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Title:
ANTIMICROBIAL COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2013/086415
Kind Code:
A1
Abstract:
Provided herein is technology relating to antimicrobial compounds and particularly, but not exclusively, to analogs of rifalazil having increased inhibition of RNA polymerase and decreased induction of human cy-tochrome P450. The compounds have an increased affinity for a bacterial RNA polymerase (e.g., a MTB RNA polymerase) and a decreased affinity for a human pregnane X receptor. Thus, in some embodiments, the steric clash of A or R with residues in the binding pocket of the human pregnane X receptor reduces an affinity of the compound for the human pregnane X receptor. Consequently, in some embodiments the steric clash thus reduces the induction (e.g., an activity) of a cytochrome P450 and/or other related proteins.

Inventors:
SHOWALTER HOLLIS D (US)
GARCIA GEORGE A (US)
XU HAO (US)
ATWAL SUMANDEEP K (US)
NAWARATHNE IROSHA (US)
KIRCHHOFF PAUL D (US)
Application Number:
PCT/US2012/068570
Publication Date:
June 13, 2013
Filing Date:
December 07, 2012
Export Citation:
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Assignee:
UNIV MICHIGAN (US)
SHOWALTER HOLLIS D (US)
GARCIA GEORGE A (US)
XU HAO (US)
ATWAL SUMANDEEP K (US)
NAWARATHNE IROSHA (US)
KIRCHHOFF PAUL D (US)
International Classes:
C07D265/34
Foreign References:
US20050197333A12005-09-08
US20030105086A12003-06-05
US6566354B12003-05-20
US20090143373A12009-06-04
US20050261262A12005-11-24
Attorney, Agent or Firm:
CASIMIR, David A. (S.C.2275 Deming Way,,Suite 31, Middleton WI, US)
Download PDF:
Claims:
CLAIMS

WE CLAIM:

1. A compound having the formula:

wherein:

R is an independent chemical moiety that:

1) increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and

2) causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor? and

A is H- or A is an independent chemical moiety that:

1) increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and

2) causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor.

2. The compound of claim 1 wherein A is H- and R is Et2NCH2CH2-. compound of claim 1 wherein A is H- and R is

The com ound of claim 1 wherein A is H- and R is

The compound of claim 1 wherein A is

/-Bu

The compound of claim 1 wherein A is selected from the group consisting of H- and

The compound of claim 6 wherein R is selected from the group consisting of

and

and wherein n = 1-3 and wherein R' is selected from the group consisting of H, methyl, ethyl, i'-Pr, and i'-Bu.

8. The compound of claim 6 wherein R is selected from the group consisting of

, and

and wherein n is 1-3.

9. The compound of claim 6 wherein R is selected from the group consisting of

N R:<N, ,N O

R Ή .

, and ^

wherein R" is selected from the roup consistin of

and n = 1-3.

10. The compound of claim 1 wherein the steric clash reduces an affinity of the compound for the human pregnane X receptor. The compound of claim 1 wherein the steric clash reduces an induction of a cytochrome P450 and other related proteins.

The compound of claim 1 wherein the A or the R interacts with a sigma factor of the RNA polymerase.

The compound of claim 1 wherein the A or the R interacts with a beta region of the RNA polymerase.

The compound of claim 1 wherein the A or the R interacts with a beta prime region of the RNA polymerase.

The compound of claim 1 wherein the steric clash comprises an interaction of the A or the R with an amino acid at a position in the primary sequence selected from the group consisting of 237, 238, 239, 240, 241, 242, and 243.

The compound of claim 1 wherein the steric clash comprises an interaction of the A or the R with an amino acid in the human pregnane X receptor selected from the group consisting of Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243.

The compound of claim 1 wherein a first MIC90 of the compound against a Mycobacterium tuberculosis is less than a second MIC90 of rifampin against the Mycobacterium tuberculosis.

The compound of claim 1 wherein a MIC90 of the compound against a

Mycobacterium tuberculosis is approximately 0.02 - 0.08 micromolar.

19. The compound of claim 1 wherein a first concentration at which the compound inhibits a rif-resistant RNA polymerase is lower than a second concentration at which rifalazil inhibits the rif-resistant RNA polymerase.

20. The compound of claim 1 wherein a first concentration at which the

compound inhibits a rif-resistant RNA polymerase is approximately one-half to one-fifth of a second concentration at which rifalazil inhibits the rif- resistant RNA polymerase.

21. The compound of claim 1 wherein a first concentration at which the

compound inhibits a rif-resistant RNA polymerase is approximately one-fifth to one-fiftieth of a second concentration at which rifalazil inhibits the rif- resistant RNA polymerase.

22. The compound of claim 19 wherein the rif-resistant RNA polymerase is a Mycobacterium tuberculosis rif-resistant RNA polymerase.

23. The compound of claim 22 wherein the Mycobacterium tuberculosis ^ rif- resistant RNA polymerase comprises a S450L substitution.

24. The compound of claim 22 wherein the Mycobacterium tuberculosis ^ rif- resistant RNA polymerase comprises a D435V substitution.

25. A method of manufacturing a compound having the formula: comprising the steps of

a) dialkylating a 2-nitroresorcinal to produce a dibenzyl ether? and b) mono-debenzylating the dibenzyl ether to produce a nitrophenol havin the formula

wherein A or R are independent chemical moieties that:

1) increase an inhibiting interaction of the compound with an RNA

polymerase relative to rifalazil; and

2) cause a steric clash of the compound with a side chain in a bindin pocket of a human pregnane X receptor.

A compound having the formula:

obtainable by a method comprising the steps of

a) dialkylating a 2-nitroresorcinal to produce a dibenzyl ether? and b) mono-debenzylating the dibenzyl ether to produce a nitrophenol having the formula

wherein A or R are independent chemical moieties that:

1) increase an inhibiting interaction of the compound with an RNA

polymerase relative to rifalazil; and

2) cause a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor.

A compound having the formula:

for use as a medicament,

wherein A or R are independent chemical moieties that:

1) increase an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and

2) cause a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor.

28. A compound having the formula:

for use as a medicament for a treatment of a bacterial infection, wherein A or R are independent chemical moieties that:

l) increase an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and 2) cause a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor.

29. The compound of claim 28 wherein the bacterial infection is caused by a rif- resistant organism.

30. The compound of claim 28 for the treatment of tuberculosis.

31. The compound of claim 28 wherein the bacterial infection is an infection with Mycobacterium tuberculosis.

32. The compound of claim 28 wherein the bacterial infection is caused by a multidrug-resistant strain of a bacterium.

33. The compound of claim 28 wherein the bacterial infection is caused by an extensively drug-resistant bacterium.

34. A compound having the formula:

for use as a medicament for a treatment of a subject infected with HIV, wherein A or R are independent chemical moieties that: 1) increase an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and

2) cause a steric clash of the compound with a side chain in a bindin pocket of a human pregnane X receptor.

The compound of claim 34 wherein the subject has AIDS.

A method for treating a subject comprising:

1) selecting a subject in need of a treatment;

2) administering to the subject a compound having the formula:

wherein A or R are independent chemical moieties that:

1) increase an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil; and

2) cause a steric clash of the compound with a side chain in a bindin pocket of a human pregnane X receptor.

The method of claim 36 wherein the subject has tuberculosis.

The method of claim 36 wherein the subject is infected with HIV.

39. The method of claim 36 wherein the subject has AIDS. The method of claim 36 wherein the subject is infected with Mycobacterium tuberculosis.

The method of claim 36 wherein the subject is infected with a rif-resistant organism.

The compound of claim 1 wherein R is

and n is 2 or 4.

Description:
ANTIMICROBIAL COMPOUNDS

FIELD OF INVENTION

Provided herein is technology relating to antimicrobial compounds and particularly, but not exclusively, to analogs of rifalazil having an increased inhibition of RNA polymerase and a decreased induction of human cytochrome P450.

BACKGROUND

Tuberculosis (TB) is a contagious and deadly disease that has reached pandemic proportions. According to the World Health Organization (WHO), 8 to 10 million new cases of TB are diagnosed each year and 2 to 3 million people die from the disease each year. Consequently, the causative agent of TB, Mycobacterium tuberculosis, is a leading cause of adult deaths from infectious disease. A high proportion of these newly diagnosed cases and deaths occurs in HIV-positive people, and a significant number of AIDS deaths in Africa is attributable to TB infections. Global population growth is increasing the disease burden and, in turn, posing a continuing health and financial burden in various parts of the world, particularly in Asia and Africa.

TB is caused predominantly by Mycobacterium tuberculosis (MTB), an obligate aerobic bacillus that divides at an extremely slow rate. The chemical composition of its cell wall includes peptidoglycans and complex lipids, in particular mycolic acids, which are a significant determinant of its virulence. The unique structure of the cell wall of MTB allows it to lie dormant for many years as a latent infection, particularly as it can grow readily inside macrophages, hiding it from the host's immune system.

While TB has grown to be a pandemic, no new TB drugs have been

introduced into clinical use in the last four decades. Furthermore, the continuing rise in multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) has contributed to the dire need for new TB antibiotics. Drugs that are active against resistant forms of TB are less potent, more toxic, and need to be taken for an extended period of time (e.g., continuously for more than 18 months). Also, the recent emergence of virtually untreatable extensively drug-resistant TB (XDR-TB) poses a new threat to TB control worldwide. One particularly difficult problem is that the effective treatment of TB in persons co-infected with HIV is complicated by drug-drug interactions.

The rifamycins are the most commonly used drugs for TB, and semisynthetic derivatives have been reported that show improved antimycobacterial activities. These include rifampin (RMP, see Figure l), which is the cornerstone of current short-term TB treatment. Amongst newer derivatives, rifalazil (RLZ, see Figure l) has proved most interesting because of its potency and relative lack of toxicity in early rodent studies. RLZ is an exceedingly potent rifamycin derivative that is 16 - 256 times more potent than RMP and is particularly effective against many of the RMP-resistant strains of MTB. Several early studies involving MTB strains with various rpoB mutations clearly indicated that the mutations identified with RMP- resistant, rifapentine-resistant, and rifabutin-resistant strains retained sensitivity to RLZ. RLZ and its benzoxazinorifamycin analogs also have showed excellent activity against other organisms with RMP-resistant mutations, including

Streptococcus pyogenes, Chlamydia trachomatis, and Chlamydia pneumonia.

In mouse in vivo efficacy studies, RLZ has been shown to be more potent than

RMP, including having activity against some RMP-resistant strains. Longer term MTB studies in combination with other agents indicated the same level of cure could be achieved with a shorter duration of treatment (e.g., at least one-half of the time) with RLZ as compared to RMP. In PK studies, RLZ has shown a high volume of distribution and produced tissue levels in rats up to 200 times those in plasma. It displayed a very long half-life (60-100 hours) in human trials.

One drawback to using the rifamycins in TB treatments is their many drug- drug interactions. This effect appears to be minimized with RLZ as shown in rat and dog studies. These studies also showed RLZ not to be an inducer of hepatic cytochrome P450 (Cyp450). Unfortunately, in a series of phase I and phase II clinical trials, RLZ proved to be quite toxic, with most adverse effects associated with a flu-like syndrome and leucopenia even at lower dose levels. Hence, its development for TB indications has been suspended.

SUMMARY

Provided herein is technology relating to antimicrobial (e.g., anti- mycobacterial, anti-TB) compounds and particularly, but not exclusively, to analogs of rifalazil having increased inhibition of RNA polymerase (e.g., through an interaction with a sigma factor or, e.g., through an interaction with another subunit of the RNA polymerase). In addition, the compounds also demonstrate decreased induction of human cytochrome P450.

Accordingly, embodiments of the technology provide compounds having the formula

wherein R is an independent chemical moiety that increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil and causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor and wherein A is H— or is an independent chemical moiety that increases an inhibiting interaction of the compound with an RNA polymerase relative to rifalazil and causes a steric clash of the compound with a side chain in a binding pocket of a human pregnane X receptor. In some embodiments, A is H- and R is Et2NCH2CH2-, in some embodiments A is H- and R is

in some embodiments, A is H- and R is

and in some embodiments A is

and R is

In some embodiments, A is selected from the group consisting of H- and . In some embodiments R comprises

herein

R' is selected from the roup consisting of H, methyl, eth l, i ' -Pr, and i ' -Bu? R"

comprises , and/or

; and n = 1-3.

The compounds have an increased affinity for a bacterial RNA polymerase (e.g., a MTB RNA polymerase) and a decreased affinity for a human pregnane X receptor. Thus, in some embodiments, the steric clash of A or R with residues in the binding pocket of the human pregnane X receptor reduces an affinity of the compound for the human pregnane X receptor. Consequently, in some embodiments the steric clash thus reduces the induction (e.g., an activity) of a cytochrome P450 and/or other related proteins. In addition, some embodiments provide that the A moiety or the R moiety interacts with a sigma factor of the RNA polymerase. In some embodiments, the A or the R interacts with a beta region of the RNA polymerase and in some embodiments the A or the R interacts with a beta prime region of the RNA polymerase. The A or R group adds steric volume to the compound, and thus in some embodiments the steric clash comprises an interaction of the A or the R with an amino acid of the human pregnane X receptor at a position in the primary sequence selected from the group consisting of 237, 238, 239, 240, 241, 242, and 243. In some embodiments the steric clash comprises an interaction of the A or the R with an amino acid in the human pregnane X receptor selected from the group consisting of Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243 (see, e.g., UniProt accession number 075469 or PDB accession number lskx).

The compounds exhibit increased potency against Mycobacterium

tuberculosis when compared to conventional compounds. Accordingly, in some embodiments the compound has a MIC90 against Mycobacterium tuberculosis that is less than the MIC90 of rifampin against Mycobacterium tuberculosis. For instance, in some embodiments the MIC90 of the compounds encompassed by the present technology against a Mycobacterium tuberculosis is approximately 0.02 - 0.08 micromolar. Furthermore, in some embodiments the compound inhibits a rif- resistant RNA polymerase at a concentration that is lower than the concentration at which rifalazil inhibits the rif-resistant RNA polymerase. For instance, in some embodiments the concentration at which the compound inhibits a rif-resistant RNA polymerase is approximately one-half to one-fifth of the concentration at which rifalazil inhibits the rif-resistant RNA polymerase. In some embodiments the concentration at which the compound inhibits a rif-resistant RNA polymerase is approximately one-fifth to one-fiftieth of the concentration at which rifalazil inhibits the rif-resistant RNA polymerase.

