WO2018035291A1

WO2018035291A1 - 1,2,4-triazolidine-3-thione derivatives and uses thereof

Info

Publication number: WO2018035291A1
Application number: PCT/US2017/047289
Authority: WO
Inventors: Christian C. MELANDER; William M. HUGGINS
Original assignee: North Carolina State University
Priority date: 2016-08-17
Filing date: 2017-08-17
Publication date: 2018-02-22

Abstract

The present invention relates to 1,2,4-triazolidine-3-thione compounds, compositions, and methods for the treatment or prevention of bacterial infections (for example, bacterial infections due to Acinetobacter).

Description

l,2,4-TRIAZOLIDINE-3-THIONE DERIVATIVES AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Serial No. 62/376,050 filed August 17, 2016, the disclosure of which is expressly incorporated herein by reference.

FIELD

The present invention relates to l,2,4-triazolidine-3-thione compounds, compositions, and methods for the treatment or prevention of bacterial infections (for example, bacterial infections due to Acinetobacter).

BACKGROUND

Antibiotic resistance has become one of the forefront issues in global health because of the emergence of multi drug resistant (MDR) bacteria. According to the Centers for Disease Control and Prevention (CDC), an estimated two million people each year acquire MDR bacterial infections, of which 23,000 are fatal. Even more alarming is the dearth of antibiotic options emerging to treat these infections. Only two antibiotics belonging to novel structural classes have been brought into the clinic in the past 40 years, daptomycin and linezolid. Of great concern is that both of these antibiotics are only effective against Gram-positive bacteria, which leaves treatment options for MDR Gram-negative bacteria as an urgent unmet medical need.

Prominent Gram-negative pathogens include Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, which along with the Gram- positive species Enterococcus faecium and Staphylococcus aureus make up the bacterial species that are often referred to as "ESKAPE" pathogens. Currently, the most effective option to treat MDR Gram-negative bacteria remains the polymyxin colistin, which has significant side effects including nephrotoxicity. These side effects, coupled with colistin's effectiveness, have made it a last resort antibiotic against MDR Gram-negative bacteria. In addition, colistin resistant strains of bacteria are being isolated with greater frequency. Of the Gram-negative ESKAPE pathogens, A. baumannii has recently come under the microscope because of its prevalence in wound infections in US servicemen that have been injured in the conflicts in Iraq and Afghanistan, as well as its ability to survive in hospital environments and to develop pan resistance to antibiotics. Thus, there is an unmet need to identify novel antibiotics that can target these ESKAPE pathogens. In particular, there is a need to develop antibiotics for the treatment of Acinetobacter infections, including multi-drug resistant (MDR) Acinetobacter infections.

The compounds, compositions, and methods disclosed herein address these and other needs.

SUMMARY

Disclosed herein are l,2,4-triazolidine-3-thione compounds and methods for treating and preventing bacterial infections. In some embodiments, compounds are disclosed for the treatment and prevention of bacterial infections due to Acinetobacter infections, for example Acinetobacter baumannii.

In one aspect of the invention, disclosed herein is a compound of Formula I:

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen;

wherein at least one of R⁴, R⁵, or R⁶ is halogen;

or a pharmaceutically acceptable salt thereof.

In one aspect of the invention, disclosed herein is a compound of Formula II:

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁵ is halogen;

or a pharmaceutically acceptable salt thereof.

In one aspect of the invention, disclosed herein is a compound of Formula

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴ is halogen;

or a pharmaceutically acceptable salt thereof. In one embodiment, the compound is:

In one embodiment, the compound is:

In one aspect, disclosed herein is a method of treating or preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula I, II, or III. In one embodiment, the Acinetobacter infection is Acinetobacter baumannii. In one embodiment, the Acinetobacter infection is a multi-drug resistant (MDR) infection. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

FIG. 1. Time kill curve for compound 5 against baumannii 5075.

FIG. 2. Time kill curve for compound 1 against baumannii 5075.

FIG. 3. Percent survival of G. mellonella after baumannii 5075 infection and compound 5 treatment.

FIG. 4. Percent survival of G mellonella after baumannii 5075 infection and compound 1 treatment.

DETAILED DESCRIPTION

Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. The following definitions are provided for the full understanding of terms used in this specification.

Terminology

As used herein, the article "a," "an," and "the" means "at least one," unless the context in which the article is used clearly indicates otherwise.

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms "substitution" or "substituted with" include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

"Z¹," "Z²," "Z³," and "Z⁴" are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.

The term "aliphatic" as used herein refers to a non-aromatic hydrocarbon group and includes branched and unbranched, alkyl, alkenyl, or alkynyl groups.

The term "alkyl" as used herein is a branched or unbranched saturated hydrocarbon group. In some embodiments, the alkyl comprises 1 to 10 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. The alkyl group can also be substituted or unsubstituted. The alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

Throughout the specification "alkyl" is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term "halogenated alkyl" specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term "alkoxyalkyl" specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term "alkylamino" specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When "alkyl" is used in one instance and a specific term such as "alkylalcohol" is used in another, it is not meant to imply that the term "alkyl" does not also refer to specific terms such as "alkylalcohol" and the like. This practice is also used for other groups described herein. That is, while a term such as "cycloalkyl" refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an "alkyl cycloalkyl." Similarly, a substituted alkoxy can be specifically referred to as, e.g., a "halogenated alkoxy," a particular substituted alkenyl can be, e.g., an "alkenyl alcohol," and the like. Again, the practice of using a general term, such as "cycloalkyl," and a specific term, such as "alkylcycloalkyl," is not meant to imply that the general term does not also include the specific term.

The term "alkoxy" as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an "alkoxy" group can be defined as— OZ¹ where Z¹ is alkyl as defined above.

The term "alkenyl" as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (Z¹Z²)C=C(Z³Z⁴) are intended to include both the E and Z isomers. This can be presumed in structural formulae herein wherein an asymmetric alkene is present, or it can be explicitly indicated by the bond symbol C=C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The term "alkynyl" as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The term "aryl" as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term "heteroaryl" is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. The term "non- heteroaryl," which is included in the term "aryl," defines a group that contains an aromatic group that does not contain a heteroatom. The aryl or heteroaryl group can be substituted or unsubstituted. The aryl or heteroaryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term "biaryl" is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term "cycloalkyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term "heterocycloalkyl" is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term "cycloalkenyl" as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, i.e., C=C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term "heterocycloalkenyl" is a type of cycloalkenyl group as defined above, and is included within the meaning of the term "cycloalkenyl," where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.

The term "cyclic group" is used herein to refer to either aryl groups, non-aryl groups {i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.

The term "aldehyde" as used herein is represented by the formula— C(0)H. Throughout this specification "C(O)" or "CO" is a short hand notation for C=0.

The terms "amine" or "amino" as used herein are represented by the formula — NZ^XZ², where Z¹ and Z² can each be substitution group as described herein, such as hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "carboxylic acid" as used herein is represented by the formula— C(0)OH. A "carboxylate" or "carboxyl" group as used herein is represented by the formula

— C(0)0^" The term "ester" as used herein is represented by the formula — OC(0)Z¹ or — C(0)OZ¹, where Z¹ can be an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "ether" as used herein is represented by the formula Z^lOZ², where Z¹ and Z² can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "ketone" as used herein is represented by the formula Ζ¾(0)Ζ², where Z¹ and Z² can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "halide" or "halogen" as used herein refers to the fluorine, chlorine, bromine, and iodine.

The term "hydroxyl" as used herein is represented by the formula— OH.

The term "nitro" as used herein is represented by the formula— N0₂.

The term "silyl" as used herein is represented by the formula— SiZ¹Z²Z³, where Z¹, Z², and Z³ can be, independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "sulfonyl" is used herein to refer to the sulfo-oxo group represented by the formula— S(0)₂Z¹, where Z¹ can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.

The term "sulfonylamino" or "sulfonamide" as used herein is represented by the formula — S(0)₂ H— .

The term "thiol" as used herein is represented by the formula— SH.

The term "thio" as used herein is represented by the formula— S— .

"R¹," "R²," "R³," "Rⁿ," etc., where n is some integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R¹ is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxyl group, an amine group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e. , attached) to the second group. For example, with the phrase "an alkyl group comprising an amino group," the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.

Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer, diastereomer, and meso compound, and a mixture of isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, and methods, examples of which are illustrated in the accompanying Examples and Figures.

Compounds

In one aspect of the invention, disclosed herein is a compound of Formula I:

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen;

wherein at least one of R⁴, R⁵, or R⁶ is halogen;

or a pharmaceutically acceptable salt thereof.

In one aspect of the invention, disclosed herein is a compound of Formula II:

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁵ is halogen;

or a pharmaceutically acceptable salt thereof.

In one aspect of the invention, disclosed herein is a compound of Formula

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring;

R⁴ is halogen;

or a pharmaceutically acceptable salt thereof. In one embodiment, R¹ is alkyl. In one embodiment, R¹ is Ci-C₆ alkyl. In one embodiment, R¹ is C1-C3 alkyl. In one embodiment, R¹ is a branched alkyl. In one embodiment, R¹ is an unbranched alkyl. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is unsubstituted. In one embodiment, R¹ is methyl. In one embodiment, R¹ is ethyl. In one embodiment, R¹ is n-propyl. In one embodiment, R¹ is isopropyl.

In one embodiment, R² is alkyl. In one embodiment, R² is Ci-C₆ alkyl. In one embodiment, R² is C1-C3 alkyl. In one embodiment, R² is a branched alkyl. In one embodiment, R² is an unbranched alkyl. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is unsubstituted. In one embodiment, R² is methyl. In one embodiment, R² is ethyl. In one embodiment, R² is n-propyl. In one embodiment, R² is isopropyl.

In one embodiment, R¹ and R² are joined to form a cycloalkyl ring. In one embodiment, the cycloalkyl is cyclopentyl. In one embodiment, the cycloalkyl is cyclohexyl. In one embodiment, the cycloalkyl is substituted. In one embodiment, the cycloalkyl is unsubstituted.

In one embodiment, R⁴ is selected from hydrogen or halogen. In one embodiment, R⁴ is hydrogen. In one embodiment, R⁴ is halogen. In one embodiment, R⁴ is CI. In one embodiment, R⁴ is Br. In one embodiment, R⁴ is F. In one embodiment, R⁴ is I.

In one embodiment, R⁵ is selected from hydrogen or halogen. In one embodiment, R⁵ is hydrogen. In one embodiment, R⁵ is halogen. In one embodiment, R⁵ is CI. In one embodiment, R⁵ is Br. In one embodiment, R⁵ is F. In one embodiment, R⁵ is I.

In one embodiment, R⁶ is selected from hydrogen or halogen. In one embodiment, R⁶ is hydrogen. In one embodiment, R⁶ is halogen. In one embodiment, R⁶ is CI. In one embodiment, R⁶ is Br. In one embodiment, R⁶ is F. In one embodiment, R⁶ is I.

In one embodiment, the compound is selected from the group consisting of:

and

In one embodiment, the compound is:

In one embodiment, the compound is:

Methods

Acinetobacter is a genus of bacteria that are strictly aerobic non-fermentative gram- negative bacilli. Acinetobacter species are widely distributed in nature and can survive for long periods of time on wet or dry surfaces. Acinetobacter species are considered to be non-pathogenic to healthy subjects, but it is becoming increasingly apparent that Acinetobacter species persist in hospital environments for a long period of time and can be responsible for nosocomial infections in compromised patients. Acinetobacter baumannii is a frequent cause of nosocomial pneumonia, especially of late-onset ventilator associated-pneumonia and it can cause various other infections including skin and wound infections, bacteraemsa, and meningitis. Acinetobacter Iwqffii has also been associated with meningitis. Other species including Acinetobacter haemofyticus, Acinetobacter joimsonii, Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, or Acinetobacter ursingii have also been linked to infection. Of particular note is the prevalence of Acinetobacter baumannii infections in serviceman stationed in the Middle East,

any Acinetobacter strains appear to be multidrug resistant, thus making the combat of Acinetobacter infections difficult. Multidrug resistance (MDR) in bacteria describes the situation where a bacterium is resistant to at least three classes of drugs, specifically in the context of bacteria, at least three classes of anti-microbial (or more specifically anti-bacterial) agents. Antibiotics in one class are functionally unrelated, stmcturallv unrelated, or both, to antibiotics in a different class. MDR in bacteria is thus often termed multiple anti -bacterial drug resistance or multiple antibiotic resistance. The terms are used interchangeably in the art and herein. Bacteria displaying multidrug resistance phenotypes (or multiple antibacterial/antibiotic drug resistance phenotypes) are referred to as MDR bacteria (or sometimes MAR bacteria). Again, these terms are used interchangeably in the art and herein.

