WO2018152408A9

WO2018152408A9 - Pleuromutilin derivatives and uses thereof

Info

Publication number: WO2018152408A9
Application number: PCT/US2018/018503
Authority: WO
Inventors: Michio Kurosu; Katsuhiko MITACHI
Original assignee: University Of Tennessee Research Foundation
Priority date: 2017-02-17
Filing date: 2018-02-16
Publication date: 2018-09-20
Also published as: WO2018152408A1

Abstract

Provided herein are pleuromutilin derivatives and analogs that are useful as antimicrobial agents against Acinetobacter baumannii and Mycobacterium.

Description

PLEUROMUTILIN DERIVATIVES AND USES THEREOF

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No 62/460,400, filed February 17, 2017. The entire content of this application is incorporated herein by reference in their entirety.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. AI094411 awarded by the National Institutes of Health. The Government has certain rights in the invention.

TECHNICAL FIELD

This disclosure relates to novel derivatives and analogs of pleuromutilin useful as antimicrobial agents. Derivatives and analogs of pleuromutilin are useful against Mycobacterium and Acinetobacter baumannii.

BACKGROUND

Acinetobacter baumannii is one of the most common healthcare-associated Gram-negative bacteria that causes a variety of diseases, ranging from pneumonia to serious blood infections, and affects people with compromised immune systems and chronic lung disease. A significant increase in the incidence of multidrug-resistant (MDR) strains has raised the profile of this emerging opportunistic pathogen. Due to its ability to survive on artificial surfaces and to resist desiccated conditions, A. baumannii survives in food, water, or soil, and it can be spread by direct contact. However, A. baumannii infections are commonly implicated in nosocomial infections, and in some countries high isolation rates of drug resistant A. baumannii have been reported from patients in the intensive care unit (ICU). Lately, high incidence of MDR A. baumannii infections has been reported in veterans in Iraq and Afghanistan. A. baumannii targets mucous membranes and injured skin area, causing necrotizing infections that lead to septicemia and death. Mortality rates from A. baumannii septicemia were reported to be 34.0-43.4% in the ICU and 16.3% outside the ICU. Clinically isolated A. baumannii strains acquired a wide array of drug resistant mechanisms including a variety of efflux mechanisms that show resistance to all commonly prescribed antibacterial drugs. The therapeutic strategies for treating these highly resistant organisms are associated with significant toxicity with the limited number of antibacterial agents (e.g. sulbactam, imipenem-cilastatin, meropenem, doripenem, amikacin, tobramycin, polymyxins (or colistin), tigecycline, and minocycline). Although a significant amount of studies have been devoted to optimizing the use of currently available agents or identifying any combination thereof, new chemical entities have not been developed for the treatment of MDR A. baumannii infection since the introduction of tigecycline in 2005. Thus, there is an urgent need for new agents for use in treating infections caused by A. baumannii.

Since the discovery of diverse structures of bacterial protein biosynthesis inhibitors (e.g. aminoglycosides, tetracyclines, chloramphenicol, linezolid, lincosamides, spectinomycin, streptogramins, mupirocin, and macrolides), rRNA is still an attractive antibacterial drug target. Pleuromutilin, a strong binder of the 23 S rRNA subunit, was first isolated in 1952 from two Basidiomycete spp. (Pleurotus mutilus and Pleurotus passeckerianus). Pleuromutilin analogs such as tiamulin and valnemulin are used in veterinary medicine. Although, activity of pleuromutilins against Gram-positive bacteria was known since 1952, the effectiveness of pleuromutilin analogs against Mycobacteria spp. was not reported until recently (Long et. al. 2009). Valnemulin interferes with the bacterial translation by binding at two ribosomal key sites known as the "A" site and the "P" site, resulting in the inhibition of peptide elongation and the cessation of bacterial growth. Oxazolidinone drugs such as linezolid bind to the "A" site and only partially overlaps with that of the pleuromutilin class.

There remains a need for pleuromutilin derivatives and methods of use thereof as antimicrobial agents.

SUMMARY

In a first aspect, the present disclosure provides a compound having structure of formula (I):

or a pharmaceutically acceptable salt thereof; wherein L is -NH- or - NHCH₂C(CH₃)2S- ; Q is a divalent amino acid residue; R is hydrogen or C₁-C₁₀ alkyl that is optionally substituted with one or more groups selected from amino, (C₁- C₆)alkylamino, di(C₁-C₆)alkylamino, hydroxy, (C₁-C₆)alkoxy, and oxo; or Q-R is - C(0)-C₁-C₆ alkyl; R^a is hydrogen and R^b is hydroxyl; or R^a is hydroxyl and R^b is hydrogen; or R^a and R^b together are an oxo substituent; or R^a and R^b together are =N- OR^c, wherein R^c is hydrogen or C₁-C₆ alkyl; and R^d is -CH₂CH₃ or -CH=CH₂.

In another aspect, provided herein is a compound selected from the compounds of Table 1, and pharmaceutically acceptable salts thereof:

Table 1







In another aspect, the present disclosure provides a pharmaceutical composition comprising any of the compounds described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition finds use in a method for treating a bacterial infection in a patient in need thereof. In some embodiments, the pharmaceutical composition together with an additional suitable therapeutic agent finds use in a method for treating a bacterial infection in a patient in need thereof.

In another aspect, the present disclosure provides a method for treating a bacterial infection in a patient in need thereof, comprising administering to the patient any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the compounds described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the method comprises administering to the patient any of the compounds as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising any of the compounds described herein, or a pharmaceutically acceptable salt thereof, together with an additional suitable therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A and FIG. IB show checkerboards for synergistic combinations of doxycycline with compound 51 or valnemulin.

FIG. 2 shows the efficacy of compound 51 in a mouse model of infection.

FIG. 3 shows pleuromutilin and representative pleuromutilin analogs. FIG. 4 shows general structures of pleuromutilin derivatives with 3- methoxyamine oxime, 3-hydroxyamine oxime, and 3-hydroxyl substituents (A, B, and C, respectively) and anti-Acinetobacter molecules 51-54.

FIG. 5 shows the synthesis scheme of pleuromutilin analogs 51-54.

FIG. 6 shows the valnemulin core structure for anti-Acinetobacter agents with

3-hydroxy, N-alkylation or -acylation, and 12-methylene saturation modifications for improved activity.

FIG. 7 shows the half-life and cytotoxicity of compound 51 versus valnemulin.

FIG. 8 shows the effect of 51-Dox 35/1 and compound 51 on survival rate in the mouse infected with A. baumannii.

FIG. 9 shows the amino acid alignment of the 50S ribosomal protein L3 (RplC) from a 1 -resistant A. baumannii strain (1^R).

FIG. 10 depicts the kill-curve of selected compounds with activity against Mycobacterium tuberculosis H37R_V

FIG. 11 shows the in vitro metabolic stability of pleuromutilin analogs (50, 41 and 42).

FIG. 12 shows the structures of pleuromutilin derivatives that are anti-Mtb molecules.

FIG. 13 shows the synthesis scheme of pleuromutilin analogs 50, 41 and 42.

FIG. 14 depicts the binding of tiamulin and linezolid on a ribosome.

FIG. 15 shows the effect of pleuromutilin analogs and representative TB drugs against intracellular Mtb CDC1551-tdTomato (a transformant Mtb CDC1551 containing tdTomato) in macrophages (J774A.1 cells).

FIG. 16 depicts the in vitro time-kill assessment of valnemulin, analogs 50, 41,

42 and the first line TB drugs (RIF and INH).

FIG. 17 shows the in vitro metabolic stability of pleuromutilin analogs (50, 41 and 42). FIG. 18 shows the concentration of compound 50 in plasma and lungs over time after intravenous administration in mice

FIG. 19 shows the amino acid alignment of the 50S ribosomal protein L3 (RplC) from a 51 -resistant A. baumannii strain (51^R).

DETAILED DESCRIPTION

Compounds of the Invention

Compounds of the invention are provided in the following aspects and embodiments, e.g., compounds of any one of formulas (I), (II), (III), (IV), (V), (VI), (VI- 1), (VI-2) and pharmaceutically acceptable salts thereof.

In one aspect, provided herein is a compound having the structure of formula

or a pharmaceutically acceptable salt thereof; wherein

L is -NH- or -NHCH₂C(CH₃)₂S-;

Q is a divalent amino acid residue;

R is hydrogen or C₁-C₁o alkyl that is optionally substituted with one or more groups selected from amino, (C₁-C₆)aIkylamino, di(C₁-C₆)alkylamino, hydroxy, (d- C₆)alkoxy, and oxo;

or

Q-R is -C(0)-C₁-C₆ alkyl;

R^a is hydrogen and R^b is hydroxyl; or

R^a is hydroxyl and R^b is hydrogen; or

R^a and R^b together are an oxo substituent; or

R^a and R^b together are =N-OR^c, wherein R^c is hydrogen or C₁-C₆ alkyl; and

R^d is -CH2CH3 or -CH=CH₂.

In an embodiment, the compound of formula (I) has the structure of formula

embodiment, the compound of formula (I) has the structure of formula

(III):

embodiment, the compound of formula (I) has the structure of formula

(IV):

embodiment, the compound of formula (I) has the structure of formula

(V):

In an embodiment, the compound of formula (I) has the structure of formula

(VI):

wherein R¹ is aryl, heteroaryl, linear or branched C₁-C₆ alkyl, or the side chain of a proteinogenic a-amino acid.

In an embodiment, the compound of formula (VI) has the structure of formula (VI-1):

embodiment, the compound of formula (VI) has the structure of formula

(VI-2):

In certain embodiments, R is -CH₂CH₃. In other embodiments, R is

CH=CH₂.

In certain embodiments, R is hydrogen. In other embodiments, R is a -(C₂- C₆)-N]¾ group, wherein the carbon chain is optionally substituted with one or more groups selected from amino, (C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, hydroxy, (d- C₆)alkoxy, and oxo.

In other embodiments, R is R², R³ or R⁴, wherein

In certain embodiments Q is alanine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, or valine.

In a certain embodiment, Q is leucine.

In an embodiment, the compound of the invention (i.e.., a compound of any one of formulas (I), (II), (III), (IV), (V), (VI), (VI- 1), (VI-2) or a pharmaceutically acce table salt thereof) is selected from the compounds of Table 1 :







In another aspect, provided herein is a compound having the structure of formula (VIII):

or a pharmaceutically acceptable salt thereof; wherein

L is -NH- or -OH

Q is hydrogen or absent;

R^a is hydrogen and R^b is hydroxyl; or

R^a is hydroxyl and R^b is hydrogen; or

R^a and R^b together are an oxo substituent; or

R^a and R^b together are =N-OR^c, wherein R^c is hydrogen or C₁-C₆ alkyl; and R^d is -CH₂CH₃ or -CH=CH₂.

In one aspect, provided herein is a compound having the structure of formula

(la):

or a pharmaceutically acceptable salt thereof; wherein

L is -NH- or -NHCH₂C(CH₃)₂S-; Q is a divalent amino acid residue;

R is hydrogen or C₁-C₁o alkyl that is optionally substituted with one or more groups selected from amino, (C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, hydroxy, (C₁- C₆)alkoxy, and oxo;

or

Q-R is -C(0)-C₁-C₆ alkyl;

R^a is hydrogen and R^b is hydroxyl; or

R^a is hydroxyl and R^b is hydrogen; or

R^a and R^b together are an oxo substituent; or

R^d is C1-C4 alkyl or C1-C4 alkenyl.

In a certain embodiment, Q is leucine.

In another aspect, provided herein is a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition further comprises an additional therapeutic agent. In a particular embodiment, the additional therapeutic agent is doxycycline.

In another particular embodiment, provided herein is a pharmaceutical composition comprising compound 51, doxycycline and a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method for treating a bacterial infection in a patient in need thereof, comprising administering to the patient a compound of the invention, or a pharmaceutical composition comprising a compound of the invention.

In an embodiment, the bacterial infection is an infection of Acinetobacter baumannii, Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Mycobacterium tuberculosis. In an embodiment, the bacterial infection is an infection of Mycobacterium tuberculosis. In an embodiment, the bacterial infection is an infection of Acinetobacter baumannii. In an embodiment of the method, the bacterial infection is an infection of Mycobacterium tuberculosis and the compound is selected from compounds 41, 42 and 50.

In an embodiment of the method, the bacterial infection is an infection of Acinetobacter baumannii and R^a is hydroxyl and R^b is hydrogen in the compound.

In an embodiment of the method, the bacterial infection is an infection of a Gram-positive bacterium, and R^d is -CH₂CH₃ in the compound.

In an embodiment, the method comprises administering to the patient a compound of the invention, and further comprises administering an additional therapeutic agent. In a particular embodiment, the additional therapeutic agent is doxycycline. In another particular embodiment, provided herein is a pharmaceutical composition comprising compound 51, doxycycline and a pharmaceutically acceptable carrier.

In another particular embodiment, provided herein is a method for treating a bacterial infection in a patient in need thereof, comprising administering to the patient compound 51 and doxycycline, or a pharmaceutical composition comprising compound 51 in combination with doxycycline. In one embodiment, compound 51 and doxycycline are administered simultaneously. In another embodiment, compound 51 and doxycycline are administered sequentially.

Definitions

As used herein, "proteinogenic a amino acid" refers to an amino acid that is incorporated biosynthetically into proteins during translation. In an embodiment, proteinogenic amino acids are selected from histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, alanine, arginine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, proline, serine, tyrosine, asparagine, selenocysteine, pyrrolysine.

As used herein, the term "divalent amino acid residue" refers to a divalent moiety comprising a carbonyl, an amino group alpha or beta to the carbonyl, and optionally a side chain comprising one or more alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl, alkoxyl, thiol, thioalkyl, amino, alkylamino, carboxyl, or carboxamido groups. In certain embodiments, a divalent amino acid residue has the formula: -C(0)-CH(R)-NH-, wherein R is a hydrogen or a side chain as described above. In a particular embodiment, R is the side chain of a proteinogenic amino acid.

As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon moiety. Preferably the alkyl comprises 1 to 20 carbon atoms, 1 to 16 carbon atoms, 1 to 10 carbon atoms, or more preferably 1 to 6 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isο-propyl, n-butyl, sec-butyl, isο-butyl, icri-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2- dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl and the like. Furthermore, the expression "C_x-C_y-alkyl", wherein x is 1-5 and y is 2-10 indicates a particular alkyl group (straight- or branched- chain) of a particular range of carbons. For example, the expression C1-C4 alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

As used herein, the term "alkoxy" refers to -O-alkyl.

The term "cycloalkyl" refers to an optionally substituted non-aromatic cyclic hydrocarbon ring, which optionally includes an alkylene linker through which the cycloalkyl may be attached. Exemplary "cycloalkyl" groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and substituted versions thereof. As used herein, the term "cycloalkyl" includes an optionally substituted fused polycyclic hydrocarbon saturated ring and aromatic ring system, namely polycyclic hydrocarbons with less than maximum number of non- cumulative double bonds, for example where a saturated hydrocarbon ring (such as a cyclopentyl ring) is fused with an aromatic ring (herein "aryl," such as a benzene ring) to form, for example, groups such as indane.

The term "aryl" refers to an optionally substituted benzene ring or to an optionally substituted fused benzene ring system, for example anthracene, phenanthrene, or naphthalene ring systems. Examples of "aryl" groups include, but are not limited to, phenyl, 2-naphthyl, 1-naphthyl, and the like.

The term "heteroaryl" refers to an optionally substituted monocyclic five to seven membered aromatic ring, or to an optionally substituted fused bicyclic aromatic ring system comprising two of such aromatic rings. These heteroaryl rings contain one or more nitrogen, sulfur, and/or oxygen atoms, where N-oxides, sulfur oxides, and dioxides are permissible heteroatom substitutions. Examples of "heteroaryl" groups used herein include, but should not be limited to, furan, thiophene, pyrrole, imidazole, pyrazole, triazole, tetrazole, thiazole, oxazole, isoxazole, oxadiazole, thiadiazole, isothiazole, pyridine, pyridazine, pyrazine, pyrimidine, quinoline, isoquinoline, benzofuran, benzothiophene, indole, indazole, benzimidizolyl, imidazopyridinyl, pyrazolopyridinyl, pyrazolopyrimidinyl, and the like.

