WO2013116823A1 - Benzimidazoles and uses thereof - Google Patents

Abstract

The present invention relates to novel 2,5,6-benzimidazole derivatives of formula I and pharmaceutically acceptable salts thereof. Another aspect of the invention relates to methods of treating a patient infected by Mycobacterium tuberculosis or Mycobacterium avium complex by administering to the patient a 2,5,6- benzimidazole derivative or a pharmaceutically acceptable salt thereof.

Description

BENZIMIDAZOLES AND USES THEREOF This invention was made with government support under grant number AI078251 awarded by the National Institute of Health. The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos.

61/594,322, filed February 2, 2012; 61/600,047, filed February 17, 2012; and 61/614,908, filed March 23, 2012, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Tuberculosis (TB) was one of the first infectious diseases to be identified. More than fifty years of research has been directed to controlling and eliminating this disease. However, the eradication of TB is still one of the most prominent challenges for basic and clinical research scientists.

Once thought to be under control, TB case reports in the U.S. increased sharply in the early 1990' s. Although, this trend has reversed and the reported numbers of new cases has steadily declined in industrialized countries, TB remains a major global public health threat. Recent statistics from the WHO estimate that there are approximately 8.4 million new cases every year with a global mortality rate of 23% or approximately 2 million deaths per year.

Poor chemotherapeutics and inadequate local-control programs contribute to the inability to manage TB and lead to the emergence of drug resistant strains of the bacteria that cause Mycobacterium tuberculosis (Mtb). A survey conducted at 58 international sites between 1996 and 1999 found exceptionally high rates of single and multidrug- resistant strains in Estonia, Latvia and Russia, and revealed that countries such as China and Iran were developing a high prevalence of multidrug -resistance (MDR-TB). See Kruuner, A., Sillastu, H., Danilovitsh, M., Levina, K., Svenson, S. B., Kallenius, G., and Hoffner, S. E. (1998) Drug resistant tuberculosis in Estonia, Int J Tuberc Lung Dis 2, 130-3. Significantly, MDR-TB is much more difficult to treat than sensitive TB, requiring administration of more expensive, second- line antibiotics for up to two years. The frequency of resistance to at least one of the first-line TB drugs (isoniazid (INH), rifampicin (RIF), pyrazinamide or ethambutol) ranged from 1.7% in Uruguay to 36.9% in Estonia. The frequency of resistance is indicative of the global problem involving not only the spread of Mtb, but also treatment.

Finally, of critical importance is the role of TB as a major opportunistic pathogen in patients with HIV/AIDS. Consequently, there is a pressing need for the development of novel TB drugs that are effective against both sensitive and resistant Mtb strains.

Likewise, new drugs are needed to treat patients infected by Mycobacterium avium complex which consists of two bacteria that are difficult to differentiate - M avium and M intracellular. Mycobacterium avium complex is a pulmonary pathogen which affects individuals who are immuno compromised, and is the most common cause of infection by nontuberculous mycobacteria in patients with AIDS.

SUMMARY OF THE INVENTION

The invention relates to a molecule having formula I:

I

wherein:

R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰, NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(O)R⁵, S(O)₂R⁵, CH(O), C(O)OR⁶, C(O)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo;

R represents alkyl, cycloalkyl, or aryl;

R represents H, alkyl, cycloalkyl, or aryl;

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰;

R⁵ represents R⁶ or OR⁶;

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl;

Y represents C(X²)R⁴, S(O)R⁵, or S(O)₂R⁵;

X¹ represents O or S;

X² represents O, S, NH, or NR⁶;

R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a

heterocyclic alkyl or a heterocyclic aryl;

R 7' and R 8° may be combined to represent a carbocyclic alkyl; alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain; cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings; aryl groups are carbocyclic or heterocyclic; carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings; heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings; halo substituents are fluoro, chloro, bromo, or iodo; each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or

substituted with one or more substituent at any position; alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁵, S(0)₂R⁵, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl; cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl; aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl; and heterocyclic alkyl and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen and sulfur; and pharmaceutically acceptable salts thereof.

The invention also relates to a method of treating a patient infected with

Mycobaterium tuberculosis, Mycobacterium avium complex, or Mycobacterium avium, the method comprising administering to the patient the compound of formula I or a pharmaceutically acceptable salt thereof.