In one aspect, disclosed herein is a method of treating or preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula I:

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen; or a pharmaceutically acceptable salt thereof.

In one aspect, disclosed herein is a method of treating or preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula II:

R¹

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁵ is independently selected from hydrogen or halogen;

or a pharmaceutically acceptable salt thereof.

In one aspect, disclosed herein is a method of treating or preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula III:

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴ is independently selected from hydrogen or halogen;

or a pharmaceutically acceptable salt thereof.

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen; wherein at least one of R⁴, R⁵, or R⁶ is halogen;

or a pharmaceutically acceptable salt thereof.

R¹

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring;

R⁵ is halogen;

or a pharmaceutically acceptable salt thereof.

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴ is halogen;

or a pharmaceutically acceptable salt thereof.

In one embodiment, R¹ is alkyl. In one embodiment, R¹ is Ci-C₆ alkyl. In one embodiment, R¹ is C1-C3 alkyl. In one embodiment, R¹ is a branched alkyl. In one embodiment, R¹ is an unbranched alkyl. In one embodiment, the alkyl is substituted. In one embodiment, the alkyl is unsubstituted. In one embodiment, R¹ is methyl. In one embodiment, R¹ is ethyl. In one embodiment, R¹ is n-propyl. In one embodiment, R¹ is isopropyl.

In one embodiment, R¹ and R² are joined to form a cycloalkyl. In one embodiment, the cycloalkyl is cyclopentyl. In one embodiment, the cycloalkyl is cyclohexyl. In one embodiment, the cycloalkyl is substituted. In one embodiment, the cycloalkyl is unsubstituted.

In one embodiment, R⁴ is selected from hydrogen or halogen. In one embodiment, R⁴ is hydrogen. In one embodiment, R⁴ is halogen. In one embodiment, R⁴ is CI. In one embodiment, R⁴ is Br. In one embodiment, R⁴ is F. In one embodiment, R⁴ is I. In one embodiment, R⁵ is selected from hydrogen or halogen. In one embodiment, R⁵ is hydrogen. In one embodiment, R⁵ is halogen. In one embodiment, R⁵ is CI. In one embodiment, R⁵ is Br. In one embodiment, R⁵ is F. In one embodiment, R⁵ is I.

In one embodiment, the Acinetobacter infection is Acinetobacter baumannii. In one embodiment, the Acinetobacter infection is a multi-drug resistant (MDR) infection.

In one embodiment, the compound is selected from the group consisting of:

In one embodiment, the compound is selected from the group consisting of:

and

In one embodiment, the compound is:

In one embodiment, the compound is:

In one embodiment, disclosed herein is a method of treating an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula I, II, or III. In one embodiment, disclosed herein is a method of preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula I, II, or III.

Combinations Therapies - Additional Antibiotics

In one embodiment, a compound of Formula I, II, or III may be administered in combination with an additional antibiotic. Classes of antibiotics and representative constituents thereof include, but are not limited to the aminoglycosides (e.g. amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin); the carbacephems (e.g. loracarbef); the 1 st generation cephalosporins (e.g. cefadroxil, cefazolin, cephalexin); 2nd generation cephalosporins (e.g. cefaclor, cefamandole, cephalexin, cefoxitin, cefprozil, cefuroxime); 3rd generation cephalosporins (e.g. cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone); 4th generation cephalosporins (e.g. cefepime); the macrolides (e.g. azithromycin, clarithromycin, dirithromycin, erythromycin, troleandomycin); the monobactams (e.g. aztreonani); the penicillins (e.g. amoxicillin, anipiciiliii, carbeniciiliii, cioxacillin, dicloxaciilin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, ticarciliin), the polypeptide antibiotics (e.g. bacitracin, colistin, polymyxin B); the quinolones (e.g. ciprofloxacin, enoxacin, gatifloxacin, levofioxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, trovafloxacin); the sulfonamides (e.g. mafenide, sulfacetamide, suifamethizole, suifasplazine, sulfisoxazole, trimethoprim-sulfamethoxazole), the tetracyclines (e.g. demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline); the glycylcyclines (e.g. tigecycline); the carbapenems (e.g. imipenem, meropeneni, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601); other antibiotics include chloramphenicol; clindamycin, etbambutol; fosfomycin, isoniazid, linezoiid; metronidazole; nitrofurantoin; pyrazinamide; quinupristin/daifopristin; rifampin; spectinomycin; and vancomycin.

In one embodiment, the additional antibiotic used is an antibiotic selected from, the macrolides, the β-lactams, which may include the carbapenems and/or monobactams and/or carbacephems, the tetracyclines, and the quinolones. In other embodiments, the antibiotic classes may include the aminoglycosides and/or the polypeptide antibiotics. In these embodiments, the antibiotic may be selected from the macrolides, the monobactams, the carbapenems, the carbacephems, the 3rd and 4th generation cephalosporins, the tetracyclines and the quinolones, and optionally the aminoglycosides and/or the polypeptide antibiotics. In more particular representative embodiments the antibiotic may be selected from macrolides, β-lactams, tetracyclines and quinolones e.g. macrolides, monobactams, carbapenems, carbacephems, 3rd and 4th generation cephalosporins, tetracyclines and quinolones. In some representative embodiments, the antibiotic may be selected from macrolides, β-lactarns and quinolones e.g. macrolides, monobactams, carbapenems, carbacephems, 3rd and 4th generation cephalosporins and quinolones. For example, the antibiotic may be selected from amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, tylosin, troleandomycin, aztreonam, imipenem, meropenem, ertapenem, doripenem, panipenem/betamipron, biapenem, PZ-601, eeflxime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin, gatifioxacm, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin. In particular, the antibiotic may selected from ceftazidime, imipenem/cilastatin, meropenem, aztreonam, oxytetracycline, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin, and it is particularly preferred that the antibiotic is selected from ceftazidime, imipenem/cilastatin, meropenem, aztreonam, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin. More preferably the antibiotic is selected from aztreonam, azithromycin, clarithromycin, di ithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin.

In other embodiments, the additional antibiotic used is tobramycin, amikacin and/or colistin. In one embodiment, the additional antibiotic is colistin.

In other embodiments, the additional antibiotic used is an aminoglycoside or a polypeptide antibiotic. In other embodiments, the antibiotic used is an antibiotic that has a positive charge under the conditions in which it will be used with the alginate oligomer, e.g. antibiotics with at least 3, e.g. at least 4, 5, 6 or 7 amino (— NH2) groups. Examples of additional antibiotics include, for example, macrolides, β-lactams, tetracyclines and quinoiones e.g. macrolides, monobactams, carbapenems, 3rd and 4th generation cephalosporins, tetracyclines and quinoiones; e.g. ceftazidime, imipenem/cilastatin, meropenem, aztreonam, oxytetracycline, azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin and ciprofloxacin.

In one embodiment, the additional antibiotic is a macrolide antibiotic and may be selected from azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, CarbomycinA, josamycin, kitasamycin, midecamicine, oleandomycin, spiramycin, troleandromycin, tylosin. Preferably the macrolide antibiotic is an azalide macrolide, preferably azithromycin, or is selected from clarithromycin, dirithromycin, erythromycin, roxithromycin or spiramycin.

Compositions

Compositions, as described herein, comprising an active compound and an excipient of some sort may be useful in a variety of medical and non-medical applications. For example, pharmaceutical compositions comprising an active compound and an excipient may be useful for the treatment or prevention of a bacterial infection, for example, an Acinetobacter infection.

Disclosed herein is a pharmaceutical composition comprising a compound of Formula I, Formula II, Formula III, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

"Excipients" include any and all solvents, diluents or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. General considerations in formulation and/or manufacture can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

Exemplary excipients include, but are not limited to, any non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as excipients include, but are not limited to, 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; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. As would be appreciated by one of skill in this art, the excipients may be chosen based on what the composition is useful for. For example, with a pharmaceutical composition or cosmetic composition, the choice of the excipient will depend on the route of administration, the agent being delivered, time course of delivery of the agent, etc., and can be administered to humans and/or to animals, orally, rectally, parenterally, intracisternally, intravaginally, intranasally, intraperitoneally, topically (as by powders, creams, ointments, or drops), bucally, or as an oral or nasal spray.

Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and combinations thereof.

Exemplary granulating and/or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include starch (e.g. cornstarch and starch paste), gelatin, sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabogalactan), alginates, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylates, waxes, water, alcohol, etc., and/or combinations thereof.

Exemplary preservatives include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

Exemplary antioxidants include alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and sodium sulfite.

Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and salts and hydrates thereof (e.g., sodium edetate, disodium edetate, trisodium edetate, calcium disodium edetate, dipotassium edetate, and the like), citric acid and salts and hydrates thereof (e.g., citric acid monohydrate), fumaric acid and salts and hydrates thereof, malic acid and salts and hydrates thereof, phosphoric acid and salts and hydrates thereof, and tartaric acid and salts and hydrates thereof. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

Exemplary antifungal preservatives include butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.

Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabi sulfite, potassium sulfite, potassium metabi sulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certain embodiments, the preservative is an anti-oxidant. In other embodiments, the preservative is a chelating agent.

Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and combinations thereof.

Exemplary lubricating agents include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary synthetic oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and combinations thereof.

Additionally, the composition may further comprise a polymer. Exemplary polymers contemplated herein include, but are not limited to, cellulosic polymers and copolymers, for example, cellulose ethers such as methylcellulose (MC), hydroxyethylcellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), methylhydroxyethylcellulose (MHEC), methylhydroxypropylcellulose (MHPC), carboxymethyl cellulose (CMC) and its various salts, including, e.g., the sodium salt, hydroxyethylcarboxymethylcellulose (HECMC) and its various salts, carboxymethylhydroxyethylcellulose (CMHEC) and its various salts, other polysaccharides and polysaccharide derivatives such as starch, dextran, dextran derivatives, chitosan, and alginic acid and its various salts, carageenan, varoius gums, including xanthan gum, guar gum, gum arabic, gum karaya, gum ghatti, konjac and gum tragacanth, glycosaminoglycans and proteoglycans such as hyaluronic acid and its salts, proteins such as gelatin, collagen, albumin, and fibrin, other polymers, for example, polyhydroxyacids such as polylactide, polyglycolide, polyl(lactide-co- glycolide) and poly(.epsilon.-caprolactone-co-glycolide)-, carboxyvinyl polymers and their salts (e.g., carbomer), polyvinylpyrrolidone (PVP), polyacrylic acid and its salts, polyacrylamide, polyacilic acid/acryl amide copolymer, polyalkylene oxides such as polyethylene oxide, polypropylene oxide, poly(ethylene oxide-propylene oxide), and a Pluronic polymer, polyoxyethylene (polyethylene glycol), polyanhydrides, polyvinylalchol, polyethyleneamine and polypyrridine, polyethylene glycol (PEG) polymers, such as PEGylated lipids (e.g., PEG-stearate, l,2-Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000], 1,2- Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000], and 1,2- Distearoyl-sn-glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000]), copolymers and salts thereof.

Additionally, the composition may further comprise an emulsifying agent. Exemplary emulsifying agents include, but are not limited to, a polyethylene glycol (PEG), a polypropylene glycol, a polyvinyl alcohol, a poly-N-vinyl pyrrolidone and copolymers thereof, poloxamer nonionic surfactants, neutral water-soluble polysaccharides (e.g., dextran, Ficoll, celluloses), non- cationic poly(meth)acrylates, non-cationic polyacrylates, such as poly(meth)acrylic acid, and esters amide and hydroxyalkyl amides thereof, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij 30]), poly(vinyl-pyrrolidone), di ethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof. In certain embodiments, the emulsifying agent is cholesterol.

Liquid compositions include emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compound, the liquid composition may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Injectable compositions, for example, injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a injectable solution, suspension, or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents for pharmaceutical or cosmetic compositions that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. Any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. In certain embodiments, the particles are suspended in a carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) Tween 80. The injectable composition can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions for rectal or vaginal administration may be in the form of suppositories which can be prepared by mixing the particles with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol, or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the particles.