As used herein, the term "amino" (alone or in combination with another term(s)) refers to -Ν1¾, or a mono- or disubstituted derivative, i.e., a secondary or tertiary amine.

An "oxo" substituent is a divalent oxygen substituent attached to another atom (e.g., a carbon) by a double bond.

Compounds of the invention may be provided as mixtures of stereoisomers as well as purified enantiomers or enantiomerically/diastereomerically enriched mixtures.

Compounds of the invention also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds described herein include and are not limited to ²H, ³H, ⁿC, ¹³C, ¹⁴C, ³⁶C1, ¹⁸F, ¹³N, ¹⁵N, ¹⁵0, ¹⁷0, ¹⁸0, and ³²P. In one embodiment, isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies. In another embodiment, substitution with heavier isotopes such as deuterium affords greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements). In yet another embodiment, substitution with positron emitting isotopes, such as ⁿC, ¹⁸F, ¹⁵0 and ¹³N, is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled compounds are prepared by any suitable method or by processes using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed.

As used herein, the term "pharmaceutically acceptable salt" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the invention within or to the patient such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the invention, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

"Treat," "treatment," or "treating," as used herein, relates to the application or administration of a therapeutic agent, i.e., a compound of the invention, to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease or disorder, a symptom of a disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

As used herein, the term "subject" refers to a human or a non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In a particular embodiment, the subject is a mammal. In another particular embodiment, the subject is human.

EXAMPLES

Example 1 : A New Combination of a Pleuromutilin Derivative and Doxycycline for Treatment of MDR Actinobacter baumannii.

Synthesis

All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. THF, CH₂CI₂, and DMF were purified via Innovative Technology's Pure-Solve System. All reactions were performed under an Argon atmosphere. All stirring was performed with an internal magnetic stirrer. Reactions were monitored by TLC using 0.25 mm coated commercial silica gel plates (EMD, Silica Gel 6OF254). TLC spots were visualized by UV light at 254 nm, or developed with ceric ammonium molybdate or anisaldehyde or copper sulfate or ninhydrin solutions by heating on a hot plate. Reactions were also monitored by using SHIMADZU LCMS-2020 with solvents: A: 0.1% formic acid in water, B: acetonitrile. Flash chromatography was performed with SiliCycle silica gel (Purasil 60 A, 230-400 Mesh). Proton magnetic resonance (¹H -NMR) spectral data were recorded on 400, and 500 MHz instruments. Carbon magnetic resonance (¹³C-NMR) spectral data were recorded on 100 and 125 MHz instruments. For all NMR spectra, chemical shifts (δΗ, δC) were quoted in parts per million (ppm), and J values were quoted in Hz. *H and ¹³C NMR spectra were calibrated with residual undeuterated solvent (CDCL: δΗ = 7.26 ppm, δC = 77.16 ppm; CD₃CN: δΗ = 1.94 ppm, δC = 1.32ppm; CD₃OD: δΗ =3.31 ppm, δC =49.00 ppm; DMSO-d₆: δΗ = 2.50 ppm, δC = 39.52 ppm; D₂O: δΗ = 4.79 ppm) as an internal reference. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = double doublets, t = triplet, q = quartet, quin = quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR) spectra were recorded on a Perkin-Elmer FT1600 spectrometer. HPLC analyses were performed with a Shimadzu LC-20AD HPLC system. All compounds were purified by reverse HPLC to be >95 % purity.

Pleuromutilin

Pleuromutilin was purchased from Nanjing Pharmatechs Co., Ltd. as a mixture of 5 and SI, and used after purification by silica gel column chromatography (hexanes/EtOAc 67 :33 to 50:50). Data for Pleuromutilin: TLC (hexanes/EtOAc 50:50) Rf = 0.40; [a]²¹ _D +0.960 (c = 4.41, CHC1₃); IR (thin film) V_max = 3448 (br), 2984, 2936, 2884, 2865 , 1731 , 1455 , 1415, 1375, 1283, 1232, 1217, 1154, 1097, 1016, 978, 933, 916, 754 cm^-1 ; ¹H NMR (400 MHz, Chloroform-d) δ 6.49 (dd, J = 17.4, 11.0 Hz, 1H), 5.83 (d, J = 8.5 Hz, 1H), 5.36 (dd, J = 11.0, 1.5 Hz, 1H), 5.21 (dd, J = 17.4, 1.6 Hz, 1H), 4.07 (d, J = 17.1 Hz, 1H), 4.01 (d, J = 17.1 Hz, 1H), 3.36 (d,J = 6.5 Hz, 1H), 2.43 (brs, 1H), 2.34 (quin, J = 7.0 Hz, 1H), 2.25 (ddd, J = 10.3 , 5.4, 2.2 Hz, 1H), 2.22 (quin, J = 9.2 Hz, 1H), 2.09 (quin, J = 8.7 Hz, 2H), 1.78 (dq, J = 14.3 , 2.8 Hz, 1H), 1.72 - 1.61 (m, 2H), 1.55 (td, J = 13.8, 3.5 Hz, 1H), 1.49 (d, J = 3.3 Hz, 1H), 1.47 - 1.45 (m, 1H), 1.43 (s, 3H), 1.38 (dq, J = 14.4, 3.8 Hz, 1H), 1.32 (d, J = 16.1 Hz, 1H), 1.17 (s, 3H), 1.12 (dd, J = 13.9, 4.5 Hz, 1H), 0.89 (d, J = 7.0 Hz, 3H), 0.70 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.88, 172.16, 138.79, 117.38, 74.54, 69.75, 61.29, 58.05, 45.40, 44.69, 43.98, 41.80, 36.57, 36.01 , 34.40, 30.36, 26.80, 26.29, 24.81, 16.61, 14.75, 11.52; HRMS (ESI+) m/z calcd for C₂₂H₃₅O₅ [M + H] 379.2484, found: 379.2438. Data for SI: TLC (hexanes/EtOAc 50:50) R_f = 0.50; IR (thin film) V_max = 3524 (br), 2982, 2927, 2882, 2866, 1733, 1472, 1463, 1456, 1414, 1371, 1248, 1152, 1117, 1020, 980, 952, 913, 772 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 6.52 (dd, J = 17.4, 11.0 Hz, 1H), 5.71 (d, J = 8.5 Hz, 1H), 5.34 (dd, J = 11.0, 1.6 Hz, 1H), 5.19 (dd, J = 17.4, 1.7 Hz, 1H), 3.35 (d, J = 6.5 Hz, 1H), 2.36 (p, J = 6.9 Hz, 1H), 2.26 (dddd, J = 19.5, 11.0, 3.3, 0.9 Hz, 1H), 2.21 (quin, J = 9.2 Hz, 1H), 2.09 (s, 1H), 2.07 - 2.01 (m, 1H), 1.97 (s, 3H), 1.76 (dq, J = 14.3, 2.9 Hz, 1H), 1.71 - 1.58 (m, 3H), 1.55 (dd, J = 13.3, 3.0 Hz, 1H), 1.46 (qd, J = 10.6, 3.2 Hz, 1H), 1.45 (s, 3H), 1.35 (dq, J = 13.7, 3.0 Hz, 1H), 1.30 (d, J = 16.0 Hz, 1H), 1.16 (s, 3H), 1.11 (dd, J = 14.1 , 4.3 Hz, 1H), 0.87 (d, J = 7.0 Hz, 3H), 0.72 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 217.27, 169.94, 139.19, 117.03, 74.59, 67.89, 58.20, 45.44, 44.96, 43.94, 41.66, 36.76, 35.97, 34.47, 30.45, 26.83, 26.19, 24.84, 21.92, 16.54, 14.86, 11.51 ; HRMS (ESI+) m/z calcd for C22H35O4 [M + H] 363.2535, found: 363.2499.

To a stirred solution of Pleuromutilin (7.48 g, 19.8 mmol) in MeOH (80 mL) was added NaBSU (1.50 g, 39.5 mmol) at 0 °C. After 8 h, the reaction mixture was quenched with aq. sat. NH₄CI. After 12 h, the reaction mixture was extracted with EtOAc and the combined organic phase was dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 70:30 to 50:50) to give S2 (7.50 g, 19.7 mmol, 99%): TLC (hexanes/EtOAc 50:50) R_f = 0.40; [μ]²¹ _D -0.169 (c = 1.15, CHC1₃); IR (thin film)

= 3454 (br), 2938, 2877, 1730, 1457, 1416, 1373, 1232, 1146, 1099, 1019, 1004, 931 , 755 cm^-1; H¹ NMR (400 MHz, Chloroform-d) δ 6.54 (dd, J = 15.6, 11.7 Hz, 1H), 5.70 (d, J = 9.3 Hz, 1H), 5.35 (d, J = 10.8 Hz, 1H), 5.20 (d, J = 17.4 Hz, 1H), 4.58 - 4.54 (m, 1H), 4.04 (s, 2H), 3.18 (d, J = 6.2 Hz, 1H), 2.47 - 2.31 (m, 1H), 2.29 - 2.21 (m, 1H), 2.20 - 2.13 (m, 1H), 2.13 - 2.03 (m, 1H), 2.02 - 1.92 (m, 1H), 1.91 - 1.79 (m, 1H), 1.73 (d, J = 14.4 Hz, 1H), 1.64 (t, J = 11.4 Hz, 1H), 1.52 (d, J = 4.5 Hz, 1H), 1.51 - 1.34 (m, 2H), 1.29 - 1.23 (m, 1H), 1.21 (s, 3H), 1.16 (s, 3H), 0.89 (d, J = 6.9 Hz, 1H), 0.82 (d, J = 6.7 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.83, 172.23, 139.34, 116.84, 74.85, 71.72, 61.31 , 50.87, 45.96, 45.18, 44.92, 41.26, 36.38, 35.62, 34.30, 32.53, 31.73, 27.61, 26.19, 17.45, 16.94, 12.20; HRMS (ESI+) m/z calcd for C₂₂H₃₇O₅ [M + H] 381.2641, found: 381.2618.

NOESY Map for S2:

To a stirred solution of S2 (7.50 g, 19.7 mmol) in CH₂CI₂ (100 mL) was added TsCl (4.51 g, 23.7 mmol) and DMAP (3.61 g, 29.6 mmol) at 0 °C. After 7 h at 0 °C, the reaction mixture was quenched with IN HC1 and extracted with EtOAc. The combined organic extract was washed with aq. sat. NaHCC^, dried over Na₂S0₄, concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc 60:40) to yield (10.5 g, 19.7 mmol, 99%): TLC (hexanes/EtOAc 50:50) R_f = 0.50; [a]²¹ _D -0.124 (c = 0.66, CHCI3); IR (thin film)

= 3562 (br), 2946, 2878, 1755, 1734, 1453, 1370, 1293, 1224, 1190, 1176, 1118, 1096, 1042, 1020, 1005, 925, 839, 814, 765, 719, 663 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 7.81 (d, J = 8.4 Hz, 2H), 7.36 - 7.33 (m, 2H), 6.45 (dd, J = 17.4, 11.0 Hz, 1H), 5.62 (d, J = 9.5 Hz, 1H), 5.31 (dd, J = 11.0, 1.6 Hz, 1H), 5.16 (dd, J = 17.4, 1.6 Hz, 1H), 4.55 (q, J = 5.1 Hz, 1H), 4.47 (s, 2H), 3.15 (dd, J = 11.1, 6.3 Hz, 1H), 2.45 (s, 3H), 2.26 - 2.19 (m, 1H), 2.14 - 2.05 (m, 2H), 1.99 - 1.90 (m, 1H), 1.84 (td, J = 13.4, 5.2 Hz, 1H), 1.73 - 1.69 (m, 1H), 1.63 (tdd, J = 16.1, 8.2, 4.1 Hz, 2H), 1.51 - 1.47 (m, 1H), 1.47 - 1.43 (m, 1H), 1.43 - 1.39 (m, 1H), 1.39 - 1.34 (m, 1H), 1.29 (d, J = 3.8 Hz, 1H), 1.19 (s, 3H), 1.13 (s, 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.61 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.73, 164.87, 145.23, 139.21 , 132.62, 129.89 (2C), 128.11 (2C), 116.84, 74.81 , 72.18, 65.16, 50.84, 45.93, 44.97, 44.86, 41.28, 36.34, 35.60, 34.27, 32.46, 31.71 , 27.55, 26.23, 21.70, 17.47, 16.88, 12.16; HRMS (ESI+) m/z calcd for C29H43O7S [M + H] 535.2730, found: 535.2742.

To a stirred solution of starting material (10.5 g, 19.7 mmol), l-amino-2- methylpropane-2-thiol hydrochloride (5.58 g, 39.4 mmol) and tetra-n-butylammonium bromide (0.64 g, 1.97 mmol) in THF (80 mL) was added IN NaOH (79.2 mL). After 4 h at 50 °C, the reaction mixture was extracted with CHCI₃. The combined organic extract was dried over Na₂S0₄, concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc 50:50 to CHCL/MeOH 75 :25) to give (8.75 g, 18.7 mmol, 95%): TLC (CHCL/MeOH 90:10) R_f = 0.20; [a]²¹ _D -0.062 (c = 0.87, CHCI3); IR (thin film) V_max = 3421 (br), 2955, 2876, 1721, 1462, 1371 , 1283, 1219, 1121, 1020, 1005, 932, 772 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 6.51 (dd, J = 17.4, 11.0 Hz, 1H), 5.60 (d, J = 9.4 Hz, 1H), 5.32 (dd,J = 11.0, 1.7 Hz, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 4.55 (t, J = 5.5 Hz, 1H), 3.16 (d, J = 6.4 Hz, 1H), 3.13 (s, 2H), 2.61 (s, 2H), 2.26 - 2.18 (m, 1H), 2.15 (t, J = 6.8 Hz, 1H), 2.08 (dd, J = 15.8, 9.3 Hz, 1H), 2.00 - 1.91 (m, 1H), 1.85 (td, J = 13.7, 4.5 Hz, 1H), 1.74 - 1.58 (m, 4H), 1.51 (d, J = 5.2 Hz, 1H), 1.48 - 1.41 (m, 2H), 1.41 - 1.33 (m, 1H), 1.24 (s, 6H), 1.23 (s, 3H), 1.14 (s, 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 169.38, 139.56, 116.66, 74.93, 71.25, 51.68, 51.01, 48.44, 46.00, 45.20, 44.86, 41.24, 36.62, 35.61, 34.31, 32.63, 31.76, 31.33, 27.66, 26.30, 26.25 (2C), 17.64, 17.21, 12.16; HRMS (ESI+) m/z calcd for C26H46NO4S [M + H] 468.3148, found: 468.3181.

