration below the MIC against Mtb grown under aerobic conditions. In all cases the values of MICLORA/MICMABA were <1 (See Table 1).

Table 1. Antibacterial Activities against M. tuberculosis and S. aureus, and MenA Enzyme Inhibitory Activities of the Selected Molecules.

Structure MIC (ng/mL.)" MenA Inhibition

Compounds M. tuberculosis M. tuberculosis S. aureus M. tuberculosis

-i R ₂ S. aureus ΙΟ ₅₀ (μΜ)

X, R , R ₂ = O, CH ₃ , H

(S)-2 X, Ri , R ₂ = O, CH ₃ , H >60 12.5 5.59 - + 7.5

(R)-2 X, Ri , R ₂ = O, CH ₃ , H >60 6.2 4.91 - + 17.0

(S)-11 X, Ri , R ₂ = O, C3H7, H >60 12.5 4.91 - + 16.0

(R)-11 X, Ri , F¾2— O, C3H7, H >60 6.2 5.59 - + 9.0

(S)-12 X, Ri , R ₂ = O, C ₃ H ₇ , CONH ₂ >60 12.5 2.94 - + 9.0

(R)-12 X, Ri , R ₂ = O, C ₃ H ₇ , CONH ₂ >60 2.31 0.85 - + 1.5

(R)-13 X, Ri , R ₂ = NOMe, C ₃ H ₇ , CONH ₂ >60 2.31 0.85 - + 1.2

(S)-3 X, R ₂ = O, H 60 12.5 1.88 25.0

(R)-3 X, R ₂ = O, H 60 12.5 1.46 20.0

(S)-14 X, R ₂ = O, CONH ₂ 60 12.5 4.93 20.0

(R)-14 X, R ₂ = O, CONH ₂ 60 12.5 4.93 15.0

(R)-15 X, R ₂ = NOMe, OH 60 6.25 1.43 15.0

(S)-4 >60 12.5 3.00 17.0 (R)-4 >60 12.5 3.00 14.0

rac-16 O, CH ₃ , CONH ₂ >60 3.25 2.83 7.5

(S)-5 O, CH ₃ , H >60 1.50 1.43 4.5

(R)-5 O, CH ₃ , H >60 6.25 2.85 7.5

(S)-16 O, C ₃ H ₇ , H >60 1.50 1.43 3.5

(R)-16 O, C ₃ H ₇ , H >60 6.25 5.20 9.5

(S)-17 O, C ₃ H ₇ , CONH ₂ >60 1.50 1.45 1.5

(S)-18 NOMe, C ₃ H ₇ , CONH ₂ >60 1.50 1.40 1.5

(

(R)-6 X, R ₂ = O, H >60 12.5 1.46 15.0

(S)-19 X, R ₂ = O, CONH ₂ >60 12.5 2.93 20.0

(R)-19 X, R ₂ = O, CONH ₂ >60 12.5 4.93 20.0

(S)-20 X, R ₂ = NOMe, OH >60 12.5 5.54 20.0

(R)-20 X, R ₂ = NOMe, OH >60 12.5 5.54 15.0

RFP ^d 0.2 1.47

INH ^e 0.1 >128

EMB ¹ 0.78 >128 In the class A molecules (R)-2 was two-fold more potent than rac-2 and (S)-2 in the MABA assays. Similar to an observed trend in MenA enzyme inhibitory assays, the carbamate (R)-12 had an over 5-fold increase in mycobactericidal activities compared to rac-2. (R)-12 exhibited a significant activity in the LORA assay; MIC value of (R)-12 is 1.7-fold more effective in killing non-replicating bacteria than rifampicin (MICLORA 1 -47 μg/mL). It is worth mentioning that (R)-12 was the most active in killing non-replicating Mtb in vitro among antimycobacterial drugs (approved by FDA) and preclinical drugs tested. Regardless of the stereochemistry of the chiral centers, the molecules in class B, C, and E did not show noticeable difference in antimycobacterial activity in the both MABA and LORA assays. The molecules in class D are regio isomers at the

benzophenone moiety of the class A molecules. Similar to the molecules in class A, pronounced effect of the stereochemistry of secondary alcohol on antimycobacterial activity was observed in the class D molecules. In contrast to the chirality effect observed in the molecules in class A, the molecules possessing ^-configuration exhibited better antimycobacterial activity than the corresponding R-configuration molecules. The effect of the carbamate group on enzyme and bacterial growth inhibitory activities was not observed in the molecules in class D; the MABA MIC value of the carbamate (S)-16 was equal to that of the corresponding alcohol (S)-17. Nonetheless, the MABA MIC for the rac-alcohol 5 could have been improved two-fold by forming the carbamate of its free alcohol; the MABA MICs of rac-5 and rac-16 were 6.25 and 3.25 μg/mL, respectively. Antimycobacterial activity was improved by increasing the hydrophobicity of the side chain (C8 vs. C6) in the molecules of class A, whereas noticeable bactericidal effect by increasing hydrophobicity of the side chain was not observed in the molecules of class D. Example 3. E. coli Growth Inhibitory Assays under Anaerobic Conditions.

M. tuberculosis or S. aureus treated with the MenA inhibitors could not be rescued completely even at higher concentrations of exogenous menaquinone (VK ₂ ). In contrast, growth inhibition of E. coli by the MenA inhibitor could be rescued by supplementation of VK ₂ (50 μΜ) under "anaerobic conditions" {vide supra). E. coli growth inhibition rescued by addition of VK ₂ may be attributed to the degree of permeability of VK ₂ through cell envelop. Although lack of activity of MenA inhibitors against Gram-negative bacteria grown under aerobic conditions have been demonstrated, all MenA inhibitors identified showed growth inhibition of E. coli at 5 μg/mL

concentrations under anaerobic conditions, and the inhibitory effect of MenA inhibitor was rescued by supplementation of VK ₂ . Therefore, these convenient cell-based assays using E. coli under anaerobic conditions can be utilized to confirm that the inhibitor molecules kill Gram-positive bacteria including Mtb by targeting MenA biosynthesis or bacterial electron transport systems.

Example 4. Oxygen Consumption Assays.

The basic concept underlying bacterial oxygen consumption assay is that changes in the rate of oxygen uptake result in a change in the oxygen concentrations. The oxygen consumption by bacterial concentrations greater than 10 cfu/mL bacteria is proportional to the concentration of bacteria. Effect of the inhibitor molecule on electron transport by the quantitation of decolorization of methylene blue, which is a well-known redox dye, has been unambiguously demonstrated. Oxygen-consumption assays of (R)-12 and (S)- 17 showed decolorization of methylene blue at concentrations (12.5 μg/mL) above the MIC of each molecule (MICs 2.31 and 1.50 μg/mL, respectively) against Mtb. Thus, (R)-12 and (R)-17 are very weak (or are not) electron transport system inhibitors, and thus, antimycobacterial activity of these molecules are attributed by targeting

menaquinone biosynthesis.

Example 5. Cytotoxicity of MenA Inhibitors.

In order to obtain insight into potential toxicity of identified inhibitor molecules, all antimycobacterial MenA inhibitors were evaluated in in vitro cytotoxicity assays using Vero monkey kidney cells and HepG2 human hepatoblastoma cells. Most MenA inhibitors possessing the benzophenone group showed IC ₅₀ of 1-6.5 μg/mL against two mammalian cell lines; selectivity indexes (IC ₅₀ in mammalian cells/MIC against Mtb) of a series of benzophenone MenA inhibitors identified in this program were less than 2. On the other hand, O-methyl oxime derivatives in class A showed approximately 10-fold less cytotoxic than the corresponding benzophenone derivatives in vitro cytotoxicity assays. (R)-13 showed an encouraging in vitro activity/toxicity ratio (a SI value >10). It is believed that electrophilicity of benzopheneone moiety can be diminished by O-methyl oxime formation of (R)-13. Thus, the benzophenone O-methyl oxime may not be a good electron acceptor that interferes with redox systems of mammalian cells.

Through asymmetric synthesis of a series of optically active molecules followed by screening these molecules by the assay methods described here, new MenA ( 1 ,4- dihydroxy-2-naphthoate prenyltransferase) biosynthesis inhibitors have been identified that showed very weak (or no) inhibitory activities of bacterial electron transport systems. A series of MenA inhibitors identified in this program exhibited antimicrobial spectrum focused against Mtb. Selective activity against Mtb is ideal in TB drug discovery due to the fact that TB chemotherapy requires long regimen, so that broad- spectrum anti-TB agents may cause resistant to other bacteria during TB chemotherapy. The carbamates (R)-12 and (R)-13 in class A showed significant growth inhibitory activities against non- replicating Mtb (0.85 μg/mL) with the MICLORA/MICMAB A/ value of 0.37

(MICLORA/MICMABA = 7.35 for rifampin). Effectiveness in non-replicating Mtb was also confirmed via assays using a modified Wayne model. Among antimycobacterial agents tested, the inhibitor (R)-12 and (R)-13 were the most active in killing non-replicating Mtb in vitro. MenA inhibitors identified in this program strongly suggested that menaquinone biosynthesis is important in maintaining mycobacterial viability under conditions of restricted oxygen. MenA inhibitors seem to be able to block the electron flow, consequently inhibiting the bacterial growth. It is conceptually very interesting that

MenA inhibitors can be developed as indirect ATP synthesis inhibitors. The assay data for the identified MenA inhibitors indicate that menaquinone biosynthesis is a unique and new antimycobacterial target. In addition, in tuberculosis, the key to shortening the long regimen lies in targeting the non-replicating persistence subpopulation. Thus, the discovery of molecules that kill non-replicating Mtb at lower concentrations than MIC against Mtb under aerobic conditions is of significance in terms of discovering new lead molecules that can be developed into new drugs to kill Mtb in any state. Moreover, resistant bacteria to the MenA inhibitors were not isolated in the mutation frequency studies. Therefore, unlike the other known bacterial molecular targets, menA shows extremely low mutation frequency. In conclusion, robustness of the in vitro assay approaches to identify selective MenA inhibitors against M. tuberculosis (summarized in Figure 2) has been

demonstrated. The MenA inhibitors described here can be synthesized in short steps with high yield, and structural modifications to improve in vitro efficacy will be achieved by modifying the hydrophobic side chain and benzophenone O-methyl oxime moieties of (R)-13.