The invention also relates to the following benzimidazole compounds:

DETAILED DESCRIPTION

The invention relates to novel benzimidazole derivatives. These benzimidazole derivatives can be used to treat a patient infected by Mycobacterium

tuberculosis, Mycobacterium avium complex, or Mycobacterium avium.

The molecules have formula I:

I

In this formula, R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰,

NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(O)R⁵, S(O)₂R⁵, CH(O), C(O)OR⁶, C(O)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo.

R 2 represents alkyl, cycloalkyl, or aryl. Preferably, R 2 is alkyl or phenyl.

R 3 represents H, alkyl, cycloalkyl, or aryl. R 3 is preferably H.

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰. Preferably, R⁴ is alkyl, aryl, or OR⁶. More preferably, R⁴ is alkyl or phenyl.

R⁵ represents R⁶ or OR⁶. Preferably, R⁵ is alkyl or phenyl.

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl. For example, R 7 may represent methyl and R 8 may represent phenyl.

Y represents C(X²)R⁴, S(O)R⁵, or S(O)₂R⁵. X¹ represents O or S. X² represents O, S, NH, or NR⁶.

R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a heterocyclic alkyl or a heterocyclic aryl.

For example, R and Y may be combined to represent a heterocyclic alkyl ring, resulting in the following structure:

Similarly, R⁹ and R¹⁰ can be combined to represent a heterocyclic aryl ring, resulting in the following structure:

In another example, R 7 and R 8 can be combined to represent a carbocyclic alkyl, resulting in the following structure: Y

Alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain. Some examples of suitable straight-chained, saturated alkyl groups include methyl, ethyl, ^-propyl, /i-butyl, rc-pentyl, /i-hexyl groups and dodecyl and hexadecyl. Preferred straight chain, saturated alkyl groups include methyl and ethyl.

Some examples of suitable branched, saturated alkyl groups include iso-propyl, iso-butyl, sec-butyl, t-butyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl (isopentyl), 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl (neopentyl), 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl groups, and 2-methyl,5-ethyldecyl. Preferred branched, saturated alkyl groups include isopropyl and t-butyl.

Some examples of unsaturated alkyl groups include ethenyl, ethynyl, propenyl, propargyl, isopropenyl, crotyl, 1-hexenyl, and 1-octenyl.

Cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings. Ring systems are monocyclic, bicyclic, tricyclic, or tetracyclic and can be bridged or non- bridged.

Some examples of carbocyclic alkyl groups include cyclobutanyl, cyclopentanyl, cyclohexanyl, and cycloheptanyl. Examples of fused carbocyclic alkyl groups include indenyl, isoindenyl. Bridged groups include bicyclo [2.2.1] heptane, bicyclco [5.2.0] nonane, and bicyclo [5.2.0] nonane.

Some examples of heterocyclic alkyl groups include pyrrolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, morpholino, and oxazolidinyl. Examples of fused heterocyclic alkyl groups include benzomorpholino, benzopyrrolidinyl, indolinyl, and benzopiperidinyl .

Aryl groups can be either carbocyclic or heterocyclic.

Carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings. A preferred unfused carbocyclic aryl group is phenyl.

Some examples of fused carbocyclic aryl groups include naphthyl, phenanthryl, anthracenyl, triphenylenyl, chrysenyl, and pyrenyl.

Heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings.

Some examples of unfused heterocyclic aryl groups include thiophenyl, furyl, pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, and pyrazinyl. Some examples of fused heterocyclic aryl groups include purinyl, 1,4- diazanaphthalenyl, indolyl, benzimidazolyl, 4,5-diazaphenanthrenyl, benzoxazolyl, isoindolyl, quinolinyl, isoquinolinyl, and benzofuranyl.

Halo substituents are fluoro, chloro, bromo, or iodo. Preferred halo substituents are fluoro, chloro, or bromo.

Each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or substituted with one or more substituent at any position. Alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁶, S(0)₂R⁶, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl.

Cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl. Aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl.

Heterocyclic alky and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen, and sulfur. In this specification, groups of various parameters containing multiple members are described. Within a group of parameters, each member may be combined with any one or more of the other members to make additional sub-groups. For example, if the members of a group are a, b, c, d, and e, additional sub-groups specifically contemplated include any two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.

In some cases, the members of a first group of parameters, e.g., a, b, c, d, and e, may be combined with the members of a second group of parameters, e.g., A, B, C, D, and E. Any member of the first group or of a sub-group thereof may be combined with any member of the second group or of a sub-group thereof to form additional groups, i.e., b with C; a and c with B, D, and E, etc.