To a stirred solution of starting material (1.65 g, 3.52 mmol), Boc-D-Val-OH

(1.15 g, 5.28 mmol), NaHCO₃ (2.96 g, 35.2 mmol) and Glyceroacetonide-Oxyma (1.20 g, 5.28 mmol) in DMF-H₂0 (9/1, 17.6 mL) was added EDCI (3.38 g, 17.6 mmol). The reaction mixture was stirred for 15 h at rt, quenched with aq. sat. NaHCO₃, and extracted with EtOAc. The combined organic extract was washed with IN HC1, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 67:33 to 50:50) to afford S3 (2.28 g, 3.41 mmol, 97%): TLC (hexanes/EtOAc 50:50) R_f = 0.40; [a]²¹ _D - 0.414 (c = 2.63, CHCI3); IR (thin film) V_max = 3332 (br), 2961 , 2937, 2876, 1705, 1663, 1521, 1504, 1456, 1391, 1367, 1288, 1248, 1165, 1019, 1005, 932, 755 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 6.93 (brs, 1H), 6.50 (dd, J = 17.4, 11.0 Hz, 1H), 5.60 (d, J = 9.3 Hz, 1H), 5.33 (d, J = 11.4 Hz, 1H), 5.17 (dd, J = 17.4, 1.3 Hz, 1H), 5.13 (brs, 1H), 4.56 (t, J = 5.9 Hz, 1H), 4.01 (dd, J = 8.7, 5.4 Hz, 1H), 3.31 (dd, J = 14.0, 6.6 Hz, 1H), 3.17 (d, J = 1.7 Hz, 2H), 3.19 - 3.12 (m, 1H), 2.24 (ddd, J = 11.5, 7.1, 4.4 Hz, 1H), 2.20 - 2.12 (m, 2H), 2.09 (dd, J = 15.8, 9.5 Hz, 1H), 1.96 (tdd, J = 14.9, 7.3, 5.4 Hz, 1H), 1.86 (td, J = 13.6, 4.6 Hz, 1H), 1.75 - 1.69 (m, 2H), 1.69 - 1.58 (m, 3H), 1.51 (d, J = 4.2 Hz, 2H), 1.45 (s, 9H), 1.41 - 1.38 (m, 2H), 1.25 (s, 3H), 1.24 (s, 3H), 1.23 (s, 3H), 1.15 (s, 3H), 0.99 (d, J = 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 7.0 Hz, 3H), 0.70 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 171.70, 170.00, 139.36, 116.81, 74.86, 71.76, 50.88, 47.54, 46.95, 45.94, 45.21, 44.84, 41.18, 36.45, 35.55, 34.25, 32.50, 31.72, 31.48, 30.95, 28.35 (3C), 27.61 ,

26.35, 26.28, 26.21, 19.36, 17.67, 17.56, 17.23, 12.19; HRMS (ESI+) m/z calcd for

C₃₆H₆₃N2O7S [M + H] 667.4356, found: 667.4331.

To a stirred solution of S3 (0.30 g, 0.46 mmol) in dioxane (0.5 niL) was added a 4N solution of HCI in dioxane (1.14 niL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by

C18 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: 50:50 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 51

(0.23 g, 0.40 mmol, 88%, retention time: 25 min): TLC (CHCl₃/MeOH 90:10) R_f =

0.30; [a]²² _D -0.201 (c = 1.61, MeOH); IR (thin film) v_max = 3395 (br), 2957, 2875,

1716, 1658, 1525, 1463, 1370, 1285, 1144, 1020, 1005, 932 cm^-1; ¹H NMR (400

MHz, Methanol-^) δ 6.40 - 6.31 (m, 1H), 5.62 (d, J = 9.2 Hz, 1H), 5.17 (q, J = 1.7

Hz, 1H), 5.15 - 5.12 (m, 1H), 4.49 (t, J = 5.3 Hz, 1H), 3.38 - 3.34 (m, 2H), 3.29 (d, J = 6.8 Hz, 2H), 3.26 - 3.21 (m, 2H), 2.36 (ddd, J = 11.8, 7.2, 4.2 Hz, 1H), 2.22 - 2.10

(m, 2H), 2.03 (quind, J = 6.9, 5.2 Hz, 1H), 1.92 (dddd, J = 14.7, 13.0, 10.5, 5.2 Hz,

2H), 1.72 - 1.57 (m, 4H), 1.50 - 1.35 (m, 3H), 1.33 (q, J = 3.8 Hz, 1H), 1.27 (s, 3H),

1.26 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H), 1.01 (d, J = 6.9 Hz, 3H), 0.95 (d, J = 6.8 Hz,

3H), 0.86 (d, J = 7.0 Hz, 3H), 0.70 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 176.36, 171.35, 141.52, 116.22, 77.39, 76.08, 73.26, 61.58, 52.21, 47.65, 47.18,

46.72, 45.95, 42.56, 37.23, 37.13, 34.59, 33.32, 33.14, 32.90, 32.51, 28.98, 28.00,

26.97, 26.91, 19.87, 17.84, 17.68, 17.53, 12.51 ; HRMS (ESI+) m/z calcd for

C₃1H55N2O5S [M + H] 567.3832, found: 567.3866.

To a stirred solution of 51 (73.2 mg, 0.13 mmol), Boc-L-Orn(Boc)-OH (63.2 mg, 0.19 mmol), NaHCO₃ (109 mg, 1.29 mmol) and Glyceroacetonide-Oxyma (59.3 mg, 0.26 mmol) in DMF-H₂0 (9/1 , 0.65 mL) was added EDCI (124 mg, 0.65 mmol). The reaction mixture was stirred for 8 h at rt, quenched with aq. sat. NaHCC^, and extracted with EtOAc. The combined organic extract was washed with IN HCI, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 60:40 to 20:80) to afford S4 (106 mg, 0.12 mmol, 91 %): TLC (hexanes/EtOAc 33 :67) R_f = 0.40; [a]²² _D -0.021 (c = 1.36, CHC1₃); IR (thin film) v_max = 3315 (br), 2962, 2934, 2874, 1699, 1649, 1522, 1513, 1457, 1391, 1366, 1281, 1250, 1220, 1170, 1020, 1005, 772 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 7.01 (brs, 1H), 6.74 (d, J = 8.4 Hz, 1H), 6.50 (dd, J = 17.4, 11.0 Hz, 1H), 5.59 (d, J = 9.4 Hz, 1H), 5.33 (dd, J = 11.0, 1.6 Hz, 1H), 5.32 (brs, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 4.71 (t, J = 6.1 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.32 (dd, J = 8.4, 5.4 Hz, 1H), 4.08 (brs, 1H), 3.39 (dd, J = 14.0, 7.0 Hz, 1H), 3.17 (d, J = 2.4 Hz, 2H), 3.17 - 3.14 (m, 1H), 3.09 (s, 2H), 3.08 - 3.04 (m, 1H), 2.28 - 2.19 (m, 2H), 2.13 (dd, J = 13.3, 6.6 Hz, 1H), 2.09 - 2.02 (m, 1H), 1.99 - 1.78 (m, 5H), 1.74 - 1.68 (m, 2H), 1.68 - 1.57 (m, 4H), 1.52 - 1.49 (m, 2H), 1.49 - 1.45 (m, 2H), 1.43 (s, 18H), 1.40 - 1.32 (m, 2H), 1.24 (s, 3H), 1.23 (s, 3H), 1.22 (s, 3H), 1.14 (s, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 172.09, 171.10, 170.84, 170.02, 156.23, 139.45, 116.75, 74.86, 71.78, 60.36, 58.53, 50.90, 47.63, 46.96, 45.95, 45.19, 44.85, 41.20, 36.46, 35.56, 34.64, 34.23, 32.49, 31.72, 31.55, 31.49, 29.69, 28.44 (3C), 28.31 (3C), 27.64, 26.31, 26.23, 25.25, 22.61, 21.00, 20.67, 19.33, 17.56, 17.24, 14.17, 14.07, 12.17; HRMS (ESI+) mlz calcd for C₄₆H₈₁N₄Oi₀S [M + H] 881.5673, found: 881.5695.

To a stirred solution of S4 (45.6 mg, 0.051 mmol) in dioxane (0.2 mL) was added a 4N solution of HCI in dioxane (0.25 mL). The reaction mixture was stirred for 2 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by C18 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: 50:50 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 52 (33.0 mg, 0.047 mmol, 93%, retention time: 13 min): TLC (CHCl₃/MeOH 90:10) Rf = 0.10; [a]²² _D +0.091 (c = 0.11, MeOH); IR (thin film) v_max = 3438 (br), 3202 (br), 2968, 1658, 1427, 1287, 1148, 1018, 724 cm^-1; ¹H NMR (400 MHz, Methanol-d4) δ 8.23 (d, J = 6.6 Hz, 1H), 6.35 (dd, J = 17.0, 11.3 Hz, 1H), 5.62 (d, J = 9.9 Hz, 1H), 5.19 - 5.14 (m, 1H), 5.13 (s, 1H), 4.49 (t, J = 5.7 Hz, 1H), 4.22 (d, J = 6.6 Hz, 1H), 4.01 (t, J = 6.1 Hz, 1H), 3.77 - 3.71 (m, 1H), 3.61 - 3.56 (m, 1H), 2.96 (t, J = 7.6 Hz, 2H), 2.41 - 2.29 (m, 1H), 2.21 - 2.05 (m, 3H), 1.99 - 1.85 (m, 4H), 1.76 - 1.57 (m, 6H), 1.56 - 1.38 (m, 5H), 1.35 - 1.29 (m, 2H), 1.26 (s, 3H), 1.26 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H), 1.05 (s, 3H), 1.03 (s, 3H), 0.86 (d, J = 6.4 Hz, 3H), 0.70 (d, J = 7.7 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 173.61, 171.56, 169.98, 141.62, 116.18, 77.39, 76.02, 73.57, 73.31 , 72.45, 62.18, 61.02, 53.82, 52.19, 47.78, 47.19, 45.94, 42.56, 40.29, 37.22, 37.16, 34.59, 32.91 , 32.52, 32.18, 31.74, 28.06, 28.04, 27.02, 26.81, 22.54, 19.91, 18.88, 17.80, 17.54, 12.53; HRMS (ESI+) m/z calcd for C₃₆H₆₅N₄0₆S [M + H] 681.4625, found: 681.4602.

To a stirred solution of 51 (73.2 mg, 0.13 mmol), Boc-Gly-OH (33.9 mg, 0.19 mmol), NaHC0₃ (108 mg, 1.29 mmol) and Glyceroacetonide-Oxyma (43.3 mg, 0.19 mmol) in DMF-H₂0 (9/1, 0.65 mL) was added EDCI (124 mg, 0.65 mmol). The reaction mixture was stirred for 12 h at rt, quenched with aq. sat. NaHC0₃, and extracted with EtOAc. The combined organic extract was washed with IN HC1, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 60:40 to 20:80) to afford S5 (83.8 mg, 0.12 mmol, 89%): TLC (hexanes/EtOAc 33 :67) R_f = 0.40; [a]²² _D -0.403 (c = 1.86, CHC1₃); IR (thin film) v_max = 3312 (br), 2963, 2937, 2876, 1708, 1657, 1541, 1522, 1456, 1390, 1368, 1282, 1251 , 1219, 1166, 1020, 1005, 934, 771 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 7.08 (t, J = 6.3 Hz, 1H), 6.84 (d, J = 8.6 Hz, 1H), 6.47 (dd, J = 17.4, 11.0 Hz, 1H), 5.59 (d, J = 9.3 Hz, 1H), 5.41 - 5.36 (m, 1H), 5.32 (dd, J = 11.0, 1.6 Hz, 1H), 5.15 (dd, J = 17.4, 1.7 Hz, 1H), 4.53 (t, J = 5.5 Hz, 1H), 4.37 (dd, J = 8.7, 5.6 Hz, 1H), 3.89 (dd, J = 16.9, 5.8 Hz, 1H), 3.78 (dd, J = 16.8, 5.6 Hz, 1H), 3.26 (dd, J = 14.0, 6.4 Hz, 1H), 3.16 (d, J = 2.2 Hz, 2H), 3.18 - 3.11 (m, 1H), 2.27 - 2.16 (m, 2H), 2.16 - 2.06 (m, 2H), 1.99 - 1.89 (m, 1H), 1.85 (td, J = 13.4, 12.5, 3.9 Hz, 1H), 1.74 - 1.67 (m, 1H), 1.67 - 1.56 (m, 3H), 1.52 - 1.46 (m, 2H), 1.43 (s, 9H), 1.41 - 1.32 (m, 2H), 1.23 (s, 3H), 1.22 (s, 3H) 1.21 (s, 3H), 1.13 (s, 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.80 (d, J = 1.0 Hz, 3H), 0.67 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 170.87, 170.23, 169.55, 139.36, 116.79, 74.84, 71.93, 58.39, 50.86, 47.55, 47.01, 45.92, 45.22, 44.82, 41.18, 36.37, 35.55, 34.19, 32.45, 31.71 , 31.49, 30.87, 28.26 (3C), 27.61, 26.34 (2C), 26.23, 19.27, 17.70, 17.49, 17.18, 12.15 ; HRMS (ESI+) mlz calcd for C38H66N3O8S [M + H] 724.4571 , found: 724.4589.

To a stirred solution of S5 (39.7 mg, 0.055 mmol) in dioxane (0.2 mL) was added a 4N solution of HCl in dioxane (0.3 mL). The reaction mixture was stirred for 2 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: 80:20 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 53 (32.6 mg, 0.052 mmol, 95%, retention time: 11 min): [a]²² _D +0.538 (c = 1.08, MeOH); IR (thin film) v_max = 3421 (br), 3320 (br), 3078 (br), 2961, 2877, 1715, 1661 , 1541, 1463, 1456, 1388, 1370, 1279, 1146, 1020, 1004, 935 cm^-1; ¹H NMR (400 MHz, Methanol-d4) δ 6.35 (dd, J = 17.2, 11.4 Hz, 1H), 5.62 (d, J = 9.4 Hz, 1H), 5.17 (s, 1H), 5.16 - 5.12 (m, 1H), 4.49 (t, J = 5.5 Hz, 1H), 4.26 (d, J = 6.6 Hz, 1H), 3.66 (s, 1H), 3.35 (s, 1H), 3.30 - 3.25 (m, 2H), 2.40 - 2.31 (m, 1H), 2.20 - 2.09 (m, 3H), 1.96 (dd, J = 13.9, 4.3 Hz, 1H), 1.92 - 1.86 (m, 1H), 1.73 - 1.56 (m, 5H), 1.49 - 1.37 (m, 3H), 1.34 - 1.28 (m, 3H), 1.26 (s, 3H), 1.25 (s, 3H), 1.25 (s, 3H), 1.13 (s, 3H), 1.01 (d, J = 6.5 Hz, 3H), 0.99 (d, J = 6.5 Hz, 3H), 0.86 (d, J = 1.0 Hz, 3H), 0.70 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 171.51, 141.55, 116.24, 77.41, 76.07, 73.31, 60.60, 52.22, 47.19, 45.96, 42.58, 37.23, 37.16, 34.59, 33.32, 32.91, 31.94, 29.11 , 29.04, 28.99, 28.04, 26.96, 26.85, 19.89, 18.49, 17.82, 17.50, 12.52; HRMS (ESI+) mlz calcd for C₃₃H₅₈N₃O₆S [M + H] 624.4046, found: 624.4081.

To a stirred solution of 51 (100 mg, 0.17 mmol) and N-Boc-2- amino acetaldehyde (53.1 mg, 0.33 mmol) in MeOH (0.83 mL) was added sodium cyanoborohydride (21.0 mg, 0.33 mmol) at 0 °C. After being stirred for 2 h at rt, quenched with aq. saturated NaHCO₃, and extracted with CHCI₃. The combined organic extract was dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (CHC13/MeOH 98:2 to 97:3) to afford S6 (104 mg, 0.15 mmol, 86%): TLC (CHCl₃/MeOH 90:10) R_f = 0.40; [a]²² _D - 0.051 (c = 4.52, CHCI3); IR (thin film) V_max = 3340 (br), 2958, 2936, 2875, 1699, 1659, 1522, 1457, 1390, 1366, 1281, 1251, 1218, 1167, 1020, 1005, 931, 754 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 7.92 (t, J = 6.1 Hz, 1H), 6.49 (dd, J = 17.4, 11.0 Hz, 1H), 5.58 (d, J = 9.4 Hz, 1H), 5.30 (brs, 1H), 5.27 (dd, J = 11.0, 1.6 Hz, 1H), 5.16 (dd, J = 17.4, 1.6 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 3.40 (dd, J = 14.0, 7.1 Hz, 1H), 3.33 - 3.23 (m, 2H), 3.06 (dd, J = 13.8, 5.0 Hz, 1H), 2.96 (d, J = 4.2 Hz, 1H), 2.82 - 2.75 (m, 1H), 2.75 - 2.68 (m, 1H), 2.28 - 2.20 (m, 1H), 2.20 - 2.05 (m, 3H), 2.00 - 1.90 (m, 1H), 1.85 (td, J = 13.4, 5.3 Hz, 1H), 1.74 - 1.66 (m, 1H), 1.66 - 1.57 (m, 2H), 1.50 (d, J = 4.2 Hz, 1H), 1.47 (d, J = 6.7 Hz, 3H), 1.42 (s, 9H), 1.39 - 1.34 (m, 2H), 1.26 (s, 3H), 1.24 (s, 3H), 1.22 (s, 3H), 1.14 (s, 3H), 1.01 (d, J = 6.9 Hz, 3H), 0.93 (d, J = 6.9 Hz, 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCI3) δ 169.92, 139.51, 116.59, 74.84, 71.83, 68.49, 50.89, 49.40, 47.07, 46.94, 45.94, 45.09, 44.87, 41.19, 40.58, 36.42, 35.59, 34.24, 32.47, 31.72, 31.59, 31.26, 28.45 (3C), 28.36, 28.33, 27.63, 26.65, 26.47, 26.25, 19.55, 17.88, 17.58, 17.27, 12.13 ; HRMS (ESI+) mlz calcd for C₃8H₆₈N₃0₇S [M + H] 710.4778, found: 710.4803.