Synthesis Examples Example 6. Chemistry. General Information.

All glasswares were oven dried, assembled hot and cooled under a stream of nitrogen before use. Reactions with air sensitive materials were carried out by standard syringe techniques. Commercially available reagents were used as received without further purification. Thin layer chromatography was performed using 0.25 mm silica gel 60 (F254, Merck) plates visualizing at 254 nm, or developed with eerie ammonium molybdate or anisaldehyde solutions by heating with a hot-air gun. Specified products were purified by flash column chromatography using silica gel 60 (230-400 mesh, Merck). IR absorptions on NaCl plates were run on a Perkin Elmer FT-IR 1600. 1H- NMR spectral data were obtained using Varian 300, 400, and 500 MHz instruments. The residual solvent signal was utilized as an internal reference. 13 C NMR spectral data were obtained using a Varian 100, 125 MHz spectrometer. Chemical shifts were reported in parts per million (ppm) downfield from TMS, using the middle resonance of CDC1 ₃ (77.0 ppm) as an internal standard. For all NMR spectra, δ values are given in ppm and J values in Hz. Mass spectra were obtained at University of Tennessee Central Instrument Facility. HPLC analyses were performed with a Shimadzu LC-20AD HPLC system. All compounds were purified by PTLC or reverse HPLC to be >95 purity whose purities were established by HPLC.

Example 7. (4-Chlorophenyl)(3-methoxyphenyl)methanone. 4-Chlorobenzoyl chloride (5.00 g, 28.58 mmol) was dissolved in CH ₂ C1 ₂ (240 mL) and cooled to 0 °C. N, O-Dimethylhydroxyl amine (3.07 g, 31.43 mmol) and triethylamine (6.36 g, 62.85 mmol) were added into the reaction mixture. After 4h at r. , the reaction mixture was quenched with water and the organic phase was washed with IN HC1. The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo to afford crude 4-chloro-N-methoxy-N-methylbenzamide (6.41 g). This was used further purification. To a stirred solution of l-iodo-3-methoxybenzene (21.48 g, 91.80 mmol) in THF (10 mL) was added isopropyl magnesium chloride (2M, 1.50 mmol) at -20 °C. After lh at the same temperature, the reaction mixture was cooled to -78 °C and 4-chloro-N- methoxy-N-methylbenzamide (100 mg, 0.50 mmol) in THF (1 mL). The reaction mixture was warmed to 0 °C over 30 min. After 3h at 0 °C, the reaction mixture was quenched with aq. sat. NH ₄ C1. The water phase was extracted with EtOAc and the combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel chromatography (4: 1, Hexanes:EtOAc) afforded (4-Chlorophenyl)(3- methoxyphenyl)methanone (97.0 mg, 79%) as a white powder; 1H NMR (500 MHz, CDCI3): δ 3.88 (s, 3H), 7.16 (dd, J = 1.5, 8 Hz, 1H), 7.31-7.35 (m, 2H), 7.40 (t, J = 8 Hz, 1H), 7.47 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 55.6, 114.4, 119.2, 122.8, 128.8, 129.5, 131.6, 136.1, 138.7, 139.1, 159.8, 195.4; LRMS (ESI) m/z: 247.0 (M+H) ⁺ .

Example 8. (4-Chlorophenyl)(3-hydroxyphenyl)methanone (21). To a stirred solution of (4-chlorophenyl)(3-methoxyphenyl)methanone (5.30 g, 21.5 mmol) in AcOH (15 mL) was added HBr (48% in water, 200 mL). The reaction mixture was gently refluxed at 125 °C for 36h. The reaction mixture was cooled to r.t. and all volatiles were distilled off. Purification by silica gel chromatography (4: 1,

Hexanes:EtOAc) afforded 21 (3.84 g, 78%) as a white solid and the unreacted starting material (15%) was recovered. 1H NMR (500 MHz, CDC1 ₃ ): δ 5.84 (s, 1H), 7.02-7.04 (m, 1H), 7.19-7.21 (m, 1H), 7.25-7.29 (m, 2H), 7.37-7.39 (m, 2H), 7.67-7.69 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 116.6, 120.4, 122.9, 128.9, 129.9, 131.8, 135.8, 138.7, 139.4, 156.2, 195.9; LRMS (ESI) m/z: 233.0 (M+H) ⁺ .

Example 9. tert-Butyl 4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidine-l- carboxylate.

To a stirred solution of (4-chlorophenyl)(3-hydroxyphenyl)methanone (21, 2.00 g, 8.71 mmol), ieri-butyl 4-(hydroxymethyl)piperidine-l-carboxylate (25, 3.75 g, 17.4 mmol), and TPP (3.42 g, 13.05 mmol) in THF (40 mL) was added DIAD (4.79 g, 13.05 mmol). After 3h, all volatiles were evaporated in vacuo. Purification by silica gel chromatography (4: 1, hexanes:EtOAc) provided ie/t-butyl 4-((3-(4- chlorobenzoyl)phenoxy)methyl)piperidine-l-carboxylate (3.61 g, 97%) as a colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.64-1.25 (m, 2H), 1.39 (s, 9H), 1.75 (d, J = 13.5 Hz, 2H), 1.87-1.90 (m, 1H), 2.67 (bs, 2H), 3.77 (d, J = 6.5 Hz, 2H), 4.08 (bs, 2H), 7.04 (dd, J = 2, 8 Hz, 1H), 7.20-7.22 (m, 2H), 7.29 (t, J = 8 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.66- 7.68 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 28.6, 29.0, 36.4, 72.7, 79.6, 115.0, 119.6, 122.8, 128.8, 129.5, 131.6, 136.0, 138.7, 139.1, 155.0, 159.2, 195.4; LRMS (ESI) m/z: 430.1 (M+H) ⁺ .

Example 10. (4-Chlorophenyl)(3-(piperidin-4-ylmethoxy)phenyl)methanone (26).

ie/t-Butyl 4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidine-l-carboxyla te (3.61 g, 8.50 mmol) was dissolved in 50% trifluoro acetic acid (TFA) in CH ₂ CI ₂ (35 mL) and stirred for 2h at r.t. All volatiles were evaporated in vacuo. The reaction mixture was dissolved in CH ₂ CI ₂ and washed with IN NaOH (twice). The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel

chromatography (4: 1, CHCl ₃ :MeOH) afforded 26 (2.66 g, 95%) as a color less oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.24-1.32 (m, 2H), 1.79 (d, J = 13 Hz, 2H), 1.86-2.10 (m, 1H), 2.79-2.64 (m, 2H), 3.11 (d, J = 12.5 Hz, 2H), 3.78 (d, J = 6.5 Hz, 2H), 7.05 (dd, J = 2, 8 Hz, 1H), 7.20-7.23 (m, 2H), 7.30 (t, J = 8 Hz, 1H), 7.39 (d, J = 9 Hz, 2H), 7.69 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 29.2, 29.9, 31.1, 36.3, 46.2, 73.2, 115.1, 119.7, 122.8, 128.8, 129.5, 131.6, 136.1, 138.7, 139.1, 159.4, 195.5; LRMS (ESI) m/z: 330.1 (M+H) ⁺ .

Example 11. (5)-(4-Chlorophenyl)(3-((l-(2-hydroxyoctyl)piperidin-4- yl)methoxy)phenyl)methanone ((S)-ll).

To a stirred solution of 26 (100.0 mg, 0.31 mmol) in CH ₂ C1 ₂ was added A1(CH ₃ ) ₃ (0.2 M in CH ₂ C1 ₂ , 0.43 mmol) at 0 °C. After 15 min., (S)-l,2-epoxyoctane (29, 60.0 mg, 0.43 mmol) was added. After 4h at r.t., the reaction mixture was quenched with aq.

NaHC0 ₃ and extracted with CH ₂ CI ₂ . The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel chromatography (3:2,

CHCl ₃ :MeOH) afforded (S)-2 (103.0 mg, 73%) as a colorless oil. [a] ²⁰ _D = +13.9 (c 1.0 in CHC1 ₃ ); 1H NMR (300 MHz, CDC1 ₃ ): δ 0.90 (s, 3H), 1.31-1.49 (m, 11H), 1.84-1.99 (m, 4H), 2.28-2.39 (m, 3H), 2.87 (d, J = 10.2 Hz, 1H), 3.10 (d, J = 10.2 Hz, 1H), 3.69 (s, 1H), 3087 (d, J = 3.9 Hz, 2H), 7.14 (d, J = 7.2 Hz, 1H), 7.29-7.49 (m, 5H), 7.77 (dd, J = 2.1, 8.4 Hz, 2H); ¹³ C NMR (75 MHz, CDC1 ₃ ): δ 13.5, 22.0, 25.0, 28.5, 28.8, 28.9, 31.2, 34.5, 35.3, 51.3, 54.7, 64.0, 65.8, 72.3, 114.4, 118.8, 122.0, 128.1, 128.8, 130.8, 135.4, 138.1, 138.3, 158.7, 194.6; HRMS (ESI ⁺ ): m/z Calcd. for C ₂₇ H ₃₆ C1N0 ₃ (M+H) ⁺ : 458.2405; found: 458.2403. The purity of (5 11 was determined to be >99 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (S)-ll was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=21.0 min and (R)-enantiomer: ¾ = 26.7 min).