In some embodiments, provided herein are compounds that are more effective than conventional compounds against drug-resistant bacteria, e.g., a drug-resistant Mycobacterium tuberculosis, e.g., having a mutant RNA polymerase. In some embodiments the compounds are effective against a rif-resistant RNA polymerase that is a Mycobacterium tuberculosis rif-resistant RNA polymerase. For instance, in some embodiments the compounds are effective against a Mycobacterium

tuberculosis rif-resistant RNA polymerase comprising a S450L substitution (e.g., a substitution of a leucine for the serine at position 450 of the native primary amino acid sequence). In some embodiments the compounds are effective against a

Mycobacterium tuberculosis rif-resistant RNA polymerase comprising a D435V substitution (e.g., a substitution of a valine for the aspartate at position 435 of the native primary amino acid sequence).

The technology provides methods of manufacturing antimicrobial compounds. In particular, provided herein are embodiments of technology providing methods of manufacturing a compound having the formula

comprising the steps of dialkylating a 2-nitroresorcinal to produce a dibenzyl ether and mono-debenzylating the dibenzyl ether to produce a nitrophenol having the formula

In some embodiments, the technology provides a compound having the formula

and which is obtainable by a method comprising the steps of dialkylating a 2- nitroresorcinal to produce a dibenzyl ether and mono-debenzylating the dibenzyl ether to produce a nitrophenol having the formula

In some embodiments the technology provides a compound having the formula

for use as a medicament. Furthermore, some embodiments provide a compound having the formula Me Me

Me

for use as a medicament for a treatment of a bacterial infection. The compounds find use in treating a wide range of bacterial infections (e.g., caused by different organisms and characterized by different clinical descriptions). For example, in some embodiments the bacterial infection is caused by a rif-resistant organism. In some embodiments, the compound finds use in treating tuberculosis. In some embodiments, the compound finds use in treating a bacterial infection that is an infection with Mycobacterium tuberculosis. In some embodiments, the bacterial infection is caused by a multidrug-resistant strain of a bacterium and in some embodiments the bacterial infection is caused by an extensively drug-resistant bacterium. The compounds find use in the treatment of subjects that have tuberculosis in combination with another disease or malady. For instance, in some embodiments the technology is related to compounds having the formula

for use as a medicament for a treatment of a subject infected with HIV, and in some embodiments the compound is used to treat a subject that has AIDS. Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the present technology will become better understood with regard to the following drawings:

Figure 1 shows the chemical structures of rifampin (RMP, Figure 1A) and related compounds including rifalazil (RZL, Figure IB).

Figure 2a shows a schematic of a synthesis known as Scheme 1 for producing embodiments of the compounds provided herein.

Figure 2b shows a schematic of a synthesis for producing embodiments of the compounds provided herein.

Figure 2c shows a schematic of a synthesis for producing embodiments of the compounds provided herein.

Figure 2d shows a schematic of a synthesis for producing embodiments of the compounds provided herein.

Figure 2e shows a schematic of a synthesis for producing embodiments of the compounds provided herein.

Figure 3 shows data plots for experiments testing the activation of hPXR by embodiments of the compounds provided herein.

Figure 4a shows plots of the linear plasma mean concentration versus time for analog 2b in single dose studies.

Figure 4b shows plots of the linear plasma mean concentration versus time for analog 2b in multiple dose studies.

Figure 5 shows a plot of the linear lung tissue mean concentration versus time for analog 2b in a multiple dose study. Figure 6 shows a schematic of a synthesis for producing embodiments of the compounds provided herein.

DETAILED DESCRIPTION

Provided herein is technology relating to antimicrobial compounds and particularly, but not exclusively, to analogs of rifalazil having increased inhibition of RNA polymerase and decreased induction of human cytochrome P450.

Definitions

To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.

Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrase "in one embodiment" as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase "in another

embodiment" as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined without departing from the scope or spirit of the invention.

In addition, as used herein, the term "or" is an inclusive "or" operator and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on additional factors not described unless the context clearly dictates otherwise. In addition, throughout the specification, the meaning of "a", "an", and "the" include plural references. The meaning of "in" includes "in" and "on."

As used herein, the term "effective amount" refers to the amount of a compound required to treat or prevent an infection. The effective amount of an active compound used for therapeutic or prophylactic treatment of conditions caused by or contributed to by a microbial infection varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

As used herein, the term "administration" or "administering" refers to a method of giving a composition to a patient, by a route such as inhalation, ocular administration, nasal instillation, parenteral administration, dermal

administration, transdermal administration, buccal administration, rectal administration, sublingual administration, perilingual administration, nasal administration, topical administration, and oral administration. Parenteral administration includes intrathecal, intraarticular, intravenous, intraperitoneal, subcutaneous, and intramuscular administration. The optimal method of administration of a drug or drug combination to treat a particular disease varies depending on various factors, e.g., the oral bioavailability of the drug(s), the anatomical location of the disease tissue, and the severity of disease.

As used herein, the term "co-administration" refers to the administration of at least two agents or therapies to a subject. In some embodiments, the co ¬ administration of two or more agents or therapies is concurrent. In other embodiments, a first agent or therapy is administered prior to a second agent or therapy. Those of skill in the art understand that the formulations or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co- administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in

embodiments where the co- administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent.

As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for therapeutic use.

The terms "pharmaceutically acceptable" or "pharmacologically acceptable", as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.

As used herein, "therapeutically effective dose" refers to an amount of a therapeutic agent sufficient to bring about a beneficial or desired clinical effect. Said dose can be administered in one or more administrations. However, the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including, but not limited to, the patient's age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired (e.g., aggressive versus conventional treatment).

As used herein, the term "treat" refers to administering a pharmaceutical composition for prophylactic or therapeutic purposes, wherein the growth of bacteria is prevented, stabilized, or inhibited; or wherein bacteria are killed. As used herein, the term "treating" refers to administering or prescribing a

pharmaceutical composition for prophylactic or therapeutic purposes. To "prevent disease" refers to prophylactic treatment of a patient who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disease. To "treat disease" or use for "therapeutic treatment" refers to administering treatment to a patient already suffering from a disease to improve the patient's condition. Thus, in the claims and embodiments, treating is the administration to an animal either for therapeutic or prophylactic purposes.

As used herein, the terms "animal", "subject", and "patient" specifically include mammals, such as a human, as well as cattle, horses, dogs, cats, and birds, but also can include many other species to which a drug is administered.

As used herein, the terms "alkyl" and the prefix "alk-" are inclusive of both straight chain and branched chain saturated or unsaturated groups, and of cyclic groups, i.e., cycloalkyl and cycloalkenyl groups. Unless otherwise specified, acyclic alkyl groups are from 1 to 6 carbons. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 8 ring carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and adamantyl groups. Alkyl groups may be substituted with one or more substituents or unsubstituted. Exemplary substituents include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halogen, alkylsilyl, hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups. When the prefix "alk" is used, the number of carbons contained in the alkyl chain is given by the range that directly precedes this term, with the number of carbons contained in the remainder of the group that includes this prefix defined elsewhere herein. For example, the term "C1-C4 alkaryl" exemplifies an aryl group of from 6 to 18 carbons (e.g., see below) attached to an alkyl group of from 1 to 4 carbons.

As used herein, the term "aryl" refers to a carbocyclic aromatic ring or ring system. Unless otherwise specified, aryl groups are from 6 to 18 carbons. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl, and indenyl groups.

As used herein, the term "heteroaryl" refers to an aromatic ring or ring system that contains at least one ring heteroatom (e.g., 0, S, Se, N, or P). Unless otherwise specified, heteroaryl groups are from 1 to 9 carbons. Heteroaryl groups include furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, oxatriazolyl, pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl, isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl, benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl, purinyl, and carbazolyl groups.

As used herein, the term "heterocycle" refers to a non- aromatic ring or ring system that contains at least one ring heteroatom (e.g., 0, S, Se, N, or P). Unless otherwise specified, heterocyclic groups are from 2 to 9 carbons. Heterocyclic groups include, for example, dihydropyrrolyl, tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl, tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl,

dihydrothiophene, tetrahydrothiophene, and morpholinyl groups.

Aryl, heteroaryl, or heterocyclic groups may be unsubstituted or substituted by one or more substituents selected from the group consisting of Ci-6 alkyl, hydroxy, halo, nitro, Ci-6 alkoxy, Ci-6 alkylthio, trifluoromethyl, Ci-6 acyl, arylcarbonyl, heteroarylcarbonyl, nitrile, Ci-6 alkoxycarbonyl, alkaryl (where the alkyl group has from 1 to 4 carbon atoms), and alkheteroaryl (where the alkyl group has from 1 to 4 carbon atoms).

As used herein, the term "alkoxy" refers to a chemical substituent of the formula -OR, where R is an alkyl group. By "aryloxy" is meant a chemical substituent of the formula -OR', where R' is an aryl group.

As used herein, the term "C x - y alkaryl" refers to a chemical substituent of formula -RR', where R is an alkyl group of x to y carbons and R' is an aryl group as defined elsewhere herein.

As used herein, the term "C x - y alkheteraryl" refers to a chemical substituent of formula RR", where R is an alkyl group of x to y carbons and R" is a heteroaryl group as defined elsewhere herein.

As used herein, the term "halide" or "halogen" or "halo" refers to bromine, chlorine, iodine, or fluorine.

As used herein, the term "non- vicinal 0, S, or N" refers to an oxygen, sulfur, or nitrogen heteroatom substituent in a linkage, where the heteroatom substituent does not form a bond to a saturated carbon that is bonded to another heteroatom.

For structural representations where the chirality of a carbon has been left unspecified it is to be presumed by one skilled in the art that either chiral form of that stereocenter is possible.

The terms "bacteria" and "bacterium" refer to prokaryotic organisms of the domain Bacteria in the three-domain system (see, e.g., Woese CR, et al., Proc Natl Acad Sci USA 1990, 87: 4576 - 79). It is intended that the terms encompass all microorganisms considered to be bacteria including Mycobacterium, Mycoplasma, Chlamydia, Actinomyces, Streptomyces, and Rickettsia. All forms of bacteria are included within this definition including cocci, bacilli, spirochetes, spheroplasts, protoplasts, etc. In some embodiments, bacteria are capable of causing disease and product degradation or spoilage. As used herein, a "pathogen" is a bacterium that is capable of causing a disease. "Strain" as used herein in reference to a microorganism describes an isolate of a microorganism (e.g., bacteria, virus, fungus, parasite) considered to be of the same species but with a unique genome and, if nucleotide changes are non- synonymous, a unique proteome differing from other strains of the same organism. Strains may differ in their non-chromosomal genetic complement. Typically, strains are the result of isolation from a different host or at a different location and time, but multiple strains of the same organism may be isolated from the same host.

As used herein, the term "bacterial infection" refers to the invasion of a host animal by pathogenic bacteria. For example, the infection may include the excessive growth of bacteria that are normally present in or on the body of an animal or growth of bacteria that are not normally present in or on the animal. More generally, a bacterial infection can be any situation in which the presence of a bacterial population(s) is damaging to a host animal. Thus, an animal is "suffering" from a bacterial infection when an excessive amount of a bacterial population is present in or on the animal's body, or when the presence of a bacterial population(s) is damaging the cells or other tissue of the animal.

As used herein, the term "persistent bacterial infection" refers to an infection that is not completely eradicated through standard treatment regimens using anti ¬ bacterial agents. Persistent bacterial infections are caused by bacteria capable of establishing a cryptic or latent phase of infection and may be classified as such by culturing the bacteria from a patient and demonstrating bacterial survival in vitro in the presence of anti-bacterial agents or by determination of anti-bacterial treatment failure in a patient. An in vivo persistent infection can be identified through the use of a reverse transcriptase polymerase chain reaction (RT-PCR) to demonstrate the presence of 16S rRNA transcripts in bacterially infected cells after treatment with anti-bacterial agents (see, e.g., Antimicrob. Agents Chemother. 12- 3288-97, 2000).

As used herein, the terms "rifampin" and "rifampicin" are interchangeable. As used herein, "rif-resistant" and "rif-resistance" and "RifR" are meant to describe a bacterium, bacteria, or an RNA polymerases that are not readily inhibited by a rifampicin or a related derivative or analog drug in the rifampicin family, including but not limited to rifampicin, rifamycin, rifapentine, rifabutin, and rifalazil.

As used herein, the term "MIC90" or "minimum inhibitory concentration, 90%" is used to refer to the lowest concentration of a compound that causes >90% growth inhibition (e.g., growth is <10% the growth in the absence of the compound). MIC90 values are used to characterize the potency of a compound and a lower MIC90 value indicates a more potent compound.

As used herein, the term "IC50" or "inhibition constant, 50%" refers to the lowest concentration of a compound that causes the activity of a biological molecule (e.g., an enzyme) to decrease to 50% of the activity of the biological molecule in the absence of the compound. IC50 values are used to characterize the potency of a compound and a lower IC50 value indicates a more potent compound.

The terms "variant" and "mutant" when used in reference to a polypeptide refer to an amino acid sequence that differs by one or more amino acids from another, usually related polypeptide. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties. One type of conservative amino acid substitution refers to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine? a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine? a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are : valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. More rarely, a variant may have "non- conservative" changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted, or deleted without abolishing biological activity may be found using computer programs well known in the art, for example, DNAStar software. Variants can be tested in functional assays. Preferred variants have fewer than 10%, and preferably fewer than 5%, and still more preferably fewer than 2% of the amino acids being changed (whether substitutions, deletions, and so on).