To a stirred solution of S6 (27.3 mg, 0.038 mmol) in dioxane (0.2 mL) was added a 4N solution of HCI in dioxane (0.4 mL). The reaction mixture was stirred for 3 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: 50:50 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 54 (21.6 mg, 0.035 mmol, 93%, retention time: 27 min): TLC (CHCL/MeOH 90:10) R_f = 0.20; [a]²² _D 0.031 (c = 0.033, MeOH); IR (thin film) V_max = 3430 (br), 2959, 2931, 2878, 1716, 1676, 1563, 1555, 1542, 1463, 1457, 1287, 1130, 1020, 1005, 933 cm^-1; ¹H NMR (500 MHz, Methanol-d4) δ 6.37 - 6.31 (m, 1H), 5.62 (d, J = 9.3 Hz, 1H), 5.16 (d, J = 4.8 Hz, 1H), 5.13 (s, 1H), 4.49 (t, J = 5.5 Hz, 1H), 3.70 - 3.64 (m, 1H), 3.46 (d, J = 14.0 Hz, 1H), 3.34 (d, J = 2.9 Hz, 2H), 3.27 (d, J = 14.1 Hz, 3H), 3.23 - 3.16 (m, 1H), 2.40 - 2.33 (m, 1H), 2.29 - 2.21 (m, 1H), 2.19 - 2.11 (m, 2H), 1.98 - 1.87 (m, 2H), 1.72 - 1.61 (m, 3H), 1.61 - 1.57 (m, 1H), 1.48 - 1.36 (m, 2H), 1.35 - 1.32 (m, 1H), 1.29 (s, 6H), 1.24 (s, 3H), 1.15 (d, J = 6.7 Hz, 3H), 1.13 (s, 3H), 1.11 (d, J = 6.7 Hz, 3H), 0.86 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 171.49, 141.64, 116.18, 77.40, 76.02, 73.45, 68.59, 52.20, 47.80, 47.19, 46.71, 45.98, 45.83, 42.59, 37.21, 37.19, 34.60, 33.31, 32.90, 32.74, 31.99, 29.00, 28.05, 27.11, 19.07, 18.76, 17.80, 17.52, 12.51 ; HRMS (ESI+) mlz calcd for C₃₃H₆₀N₃O5S [M + H] 610.4254, found 610.4245.

Materials and Methods

Bacterial Strains and Growth of Bacteria. Acinetobacter baumannii (ATCC 19606, ATCC 1793), MDR A. baumannii (ATCC BAA 18002) Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 8047), and Escherichia coli (ATCC 10798), were purchased from American Type Culture Collection (ATCC). Mycobacterium tuberculosis (H₃7RV) was obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). Single colonies of A. baumannii, P. aeruginosa, K. pneumoniae, S. aureus, and E. coli were grown on tryptic soy agar for 24 h at 37 °C in a static incubator and cultured in tryptic soy broth until log phase to be an optical density (OD) of 0.4-0.5. The OD was monitored at 600 nm using a 96-well microplate reader. A single colony of M. tuberculosis was obtained on Difco Middlebrook 7H10 nutrient agar enriched with 10% oleic acid, albumin, dextrose, and catalase (OADC) for M. tuberculosis by incubating for 15 days. Seed cultures and larger cultures were obtained using Middlebrook 7H9 broth enriched with OADC (for M. tuberculosis) by incubating for 15 days at 37 °C in a shaking incubator (200rpm).

Synergistic Effect of Doxycycline with Compound 51 or Valnemulin. The synergistic or antagonistic activities of doxycycline with analog 51 or valnemulin were assessed in vitro via micro dilution broth checkerboard technique. The FIC index was calculated according to the following equation.

∑FIC=FICA+FICB=CA/MICA+C_B/MICB where, MIC_A and MIC_B: MIC of drugs A and

B, CA and CB=concentrations of drugs A and B used in combination. In these interaction studies,∑FIC of less than 0.5 represents synergistic activity. Synergistic or antagonistic activities of compound 51 with doxycycline are shown in FIG. 1A; synergistic or antagonistic activities of valnemulin with doxycycline are shown in

FIG. IB.

FIG. 1 : MIC of analog 51 is 12.5 μg/mL and of doxycycline is 0.2 μg/mL. Horizontal values represent concentrations of analog 51 or Valnemulin and vertical values represent concentrations of doxycycline. The values inside each cell is the fractional inhibitory concentration (∑FIC) for that combination.

Microsomal Stability and Protein Binding. Pooled Sprague-Dawley rat liver microsomes were purchased from Corning Life Sciences (Oneonta, NY, USA). Microsomes ((20 mg/mL) were thawed on ice and diluted using phosphate buffer (100 mM, pH: 7.4), resulting in a protein concentration of 1 mg/mL. Stock solutions (10 mg/L) of 1, valnemulin and verapamil (positive control) was prepared in DMSO (50%). A final concentration of 500 ng/niL was used for incubation with microsomes. NADPH (final concentration: 1 mM) was used as a co-factor. All the above solutions except NADPH were added to individual wells (12-well) in triplicate and were allowed to equilibrate for 5 min at 37 °C. NADPH was then added. 50 aliquots in triplicate were drawn from the incubation mixture at 0, 5, 10, 20, 30, 45 and 60 min and immediately the reaction was quenched by addition of ice-cold methanol (4- volumes). Analysis was performed by LC-MS. The samples containing methanol was lyophilized to remove all volatiles. The residue was dissolved in IN HC1 aq. (10uL) and MeOH (40uL). The resulted solution (20uL) was injected to LC- MS. MS solvent 90:10 acetonitrile/0.05% formic acid in water. Flow rate: 0.5mL/min.

Protein Binding. Plasma protein binding of compound 51 and verapamil (reference) was determined by equilibrium dialysis. The ready to use red device inserts (MW cutoff 6000-8000 D, RED® device, Thermo Scientific, Rockford, USA) containing plasma and buffer chambers for dialysis was used. The inserts were placed in a base plate. High and low concentrations (5000 ng/niL and 500 ng/mL) of 1 and reference were prepared in rat plasma (Innovative Grade US Origin Sprague-Dawley Rat Plasma' (anticoagulant: Lithium Heparin), catalog# IGRT-N) and an aliquot of 300 was added in the plasma chamber in duplicate. A 500 aliquot of blank isotonic phosphate buffer, pH 7.4 was added to buffer chamber of dialysis device. The base plate was covered with sealing tape and incubated at 37 °C at approximately 100 r.p.m on an orbital shaker for 4 h to achieve equilibrium. At the end of incubation, 50 aliquots were withdrawn from plasma and buffer chamber and add

50 μL of buffer to plasma sample and 50 μL of plasma to buffer sample. Four volumes of internal standard spiked methanol were added, vortexed for 30 seconds and centrifuged at 1000 rpm for 5 min. The samples containing methanol were lyophilized to remove all volatiles. The residue was dissolved in IN HC1 aq. (IOuL) and MeOH (40uL). The resulted solution (20uL) was injected to LC-MS. MS solvent 90:10 acetonitrile/0.05% formic acid in water. Flow rate: 0.5mL/min. The free fraction of the drug was calculated as ratio of the concentrations in the buffer and in plasma. The results were expressed in terms of % bound to plasma proteins.

Efficacy of 1 in Mouse Model of Infection. The in vivo efficacy of compound

51 and 51-Dox 35/1 was evaluated in a mouse septicemia model using the C57BL/6 mice and A. baumannii ATCC 1793 strain (at a dose that lead to 75% of death). One hour after the infection, the molecules (51, 51-Dox 35/1, and tobramycin (reference)) were administered intraperitoneally (IP) at single doses (from 2 to 60 mg/kg) (FIG. 2). A. baumannii (ATCC19606) cultured at 37 °C were collected by centrifugation and infected into C57BL/6 mice to create a mouse septicemia model. One hour after the infection (at a dose that lead to 75% of death), the molecules (51, 51-Dox 35/1 , and tobramycin (reference)) were administered intraperitoneally (IP) at single dose (from 2 to 60 mg/kg). Mice were monitored for 5 days and death was defined as the end point.

Cytotoxicity Assays. Selected molecules were tested for cytotoxicity (IC₅₀) in Vero cells via a MTT colorimetric assay. Vero cell line was cultured in Complete eagle" s minimum essential growth medium (EMEM) containing L-glutamine, sodium pyruvate, minimum essential amino acids, penicillin-streptomycin and 10% fetal bovine serum. After 72 h of exposure of molecules to this cell line at concentrations ranging from 0.78 to 200 μg/mL, the culture medium was changed to complete EMEM without phenol red before addition of yellow tetrazolium dye; MTT. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by BioTek Synergy HT Spectrophotometer.

Cytotoxicity assays were performed using Vero monkey kidney (ATCC CCL- 81) and HepG2 human hepatoblastoma cell (ATCC HB-8065) lines. Vero or HepG2 cells were cultured in 75 mm flasks and transferred to 96-well cell culture plates using ATCC-formulated Eagle's minimum essential medium containing 10% FBS, and penicillin-streptomycin. Serially diluted aliquots of each test compound at concentrations ranging from 0.78-200 μg/mL were added to the cells. Control compounds with known toxicity such as tunicamycin, colistin or tobramycin were included on each plate. The plates were incubated and cytotoxic effects were determined via the MTT assay.

Identification of Resistant Mechanism of A. baumannii to analog 51.

Sequence of A. baumannii 50S ribosomal subunit protein L3 (rplC)

TTAACAAAGATGCGCGTGACCAGTACGAAATCCGCACCTACAAACGTTTGATCGACA TCG TTCAACCTACAGATAAAACTGTTGATGCATTGATGAAGTTAGATCTTGCAGCTGGTG TTG

AT G T T c AG AT T AAT T AAC GAT T AGT T AAT T AGGC

CGC

TTTTTTAGAGGTTTATGCACATGGCTATTGGTTTAGTCGGTCGCAAATGTGGTATGA CTC

GCATCTTTACAGATGCTGGTGTTTCTGTACCTGTTACAGTCATCGAAGTCGATCCAA ACC

GCATTACGCAAATCAAAACACTTGAAACTGATGGTTATCAAGCTGTTCAAGTAACTA CTG GCGAACGTCGCGAGTCTCGCGTAACTAACGCTCAAAAAGGTCACTTCGCTAAAGCGG GTG

TTGCTGCTGGTCGTTTAGTTAAAGAGTTTCGTGTTACTGAAGCTGAGCTTGAAGGCC GTG

AAGTTGGCGGTACTATTGGCGTTGATTTGTTCACAGTTGGTCAAATTGTTGACGTAA CTG

GTCAATCAAAAGGTAAAGGTTTCCAAGGTGGTGTTAAACGTTGGAATTTCCGTACCC AAG

ATGCTACTCACGGTAACTCTGTTTCTCACCGTGTTTTAGGTTCTACAGGTCAAAACC AAA CTCCTGGACGCGTGTTCAAAGGCAAAAAAATGGCTGGTCACTTAGGTGATGAACGCG TAA

CAGTACAAGGTCTTGAAATCGTATCTGTTGACACTGAACGTTCAGTTTTGGTTGTTA AGG

GTGCAATTCCTGGTGCAACTGGCGGTGACGTTATCGTACGTCCTACCATCAAGGCCT GAG

G G GAAAT AC c G T G AAT T T ΑΑΑΑ ΑΤ TGTCTGAAGT

AGC

TTTCGGACGTGAATTTAACGAAGCTCTTGTACACCAAGTTGTTACTGCTTACTTAGC AGG TGGTCGTCAAGGTACTCGTGCTCACAAATCACGTGCAGACGTTTCTGGCGGTGGTAA AAA ACCATTCCGTCAAAAAGGT

Abrp 1 CupPrimer

CTTTGGGTTAAGGCTTTCGG

AbrplCdn

CAACAGCAGAGCCGGAAACAG

RplC Sequence of Analog 51 Resistant Mutant (51R) of A. baumannii ATGGCTATTGGTTTAGTCGGTCGCAAATGTGGTATGACTCGCATCTT

TACAGATGCTGGTGTTTCTGTACCTGTTACAGTCATCGAAGTCGATC CAAACCGCATTACGCAAACACTTGAAACTGATGGTTATCAAGCTGTT CAAGTAACTACTGGTGAACGTCGCGAGTCTCGCGTAACTAACGCTCA AAAAGGTCACTTCGCTAAAGCGGGTGTTGCTGCTGGTCGTTTAGTTA AAGAGTTTCGTGTTACTGAAGCTGAGCTTGAAGGCCGTGAAGTTGGC GGTACTATTGGCGTTGATTTGTTCACAGTTGGTCAAATTGTTGACGT AACTGGTCAATCAAAAGGTAAAGGTTTCCAAGGTGGTGTTAAACGTT GGAATTTCCGTACCCAAGATGCTACTCACGGTAACTCTGTTTCTCAC CGTGTTTTAGGTTCTACAGGTCAAAA|CAAACTCCTGGACGCGTGTT

CAAAGGCAAAAAAATGGCTGGTCACTTAGGTGATGAACGCGTAACAG TACAAGGTCTTGAAATCGTATCTGTTGACACTGAACGTTCAGTTTTG GTTGTTAAGGGTGCAATTCCTGGTGCAACTGGCGGTGACGTTATCGT ACGTCCTACCATCAAGGCCTGAGGGGAAATACCGTGAATTTAA

Blast against Wild type and Mutant A. baumannii

Query 7 MAIGLVGRKCGMTRIFTDAGVSVPVTVIEVDPNRITQIKTLETDGYQAVQVTTGERRESR 186

MAIGLVGRKCGMTRIFTDAGVSVPVTVIEVDPNRITQIKTLETDGYQAVQVTTGERRESR

Sbjct 1 MAIGLVGRKCGMTRIFTDAGVSVPVTVIEVDPNRITQIKTLETDGYQAVQVTTGERRESR 60

Query 187 VTNAQKGHFAKAGVAAGRLVKEFRVTEAELEGREVGGTI GVDLFTVGQIVDVTGQSKGKG 366

VTNAQKGHFAKAGVAAGRLVKEFRVTEAELEGREVGGTI GVDLFTVGQIVDVTGQSKGKG

Sbjct 61 VTNAQKGHFAKAGVAAGRLVKEFRVTEAELEGREVGGTI GVDLFTVGQIVDVTGQSKGKG 120

Query 367 FQGGVKRWNFRTQDATHGNSVSHRVLGSTGQIQTPGRVFKGKKMAGHLGDERVTVQGLE I 546

FQGGVKRWNFRTQDATHGNSVSHRVLGSTGQ QTPGRVFKGKKMAGHLGDERVTVQGLE I

sbj ct 12 1 FQGGVKRWNFRTQDATHGNSVSHRVLGSTGQ|QTPGRVFKGKKMAGHLGDERVTVQGLE I

180

Query 547 VSVDTERSVLVVKGAIPGATGGDVIVRPT IKA 642

VSVDTERSVLVVKGAIPGATGGDVIVRPT IKA

Sbjct 181 VSVDTERSVLVVKGAIPGATGGDVIVRPT IKA 212

Gene Analyses of Resistant Strain of A. baumannii against 51. Spontaneous resistant mutants of A. baumannii ATCC 19606 with decreased sensitivity towards compound 51 were isolated by sub-culturing bacteria on agar plates with concentrations of 51 at MIC and above. Resistant mutants were isolated at 16 x MIC of compound 51. Spontaneous resistant mutants with decreased sensitivity against rifampicin were isolated at 32 x MIC of rifampicin (control). The chromosomal DNAs from the resistant mutant (1^R) and wild-type Acinetobacter baumannii ATCC19606 were isolated. The rplC gene fragment was amplified using A. baumannii rplC specific primers (AbrplCupPrimer 5' CTTTGGGTTAAGGCTTTCGG 3' and Abrplcdn

5 ' CAACAGCAGAGCCGGA AACAG 3 '), purified, and DNA sequenced. The DNA sequencing result was used to blast against rplC DNA sequence of A. baumannii in NIH Genome database.