Example 12. (R)-(4-chlorophenyl)(3-((l-(2-hydroxyoctyl)piperidin-4- yl)methoxy)phenyl)methanone ((R)-ll).

Colorless oil. [a] ²⁰ _D = - 12.8 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.81 (t, J = 7 Hz, 3H), 1.18- 1.42 (m, 12H), 1.76- 1.78 (m, 3H), 1.90 (t, J = 11 Hz, 1H), 2.17- 2.28 (m, 3H), 2.77 (d, J = 11 Hz, 1H), 3.00 (d, J = 11.5 Hz, 1H), 3.58-3.62 (m, 1H), 3.78 (d, J = 6 Hz, 2H), 7.05 (dd, J = 2, 8 Hz, 1H), 7.19-7.23 (m, 2H), 7.30 (t, J = 8 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): 514.3, 14.3, 22.8, 22.9, 25.9, 29.3, 29.6, 29.7, 32.1, 32.1, 34.5, 35.3, 36.1, 52.0, 55.5, 64.7, 66.5, 73.0, 110.2, 115.1, 119.7, 122.8, 128.9, 129.6, 131.7, 136.2, 138.8, 139.1, 159.4, 195.5. HRMS (ESI ⁺ ): m/z Calcd. for C ₂₇ H ₃₆ C1N0 ₃ (M+H) ⁺ : 458.2404; found: 458.2403. The purity of (R)-ll was determined to be >99 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (R)-ll was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=21.0 min and (R)-enantiomer: ¾ = 26.7 min).

Example 13. (5)-l-(4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidin-l-yl) octan-2- yl carbamate ((5 12).

To a stirred solution of (5 11 (10 mg, 0.02 mmol) and DMAP (5.0 mg, 0.04 mmol) in CH ₂ C1 ₂ was added trimethylsilylisocyanate (TMSNCO) (7.0 mg, 0.06 mmol). After 4h at r. , the reaction mixture was quenched with aq. NaHC0 ₃ , and extracted with CH ₂ C1 ₂ . The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel chromatography (9: 1, CHCl ₃ :MeOH) afforded (S)-12 (7.0 mg, 63%) as a colorless oil. [a] ²⁰ _D = +9.4° (c 0.9 in CHC1 ₃ ); 1H NMR (300 MHz, CDC1 ₃ ): δ 0.89 (d, J = 1.2 Hz, 3H), 1.31- 1.58 (m, 12H), 1.90 (d, J = 10.8 Hz, 3H), 2.09 (m, 1H), 2.37-2.44 (m, 3H), 2.96 (m, 1H), 3.15 (m, 1Η)3.76-3.89 (m, 3H), 7.13-7.16 (m, 1H), 7.28-7.50 (m, 5H), 7.78(dd, J = 1.8, 8.4 Hz, 2H); ¹³ C NMR (75 MHz, CDC1 ₃ ): δ 13.5, 22.0, 25.0, 28.1, 28.4, 28.9, 29.1, 31.2, 34.5, 35.1, 51.5, 54.7, 64.1, 65.7, 72.1, 114.5, 118.8, 122.1, 128.1, 128.8, 130.8, 135.4, 138.1, 138.4, 158.6, 194.6; MS (ESI ⁺ ): m/z Calculated for (M+H) ⁺ : 501.2400; found: 501.2309. The purity of (S)-12 was determined to be >99% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (S)-12 was determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=18.0 min and (R)-enantiomer: ¾ = 20.0 min).

Example 14. (R)-l-(4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidin-l-yl) octan-2- yl carbamate ((R)-12).

A colorless oil. [a] ²⁰ _D = +-8.6 (c 0.8 in CHC1 ₃ ); 1H NMR (300 MHz, CDC1 ₃ ): δ 0.88-0.97 (m, 3H), 1.28- 1.49 (m, 13H), 1.93 (d, J = 10.8 Hz, 3H), 2.21 (s, 2H), 2.49 (s, 2H), 3.10 (s, 1H), 3.27 (s, 1H), 3.90 (d, J = 5.4 Hz, 2H), 7.13-7.15 (m, 1H), 7.25-7.50 (m, 5H), 7.76-7.79 (m, 2H); ¹³ C NMR (75 MHz, CDC1 ₃ ): δ 13.5, 22.0, 23.3, 24.9, 27.7, 28.8, 29.1, 31.2, 34.5, 34.9, 51.7, 54.7, 64.1, 65.6, 71.9, 114.5, 118.8, 122.1, 128.1, 128.8, 130.8, 138.2, 138.4, 158.5, 194.7; HRMS (ESI ⁺ ): m/z Calculated for C ₂₈ H ₃₇ C1N ₂ 0 ₄ (M+H) ⁺ : 501.2400; found: 501.2309. The purity of (R)-12 was determined to be >99% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm;

CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-12 was determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=18.0 min and (R)-enantiomer: ¾ = 20.0 min).

Example 15. l-(3-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)octan-l-on e (44).

To a stirred solution of ieri-butyl-(3-iodo-benzyloxy)-dimethylsilane (42, 0.70 g, 2.0 mmol) in THF (3 mL) was added isopropylmagnesium chloride (2M in THF, 3.0 mmol) at 0 °C. After 2.5h, the reaction mixture was cooled to -78 °C and N-methoxy-N- methyloctanamide (43, 0.12 g, 0.66 mmol) in THF (1 mL) was added. After 3h at 0 °C, the reaction mixture was quenched with aq. NH ₄ C1. The water phase was extracted with CH ₂ CI ₂ . The combined organic phase was dried over Na ₂ S04 and evaporated in vacuo. Purification by silica gel chromatography (9.8:0.2, Hexanes:EtOAc) afforded 44 (0.21 g, 91%) as a colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 0.11 (s, 6H), 0.88 (t, J = 7.5 Hz, 3H), 0.96 (s, 9H), 1.29- 1.39 (m, 8H), 1.73 (d, J = 7 Hz, 2H), 2.95 (t, J = 7 Hz, 2H), 4.79 (s, 2H), 7.42 (t, J = 7 Hz, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.84 (d, J = 8 Hz, 1H), 7.91 (s, 1H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ -5.3, 14.1, 18.4, 22.6, 24.5, 25.9, 29.1, 29.4, 31.7, 38.7, 64.5, 125.6, 126.7, 128.5, 130.4, 137.1, 142.0, 200.7; LRMS (ESI) m/z: 336.2 (M+H) ⁺ .

Example 15. (R)-l-(3-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)octan- l-ol.

To a stirred solution of 44 (0.2 g, 0.57 mmol) in THF (2 mL) at -78 °C was added (S)-2-methyl-CBS (0.12 g, 0.43 mmol) and BH ₃ -Me ₂ S (43.0 mg, 0.57 mmol). The reaction mixture was kept at - 15 °C for an additional 1.5h and quenched with MeOH followed by water. The water phase was extracted with EtOAc. The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel chromatography (9: 1, Hexanes:EtOAc) afforded (R)- l-(3-(((tert- butyldimethylsilyl)oxy)methyl)phenyl)octan- l-ol (180.0 mg, 89%) as a colorless oil. [a] ²⁰ _D = +35.1 (c 1 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.10 (s, 6H), 0.87 (t, J = 6.5 Hz, 3H), 0.94 (s, 9H), 1.25- 1.31 (m, 9H), 1.37-1.42 (m, 1H), 1.67- 1.83 (m, 3H), 4.66 (t, J = 6.5 Hz, 1H), 4.74 (s, 3H), 7.21-7.26 (m, 2H), 7.29-7.32 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ -5.2, 14.1, 18.4, 22.6, 25.8, 26.0, 29.2, 29.5, 31.8, 39.1, 65.0, 74.8, 123.6, 124.5, 125.3, 128.3, 141.7, 144.9; LRMS (ESI) m/z: 338.2 (M+H) ⁺ .

Example 16. (5)-l-(3-(((tert-Butyldimethylsilyl)oxy)methyl)phenyl)octan- l-ol.

Colorless oil. [a] ²⁰ _D = -34.8 (c 1 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.10

(s, 6H), 0.87 (t, J = 6.5 Hz, 3H), 0.94 (s, 9H), 1.25- 1.31 (m, 9H), 1.37- 1.42 (m, 1H), 1.67- 1.83 (m, 3H), 4.66 (t, J = 6.5 Hz, 1H), 4.74 (s, 3H), 7.21-7.26 (m, 2H), 7.29-7.32 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ -5.2, 14.1, 18.4, 22.6, 25.8, 26.0, 29.2, 29.5, 31.8, 39.1, 65.0, 74.8, 123.6, 124.5, 125.3, 128.3, 141.7, 144.9; LRMS (ESI) m/z: 338.2 (M+H) ⁺ .