The nomenclature used to describe variants of nucleic acids or proteins specifies the type of mutation and base or amino acid changes. For a nucleotide substitution (e.g., 76A>T), the number is the position of the nucleotide from the 5' end, the first letter represents the wild type nucleotide and the second letter represents the nucleotide which replaced the wild type. In the given example, the adenine at the 76th position was replaced by a thymine. If it becomes necessary to differentiate between mutations in genomic DNA, mitochondrial DNA,

complementary DNA (cDNA), and RNA, a simple convention is used. For example, if the 100th base of a nucleotide sequence is mutated from G to C, then it would be written as g.l00G>C if the mutation occurred in genomic DNA, m.l00G>C if the mutation occurred in mitochondrial DNA, c.l00G>C if the mutation occurred in cDNA, or r.l00g>c if the mutation occurred in RNA. For amino acid substitution (e.g., D111E), the first letter is the one letter code of the wild type amino acid, the number is the position of the amino acid from the N-terminus, and the second letter is the one letter code of the amino acid present in the mutation. Nonsense mutations are represented with an X for the second amino acid (e.g. D111X). For amino acid deletions (e.g. AF508, F508del), the Greek letter Δ (delta) or the letters "del" indicate a deletion. The letter refers to the amino acid present in the wild type and the number is the position from the N terminus of the amino acid where it is present in the wild type.

The term "domain" when used in reference to a polypeptide refers to a subsection of the polypeptide that possesses a unique structural and/or functional characteristic; typically, this characteristic is similar across diverse polypeptides. The subsection typically comprises contiguous amino acids, although it may also comprise amino acids that are far apart in the primary sequence but that act in concert or that are in close proximity in the tertiary or quaternary structure due to folding or other configurations.

The term "gene" refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA or a polypeptide or its precursor (e.g., proinsulin). A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term "portion" when used in reference to a gene refers to fragments of that gene. The fragments may range in size from a few nucleotides to the entire gene sequence minus one nucleotide. Thus, "a nucleotide comprising at least a portion of a gene" may comprise fragments of the gene or the entire gene.

The term "gene" also encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences that are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences that are located 3' or downstream of the coding region and that are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. The mRNA functions during translation to specify the sequence or order of amino acids in a nascent polypeptide. Genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers that control or influence the transcription of the gene. The 3' flanking region may contain sequences that direct the termination of transcription or posttranscriptional cleavage.

The term "wild-type" when made in reference to a gene refers to a gene that has the characteristics of a gene isolated from a naturally occurring source. The term "wild-type" when made in reference to a gene product refers to a gene product that has the characteristics of a gene product isolated from a naturally occurring source. The term "naturally-occurring" as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man in the laboratory is naturally-occurring. A wild-type gene is frequently that gene which is most frequently observed in a population and is thus arbitrarily designated the "normal" or "wild-type" form of the gene. Strains of an organism may comprise naturally- occurring variants that differ from the wild-type form. In contrast, the term "modified" or "mutant" when made in reference to a gene or to a gene product refers, respectively, to a gene or to a gene product that displays modifications in sequence or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.

The term "sample" is used in its broadest sense. In one sense it can refer to an animal cell or tissue. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from plants or animals (including humans) and encompass fluids, solids, tissues, and gases. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. These examples are not to be construed as limiting the sample types applicable to the present invention.

As used herein, the term "in vitro" refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments may include, but are not limited to, test tubes and cell cultures. The term "in vivo" refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment. Embodiments of the technology

Although the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation. Analog Design

Experiments conducted during the development of embodiments of the technology described herein employed molecular modeling to identify candidate

0

compounds. The modeling was based on the 2.5 A resolution structure of rifabutin (RFB) in complex with the Thermus thermophilus RNAP holoenzyme (PDB ID: 2a68) (Artsimovitch, I. et al. Cell 2005, 122 (3): 351-63). The rifamycin-binding site is highly conserved among bacteria; therefore this structure provides a basis for understanding how rifamycin analogs interact with the MTB RNA polymerase.

The crystal structure (PDB accession number 2a68) contains two complete complexes. Observed differences between these two complexes and what is seen in the related complexes present in a structure of rifapentine in complex with the T. thermophilus RNAP holoenzyme (PDB accession number 2a69) suggest that, at least in the free holoenzyme, the sigma factor hairpin loop exists in two distinct physiologically relevant conformations.

The modeling studies described herein indicate that there are a number of analogs with a range of size, flexibility, and spatial variation that interact with the sigma hairpin loop and other portions of the RNAP complex. These variations are further extended by the differences in attachment points on the planned subclasses. These analogs were chosen to increase drug potency by doing one or more of the following: (a) making additional contacts with the sigma factor, beta, or beta prime regions of the RNAP and/or (b) interfering with the binding of the sigma factor or further occluding the channel. During the development of embodiments of the technology provided herein, sets of rifamycin analogs that are representative of the structure-based design approaches were synthesized and others were designed for synthesis (see, e.g., Figure l). Synthesized compounds were tested for potency, toxicity, and Cyp450 induction.

Modeling the RLZ/RNAP complex displays the presence of interaction surfaces at 4.5 A between the analog tail and residues of the surrounding RNAP. The models show that these analogs have the potential to interact with different regions of RNAP and can participate in additional contacts with RNAP sidechains that may have different potency profiles.

RMP is the largest activator of hPXR currently known and is a potent activator of hPXR (Chrencik, J. E. et al. Mol Endocrinol 2005, 19: 1125-34). RMP fills the ligand-binding pocket very well. Differences in RMP activation of the human versus the mouse PXR are interpreted in light of structural information. For example, Leu308 in the human PXR is replaced by Phe in the mouse and Ser247 is replaced by Trp in the mouse. These changes help explain the differential RMP activation of the human and mouse PXRs, e.g., due to impaired binding of RMP to the mouse PXR. The relative potency of CYP3A4 induction is RMP > rifapentine > rifabutin > RLZ. Modeling the hPXR binding site with these four rifamycins in spatial relation to resolved hPXR residues (see Examples for details of modeling) shows that there are seven hPXR residues, Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243, in very close proximity to the synthetic branch point for the embodiments of the analogs designed herein. These residues are resolved in each of the five hPXR structures and, other than one of the Phe-237 rings, have fairly conserved relative coordinates. This, along with the presence of Pro-241, would suggest that these residues are located in a more rigid region of the hPXR ligand-binding pocket.

The hPXR ligand-binding pocket is large, flexible, and capable of adapting itself to bind a large variety of ligands. The modeling suggests that the proposed analogs designed to target RNAP sigma factors have the added benefit of overwhelming the normally promiscuous hPXR binding pocket. It appears that the tails of the designed analogs prevent binding of the compounds to hPXR by projecting into rigid, sterically encumbered regions of hPXR, although an understanding of the mechanism is not needed to practice the technology described herein, nor is the technology limited by any particular mechanism of action.

Impeded binding of compounds to hPXR is contemplated to reduce induction of CYP450.

Accordingly, in some embodiments the analog is a compound having the formula

In some embodiments, A is H- and R is Et2NCH2CH2-, in some embodiments A is H- and R is in some embodiments, A is H- and R is

and in some embodiments A is

and R is

The A or R group adds steric volume to the compound, and thus in some embodiments the steric clash comprises an interaction of the A or the R with an amino acid of the human pregnane X receptor at a position in the primary sequence selected from the group consisting of 237, 238, 239, 240, 241, 242, and 243. In some embodiments the steric clash comprises an interaction of the A or the R with an amino acid in the human pregnane X receptor selected from the group consisting of Phe-237, Ser-238, Leu-239, Leu-240, Pro-241, His-242, and Met-243 (see, e.g., UniProt accession number 075469 or PDB accession number lskx).

In some embodiments, compounds having the formula

are synthesized by dialkylating a 2-nitroresorcinal to produce a dibenzyl ether and mono-debenzylating the dibenzyl ether to produce a nitrophenol having the formula

Additionally, some embodiments provide a compound

wherein R is a moiety defined as one of the following

and wherein n = 1-3 and R' = H, methyl, ethyl, 1-methylethyl (e.g., isaCs i or i ' -Pr), or 2-methylpropyl (e.g., i ' scrC iHg or i ' -Bu). Furthermore, in some embodiments, R is a moiet defined as one of the following:

wherein n = 1-3; in some embodiments, R is a moiety defined as one of the following wherein n = 1- " is a moiet defined as one of the following

wherein n = 1-3.

In some embodiments, it is contemplated that the compounds according to the technology have a structure according to one of the following:

Analog synthesis

The synthetic route utilized to make the target "one-armed" compounds 2tr2d is shown in Figure 2a. Additional schemes contemplated for synthesizing rifamycin derivatives are provided in Figures 2b-2e and Figure 6. The RLZ literature (e.g., Yamane, T. et al. Chem Pharm Bull (Tokyo) 1993, 41: 148-55) suggested a strategy of annulating the benzoxazino moiety onto rifamycin S (12) with a suitably protected monoether (e.g., TBS) of 2-aminoresorcinol, followed by ether deprotection and then side chain installation off the nascent phenol by any number of alkylation methodologies. This was investigated, but yields were very poor and the scope of alkylation possibilities was quite limited.

Instead, in some embodiments, the present technology provides a fully tethered 2-aminoresorcinol monoether that is annulated onto the rifamycin S framework in a single step. This approach allowed the design of a wide range of tethers off the "southeastern" part of the rifalazil-type template and, more importantly, minimized difficult synthetic transformations and product

purifications involving the complex rifamycin S core to a single last step. Thus, a robust procedure was developed to produce intermediate 5, which would serve as a key starting material for introduction of the chosen tethers.

Additionally, while there are two reports for the synthesis of this compound

(Dhingra, K. et al. Chemical Communications 2008 Aug 27 (29): 3444-46; Binggeli, A. et al., U.S. Pat. Appl. Ser. No. 11/515,041), neither was deemed to be a practical solution for the required synthesis. Instead, a novel two-step procedure was developed. Dialkylation of 2-nitroresorcinol (3), similar to the literature procedure (Havera, H. J. et al. U.S. Pat. No. 3995041), gave a 95% yield of dibenzyl ether 4 which was then cleanly mono-debenzylated to nitrophenol 5 (Dhingra, K. et al. Chemical Communications 2008 Aug 27 (29): 3444-46) in 82% yield. Intermediate 5 provided a basis for synthesis of the target tethers. Phenolic alkylation with 2- (diethylaminoethyl) ethyl chloride hydrochloride under standard conditions provided 6 in 87% yield. Hydrogenation of 6 utilizing Pearlman's catalyst simultaneously reduced the nitro function and hydrogenolyzed the benzyl protecting group to give the 2-aminoresorcinol ether 7 in 81% yield. A similar sequence of reactions was followed to provide ethers 11a, lib. Alkylation of 5 with 1,4-dibromobutane gave 8 in 93% yield, which was subsequently aminated with two mo no -substituted piperazines to provide compounds 9a and 9b in 68% and 99% yields, respectively, t Boc deprotection of 9b followed by acylation of 9c with 2-(lH-imidazol-l-yl)acetic acid provided 10, the amide congener of 9a, in 72% yield. Hydrogenation of 9a and 10 was conducted as described for 6 to provide the remaining 2-aminoresorcinol ethers 11a and lib, respectively, in nearly quantitative yields. Each 2- aminoresorcinol ether (7, 11a, lib) was then annulated onto rifamycin S (12) to provide target compounds 2b - 2d in 35 - 74% yields following a two- stage purification utilizing medium pressure and then preparative plate silica gel chromatography.

A single "two-armed" RLZ congener (2e) was synthesized by condensation of 2d with Visobutylpiperazine under oxidative conditions as described for the synthesis of RLZ (2a) (Yamane, T. et al. Chem Pharm Bull (Tokyo) 1993, 41: 148- 55). This reaction gave a 74% yield of 2e following rigorous purification. The structural assignments of 2b - 2e were supported by diagnostic peaks in the Ή NMR spectra and by chemical ionization (CI) and high resolution (HR) mass spectrometry.

In some embodiments, compounds are contemplated that are synthesized according to a schema such as

wherein the step of reacting with Et 3 N/HC0 2 H, Pd(OAc) 2 , and PPh 3 in DMF (e.g., as marked with an asterisk above) is performed in some embodiments according to the method of Cabri, et al. as described in J. Org. Chem. 55: 350-353 (1990), incorporated herein by reference. These compounds are contemplated to have activities within the scope of the technology and find use in applications as described herein.

Pharmaceutical formulations

With respect to administering a rifalazil or rifampin analog to a subject, it is contemplated that the compounds be administered in a pharmaceutically effective amount. One of ordinary skill recognizes that a pharmaceutically effective amount varies depending on the therapeutic agent used, the subject's age, condition, and sex, and on the extent of the disease in the subject. Generally, the dosage should not be so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. The dosage can also be adjusted by the individual physician or veterinarian to achieve the desired therapeutic goal.

As used herein, the actual amount encompassed by the term

"pharmaceutically effective amount" will depend on the route of administration, the type of subject being treated, and the physical characteristics of the specific subject under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical, veterinary, and other related arts. This amount and the method of administration can be tailored to achieve optimal efficacy but will depend on such factors as weight, diet, concurrent medication, and other factors that those skilled in the art will recognize.

In some embodiments, a rifalazil or rifampin analog according to the technology provided herein, a derivative thereof, or a pharmaceutically acceptable salt thereof, is administered in a pharmaceutically effective amount. In some embodiments, a rifalazil or rifampin analog, a derivative thereof, or a

pharmaceutically acceptable salt thereof, is administered in a therapeutically effective dose.

The dosage amount and frequency are selected to create an effective level of the compound without substantially harmful effects. When administered orally or intravenously, the dosage of a rifampin analog or related compounds will generally range from 0.001 to 10,000 mg/kg/day or dose (e.g., 0.01 to 1000 mg/kg/day or dose? 0.1 to 100 mg/kg/day or dose).