Results

Chemistry and SAR. Pleuromutilin and representative pleuromutilin analogs are shown in FIG. 3. New drugs for drug resistant Gram- negative organisms are a major unmet clinical need for new antibiotic agents. In order to identify new pleuromutilin derivatives that expand the spectrum of activity against Gram-negative organisms, a 50-membered library was generated whose structure contained the methoxyamine-oxime (A), hydoxyamine-oxime (B), or 3-hydroxy (C) core structure; those structures were further diversified by the reduction of double bond (exposition), amide-formations, and reductive aminations (see, above). The generated molecules were evaluated in the growth inhibitory assay against A. baumannii strain (ATCC19606). Four mti-Acinetobacter pleuromutilin analogs (51-54) were identified which exhibited the MIC₅₀ value less than <12.5 μg/mL (FIG. 4).

In order to confirm mti-Acinetobacter activity of analogs 51-54, these molecules were resynthesized. Their syntheses are illustrated in FIG. 5. The analogs synthesized in FIG. 5 were purified via reverse-phase HPLC, and their MIC values were determined against drug sensitive A. baumannii. The analogs 53-54 exhibited the MIC₅₀ and MIC₉₀ values of 3.13 and >12.5 μg/mL, respectively, suggesting that they are bacteriostatic molecules. Analog 51 displayed the MIC₅₀, MIC₉₀, and MIC₁₀₀ value of 1.75, 3.13 and 6.25, respectively, indicating its bactericidal activity (Table 2). In vitro bacterial growth inhibitory activity of compound 51 was comparable to anti- Acinetobacter drugs such as tobramycin and colistin (polymyxin E). Table 2 shows the MICs of analogs 51-54, representative antibacterial agents (clinically used), and combination of 51-doxycycline. ^aThe broth dilution method was used.; MIC₁₀₀ 2.00 μg/mL for 51-Dox 35/1.; A. baumannii: Acinetobacter baumannii.; K. pneumonia: Klebsiella pneumonia.; E. coli: Escherichia colt; P. aeruginosa: Pseudomonas aeruginosa.; S. aureus: Staphylococcus aureus.; M. tuberculosis: Mycobacterium tuberculosis. Table 2

The identified molecules 51-54 are the C3-reduced analogs of valnemulin

(FIG. 3). Analog 51 exhibited superior MIC₅₀ and MIC₉₀ level to those of valnemulin.

The analog 51 killed an MDR strain, A. baumannii (ATCC BAA-1800) at 6.25-12.5 μg/mL concentration, albeit tobramycin and colistin did not kill the same strain at the concentrations effective against a drug-susceptible strain (entry 2 in Table 2). Based on bacterial growth inhibitory assays of the pleuromutilin analogs against batteries of

Gram-negative and -positive bacteria, the following structure-activity relationship

(SAR) was realized. Alkylation or acylation of the D-valine amino group decreases bactericidal activity, although the modification with a variety of functional groups (R

in FIG. 6) is possible to retain the bacteriostatic activity. Hydrogenation of the C12- vinyl group increases activity against Gram-positive bacteria. The C3-hydroxy group increases in not only bactericidal activity against A. baumannii, but also pharmacological property such as water- solubility (1.5 times greater than valnemulin) and metabolic stability (vide infra).

Synergistic effect of Compound 51 with doxycycline. The synergistic or antagonistic activities of compound 51 were assessed in vitro via micro dilution broth checkerboard technique (Hsieh et al., Diagn. Microbiol. Infec. Dis. 1993, 16:343-349;

Ohrt et al., Antimicrob. Agents. Chemother. 2002, 46:2518-2524; and Siricilla et al., . J Antibiot. 2014, 68:271-278). In the checkerboard analyses of a combination of compound 51 and anti-Acinetobacter drugs (tobramycin, gentamycin, tigecycline, minocycline, doxycycline, rifampicin, and polymyxin), compound 51 displayed strong synergistic effects with doxycycline in a wide range of concentrations. Table 3 summarizes the results of FIC index analyses for a combination of compound 51 plus doxycycline and valnemulin plus doxycycline that showed synergistic combination

(∑FIC < 0.5). The FIC index range of 0.16 to 0.50 was observed for 8 combinations of two molecules out of 96 different concentrations (entries 1-8 in Table 3). Compared to a wide range of synergistic effects observed for compound 51, valnemulin showed synergistic effect at two combinations with doxycycline (entry 9). It was demonstrated that 60/1, 35/1, and 2/1 ratio of compound 51 and doxycycline (51-Dox 60/1 , 51-Dox 35/1, and 51-Dox 2/1) killed a drug susceptible A. baumannii with the MIC₉₀ of 0.78 μg/mL (entries 3-5 in Table 3, also see Table 2). These combinations killed the rifampicin (32xMIC)- and compound 51 (16xMIC) -resistant strains at 0.78 and 3.13 μg/mL concentration in 24 h. Significantly, 51-Dox 60/1, 51Dox 35/1, and 51-Dox 2/1 killed MDR A. baumannii (ATCC BAA- 1800) with much lower concentrations (MIC₉₀ of 3.13-6.25 μg/mL) than the MIC values of the individual molecules (MIC₉₀ >25 and 12.5 for Dox and analog 51, respectively). Table 3 shows the fractional inhibitory concentration of a combination of doxycycline and compound 51 or valnemulin. ^a∑FIC index for the wells at growth-no growth interface. ^bThe MIC values of molecule 51, valnemulin, and doxycycline against A. baumannii (ATCC 19606) are 6.25, 12.5, and 0.20 μg/mL, respectively. ^cCA and CB are concentrations of A and B. ^d∑FIC is the sum of fractional inhibitory concentration calculated by the equation∑FIC = FIC_A + FIC_B = C_A/MIC_A + C_B/MIC_B.

Table 3

In vitro metabolic stability and toxicity of Compound 51. Despite widespread use of valnemulin in veterinary fields, its metabolic profile has not been reported until recently. Several groups reported that the half-lives of valnemulin in plasma in vivo and ex-vivo are relatively short (1.3-2.9 h), suggesting challenges in its application for systemic antimicrobial therapy. In the current in vitro metabolic stability testing (McGinnity et al., Drug Metab. Dispos. 2004, 32:1247-1253), a striking difference in half-life ( t_½ ) was observed between compound 51 and valnemulin in rat liver microsomes; t_½ of valnemulin was 1.29 min., on the other hand, t_½ of compound 51 was >60 min. As such, in vitro half-life was significantly extended by reduction of the C3-carbonyl group of valnemulin. In vitro cytotoxicity against mammalian cells (e.g. Vero cells) of compound 51 (IC₅₀ 45.3 μg/mL) was 2.85 times less toxic than that of valnemulin (IC50 15.9 μg/mL) (FIG. 7).

In vivo effect of 51-Dox 35/1. As summarized above, the favorable in vitro physicochemical properties of analog 51 over valnemulin were realized. In addition, it was determined that compound 51 binds to the rat plasma protein with 76.9% (PPB), providing insights into the in vivo efficacy of compound 51 in infected animal models. The in vivo efficacy of compound 51 and 51-Dox 35/1 was evaluated in a mouse septicemia model using the C57BL/6 mice and A. baumannii (ATCC19606) strain (at a dose that lead to 75% of death). One hour after the infection, the molecules (analog 51, 51-Dox 35/1, or tobramycin) were administered intraperitoneally (IP) at single dose (from 2 to 60 mg/kg) (FIG. 8). At the 10, 20, and 60 mg/kg 51-Dox 35/1 dose, 100% of the mice survived, while all mice in the control group succumbed to infection in two days. The reference molecule, tobramycin survived the mice at 6.0 mg/kg dose IP. 100% of the mice in the group administered 4.0 mg/kg 51-Dox 35/1 were rescued mortality of the A. baumannii infection. At the 2.0 mg/kg 51-Dox 35/1 dose, 60% of the mice survived. Similarly, analog 51 showed effectiveness in the same in vivo studies, but required higher dosage than that with 51- Dox35/l. In FIG. 8, The C57BL/6 mice were infected intraperitoneally with A. baumannii (ATCC19606) strain at a dose that led to >75% of death in a day. The test molecules were intraperitoneally administered once after 1 h of the infection. Mortality was monitored for 5 days for all groups (P<0.05). A resistant mechanism of A. baumannii against 51. The pleuromutilin derivatives target the peptidyl transfer center of the 50S ribosomal protein L3 (rplC), inhibiting protein biosynthesis. Multiple mutations in rplC of S. aureus have been reported that can define a region of rplC capable of causing decreased susceptibility of the pleuromutilin derivative in S. aureus. In order to identify a potential mechanism of resistance to compound 51, chromosomal DNA was isolated from the resistant mutant (51^R, 16xMIC) and its parental wild-type control A. baumannii (ATCC 19606). The rplC gene fragment was amplified using A. baumannii rplC specific primers and sequenced. The DNA sequencing results were blasted against rplC DNA sequence of A. baumannii in the NIH genetic sequence database. The DNA sequence alignment revealed a C456A signal nucleotide mutation, which corresponded to N152K mutation in the protein sequence of RplC (FIG. 9). Interestingly, 51-Dox 35/1 and 51-Dox 2/1 effectively killed A. baumannii mutant 51^R with the MIC₁oo value of 3.13-6.26 μg/mL (vide supra). Any other tetracyclines such as minocycline, tigecycline, and demeclocycline did not exhibit the same effect as observed with doxycycline (Dox) against A. baumannii mutant 51^R. In FIG. 9, 51^R is the Query and wild-type control is the Sbjct: the highlighted amino acid represents the site mutation in RplC.

Spontaneous mutation frequency. The frequency that an A. baumannii strain spontaneously developed resistance to 51-Dox 35/1 was evaluated by applying the culture of A. baumannii (ATCC19606) strain to agar media containing 51-Dox 35/1 at concentrations 4- and 8-fold the MIC₁oo (2.0 μg/mL) on agar media. There was no colony on the plate containing 4xMIC after 48 h incubation when 1 x 10⁹ CFU bacteria were plated. Two colonies were identified on the plate containing 8xMIC when applied 1 x 10¹⁰ CFU bacteria. Two strains isolated in these experiments did not grow on the agar plates containing 51-Dox35/l at the 4x and 8x MIC concentrations. Thus, calculated spontaneous resistance mutation frequency of 51- Dox35/l is less than 1 x 10^-10 for the ATCC19606 strain. Compound 51 alone showed spontaneous resistant mutants for the same strain with the mutation frequency of 1 x 10^-8 at 4xMIC concentration. Example 2: Novel Pleuromutilin Analogs as Protein Biosynthesis Inhibitors.

Synthesis

All chemicals were purchased from commercial sources and used without further purification unless otherwise noted. THF, CH₂CI₂, and DMF were purified via Innovative Technology's Pure-Solve System. All reactions were performed under an Argon atmosphere. All stirring was performed with an internal magnetic stirrer. Reactions were monitored by TLC using 0.25 mm coated commercial silica gel plates (EMD, Silica Gel 6OF254). TLC spots were visualized by UV light at 254 nm, or developed with ceric ammonium molybdate or anisaldehyde or copper sulfate or ninhydrin solutions by heating on a hot plate. Reactions were also monitored by using SHIMADZU LCMS-2020 with solvents: A: 0.1% formic acid in water, B: acetonitrile. Flash chromatography was performed with SiliCycle silica gel (Purasil 60 A, 230-400 Mesh). Proton magnetic resonance ( ¹H -NMR) spectral data were recorded on 400, and 500 MHz instruments. Carbon magnetic resonance (¹³C-NMR) spectral data were recorded on 100 and 125 MHz instruments. For all NMR spectra, chemical shifts (δΗ, δC) were quoted in parts per million (ppm), and valuJes were quoted in Hz. ¹ H and ¹³C NMR spectra were calibrated with residual undeuterated solvent (CDCL: δΗ = 7.26 ppm, δC = 77.16 ppm; CD₃CN: δΗ = 1.94 ppm, δC = 1.32ppm; CD₃OD: δΗ =3.31 ppm, δC =49.00 ppm; DMSO-d₆: δΗ = 2.50 ppm, δC = 39.52 ppm; D₂O: δΗ = 4.79 ppm) as an internal reference. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, dd = double doublets, t = triplet, q = quartet, quin = quintet, hept = heptet, m = multiplet, br = broad. Infrared (IR) spectra were recorded on a Perkin- Elmer FT 1600 spectrometer. HPLC analyses were performed with a Shimadzu LC-20AD HPLC system. All compounds were purified by reverse HPLC to be >95% purity.

Pleuromutilin To a stirred solution of Pleuromutilin (2.00 g, 5.28 mmol) in CH₂C1₂ (26.4 mL) was added TsCl (1.21 g, 6.34 mmol) and DMAP (1.94 g, 15.9 mmol) at 0 °C.

After 4 h at 0 °C, the reaction mixture was quenched with IN HCl and extracted with

EtOAc. The combined organic extract was washed with aq. sat. NaHCCh, dried over

Na₂S0₄, concentrated in vacuo. The crude product was purified by silica gel column chromatography (hexanes/EtOAc 60:40) to yield the product (2.13 g, 3.99 mmol,

76%): TLC (hexanes/EtOAc 50:50) R_f = 0.50; [a]²⁰ _D +0.355 (c = 1.81, CHC1₃); IR

(thin film) V_max = 3566 (br), 2984, 2937, 2883, 2865, 1757, 1732, 1598, 1455, 1368,

1291, 1223, 1190, 1176, 1117, 1096, 1038, 1019, 815, 753, 719, 663 cm^-1; ¾ NMR

(400 MHz, Chloroform-d) δ 7.80 (d, J = 8.4 Hz, 2H), 7.35 - 7.32 (m, 2H), 6.39 (dd, J = 17.4, 11.0 Hz, 1H), 5.75 (d, J = 8.5 Hz, 1H), 5.31 (dd, J = 11.0, 1.5 Hz, 1H), 5.17

(dd, J = 17.4, 1.6 Hz, 1H), 4.46 (s, 2H), 3.33 (dd, J = 10.5, 6.5 Hz, 1H), 2.44 (s, 3H),

2.29 - 2.13 (m, 3H), 2.08 - 2.05 (m, 1H), 2.01 (d, J = 8.7 Hz, 1H), 1.74 (dq, J = 14.4,

3.0 Hz, 1H), 1.68 - 1.58 (m, 1H), 1.53 - 1.41 (m, 2H), 1.39 (s, 3H), 1.36 - 1.33 (m,

1H), 1.33 - 1.27 (m, 1H), 1.23 (d, J = 15.9 Hz, 1H), 1.14 (s, 3H), 1.08 (dd, J = 13.9,

4.5 Hz, 1H), 0.86 (d, J = 6.9 Hz, 3H), 0.61 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz,

CDC1₃) δ 216.63, 164.81, 145.24, 138.70, 132.60, 129.87 (2C), 128.04 (2C), 117.29,

74.49, 70.27, 65.01 , 57.98, 45.35, 44.48, 43.94, 41.81 , 36.51, 36.00, 34.36, 30.30,

26.74, 26.39, 24.78, 21.64, 16.48, 14.72, 11.42; HRMS (ESI+) m/z calcd for

C₂₉H₄₁0₇S [M + H] 533.2573, found: 533.2546.