Example 17. (R)-l-(3-(Hydroxymethyl)phenyl)octyl acetate. (R)- l-(3-(((ieri-butyldimethylsilyl)oxy)methyl)phenyl)octan- l-ol (70 mg, 0.2 mmol) was dissolved in pyridine (1 mL) and acetic anhydride (1 mL). After 5h at r.t., all volatiles were evaporated in vaccuo. Purification by silica gel chromatography (9.5:0.5, Hexanes:EtOAc) afforded (R)- l-(3-(((ieri-butyldimethylsilyl)oxy)methyl)phenyl)octyl acetate (78.4 mg, 100%) as a colorless oil. To a stirred solution of (R)- l-(3-(((tert- butyldimethylsilyl)oxy)methyl)phenyl)octyl acetate (25 mg, 0.06 mmol) in THF (0.5 mL) was added TBAF (1M in THF, 0.12 mmol). After 4h at r.t., the reaction was quenched with water. The water phase was extracted with EtOAc, and the combined extract was washed with brine, dried over Na ₂ S0 ₄ , and concentrated in vacuo. Purification by silica gel chromatography (7:3, Hexane:EtOAc) afforded (R)- l-(3-

20

(hydro xymethyl)phenyl)octyl acetate (14.0 mg, 79%) as a colorless oil. [a] D = +38.0 (c 0.7 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, J = 7 Hz, 3H), 1.20- 1.34 (m, 10H), 1.73- 1.79 (m, 1H), 1.86- 1.93 (m, 1H), 2.06 (s, 3H), 4.70 (s, 2H), 5.71 (t, J = 7 Hz, 1H), 7.24-7.29 (m, 2H), 7.32-7.35 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 21.3, 22.6, 25.6, 29.1, 29.3, 31.8, 36.3, 65.3, 76.2, 125.1, 125.8, 126.4, 128.7, 141.1, 141.3, 170.5; LRMS (ESI) m/z: 279.2 (M+H) ⁺ .

Example 18. (S)-l-(3-(Hydroxymethyl)phenyl)octyl acetate.

Colorless oil. [a] ²⁰ _D = -35.0 (c 0.8 in CHCI ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, J = 7 Hz, 3H), 1.20- 1.34 (m, 10H), 1.73- 1.79 (m, 1H), 1.86- 1.93 (m, 1H), 2.06 (s, 3H), 4.70 (s, 2H), 5.71 (t, J = 7 Hz, 1H), 7.24-7.29 (m, 2H), 7.32-7.35 (m, 2H); ¹³ C NMR (125 MHz, CDCI ₃ ): δ 14.1, 21.3, 22.6, 25.6, 29.1, 29.3, 31.8, 36.3, 65.3, 76.2, 125.1, 125.8, 126.4, 128.7, 141.1, 141.3, 170.5; LRMS (ESI) m/z: 279.2 (M+H) ⁺ .

Example 19. (R)-l-(3-((4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidin-l - yl)methyl)phenyl)octyl acetate ((R)-46).

To a stirred solution of DMSO (113.0 mg, 1.44 mmol) in CH ₂ C1 ₂ (2 mL) at -78 °C was added oxalyl chloride (91.0 mg, 0.72 mmol). After 30 min., (R)- l-(3- (hydroxymethyl)phenyl)octyl acetate (100.0 mg, 0.36 mmol) in CH ₂ C1 ₂ (0.5 mL) was added. After 30 min. at -78 °C, Et ₃ N (220.0 mg, 2.16 mmol) was added and the reaction mixture was warmed to r.t. over lh. The reaction mixture was quenched with water and the water phase was extracted with CH ₂ C1 ₂ . The combined extract was washed with brine, dried over Na ₂ S ₂ 0 ₄ , and evaporated in vacuo. Purification by silica gel chromatography afforded the corresponding aldehyde (R)- l-(3-formylphenyl)octyl acetate ((R)-45, 95.0 mg) as a colorless oil. To a stirred solution of (R)-45 (95.0 mg, 0.34 mmol) and (4-chlorophenyl)(3-(piperidin-4-ylmethoxy)phenyl)methanone (26, 98.7 mg, 0.30 mmol) in CH ₂ C1 ₂ was added NaBH(OAc) ₃ (150.0 mg, 0.72 mmol). After 8h at r. , the reaction was quenched with aq. sat. NaHC0 ₃ and the water phase was extracted with CH ₂ C1 ₂ . The combined extract was washed with brine, dried over Na ₂ S ₂ 0 ₄ , and evaporated in vacuo. Purification by silica gel chromatography (3:7, Hexanes:EtOAc) afforded (R)-46. (170.0 mg, 96%) as a colorless oil. [a] ²⁰ _D = - 17.9 (c 0.8 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.86 (t, J = 7 Hz, 3H), 1.23- 1.29 (m, 10H), 1.43- 1.45 (m, 2H), 1.73- 1.91 (m, 5H), 1.99-2.04 (m, 2H), 2.07 (s, 3H), 2.93 (d, J = 10 Hz, 2H), 3.53 (d, J = 1.5 Hz, 2H), 3.85 (d, J = 6 Hz, 2H), 5.72 (t, J = 7 Hz, 1H), 7.12 (dd, J = 1.5, 7.5 Hz, 1H), 7.21-7.30 (m, 6H), 7.36 (t, J = 8 Hz, 1H), 7.45 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 21.4, 22.6, 25.6, 29.0, 31.8, 35.9, 36.4, 53.3, 53.3, 63.2, 72.9, 76.2, 114.9, 119.5, 122.5, 125.1, 127.3, 128.3, 128.6, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 140.8, 159.2, 170.4, 195.3; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₆ H ₄₄ C1N0 ₄ (M+H) ⁺ : 590.3010; found: 590.3015.

Example 20. (R)-(4-chlorophenyl)(3-((l-(3-(l-hydroxyoctyl)benzyl)piperid in-4- yl)methoxy)phenyl) methanone ((R)-3).

To a stirred solution of (R)-46 (20.0 mg, 0.03 mmol) in acetonitrile (1 mL) was added IN NaOH (1 mL). After 2h at r.t., the reaction mixture was quenched with water and extracted with CHC1 ₃ . The combined organic phase was washed with brine, Na ₂ S ₂ 0 ₄ , and evaporated in vacuo. Purification by silica gel chromatography (1 :3,

Hexanes:EtOAc) afforded (R)-3 (10.0 mg, 54%) as a colorless oil. [a] ²⁰ _D = +25.8 (c 0.8 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.86 (t, J = 7 Hz, 3H), 1.21- 1.28 (m, 10H), 1.40- 1.47 (m, 3H), 1.68- 1.82 (m, 5H), 2.02 (t, J = 12 Hz, 2H), 2.94 (d, J = 11 Hz, 2H), 3.53 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.66 (t, J = 7 Hz, 1H), 7.12 (dd, J = 2.5, 8 Hz, 1H), 7.22-7.31 (m, 6H), 7.36 (t, J = 8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 29.0, 29.2, 29.5, 31.8, 35.9, 39.2, 45.6, 53.3, 53.3, 63.3, 72.9, 74.7, 114.9, 119.5, 122.6, 124.6, 126.8, 128.3, 128.4, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 145.0, 159.2, 195.3. HRMS (ESI ⁺ ): m/z Calcd. for C ₃₄ H ₄₂ C1N0 ₃ (M+H) ⁺ : 548.2906; found: 548.2909. The purity of (R)-3 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-3 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=15.0 min and (R)-enantiomer: ¾ = 16.0 min).

Example 21. (5)-(4-chlorophenyl)(3-((l-(3-(l-hydroxyoctyl)benzyl)piperid in-4- yl)methoxy)phenyl) methanone ((R)-3).

Colorless oil, [a] ²⁰ _D = -25.6 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 1H NMR (500 MHz, CDC1 ₃ ): δ 0.86 (t, J = 7 Hz, 3H), 1.21- 1.28 (m, 10H), 1.40- 1.47 (m, 3H), 1.68- 1.82 (m, 5H), 2.02 (t, J = 12 Hz, 2H), 2.94 (d, J = 11 Hz, 2H), 3.53 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.66 (t, J = 7 Hz, 1H), 7.12 (dd, J = 2.5, 8 Hz, 1H), 7.22-7.31 (m, 6H), 7.36 (t, J = 8 Hz, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 29.0, 29.2, 29.5, 31.8, 35.9, 39.2, 45.6, 53.3, 53.3, 63.3, 72.9, 74.7, 114.9, 119.5, 122.6, 124.6, 126.8, 128.3, 128.4, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 145.0, 159.2, 195.3. HRMS (ESI ⁺ ): m/z Calcd. for C ₃₄ H ₄₂ C1N0 ₃ (M+H) ⁺ : 548.2907; found: 548.3001. The purity of (5 3 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm;

CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (5 3 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=15.0 min and (R)-enantiomer: ¾ = 16.0 min).

Example 22. (R)-l-(3-((4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidin-l - yl)methyl)phenyl)octyl carbamate ((R)-14).

To a stirred solution of (R)-3 (20.0 mg, 0.04 mmol) and DMAP (100.0 mg, 0.08 mmol) in CH ₂ C1 ₂ (0.5 mL) was added TMSNCO (100.0 mg, 0.09 mmol). After 4h at r.t., all volatiles were evaporated in vauo. Purification by silica gel chromatography (9: 1, CHCl ₃ :MeOH) afforded (R)-14. (17.3 mg, 80%) as a colorless oil. [a] ²⁰ _D = +21.1 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.85 (t, J = 7 Hz, 3H), 1.26 (m, 9H), 1.35- 1.46 (m, 3H), 1.59- 1.74 (m, 2H), 1.82 (d, J = 10.5 Hz, 3H), 2.02 (m, 2H), 2.94 (d, J = 8 Hz, 2H), 3.54 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.60 (q, J = 5 Hz, 1H), 7.11-7.12 (m, 1H), 7.18- 7.19 (m, 2H), 7.24-7.29 (m, 4H), 7.36 (t, J = 8.5 Hz, 1H), 7.46 (d, J = 8 Hz, 2H), 7.75 (d, J = 9 Hz, 2H); C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 26.0, 29.0, 29.3, 29.5, 31.9, 35.9, 40.7, 53.2, 72.9, 75.1, 114.9, 119.4, 122.5, 126.8, 128.0, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 159.2, 195.3; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H4 ₃ C1N ₂ 04 (M+H) ⁺ :

590.2901 ; found: 590.2898. The purity of (R)-14 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm;

CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-14 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=14.0 min and (R)-enantiomer: ¾ = 15.0 min).