Solid compositions include capsules, tablets, pills, powders, and granules. In such solid compositions, the particles are mixed with at least one excipient and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Tablets, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Compositions for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The active compound is admixed with an excipient and any needed preservatives or buffers as may be required.

The ointments, pastes, creams, and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to the active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.

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

The active ingredient may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the active ingredient will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular active ingredient, its mode of administration, its mode of activity, and the like. The active ingredient, whether the active compound itself, or the active compound in combination with an agent, is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the active ingredient will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the active ingredient employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific active ingredient employed; the duration of the treatment; drugs used in combination or coincidental with the specific active ingredient employed; and like factors well known in the medical arts.

The active ingredient may be administered by any route. In some embodiments, the active ingredient is administered via a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In general the most appropriate route of administration will depend upon a variety of factors including the nature of the active ingredient (e.g., its stability in the environment of the gastrointestinal tract), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc. The exact amount of an active ingredient required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

EXAMPLES

Example 1. l,2,4-triazolidine-3-thiones for prevention and treatment of multi-drug resistant Acinetobacter baumannii

Antibiotic resistance has become one of the forefront issues in global health because of the emergence of multi drug resistant (MDR) bacteria. According to the Centers for Disease Control and Prevention (CDC), an estimated two million people each year acquire MDR bacterial infections, of which 23,000 are fatal.¹ Even more alarming is the dearth of antibiotic options emerging to treat these infections. Only two antibiotics belonging to novel structural classes have been brought into the clinic in the past 40 years, daptomycin and linezolid.² Of great concern is that both of these antibiotics are only effective against Gram-positive bacteria, which leaves treatment options for MDR Gram-negative bacteria as an urgent unmet medical need.

Prominent Gram-negative pathogens include Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, which along with the Gram- positive species Enterococcus faecium and Staphylococcus aureus make up the bacterial species that are often referred to as "ESKAPE" pathogens.³ Currently, the most effective option to treat MDR Gram-negative bacteria remains the polymyxin colistin, which has significant side effects including nephrotoxicity⁴. These side effects, coupled with colistin's effectiveness, have made it a last resort antibiotic against MDR Gram-negative bacteria. Worryingly, colistin resistant strains of bacteria are being isolated with greater frequency⁵ Of the Gram-negative ESKAPE pathogens, A. baumannii has recently come under the microscope because of its prevalence in wound infections in US servicemen that have been injured in the conflicts in Iraq and Afghanistan⁶, as well as its ability to survive in hospital environments and to develop pan resistance to antibiotics.⁷ In 2009, compound 1 was reported to have antifungal activity against Candida

albicans .^[8]

Structure of compound 1.

Recently, genomic sequencing has revealed that A. baumannii has similar biosynthetic machinery to C. albicans. Compound 1 was investigated for antibiotic activity against the MDR A. baumannii strain AB5075^[9], and a minimum inhibitory concentration (MIC) of 8 μg/mL was observed. Analogues were synthesized of the l,2,4-triazolidine-3-thione core structure with variations introduced at the 2 and 5 positions (Scheme 1) to identify a new, more biologically active compound.

Disclosed herein is the synthesis of analogues of l,2,4-triazolidine-3-thiones as well as their antibiotic activity against MDR baumannii. A compound was synthesized that has an MIC of up to four-fold lower against multiple strains of MDR A. baumannii compared to compound 1 and shows activity in a Galleria mellonella model of infection, unlike parent compound 1 which does not show activity in the Galleria mellonella model of infection.

The l,2,4-triazolidine-3-thione scaffold is accessible through a three component reaction between ketones or aldehydes, hydrazines and potassium thiocyanate in hydrochloric acid for 16 hours in the dark (Scheme 1). The resulting triazolidines typically precipitate during the reaction, allowing for simple filtration followed by recrystallization from methanol to deliver purified material.⁸' ⁹ Moreover, this synthetic procedure allows for modifications to be made at the N-2 and C-5 positions on the ring, thus allowing assessment of l,2,4-triazolidine-3-thione analogs for structure-activity relationship studies.

2a-o R³ = Ph

3a-c 4a-o R =R²

Scheme 1. Synthesis of l,2,4-triazolidine-3-thiones. Initially, the effect of substitution was evaluated at carbon 5 by varying the identity of the aliphatic substituents. A series of 14 compounds that were mono- or di- substituted with alkyl or aryl groups were synthesized and screened for antibiotic activity against AB5075 under standard CLSI broth microdilution conditions (Table 1). A. baumannii strain 5075 is a MDR primary clinical isolate and was chosen to evaluate the l,2,4-triazolidine-3-thiones because of its virulence and MDR properties.¹⁰ Both the dimethyl (compound 2a) and diethyl (compound 2e) derivatives exhibited identical activity to the parent compound. Compounds possessing larger alkyl groups than that of the parent compound exhibited increased MICs in all cases tested here (compounds 2h, 2i, 2j). Branched isopropyl groups also yielded either no change, or an increase in MIC (compounds 2b, 2f). Replacing one of the alkyl groups with a hydrogen atom (compounds 2d, 2g, 2k), as well as replacing one of the alkyl groups with an aromatic ring (compounds 2c, 21) increased the MIC. The effect of constraining the substituents as a spirocycle was also investigated (compounds 2m, 2n); however, these changes delivered analogs with identical activity as the parent compound.

Table 1. MIC values for carbon 5 modifications against baumannii 5075, where R³=Ph.

'All concentrations are in μg/mL.

As an increase in antibiotic activity through modification of the 5-position was not observed, substitution at the N-2 position of the ring was tested next. Benzyl, cyclohexyl, and 2- pyridinyl derivatives of the diethyl substituted derivative (Table 2) were synthesized. The diethyl substituted derivatives were chosen instead of the parent methyl ethyl substituted derivatives to avoid racemic mixtures. All of the substitutions of the N-2 position led to an increase in MIC, which led to the conclusion that the phenyl ring at the N-2 position was required for activity. Table 2. MIC values for aliphatic position 2 modifications against A. baumannii 5075, where R¹⁼R²= Et

All concentrations are in μg/mL.

Given the significant impact that changing the 2-phenyl substituent to a 2-benzyl had upon activity, substitution of the phenyl ring itself was investigated to determine if it had any effect on the MIC (Table 3). The 4-chloro derivative 4a exhibited increased activity compared to the parent compound, with an MIC of 2 μg/mL. Encouraged by this result, additional halogenated derivatives 4b, 4c, and 4d were prepared, however an increase in MIC was observed for these compounds. Compounds 4e-4h were prepared to investigate the effect of fluoro and chloro substitution at the 2 and 3 positions on the phenyl ring, with only an increase in MIC observed. However, MIC values at the 3 position were lower than those observed for substitutions at the 2 position. Table 3. MIC values for aromatic position 2 modifications against A. baumannii 5075, where R¹⁼R²= Et

Compound _R3= MIC

4a (4-Chlorophenyl) 2

4b (4-Bromophenyl) 4

4c (4-Iodophenyl) 8

4(1 (4-Fluorophenyl) 4

4e (2-Fluorophenyl) 32

4f (3 -Fluorophenyl) 8

4g (2-Chlorophenyl) 128

4h (3 -Chlorophenyl) 8

4i (3-Chloro-4-Fluoro) 32

4j (3 , 5 -Difluorophenyl) >128

4k (3,4-Dichlorophenyl) >128

41 (4-Isopropylphenyl) >128

4m (4-Nitrophenyl) >128

4n (4-Cyanophenyl) >128

4o (4-

Trifluoromethylphenyl) >128

'All concentrations are in μg/mL.

Di substitution was also explored, with 3,4 and 3,5 halogen substitution patterns, however this also resulted in an increase in MIC (compounds 4i-4k). Because substitution at the 4-position of the phenyl ring produced the most active compounds for the halogenated derivatives, the next set of compounds to be synthesized included a variety of compounds with either electron withdrawing or electron donating groups at the para position. As shown with compounds 41-4o, the substitutions tested here, other than a halogen, were not tolerated.

With a more active derivative, 4a, identified, compound 4a was tested for activity against multiple strains of MDR A. baumannii (representative examples shown in Table 4). The 4- chlorophenyl analogue of compound 4a, compound 5, was also prepared to investigate whether the lipophilicity of the compounds could be tuned without affecting the activity of the compounds. Compound 4a has a predicted log D value of 3.85, while compound 5 has a predicted log D value of 2.58 at pH 7.

4a 5

Structures of compounds 4a and 5.

All three compounds retained activity against multiple strains of A. baumannii, while compound 5 was more active against several strains of A. baumannii than either compound 4a or the parent compound 1.

Table 4. MIC results for Compounds 1, 4a, and 5 against multiple MDR strains of baumannii.

All concentrations are in μg/mL.

8a R = Me

©b R = Bn

Scheme 2. Alkylation of the thiol moiety After the identification of Compound 5 as the most active analogue against several A. baumannii strains, further structure activity relationships of compound 5 were investigated, beginning with the effect of alkylating the sulfur atom at the C-3 position. Briefly, compound 5 was reacted with the corresponding alkyl halide in methanol at room temperature overnight to yield the thioethers 6a-b (Scheme 2). The methyl and benzyl analogues were prepared, and the observed MIC for both compounds was greater than 128 μg/mL, showing the necessity of the unsubstituted sulfur atom for biological activity. To further probe the S AR of the 1 ,2,4-triazolidine- 3-thione core, effect of alkylation of the N-l position of compound 5 was also investigated (Scheme 3).

Scheme 3. Synthetic route to allow for the alkylation of N-V Reagents and conditions: (a) Alloc- Cl, DMAP, Et₃N, THF rt, 16 h; (b) RX, NaH, DMF 0° C to rt, 4 h; (c) NaBH₄, Pd(PPh₃)₄, EtOH 0° C to rt, lh (d) 12 N HC1 (pH 2.5-3), rt, 4h

In order to alkylate the N-l position, the thiol was protected with an alloc protecting group. The N-l position was then alkylated using sodium hydride and the corresponding alkyl halide. The alloc group was removed using sodium borohydride and tetrakis(triphenylphosphine)palladium(0) in ethanol at 0 °C, and the solution was then acidified with 12 N HC1 at room temperature (Cvetovic, R. J., et al. Journal of Organic Chemistry 1994, 59, 7704-7708). The MICs of compounds 9a-b were all greater than 128 μg/mL, demonstrating the need for the free N-H at the N-l position on the l,2,4-triazolidine-3-thione ring for biological activity.

The final structural modification that was investigated was to move the 4-chlorophenyl substituent from the N-2 position to the N-4 position of compound 5 (Scheme 4).

Scheme 4. Synthesis of the N-4 substituted l,2,4-triazolidine-3-thione ring. The reverse l,2,4-triazolidine-3-thione scaffold is accessed through the reaction of N-(4- chlorophenyl) hydrazinecarbothioamide with acetone in hydrochloric acid for 16 hours in the dark (Scheme 4). As with compounds l-4o, the resulting triazolidine precipitated during the reaction, allowing for simple filtration followed by recrystallization from methanol to deliver purified material.¹²' ¹³ The observed MIC of compound 10 was greater than 128 μg/mL, which is a greater than 64-fold increase in MIC compared to compound 5, indicating that the original substitution pattern of the l,2,4-triazolidine-3-thiones is the more active motif.

Compounds 1 and 5 were investigated further to determine if they were acting in a bactericidal or bacteriostatic manner by constructing time kill curves. An antibiotic is defined as bactericidal when it kills >99.9% of bacteria at a concentration no greater than four times the MIC.¹¹ As seen in the time kill curve below (Figure 1), compound 5 is bactericidal, effecting a greater than six log reduction in colony forming units per milliliter (CFU/mL) at double the MIC and greater. The results for compound 1 are shown in Figure 2.

In C. albicans, compound 1 distorts the ratio of unsaturated to saturated fatty acids likely by inhibiting the fatty acid desaturase gene OLEl.¹² As previously mentioned, C. albicans and A. baumonnii were recently found to possess similar biosynthetic machinery, and it was established that fatty acid supplementation of media abrogated the bactericidal effects of compound 1 against A. baumonnii. In order to test whether compound 5 exhibited similar behavior, an MIC assay under fatty acid supplementation was performed. As with compound 1, when the media was supplemented with 0.2% linoleic acid, the activity of compound 5 against baumonnii 5075 was completely abolished with an observed MIC of >128 μg/mL.