Methods of administering a pharmaceutically effective amount include, without limitation, administration in parenteral, oral, intraperitoneal, intranasal, topical, sublingual, rectal, and vaginal forms. Parenteral routes of administration include, for example, subcutaneous, intravenous, intramuscular, intrastemal injection, and infusion routes. In some embodiments, a rifampin analog, a derivative thereof, or a pharmaceutically acceptable salt thereof, is administered orally.

Pharmaceutical compositions preferably comprise one or more compounds of the present invention associated with one or more pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutically acceptable carriers are known in the art such as those described in, for example, Remingtons Pharmaceutical

Sciences, Mack Publishing Co. (A. R. Gennaro ed., 1985).

In some embodiments, a single dose of a rifalazil or rifampin analog or related compounds is administered to a subject. In other embodiments, multiple doses are administered over two or more time points, separated by hours, days, weeks, etc. In some embodiments, compounds are administered over a long period of time (e.g., chronically), for example, for a period of months or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months or years? e.g., for the lifetime of the subject). In such embodiments, compounds may be taken on a regular scheduled basis (e.g., daily, weekly, etc.) for the duration of the extended period.

In some embodiments, a rifalazil or rifampin analog or a related compound is co -administered with another compound or more than one other compound (e.g., 2 or 3 or more other compounds). In some embodiments, the rifalazil or rifampin analog or related compound is co-administered with an antibiotic such as, e.g., isoniazid, pyrazinamide, ethambutol, or streptomycin. In some embodiments, the antibiotic co-administered with the rifampicin or rifampin analog is an

aminoglycoside (e.g., amikacin, kanamycin), a polypeptide (e.g., capreomycin, viomycin, enviomycin), a fluoroquinolone (e.g., ciprofloxacin, levofloxacin,

moxifloxacin), a thioamide (e.g., ethionamide, prothionamide, tiocarlide),

cycloserine, or p-aminosalicylic acid. In some embodiments, the antibiotic co ¬ administered with a rifalazil or rifampin analog is rifabutin, a macrolide (e.g., clarithromycin), linezolid, thioacetazone, thioridazine, arginine, vitamin D, or R207910. In some embodiments, a steroid (e.g., a corticosteroid (e.g., prednisolone or dexamethasone)) is co-administered with the rifalazil or rifampin analog.

Thalidomide is co- administered with a rifalazil or rifampin analog in some embodiments. In some embodiments, interferon-γ is co-administered; in some embodiments, meropenem, morinamide, terizidone, or clavulanic acid is co ¬ administered. In some embodiments, co-amoxiclav, clofazimine, prochlorperazine, metronidazole, or PA-824 is co-administered with a rifalazil or rifampin analog or related compound. In some embodiments, Dzherelo, Anemin, Svitanok, Lizorm, Immunoxel, or Immunitor is co-administered with a rifalazil or rifampin analog.

Kits

The technology provided herein also includes kits for use in the instant methods. Kits of the technology comprise one or more containers comprising rifalazil or rifampin, a derivative thereof, or a pharmaceutically acceptable salt thereof, and/or a second agent, and in some variations further comprise instructions for use in accordance with any of the methods provided herein. The kit may further comprise a description of selecting an individual suitable for treatment. Instructions supplied in the kits of the technology are typically written instructions on a label or package insert (e.g., a paper insert included with the kit), but machine -readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also contemplated. Examples

Methods and materials

Computational Modeling of Rifamycin-RNAP Complexes

The structure of 2a68 was used as the starting point in the modeling studies. The following modifications were made to the structure prior to its use. All water molecules and metals greater than 12 A from rifabutin (RFB) were removed. Four magnesium ions within 12 A were converted to water molecules. Partially missing residues were repaired. Connection points for completely missing residues were greater than 35 A from RFB and they were thus kept fixed in space during the energy minimizations. All N and C termini, either real or as a result of missing residues, were acetylated and amidated, respectively. RFB was removed from the complex. Hydrogen atoms were added to the proteins and the force field set to

AMBER99 and charged.

A series of energy minimizations were then conducted to relax the positions of the modified atoms using the AMBER99 force field to gradients of 0.01. Positions of hydrogen atoms were first relaxed with energy minimization. Repaired residues except for their C alpha atoms, termini, and hydrogen atoms were then relaxed.

Lastly, the complete repaired residues, termini, and hydrogen atoms were relaxed.

Resulting conformations of the repaired residues and termini were checked. Bond orders for RFB were corrected, hydrogen atoms added, and the force field set to MMFF94x and charged. Positions of hydrogen atoms were relaxed with energy minimization using the MMFF94x force field to a gradient of 0.01. RFB was returned to the complex in its original pose.

A second series of energy minimizations were then conducted using the

MMFF94x force field to gradients of 0.01. Positions of hydrogen atoms were first relaxed followed by positions of hydrogen atoms and all water molecules. Atoms of the RFB were then included in the minimizations. Lastly, positions of all RFB, water, and hydrogen atoms and protein residues having one or more atoms within

12 A from RFB were relaxed with energy minimization. The naphthalene ring of

RFB drifted approximately 1 A toward the cleft of the complex from its

crystallographic position with relatively minor movements of the protein residues well within the 2.5 A resolution of the starting structure.

Complexes for proposed analogs were generated from the modified RFB complex as follows : All water molecules present in the structure were removed. All residues not having one or more atoms within approximately 20 A of the RFB were deleted. The RFB structure was modified to generate the proposed analog. Atoms of the RFB, which were not modified in the generation of the proposed analog, were initially fixed in space and treated as part of the RNAP holoenzyme. Modifications were made to the RFB structure to produce the proposed analog while keeping the unmodified atoms of RFB fixed in relation to the RNAP complex. A LowModeMD (Labute, P. J. Chem. Inf. Model. 2010, 50: 792-800) conformational search algorithm with energy minimization was then employed to generate plausible poses (conformations) of the modified portions of RFB. The LowModeMD search was conducted in MOE (Chemical Computing Group, Montreal) using default settings.

Hydrogen atoms, modified portions of RFB and protein side chains within ~16 A of the modified portions were allowed to move during the conformational search and energy minimizations. Generated poses were ranked by interaction energies and duplicate poses based on a RMSD cutoff removed.

The lowest energy pose was then selected and the truncated RNAP complex soaked with water to a surrounding distance of 6 A. As described above, a series of energy minimizations was then conducted using the MMFF94x force field to relax the complex. First, hydrogen atoms and then the water molecules were allowed to relax while the entire analog and all of the RNAP atoms were held fixed. Second, the modified portions of RFB and side chains of RNAP within 16 A were also allowed to relax with the water molecules and hydrogen atoms. Finally, the entire analog molecule, residues of RNAP within 16 A, and the water molecules and hydrogen atoms were allowed to relax. The relaxed complexes were then examined to determine how the proposed analog may interact with the sigma factor or other portions of RNAP (specifically the beta and beta prime subunits). Computa tional Modeling of Rifamycin -hPXR Complexes

3D structures were generated using the 2.5 A resolution crystal structure for Thermus thermophilus RNAP in complex with rifabutin (RFB) obtained from the Protein Data Bank (PDB accession number 2a68). As described above, coordinates for RFB were relaxed in the presence of the RNAP using a series of energy minimizations with decreasing constraints on the surrounding protein atoms and water molecules. Coordinates for RMP, rifapentine, and RLZ were created and relaxed in the same fashion after modifying RFB. The 2.8 A resolution crystal structure of hPXR in complex with rifampin was retrieved from the Protein Data Bank (PDB accession number lskx). In this structure, the l-amino-4- methylpiperazine tail of RMP and three hPXR loops adjacent to the binding pocket (residues 178-209, 229-235, and 310-317) are disordered and unresolved in the structure. The naphthalene portions of the four rifamycins generated from the 2a68 structure were overlayed onto the naphthalene portion of RMP in complex with hPXR structure lskx. In addition to the complex with RMP, four other relatively complete hPXR structures are available. The hPXR apo structure (PDB accession number lilg) and hPXR complexes with SR12813 (PDB accession number lilh), hyperforin (PDB accession number lml3), and colupulone (PDB accession number 2qnv) were obtained from the Protein Data Bank. These four hPXR complexes were superimposed onto the hPXR structure of lskx containing the four modeled-in rifamycins. Coordinates were not relaxed with energy minimization due to many missing residues.

Chemical Syntheses

All reagents were commercially available and used without further purification. Melting points were determined in open capillary tubes on a

Laboratory Devices Mel-Temp apparatus and are uncorrected. Ή and 13 C NMR spectra were obtained on Bruker 500 MHz spectrometers with CDCI3 or d6 _ DMSO as solvent and chemical shifts are reported relative to the residual solvent peak in δ (ppm). Mass spectrometry analysis was performed using a Waters LCT time-of- flight mass spectrometry instrument. High resolution mass spectrometry (HRMS) analysis was performed on an Agilent Q-TOF system. Analytical HPLC was performed on a Perkin Elmer Series 200 system with an Agilent Eclipse plus C18 (4.6 + 7.5 mm, 3.5 mm particle size) column. The mobile phase was a 15 min binary gradient of acetonitrile (containing 0.1 % TFA) and water (20-90%). Thin-layer chromatography (TLC) was performed on silica gel GHLF plates (250 microns) purchased from Analtech. Extraction solutions were dried over MgSC prior to concentration.

(((2-Nitro-l,3-phenylene)bis(oxy))bis(methylene))dibenzen e (4). A mixture of 2- nitroresorcinol (3; 2.0 g, 12.9 mmol), CS2CO3 (10.5 g, 32.2 mmol), benzyl bromide (3.39 ml, 28.4 mmol) and DMF (35 mL) was stirred at room temperature for 12 h. The mixture was diluted with ethyl acetate and washed sequentially with 1% aq HC1 and brine. The organic phase was dried and concentrated to leave a yellow oil, which was diluted with 2-propanol to precipitate pure product. The solids were collected to leave 4 (4.35 g, 95%) as light yellow crystals: mp 87.5 - 88°C (lit 80°C); R 0.26 (hexanes : ethyl acetate, 5 : l); Ή NMR (CDCla) δ 7.3 (m, 10 H), 7.23 (t, J- 8.5 Hz, 1 H), 6.64 (d, J= 8.5, 2 H), 5.16 (s, 4 H); i3 C NMR (CDCI3) δ 150.9, 135.6, 130.9, 128.7, 128.2, 127.0, 106.2, 71.0; MS (ES + ) m/z 358.1 (M+Na) + . 3-(Benzyloxy)-2-nitrophenol (5). A solution of dibenzyl ether 4 (3.0 g, 8.95 mmol) in dichloromethane (80 mL) at -78°C was treated drop- wise with boron trichloride (13 mL, 1 M in heptane) during which the color changed to dark purple. The reaction was monitored by TLC and stirred at -78°C until all starting material was consumed (l h). Methanol (5 mL) was added drop-wise and the mixture was brought to room temperature, cautiously diluted with 5% aq sodium bicarbonate, and then extracted with dichloromethane (2x). The combined extracts were dried and concentrated to an orange oil that was purified by flash silica gel chromatography eluting with hexanes : ethyl acetate (5 : l). Product fractions were pooled and concentrated to give 5 (1.79 g, 82%) as a bright yellow solid: mp 67 - 67.5 °C; R/0.24 (hexanes : ethyl acetate, 5 : l); Ή NMR (CDCla) δ 10.18 (brs, 1 H), 7.4 (m, 2 H), 7.3 (m, 4 H), 6.72 (d, J= 8.5 Hz, 2 H), 6.6 (d, J= 8.5 Hz, 2 H), 5.21 (s, 2 H); 13 C NMR (CDCI3) δ 155.7, 154.7, 135.6, 135.4, 128.7, 128.2, 126.9, 111.0, 105.1, 71.4; MS (ES + ) m/z 268.0 (M+Na) + . 2-(3-(Benzyloxy)-2-nitrophenoxy)-N,N-diethylethanamine (6). A mixture of nitrophenol 5 (1.31 g, 5.4 mmol), 2-(diethylaminoethyl)ethyl chloride hydrochloride (1.2 g, 7 mmol), Cs 2 C0 3 (4.37 g, 13.4 mmol) and acetone (20 mL) was stirred at 50°C for 3 h. The mixture was filtered and the filtrate was concentrated to a residue that purified by flash silica gel chromatography eluting with hexanes : ethyl acetate (5 : l). Product fractions were pooled and concentrated to leave 6 (1.71 g, 93%) as a light yellow oil: R = 0.22 (CH 2 C1 2 : methanol, 95 : 5); Ή NMR (CDCla) δ 7.3 (m, 6 H), 6.62, (dd, J = 3.6 Hz, J 2 = 14.1 Hz, 2 H), 5.16 (s, 2 H), 4.1 (t, J= 10.5 Hz, 2 H), 2.8 (t, J= 10.5 Hz, 2 H), 2.6 (q, J= 11.9 Hz, 4 H), 1.0 (t, e / ' = 11.9 Hz, 6 H); 13 C NMR (CDCI3) δ 151.2, 150.8, 135.6, 130.9, 128.6, 128.2, 127.0, 105.9, 105.6, 70.9, 68.5, 51.2, 47.9, 11.9; MS (ES + ) m/z 245.1 (M+H) + .