Example 23. (5)-l-(3-((4-((3-(4-chlorobenzoyl)phenoxy)methyl)piperidin-l - yl)methyl)phenyl)octyl carbamate (5) 14.

Colorless oil. [a] ²⁰ _D = -21.9 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.85 (t, J = 7 Hz, 3H), 1.26 (m, 9H), 1.35- 1.46 (m, 3H), 1.59- 1.74 (m, 2H), 1.82 (d, J = 10.5 Hz, 3H), 2.02 (m, 2H), 2.94 (d, J = 8 Hz, 2H), 3.54 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.60 (q, J = 5 Hz, 1H), 7.11-7.12 (m, 1H), 7.18-7.19 (m, 2H), 7.24-7.29 (m, 4H), 7.36 (t, J = 8.5 Hz, 1H), 7.46 (d, J = 8 Hz, 2H), 7.75 (d, J = 9 Hz, 2H). ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 26.0, 29.0, 29.3, 29.5, 31.9, 35.9, 40.7, 53.2, 72.9, 75.1, 114.9, 119.4, 122.5, 126.8, 128.0, 128.6, 129.3, 131.4, 135.9, 138.5, 138.9, 159.2, 195.3; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₃ C1N ₂ 0 ₄ (M+H) ⁺ : 590.2901 ; found: 590.2899. The purity of (5 14 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (5 14 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (S)- enantiomer: ¾=14.0 min and (R)-enantiomer: ¾ = 15.0 min).

Example 24. (4-Chlorophenyl)(3-((3-vinylbenzyl)oxy)phenyl)methanone.

To a stirred solution of (4-chlorophenyl)(3-hydroxyphenyl)methanone (21, 26.0 mg, 1.13 mmol), (3-vinylphenyl)methanol (48, 230.0 mg, 1.7 mmol), TPP (0.45 g, 1.7 mmol) in THF (10 mL) was added DIAD (0.62 g, 1.7 mmol). After 4h at r. , all volatiles were evaporated in vacuo to afford the crude product. Purification by silica gel chromatography (4: 1, hexanes:EtOAc) provided (4-chlorophenyl)(3-((3- vinylbenzyl)oxy)phenyl)methanone (334.3 mg, 85%) as a yellow oil. 1H NMR (500 MHz, CDCI3): δ 5.12 (s, 3H), 5.28 (d, J = 10.5 Hz, 1H), 5.77 (d, J = 17.5 Hz, 1H), 6.73 (q, J = 11 Hz, 1H), 7.22 (dd, J = 2.5, 8.5 Hz, 1H), 7.31-7.46 (m, 8H), 7.71-7.73 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 69.9, 70.1, 70.4, 114.3, 114.4, 114.5, 114.5, 114.5, 114.6, 115.3, 115.6, 119.8, 120.0, 122.7, 123.0, 125.2, 125.5, 125.8, 126.1, 126.7, 127.0, 128.5, 128.8, 129.0, 129.4, 129.6, 131.3, 131.5, 135.8, 136.5, 136.5, 136.8, 138.1, 138.5, 138.9, 158.7, 195.1 ; LRMS (ESI) m/z: 349.1 (M+H) ⁺ .

Example 25. (4-Chlorophenyl)(3-((3-(oxiran-2-yl)benzyl)oxy)phenyl)methan one

(rac-49).

To a stirred solution of (4-chlorophenyl)(3-((3- vinylbenzyl)oxy)phenyl)methanone (250.0 mg, 0.70 mmol) in acetonitrile (60 mL) was added Na ₂ EDTA (0.09%, 8 mL), oxone (86.0 mg, 0.28 mmol), NaHC0 ₃ (0.056 g, 0.67 mmol), and trifhioroacetone (250.0 mg, 2.24 mmol) at 0 °C. After lh, an additional oxone (86.0 mg, 0.28 mmol) and NaHC0 ₃ (56.0 mg, 0.67 mmol) were added every lh; this process was repeated 8 times during which the reaction temperature was kept at 0 °C. After completion of the reaction all volatiles were evaporated in vacuo. The residue was dissolved in water and the water phase was extracted with CH ₂ C1 ₂ . The combined organic phase was dried over Na ₂ S0 ₄ and evaporated in vacuo. Purification by silica gel chromatography (9: 1, Hexane:EtOAc) afford rac-49 (148.0 mg, 74%) as a colorless oil. rac-49 was resolute via Jocobsen' s kinetic resolution with (S^-salene Co(III) or (R,R)- salene Co(III) to afford (R)-49 and (5 49, respectively. (R)-49. [a] ²⁰ _D = +11.3° (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 2.79 (dd, J = 2.5, 5.5 Hz, 1H), 3.15 (t, J = 4 Hz, 1H), 3.88 (t, J = 3 Hz, 1H), 5.10 (s, 2H), 7.21 (dd, J = 4.5, 8 Hz, 1H), 7.26-7.27 (m, 1H), 7.33-7.41 (m, 6H), 7.44 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 51.5, 52.4, 70.2, 115.6, 120.0, 123.1, 124.7, 125.5, 127.5, 128.8, 129.1, 129.7, 131.6, 136.0, 137.1, 138.5, 138.8, 139.1, 158.8, 195.3. LRMS (ESI) m/z: 365.1 (M+H) ⁺ . (S 49. Colorless oil. [a] ²⁰ _D = -9.3° (c 0.5 in CHC1 ₃ ).

Example 26. (R)-(4-Chlorophenyl)(3-((3-(2-hydroxy-l-((4- methoxyphenethyl)amino)ethyl)benzyl)oxy) phenyl)methanone ((R)-4).

A mixture of (R)-49 (700.0 mg, 0.2 mmol), /7-methoxyphenethyl amine (30.0 mg, 0.20 mmol) and Zn(C10 ₄ ) ₂ .6H ₂ 0 (10.0 mg) is heated at 90 °C for lh. Purification by silica gel chromatography (7:3, Hexanes:EtOAc) afforded (R)-4 (27.0 mg, 27%) as a colorless oil and the corresponding regiodisomer (15%). (R)-4. [a] D = +20.3 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 2.68-2.76 (m, 3H), 2.84-2.95 (m, 3H), 3.78 (s, 3H), 4.72 (dd, J = 3.5, 9.5 Hz, 1H), 5.09 (s, 2H), 6.83 (d, J = 8 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.20 (dd, J = 1.5, 8 Hz, 1H), 7.32-7.44 (m, 9H), 7.72 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 30.9, 35.2, 50.7, 55.3, 56.8, 70.2, 71.2, 114.0, 115.5, 119.8, 122.9, 124.9, 125.7, 126.7, 128.6, 128.7, 129.5, 129.6, 131.4, 135.8, 136.6, 138.5, 138.9, 143.0, 158.2, 158.7, 195.2; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₁ H ₃₀ CINO ₄ (M+H) ⁺ :

516.1905; found: 516.2101. The purity of (R)-4 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm;

CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-4 was determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=13.5 min and (R)-enantiomer: ¾ = 15.0 min).

Example 27. (S)-(4-Chlorophenyl)(3-((3-(2-hydroxy-l-((4- methoxyphenethyl)amino)ethyl)benzyl)oxy) phenyl)methanone ((5 4).

A colorless oil. [a] ²⁰ _D = - 19.5 (c 0.5 in CHCI ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 2.68-2.76 (m, 3H), 2.84-2.95 (m, 3H), 3.78 (s, 3H), 4.72 (dd, J = 3.5, 9.5 Hz, 1H), 5.09 (s, 2H), 6.83 (d, J = 8 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.20 (dd, J = 1.5, 8 Hz, 1H), 7.32-7.44 (m, 9H), 7.72 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 30.9, 35.2, 50.7, 55.3, 56.8, 70.2, 71.2, 114.0, 115.5, 119.8, 122.9, 124.9, 125.7, 126.7, 128.6, 128.7, 129.5, 129.6, 131.4, 135.8, 136.6, 138.5, 138.9, 143.0, 158.2, 158.7, 195.2; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₁ H ₃₀ CINO ₄ (M+H) ⁺ : 516.1895; found: 516.19051. The purity of (S)-4 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (5 4 was determined to be >99% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=13.5 min and (R)-enantiomer: ¾ = 15.0 min).

Example 28. tert-Butyl 4-((3-chloro-4-(4-chlorobenzoyl)phenoxy)methyl)piperidine-

1-carboxylate.

Colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.29 (m, 2H), 1.48 (s, 9H), 1.83 (d,

12.5 Hz, 2H), 1.98 (s, 1H), 2.76 (bs, 2H), 3.86 (d, J = 6.5 Hz, 2H), 4.18 (bs, 2H), 6.87 (dd, J = 1, 8.5 Hz, 1H), 6.97 (d, J = 1.5 Hz, 1H), 7.34 (d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 8.5 Hz); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 28.6, 28.9, 36.2, 72.9, 79.5, 113.2, 116.2, 128.9, 130.2, 131.4, 131.4, 133.3, 135.8, 139.8, 154.9, 161.3, 193.7; LRMS (ESI) nVz: 465.4 (M+H) ⁺ .

Example 29. (2-Chloro-4-(piperidin-4-ylmethoxy)phenyl)(4- chlorophenyl)methanone (50).

Colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.38-1.29 (m, 2H), 1.84 (s, 2H), 2.01- 1.95 (m, 1H), 2.70 (m, 2H), 3.18 (d, J = 12 Hz, 2H), 3.87 (d, J = 6.5 Hz, 2H), 6.89 (dd, J = 2, 8.5 Hz, 1H), 6.99 (d, J = 2.5 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.45 (d, J = 8.5 Hz, 2H), 7.75 (d, J = 8.5 Hz, 2H). ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 30.2, 36.5, 46.4, 73.6, 113.3, 116.3, 129.0, 130.2, 131.5, 131.6, 133.5, 136.0, 140.0, 161.6, 193.9. LRMS (ESI) m/z: 364.3 (M+H) ⁺ .

Example 30. (R)-(2-chloro-4-((l-(2-hydroxyoctyl)piperidin-4-yl)methoxy)p henyl)(4- chlorophenyl)methanone ((R)-16).

Colorless oil. [a] ²⁰ _D = +28.6 (c 1.0 in CHC1 ₃ ); 1H NMR (300 MHz, CDC1 ₃ ): δ

0.90 (bs, 3H), 1.31- 1.56 (m, 12H), 1.88 (d, J = 10.5 Hz, 3H), 2.08 (bs, 1H), 2.36-2.44 (m, 3H), 2.94-3.18 (m, 2H), 3.75 (s, 1H), 3.88 (d, J = 4.5 Hz, 2H), 6.87-6.90 (m, 1H), 6.99 (t, J = 2.1 Hz, 1H), 7.34-7.46 (m, 3H), 7.73-7.76 (m, 2H); ¹³ C NMR (75 MHz, CDC1 ₃ ): δ 8.3, 13.5, 22.0, 25.0, 28.1, 28.4, 28.9, 31.2, 34.5, 35.1, 45.4, 51.4, 54.6, 64.0, 65.7, 72.4, 112.5, 115.7, 128.3, 129.7, 130.7, 130.8, 132.7, 135.2, 139.2, 160.7, 194.0; MS (ESI ⁺ ): m/z Calcd. for C ₂₇ H ₃₅ C1 ₂ N0 ₃ (M+H) ⁺ : 492.2063; found: 492.2068. The purity of (R)-16 was determined to be >95 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (R)-16 was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=16.5 min and (R)-enantiomer: ¾ = 17.5 min).

Example 31. (R)-l-(4-((3-chloro-4-(4-chlorobenzoyl)phenoxy)methyl)piperi din-l- yl)octan-2-yl carbamate ((R)-17).

Colorless oil. [a] ²⁰ _D = +21.3 (c 1.0 in CHC1 ₃ ); 1H NMR (300 MHz, CDC1 ₃ ): δ 0.9 (bs, 3H), 1.31- 1.50 (m, 12H), 1.85-2.00 (m, 4H), 2.29-2.40 (m, 3H), 2.88 (d, J = 10.8 Hz, 1H), 3.11 (d, J = 10.8 Hz, 1H), 3.70 (s, 1H), 3.87 (d, J = 5.7 Hz, 2H), 6.87-6.99 (m, 2H), 7.28-7.46 (m, 3H), 7.73-7.76 (m, 2H); C NMR (75 MHz, CDC1 ₃ ): 513.5, 22.0, 25.0, 28.4, 28.7, 28.9, 31.3, 34.5, 35.2, 51.3, 54.6, 64.0, 65.8, 72.5, 112.6, 115.7, 128.3, 129.7, 130.7, 130.8, 132.7, 135.3, 139.2, 160.8, 193.0; HRMS (ESI+): m/z Calcd. for

C ₂₈ H ₃₆ Cl ₂ N ₂ 04 (M+H) ⁺ : 535.2106; found: 535.2109. The purity of (R)-17 was determined to be >95 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (R)-17 was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (S)- enantiomer: ¾=18.0 min and (R)-enantiomer: ¾ = 19.5 min).

Example 32. (5)-(2-Chloro-4-((l-(3-(l-hydroxyoctyl)benzyl)piperidin-4- yl)methoxy)phenyl)(4-chlorophenyl) methanone ((5)-6).

Colorless oil. [a] ²⁰ _D = -31.7 (c 0.2 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, J = 6.5 Hz, 3H), 1.25- 1.50 (m, 12H), 1.68- 1.86 (m, 5H), 2.05 (t, J = 11.5 Hz, 3H), 2.97 (d, J = 11.5 Hz, 2H), 3.56 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.67 (t, J = 7 Hz, 1H), 6.86 (dd, J = 2, 8.5 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 7.24- 7.26 (m, 2H), 7.29-7.35 (m, 3H), 7.42 (d, J = 8.5 Hz, 2H), 7.73 (d, J = 9 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 28.8, 29.3, 29.5, 31.8, 35.7, 39.2, 53.2, 53.2, 63.3, 73.1, 76.6, 113.1, 116.1, 124.7, 126.8, 128.4, 128.4, 128.8, 030.0, 131.3, 131.4, 133.2, 135.8, 139.7, 145.0, 161.4, 193.7; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₄ H ₄ iCl ₂ N0 ₃ (M+H) ⁺ : 582.2504; found: 582.2302. The purity of (5 6 was determined to be >95 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm;

CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (5 6 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=14.5 min and (R)-enantiomer: ¾ = 15.5 min).

Example 33. (R)-(2-Chloro-4-((l-(3-(l-hydroxyoctyl)benzyl)piperidin-4- yl)methoxy)phenyl)(4-chlorophenyl) methanone ((R)-6).

Colorless oil. [a] ²⁰ _D = +32.5 (c 0.2 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, / = 6.5 Hz, 3H), 1.25- 1.50 (m, 12H), 1.68-1.86 (m, 5H), 2.05 (t, / = 11.5 Hz, 3H), 2.97 (d, J = 11.5 Hz, 2H), 3.56 (s, 2H), 3.85 (d, J = 6 Hz, 2H), 4.67 (t, J = 7 Hz, 1H), 6.86 (dd, J = 2, 8.5 Hz, 1H), 6.96 (d, J = 2.5 Hz, 1H), 7.24-7.26 (m, 2H), 7.29-7.35 (m, 3H), 7.42 (d, J = 8.5 Hz, 2H), 7.73 (d, / = 9 Hz, 2H); C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 28.8, 29.3, 29.5, 31.8, 35.7, 39.2, 53.2, 53.2, 63.3, 73.1, 76.6, 113.1, 116.1, 124.7, 126.8, 128.4, 128.4, 128.8, 030.0, 131.3, 131.4, 133.2, 135.8, 139.7, 145.0, 161.4, 193.7; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₄ H ₄ iCl ₂ N0 ₃ (M+H) ⁺ : 582.2508; found: 582.2509. The purity of (R)-6 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-6 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=14.5 min and (R)-enantiomer: ¾ = 15.5 min).

Example 34. (5)-l-(3-((4-((3-chloro-4-(4-chlorobenzoyl)phenoxy)methyl)pi peridin- l-yl)methyl)phenyl)octyl carbamate ((5) 19).

Colorless oil. [a] ²⁰ _D = -26.6 (c 0.2 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, / = 6.5 Hz, 3H), 1.29- 1.42 (m, 13H), 1.68- 1.88 (m, 6H), 2.17 (bs, 2H), 3.08-3.11 (m, 1H), 3.67 (bs, 1H), 3.87 (d, / = 6 Hz, 2H), 4.68 (q, / = 1.5 Hz, 1H), 6.85 (dd, / = 2.5, 9 Hz, 1H), 6.96 (d, / = 2.5 Hz, 1H), 7.28-7.43 (m, 7H), 7.71-7.74 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 16.6, 20.0, 22.6, 24.5, 25.9, 29.5, 31.8, 39.3, 113.0, 116.2, 128.9, 131.3, 131.4, 133.2, 135.6, 139.8, 159.7, 192.4, 193.7; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₂ C1 ₂ N ₂ 0 ₄ (M+H) ⁺ : 625.2508; found:

625.2510. The purity of (S)-19 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (S)-19 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=12.5 min and (R)-enantiomer: ¾ = 14.0 min).

Example 35. (R)-l-(3-((4-((3-chloro-4-(4-chlorobenzoyl)phenoxy)methyl)pi peridin- l-yl)methyl)phenyl)octyl carbamate ((R)-19).

Colorless oil. [a] ²⁰ _D +26.3 (c 0.2 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, / = 6.5 Hz, 3H), 1.29- 1.42 (m, 13H), 1.68- 1.88 (m, 6H), 2.17 (bs, 2H), 3.08-3.11 (m, 1H), 3.67 (bs, 1H), 3.87 (d, / = 6 Hz, 2H), 4.68 (q, / = 1.5 Hz, 1H), 6.85 (dd, / = 2.5, 9 Hz, 1H), 6.96 (d, / = 2.5 Hz, 1H), 7.28-7.43 (m, 7H), 7.71-7.74 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 16.6, 20.0, 22.6, 24.5, 25.9, 29.5, 31.8, 39.3, 113.0, 116.2, 128.9, 131.3, 131.4, 133.2, 135.6, 139.8, 159.7, 192.4, 193.7; MS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₂ CI ₂ N ₂ O ₄ (M+H) ⁺ : 625.2508; found: 625.2506. The purity of (R)-19 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-19 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (S)- enantiomer: ¾=12.5 min and (R)-enantiomer: ¾ = 14.0 min).

Example 36. (4-Chlorophenyl)(3-hydroxyphenyl)methanone O-methyl oxime (22).

To a stirred solution of 21 (800.0 mg, 3.5 mmol) in pyridine (30 mL) was added O-methylhydroxylamine hydrochloride (880 mg, 10.5 mmol). The reaction mixture was heated to 105 °C for 16h. All volatiles were evaporated in vacuo. Purification by silica gel chromatography (7:3, Hexanes:EtOAc) afforded 22 (780.0 mg, 86%) as a colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 3.96 (s, 3H), 6.77-6.86 (m, 2H), 6.92-6.96 (m, 1H), 7.24-7.28 (m, 3H), 7.37-7.42 (m, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 62.5, 62.5, 114.5, 116.1, 116.3, 116.8, 120.5, 121.3, 128.5, 128.5, 129.1, 129.6, 129.6, 130.8, 131.4, 134.2, 134.6, 135.0, 135.5, 137.3, 155.6, 155.6, 155.8, 155.8; LRMS (ESI) m/z:

262.026(M+H) ⁺ .