In order to probe the antibiotic spectrum of compound 5, MICs against representative strains of Escherichia coli, P. aeruginosa, K. pneumoniae, and methicillin resistant S. aureus (MRSA) were recorded. An MIC of >128 μg/mL was observed for all bacterial species tested here, other than A. baumonnii, establishing that compound 5 is a narrow spectrum antibiotic.

After finding that compound 5 was most active, activity was evaluated in a G mellonella infection model with baumonnii 5075.¹⁰ In G. mellonella, 30 min after worms were inoculated with 6.0 X 10⁵ CFU of AB5075, they received a 5 ΐ treatment injection of 50 or 100 mg/kg compound 1 or 5. As documented previously, compound 1 was found to be inactive against A. baumannii 5075 in this model (Figure 4). However, compound 5 exhibited modest in vivo activity, with 50 mg/kg of compound 5 saving 22% of worms after 6 days, and 100 mg/kg saving 32% of worms after 6 days. In comparison, 80% of worms died after 2 days, and 100% of untreated, infected worms were dead after 6 days (Figure 3).

In conclusion, l,2,4-triazolidine-3-thiones were identified as possessing antibiotic activity against MDR A. baumannii. Three libraries of compounds were then synthesized to identify a compound with increased activity. After making modifications at the C-5 and N-2 positions on the l,2,4-triazolidine-3-thione core structure to no effect, substitutions were made to the phenyl ring at position 2 to yield compound 4a, which displayed a four-fold improvement in MIC against baumannii compared to compound 1. Compound 5 was also synthesized and tested against multiple strains of A. baumannii along with compound 1 and compound 4a in order to ascertain whether the lipophilicity could be tuned without effecting the MIC. Compound 5 displayed an up to four-fold improvement against multiple strains of baumannii compared with compound 1, as well as returning a lower MIC than compound 4a in most cases. Compound 5 was found to be bactericidal, and an m-vivo study was conducted with compound 5 against A. baumannii 5075 using G. mellonella. The m-vivo study demonstrated modest, single dose activity. Further structural modifications and biological evaluations against A. baumannii are underway using compound 5 as our current lead in an attempt to further augment activity.

Methods

All reagents used for chemical synthesis were purchased from commercially available sources and used without further purification. Chromatography was performed using 60 A mesh standard grade silica gel from Sorbtech. NMR solvents were obtained from Cambridge Isotope Laboratories and used as is. All ¾ NMR (300 or 400 MHz) and ¹³C NMR (75 or 100 MHz) spectra were recorded at 25 °C on Varian Mercury spectrometers. Chemical shifts (δ) are given in parts per million relative to tetramethylsilane or the respective NMR solvent; coupling constants (J) are in hertz (Hz). Abbreviations used are s, singlet; brs, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; m, multiplet. Mass spectra were obtained at the NCSU Department of Chemistry Mass Spectrometry Facility. Infrared spectra were obtained on an FT/IR- 4100 spectrophotometer (v_max in cm^"1). UV absorbance was recorded on a Genesys 10 scanning UV/visible spectrophotometer (λ_ω3χ in nm). The purities of the tested compounds were all verified to be >95% by LC-MS analysis on a Shimadzu LC-MS 2020 with Kinetex, 2.6 mm, C18 50 2.10 mm.

General Synthetic Procedure for l,2,4-triazoline-3-thiones (Compounds l-3b, 4a-4o):

To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and the corresponding hydrazine (3 mmol). The desired ketone or aldehyde (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol¹ to give the desired product.²

5,5-diethyl-2-(pyridin-2-yl)-l,2,4-triazolidine-3-thione (Compound 3c):

To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and the 2-hydrazinopyridine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The oil that formed in solution was extracted with DCM and the aqueous layer was removed. The organic layer was then washed with a saturated solutions of sodium bicarbonate (2 x 25 mL) and brine (2 x 25 mL). The organic layer was then dried with magnesium sulfate, filtered, and the solvent was removed under reduced pressure. The product was then purified by flash chromatography (1-3% MeOH sat. w/ ¾/DCM) to yield 5,5-diethyl-2-(pyridin-2-yl)-l,2,4- triazolidine-3-thione as a brown oil.

General Synthetic Procedure for S-methylation of l,2,4-triazolidine-3-thiones (Compound 6a):

To a solution of compound 5 (0.32 g, 1.32 mmol) in methanol (25 mL) under nitrogen at room temperature, methyl iodide (0.23 g, 1.59 mmol, 1.20 eq) was added dropwise to the solution. The reaction was allowed to stir overnight for 16 hours at room temperature. The solvent was removed under reduced pressure, and the crude solid was recrystallized with dichloromethane to yield l-(4-chlorophenyl)-3,3-dimethyl-5-(methylthio)-2,3-dihydro-lH-l,2,4-triazole as an off- white solid.

General Synthetic Procedure for S-benzylation of l,2,4-triazolidine-3-thiones (Compound 6b):

To a solution of compound 5 (0.27 g, 1.12 mmol) in methanol (25 mL) under nitrogen at room temperature, benzyl bromide (0.23 g, 1.34 mmol, 1.20 eq) was added dropwise to the solution. The reaction was allowed to stir overnight for 16 hours at room temperature. The solvent was removed under reduced pressure, and the crude solid was purified using flash column chromatography (10-40% EtOAc/Hex) to yield 5-(benzylthio)-l-(4-chlorophenyl)-3,3-dimethyl-

2,3-dihydro-lH-l,2,4-triazole as a brown oil.

General Synthetic Procedure for S-alloc protection of l,2,4-triazolidine-3-thiones (Compound 7):

To a solution of compound 5 (2.07 mmol), DMAP (0.1 eq) and triethyl amine (4.14 mmol) in THF (20 mL) under nitrogen at room temperature, allyl chloroformate (2.27 mmol) was added dropwise. The reaction was allowed to stir overnight for 16 hours at room temperature. The solution was extracted with dichlorom ethane (2 x 40 mL), and washed with IN HCl (35 mL), saturated NaHCCb (35 mL), and brine (35 mL), dried (MgS0₄) and concentrated under reduced pressure. The residue was purified using flash column chromatography (5-20% EtO Ac/Hex) to yield O-allyl S-(2-(4-chlorophenyl)-5,5-dimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate as an orange-white solid.

General Synthetic Procedure for N-l alkylation of S-alloc protected l,2,4-triazolidine-3- thiones (Compounds 8a-c):

To a solution of compound 6 (1.31 mmol) in DMF (12 mL) at 0° C under nitrogen was added sodium hydride (1.44 mmol) and the mixture was allowed to stir for 10 minutes. The desired alkyl halide (1.44 mmol) was added dropwise and the reaction was allowed to stir for 4 hours warming to room temperature. The reaction was then quenched with water (15 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then washed with IN HCl (1 x 20 mL), brine (2 x 20 mL), dried (MgS0₄), and concentrated under reduced pressure. The residue was then purified using flash column chromatography (5-15% EtO Ac/Hex) to yield the desired product.

General Synthetic Procedure for the S-alloc deprotection of N-l alkylated 1,2,4-triazoidine- 3-thiones (Compounds 9a-c):

To a solution of alloc protected intermediate compound 8 (0.31 mmol) in EtOH (10 mL) at 0° C under nitrogen was added tetrakis(triphenylphosphine)palladium (0) (0.001 mmol) and sodium borohydride (0.63 mmol). After 1 hour, the reaction was acidified to pH 2.5-3 using 12 N HCl, and the reaction was allowed to stir for 4 hours. After completion, the reaction was extracted with 1 : 1 EtOAC/Hex (2 x 20 mL). The combined organic layers were washed with water (20 mL), saturated NaHCCb (20 mL), brine (20 mL), dried (MgS0₄) and concentrated under reduced pressure. The residue was then purified using flash chromatography (10-25%) EtO Ac/Hex) to the desired product.³ General Synthetic Procedure for l,2,4-triazoline-3-thione (Compound 10):

To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was N-(4- chlorophenyl)hydrazinecarbothioamide (3 mmol). Acetone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol¹ to yield 4-(4-chlorophenyl)-5,5-dimethyl-l,2,4- triazolidine-3-thione as a white solid.²

Broth Microdilution Method for MIC determination

Day cultures (6 h) were subcultured to 5 x 10⁵ CFU/mL in Mueller- Hinton II broth cation adjusted (CAMHB). Aliquots (1 mL) were placed in culture tubes, and compound was added from 128 μ§/ ιί stock samples in DMSO, such that the compound concentration equaled the highest concentration tested (128 μ§/πΛ or 64 μ§/ ιί). Samples were then aliquoted (200 pL) into the first wells of a 96-well plate, with all remaining wells being filled with 100 μΐ. of initial bacterial subculture. Row 1 wells were mixed five times, before 100 μΐ. was transferred to row 2. Row 2 was then mixed five times, and 100 μΐ. was transferred to row 3. This process was repeated until the final row had been mixed; this served to serially dilute the compound. Plates were then covered with GLAD Press n' Seal and incubated under stationary conditions at 37 °C for 16 h. MIC values were then recorded as the lowest concentration at which no bacterial growth was observed.

Fatty Acid Supplementation Assay

Day cultures (6 h) were subcultured to 5 x 10⁵ CFU/mL in Mueller- Hinton II broth cation adjusted (CAMHB) supplemented with 0.02% linoleic acid. Aliquots (1 mL) were placed in culture tubes, and compound was added from 128 μg/mL stock samples in DMSO, such that the compound concentration equaled the highest concentration tested (128 μg/mL). Samples were then aliquoted (200 μL) into the first wells of a 96-well plate, with all remaining wells being filled with 100 μL of initial bacterial subculture. Row 1 wells were mixed five times, before 100 μL was transferred to row 2. Row 2 was then mixed five times, and 100 μL was transferred to row 3. This process was repeated until the final row had been mixed; this served to serially dilute the compound. Plates were then covered with GLAD Press n' Seal and incubated under stationary conditions at 37 °C for 16 h. MIC values were then recorded as the lowest concentration at which no bacterial growth was observed.

Checkerboard assay

MHB was inoculated with A. baumannii (5 x 10⁵ CFU/ml) and 100 mL aliquots were distributed to all wells of a 96-well plate except for well la. Inoculated MHB (200 mL) containing a selected compound (at a concentration for 2x the highest concentration being tested) was added to well la, and 100 mL of the same sample was added to wells 2a-12a. Column A cells were mixed 6-8 times, and then 100 mL was withdrawn and transferred to column B. This process was repeated up to column G (column H was not mixed to determine the MIC of the antibiotic alone). Inoculated media (100 mL) containing antibiotic at 2x the highest concentration being tested was placed in wells Al-Hl and serially diluted, all the way until row 1 1 (row 12 was not mixed to determine the MIC of the compound alone). The plates were covered and sealed with GLAD Press'n Seal, and incubated under stationary conditions at 37 C. After 16 h the MIC values of both compound and antibiotic were recorded, as well as combination. The∑FIC values were calculated as follows: ∑F1C ¼ FICcmpd + FICantibiotic, where FIC_Cmpd=[MIC_Cmpd in combination]/[MIC_cmpd alone], and FICantibiotic ¼ [MICantibiotic in combination]/[MIC_antibiotic alone]. The combination is considered synergistic if∑FIC < 0.5, indifferent if 0.5 <∑FIC < 2, and antagonistic if∑FIC > 2.

Synthesis of Table 1 Compounds

5-ethyl-5-methyl-2-phenyl-l,2,4-triazolidine-3-thione (1): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 2-butanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-ethyl-5-methyl-2-phenyl-l,2,4-triazolidine-3-thione as a white-orange solid (m.p = 1 12 °C, 64%). ¾ (400 MHz, CDCb) δ 7.96 (d, J=8.4, 2H), 7.81 (brs, 1H), 7.36 (t, J=7.6, 2H), 7.17 (t, J=7.6, 2H), 4.99 (s, 1H), 1.77 (m, J=7.6, 2H), 1.46 (s, 3H), 1.02 (t, J=7.6, 3H) ppm; ¹³C MR (100 MHz,CDCl₃) δ 1 17.0, 139.2, 128.4, 125.6, 122.3, 77.8, 31.8, 28.3, 8.3 ppm; JK v_max (cm^"1) 3155, 2966, 1492, 1386; HRMS (ESI) calcd for CnHi₅N₃S [M+H]⁺ d 222.10546.