2-Amino-3-(2-(diethylamino)ethoxy)phenol (7). 2-(Diethylamino)ethyl ether 6 (1.8 g, 5.2 mmol) was dissolved in 10% acetic acid in methanol (50 mL) in a 250 mL Parr hydrogenation bottle. Catalyst (20% Pd(OH) 2 /C, 0.1 g) was added and the mixture was hydrogenated at 40 psi H 2 for ~20 h. The reaction mixture was rapidly filtered over Celite®, and the filtrate was concentrated and diluted with ethyl acetate. The solution was washed with 5% aq sodium carbonate, dried, and concentrated to a brown solid that was triturated in hot hexanes. The solids were collected and dried to leave 7 (0.95 g, 81%): mp 91 - 91.5°C; R/0.21 (CH 2 C1 2 : methanol, 85 : 15); Ή NMR (CDCI3) δ 6.55 (t, J= 7.7 Hz, 1 H), 6.4 (m, 2 H), 4.07 (t, J= 5.8 Hz, 2 H), 2.91 (t, J= 5.8 Hz, 2 H), 2.7 (m, 4 H), 1.1 (t, J= 7.1 Hz, 6 H); 13 C NMR (CDCla) δ 148.0, 145.3, 124.9, 117.8, 109.0, 104.4, 66.5, 51.9, 47.4, 11. l; MS (ES + ) m/z 225.1 (M+H) + . l-(Benzyloxy)-3-(4-bromobutoxy)-2-nitrobenzene (8). To a mixture of DMF (5 mL), 1,4-dibromobutane (5 mL) and Cs 2 C03 (1.66 g, 5.1 mmol) was added slowly a solution of nitrophenol 5 (0.5 g, 2.0 mmol) in DMF (5 mL). The mixture was stirred at room temperature for 16 h, and then DMF was removed in vacuo to leave an oil that was distributed between 1% aq HC1 and ethyl acetate. The organic phase was dried and concentrated to a light yellow oil that was purified by flash silica gel chromatography eluting with hexanes : ethyl acetate (6 : l). Product fractions were pooled and concentrated to give 8 (0.702 g, 93%) as a light yellow oil: R 0.45

(hexanes : ethyl acetate, 2 : l); Ή NMR (CDCla) δ 7.36 (m, 5 H), 7.26 (m, 1 H), 6.61 (m, 2 H), 5.16 (s, 2 H), 4.08 (t, J= 5.8 Hz, 2 H), 3.46 (t, J= 6.3 Hz, 2 H), 2.02 (m, 2 H), 1.92 (m, 2 H); 13 C NMR (CDCla) δ 151.1, 150.9, 135.6, 131.0, 128.7, 128.2, 127.0, 106.1, 105.6, 70.9, 68.4, 33.4, 28.9, 27.5; MS (ES + ) m/z 401.9, 403.9 (M+Na) + . l-(2-(lH-Imidazol-l-yl)ethyl)-4-(4-(3-(benzyloxy)-2-nitrophe noxy)butyl)piperazine (9a). A solution of bromobutyl ether 8 (l.O g, 2.6 mmol), l- [2-(lH-imidazol- l _ yl)ethyl]piperazine (0.52 g, 2.9 mmol; Oakwood Products Inc.), N,N- diisopropylethylamine (5 mL) and acetonitrile (18 mL) was heated at reflux overnight. The solution was concentrated and the residue was distributed between dichloromethane and 5% aq sodium carbonate. The organic phase was dried and concentrated to an orange oil that was purified by flash silica gel chromatography eluting with dichloromethane : methanol : NH4OH (90 : 10 : 0.5). Product fractions were pooled and concentrated to leave 9a (0.86 g, 68%) as an oil: i H NMR (CDCI3) δ 7.53 (s, 1 H), 7.36-7.22 (m, 6 H), 7.03 (s, 1 H), 6.97 (d, J= 1.1 Hz, 1 H), 6.61 (m, 1 H), 5.15 (s, 1 H), 4.05 (t, J= 6.3 Hz, 2 H), 4.01 (t, J= 6.5 Hz, 2 H), 2.67 (t, J= 6.5 Hz, 2 H), 2.48 (bs, 8 H), 2.36 (t, J= 6.5 Hz, 2 H), 1.79 (m, 2 H), 1.61 (m, 2 H); 13 C NMR (CDCI3) δ 151.3, 150.8, 137.4, 135.6, 132.8, 130.9, 129.2, 128.7, 128.2, 127.0, 119.3, 105.8, 105.6, 70.9, 69.3, 58.6, 57.8, 53.3, 53.0, 44.7, 26.9, 23.1; MS (ES + ) ^ 480.1 (M+H) + . tert-Butyl 4-(4-(3-(benzyloxy)-2-nitrophenoxy)butyl)piperazine-l-carbox ylate (9b). A solution of bromobutyl ether 8 (0.4 g, 1.05 mmol), 1-Boc-piperazine (0.282 g, 1.514 mmol) ^AMiisopropylethylamine (4 mL) and acetonitrile (10 mL) was heated at reflux for 12 h. The solution was concentrated and distributed between ethyl acetate and brine. The organic phase was dried and concentrated to residue that was purified by flash silica gel chromatography eluting with ethyl acetate. Product fractions were pooled and concentrated to leave 9b (0.505 g, 99%) : Ή NMR (CDCI3) δ 7.36 (m, 4H), 7.31 (m, 1 H), 7.24 (m, 1 H), 6.61 (m, 2 H), 5.16 (s, 2 H), 4.06 (t, J= 6.2 Hz, 2 H), 3.41 (t, J= 7.0 Hz, 4 H), 2.36 (t, J= 7.0 Hz, 4 H), 1.80 (m, 2 H), 1.62 (m, 2 H), 1.46 (s, 9 H), 1.26 (t, J= 7.2 Hz, 2 H); i3 C NMR (CDCI3) δ 154.9, 151.5, 150.9, 135.8, 131.0, 128.8, 128.3, 127.1, 106.0, 105.7, 79.7, 71.1, 69.4, 60.5, 58.1, 53.1, 28.6, 27.0, 23.2, 21.2, 14.3; MS (ES + ) ^ 486.1 (M+H) + . l-(4-(4-(3-(Benzyloxy)-2 itrophenoxy)buty piperazin-l-yl)"2-(lH-imidazol-l- yl)ethanone (10). Trifluoro acetic acid (2 mL) was added dropwise to a solution of 9b (0.505 g, 1.04 mmol) in dichloromethane (8 mL), and the resultant mixture was stirred at room temperature for 3 h. The solution was concentrated to leave 9c (0.52 g, quantitative) as the crystalline trifluoroacetate salt. This was then dissolved into DMF (10 mL) and ^A iisopropylethylamine (3 mL), and the mixture was stirred at room temperature for 10 min followed by treatment with l-ethyl-3 _ [3- dimethylamino propyl] carbodiimide hydrochloride (EDC HC1; 0.22 g, 1.14 mmol), N- hydroxybenzotriazole (HOBt; 0.175 g, 1.14 mmol) and 2-(lH-imidazol-l-yl)acetic acid (0.197 g, 1.56 mmol; Tokyo Chemical Industry Co. Ltd.). After stirring under N2 for 16 h, DMF was removed in vacuo and the residue was distributed between dichloromethane and 5% aq sodium carbonate. The organic phase was dried and concentrated to an oil that was purified by flash silica gel chromatography eluting with dichloromethane : methanol : NH4OH (95 : 5 : 0.5). Product fractions were pooled and concentrated to give 10 (0.37 g, 72%) as a yellow oil: Ή NMR (CDCI3) δ 7.49 (s, 1 H), 7.36 (m, 5 H), 7.26 (m, 1 H), 7.09 (s, 1 H), 6.95 (s, 1 H), 6.61 (m, 1H), 5.16 (s, 2 H), 4.75 (s, 2 H), 4.07 (t, J= 5.9 Hz, 2 H), 3.62 (m, 2 H), 3.44 (m, 2 H), 2.42 (t, J= 4.9 Hz, 4 H), 2.39 (t, J= 7.2 Hz, 2 H), 1.81 (m, 2 H), 1.62 (m, 2 H); 13 C NMR (CDCI3) δ 164.4, 151.3, 150.8, 138.0, 135.6, 131.0, 129.5, 128.7, 128.2, 127.0, 120.1, 105.9, 105.5, 70.9, 69.1, 57.6, 52.6, 47.9, 45.1, 42.3, 26.7, 22.9; MS (ES + ) m/z 494.1 (M+H) + .

3-(4-(4-(2-(l.ffImidazol-l-y ethy piperazin-l-yl)butoxy)-2-aminophenol (lla).

Compound 9a (0.86 g, 1.8 mmol) was dissolved in a mixture of 10% aq HC1 (10 mL) and methanol (90 mL) in a Parr hydrogenation bottle. Catalyst (20% PdiOHVC, 0.05 g) was added and the mixture was hydrogenated at 40 psi ¾ for ~40 h. The reaction mixture was rapidly filtered over Celite® and the filtrate was concentrated and diluted with ethyl acetate. The solution was washed with 5% aq sodium carbonate, dried, and concentrated to give lla (0.61 g, 95%) as a brown solid: i H NMR (CDCI3) δ 7.56 (s, 1 H), 7.05 (s, 1 H), 6.96 (s, 1 H), 6.52 (t, J= 7.2 Hz, 1 H), 6.44 (s, 1 H), 6.37 (d, J= 8.0 Hz, 1 H), 3.98 (m, 4 H), 2.66 (t, J= 6.2 Hz, 2 H), 2.49 (bs, 8 H), 2.41 (t, J= 6.5 Hz, 2 H), 1.78 1.68 (m, 2 H); 13 C NMR (CDCI3) δ 147.6, 145.5, 128.6, 124.9, 119.4, 117.3, 108.7, 103.7, 68.1, 58.4, 58.2, 53.0, 50.4, 44.7, 27.5, 23.3; MS (ES + ) m/z 360.1 (M+H) + . l-(4-(4-(2-Amino-3-hydroxyphenoxy)butyl)piperazin-l-yl)-2-(l H-imidazol-l- yl)ethanone (lib). Compound 10 (0.37 g, 0.75 mmol) was dissolved in a mixture of 10% aq HC1 (5 mL) and methanol (45 mL) in a Parr hydrogenation bottle. Catalyst (20% PdCOHVC, 0.02 g) was added and the mixture was hydrogenated at 40 psi ¾ for ~40 h. Workup as described above for the synthesis of 11a gave lib (0.27 g, 98%) as a brown solid: Ή NMR (CD3OD) δ 7.78 (s, 1 H), 7.13 (s, 1 H), 7.06 (s, 1 H), 6.58 (m, 1 H), 6.43 (m, 2 H), 5.08 (s, 2 H), 4.03 (t, J= 5.9 Hz, 2 H), 3.66 (s, 2 H), 3.62 (s, 2 H), 2.75 (s, 2 H), 2.65 (m, 4 H), 1.82 (m, 4 H); i3 C NMR (CD3OD) δ 174.8, 165.8, 148.0, 145.8, 138.1, 126.0, 123.0, 121.3, 118.2, 107.8, 103.5, 67.7, 57.3, 52.1, 51.8, 43.5, 40.9, 26.8, 22.2, 20.1; MS (ES + ) m/z 374.1 (M+H) + .

Benzoxazinorifamycin (2b). A mixture of aminophenol 7 (0.336 g, 1.5 mmol), rifamycin S (12; 2.085 g, 3 mmol) and 1,4-dioxane (20 mL) was stirred at room temperature overnight. The mixture was then concentrated to a black solid that was dissolved in 20 mL of methanol and treated with MnC>2 (0.3 g, 3.45 mmol). The mixture was stirred at room temperature for 30 min, filtered over Celite® and the filtrate concentrated to a dark residue that was purified by flash silica gel chromatography eluting with dichloromethane : methanol (95 : 5 to 90 : 10). Product fractions were pooled and concentrated to give partially purified 2b as a deep purple solid. Further purification by preparative TLC was conducted on a 20 _ mg scale, eluting the plate with dichloromethane : methanol (90 : 10). The yield was ~55% : R 0.58 (dichloromethane : methanol, 85 : 15); HPLC fa 6.1 min (95.4% purity); Ή NMR (CDCI3) δ 7.48 (t, J= 8.4 Hz, 1 H), 6.92 (d, J= 8.2 Hz, 2 H), 5.98 (d, J= 15.4 Hz, 2 H), 5.14 (bs, 2 H), 4.97 (m, 1 H), 4.41 (t, J= 11.6, 2 H), 3.29 (s, 2 H), 3.13 (s, 1 H), 3.08 (s, 3 H), 3.01 (d, J= 9.3, 2 H), 2.65 (m, 4 H), 2.29 (s, 3 H), 2.09 (s, 3 H), 2.05 (s, 3 H), 1.79 (s, 3 H), 1.70 (m, 2 H), 1.60 (m, 2 H), 1.25 (s, 3 H), 1.15 (t, J= 7.1 Hz, 6 H), 0.94 (d, J= 7.1 Hz, 3 H), 0.76 (d, J= 6.9 Hz, 3 H), 0.63 (d, J= 7.1 Hz, 3 H); MS (ES + ) m/z 900.1 (M+H) + ; HRMS (MALDI) calcd for C49H61N3O13 [(M + H) + ],

900.4277; found 900.4269.

Benzoxazinorifamycin (2c). A mixture of aminophenol 11a (80 mg, 0.22 mmol), rifamycin S (12; 220 mg, 0.32 mmol) and 1,2-dichloroethane (10 mL) was stirred at room temperature for 16 h. The reaction mixture was then concentrated to a black solid that was dissolved in 10 mL of methanol and treated with MnC>2 (80 mg, 0.92 mmol). The mixture was stirred at room temperature for 30 min, filtered over Celite®, and concentrated to a dark residue that was purified by flash silica gel chromatography eluting with dichloromethane : methanol : NH4OH (94 : 6 : 0.5). Product fractions were pooled and concentrated to give a solid that was further purification by preparative TLC, eluting the plate with dichloromethane : methanol (92 : 8). The product band was processed to give 2c (170 mg, 74%) as a dark purple solid: HPLC 5.15 min (91.7% purity); Ή NMR (CDCI3) δ 7.56 (s, 1 H), 7.48 (t, J=

8.4 Hz, 1 H), 7.04 (s, 1 H), 6.98 (s, 1 H), 6.92 (d, J= 8.3 Hz, 1 H), 6.80 (d, J= 8.2 Hz, 1 H), 5.97 (m, 2 H), 5.12 (bs, 2 H), 4.94 (m, 1 H), 4.24 (t, J= 5.6 Hz, 2 H), 4.03 (t, J=

6.5 Hz, 2 H), 3.09 (s, 3 H), 3.01 (d, J= 9.3 Hz, 1 H), 2.68 (m, 2 H), 2.52 (bs, 11 H), 2.29, (s, 3 H), 2.09 (s, 3 H), 2.05 (s, 6 H), 1.80 (s, 5 H), 1.69 (m, 2 H), 1.60 (m, 2 H),

1.25 (s, 3 H), 0.93 (s, 3 H), 0.75 (s, 3 H), 0.64 (s, 3 H); MS (ES + ) m/z 1035.1 (M+H) + ; HRMS (MALDI) calcd for C49H61N3O13 [(M + H) + ], 1035.5074; found 1035.5095.