Example 37. (2-Chloro-4-hydroxyphenyl)(4-chlorophenyl)methanone O-methyl oxime (24).

To a stirred solution of 23 (500.0 mg, 1.87 mmol) in pyridine (15 mL) was added

O-methylhydroxylamine hydrochloride (470.0 mg, 5.60 mmol). The reaction mixture was heated to 105 °C for 16h. All volatiles were evaporated in vacuo. Purification by silica gel chromatography (7:3, Hexanes:EtOAc) afforded 24 (520.0 mg, 93%) as a colorless oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 3.99 (s, 3H), 6.80 (dd, J = 2.5, 8.5 Hz, 1H), 6.95 (d, J = 2 Hz, 1H), 7.03 (d, J = 8Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 62.7, 114.4, 116.8, 124.8, 128.2, 128.7, 130.7, 133.2, 133.6, 135.5, 153.4, 156.7; LRMS (ESI) m/z: 296.02(M+H) ⁺ .

Example 38. tert-Butyl 4-((3-((4- chlorophenyl)(methoxyimino)methyl)phenoxy)methyl)piperidine- l-carboxylate.

To a stirred solution of 22 (0.93 g, 3.59 mmol), iert-butyl 4-

(hydroxymethyl)piperidine-l-carboxylate (25, 1.55 g, 7.18 mmol), and TPP (1.41 g, 5.38 mmol) in THF (8 mL) was added DIAD (1.98 g, 5.38 mmol). After 4h at r.t., all volatiles were evaporated in vauo. Purification by silica gel chromatography (9: 1,

Hexanes:EtOAc) provided ie/t-butyl 4-((3-((4- chlorophenyl)(methoxyimino)methyl)phenoxy)methyl)piperidine- l-carboxylate (1.28 g, 78%; a mixture of isomers) as a yellow color liquid. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.22- 1.31 (m, 2H), 1.46 (s, 9H), 1.81 (d, J = 13 Hz, 2H), 1.93-1.95 (m, 1H), 2.74 (bs, 2H), 3.79 (dd, J = 2.5, 6.5 Hz, 2H), 3.98 (s, 3H), 4.15 (bs, 2H), 6.82-7.04 (m, 3H), 7.20-7.44 (m, 4H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 28.5, 28.9, 36.3, 62.5, 62.6, 72.3, 72.3, 79.4,

113.6, 115.0, 115.1, 120.6, 121.4, 128.4, 128.5, 129.0, 129.3, 129.4, 130.7, 131.5, 134.1, 134.7, 134.9, 135.4, 137.4, 154.9, 155.5, 158.8, 159.0; LRMS (ESI) m/z: 459.20(M+H) ⁺ .

Example 39. (4-Chlorophenyl)(3-(piperidin-4-ylmethoxy)phenyl)methanone O- methyl oxime (27).

ie/t-butyl 4-((3-((4- chlorophenyl)(methoxyimino)methyl)phenoxy)methyl)piperidine- 1-carboxylate (0.6 g, 1.3 mmol) was subjected to 50% TFA in CH ₂ C1 ₂ (7 mL). After lh at r.t., all volatiles were evaporated in vacuo. The residue was dissolved in CHC1 ₃ and washed with IN NaOH. The combined CHC1 ₃ was washed with brine, dried over Na ₂ S0 ₄ , and evaporated in vacuo. Purification by silica gel chromatography (4: 1, CHCl ₃ :MeOH) afforded 27 (0.56 g, 79%) as a yellow oil. 1H NMR (500 MHz, CDC1 ₃ ): δ 1.74(q, J = 12 Hz, 2H), 2.05 (t, J = 14 Hz, 3H), 2.95 (q, J = 11.5 Hz, 2H), 3.47 (d, J = 12.5 Hz, 2H), 3.83 (d, J = 5.5 Hz, 2H), 3.98 (s, 3H), 6.82-7.04 (m, 3H), 7.21-7.43 (m, 5H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 25.7, 34.4, 43.7, 62.6, 62.6, 71.2, 71.3, 113.4, 115.0, 115.1, 115.7, 121.0,

121.7, 128.5, 128.5, 129.0, 129.5, 130.7, 131.4, 134.2, 134.6, 134.9, 135.4, 137.5, 155.3, 155.3, 158.4, 158.6; LRMS (ESI) m/z: 459.14(M+H) ⁺ .

Example 40. (R)-l-(4-((3-((4-

Chlorophenyl)(methoxyimino)methyl)phenoxy)methyl)piperidi n-l-yl)octan-2-yl carbamate ((R)-13).

To a stirred solution of 27 (180 mg, 0.50 mmol) in CH ₂ C1 ₂ was added A1(CH ₃ ) ₃ (0.2 M in CH ₂ C1 ₂ , 1.50 mmol). After 15 min. at 0 °C, (R)-l,2-epoxyoctane ((R)-29, 220.0 mg, 1.75 mmol) in CH ₂ C1 ₂ (0.5 mL) was added. After 4h at 0 °C, the reaction mixture was quenched with aq. sat. NaHC0 ₃ . The water phase was extracted with CH ₂ C1 ₂ and the combined extract was dried over Na ₂ S0 ₄ , and evaporated in vacuo. Purification by silica gel chromatography (4: 1, CHCl ₃ :MeOH) afforded (R)-(4-chlorophenyl)(3-((l-(2- hydroxyoctyl)piperidin-4-yl)methoxy)phenyl)methanone O-methyl oxime ((R)-30, 195.5 mg, 80%) as a colorless oil. To a stirred solution of (R)-(4-chlorophenyl)(3-((l-(2- hydroxyoctyl)piperidin-4-yl)methoxy)phenyl)methanone O-methyl oxime ((R)-30, 40.0 mg, 0.08 mmol) and DMAP (33.0 mg, 0.27 mmol) in CH ₂ C1 ₂ (1 mL) was added

TMSNCO (30.0 mg, 0.27 mmol). After 4h at r.t, all volatiles were evaporated in vacuo. Purification by silica gel chromatography (9: 1, CHCl ₃ :MeOH) afforded (R)-13 (30.0 mg, 69%) as a colorless oil. [a] ²⁰ _D +105 (c 0.2 in CHC1 ₃ ); 1H NMR (400 MHz, CDC1 ₃ ): δ 0.87 (d, J = 6.8 Hz, 3H), 1.26- 1.53 (m, 11H), 1.86 (d, J = 11.6 Hz, 2H), 2.06 (t, J = 9.6 Hz, 1H), 2.39 (t, J = 11.6 Hz, 2H), 2.96 (d, J = 8.4 Hz, 1H), 3.16 (d, J = 10 Hz, 1H), 3.74-3.80 (m, 3H), 3.97 (s, 2H), 1.82-7.04 (m, 2H), 7.13-7.44 (m, 6H); ¹³ C NMR (100 MHz, CDC1 ₃ ): δ 14.1, 22.6, 25.6, 26.5, 28.6, 28.9, 29.2, 29.4, 31.8, 35.0, 35.7, 52.0, 55.3, 60.5, 62.5, 62.6, 64.5, 66.1, 72.3, 113.6, 115.0, 115.1, 115.6, 120.6, 121.3, 127.8, 128.4, 128.5, 129.0, 129.3, 129.4, 130.7, 131.5, 134.1, 134.7, 134.8, 135.3, 137.4, 155.5, 155.5, 158.8, 159.0. MS (ESI ⁺ ): m/z Calcd. for C ₂₉ H ₄ oClN ₃ 0 ₄ (M+H) ⁺ : 530.2706; found:

530.2801. The purity of (R)-13 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-13 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=l .0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=20.5 min and (R)-enantiomer: ¾ = 21.5 min).

Example 41. (R)-(4-chlorophenyl)(3-((l-(3-(l-hydroxyoctyl)benzyl)piperid in-4- yl)methoxy)phenyl)methanone O-methyl oxime ((R)-15).

Colorless oil. [a] ²⁰ _D +30.5 (c 0.2 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.86

(t, J = 6.5 Hz, 3H), 1.24- 1.43 (m, 12H), 1.67- 1.80 (m, 5H), 1.99 (t, J = 11.5 Hz, 2H), 2.91 (d, J = 11.5 Hz, 2H), 3.51 (s, 2H), 3.77 (d, J = 4 Hz, 2H), 3.97 (s, 3H), 4.65 (t, J = 7 Hz, 1H), 6.81-7.03 (m, 3H), 7.19-7.43 (m, 9H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 29.0, 29.3, 29.5, 31.8, 35.9, 39.2, 53.3, 53.4, 62.5, 62.6, 63.4, 72.7, 74.6, 113.7, 115.0, 115.1, 115.6, 120.5, 121.2, 124.6, 126.8, 128.3, 128.4, 128.4, 128.5, 129.0, 129.3, 129.3, 130.7, 131.5, 134.1, 134.7, 134.8, 135.3, 137.4, 138.4, 145.0, 155.5, 155.6, 159.0, 159.1 ; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₅ C1N ₂ 0 ₃ (M+H) ⁺ : 577.3105; found: 577.3106. The purity of (R)-15 was determined to be >95 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (R)-15 was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 20/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾ = 20.5 min and (R)-enantiomer: ¾ = 21.0 min)..