5,5-dimethyl-2-phenyl-l,2,4-triazolidine-3-thione (2b): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Acetone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-dimethyl-2-phenyl-l,2,4-triazolidine-3-thione as a white-yellowish solid (m.p = 131 °C, 60%). ¾ (400 MHz, CDCb) δ 8.05 (brs, 1H), 7.92 (d, J=8.4, 2H), 7.35 (t, J=8.4, 2H), 7.17 (t, J=7.2, 1H), 5.14 (s, 1H), 1.47 (s, 6H) ppm;¹³C NMR (100 MHz, CDCb) δ 177.5, 139.2, 128.5, 125.7, 122.4, 75.3, 25.5 ppm; JR v_max (cm^"1) 3159, 2967, 1499, 1384, 740; HRMS

10H13N3S [M+H]⁺ 208.09029, found 208.09029.

5-isopropyl-5-methyl-2-phenyl-l,2,4-triazolidine-3-thione (2c): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 3-methyl-2-butanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-isopropyl-5-methyl-2-phenyl-l,2,4-triazolidine-

3-thione as a white-orange solid (m.p = 142 °C, 46%). ¾ (400 MHz, CDCb) δ 7.95 (t, J=7.6, 2H0, 7.84 (brs, 1H), 7.359 (t, J=7.6, 2H0, 7.17 (t, J=7.2, 1H), 4.94 (s, 1H), 1.99 (m, J=6.8, 1H), 1.41 (s, 3H), 1.01 (d, J=6.8, 6H) ppm; ¹³C MR (100 MHz, DMSO^) δ 175.4, 140.1, 127.9, 123.8, 121.0, 78.7, 36.0, 19.7, 17.0, 16.6 ppm; IR v_max (cm^"1) 3150, 2961, 1498, 1380, 745; HRMS (ESI) calcd

[M+H]⁺ 236.12159, found 236.12153.

5-methyl-2-phenyl-l,2,4-triazolidine-3-thione (2e): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Acetaldehyde (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-methyl-2-phenyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 132 °C, 52%). ¾ (300 MHz, DMSO-i&) δ 10.51 (s, 1H), 8.99 (s, 1H), 7.30 (t, J=8.1, 2H), 7.03 (d, J=6.3, 3H), 5.10 (d, J=5.1, 1H) ppm, 1.35 (d, J=5.7, 3H) ppm; ¹³C NMR (75 MHz, DMSO-d₆) δ 177.3, 151.0, 129.0, 122.7, 115.9, 78.9, 22.5 ppm; IR v_max (cm^"1) 3100, 2974, 1603, 693; HRMS (ESI) calcd for C9H11N3S [M+H]⁺ 194.07464, found 194.07449.

5,5-diethyl-2-phenyl-l,2,4-triazolidine-3-thione (2f): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-phenyl-l,2,4-triazolidine-3-thione a white solid (m.p = 115 °C,

57%). ¾ (400 MHz, CDCb) 8.48 (s, 1H), 7.94 (d, J=8, 2H), 7.31 (t, J=8, 2H), 7.11 (t, J=7.2, 1H), 4.99 (s, 1H), 1.76-1.62 (m, 4H), 0.94 (t, J=7.6, 6H) ppm;¹³C MR (100 MHz,CDCl₃) δ 176.3, 139.1, 128.3, 125.3, 122.1, 80.2, 29.4, 7.9 ppm; IR v_max (cm^"1) 3157, 2964, 1492, 1384, 748; HRMS

C12H17N3S [M+H]⁺ 236.12159, found 236.12103.

5-ethyl-2-phenyl-l,2,4-triazolidine-3-thione (2h): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Priopionaldehyde (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-ethyl-2-phenyl-l,2,4-triazolidine-3-thione white-yellowish solid (m.p = 114 °C, 43%). ¾ (400 MHz, CDCb) δ 7.95 (dd, Ji=1.2, J₂=7.6, 2H), 7.51 (brs, 1H), 7.36 (t, J=7.2, 2H), 7.18 (t, J=7.2, 1H), 5.18 (brs, 1H), 4.74-4.68 (m, 1H), 1.78-1.67 (m, 2H), 1.02 (t, J=7.2, 3H) ppm;¹³C MR (100 MHz, DMSO-d₆) δ 176.4, 139.9, 127.9, 124.1, 121.3, 71.8, 27.0, 8.5 ppm; IR Vmax (cm^"1) 3109, 2971, 1599, 687; HRMS (ESI) calcd for C10H13N3S [M+H]⁺ 208.09029, found 208.08995.

5-methyl-2-phenyl-5-propyl-l,2,4-triazolidine-3-thione (2i): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 2-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-methyl-2-phenyl-5-propyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 108 °C, 55%). ¾ (300 MHz, CDCb) δ 7.97-7.94 (m, 2H), 7.43 (brs, 1H), 7.37 (t, J=7.5, 2H), 7.18 (t, J=7.5), 4.94 (brs, 1H), 1.75-1.69 (m, 2H), 1.54-1.43 (m, 5H), 0.96 (t, J=7.2, 3H) ppm; ¹³C NMR (100 MHz, CD₃OD) δ 178.2, 141.1, 129.1, 126.1, 123.5, 77.9, 42.6, 23.6, 18.1, 14.7 ppm; JK v_max (cm^"1) 3155, 2962, 1491, 1386, 750; HRMS (ESI) calcd for C12H17N3S +H]⁺ 236.12159, 136.12128 found.

2-phenyl-5-propyl-l,2,4-triazolidine-3-thione (21): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Butyraldehyde(3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-phenyl-5-propyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 157 °C,

50%). ¾ (300 MHz, DMSO-i&) δ 10.49 (s, 1H), 9.06 (s, 1H), 7.31 (t, J=7.2, 2H), 7.00 (m, 3H), 4.96 (s, 1H), 1.60-1.44 (m, 4H), 0.95 (t, J=6.6, 3H)ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 177.5, 151.6, 129.1, 122.6, 1 15.9, 82.4, 37.9, 16.6, 13.8 ppm; IR v_max (cm^"1) 31 12, 2978, 1592, 683; HRMS (ESI) calcd for C11H15N3S [M+H]⁺ 222.10594, found 222.10525.

2-phenyl-l,2,4-triazaspiro[4.5]decane-3-thione (2o): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Cyclohexanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-phenyl-l,2,4-triazaspiro[4.5]decane-3-thione as a white-brownish solid (m.p = 141 °C, 62%). ¾ (400 MHz, CDCb) δ 7.98 (brs, 1H), 7.94-7.87 (m, 2H), 7.43-7.29 (m, 2H), 7.22-7.15 (m, 1H), 4.92 (s, 1H), 1.81-1.77 (m, 4H), 1.68-1.63 (m, 4H), 1.46 (t, J=5.2, 2H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 176.1, 140.4, 127.9, 124.0, 121.3, 75.8, 34.4, 24.7, 22.1 ppm; IR v_max (cm-¹) 3165, 2964, 1510, 1386, 739; HRMS (ESI) calcd for C13H17N3S [M+H]⁺ 248.12159, found 248.12115.

Synthesis of Compounds

5-methyl-2,5-diphenyl-l,2,4-triazolidine-3-thione (2d): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Acetophenone(3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-methyl-2,5-diphenyl-l,2,4-triazolidine-3-thione as a white-orange solid (m.p = 120 °C, 64%). ¾ (300 MHz, CDCb) δ 7.93-7.89 (m, 2H), 7.50-7.39 (m, 5H), 7.33-7.29 (m, 2H), 7.04-6.99 (m, 1H), 2.25 (s, 3H) ppm;¹³C NMR (100 MHz, CDCb) δ 145.3, 141.2, 139.1, 129.3, 128.3, 127.9, 125.5, 120.2, 113.2, 11.8 ppm; IR v_max (cm^"1) 3159, 2965, 1492, 1382, 749; HRMS

15H15N3S [M+H]⁺ 270.10594, found 270.10561.

5-ethyl-5-isopropyl-2-phenyl-l,2,4-triazolidine-3-thione (2g): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 2-methyl-3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-ethyl-5-isopropyl-2-phenyl-l,2,4-triazolidine-3- thione as a yellowish solid (m.p = 121 °C, 48%). ¾ (400 MHz, CDCb) δ 8.42 (brs, 1H), 8.01 (d, J=8.4, 2H), 7.36(t, J=8.4, 2H), 7.16 (t, J=6.4, 1H), 2.01 (m, J=7.2, 1H), 1.78 (m, J=7.2, 2H), 1.01 (t, J=7.2, 9H) ppm;¹³C MR (100 MHz, DMSO-d₆) 5175.0, 140.0, 127.8, 123.7, 120.9, 80.8, 34.9, 27.4, 16.6, 16.4, 7.6 ppm; IR v_max (cm^"1) 3162, 2959, 1500, 1393, 746; HRMS (ESI) calcd for C13H19N3S [M+H]⁺ 250.13724, found 240.13682.

5-ethyl-2-phenyl-5-propyl-l,2,4-triazolidine-3-thione (2j): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 3-hexanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-ethyl-2-phenyl-5-propyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 127 °C, 55%). ¾ (400 MHz, CDCb) δ 8.30 (brs, 1H), 7.94 (d, J=7.6, 2H), 7.328 (d, J=7.6, 2H), 7.133 (t, J=6.8, 1H), 4.95 (s, 1H), 1.78-1.58 (m, 4H), 1.48-1.32 (m, 2H), 1.01-0.82 (m, 6H) ppm; ¹³C MR (100 MHz, DMSO^) δ 175.7, 140.1, 127.9, 123.9, 121.1, 78.6, 38.8, 29.6, 16.2, 14.3, 7.7 ppm; IR v_max (cm^"1) 3158, 2970, 1489, 1390, 739; HRMS (ESI) calcd

+H]⁺ 250.13724, found 250.13656.

2-phenyl-5,5-dipropyl-l,2,4-triazolidine-3-thione (2k): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). 4-heptanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-phenyl-5,5-dipropyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 128 °C, 40%). ¾ (300 MHz,CDCl₃) δ 7.98-7.934 (m, 2H), 7.82 (brs, 1H), 7.37 (t, J=7.5, 2H), 7.20- 7.15 (m, 1H), 4.75 (brs, 1H), 1.74-1.66 (m, 4H), 1.50-1.40 (m, 4H), 0.95 (t, J=6.9, 6H); ppm; ¹³C NMR QOO MHz, CD₃OD) δ 177.7, 141.1, 129.1, 126.3, 123.8, 80.1, 40.8, 17.8, 14.7 ppm; IR v_max (cm^"1) 3150, 2963, 1491, 1382, 741; HRMS (ESI) calcd for C14H21N3S [M+H]⁺ 264.15289, found

2,5-diphenyl-l,2,4-triazolidine-3-thione (2m): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Benzaldehyde (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5-methyl-2,5-diphenyl-l,2,4-triazolidine-3-thione as a white-brown solid (m.p = 140 °C, 60%). ¾ (300 MHz, DMSO- d₆) δ 10.39 (s, 1H), 7.69 (s, 1H), 7.42 (d, 2H), 7.31 (t, 2H), 7.28 (m,

3H), 7.23 (d, 2H), 6.78 (t, 1H) ppm; C MR (100 MHz, DMSO-d₆) δ 145.5, 136.6, 136.1, 129.3, 128.8, 128.1, 125.8, 118.9, 112.2 ppm; IR v_max (cm^"1) 3157, 1487, 1370, 749; HRMS (ESI) calcd

[M+H]⁺ 256.09029, found 256.08970.

2-phenyl-l,2,4-triazaspiro[4.4]nonane-3-thione (2n): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and phenyl hydrazine (3 mmol). Cyclopentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-phenyl-l,2,4-triazaspiro[4.4]nonane-3-thione as a pink solid (m.p = 130 °C, 70%). ¾ (400 MHz, CDCb) δ 8.05 (brs, 1H), 7.93 (d, J=8, 2H), 7.36 (t, J=8, 2H), 7.17 (t, J=6, 1H), 5.07 (brs, 1H), 1.96-1.81 (m, 4H), 1.76-1.69 (m, 4H) ppm; 13C NMR (100 MHz, DMSO-i&) δ 176.3, 140.0, 127.9, 124.0, 121.3, 83.8, 35.8, 22.9 ppm; IR v_max (cm^"1) 3158, 2970, 1507, 1381, 732; HRMS (ESI) calcd for C12H15N3S [M+H]⁺ 234.10594, found 234.10583.