To a stirred solution of starting material (196 mg, 0.37 mmol), l-amino-2- methylpropane-2-thiol hydrochloride (104 mg, 0.74 mmol) and tetra-n- butylammonium bromide (11.9 mg, 0.037 mmol) in THF (1.5 mL) was added IN

NaOH (1.5 mL). After 3 h at 50 °C, the reaction mixture was extracted with CHCI₃

(1, 2). The combined organic extract was dried over Na₂S0₄, concentrated in vacuo.

The crude product was purified by silica gel column chromatography (hexanes/EtOAc

50:50 to CHCL/MeOH 75 :25) to give the product (140 mg, 0.30 mmol, 82%): TLC (CHCl₃/MeOH 90:10) R_f = 0.20; [a]²⁰ _D +0.335 (c = 1.49, CHC1₃); IR (thin film) V_max

= 3433 (br), 2928, 2881, 2864, 1726, 1631, 1524, 1457, 1412, 1375, 1281 , 1152,

1119, 1033, 1010, 980, 954, 938, 916, 816, 754, 683 cm^-1; H NM¹ R (400 MHz,

Chloroform-d) δ 6.48 (dd, J = 17.4, 11.0 Hz, 1H), 5.76 (d, J = 8.4 Hz, 1H), 5.36 (dd, J = 10.9, 1.5 Hz, 1H), 5.21 (dd, J = 17.4, 1.6 Hz, 1H), 3.36 (d, J = 6.5 Hz, 1H), 3.15 (d,

J = 2.3 Hz, 2H), 2.63 (s, 2H), 2.38 - 2.30 (m, 1H), 2.32 - 2.14 (m, 2H), 2.13 - 2.03

(m, 2H), 1.87 (brs, 2H), 1.77 (dq, J = 14.4, 3.1 Hz, 1H), 1.71 - 1.61 (m, 2H), 1.60 - 1.50 (m, 2H), 1.50 - 1.47 (m, 1H), 1.46 (s, 3H), 1.41 - 1.33 (m, 1H), 1.26 (s, 6H),

1.17 (s, 3H), 1.11 (dd, J = 14.0, 4.3 Hz, 1H), 0.88 (d, J = 7.0 Hz, 3H), 0.73 (d, J = 6.9

Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.94, 169.57, 139.01, 117.30, 74.64,

69.52, 58.21, 51.42, 48.22, 45.47, 44.78, 43.95, 41.82, 36.79, 36.03, 34.46, 31.32,

30.46, 26.87, 26.34, 26.33, 26.29, 24.87, 16.86, 14.92, 11.50; HRMS (ESI+) mJz calcd for C₂₆H₄₄NO₄S [M + H] 466.2991 , found: 466.3016.

To a stirred solution of amine (62.1 mg, 0.13 mmol), Boc-D-Leu-OH (46.2 mg, 0.20 mmol), NaHCO₃ (112 mg, 1.33 mmol) and Glyceroacetonide-Oxyma (45.6 g, 1.20 mmol) in DMF-H₂0 (9/1 , 0.7 mL) was added EDCI (128 mg, 0.67 mmol).

The reaction mixture was stirred for 5 h at rt, quenched with aq. sat. NaHC0₃, and extracted with EtOAc. The combined organic extract was washed with IN HC1, brine, dried over Na₂S0₄ and concentrated in vacuo (3,4,5). The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 75 :25 to 60:40) to afford the product (55.2 mg, 0.081 mmol, 61 %): TLC (hexanes/EtOAc 50:50) R_f =

0.40; [a]²⁰D +0.497 (c = 2.32, CHCI3); IR (thin film) V_max = 3322 (br), 2957, 2931,

2868, 1722, 1662, 1525, 1454, 1388, 1366, 1279, 1247, 1164, 1116, 1018, 980, 917,

752 cm^-1; ¹H NMR (400 MHz, Chloroform-d) δ 7.06 (t, J = 6.2 Hz, 1H), 6.45 (dd, J =

17.4, 11.0 Hz, 1H), 5.75 (d, J = 8.4 Hz, 1H), 5.31 (dd, J = 11.0, 1.6 Hz, 1H), 5.19 (dd,

J = 17.4, 1.6 Hz, 1H), 4.99 (d, J = 8.2 Hz, 1H), 4.19 - 4.11 (m, 1H), 3.39 - 3.32 (m,

1H), 3.21 (t, J = 6.2 Hz, 2H), 3.17 (d, J = 2.5 Hz, 2H), 2.36 - 2.28 (m, 1H), 2.28 - 2.15 (m, 2H), 2.13 - 2.05 (m, 2H), 1.80 - 1.73 (m, 1H), 1.72 - 1.59 (m, 3H), 1.59 - 1.46 (m, 2H), 1.44 (s, 3H), 1.43 (s, 9H), 1.40 - 1.33 (m, 1H), 1.29 (d, J = 16.1 Hz,

1H), 1.23 (s, 6H), 1.16 (s, 3H), 1.11 (dd, J = 13.9, 4.4 Hz, 1H), 0.95 (dd, J = 6.3, 1.7

Hz, 6H), 0.87 (d, J = 7.0 Hz, 3H), 0.71 (d, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz,

CDC1₃) δ 216.91, 172.89, 170.05, 138.89, 117.32, 74.59, 69.78, 58.14, 47.60, 47.08,

45.42, 44.81, 43.92, 41.77, 36.69, 36.00, 34.41 , 31.44, 30.39, 28.33 (3C), 26.82,

26.33, 26.26, 26.17, 24.83, 24.81, 22.92, 16.81, 14.86, 11.50; HRMS (ESI+) m/z calcd for C37H63N2O7S [M + H] 679.4356, found: 679.4328.

To a stirred solution of starting material (18.7 mg, 0.027 mmol) in dioxane

(0.2 mL) was added a 4N solution of HCI in dioxane (0.8 mL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: a gradient elution of 70:30 to 100:0 MeOH : H₂0 over

20 min, flow rate: 2.0 mL/min, UV: 220 nm] to afford 41 (11.9 mg, 0.021 mmol,

75%, retention time: 18 min): TLC (CHCl₃/MeOH 90: 10) R_f = 0.20; [a]²¹ _D +0.125 (c

= 0.50, MeOH); IR (thin film) v_max = 3354 (br), 2955, 2932, 2867, 1729, 1659, 1522,

1464, 1414, 1386, 1368, 1282, 1144, 1117, 1019, 981, 953, 939, 917 cm^-1; ¹H NMR

(400 MHz, Methanol-d₄) δ 6.32 (dd, J = 17.9, 10.8 Hz, 1H), 5.75 (d, J = 8.3 Hz, 1H),

5.17 (q, J = 1.6 Hz, 1H), 5.14 (q, J = 1.6 Hz, 1H), 3.50 (d, J = 6.1 Hz, 1H), 3.39 (dd, J = 8.2, 6.1 Hz, 1H), 3.28 (d, J = 5.9 Hz, 2H), 2.39 - 2.32 (m, 2H), 2.32 - 2.22 (m, 1H),

2.16 (ddd, J = 16.0, 8.9, 4.9 Hz, 2H), 1.85 - 1.79 (m, 1H), 1.76 (ddd, J = 13.1, 6.6, 1.5

Hz, 1H), 1.69 (dt, J = 11.2, 2.2 Hz, 1H), 1.66 - 1.54 (m, 4H), 1.48 (d, J = 2.6 Hz, 1H),

1.46 (s, 3H), 1.44 - 1.32 (m, 4H), 1.32 - 1.28 (m, 1H), 1.27 (s, 3H), 1.25 (s, 3H), 1.15

(s, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.95 (d, J = 6.5 Hz, 3H), 0.93 (d, J = 7.0 Hz, 3H),

0.73 (d, J = 6.6 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 219.60, 178.24, 171.39,

141.16, 116.62, 75.46, 71.45, 59.32, 54.73, 47.81, 47.79, 47.77, 46.81, 45.99, 45.76,

45.25, 43.14, 38.16, 37.72, 35.29, 31.52, 28.17, 28.05, 26.90, 26.79, 25.92, 25.85, 23.47, 22.52, 17.13, 15.41, 11.77; HRMS (ESI+) m/z calcd for C₃2H55N2O5S [M + H] 579.3832, found: 579.3864.

To a stirred solution of starting material (19.1 mg, 0.28 mmol) in

MeOH/EtOAc (1 :1, 3.0 mL) was added Pd/C (10 wt %, 5.0 mg). H₂ gas was introduced and the reaction mixture was stirred for 20 h under H₂. The solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 67:33 to 60:40) to obtain the product (16.4 mg, 0.024 mmol, 85%): TLC (hexanes/EtOAc 50:50) R_f = 0.50; [a]²¹ _D +0.067 (c = 0.41, CHCI₃); IR (thin film) v_max = 3323 (br), 2958, 2931, 2871 , 1724, 1666, 1526, 1462, 1385, 1367, 1281, 1167, 1116, 1046, 972, 755 cm^-1; 1H NMR (400 MHz, Chloroform-d) δ 7.09 (t, J = 6.2 Hz, 1H), 5.62 (d, J = 8.2 Hz, 1H), 4.99 (d, J = 8.2 Hz, 1H), 4.20 - 4.11 (m, 1H), 3.41 (d, J = 5.9 Hz, 1H), 3.29 (dd, J = 14.0, 6.5 Hz, 1H), 3.22 - 3.15 (m, 1H), 3.18 (s, 2H), 2.39 (quin, J = 6.9 Hz, 1H), 2.23 (dd, J = 7.7, 2.9 Hz, 1H), 2.21 - 2.15 (m, 1H), 2.10 (d, J = 2.7 Hz, 1H), 1.81 - 1.77 (m, 1H), 1.74 (dd, J = 7.8, 4.3 Hz, 1H), 1.71 - 1.67 (m, 1H), 1.67 - 1.62 (m, 1H), 1.61 - 1.58 (m, 1H), 1.58 - 1.54 (m, 1H), 1.54 - 1.46 (m, 3H), 1.44 (s, 9H), 1.42 (s, 3H), 1.39 - 1.36 (m, 1H), 1.32 (d, J = 16.2 Hz, 1H), 1.25 (s, 6H), 1.11 (td, J = 14.0, 4.3 Hz, 1H), 0.97 (s, 3H), 0.96 (s, 6H), 0.94 (d, J = 7.3 Hz, 3H), 0.75 (t, J = 7.4 Hz, 3H), 0.69 (d, J = 6.9 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.98, 172.69, 170.29, 70.12, 60.37, 58.46, 47.63, 47.14, 45.52, 41.84, 40.99, 40.88, 36.68, 34.43, 34.29, 31.31, 30.21, 28.34 (3C), 26.85, 26.38, 26.25, 26.09, 24.90, 24.81, 22.97, 20.65, 16.69, 14.86, 14.18, 11.01, 8.31 ; HRMS (ESI+) m/z calcd for C37H65N2O7S [M + H] 681.4512, found: 681.4533.

To a stirred solution of starting material (11.7 mg, 0.017 mmol) in dioxane (0.2 mL) was added a 4N solution of HC1 in dioxane (0.3 mL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: a gradient elution of 25 :75 to 55 :45 MeOH : H₂O over 20 min then 55:45 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 mo] to afford 42 (7.5 mg, 0.013 mmol, 75%, retention time: 29 min): TLC (CHCl₃/MeOH 90:10) R_f = 0.20; [a]²¹ _D +0.071 (c = 0.27, MeOH); IR (thin film) = 3344 (br), 2958, 2932, 2871, 1732, 1664, 1521, 1464, 1384, 1368, 1282, 1148, 1117 cm^-1; ¹H NMR (400 MHz, Methanol-d4) δ 5.67 (d, J = 8.2 Hz, 1H), 3.45 (d, J = 6.0 Hz, 1H), 3.39 (dd, J = 8.2, 6.1 Hz, 1H), 3.35 (s, 1H), 3.29 (d, J = 3.2 Hz, 1H), 2.41 - 2.31 (m, 2H), 2.26 (dddd, J = 19.3, 11.1, 2.6, 1.3 Hz, 1H), 2.14 (dt, J = 19.2, 9.3 Hz, 1H), 1.85 - 1.72 (m, 4H), 1.69 (dt, J = 11.2, 2.1 Hz, 1H), 1.65 - 1.49 (m, 5H), 1.47 (d, J = 2.5 Hz, 1H), 1.45 (s, 3H), 1.40 (d, J = 1.2 Hz, 1H), 1.39 - 1.32 (m, 3H), 1.28 (s, 3H), 1.26 (s, 3H), 1.14 (td, J = 14.2, 4.6 Hz, 1H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.5 Hz, 3H), 0.94 (s, 3H), 0.91 (s, 3H), 0.74 (t, J = 7.4 Hz, 3H), 0.73 (d, J = 6.8 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 219.67, 178.22, 171.80, 76.65, 71.41, 59.40, 54.73, 47.86, 47.84, 46.84, 45.78, 43.07, 41.94, 41.72, 38.16, 35.90, 35.29, 32.31, 31.46, 28.15, 26.88, 26.78, 26.74, 25.93, 25.70, 23.48, 22.51, 21.44, 17.30, 15.43, 11.71, 8.81 ; HRMS (ESI+) mlz calcd for C₃2H57N2O5S [M + H] 581.3988, found: 581.3958.

To a stirred solution of amine (83.1 mg, 0.18 mmol), Boc-D-Leu-OH (61.6 mg, 0.27 mmol), NaHC0₃ (149 mg, 1.78 mmol) and Glyceroacetonide-Oxyma (60.8 mg, 0.27 mmol) in DMF-H₂0 (9/1, 0.9 mL) was added EDCI (170 mg, 0.89 mmol) (3,4,5). The reaction mixture was stirred for 5 h at rt, quenched with aq. sat. NaHC0₃, and extracted with EtOAc. The combined organic extract was washed with IN HC1, brine, dried over Na₂S0₄ and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 67:33 to 60:40) to afford the product (108 mg, 0.158 mmol, 89%): TLC (hexanes/EtOAc 50:50) R_f = 0.40; [αΓο -0.087 (c = 3.10, CHC1₃); IR (thin film) V_max = 3323 (br), 2956, 2934, 2872, 1704, 1666, 1526, 1455, 1390, 1367, 1286, 1249, 1166, 1120, 1020, 1005, 933, 755 cm^-1; H¹ NMR (400 MHz, Chloroform-d) δ 7.11 - 7.02 (m, 1H), 6.49 (dd, J = 17.3, 11.0 Hz, 1H), 5.61 (d, J = 9.3 Hz, 1H), 5.31 (dd, J = 10.9, 1.7 Hz, 1H), 5.16 (dd, J = 17.4, 1.7 Hz, 1H), 4.98 (d, J = 8.1 Hz, 1H), 4.55 (t, J = 5.5 Hz, 1H), 4.19 - 4.10 (m, 1H), 3.22 (dd, J = 11.5, 6.1 Hz, 1H), 3.17 (s, 2H), 2.24 (ddd, J = 13.8, 6.8, 3.2 Hz, 1H), 2.17 - 2.04 (m, 2H), 1.94 (dddd, J = 16.3, 10.3, 7.4, 3.6 Hz, 1H), 1.89 - 1.80 (m, 1H), 1.76 - 1.57 (m, 4H), 1.54 - 1.48 (m, 1H), 1.44 (s, 12H), 1.27 - 1.21 (m, 12H), 1.14 (s, 3H), 0.96 (d, J = 6.0 Hz, 6H), 0.80 (d, J = 7.0 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDC1₃) δ 216.96, 172.84, 170.06, 139.42, 116.78, 74.86, 71.67, 60.38, 50.89, 47.55, 47.06, 45.94, 44.81 , 41.17, 36.44, 35.56, 34.24, 32.50, 31.72, 31.46, 28.34 (3C), 27.61, 26.21 (2C), 24.81, 22.95, 21.03, 17.54, 17.20, 14.17, 12.18; HRMS (ESI+) m/z calcd for CsvH^OvS [M + H] 681.4512, found: 681.4535.