For example, in the present invention, groups of various parameters are defined (e.g. R¹, R², R³, R⁴, X). Each group contains multiple members. For example, R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰. Each member may be combined with each other member to form additional sub-groups, e.g., H, SR⁶, NH₂, and NHR⁶; R⁶, NHR⁶, and NR⁹R¹⁰; H, R⁶, OR⁶, NH₂, and NR⁹R¹⁰.

The instant invention further contemplates embodiments in which each element listed under one group may be combined with each and every element listed under any

1 2

other group. For example, X is identified above as representing O or S. R is identified above as being alkyl, cycloalkyl, or aryl. Each element of X (O or S) can be combined with each and every element of R (alkyl, cycloalkyl, or aryl). For example, in one embodiment, X 1 may be O and R2 may be ethyl. Alternatively, X 1 may be S and R2 may be phenyl, etc. Similarly, a third parameter is R , in which the elements are defined as H, alkyl, cycloalkyl, or aryl. Each of the above embodiments may be combined with each and every element of R 3. For example, in the embodiment wherein X is O and R 2 is methyl, R 3 may be H (or any other chemical moiety within the element of R 3 ).

The compounds of this invention are limited to those that are chemically feasible and stable. Therefore, a combination of substituents or variables in the compounds described above is permissible only if such a combination results in a stable or

chemically feasible compound. A stable compound or chemically feasible compound is one in which the chemical structure is not substantially altered when kept at a

temperature of 40°C or less, in the absence of moisture or other chemically reactive conditions, for at least a week.

Pharmaceutically acceptable salts

The present invention also relates to pharmaceutically acceptable salts of the benzimidazole derivatives. The pharmaceutically acceptable salts include the

conventional non-toxic salts of the benzimidazole derivatives as formed, e.g., from nontoxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy- benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

The pharmaceutically acceptable salts of the benzimidazole derivatives of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared either by ion exchange chromatography or by reacting the free base with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid in a suitable solvent or various combinations of solvents.

Synthesis of the benzimidazole derivatives The benzimidazoles of the present invention can be synthesized by methods known in the art. The following scheme represents one approach to the synthesis of the compounds of formula I, wherein X¹ is O. Scheme 1

General Procedure for the synthesis of novel 2,5,6- trisubstituted benzimidazoles (5) bearing an ether linkage at the 6-position

N H₂ R²OH/ (1 .2 eq.)

N0₂ 1 M KOH, THF,

or KoCO . Acetone

R¹ = cyclo hexyl

2b R² = butyl ,

2c R² = phenly ,

2 = 4-flu ro phenyl

SnCI₂ 2H ₂0 (7 eq .)

EtOH , reflux

Y = RCO , ROCO, RNH CO, RN CS , RSO,

The first step involves the nucleophilic aromatic substitution of commercially available 2,4-dinitro-5-fluoroaniline byalcohol in the presence of 1 M of KOH or K₂CO₃ as base to give compound 2. The acylation of compound 2 with the cyclohexanecarbonyl chloride generated the intermediate 3. Tin (II) chloride mediated reduction and

cyclization of intermediate 3 formed benzimidazole intermediates 4. The final

intermediates (0.01 mM) were dissolved in dichloromethane and transferred into ninety- six well plates. Twelve different acylating agents, isocyanates, and thiocyanates (1.1 eq.) were dissolved in dichloromethane and added to the individual wells. The plates were slowly shaken for a day. Aminomethylated resins (10 eq.) were added and shaken for a day to remove excess reagent. Filtering of the resins with dichloromethane produces 4 libraries of tribsustituted benzimidazoles 5.

The following scheme represents one approach to the synthesis of the compounds of formula I, wherein X¹ is S. Scheme 2

General Procedure for the synthesis of novel 2,5,6- trisubstituted benzimidazoles (9), bearing a sulfide Linkage at the 6-position R²SH (1 .2 eq.)

N0₂ 1 M KOH THF,

or K₂uu₃, Acelone

6a R² = butyl, R¹ = cyclohexyl

6b R² = phenly,

6c R² = 4-f luorophenyl,

6d R² = benzyl

R²S- 1. T I r raannssfTeerr e eaacchn to 9 y6b--wweelili p pliaatieess R²S.