2-benzyl-5,5-diethyl-l,2,4-triazolidine-3-thione (3a): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and benzyl hydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-benzyl-5,5-diethyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 118 °C, 43%). ¾ (400 MHz, CDCb) δ 7.41-7.30 (m, 5H), 7.10 (brs, 1H), 1.68-1.54 (m, 4H), 0.87 (t, J=7.6, 6H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 177.6, 137.2, 128.2, 127.9, 127.2, 79.3, 50.3, 28.9, 7.6 ppm; JR v_max (cm^"1) 3223, 2967, 1469, 1421, 735; HRMS (ESI) calcd

[M+H]⁺ 250.13724, found 250.13674.

2-cyclohexyl-5,5-diethyl-l,2,4-triazolidine-3-thione (3b): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and cyclohexyl hydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-cyclohexyl-5,5-diethyl-l,2,4-triazolidine-3- thione as a white solid (m.p = 162 °C, 39%). ¾ (400 MHz, CDCb) δ 4.37-4.31 (m, 1H), 4.07 (s,

1H), 1.79 (d, J=10.8, 4H), 1.72-1.48 (m, 6H), 1.40-1.14 (m, 4H), 1.12-1.06 (m, 2H), 0.90 (t, J=7.6, 6H);¹³C NMR (100 MHz, CDCb) δ 176.7, 80.5, 55.3, 29.5, 28.7, 25.3, 7.9 ppm; IR v_max (cm^"1)

6, 1149; HRMS (ESI) calcd for C12H23N3S [M+H]⁺ 242.16855, found 242.16791.

5,5-diethyl-2-(pyridin-2-yl)-l,2,4-triazolidine-3-thione (3c): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 2- hydrazinopyridine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The oil that formed was extracted with dichloromethane and washed with brine (25 mL), saturated sodium bicarbonate (25 mL), and finally brine (25 mL) . The organic layer was then dried over magnesium sulfate, and the solvent was removed under reduced pressure. The crude mixture was purified using flash chromatography (1-3% MeOH sat. with H3/DCM) to yield 5,5-diethyl-2-(pyridin-2-yl)-l,2,4- triazolidine-3-thione as a brown oil (10%). ¾ (400 MHz, CDCb) δ 8.07-8.04 (m, 1H), 7.88 (brs, 1H), 7.52 (td, Ji=0.3, J₂=6.0, 1H), 7.23 (d, J=7.5, 1H), 6.69-6.65 (m, 1H), 2.28 (sext, J=7.5, 4H), 1.12 (q, J=7.5, 6H) ppm; ¹³C (100 MHz, CDCb) δ 157.7, 153.9, 146.9, 138.1, 115.1, 107.4, 29.7, 21.9, 10.9, 9.7 ppm; UV ( max nm) 325; IR vmax (cm^"1) 3341, 2974, 1599, 1441, 767; HRMS (ESI)

[M-H]^" 235.10229, found 235.10130.

2-(4-chlorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4a): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- chlorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(4-chlorophenyl)-5,5-diethyl-l,2,4- triazolidine-3-thione as a white-brownish solid (m.p = 157 °C, 63%). ¾ (300 MHz, CDCb) δ 8.00 (d, J=9.3, 2H), 7.49 (brs, 1H), 7.32 (d, J=9, 2H), 1.74 (m, 4H), 1.00 (t, J=7.5, 6H); ¹³C MR (100 MHz, CDCb) δ 176.9, 137.9, 130.6, 128.5, 123.0, 80.3, 29.6, 8.0 ppm; IR v_max (cm^"1) 3180, 2971,

MS (ESI) calcd for Ci₂Hi₆ClN₃S [M+H]⁺ 270.08262, found 270.08192.

2-(4-bromophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4b): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- bromophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(4-bromophenyl)-5,5-diethyl-l,2,4- triazolidine-3-thione as a white-brown solid (m.p = 161 °C, 53%). ¾ (400 MHz, DMSO-<f₆) δ 9.44 (s, 1H), 8.09-7.96 (m, 2H), 7.56-7.44 (m, 2H), 6.59 (s, 1H), 1.65-1.54 (m, 4H), 0.88 (t, J=7.2, 6H) ppm; ¹³C NMR (100 MHz, DMSO^) δ 175.7, 139.5, 130.6, 122.5, 115.6, 79.1, 29.1, 7.6 ppm; IR v_max (cm^"1) 3180, 2973, 1501, 1379, 816; HRMS (ESI) calcd for Ci₂Hi₆BrN₃S [M+H]⁺

314.3141.

5,5-diethyl-2-(4-iodophenyl)-l,2,4-triazolidine-3-thione (4c): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- iodophenylhydrazine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol yield 5,5-diethyl-2-(4-iodophenyl)-l,2,4-triazolidine-3- thione as a white-orange solid (m.p = 161 °C, 43%). ¾ (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 7.91 (d, J=8.8, 2H), 7.67 (d, J=8.8, 2H), 6.57 (s, 1H), 1.62-1.57 (m, 4H), 0.879 (t, J=7.2, 3H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 175.6, 140.0, 136.5, 122.7, 87.6, 79.0, 29.1, 7.6 ppm; IR v_max (cm^" ^l) 3180, 2970, 1498, 1376, 815; HRMS (ESI) calcd for Ci₂Hi₆IN₃S [M+H]⁺ 362.01824, found

5,5-diethyl-2-(4-fluorophenyl)-l,2,4-triazolidine-3-thione (4d): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- fluorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(4-fluorophenyl)-l,2,4- triazolidine-3-thione as a white solid (m.p = 148 °C, 40%). ¾ (400 MHz, DMSO^) δ 9.30 (s, 1H), 7.99 (t, J=8.8, 2H), 7.17 (t, J=8.8, 2H), 6.57 (s, 1H), 1.62 (q, J=7.6, 4H), 0.89 (t, J=7.6, 6H) ppm;¹³C NMR (100 MHz, DMSO-d₆) δ 176.7, 135.2, 124.4, 124.3, 115.3, 115.1, 80.3, 29.5, 8.0 ppm; IR v_max (cm^"1) 3178, 2972, 1498, 1382, 820; HRMS (ESI) calcd for Ci₂Hi₆FN₃S [M+H]⁺ nd 254.11180.

5,5-diethyl-2-(2-fluorophenyl)-l,2,4-triazolidine-3-thione (4e): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 2- fluorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(2-fluorophenyl)-l,2,4- triazolidine-3-thione as a white solid (m.p = 158 °C, 47%). ¾ (300 MHz, CDC1₃) δ 7.91 (brs, 1H), 7.65 (m, 1H), 7.31 (m, 1H), 7.17 (m, 2H), 1.81 (m, J=7.5, 4H), 1.02 (t, J=7.5, 6H) ppm; ¹³C MR (100 MHz, DMSO-i&) δ 177.9, 158.5, 156.0, 129.7, 129.3, 129.2, 127.6, 127.5, 124.2, 116.4, 116.2, 80.8, 28.6, 7.7 ppm; IR v_max (cm^"1) 3157, 2969, 1490, 1405, 908, 764; HRMS (ESI) calcd

+H]⁺ 254.11217, found 254.11191.

5,5-diethyl-2-(3-fluorophenyl)-l,2,4-triazolidine-3-thione (4f): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 3- fluorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(3-fluorophenyl)-l,2,4- triazolidine-3-thione as a white solid (m.p = 120 °C, 60%). ¾ (400 MHz, DMSO^) δ 9.52 (s, 1H), 8.10-8.05 (m, 1H), 7.91-7.88 (m, 1H), 7.36 (q, J=6.8, 1H), 6.92-6.87 (m, 1H), 6.59 (s, 1H), 1.50 (q, J=7.6, 4H), 0.89 (t, J=8, 6H); ¹³C MR (100 MHz, DMSO-d₆) δ 175.8, 162.6, 160.2, 141.9, 141.8, 129.6, 129.5, 115.9, 110.0, 109.8, 107.0, 106.8, 79.1, 29.1, 7.6 ppm; IR v_max (cm^"1) 3179, 2968, 1585, 1481, 771; HRMS (ESI) calcd for Ci₂Hi₆FN₃S [M+H]⁺ 254.11217, found 254.11164.

2-(2-chlorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4g): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 2- chlorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(2-chlorophenyl)-5,5-diethyl-l,2,4- triazolidine-3-thione as a tan solid (m.p = 178 °C, 54%). ¾ (400 MHz, CDCb) δ 8.29 (brs, 1H), 7.58 (dt, Ji=2, J₂=6.4, 1H), 7.48 (dt, Ji=2, J₂=6.4, 1H), 7.36-7.31 (m, 2H), 1.89-1.73 (m, 4H), 1.022 (t, J=7.2, 6H) ppm;¹³C NMR (100 MHz, DMSO-d₆) δ 177.9, 137.0, 132.5, 131.1, 129.9, 129.5, 127.5, 80.9, 28.0, 7.7 ppm; IR v_max (cm^"1) 3156, 2971, 1488, 1402, 908, 767; HRMS (ESI) calcd

+H]⁺ 270.08262, found 270.08252.

2-(3-chlorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4h): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 3- chlorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(3-chlorophenyl)-5,5-diethyl-l,2,4- triazolidine-3-thione as a white-yellow solid (m.p = 110 °C, 44%). ¾ (400 MHz, DMSO^) δ 9.58-9.45 (m, 1H), 8.28-8.16 (m, 1H), 8.07-7.99 (m, 1H), 7.37-7.27 (m, 1H), 7.13-7.06 (m, 1H), 6.60(brs, 1H), 1.66-1.54 (m, 4H), 0.95-0.81 (m, 6H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 175.8, 141.5, 132.2, 129.5, 123.0, 119.7, 118.6, 79.1, 29.1, 7.6 ppm; IR v_max (cm^"1) 3180, 2966, 1 MS (ESI) calcd for Ci₂Hi₆ClN₃S [M+H]⁺ 270.08262, found 270.08220.

2-(3-chloro-4-fluorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4i): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 3-chloro-4-fluorophenylhydrazine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(3-chloro-4-fluorophenyl)-5,5-diethyl- l,2,4-triazolidine-3-thione as a tan solid (m.p = 128 °C, 60%). ¾ (300 MHz, CDCb) δ 8.11 (dd, Ji=2.7, J₂=6.6, 1H), 7.98-7.93 (m, 2H), 7.10 (t, J=8.7, 1H), 1.86-1.65 (m, 4H), 0.99 (t, J=7.5, 6H) ppm; ¹³C MR (100 MHz, DMSO^) δ 175.8, 154.6, 152.1, 137.3, 121.9, 120.9, 120.8, 118.4, 118.2, 116.1, 115.9, 79.2, 29.1, 7.6 ppm; IR vmax (cm^"1) 3180, 2970, 1474, 1368, 974; HRMS (ESI) calcd for C12H15CIFN3S [M+H]⁺ 288.07320, found 288.07261.

2-(3,5-difluorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4j): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 3,5-difluorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(3,5- difluorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 128 °C, 28%). ¾

(400 MHz, DMSO-i&) δ 9.76 (s, 1H), 7.97 (d, J=12.4, 2H), 6.94-6.84 (m, 1H), 6.62 (brs, 1H), 1.66-1.54 (m, 4H), 0.88 (t, J=8, 6H) ;¹³C NMR (100 MHz, DMSO^) δ 175.9, 163.0, 162.9, 160.6, 160.5, 142.6, 102.4, 102.1, 98.3, 98.0, 97.8, 79.2, 29.1, 7.6 ppm; IR v_max (cm^"1) 3180, 2971, 1627,

(ESI) calcd for Ci₂Hi₅F₂N₃S [M+H]⁺ 272.10275, found 272.10212.