To a stirred solution of starting material (31.1 mg, 0.046 mmol) in MeOH/EtOAc (1 : 1, 3.0 mL) was added Pd/C (10 wt %, 8.0 mg). H₂ gas was introduced and the reaction mixture was stirred for 14 h under H₂. The solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by silica gel column chromatography (hexanes/EtOAc 75:25 to 60:40) to obtain the product (22.0 mg, 0.032 mmol, 71%): TLC (hexanes/EtOAc 67:33) R_f = 0.30; [a]²¹ _D - 0.063 (c = 0.61, CHCI₃); IR (thin film) V_max = 3323 (br), 2957, 2933, 2873, 1697, 1666, 1526, 1462, 1367, 1285, 1250, 1219, 1166, 1119, 1046, 1021, 1004, 971, 931 , 771 cm^-1; H¹ NMR (400 MHz, Chloroform-d) δ 7.11 (d, J = 5.8 Hz, 1H), 5.49 (d, J = 9.2 Hz, 1H), 4.99 (d, J = 8.2 Hz, 1H), 4.53 (t, J = 5.6 Hz, 1H), 4.20 - 4.11 (m, 1H), 3.23 (dd, J = 6.1 , 3.8 Hz, 2H), 3.18 (s, 2H), 2.26 - 2.15 (m, 1H), 1.96 (ddt, J = 14.3, 11.9, 6.2 Hz, 1H), 1.87 - 1.59 (m, 4H), 1.58 - 1.47 (m, 1H), 1.44 (s, 12H), 1.40 - 1.32 (m, 1H), 1.27 - 1.22 (m, 9H), 1.20 (s, 3H), 0.96 (d, J = 6.3 Hz, 9H), 0.92 (s, 3H), 0.86 (s, 3H), 0.76 (t, J = 7.4 Hz, 3H), 0.67 (d, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCI₃) δ 217.00, 172.72, 170.18, 72.10, 60.37, 58.46, 50.99, 47.62, 47.08, 45.98, 41.61, 41.40, 41.35, 36.47, 34.22, 33.83, 32.12, 31.65, 31.41 , 28.35 (3C), 27.62,

26.34, 26.29, 26.14, 24.81, 22.98, 20.73, 17.62, 16.96, 14.85, 14.18, 11.68, 8.34;

HRMS ESI+) mlz calcd for C37H67N2O7S [M + H] 683.4669, found: 683.4687.

To a stirred solution of starting material (15.3 mg, 0.022 mmol) in dioxane

(0.2 niL) was added a 4N solution of HC1 in dioxane (0.4 mL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: a gradient elution of 25 :75 to 55 :45 MeOH : H₂O over

20 min then 55:45 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 50

(10.1 mg, 0.017 mmol, 77%, retention time: 25 min): TLC (CHCl₃/MeOH 90:10) R_f =

0.30; [a]²¹ _D +0.004 (c = 0.81 , MeOH); IR (thin film) v_max = 3432 (br), 2958, 2876,

1680, 1562, 1464, 1370, 1290, 1144, 1020, 1001, 970, 930 cm^-1; ¹H NMR (400 MHz,

Methanol-^) δ 5.58 (d, J = 9.0 Hz, 1H), 4.48 (t, J = 5.6 Hz, 1H), 3.94 (dd, J = 8.2, 5.6

Hz, 1H), 3.38 (dd, J = 14.8, 4.5 Hz, 2H), 3.35 (s, 2H), 3.29 - 3.23 (m, 2H), 2.36 (tq, J = 11.2, 7.0, 5.6 Hz, 1H), 2.17 (quin, J = 6.8 Hz, 1H), 1.94 (dd, J = 12.9, 3.7 Hz, 1H),

1.91 - 1.86 (m, 1H), 1.84 - 1.73 (m, 4H), 1.73 - 1.64 (m, 3H), 1.64 - 1.56 (m, 2H),

1.52 (dd, J = 14.1, 7.4 Hz, 1H), 1.48 - 1.41 (m, 1H), 1.41 - 1.35 (m, 1H), 1.35 - 1.32

(m, 1H), 1.30 (s, 3H), 1.28 (s, 3H), 1.23 (s, 3H), 1.05 (d, J = 4.8 Hz, 3H), 1.04 (d, J =

4.9 Hz, 3H), 0.91 (s, 3H), 0.86 (d, J = 7.0 Hz, 3H), 0.74 (t, J = 7.4 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 172.24, 170.72, 77.33, 77.17, 73.47,

53.14, 52.19, 47.83, 47.17, 42.66, 42.55, 42.04, 42.01 , 37.20, 35.42, 34.59, 33.16,

32.66, 32.44, 29.06, 26.95, 26.91, 26.68, 25.56, 23.13, 22.19, 21.47, 17.77, 17.54,

12.52, 8.79; HRMS (ESI+) mlz calcd for C32H59N2O5S [M + H] 583.4145, found:

583.4166.

To a stirred solution of starting material (11.3 mg, 0.016 mmol) in dioxane

(0.4 mL) was added a 4N solution of HC1 in dioxane (0.6 mL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: a gradient elution of 70:30 to 100:0 MeOH : H₂O over

20 min then MeOH, flow rate: 2.0 mL/min, UV: 220 nm] to afford 45 (5.6 mg, 0.009 mmol, 58%, retention time: 25.8 min): H N¹MR (400 MHz, Methanol-d 4) δ 5.79 (d, J = 8.1 Hz, 1H), 3.80 (s, 3H), 3.47 (dd, J = 8.3, 6.1 Hz, 1H), 3.40 - 3.34 (m, 2H), 3.28

(d, J = 13.0 Hz, 1H), 2.56 (s, 1H), 2.54 - 2.43 (m, 1H), 2.35 (q, J = 6.7 Hz, 1H), 2.27

(ddd, J = 19.3, 10.6, 1.8 Hz, 1H), 2.02 (ddd, J = 11.0, 7.2, 3.7 Hz, 1H), 1.86 (dd, J =

16.1, 8.3 Hz, 1H), 1.82 - 1.72 (m, 2H), 1.70 - 1.51 (m, 4H), 1.49 (s, 3H), 1.48 - 1.40

(m, 2H), 1.40 - 1.32 (m, 3H), 1.28 (d, J = 8.4 Hz, 6H), 1.26 - 1.21 (m, 1H), 1.17 (dd,

J = 13.8, 4.2 Hz, 1H), 0.99 (d, J = 6.5 Hz, 3H), 0.97 (d, J = 6.5 Hz, 3H), 0.93 (s, 3H),

0.87 (s, 3H), 0.74 (t, J = 7.4 Hz, 6H); ¹³C NMR (101 MHz, MeOD) δ 171.94, 167.67,

76.54, 72.15, 61.76, 54.51, 53.85, 47.87, 45.22, 43.55, 41.85, 41.59, 36.37, 36.02,

32.38, 30.41, 28.55, 28.41, 26.89, 26.80, 26.71, 25.88, 24.65, 23.44, 22.46, 21.44,

17.36, 17.18, 12.79, 8.81.

To a stirred solution of starting material (15.0 mg, 0.021 mmol) in dioxane (0.4 mL) was added a 4N solution of HC1 in dioxane (0.6 mL). The reaction mixture was stirred for 1 h at rt, and all volatiles were evaporated in vacuo. The crude mixture was purified by CI 8 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μιη, 250 x 10 mm), solvents: a gradient elution of 70:30 to 100: 0 MeOH : H₂O over 20 min then MeOH, flow rate: 2.0 mL/min, UV: 220 nm] to afford 44 (10.3 mg, 0.017 mmol, 80%, retention time: 21 min): H N¹ MR (400 MHz, Methanol-d 4) δ 6.38 - 6.27 (m, 1H), 5.87 (d, J = 8.2 Hz, 1H), 5.14 (dq, J = 14.1 , 1.6 Hz, 2H), 3.80 (s, 3H), 3.41 (d, J = 6.1 Hz, 1H), 3.40 - 3.38 (m, 1H), 3.29 - 3.26 (m, 2H), 2.59 (s, 1H), 2.50 (dtd, J = 19.2, 9.6, 1.2 Hz, 1H), 2.31 (ddt, J = 18.5, 9.7, 7.4 Hz, 2H), 2.22 (dd, J = 14.5, 6.9 Hz, 1H), 2.08 - 1.96 (m, 1H), 1.76 (ddt, J = 13.1 , 7.7, 6.5 Hz, 1H), 1.67 (dt, J = 13.6, 2.7 Hz, 1H), 1.63 - 1.55 (m, 2H), 1.50 (s, 3H), 1.49 - 1.47 (m, 1H), 1.46 - 1.42 (m, 1H), 1.42 - 1.38 (m, 1H), 1.36 (d, J = 5.1 Hz, 1H), 1.33 - 1.31 (m, 1H), 1.31 - 1.28 (m, 1H), 1.27 (s, 3H), 1.25 (s, 3H), 1.20 (d, J = 4.5 Hz, 1H), 1.15 (s, 3H), 0.98 (d, J = 6.6 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.89 (d, J = 7.0 Hz, 3H), 0.73 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 178.03, 171.47, 167.64, 141.28, 116.51, 75.36, 72.09, 61.77, 54.70, 53.81, 47.81, 45.99, 45.68, 45.15, 43.63, 37.81, 36.36, 32.51 , 30.48, 28.72, 28.30, 28.11, 26.91, 26.79, 25.92, 24.67, 23.47, 22.51, 17.19, 17.18, 12.84.

To a stirred solution of 51 (11.1 mg, 0.020 mmol) in MeOH (5.0 mL) was added Pd/C (10 wt %, 5.0 mg). H₂ gas was introduced and the reaction mixture was stirred for 11 h under H₂. The solution was filtered through Celite and concentrated in vacuo. The crude mixture was purified by C18 reverse-phase HPLC [column: HYPERSIL GOLD™ (175 A, 12 μηι, 250 x 10 mm), solvents: a gradient elution of 25:75 to 55 :45 MeOH : H₂0 over 20 min then 55 :45 MeOH : H₂0, flow rate: 2.0 mL/min, UV: 220 nm] to afford 40 (10.1 mg, 0.018 mmol, 91%, retention time: 23.7 min): 1H NMR (400 MHz, Methanol-d4) δ 5.55 (d, J = 8.9 Hz, 1H), 4.47 (t, J = 5.7 Hz, 1H), 3.73 (d, J = 5.3 Hz, 1H), 3.51 (d, J = 14.0 Hz, 1H), 3.36 (d, J = 14.9 Hz, 1H), 3.27 - 3.18 (m, 2H), 2.36 (tq, J = 11.2, 6.6 Hz, 1H), 2.24 (dt, J = 13.2, 6.7 Hz, 1H), 2.16 (p, J = 6.6 Hz, 1H), 1.91 (tdd, J = 14.2, 9.8, 5.3 Hz, 2H), 1.77 (dt, J = 14.8, 8.3 Hz, 2H), 1.72 - 1.60 (m, 3H), 1.57 (d, J = 5.1 Hz, 1H), 1.52 (dd, J = 14.1 , 7.4 Hz, 1H), 1.47 - 1.41 (m, 1H), 1.39 (d, J = 4.0 Hz, 1H), 1.31 (s, 3H), 1.29 (s, 3H), 1.26 (s, 1H), 1.23 (s, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.09 (d, J = 7.0 Hz, 3H), 0.90 (s, 3H), 0.86 (d, J = 6.9 Hz, 3H), 0.74 (t, J = 7.3 Hz, 3H), 0.69 (d, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, MeOD) δ 171.84, 169.61, 77.35, 77.20, 73.40, 59.93, 52.20, 47.65, 47.18, 42.68, 42.56, 42.10, 37.22, 35.40, 34.59, 33.16, 32.67, 32.53, 31.62, 29.06, 27.05, 26.92, 26.85, 21.43, 19.03, 17.90, 17.81, 17.58, 12.48, 8.81.

Materials and Methods

Bacterial strains and growth of bacteria. Mycobacterium tuberculosis (H₃7RV) were obtained through BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). Mycobacterium smegmatis (ATCC 607), Mycobacterium bovis (BCG), Staphylococcus aureus (ATCC 25923, BAA 2094, BAA 44, and BAA 1683), Enterococcus faecium (ATCC 349), Enterococcus faecalis (ATCC 19433), Streptococcus pneumoniae (ATCC 6301), Streptococcus salivarius (ATCC 6301), Bacillus subtilis (ATCC 6051), Clostridium difficile (ATCC 43596), Klebsielle pneumoniae (ATCC 8047), Pseudomonas aeruginosa (ATCC 27853), Acinetobacter baumannii (ATCC 19606), and Escherichia coli (ATCC 10798) were obtained from American Type Culture Collection (ATCC).

Bacterial Culture Conditions. A single colony of each Mycobacterium strain (M. tuberculosis H₃7RV, M. bovis (BCG) and M. smegmatis ATCC 607) was obtained on a Difco Middlebrook 7H10 nutrient agar enriched with 10% oleic acid, albumin, dextrose and catalase (OADC for M. tuberculosis), albumin, dextrose and catalase (ADC for M. smegmatis and M. bovis). Tryptic Soy agar was used for all other strains except C. difficile. A single colony of C. difficile was obtained on modified reinforced clostridial agar that was prepared anaerobically.

Seed cultures of each Mycobacterium strain were obtained in Middlebrook 7H9 broth enriched with OADC (for M. tuberculosis), ADC (for M. smegmatis and M. bovis). Tryptic Soy broth was used for all other strains except C. difficile. C. difficile was cultured in modified reinforced clostridial broth that was prepared anaerobically in an anerobic chamber under 10% H₂, 5% CO2, and 85% N2 conditions. Flasks containing each bacterial strain was incubated to mid-log phase in their respective culture media, in a shaking incubator at 37 °C with a shaking speed of 200 rpm and cultured to mid-log phase (Optical density - 0.5). The optical density was monitored at 600 nm using a microplate reader (Biotek Synergy XT).

Minimum Inhibitory Concentration Assays: Microplate Alamar Blue Assay (MABA). These were performed according to the published protocol (Collins et al., Antimicrob. Agents. Chemother. 1997, 41(5): 1004-1009). Bacterial cultures at 0.5 optical density, was treated with serial dilutions of inhibitors in aerobic conditions and incubated at 37 °C for 15 days for M. tuberculosis (H37Rv) and M. bovis (BCG). Incubation time was 48h for M. smegmatis (ATCC 607). All other bacteria were incubated for 24 h. After incubation, 20 μ L of resazurin (stock-0.02%) was added and incubated on a shaking incubator at 37 °C for 4 h for M. tuberculosis and M. bovis. For all other bacteria 20μ L was added from a 0.08% resazurin stock solution and left for 1 h. The lowest concentration at which the color of resazurin was completely retained as blue was read as the MIC₁₀₀ (Pink = Growth, Blue = No growth). The absorbance measurements were also performed using a Biotek Synergy XT (Winooski, VT, USA), 96 well plate reader at 570 nm and 600 nm. Assay plates for C. difficile was incubated for 24 h in the anaerobic conditions and the optical density was measured at the end of incubation and wells with no visible growth was considered MIC.

Minimum Inhibitory Concentration Assays: Luminescence-based Low- oxygen-recovery Assay (LORA). These assays were performed according to the reported procedures in the facility of Illinois TB Research Institute (Cho et al., Antimicrob. Agents. Chemother. 2007, 51(4):1380-1385). In brief, M. tuberculosis H37Rv cells were transformed by mixing at least 1 μg of the purified plasmid, pFCA- luxAB and incubating at room temperature for 30 min, followed by electroporation (Snewin et al., Infect. Immun. 1999, 67(9):4586-4593). M.tuberculosis pFCA-luxAB strain cultured was diluted in Middlebrook 7H12 broth, and sonicated for 15 s. The cultures were diluted to obtain an A570 of 0.03 to 0.05 and 3,000 to 7,000 RLUs per 100 μΐ. Twofold serial dilutions of antimicrobial agents were prepared in black 96- well microtiter plates (100 μΐ), and 100 μΐ of the cell suspension was added. The microplate was placed under anaerobic conditions (oxygen concentration, less than 0.16%) by using an Anoxomat model WS-8080 (MART Microbiology) and three cycles of evacuation and filling with a mixture of 10% H₂, 5% CO₂, and 85% N₂. Incubation was continued for 10 days, and transferred to an ambient gaseous condition (5% CO₂- enriched air) incubator for a 28 h "recovery." 100 μΐ culture was transferred to white 96-well microtiter plates for determination of luminescence.