SnCI₂ 2H₂0 (7 eq.) 2. RCOCI/ROSu/RNCO/ RNCS, or RS0₂CI

4 M HCI, EtOH ^H2N 3. NH₂.scavenge

reflux 8

Y = RCO, ROCO, RN HCO, RNCS, RS0₂

The first step involves the nucleophilic aromatic substitution of commercially available 2,4-dinitro-5-fluoroaniline(l) by thiol in the presence of 1 M of KOH or K₂C0₃ as base to give compound 6. The acylation of compound 6 with the cyclohexanecarbonyl chloride generated the intermediate 7. Tin (II) chloride mediated reduction and

cyclization of intermediate 7 formed benzimidazole intermediates 8. The final

intermediates (0.01 mM) were dissolved in dichloromethane and transferred into ninety- six well plates. Twelve different acylating agents, isocyanates, and thiocyanates (1.1 eq.) were dissolved in dichloromethane and added to the individual wells. The plates were slowly shaken for a day. Aminomethylated resins (10 eq.) were added and shaken for a day to remove excess reagent. Filtering of the resins with dichloromethane produces 4 libraries of tribsustituted benzimidazoles 9.

The compounds were analyzed for purity and confirmation of structure using liquid chromatography- mass spectrometry/ UV spectroscopy according to the procedure outlined below. Scheme 3

General Procedure for the library of 2,5,6- trisubstituted benzimizoles 5 and 9

1 . Transfer each to 96-well plates

2. RCOCI/ROSu/RNCO/ RNCS, or RS0₂CI

3. NH₂. scavenger

4/8

Y = RCO, ROCO, RNHCO,

RNCS, RS0₂

The final intermediates 4/8 (0.01 mmol) were dissolved in dichloromethane and transferred into ninety-six well plates. Twelve different acylating agents, isocyanates, and isothiocyanates (1.1 eq.) were dissolved in dichloromethane and added to the individual wells. The plates were slowly shaken for a day. Aminomethylated

polystyrene resin EHL (200-400 mesh) 2% DVB (10 eq.) was added to scavenge excess or unreacted acylating agents, isocyanates and isothiocyanate. After reacting for 24 hours with shaking to remove excess reagent. Filtering of the resins with dichloromethane provided 4 libraries of 2,5,6-tribsustituted benzimidazoles 5/9. Products in the 96 wells were analyzed by LC-MS/UV for their purity and confirmation of structure. The purity was observed to be in the range of 80-90 %. Uses of the benzimidazole derivatives

The invention also relates to a method of treating a patient infected with

Mycobacterium tuberculosis or Mycobacterium avium complex, which includes

Mycobacterium avium and Mycobacterium intracellular e. The method comprises administering to the patient the compound of formula (I) or a pharmaceutically acceptable salt thereof.

The method and compounds of the invention may be employed alone, or in combination with other anti-bacterial agents. Other anti-bacterial agents include isoniazid, rifampin, pyrazinamide, rifabutin, streptomycin and ciprofloxacin. The combination of these anti-bacterial agents and the compounds of the invention will provide new agents for the treatment of tuberculosis, including MDR-TB and XDR-TB, and pulmonary MAC disease.

An effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof as used herein is any amount effective to treat a patient infected by Mtb or Mycobacterium avium complex. Modes of administration and doses can be determined by those having skill in the art. An effective amount of the compound will vary with the group of patients (age, sex, weight, etc.), the nature and severity of the condition to be treated, the particular compound administered, and its route of

administration. Amounts suitable for administration to humans are routinely determined by physicians and clinicians during clinical trials.

The minimum dose of the compound is the lowest dose at which efficacy is observed. For example, the minimum dose of the compound may be about O.lmg/kg/day, about 1 mg/kg/day, or about 3 mg/kg/day.

The maximum dose of the compound is the highest dose at which efficacy is observed in a patient, and side effects are tolerable. For example, the maximum dose of the compound may be about 10 mg/kg/day, about 9 mg/kg/day, or about 8 mg/kg/day. In another embodiment, the maximum dose of the compound may be up to about 50 mg/kg/day.

A benzimidazole derivative useful in the methods of the present invention may be administered by any method known in the art. Some examples of suitable modes of administration include oral and systemic administration. Systemic administration can be enteral or parenteral. Liquid or solid (e.g., tablets, gelatin capsules) formulations can be employed.