2-(3,4-dichlorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione (4k): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 3,4-dichlorophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(3,4- dichlorophenyl)-5,5-diethyl-l,2,4-triazolidine-3-thione as an orange solid (m.p = 120 °C, 59%). ¾ (400 MHz, CDCb) δ 8.20 (t, J=2.8, 1H), 8.04 (dt, Ji=2.8, J₂=8.8, 1H), 7.94 (brs, 1H),7.390 (dd, Ji=2, J₂=8.8, 1H), 4.91 (s, 1H), 1.83-1.65 (m, 4H), 0.99 (t, J=7.2, 6H) ppm;¹³C NMR (100 MHz, CDCb) 5 176.9, 138.8, 132.1, 129.9, 128.3, 122.7, 120.4, 80.5, 29.5, 8.0 ppm; JR v_max (cm^"1) 3176, 2971, 1476, 1370, 976; HRMS (ESI) calcd for Ci₂Hi₅Cl₂N₃S [M+H]⁺ 304.04365, found

5,5-diethyl-2-(4-isopropylphenyl)-l,2,4-triazolidine-3-thione (41): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4-isopropylphenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(4-isopropylphenyl)- l,2,4-triazolidine-3-thione as a white solid (m.p = 152 °C, 64%). ¾ (400 MHz, CDCb) δ 8.02 (s, 1H), 7.84-7.82 (dd, Ji=1.2, J₂=5.1, 2H), 7.23-7.21 (dd, Ji=1.2, J₂=5.1, 2H), 2.90 (sep, J=7.2, 1H), 1.83-1.68 (m, 4H), 1.24 (d, J=7.2, 6H), 1.00 (t, J=7.6, 6H) ppm; ¹³C NMR (100 MHz, CDCb) δ 176.4, 146.4, 136.8, 126.4, 122.5, 80.2, 33.7, 29.5, 24.0, 8.0 ppm; IR v_max (cm^"1) 3183, 2959, 1511,

SI) calcd for Ci₅H₂₃N₃S [M+H]⁺ 278.16855, found 278.16799.

5,5-diethyl-2-(4-nitrophenyl)-l,2,4-triazolidine-3-thione (4m): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- nitrophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(4-nitrophenyl)-l,2,4- triazolidine-3-thione as a bright yellow solid (m.p = 134 °C, 66%). ¾ (400 MHz, CDCb) δ 8.12 (d, J=9.2, 2H), 7.76 (s, 1H), 7.06 (d, J=9.2, 2H), 2.41-2.24 (m, 4H), 1.24-1.07 (m, 6H) ppm;¹³C NMR (100 MHz, CDC1₃) 5 156.5, 150.9, 139.5, 126.2, 111.6, 29.7, 21.8, 10.7, 9.8 ppm; IR v_max (cm^"1) 3330, 2975, 1599, 1310, 846; HRMS (ESI) calcd for C12H16N4O2S [M+H]⁺ 281.10667, found 281.10606.

4-(3,3-diethyl-5-thioxo-l,2,4-triazolidin-l-yl)benzonitrile (4n): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- cyanophenylhydrazine hydrochloride (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 4-(3,3-diethyl-5-thioxo-l,2,4- triazolidin-l-yl)benzonitrile as a white-brown solid (m.p = 167 °C, 58%). ¾ (400 MHz, DMSO- d₆) δ 9.80 (s, 1H), 8.39 (d, J=7.2, 2H), 7.78 (d, J=7.2, 2H), 6.68 (s, 1H), 1.63-1.58 (m, 4H), 0.88 (dd, Ji=5.6, J₂=7.6, 6H) ppm; ¹³C MR (100 MHz, DMSO-d₆) 5 176.0, 144.0, 132.3, 119.4, 119.1, 104.6, 79.2, 29.1, 7.6 ppm; IR Vmax (cm^"1) 3180, 2935, 2222, 1504, 1370, 822; HRMS (ESI) calcd

+H]⁺ 261.11684, found 261.11643.

5,5-diethyl-2-(4-(trifluoromethyl)phenyl)-l,2,4-triazolidine-3-thione (4o): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4-(trifluoromethyl)phenylhydrazine (3 mmol). 3-pentanone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 5,5-diethyl-2-(4- (trifluoromethyl)phenyl)-l,2,4-triazolidine-3-thione as an orange solid (m.p = 167 °C, 44%). ¾

(400 MHz, DMSO-de) δ 9.67 (s, 1H), 8.36 (d, J=8.4, 2H), 7.70 (d, J=8.4, 2H), 6.66 (s, 1H), 1.65 (q, J=7.2, 4H), 0.89 (t, J=7.2, 6H) ppm; ¹³C NMR (100 MHz, DMSO-i&) δ 176.1, 143.6, 125.8, 125.1, 123.3, 123.1, 123.0, 119.7, 79.2, 29.1, 7.6 ppm; IR v_max (cm^"1) 3184, 2935, 1488, 1327, 1121, 830; HRMS (ESI) calcd for Ci₃Hi₆F₃N₃S [M+H]⁺ 304.10898, found 304.10836.

2-(4-chlorophenyl)-5,5-dimethyl-l,2,4-triazolidine-3-thione (5): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added potassium thiocyanate (0.291 g, 3 mmol) and 4- chlorophenylhydrazine hydrochloride (3 mmol). Acetone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 2-(4-chlorophenyl)-5,5-dimethyl-l,2,4- triazolidine-3-thione as a white-brown solid (m.p = 150 °C, 47%). ¾ (300 MHz, CDCb) δ 9.38 (s, 1H), 8.04 (dd, Ji=1.2, J₂=5.1, 2H), 7.39 (dd, Ji=1.2, J₂=5.1, 2H), 1.34 (s, 6H) ppm; ¹³C NMR (100 MHz, DMSO-i&) δ 176.3, 139.1, 127.8, 127.7, 122.4, 74.1, 24.9 ppm; IR v_max (cm^"1) 3178,

813; HRMS (ESI) calcd for CioHi₂ClN₃S [M+H]⁺ 242.05132, found 242.05107.

l-(4-chlorophenyl)-3,3-dimethyl-5-(methylthio)-2,3-dihydro-lH-l,2,4-triazole (6a): To a solution of compound 5 (0.32 g, 1.32 mmol) in methanol (25 mL) under nitrogen at room temperature, methyl iodide (0.23 g, 1.59 mmol, 1.20 eq) was added dropwise to the solution. The reaction was allowed to stir overnight for 16 hours at room temperature. The solvent was removed under reduced pressure, and the crude solid was recrystallized with dichloromethane to yield l-(4- chlorophenyl)-3,3-dimethyl-5-(methylthio)-2,3-dihydro-lH-l,2,4-triazole as an off-white solid (m.p = 165 °C, 69%). ¾ (300 MHz, CD₃OD) δ 7.59-7.55 (m, 4H), 2.77 (s, 3H), 1.67 (s, 6H) ppm; ¹³C NMR (100 MHz, DMSO-i¾) δ 165.2, 134.6, 133.2, 129.6, 126.2, 79.9, 25.0, 15.9 ppm; IR v_max (cm^"1) 3101, 2965, 2323, 1504, 744; HRMS (ESI) calcd for CnHi₄ClN₃S [M+H]⁺ 256.06697, found 256.06699.

5-(benzylthio)-l-(4-chlorophenyl)-3,3-dimethyl-2,3-dihydro-lH-l,2,4-triazole (6b): To a solution of compound 5 (0.27 g, 1.12 mmol) in methanol (25 mL) under nitrogen at room temperature, benzyl bromide (0.23 g, 1.34 mmol, 1.20 eq) was added dropwise to the solution. The reaction was allowed to stir overnight for 16 hours at room temperature. The solvent was removed under reduced pressure, and the crude solid was purified using flash column chromatography (10-40% EtOAc/Hex) to yield 5-(benzylthio)-l-(4-chlorophenyl)-3,3-dimethyl- 2,3-dihydro-lH-l,2,4-triazole as a brown solid (m.p = 112 °C, 75%). ¾ (400 MHz, CDCb) δ 7.50- 7.30 (m, 9H), 4.33 (s, 2H), 1.45 (s, 6H) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 161.0, 153.4, 134.6 132.0, 129.1, 128.9, 128.5, 127.9, 125.4, 80.5, 36.8, 25.0 ppm; IR v_max (cm^"1) 3087, 2970, 1489, 1010, 697; HRMS (ESI) calcd for C17H18CIN3S [M+H]⁺ 332.09827, found 332.09845.

O-allyl S-(2-(4-chlorophenyl)-5,5-dimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate (7): To a solution of compound 5 (0.5 g, 2.07 mmol), DMAP (0.026 g, 0.1 eq) and triethyl amine (0.419 g, 4.14 mmol, 2 eq) in THF (20 mL) under nitrogen at room temperature, allyl chloroformate (0.27 g, 2.27 mmol, 1.1 eq) was added dropwise. The reaction was allowed to stir overnight for 16 hours at room temperature. The solution was extracted with dichlorom ethane (2 x 40 mL), and washed with IN HC1 (35 mL), saturated NaHC0₃ (35 mL), and brine (35 mL), dried (MgS0₄) and concentrated under reduced pressure. The residue was purified using flash column chromatography (5-20% EtOAc/Hex) to yield O-allyl S-(2-(4-chlorophenyl)-5,5- dimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate as an orange-white solid (m.p = 95 °C, 20%). ¾ (300 MHz, CDCb) δ 7.84 (d, J=9, 2H), 7.31 (d, J=9, 2H), 6.00-5.90 (m, 1H), 5.40 (dd, Ji=1.5, J₂=17.4, 1H), 5.30 (dd, Ji=1.5, J₂=17.4, 1H), 4.72 (d, J=5.7, 2H), 1.64 (s, 6H) ppm; ¹³C NMR (100 MHz, DMSO^) δ 173.5, 150.6, 137.195, 131.6, 130.9, 128.1, 124.9, 119.4, 79.7, 67.3, 23.2 ppm; IR v_max (cm^"1) 3175, 2988, 1714, 1301, 708; HRMS (ESI) calcd for C_I4H_I6C1N₃0₂S [M+H]⁺ 326.07425, found 326.07264.

O-allyl S-(2-(4-chlorophenyl)-l,5,5-trimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate (8a): To a solution of compound 7 (0.426 g, 1.31 mmol) in DMF (12 mL) at 0° C under nitrogen was added sodium hydride (0.057 g, 1.44 mmol, 1.1 eq) and the mixture was allowed to stir for 10 minutes. Methyl iodide (0.204 g, 1.43 mmol, 1.1 eq) was added dropwise and the reaction was allowed to stir for 4 hours warming to room temperature. The reaction was then quenched with water (15 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then washed with IN HCl (1 x 20 mL), brine (2 x 20 mL), dried (MgS0₄), and concentrated under reduced pressure. The residue was then purified using flash column chromatography (5-15% EtOAc/Hex) to yield O-allyl S-(2-(4-chlorophenyl)-l,5,5-trimethyl-2,5- dihydro-lH-l,2,4-triazol-3-yl) carbonothioate as a white solid (m.p = 97 °C, 19%). 1H (300 MHz, CD₃OD) δ 7.50 (d, J=6.6, 2H), 7.38 (d, J=6.6, 2H), 6.03-5.89 (m, 1H), 5.40 (dd, Ji=1.8, J₂=17.4, 1H), 5.28 (dd, Ji=1.8, J₂=17.4, 1H), 4.52 (d, J=6.9, 2H), 2.39 (s, 3H), 2.13 (s, 3H), 1.96 (s, 3H) ppm; ¹³C NMR (100 MHz, CDCb) δ 177.6, 163.2, 159.1, 140.6, 133.3, 132.6, 129.2, 128.3, 117.9, 66.4, 24.8, 20.7, 15.2 ppm; IR v_max (cm^"1) 3064, 2964, 1689, 1212, 658; HRMS (ESI) calcd for C_I5H_I8C1N₃0₂S [M+H]⁺ 340.08810, found 340.08855.