Cytotoxicity Assays. Selected molecules were tested for cytotoxicity (IC₅₀) in Vero cells via a MTT colorimetric assay. Vero cell line was cultured in Complete eagle's minimum essential growth medium (EMEM) containing L-glutamine, sodium pyruvate, minimum essential amino acids, penicillin-streptomycin and 10% fetal bovine serum. After 72 h of exposure of molecules to this cell line at concentrations ranging from 0.78 to 200 μg/mL, the culture medium was changed to complete EMEM without phenol red before addition of yellow tetrazolium dye; MTT. Viability was assessed on the basis of cellular conversion of MTT into a purple formazan product. The absorbance of the colored formazan product was measured at 570 nm by BioTek Synergy HT Spectrophotometer.

Table 4

Table 4 shows the cytotoxicities (IC₅₀) of selected compounds in Vero monkey kidney cells and the anti-Mtb activity and cytotoxicity of pleuromutilin analogs. ^aThe microplate alamar blue assay method was used; ^bLow oxygen recovery assay.

Table 5

Table 5 continued

Table 5 shows the spectrum of activity of analogs 50, 41 and 42. ^aThe microplate alamar blue assay method was used.

Killing Effect Against Intracellular M. tuberculosis. J774A.1 cells were seeded at 2.5 x 10⁵ cells/well in 24-well dishes or 1 x 10⁵ cells/well in 8-well chamber slides and incubated overnight at 37 °C in DMEM. A transformant M. tuberculosis CDC1551 expressing tdTomato was grown in 7H9 Middlebrook medium supplemented with OADC. The M. tuberculosis cells were harvested at an optical density of 0.5, washed and re-suspended in saline. J774A.1 cells were maintained in cell culture medium and were infected by M. tuberculosis (10⁶ bacteria in 0.2 mL of media): a multiplicity of infection (MOI) of «10 (bacteria/cell). The extracellular bacteria were removed by washing with PBS. The infected macrophages were treated with antibacterial agents at x2 and x4 MIC concentrations and the relative intensity of the fluorescence was measured [emission wavelength (581 nm)] via UV-vis spectroscopy in 24, 48, and 72 h for inhibition of intracellular bacterial growth. Surviving M. tuberculosis cells were confirmed by CFU method (Kong et al., Proc. Natl Acad. Sci. U.S.A. 2010, 107(27):12239-12244).

Kill-curve Graph: Determination of Colony Forming Units per Milliliter. M. tuberculosis H₃₇Rv cultures at mid-log phase (OD=0.5) were diluted to OD=0.25 and treated with inhibitor molecules at MIC, x2 MIC and x4 MIC. Each culture well was diluted 10, 100, 1000 and 10,000 fold every 24 h and 20μ1_ from each dilution was plated on 7H10 agar plates supplemented with OADC enrichment. Plates were incubated for 15 days in a static incubator at 37 °C and colonies were counted (FIG. 10).

Microsomal Stability. Pooled Sprague-Dawley rat liver microsomes were purchased from Corning Life Sciences (Oneonta, NY, USA). Microsomes ((20 mg/mL) were thawed on ice and diluted using phosphate buffer (100 mM, pH: 7.4), resulting in a protein concentration of 1 mg/mL. Stock solutions (10 mg/L) of analogs 50, 41, 42, valnemulin and verapamil (positive control) were prepared in DMSO (50%). A final concentration of 500 ng/niL was used for incubation with microsomes. NADPH (final concentration: 1 mM) was used as a co-factor. All the above solutions except NADPH were added to individual wells (12-well) in triplicate and were allowed to equilibrate for 5 min at 37°C. NADPH was then added. 50 aliquots in triplicate were drawn from the incubation mixture at 0, 5, 10, 20, 30, 45 and 60 min and immediately the reaction was quenched by addition of ice-cold methanol (4 volumes) (McGinnity et al., Drug Metab. And Dispos. 2004, 32:1247- 1253). The samples containing methanol was lyophilized to remove all volatiles. The residue was dissolved in IN HC1 aq. (ΙΟμί) and MeOH (40μί). The resulting solution (20μμί) was injected to LC-MS. MS solvent 90:10 acetonitrile/0.05% formic acid in water. Flow rate: 0.5mL/min (10). In FIG. 11, Verapamil was utilized as a control compound, and the half-life (t_1/2) of verapamil was determined to be 10 min.

Results

Effectiveness of Pleuromutilin Analogs Against Replicating and Non- replicating Mtb. To identify new pleuromutilin analogs with activity towards Mtb, over 50 analogs whose structures contained the methoxyamine-oxime (FIG. 12, A), hydroxylamine-oxime (FIG. 12, B), or 3-hydroxy (FIG. 12, C) core structure were generated. These structures were further diversified by the reduction of the double bond (C16 position), amide formations and reductive aminations. The generated molecules were evaluated in the growth inhibitory assays against Mtb ( H₃₇Rv). Eight pleuromutilin analogs were identified which exhibited the MIC₁oo value <12 μg /mL against Mtb (FIG. 12, D). In FIG. 12, the structures were diversified by the reduction of the double bond (C16 position), amide formations and reductive aminations at C14-side chain and reduction at C3 position. FIG. 13 shows the syntheses of pleuromutilin analogs 50, 41 and 42 with the following conditions: a) NaBFU, MeOH, 0 °C. b) TsCl, DMAP, CH₂C1₂, 0 °C. c) l-amino-2-methylpropane-2-thiol, 1M NaOH, nBu₄NBr, THF/H₂0 (1 : 1), 50 °C. d) Boc-D-Leu-OH, Glyceroacetonide-Oxyma, EDCI, NaHC0₃, DMF-H₂0 (9/1). e) H₂, Pd/C, MeOH-EtOAc. f) HC1, Dioxane.

The MIC values of the selected analogs were determined against Mtb H₃₇Rv in both aerobic and anaerobic conditions via MABA and LORA assays respectively. Compounds 50, 41 and 42 exhibited lowest MIC₁oo values compared to the other hit compounds with MABA MIC₁oo of 0.78-l^g/ml and LORA MIC 1.04-1.98 μg/ml (Table 5). The ratio of MICLORA/MICMABA for the three compounds was between 1.27 - 1.45 which is closer to the ideal value 1. The identified molecules are analogs of valnemulin and they exhibited a superior inhibitory profile against replicating and non-replicating Mtb H₃₇Rv compared to valnemulin and pleuromutilin itself. Valnemulin interfere with the bacterial translation by binding at two ribosomal key sites known as the "A" site and the "P" site, resulting in the inhibition of peptide elongation and the cessation of bacterial growth. Oxazolidinone drugs such as linezolid binds to the "A" site and only partially overlaps with that of the pleuromutilin class. FIG. 14 shows binding sites of tiamulin and linezolid on bacterial 5 OS ribosomal subuinit. Pleuromutilins bind to the peptidyl transferase center preventing the elongation of nascent peptide. Tiamulin interferes with the correct positioning of both A- and P-site substrates.

In order to obtain insights into potential toxicity of identified inhibitor molecules, all antimycobacterial pleuromutilin analogs were evaluated in in vitro cytotoxicity assays against Vero monkey kidney cells. The toxicity profile of analogs 50, 41 and 42 was similar to that of valnemulin.

Effectiveness of Analogs 50, 41, 42 Against Intracellular Mtb. The activity of the three analogs against intracellular Mtb was evaluated. Murine macrophage cell J774A.1 infected by a transformant Mtb CDC1551 containing tdTomato (MOI = 10) were treated with compounds 50, 41 and 42 (at x2 and x4 MICLORA). After 24, 48, and 72 h of incubation, the relative intensity of the fluorescence was measured (emission wavelength (581 nm)) via UV-vis spectroscopy (FIGs. 15A-B). Alternatively, the lysates were tenfold serially diluted in 7H10-S broth and inoculated on 7H11-S plates to determine the number of viable cell-associated Mtb to confirm the bactericidal effect of the analogs against intracellular Mtb in 72 h. The analogs killed Mtb in infected macrophages at x2 MICLORA concentrations within 72 h. FIGs. 15A-B showed clearly that compounds 50, 41 and 42 kill intracellular Mtb better than rifampicin. Bactericidal effect of compounds against the intracellular Mtb was distinguished from that of rifampicin from 48 h. INH did not kill intracellular Mtb at x2 and x4 MIC. In FIG. 15, A: Time-kill curve for intracellular Mtb at 2x MIC concentration; B : Time-kill curve for intracellular Mtb at 4x MIC concentration.

Rapid Antimvcobactericidal Activity of Compounds 50, 41 and 42. The time- kill experiments were performed at two and fourfold the MIC of compounds 50, 41 and 42 and two first-line TB drugs (RIF and INH)). Viable cell counting was performed at every 24 h for 14 days. CFUs were counted after 15 days of incubation at 37 °C. The rate of killing of analogs against Mtb was compared directly with the reference molecules, and the time-kill assessments at x2 MIC concentrations are shown in FIG. 16. Analogs 50, 41 and 42 killed 50% of Mtb at x2 MIC. This killing profile of new analogs was similar to that of RIF and INH which required 7 days to kill 50% of Mtb at 0.4 μg ml^-1 (x2 MIC) and 1.0 μg ml^-1 (x2 MIC), respectively. Spectrum of Activity of Compounds 50, 41 and 42. As summarized in Table 6, the three pleuromutilin analogs (50, 41 and 42) were tested in growth inhibitory assays against several Gram-positive and -negative bacteria including Mycobacterium spp. As summarized, compounds 50, 41 and 42 inhibitors identified in this program kills Mycobacterium species selectively and are especially effective in killing Mtb at low concentrations. In Table 6, ^aThe microplate alamar blue assay method was used.

In vitro Metabolic Stability of Compounds 50, 41, and 42. In the current study of in vitro metabolism of the three potent pleuromutilin analogs in rat liver microsomes, a striking difference in half-life (t ½) between C3 hydroxyl containing compound 50 and C3 carbonyl containing compounds 41 and 42 was observed. T_1/2 of compound 50 was > 90 min, on the other hand, t ½ of compounds 41 and 42 were 1.29 min. As such, in vitro half- life was significantly extended by reduction of the C3 -carbonyl group of valnemulin. The same trend of increased in vitro half- life by reducing the C3 -carbonyl group of valnemulin to hydroxyl group in our lead compound (50) against Gram-negative bacteria, A. baumannii was also observed (FIG. 17). In FIG. 17, verapamil was utilized as a control compound, and the half-life (t_1/2) of verapamil was determined to be 10 min.

The concentration of compound 50 was measured in mice plasma and lungs over time post-intravenous injection. The concentration of compound 50 was at near- zero levels in plasma 10 h after injection, whereas the concentration remained constant in lungs around 2000 ng/g up to a day later (FIG 18). The metabolic stability and other pharmacological properties of compound 50 are summarized in Table 7 below.

Table 7

Genetic Analysis of Compound 51 to Study the Resistance Mechanism. The pleuromutilin derivatives target the peptidyl transferase center of the 50S ribosomal protein L3 (rplC), inhibiting protein biosynthesis. Multiple mutations in rplC of S. aureus were found that can define a region of rplC capable of causing decreased susceptibility of the pleuromutilin derivative in S. aureus. In an experiment of valnemulin analogs against a Gram-negative bacterium; Acinetobacter baumannii yielded a lead compound 51 that has also displayed moderate activity against Mtb (Entry 5 in Table 6). In order to identify a potential mechanism of resistance to 51, the chromosomal DNA was isolated from the resistant mutant (51^R, 16xMIC) and its parental wild-type control A. baumannii (ATCC19606). The rplC gene fragment was amplified using A. baumannii rplC specific primers and sequenced. The DNA sequencing results were blasted against rplC DNA sequence of A. baumannii in the NIH genetic sequence database. The DNA sequence alignment revealed a C456A single nucleotide mutation, which corresponded to N152K mutation in the protein sequence of RplC (FIG. 19). In FIG. 19, 51^R (Query) and wild-type control (Sbjct): the shaded amino acid represents the site mutation in RplC. Analog 50 is structurally very similar to analog 51 with a leucine moiety at the C14 side chain and saturated C16 position. Compound 51 has a valine moiety at C14 and unsaturated C16 (FIG. 12). The target of compound 50 has seen to be similar to that of compound 51 via transcription translation coupled luciferase reporter assays showing inhibition of protein biosynthesis of 55-67% at O.^g/mL for both compounds. The fact that the gene analyses of A. baumannii strain that is resistant to a compound; 51 that is structurally similar with same mode of action to compound 50 suggests that compound 50 could have a similar mechanism of resistance.

Claims

1. A compound havin the structure of formula (I):

or a pharmaceutically acceptable salt thereof; wherein

L is -NH- or -NHCH₂C(CH₃)₂S-;

Q is a divalent amino acid residue;

R is hydrogen or C₁- C₁₀ alkyl that is optionally substituted with one or more groups selected from amino, (C₁-C₆)alkylamino, di(C₁-C₆)alkylamino, hydroxy, (C₁- C₆)alkoxy, and oxo;

or

Q-R is -C(0)-C₁-C₆ alkyl;

R^a is hydrogen and R^b is hydroxyl; or

R^a is hydroxyl and R^b is hydrogen; or

R^a and R^b together are an oxo substituent; or

R^d is -CH₂CH₃ or -CH=CH₂.

2. The compound of claim 1 having the structure of formula (II):

(II).

3. The compound of claim 1 having the structure of formula (III):

4. The compound of claim 1 having the structure of formula (IV):

5. The compound of claim 1 having the structure of formula (V):

6. The compound of claim 5 having the structure of formula (VI):

7. The compound of claim 6 having the structure of formula (VI- 1):

The com ound of claim 6 having the structure of formula (VI-2):

9. The compound of any one of claims 1-8, wherein R is -CH₂CH₃.

The compound of any one of claims 1-8, wherein R is -CH=CH;

The compound of any one of claims 1-8, wherein R is hydj

12. The compound of any one of claims 1-8, wherein R is a -(C₂-C₆)-NH₂ group, wherein the carbon chain is optionally substituted with one or more groups selected from amino, (C₁-C₆)aIkylamino, di(C₁-C₆)a]kylamino, hydroxy, (C₁-C₆)alkoxy, and oxo.

13. The compound of claim 12, wherein R is R², R³ or R⁴, wherein

14. A compound selected from the group of

74

75

76

and pharmaceutically acceptable salts thereof.

15. A pharmaceutical composition comprising a compound of any one of claims 1- 14, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition of claim 15, further comprising an additional therapeutic agent.

17. The pharmaceutical composition of claim 16, wherein the additional therapeutic agent is doxycycline.

18. A method for treating a bacterial infection in a patient in need thereof, comprising administering to the patient a compound of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 15-17.

19. The method of claim 18 wherein the bacterial infection is an infection of Acinetobacter baumannii, Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, or Mycobacterium tuberculosis.

20. The method of claim 19 wherein the bacterial infection is an infection of Mycobacterium tuberculosis.

21. The method of claim 19 wherein the bacterial infection is an infection of Acinetobacter baumannii.

22. The method of claim 20, wherein the compound is selected from compounds 41, 42 and 50.

23. The method of claim 21, wherein R^a is hydro xyl and R^b is hydrogen in the compound.

24. The method of claim 18, wherein the bacterial infection is an infection of a Gram-positive bacterium, and wherein R^d is -CH₂CH₃ in the compound.

25. The method of claim 18, wherein the method comprises administering to the patient a compound of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, and further comprises administering an additional therapeutic agent.

26. The method of claim 25, wherein the additional therapeutic agent is doxycycline.