Example 42. (R)-l-(4-((3-chloro-4-((4- chlorophenyl)(methoxyimino)methyl)phenoxy)methyl)piperidin-l -yl)octan-2-yl carbamate ((R)-18).

Colorless oil. [a] ²⁰ _D +21.5 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 0.89

(t, J = 7 Hz, 3H), 1.29- 1.51 (m, 13H), 1.78- 1.85 (m, 3H), 1.95-2.08 (m, 1H), 2.23-2.36 (m, 3H), 2.92 (bs, 1H), 3.67-3.75 (1H), 3.82 (t, / = 6 Hz, 2H), 3.98 (s, 3H), 6.88 (dd, / = 1, 8 Hz, , 1H), 7.01 (d, / = 2 Hz, 1H), 7.07 (dd, J = 3, 9 Hz, 1H), 7.29 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 14.1, 22.6, 22.7, 25.6, 25.7, 29.0, 29.1, 29.3, 29.4, 29.5, 30.9, 31.8, 31.9, 35.0, 35.8, 36.1, 51.7, 54.3, 54.4, 55.2, 62.7, 64.5, 65.8, 66.3, 70.6, 72.8, 73.1, 113.5, 113.5, 115.4, 124.6, 124.7, 128.1, 128.6, 130.5, 130.5, 133.2, 133.2, 133.8, 133.8, 135.4, 153.1, 153.2, 159.9, 160.0; HRMS (ESI ⁺ ): m/z Calculated for C ₂₉ H ₃₉ C1 ₂ N ₃ 0 ₄ (M+H) ⁺ : 564.2308; found: 564.2310. The purity of (R)-18 was determined to be >95 by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05 TFA in H ₂ 0 = 25/1). The optical purity of (R)-18 was determined to be >99 by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 22/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (^-enantiomer: ¾=20.5 min and (R)-enantiomer: ¾ = 21.0 min).

Example 43. (5)-(2-Chloro-4-((l-(3-(l-hydroxyoctyl)benzyl)piperidin-4- yl)methoxy)phenyl)(4-chlorophenyl) methanone O-methyl oxime ((S)-20).

Colorless oil. [a] ²⁰ _D -30.5 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, / = 7 Hz, 3H), 1.25- 1.46 (m, 12H), 1.68- 1.74 (m, 1H), 1.81 (d, / = 10.5 Hz, 4H), 2.02 (t, / = 9.5 Hz, 2H), 2.94 (d, / = 10.5 Hz, 2H), 3.53 (s, 2H), 3.81 (d, / = 6 Hz, 2H), 3.97 (s, 3H), 4.67 (t, / = 6.5 Hz, 1H), 6.86 (dd, / = 2.5, 8.5 Hz, 1H), 7.00 (d, / = 2 Hz, 1H), 7.06 (d, / = 8.5 Hz, 1H), 7.23-7.32 (m, 6H), 7.41 (d, / = 8 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 29.0, 29.3, 29.5, 31.8, 35.8, 39.2, 45.9, 53.3, 53.3, 62.7, 63.4, 72.9, 74.7, 113.5, 115.4, 124.6, 126.8, 128.1, 128.3, 128.4, 128.6, 130.5, 133.2, 133.8, 135.8, 145.0, 153.2, 160.0; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₄ CI ₂ N ₂ O ₃ (M+H) ⁺ : 611.2712; found: 611.2717. The purity of (5 20 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (5 20 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 25/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (S)- enantiomer: ¾=20.0 min and (R)-enantiomer: ¾ = 21.5 min).

Example 44. (R)-(2-Chloro-4-((l-(3-(l-hydroxyoctyl)benzyl)piperidin-4- yl)methoxy)phenyl)(4-chlorophenyl) methanone O-methyl oxime ((R)-20).

Colorless oil. [a] ²⁰ _D +29.5 (c 0.5 in CHC1 ₃ ); 1H NMR (500 MHz, CDC1 ₃ ): δ 1H NMR (500 MHz, CDC1 ₃ ): δ 0.87 (t, J = 7 Hz, 3H), 1.25-1.46 (m, 12H), 1.68-1.74 (m, 1H), 1.81 (d, J = 10.5 Hz, 4H), 2.02 (t, J = 9.5 Hz, 2H), 2.94 (d, J = 10.5 Hz, 2H), 3.53 (s, 2H), 3.81 (d, J = 6 Hz, 2H), 3.97 (s, 3H), 4.67 (t, J= 6.5 Hz, 1H), 6.86 (dd, J = 2.5, 8.5 Hz, 1H), 7.00 (d, J = 2 Hz, 1H), 7.06 (d, J = 8.5 Hz, 1H), 7.23-7.32 (m, 6H), 7.41 (d, J = 8 Hz, 2H); ¹³ C NMR (125 MHz, CDC1 ₃ ): δ 14.1, 22.7, 25.9, 29.0, 29.3, 29.5, 31.8, 35.8, 39.2, 45.9, 53.3, 53.3, 62.7, 63.4, 72.9, 74.7, 113.5, 115.4, 124.6, 126.8, 128.1, 128.3, 128.4, 128.6, 130.5, 133.2, 133.8, 135.8, 145.0, 153.2, 160.0; HRMS (ESI ⁺ ): m/z Calcd. for C ₃₅ H ₄₄ Cl ₂ N ₂ 0 ₃ (M+H) ⁺ : 611.2712; found: 611.2717. The purity of (R)-20 was determined to be >95% by reverse HPLC analysis (Phenominex kinetex 2.6 μ C18 100A, 100 x 4.60 mm; CH ₃ CN/0.05%TFA in H ₂ 0 = 25/1). The optical purity of (R)-20 was determined to be 95% by HPLC analyses (Daicel Chiralcel OD-H (0.46 cm x 25 cm; Hexanes/tBuOH = 25/1 with flow rate=1.0 mL/min. and a UV detector at 245 nm; (S)- enantiomer: ¾=20.0 min and (R)-enantiomer: ¾ = 21.5 min).

Example 45. Experimental Procedures.

Preparation ofMenA containing Membrane Fraction.

M. tuberculosis was grown to mid- log phase in Difco Middlebrook 7H9 nutrient broth (enriched with OADC) and then the cells were harvested by centrifugation at 4 °C followed by washing with 0.9% saline solution (thrice) through centrifugation. The washed cell pellets were suspended in homogenization buffer (containing 50 mM MOPS of pH = 8, 0.25 M sucrose, 10 mM MgCl ₂ and 5 mM 2-Marcaptoethanol) and disrupted by probe sonication on ice (10 cycles of 60s on and 90s off). The resulting suspension was then centrifuged at 15,000 g for 15 min at 4 °C. The pellet was discarded and the supernatant was centrifuged again at 200,000 g at 4 °C for lh. The resulting supernatant contain the membrane bound Men A. M. smegmatis and S. aureus MenA containing membrane fractions were obtained by the same procedures. ⁴¹

MenA Enzyme Inhibitory Assay (IC50).

The substrate l,4-dihydroxy-2-napthanoic acid (DHNA) (500 μΜ; 20 μΙ_), MgCl ₂ (5 μΜ; 20 μΐ.); CHAPS (0.1%; 20 μΐ), Tris-buffer (pH = 8; 20 μΙ_), membrane fraction containing Men A (100 μΐ), inhibitors (0- 60 μg/mL of DMSO) and

fernesylpyrophosphate (200 μΜ, 40 μί) were mixed together and incubated for 2h at 37 °C. After the incubation, the reaction mixture was quenched with 0.1 M AcOH in MeOH and the reaction mixture was extracted with hexanes (thrice). The organic portions were then concentrated and diluted with MeOH (300 μί). From each set of enzymatic reaction mixture 20 μΐ was injected into the HPLC (CH ₃ CN:0.05%TFA in H ₂ 0 = 90: 10, UV: 325 nm, flow rate: 1.5 mL/min) and the area of the peak for DMMK was quantified to obtain the IC ₅₀ value for different inhibitor molecules.

Minimum Inhibitory Concentration Assays (MABA and LORA).

These were performed according to the published protocol. A preliminary screening was conducted at 12.5 μg/mL against M. tuberculosis H ₃₇ Rv (ATCC 27294), at 60 μg/mL against S. aureus Seattle 1945 (ATCC 25923), and at 125 mg/rnL against E. coli Seattle 1946 (ATCC 25922). Compounds demonstrated at least 90% inhibitions in the preliminary screen were retested at lower concentrations to determine the MIC.

E. coli Growth Inhibitory Assays under Anaerobic Conditions.

E. coli was grown with inhibitor molecule (5 μg/mL) under anaerobic conditions in the modified E. coli growth media (see Supporting Information), and the growth curve was monitored photometrically by reading the optical density at 600 nm. E. coli growth rescue studies were performed by supplementing VK ₂ (50 μΜ).

Oxygen Consumption Assays.

In sterile glass vial 1.5 mL of M. tuberculosis or M. Smegmatis (grown to 0.5 OD value) was placed. Inhibitor molecule (80 μί, total concentration ranged from 5-100 μg/mL) and 0.01% methylene blue (100 μί) was added into the culture solution. The culture vials were kept at 37 °C for 22h. The effective concentration of the inhibitor molecule at which the electron transport systems was determined by comparing the color intensity with the control vial (no inhibitor).

Determination oflCso in Vero and HepG2 cells.

Selected molecules were tested for cytotoxicity (IC ₅₀ ) in Vero and HepG2 cells at concentrations 10 times the MIC for M. tuberculosis f½Rv. After 72h of exposure of molecule to these cell lines, viability was assessed on the basis of cellular conversion of MTT into a formazan product. Selectivity index (SI = IC ₅₀ /MIC) was determines. SI >10 was considered to be significant in this program.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Incorporation by Reference

The entire contents of all patents, published patent applications and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

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