O-allyl S-(l-butyl-2-(4-chlorophenyl)-5,5-dimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate (8b): To a solution of compound 7 (0.414 g, 1.27 mmol) in DMF (12 mL) at 0° C under nitrogen was added sodium hydride (0.056 g, 1.40 mmol, 1.1 eq) and the mixture was allowed to stir for 10 minutes. Butyl iodide (0.204 g, 1.43 mmol, 1.1 eq) was added dropwise and the reaction was allowed to stir for 4 hours warming to room temperature. The reaction was then quenched with water (15 mL) and extracted with ethyl acetate (3 x 20 mL). The combined organic layers were then washed with IN HCl (1 x 20 mL), brine (2 x 20 mL), dried (MgS0₄), and concentrated under reduced pressure. The residue was then purified using flash column chromatography (5-15% EtOAc/Hex) to yield O-allyl S-(l-butyl-2-(4-chlorophenyl)-5,5- dimethyl-2,5-dihydro-lH-l,2,4-triazol-3-yl) carbonothioate as a pale yellow oil (49%). 1H (300 MHz, CD₃OD) δ 7.46 (d, J=8.7, 2H), 7.33 (d, J=8.7, 2H), 5.98-5.89 (m, 1H), 5.37 (dd, Ji=1.5, J₂=17.1, 1H), 5.24 (dd, Ji=1.5, J₂=17.1, 1H), 4.48 (d, J=6, 2H), 2.93 (t, J=7.5, 2H), 2.09 (s, 3H), 1.93 (s, 3H), 1.65-1.57 (m, 2H), 1.43-1.36 (m, 2H), 0.93 (t, J=7.2, 3H) ppm; ¹³C NMR (100 MHz, CDCb) δ 177.6, 162.9, 159.3, 140.8, 133.4, 132.8, 129.3, 128.6, 117.9, 66.5, 32.3, 30.6, 25.0, 22.0, 20.9, 13.6 ppm; UV ( max nm) 329; IR v_max (cm-¹) 3062, 2957, 1683, 1207, 667; HRMS (ESI) calcd for Ci₈H₂₄ClN₃0₂S [M+H]⁺ 382.13505, found 382.13488.

2-(4-chlorophenyl)-l,5,5-trimethyl-l,2,4-triazolidine-3-thione (9a): To a solution of compound 7a (0.156 g, 0.46 mmol) in EtOH (10 mL) at 0° C under nitrogen was added tetrakis(triphenylphosphine)palladium (0) (0.002 g, 0.002 mmol, 0.004 eq) and sodium borohydride (0.035 g, 0.92 mmol, 2 eq). After 1 hour, the reaction was acidified to pH 2.5-3 using 12 N HC1, and the reaction was allowed to stir for 4 hours. After completion, the reaction was extracted with 1 : 1 EtOAC/Hex (2 x 20 mL). The combined organic layers were washed with water (20 mL), saturated NaHC0₃ (20 mL), brine (20 mL), dried (MgS0₄) and concentrated under reduced pressure. The residue was then purified using flash chromatography (10-25% EtOAc/Hex) to yield 2-(4-chlorophenyl)-l,5,5-trimethyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 78 °C, 22%). 1H (300 MHz, CDC1₃) δ 7.32-7.26 (m, 4H), 4.60 (brs, 1H), 2.51 (s, 3H), 1.44 (s, 6H) ppm; ¹³C NMR (100 MHz, CDC1₃) δ 160.9, 141.5, 129.5, 128.9, 122.2, 85.5, 27.7, lS.e ppmi lR vmax cm-^ lSO, 2962, 1482, 1194, 1039, 827;HRMS (ESI) calcd for CnHi₄ClN₃S +H]⁺ 256.06697, found 256.06706.

l-butyl-2-(4-chlorophenyl)-5,5-dimethyl-l,2,4-triazolidine-3-thione (9b): To a solution of compound 7b (0.120 g, 0.31 mmol) in EtOH (10 mL) at 0° C under nitrogen was added tetrakis(triphenylphosphine)palladium (0) (0.001 g, 0.001 mmol, 0.004 eq) and sodium borohydride (0.023 g, 0.63 mmol, 2 eq). After 1 hour, the reaction was acidified to pH 2.5-3 using 12 N HC1, and the reaction was allowed to stir for 4 hours. After completion, the reaction was extracted with 1 : 1 EtOAC/Hex (2 x 20 mL). The combined organic layers were washed with water (20 mL), saturated NaHC0₃ (20 mL), brine (20 mL), dried (MgS0₄) and concentrated under reduced pressure. The residue was then purified using flash chromatography (10-25%) EtOAc/Hex) to yield l-butyl-2-(4-chlorophenyl)-5,5-dimethyl-l,2,4-triazolidine-3-thione as a yellow oil (24%). 1H (300 MHz, CDC1₃) δ 7.33-7.26 (m, 4H), 4.51 (brs, 1H), 3.09 (t, J=6.9, 2H), 1.70-1.62 (m, 2H), 1.45-1.38 (m, 9H), 0.91 (t, J=7.2, 3H) ppm; ¹³C NMR (175MHz, CDC1₃) δ 160.1, 141.6, 129.4, 128.9, 122.4, 85.4, 32.9, 30.9, 27.6, 22.0, 13.78 ppm; UV _max nm) 325; JR Vmax (cm^"1) 3174, 2958, 1487, 1180, 1051, 825; HRMS (ESI) calcd for Ci₄H₂₀ClN₃S [M+H]⁺

298.11416.

4-(4-chlorophenyl)-5,5-dimethyl-l,2,4-triazolidine-3-thione (10): To a solution of hydrochloric acid (12 mL, 0.25 M, 3 mmol) was added N-(4-chlorophenyl)hydrazinecarbothioamide (3 mmol) Acetone (3 mmol) was added to the solution dropwise. The reaction was allowed to stir in the dark for 16 hours at room temperature. The precipitate that formed was filtered using vacuum filtration and washed with water four times. The precipitate was then recrystallized with methanol to yield 4-(4-chlorophenyl)-5,5-dimethyl-l,2,4-triazolidine-3-thione as a white solid (m.p = 135 °C, 84%). 1H (300 MHz, CDCb) δ 9.24 (brs, 1H), 8.64 (brs, 1H), 7.61 (d, J=8.7, 2H), 7.33 (d, J=8.7, 2H), 4.78 (s, 1H), 2.05 (s, 3H), 1.94 (s, 3H) ppm; ¹³C MR (100 MHz, CDCb) δ 175.9, 150.7, 136.5, 130.8, 128.5, 125.3, 25.2, 17.1 ppm; IR v_max (cm^"1) 3269, 2990, 1483, 1100, 592; HRMS (ESI) calcd for C10H12CIN3S [M+H]⁺ 242.05132, found 242.05142.

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The compositions and methods of the appended claims are not limited in scope by the specific compositions and methods described herein, which are intended as illustrations of a few aspects of the claims. Any compositions and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the compositions and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compositions and method steps disclosed herein are specifically described, other combinations of the compositions and method steps also are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated.

The term "comprising" and variations thereof as used herein is used synonymously with the term "including" and variations thereof and are open, non-limiting terms. Although the terms "comprising" and "including" have been used herein to describe various embodiments, the terms "consisting essentially of and "consisting of can be used in place of "comprising" and "including" to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

We claim:

1. A compound of Formula I:

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen; wherein at least one of R⁴, R⁵, or R⁶ is halogen;

or a pharmaceutically acceptable salt thereof.

The compound of claim 1, wherein R¹ is methyl.

The compound of claim 1, wherein R¹ is ethyl.

The compound of any one of claims 1 to 3, wherein R² is methyl. The compound of any one of claims 1 to 3, wherein R² is ethyl. The compound of any one of claims 1 to 5, wherein R⁴ is hydrogen. The compound of any one of claims 1 to 6, wherein R⁵ is CI.

8. The compound of any one of claims 1 to 6, wherein R⁵ is Br.

9. The compound of any one of claims 1 to 6, wherein R⁵ is F.

10. The compound of any one of claims 1 to 6, wherein R⁵ is I.

11. The compound of any one of claims 1 to 10, wherein R⁶ is hydrogen.

12. The compound of claim 1, having the formula:

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁵ is halogen;

or a pharmaceutically acceptable salt thereof.

13. The compound of claim 12, wherein R¹ is methyl.

14. The compound of claim 12, wherein R¹ is ethyl.

15. The compound of any one of claims 12 to 14, wherein R² is methyl.

16. The compound of any one of claims 12 to 14, wherein R² is ethyl.

17. The compound of any one of claims 12 to 16, wherein R⁵ is CI.

18. The compound of any one of claims 12 to 16, wherein R⁵ is Br.

19. The compound of any one of claims 12 to 16, wherein R⁵ is F.

20. The compound of any one of claims 12 to 16, wherein R⁵ is I.

21. The compound of claim 1, having the formula:

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴ is halogen;

or a pharmaceutically acceptable salt thereof.

22. The compound of claim 21, wherein R¹ is methyl.

23. The compound of claim 21, wherein R¹ is ethyl.

24. The compound of any one of claims 21 to 23, wherein R² is methyl.

25. The compound of any one of claims 21 to 23, wherein R² is ethyl.

26. The compound of any one of claims 21 to 25, wherein R⁴ is CI.

27. The compound of any one of claims 21 to 25, wherein R⁴ is Br.

28. The compound of any one of claims 21 to 25, wherein R⁴ is F.

29. The compound of any one of claims 21 to 25, wherein R⁴ is I.

30. The compound of claim 1 , selected from the group consisting of:

and

31. The compound of claim 30 having the formula:

32. The compound of claim 1 having the formula:

33. A method of treating or preventing an Acinetobacter infection, comprising administering to a patient in need thereof, an effective amount of a compound of Formula I:

Formula I

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴, R⁵, and R⁶ are independently selected from hydrogen or halogen;

or a pharmaceutically acceptable salt thereof.

34. The method of claim 33, wherein R¹ is methyl.

35. The method of claim 33, wherein R¹ is ethyl.

36. The method of any one of claims 33 to 35, wherein R² is methyl.

37. The method of any one of claims 33 to 35, wherein R² is ethyl.

38. The method of any one of claims 33 to 37, wherein R⁴ is hydrogen.

39. The method of any one of claims 33 to 38, wherein R⁵ is halogen.

40. The method of claim 39, wherein R⁵ is CI.

41. The method of claim 39, wherein R⁵ is Br.

42. The method of claim 39, wherein R⁵ is F.

43. The method of claim 39, wherein R⁵ is I.

44. The method of any one of claims 33 to 43, wherein R⁶ is hydrogen.

45. The method of any one of claims 33 to 44, wherein the Acinetobacter infection is

Acinetobacter baumannii.

46. The method of claim 33, wherein the compound has the formula:

Formula II

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁵ is independently selected from hydrogen or halogen;

or a pharmaceutically acceptable salt thereof.

47. The method of claim 46, wherein R¹ is methyl.

48. The method of claim 46, wherein R¹ is ethyl.

49. The method of any one of claims 46 to 48, wherein R² is methyl.

50. The method of any one of claims 46 to 48, wherein R² is ethyl.

51. The method of any one of claims 46 to 50, wherein R⁵ is halogen.

52. The method of claim 51, wherein R⁵ is CI.

53. The method of claim 51, wherein R⁵ is Br.

54. The method of claim 51, wherein R⁵ is F.

55. The method of claim 51, wherein R⁵ is I.

56. The method of any one of claims 46 to 55, wherein the Acinetobacter infection is Acinetobacter baumannii.

57. The method of claim 33, wherein the compound has the formula:

Formula III

wherein:

R¹ and R² are independently alkyl; or

R¹ and R² may be joined to form a cycloalkyl ring; and

R⁴ is independently selected from hydrogen or halogen;

or a pharmaceutically acceptable salt thereof.

58. The method of claim 57, wherein R¹ is methyl.

59. The method of claim 57, wherein R¹ is ethyl.

60. The method of any one of claims 57 to 59, wherein R² is methyl.

61. The method of any one of claims 57 to 59, wherein R² is ethyl.

62. The method of any one of claims 57 to 61, wherein R⁴ is halogen.

63. The method of claim 62, wherein R⁴ is CI.

64. The method of claim 62, wherein R⁴ is Br.

65. The method of claim 62, wherein R⁴ is F.

66. The method of claim 62, wherein R⁴ is I.

67. The method of any one of claims 57 to 66, wherein the Acinetobacter infection is Acinetobacter baumannii.

75

69. The method of claim 33, wherein the compound is selected from the group consisting of:

and

70. The method of claim 69, wherein the compound is

71. The method of claim 33, wherein the compound is

72. The method of any one of claims 68 to 71, wherein the Acimlobacter infection is Acinetobacter baumannii.