Benzoxazinorifamycin (2d). Reaction of a mixture of aminophenol lib (30 mg, 0.08 mmol), rifamycin S (12; 102 mg, 0.15 mmol) and 1,2-dichloroethane (4 mL) and subsequent purification was carried out exactly as described above for the synthesis of 2c to provide 2d (29 mg, 34.5%) as a dark purple solid: HPLC ti = 5.11 min (91.8% purity); Ή NMR (CDCI3) δ 7.50 (m, 2 H), 7.10 (s, 1 H), 6.95 (m, 2 H), 6.87 (d, J= 8.3 Hz, 1 H), 5.98 (s, 1 H), 5.96 (s, 1 H), 5.30 (s, 2 H), 4.95 (m, 1 H), 4.76 (m, 2 H), 4.25 (m, 2 H), 3.68 (s, 1 H), 3.63 (s, 1 H), 3.49 (s, 1 H), 3.44 (s, 1 H), 3.11 (s, 3 H), 3.03 (s, 1 H), 2.58 (m, 2 H), 2.51 (m, 4 H), 2.29 (s, 3 H), 2.09 (s, 3 H), 2.06 (s, 3 H), 1.86 (d, J = 8.5 Hz, 1 H), 1.80 (s, 3 H), 1.68 (s, 2 H), 1.61 (bs, 9 H), 1.25 (s, 3 H), 0.94 (s, 3 H), 0.75 (s, 3 H), 0.65 (s, 3 H). MS (ES + ) m/z 1049.2 (M+H) + ; HRMS (MALDI) calcd for C49H61N3O13 [(M + H) + ], 1049.4866; found 1049.4857.

RLZ analog (2e). A mixture of benzoxazinorifamycin 2d (5.5 mg, 0.005 mmol), l-(2- methylpropy piperazine (2.3 mg, 0.016 mmol; Oakwood Products Inc.), Mn02 (5 mg, 0.055 mmol) and DMSO (0.5 mL) was stirred at room temperature for 24 h and then filtered over Celite ® . The filtrate was concentrated and purified by preparative TLC, eluting with dichloromethane : methanol (90 : 10). The product band was processed to give 2e (4.6 mg, 74%) as a dark blue solid: HPLC 5.33 min (95.3% purity); MS (ES + ) m/z 1189.5 (M+H) + ; HRMS (MALDI) calcd for C49H61N3O13 [(M + H) + ],

1189.6180; found 1189.6193. MTB MIC 90 Assays

All compounds were evaluated for MIC90 vs. MTB H37RV using the microplate Alamar Blue assay (MABA) except that 7H12 media was used (replacing 7H9 + glycerol + casitone + OADC). The use of this and other redox reagents such as MTT have shown excellent correlation with colony-forming unit (CFU) _ based and radiometric analyses of mycobacterial growth in many laboratories. The MIC is defined as the lowest concentration effecting a reduction in fluorescence (or luminescence) of 90% relative to controls. Isoniazid and rifampin are included as positive quality control compounds with expected MIC ranges of 0.025-0.1 and 0.06-0.125 μg/mL, respectively.

The Low Oxygen Recovery (LORA) in vitro assay was designed to detect compounds which may have the potential for shortening the duration of therapy through (more) efficient killing of the non-replicating persistor (NRP) population. The assay involves l) adaptation of MTB to low oxygen through gradual, monitored, self-depletion of oxygen during culture in a sealed fermenter, 2) exposure for 10 days of the low-oxygen adapted culture to test compounds in microplates that are maintained under an anaerobic environment, thus precluding growth, and 3) subsequent evaluation of MTB viability as determined by the ability to recover. Recovery/viability is determined either a) by (aerobic) subculture onto solid, drug- free media and determination of colony forming units or b) by the extent to which a luciferase-expressing strain can recover the ability to produce luminescence.

Compounds such as isoniazid and ethambutol that are considered to be devoid of "sterilizing activity" are inactive in this assay while the rifamycins and the more potent fluoroquinolones, which do appear to eliminate some proportion of the persistor population and thus can affect treatment duration, are active, albeit at concentrations higher than the MICs for replicating cultures. Correlation between the CFU and luminescence readout is good with the exception of the fluoroquinolone class for which luminescence underestimates absolute activity but not relative activity. Expression and Purification ofMTB RNAP (WT and Riffi mutants)

The wild-type MTB RNAP and the RifR mutants were prepared as previously described with minor alterations. See, e.g., Gill, S. K. & Garcia, G. A. Tuberculosis 2011, 91 : 361-9. For cell lysis, the sonication method was preferred over the freeze/thaw method. For the remainder of the purification steps, the protocol outlined by Gill and Garcia was followed.

Cloning, Expression and Purification ofMTB SigA

The pAvitag vector (modified pMSCG7 vector with an Avitag introduced between Bglll and Kpnl sites) was linearized with Sspl at 37°C for 1 h and the reaction product was purified using the Qiagen PCR kit. The linearized pAvitag vector (1.6-2.0 μg) was treated with T4 DNA polymerase in 10x T4 polymerase buffer, 5 mM DTT, and 4 mM dGTP in a final reaction volume of 60 \iL. The reaction was incubated for 30 min at 22°C and then for 20 min at 75°C before being stored at -20°C. PCR primers were designed to amplify the Rv2703/sigA gene encoding SigA from pSROl. The primers included an overhang sequence that complemented the vector Ligation Independent Cloning (LIC) overhangs. The sigA gene was purified via Qiagen PCR kit. The purified PCR product (0.2 pmol) was incubated with T4 DNA polymerase, 5 mM DTT, 4 mM dCTP, 10x T4 DNA polymerase in a final reaction volume of 20 iL. The reaction was incubated for 30 min at 22°C and then for 20 min at 75°C and stored at -20°C. The treated sigA was incubated with treated pAvitag vector (-0.2 pmol) for 10 min at 22°C. Then 6.25 mM EDTA was added followed by incubation at 22°C for 5 min before reducing the temperature to 4°C. The annealed pAvitag vector containing sigA was transformed into BL21(DE3) CodonPlus RIPL cells.

For the expression of SigA protein in BL2l(DE3) CodonPlus RIPL cells, the cells were grown in 500 mL of 2x TY liquid cultures containing 100 μg/mL

carbenicillin and 30 μg/mL chloramphenicol at 37°C with vigorous shaking until cell density reached Αβοοηπι = 0.5-0.6. The protein was induced by the addition of isopropyl β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The cultures were allowed to incubate for an additional 20-24 hours at 19°C. The cells were harvested by centrifugation (6000xg 15 min, 4°C). The cell pellet of each 500-mL culture was re-suspended in 10 mL of Ni 2+ -NTA bind buffer (300 mM NaCl, 50 mM NaL PO i, 10 mM imidazole, pH 8.0). The freeze/thaw method was followed to lyse the cells, and it was repeated a total of three times. The sample was supplemented with 10 iL of Lysonase™ Bioprocessing Reagent and 100 micromolar of

phenylmethyl-sulfonyl fluoride (PMSF), and then the resulting lysate was cleared by centrifugation (21,000xg 30 min, 4°C). All further purification steps were performed at 4°C. The lysate was incubated with 2 mL Ni 2+ -NTA His ' Bind Resin overnight with gentle shaking. Each supernatant/resin mixture was applied to individual columns. The columns were washed twice with 4 mL of Ni 2+ -NTA wash buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 20 mM imidazole, pH 8.0), and the protein was then eluted in 6 mL of Ni 2+ -NTA elute buffer (300 mM NaCl, 50 mM NaH 2 P0 4 , 250 mM imidazole, pH 8.0). The protein was concentrated to a final volume of -500iL and then sterile-filtered with 0.22-micromolar syringe before being applied to a HiPrep 16/60 Sephacryl S-200 HR (GE Healthcare) column using RNAP storage buffer (10 mM Tris-HCl (pH 7.9), 0.1 mM EDTA, 0.1 mM DTT, 0.1 M NaCl) as the running buffer. The fractions containing SigA were pooled together and

concentrated to a final volume of -500 μίι using Amicon Centrifugal Filter Units (MWCO=10 kDa). The enzyme was mixed with one volume of 100% glycerol and stored in liquid nitrogen. The final concentration of the enzyme was determined via Bradford assay using the BkrRad Protein Assay Kit.

In Vitro Transcriptional Activity of MTB RNAPs and Dose Response Curves

Dose response studies with RLZ (2a) and analogs (2b-2e) were performed via rolling circle transcription assay as described previously to determine the IC50 values (See, e.g., Gill, S. K. & Garcia, G. A. Tuberculosis 2011, 91: 361-9). Each of the compounds was tested in duplicate (n=2). The concentration range used for the wild-type MTB RNAP (+/- SigA) was 1.56-100 nM for the RFL and the analogs (2a- 2e). The concentration ranges used for MTB RNAP (D435V) with SigA were as follows : for 2a and 2e (39.1-2500 micromolar); for 2b _ 2d (1.25-80 micromolar). The concentration ranges used for MTB RNAP (H445Y) with SigA were as follows: for 2a and 2e (20.5-5000 micromolar); for 2b 2d (8.2-2000 micromolar). The

concentration ranges used for MTB RNAP (S450L) with SigA were as follows: for 2a (8.2-2000 micromolar); for 2b (3.3-800 micromolar); for 2c and 2d (1.64-400 micromolar); for 2e (6.55-1600 micromolar). The final concentration of the wild-type MTB RNAP was 10 nM and the final concentrations of the mutant RNAPs were 100 nM. The core RNAP and SigA were incubated for 30 min on ice in lx RNAP reaction buffer (40 mM Tris-HCl (pH 8.0), 50 mM KC1, 10 mM MgCl 2 , 0.01% Triton X-100) before adding the analog and DNA nanocircle template (80 nM). Each reaction was initiated upon the addition of NTP solution (500 micromolar of each NTP). The IC50 values were determined via non-linear regression to a modified four parameter logistic equation. Human Pregnane X Receptor (hPXR) Activation Assay

To assess the ability of specific rifamycins to activate the human pregnane X receptor (hPXR), the hPXR activation assay system from Puracyp, Inc. was used. The manufacturer's protocol was followed for the 96 well plate assay. Briefly, the DPX2 cells were thawed in a 37°C water bath and mixed thoroughly with culture media. Then 100 μίι of cell mixture was transferred into each well and the plate was incubated overnight in a 5% CO2 incubator at 37°C. The following day, the dosing media was thawed in a 37"C water bath. The dilutions of RLZ and analogs (2a-2e) and RMP (l, positive control) were prepared as described in the manual. The 96 well plate was removed from the incubator and liquid from each well was discarded before adding 100 μίι of the dilutions to the specific wells. Each dilution of the rifamycin derivative was tested in duplicate. The plate was placed in the 5%

C02/37°C incubator again for 24 h. The next day, the CellTiter-Fluor Buffer and CellTiter- Fluor™ were thawed at room temperature before adding 5 μίι of

CellTiter- Fluor™ to 10 mL of CellTiter- Fluor Buffer. The wells of the 96 well plate were emptied again and 100 μίι of CellTiter-Fluor™ reagent was added to each well. The plate was incubated for 1 h in the 5% C02 37°C incubator. A Synergy HI Hybrid Multi-Mode Microplate Reader (BioTek) was used to measure fluorescence (λ βχ =390 nm; em =505 nm). To obtain luminescence readings, the contents of ONE- Glo™ Assay Buffer were added to the ONE-Glo™ Assay Substrate and then 100 μΐ, of mixture was transferred into each well. The plate was read after 5 min where the luminometer was set for 5 sec pre-shake with 5 sec/well read time. The Relative Luminescence Units (RLU) and Relative Fluorescence Units (RFU) were

determined as outlined under the "Quantitation of PXR Receptor Activation" section of the manual. The normalized luciferase activity (RLU/RFU) was divided by the normalized DMSO control to represent the data as "fold activation" relative to the control. The replicate data points were averaged and both the original data points and the average values were plotted as a function of log concentration versus PXR activation. The average values were then fit by non-linear regression to a modified four parameter logistic equation using Kaleidagraph (Synergy Software, Essex, VT). The model is represented by Equation 1 y = 1 + [(M3 - 1)/(1 + 10K Mi - M ° )*M2] )] (l) where M3 is the ECMAX, 1 is the lower limit of the assay, MO is the log of the rifamycin concentration, Ml is the log of the EC50, and M2 is the Hill slope. The data were normalized such that the lower limit was set to 1. Ml, M2, and M3 were fit by the regression.

Microsome stability assays

Test compound stock solutions were prepared at 200 micromolar in

acetonitrile. Two microliters were added to 198 μΐ PBS containing 1 mg/ml human or mouse microsomes. After mixing, 25 μΐ aliquots were dispensed in triplicate into 96-well plates. Control and reaction wells received 25 μΐ of PBS and of 2 mM

NADPH in PBS, respectively. Plates were incubated for 30 minutes at 37°C with shaking at 600 rpm. Internal standard solution (150 μΐ) was added to each well to quench the reaction. For controls, quenching was done prior to incubation. Plates were centrifuged at 4000 x gior 30 minutes at 4°C and the supernatant was collected for analysis. The percentage of a compound remaining and the half-life of a compound in microsomes are calculated from the measured concentrations of compound in the test reaction (Reaction) and the control reaction (Control) according to the following formulae: Percentage remaining = (Reaction / Control) x 100 (2)

Half-life = - (incubation time * In 2) / ln(Percentage remaining / 100) (3) Pharmacokinetics of Analog 2b

Previously described methods were followed (see, e.g., Jayaram, R. et al., Pharmacokinetics-pharmacodynamics of rifampin in an aerosol infection model of tuberculosis. Antimicrobial Agents and Chemotherapy 2003, 47(7), 2118-24;

Jayaram, R. et al., Isoniazid pharmacokinetics-pharmacodynamics in an aerosol infection model of tuberculosis. Antimicrobial Agents and Chemotherapy 2004, 48(8), 2951-7; Shudo, J. et al., In vivo assessment of oral administration of probucol nanoparticles in rats. Biol Pharm Bull2008, 31(2), 321-5, each incorporated herein by reference for all purposes). For the single dose studies, analog 2b was prepared in 0.5% CMC at 1 mg/ml. Healthy female BALB/c mice were administered 10 mg/kg of the suspension via oral gavage. Two mice per time point were used and 0.4 mg/kg fentanyl was given 15 minutes prior to bleeding by intraperitoneal injection. For each mouse, at least 100 μΐ of venous blood was collected via retro-orbital bleeding in BD Vacutainer® spray-coated K2EDTA tubes at 0.5, 1, 2, 4, 8, and 24 hours post- dose. Tubes were inverted several times and kept on ice. Blood was transferred to polypropylene tubes and centrifuged at 4,000 x gior 30 minutes at 4°C. The harvested plasma was transferred to new polypropylene tubes and stored at -80°C until analysis. To each sample, a 3x volume of chilled acetonitrile was added containing 0.2 micromolar internal standard (IS). The solution was vortexed and then subsequently centrifuged at 10,000 x gior 15 minutes. Calibration standard samples were prepared by spiking the stock solution of analog 2b in acetonitrile into mouse plasma to yield the following concentrations^ 0.097656, 0.195313, 0.390625, 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, and 50 micromolar.

Supernatant was injected into an LC-MS/MS for analysis. In addition, a blank (blank plasma extracted with a 3 x volume of IS) and a double blank (blank plasma extracted with a 3 x volume of pure acetonitrile) were prepared. The concentration of 2b in a blood sample for each time point was then determined.

For the multiple-dose study, mice were dosed once daily for 5 consecutive days by oral gavage. Blood samples were collected at time points of 0.5, 1, 2, 4, 8, and 24 hours and analyzed in the same way as in the single-dose study. After collecting the blood, the mice were sacrificed by carbon dioxide asphyxiation. Lung tissue was aseptically removed, rinsed in 3 ml PBS, air dried on sterilized gauze pads, weighed, and suspended in 4 x (w/v solvent/tissue) PBS buffer. Lung tissue was homogenized, mixed, and extracted with 3 x acetonitrile containing internal standard at 0.2 micromolar and centrifuged at 10,000 x gior 15 minutes at 4°C. The supernatant was collected for LC-MS/MS analysis. Calibration standard lung samples were prepared by spiking the stock solution of compound (in MeOH or MeCN) into homogenized mouse lungs and extracting with a 3 x volume of acetonitrile to yield the following concentrations: 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 micromolar. In addition, a blank (blank lung tissue extracted with a 3 x volume of IS) and a double blank (blank lung tissue extracted with a 3 x volume of pure acetonitrile) were prepared. The concentration of 2b in lung tissue for each time point was then determined. Example 1

Activity Against M. tuberculosis (H37RV) in Cell Culture

During the development of embodiments of the technology provided herein, the compounds were screened in assays to quantify their antitubercular activity under both aerobic and anaerobic conditions (Table l). Briefly, the 8 _ day microplate- based assay using Alamar blue reagent (added on day 7) for determination of growth (MABA) (see, e.g., Collins, L. & Franzblau, S. Antimicrob. Agents

Chemother. 1997, 41: 1004-09) gives an assessment of activity against replicating MTB, while the 11 -day high-throughput, luminescence-based low-oxygen-recovery assay (LORA) (see, e.g., Cho, S. H. et al. Antimicrob. Agents Chemother. 2007, 51: 1380-85) measures activity against bacteria in a non-replicating state that models clinical persistence. Minimum inhibitory concentration (MIC90) is defined as the lowest compound concentration effecting > 90% growth inhibition.

Under aerobic conditions (MABA), all newly synthesized compounds 2b - 2e displayed good activities, with MIC90 values of 0.02 - 0.08 micromolar, relative to RMP (l; 0.13 micromolar), but are less potent than RLZ (2a; < 0.004 micromolar). On the other hand, activity under anaerobic conditions (LORA) for "one armed" compounds 2b - 2d (MIC90 values of 0.35 - 0.40 micromolar) is essentially

equivalent to RMP, and these range from 4 - 18.5 fold higher than in the MABA. In contrast, LORA activity for RLZ analog 2e is strikingly poor being essentially inactive (MIC90 > 6.72 micromolar). The addition of the RLZ side chain into the 5' position of 2d introduces another basic moiety, which may impede transport across the cell membrane of the non-replicating bacterial strain of the LORA.

Table 1. MIC 90 values (micromolar) of benzoxazinorifamycin analogs versus MTB

Example 2

In vitro Inhibition of Wild-type and RMP-resistant Mutant MTB RNAPs

During the development of embodiments of the technology provided herein, inhibition constants (IC50) for RLZ and the analogs against the wild-type MTB RNAP and three rif-resistant (RifR) MTB RNAP mutants were determined via dose- response assays. Each compound was tested in duplicate dilution series ranging over the concentrations as specified below. The data were plotted as the logarithm of the rifamycin or analog concentration versus the activity relative to the activity without the RLZ or analog (expressed as a percentage). The data were then fit by nonlinear regression. The log IC50 values and the standard errors of the fit are reported in Table 2. The log IC50 values are such that the IC50 values are in units of micromolar. Negative log IC50 values reflect IC50 values less than in the micromolar range (e.g., in the nM range). Values were fit to a four-parameter logistic regression model with the top and bottom limits set at 100 and 0 respectively. The average Hill slope is 1.02. Table 2. Log IC 50 values and standard errors for rifalazil & analogs against RNAP

The standard errors of the loglCso values roughly translate into a 20-25% error in the IC50 values. The apparent IC50 values are listed in Table 3.

Table 3. Apparent IC 50 values (micromolar) for rifalazil & analogs against RNAP

All of the rifamycins inhibit the wild-type MTB RNAP in the 10 "9 M (nM) range. The lower limit of detection for this assay is an IC50 of -5-10 nM, thus it is possible that these rifamycins have true IC50 values lower than reported in Table 3. The IC50 values for RLZ with the RifR mutants of MTB were much higher, in the 10 " 4 M (-100 micromolar) range. The most frequently observed MTB RifR mutant in clinical isolates, S450L, was inhibited at 2- to 5-fold lower concentrations of 2b, 2c & 2e relative to RLZ with 2d being essentially the same as RLZ. The MTB D435V mutant was inhibited at 5" to 50-fold lower concentrations of 2b, 2c, 2d & 2e relative to RLZ. Interestingly, the H445Y mutant appears to retain resistance to all tested rifamycins. These results demonstrate that the potency of rifamycins towards RifR MTB RNAPs can be improved.

Example 3

Activation ofhPXR

Use of conventional rifamycins in humans is limited by their potent activation of the human pregnane X receptor (hPXR). Experiments were performed during the development of embodiments of the technology provided herein to measure the activation of hPXR by RLZ, RMP, and the analogs. These experiments were performed using a commercial kit as prescribed by the supplier (Puracyp, Inc.). Figure 3 shows the dose-response plots for these assays. RMP, as previously known, exhibits a high maximal degree of activation (~12-fold) and an EC50 of ~2 micromolar. RLZ has been reported to have essentially no ability to activate hPXR. The data confirm that at concentrations lower than 100 micromolar, RLZ exhibits no activation of hPXR. At 100 micromolar, RLZ does show ~2-fold activation;

however, it also shows ~2-fold loss of cell viability (suggesting cytotoxicity) at 100 micromolar. The loss of cell viability is shown in Table 4, where the mean relative fluorescence units are indicative of cell viability.

Analog 2d was fit to a dose-response curve that revealed a 6-fold maximal activation of hPXR and an EC50 of ~6 micromolar (Figure 3). This analog also starts to exhibit loss of cell viability at 25 micromolar (Table 4), such that the 100 micromolar data point was not used in the dose-response curve fit. Analog 2e shows hPXR activity very similar to that of RLZ, with no activation nor loss of cell viability below 25 micromolar and ~3-fold activation and -25% loss of cell viability at 100 micromolar. Analogs 2b and 2c were essentially identical with ~3-fold hPXR activation at 6.25 micromolar and dramatic loss of cell viability above 6.25 micromolar (such that no hPXR activation was seen). Table 4. Human pregnane X receptor (hPXR) activation

The modeling suggests that the additional bulk of the analogs reduces binding of the compounds to hPXR due to steric clashes with the binding pocket. Without being bound by any particular theory, it is likely that the flexible side chains in the binding pocket may allow for the side chains to adopt conformations that minimize this clash. The observation that 2e is very similar to RLZ in hPXR activation is consistent with this hypothesis since 2e is a very close analog of RLZ and has two added groups, thereby reducing their degrees of freedom. The lower toxicities of 2d and 2e show that improved rifamycin analogs are provided by the compositions and methods described herein. In these experiments, the value for a 1% DMSO Control was 67021 and for a dosing media control was 59992.

Example 4

Microsome Stability and Pharmacokinetics

During the development of embodiments of the technology, rifalazil and analog 2b were evaluated for metabolic stability in human microsomes. The data produced by the experiments demonstrated that each is relatively stable with estimated half-lives of 65 and 54 minutes, respectively (Table 5). Similarly, the estimated half- life of rifalazil in mouse microsomes is 53 minutes while that of the analog 2b is 141 minutes.

The pharmacokinetics of analog 2b (Figure 6) was assessed using a suspension prepared in 0.05% carboxymethylcellulose (CMC). In the single dose study, analog 2b was detected in the blood but the signal was below the lower limit of quantification. The C ma x was 0.0185 micromolar at a T ma x of 1 hour. However, analog 2b appears to accumulate in the blood of mice that were dosed once daily for 5 consecutive days with a C ma x of 1.74 micromolar at a T ma x of 2 hours, which is 100" fold higher than that observed after a single oral dose. In the lung tissue of these mice, analog 2b was also detected at a concentration of 1.79 micrograms/g (around 0.4 micromolar), but only exceeded the MIC of 0.02 micromolar for about 4 hours (Figure 7).

Table 5. Compound Remaining and Half-life of Selected Benzoxazinorifamycins after a 30 minute incubation in Human and Mouse Microsomes

All publications and patents mentioned in the above specification are herein incorporated by reference in their entirety for all purposes. Various modifications and variations of the described compositions, methods, and uses of the technology will be apparent to those skilled in the art without departing from the scope and spirit of the technology as described. Although the technology has been described in connection with specific exemplary embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in pharmacology, biochemistry, medical science, or related fields are intended to be within the scope of the following claims. Example 5

Synthesis and testing of dansyl rifalazil compounds

During the development of embodiments of the technology provided herein, rifalazil compounds were synthesized comprising a dansyl moiety. For the synthesis, all reagents were commercially available and used without further purification. Routine Ή and 13 C NMR spectra were obtained on a Varian

spectrometer at 400 MHz for Ή and 101 MHz for 13 C with CDC1 3 , CD3OD, or DMSO-ofe as solvents. Chemical shifts are reported relative to the residual solvent peak in δ (ppm). Mass spectra were recorded on a Micromass LCT time-of-flight instrument utilizing the electrospray ionization mode. Compound purity was assessed by analytical HPLC, which was performed on an Agilent 1100 series system with an Agilent Eclipse plus C18 (4.6 mm x 7.5 mm, 3.5 mm particle size) column. The mobile phase was an 11 -minute binary gradient of acetonitrile

(containing 0.1% TFA) and water (10 ~ 90%). Thin-layer chromatography (TLC) was performed on silica gel GHLF plates (250 μπι) purchased from Analtech. Extraction solutions were dried over MgSC or Na2SC>4 prior to concentration. Rifamycin S was obtained from AAPharmaSyn (Ann Arbor, MI). Other reagents and solvents were purchased from common vendors and were used without purification unless indicated otherwise. A schematic describing a synthesis of embodiments of the dansyl compounds is provided in Figure 6. In some embodiments, the following synthetic steps are provided: l-Benzyloxy-3-(2-bromoethoxy)-2-nitrobenzene (39). A mixture of nitrophenol 5 (1.226 g, 5.0 mmol), 1,2-dibromoethane (6.43 mL, 74.6 mmol), Cs 2 C0 3 (4.07 g, 12.5 mmol) and DMF (10 mL) was stirred at room temperature for 12 h. DMF was removed in vacuo to leave an oil that was distributed between 1% aq HCl and ethyl acetate. The organic phase was dried and concentrated to a residue that was purified by flash silica gel chromatography. Product fractions were pooled and concentrated to give 39 (1.56 g, 89%) as a light yellow solid, mp 83 °C, following crystallization from ethyl acetate: Ή NMR (400 MHz, CDCI3) δ 7.38 - 7.34 (m, 3H), 7.24 - 7.32 (m, 3H), 6.68 (d, J= 8.6, 1H), 6.60 (d, J= 8.5, 1H), 5.16 (s, 2H), 4.35 (t, J = 6.6, 2H), 3.58 (t, J= 6.5, 2H) ; MS (ES + ) m/z 352.2 [M+H] + .

2-(3-Benzyloxy-2-nitrophenoxy)-N-methylethan-l-amine (40a). A solution of bromoethyl ether (39; 500 mg, 1.4 mmol), methylamine (4 niL, 33 wt % solution in EtOH), 14 mmol) and acetonitrile (10 mL) was stirred at room temperature for 24 h. Upon completion of the reaction, solvent was evaporated. The solution was concentrated and the residue was partitioned between water and ethyl acetate (3x). The combined organic fractions were washed with saturated brine (2x), dried over anhydrous sodium sulfate, and concentrated.

Purification by flash silica gel chromatography (gradient elution with 100 : 0; 80 ; 20; 50 : 50; 0 ; 100 ethyl acetate and methanol, respectively), combination of product fractions, and concentration left 40a (326 mg, 76%) as a pale brown oil : Ή NMR (CDCla) δ 7.51 - 7.07 (m, 5 H), 6.62 (t, J= 8.0 Hz, 2 H), 5.13 (s, J= 6.6 Hz, 2 H), 4.14 (dd, J= 12.2, 7.2 Hz, 2 H), 2.92 (br. s, 2 H), 2.46 (s, 3 H), 1.26 (br. s, 1 H); 13 C NMR (CDCla) δ 151.21, 150.86, 135.56, 131.02, 128.63, 128.18, 126.98, 106.18, 105.82, 69.08, 50.28, 36.28, 29.67; MS (ES + ) m/z 303.1 [M+H] + , 325.1 [M+Na] + , 599.2

[2M+H] + , 627.2 [2M+Na] + . 4-(3-(Benzyloxy)-2-nitrophenoxy)-JVmethylbutan-l-amine (40b). A solution of bromobutyl ether 8 (0.63 g, 1.7 mmol), methylamine (8 mL of 33% solution in EtOH, 66.5 mmol), A^iV-diisopropylethylamine (6 mL), and acetonitrile (20 mL) was stirred at room temperature overnight in a sealed flask. The solution was concentrated and the residue was distributed between dichloromethane and 5% aqueous sodium carbonate. The organic phase was dried and concentrated to an orange oil that was purified by flash silica gel chromatography eluting with dichloromethane : methanol : NH4OH (90 ; 10 : 0.5). Product fractions were combined and concentrated to leave 40b (0.45 g, 83%) as an oil: Ή NMR (DMSO-afe) δ 7.40-7.32 (m, 6 H), 6.95 (d, J= 8 Hz, 1H), 6.88 (d, J= 8 Hz, 1H), 5.25 (s, 2 H), 4.11 (t, J= 6 Hz, 2H), 2.55 (t, J= 6 Hz, 2H), 2.31 (s, 3 H), 1.71-1.66 (m, 2 H), 1.54-1.46 (m, 2 H); i3 C NMR (DMSO-afe) δ 150.8, 150.4, 136.4, 132.0, 128.9, 128.6, 127.8, 110.0, 106.7, 106.6, 70.7, 69.4, 50.7, 35.6, 26.5, 25.0; MS (ES + ) m/z 331.2 (M + H) + .

N- (2 - (3 - Benzyloxy - 2 -nitrophenoxy) ethyl] - 5- (dimethy lamino) -N- methylnaphthalene- 1- sulfonamide (41a). A mixture of amine 40a (200 mg, 0.66 mmol), dansyl chloride (178 mg, 0.66 mmol), pyridine (530 μΐ ^ , 6.6 mmol), and dichloromethane (6 mL) was stirred at room temperature for 8 h. The mixture was partitioned between water and ethyl acetate (3x). The combined organic fractions were washed with saturated brine (2x), dried, and concentrated. Purification by flash silica gel chromatography (gradient elution with 50^50; 0^100 hexane and ethyl acetate, respectively) gave 41a (354 mg, quantitative) as a brown solid: Ή NMR (CDC1 3 ) δ 8.46 (d, J= 8.5, 1 H), 8.24 (d, J= 8.7, 1 H), 8.05 (d, J= 7.3, 1 H), 7.44 (dt, J= 13.9, 7.9, 2 H), 7.34 - 7.13 (m, 1 H), 7.16 - 6.99 (m, 6 H), 6.58 (d, J= 8.5, 1 H), 6.46 (d, J= 8.5, 1 H), 5.09 (s, 2 H), 4.16 (t, J= 5.3, 2 H), 3.55 (t, J= 5.3, 2 H), 2.92 (s, 3 H), 2.81 (s, 6 H); i3 C NMR (CDCI3) δ 151.73, 150.85, 150.63,135.50, 134.15, 130.11, 128.65, 126.98, 123.11,

119.39, 115.26, 106.52, 105.33, 71.01, 69.25, 48.82, 45.38, 36.78; MS (ES + ) ^ 536.2 [M+H] + , 558.2 [M+Na] + .

N- (4- (3 - (Benzyloxy) - 2 - nitrophenoxy)butyl) - 5 - (dimethy lamino) -N- methylnaphthalene-1 -sulfonamide (41b). A mixture of the substituted amine 40b (0.44 g, 1.3 mmol), dansyl chloride (0.36 g, 1.3 mmol), and pyridine (l.l mL, 13.4 mmol) in dichloromethane (20 mL) was stirred at rt overnight. The mixture was distributed between water and ethyl acetate. The organic phase was then dried, concentrated, and purified by flash silica gel chromatography eluting with hexane/ethyl acetate (5 ; l). Product fractions were pooled and concentrated to give 41b (0.33 g, 44%) as an oil: Ή NMR (CDCI3) δ 8.53 (d, J= 8 Hz, 1H), 8.34 (d, J= 8 Hz, 1H), 8.12 (d, J= 8 Hz, 1H), 7.51 (m, 2 H), 7.36-7.24 (m, 6 H), 7.17 (d, J= 8 Hz, 1H), 6.62 (d, J= 8 Hz, 1H), 6.57 (d, J= 8 Hz, 1H), 5.16 (s, 2 H), 4.02 (t, J= 6 Hz, 2H), 3.25 (t, J= 6 Hz, 2H), 2.28 (s, 9 H), 2.81 (s, 3 H), 1.76-1.62 (m, 4 H); 13 C NMR (CDCI3) δ 151.6, 151.2, 150.8, 135.6, 134.1, 130.9, 130.2, 130.0, 129.6, 128.6, 128.2, 128.0, 127.0, 123.1, 119.7, 115.2, 110.0, 105.9, 105.7, 70.9, 68.4, 48.9, 45.4, 33.8, 25.6, 23.6; MS (ES + ) m/z 586.3 (M + H) + .

N- (2 - (2 - Amino - 3 -hy droxyphenoxy)ethyl- 5 - (dimethylamino) -N- methylnaphthalene- 1 - sulfonamide (42a). To a solution of dansyl sulfonamide (41a) (67 mg, 125 μιηοΐ) in glacial acetic acid (8 mL) in a Parr hydrogenation bottle was added 20% Pd(OH)2/C (20 mg). The resultant mixture was hydrogenated at 45 psi ¾ for 17 h. The reaction mixture was rapidly filtered over Celite®, and the filtrate was concentrated. The residue was dissolved in methanol and concentrated over several cycles until almost all the acetic acid was gone. Crude product 42a (43 mg, 83%) was used in the next step without purification: MS (ES + ) m/z 416.2 [M+H] + , 438.2 [M+Na] + .

N- (4- (2 - Amino - 3 -hy droxyphenoxy)butyl) - 5 - (dimethylamino) -N- methylnaphthalene- 1-sulfonamide (42b). Dansyl sulfonamide 41b (0.30 g, 0.54 mmol) was dissolved in a mixture of 1 N aqueous HC1 (5 mL) and methanol (45 mL) in a 250 mL Parr hydrogenation bottle. Catalyst (20% PdiOH C, 0.03 g) was added and the mixture was hydrogenated at 40 psi H2 for 24 h. The reaction mixture was rapidly filtered over Celite®, and the filtrate was concentrated and diluted with ethyl acetate. The solution was washed with 5% aqueous sodium carbonate, dried, and concentrated to an oil that was purified by silica gel fast flash chromatography eluting with hexane/ethyl acetate (l ; l). Product fractions were pooled and concentrated to leave 42b (0.24 g, 98%) as a solid: Ή NMR (CDCI3) δ 8.53 (d, J= 8 Hz, 1H), 8.34 (d, J= 8 Hz, 1H), 8.15 (d, J= 8 Hz, 1H), 7.51 (m, 2 H), 7.17 (d, J= 8 Hz, 1H), 6.59 (d, J= 8 Hz, 1H), 6.43-6.36 (m, 2 H), 3.92 (t, J= 6 Hz, 2H), 3.28 (t, J= 6 Hz, 2H), 2.87 (s, 9 H), 2.84 (s, 3 H), 1.80-1.70 (m, 4 H); i 3 C NMR (CDCI3) δ 151.6, 148.3, 145.0, 134.1, 130.3, 129.8, 128.0, 123.6, 123.1, 119.6, 118.4, 115.2, 108.5, 104.5, 67.6, 49.2, 45.4, 33.9, 26.2, 24.2; MS (ES + ) ^444.3 (M + H) + .

Benzoxazinorifamycin dansyl probe 43a. A solution of dansyl aminophenol 42a (43 mg, 103 μιηοΐ), rifamycin S (12; 286 mg, 412 μιηοΐ), and 1,2-dichloroethane (5 mL) was stirred in the dark at room temperature for 60 h. The mixture was concentrated to a crude solid that was purified by flash silica gel chromatography (gradient elution from 90=10 to 40=60 hexane:ethyl acetate and then 80=20 to 0=100

ethylacetate : methanol, respectively). Product fractions were pooled and

concentrated to provide 43a (13 mg, 67%; 72% purity by HPLC) as a deep purple solid: MS (ES + ) m/z l091A [M+H] + , 1113.3 [M+Na] + , 1059.3 [M+H-MeOH] + , 999.3 [M+H-MeOH-AcOH] + .

Benzoxazinorifamycin dansyl probe 43b. A mixture of dansyl aminophenol 42b (0.24 g, 0.541 mmol), rifamycin S (12; 0.75 g, 1.1 mmol), and 1,2-dichloroethane (20 mL) was stirred at room temperature overnight. The mixture was then concentrated to a black solid that was dissolved in 20 mL of methanol and treated with MnC>2 (0.3 g, 3.5 mmol). The mixture was stirred at room temperature for 30 min, filtered over Celite®, and the filtrate concentrated to a dark residue that was purified by flash silica gel chromatography, eluting with hexane/ethyl acetate (l ; l to 1 ; 2). Product fractions were pooled and concentrated to give 43b (168 mg, 28%) as a deep purple solid: R f = 0.44 (ethyl acetate); HPLC = 8.69 min (94% purity); MS (ES + ) m/z 1119.8 (M + H) + . After synthesis, data were collected in experiments to test the activity of dansyl rifalazil compounds, e.g., against RNA polymerase and bacteria. Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO). Kool™ NC-45™ Universal RNA Polymerase template was from Epicentre (Madison, WI). Carbenicillin (disodium salt), corning microplates, bactotryptone, and yeast extract were from Fisher Scientific (Hampton, NH). The E. coJi ' Bh21 (DE3) CodonPlus-RIPL and Epicurian coli XL2-Blue Ultracompetent cells were from Agilent Technologies (Santa Clara, CA). QuantHT™ RiboGreen RNA Reagent and RNaseOUT™ Recombinant Ribonuclease Inhibitor were from Invitrogen (Carlsbad, CA). The Ni-NTA His»Bind ® resin was from Novagen (San Diego, CA). The nucleotide triphosphates (NTPs) were from Roche Applied Science (Indianapolis, IN). PhastGel Precast Gels and SDS Buffer Strips were from VWR (Arlington Heights, IL). The BkrRad Protein Assay kit was from BkrRad (Hercules, CA). The EC2880 strain (permeable strain with toJC ~ and imp- mutations) was a generous gift from Dr. Michael Hubband (Pfizer Scientific). Rifamycin S was from AAPharmaSyn LLC. Rifampin was from Roche Scientific. Dansyl chloride and dansyl amide were from Sigma-Aldrich.

Wild-type and RifR MTB RNAPs and MTB SigA were prepared as previously described in Gill, et al. (2012) "Structure-based Design of Novel

Benzoxazinorifamycins with Potent Binding Affinity to Wild-type and Rifampin- resistant Mutant Mycobacterium tuberculosis RNA polymerase." Journal of

Medicinal Chemistry. RNAP activity and inhibition were estimated by the production of RNA synthesis as described (Gill 2012). The IC50 values were determined via dose response studies (Table 6). Compound 43b was tested in duplicate (n=2). The final concentration of the WT MTB RNAP was 10 nM, whereas the final concentration of the RifR RNAPs was 100 nM in the reactions. Compound 43b was also evaluated for MIC90 vs. MTB H37RV using the Microplate Alamar Blue Assay (MABA) and the Low Oxygen Recovery Assay (LORA) (Cho et al. (2007) "Low-Oxygen-Recovery Assay for High-Throughput Screening of Compounds against Nonreplicating Mycobacterium tuberculosis" Antimicrob. Agents

Chemother. 51(4): 1380-1385; Collins, L. and S. G. Franzblau (1997) "Microplate alamar blue assay versus BACTEC 460 system for high- throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium"

Antimicrob. Agents Chemother. 41(5): 1004-1009; Gill (2012)). Additionally, the MIC90 values were determined for E. coliby the microdilution method described previously (Wiegand et al. (2008) "Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances" Nature Protocols 3(2): 163-175; Gill, S. K. and G. A. Garcia (2011) "Rifamycin inhibition of WT and Rif-resistant Mycobacterium tuberculosis and Escherichia coli RNA polymerases in vitro" Tuberculosis 91(5): 361-369). Results of the experiments showed that rifamycin 43b binds tightly to the WT MTB RNAP (with and without sigma factor, σ Α ) with the IC50 values in the 10 "9 M (nM) range, whereas the RifR MTB RNAPs (+σ Α ) were inhibited at higher concentrations with the IC50 values in the 10 -6 M (μΜ) range. Due to the availability of the in vitro rolling circle transcription assay, the IC50 values of compound 43b were determined against WT and RifR MTB RNAPs (Table 6).

Table 6. In vitro RNAP IC 50 Values (μΜ) for 43b

The MIC90 values are also reported against the MTB virulent strain (H37RV) and the E coli strains (Table 7). 43b inhibited the MTB with MIC90 values 0.009 μΜ and 0.93 μΜ for MABA and LORA, respectively. The MIC90 values were higher for E. coli strains including the fo/Cknockout strain (EC2880).

Table 7. 43b MIC 90 values against MTB and E. coli