Parenteral administration of the benzimidazole derivative include, for example intravenous, intramuscular, and subcutaneous injections. For instance, a chemical compound may be administered to a patient by sustained release, as is known in the art. Sustained release administration is a method of drug delivery to achieve a certain level of the drug over a particular period of time. Other routes of administration include oral, topical, intrabronchial, or intranasal administration. For oral administration, liquid or solid formulations may be used. Some examples of formulations suitable for oral administration include tablets, gelatin capsules, pills, troches, elixirs, suspensions, syrups, and wafers. Intrabronchial administration can include an inhaler spray. For intranasal administration, administration of a chemical compound can be accomplished by a nebulizer or liquid mist.

The chemical compound can be formulated in a suitable pharmaceutical carrier. In this specification, a pharmaceutical carrier is considered to be synonymous with a vehicle or an excipient as is understood by practitioners in the art. Examples of carriers include starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums and glycols.

The chemical compound can be formulated into a composition containing one or more of the following: a stabilizer, a surfactant, preferably a nonionic surfactant, and optionally a salt and/or a buffering agent.

The stabilizer may, for example, be an amino acid, such as for instance, glycine; or an oligosaccharide, such as for example, sucrose, tetralose, lactose or a dextran.

Alternatively, the stabilizer may be a sugar alcohol, such as for instance, mannitol; or a combination thereof. Preferably the stabilizer or combination of stabilizers constitutes from about 0.1% to about 10% weight for weight of the chemical compound.

The surfactant is preferably a nonionic surfactant, such as a polysorbate. Some examples of suitable surfactants include Tween 20, Tween 80; a polyethylene glycol or a polyoxyethylene polyoxypropylene glycol, such as Pluronic F-68 at from about 0.001% (w/v) to about 10% (w/v). Other preferred surfactants include Solutol H-15 and

Cremophore EL.

The salt or buffering agent may be any salt or buffering agent, such as for example sodium chloride, or sodium/potassium phosphate, respectively. Preferably, the buffering agent maintains the pH of the chemical compound formulation in the range of about 5.5 to about 7.5. The salt and/or buffering agent is also useful to maintain the osmolality at a level suitable for administration to a patient. Preferably the salt or buffering agent is present at a roughly isotonic concentration of about 150 mM to about 300 mM.

The chemical compound can be formulated into a composition which may additionally contain one or more conventional additives. Some examples of such additives include a solubilizer such as, for example, glycerol; an antioxidant such as for example, benzalkonium chloride (a mixture of quaternary ammonium compounds, known as "quart"), benzyl alcohol, chloretone or chlorobutanol; anaesthetic agent such as, for example a morphine derivative; or an isotonic agent etc. As a further precaution against oxidation or other spoilage, the composition may be stored under nitrogen gas in vials sealed with impermeable stoppers.

EXAMPLES

Examples have been set forth below for the purposes of illustration and to describe the best mode of the invention at the present time. The scope of the invention not to be in any way limited by the examples set forth herein.

EXAMPLE I. Synthetic Procedures for Novel 2,5,6- Benzimidazoles 1. 5-ethoxy-2,4-dinitrobenzenamine (2a)

A solution of 1 M KOH was added drop wise to a magnetically stirred solution of 2,4- dinitro-5-fluoroaniline (2.0 g, 9.95 mmol) and ethanol (1.74 mL, 3.0 eq.) in 40 mL of THF for 30 minutes at room temperature. The solvent was evaporated on a rotary evaporator to get rid of THF. The residue was washed with water and methylene chloride three times. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 2a as a yellow solid (2.07 g, 92 % yield): 1H NMR (300 MHz, CDC1₃); δ 1.55 (t, 3 H, /= 7.2 Hz), δ 4.18 (q, 2 H, /= 6.6 Hz), δ 6.22 (s, 1 H), δ 8.96 (s, 1 H)

2. N-(5-ethoxy-2,4-dinitrophenyl)cyclohexanecarboxamide (3a)

To a solution of 2a (1.0 g, 4.40 mmol) in 7 mL of MeCN, cyclohexanecarbonyl chloride (1.4 mL, 1.3 eq.) and pyridine (0.35 mL, 2.0 eq.) as a base were added and magnetically stirred. The mixture was carried in the microwave at 100 °C for 1 hour. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford the product compound 3a as yellow solid (1.44 g , 97 % yield): 1H NMR (300 MHz, CDC1₃); δ 1.75 (m, 2 H), δ 1.89 (m, 2 H), δ 2.05 (m, 2 H), δ 2.41 (m, 2 H), δ 4.33 (q, 2 H, /= 7.2 Hz), δ 8.83 (s, 1 H), δ 8.96 (s, 1 H), δ 10.98 (s, 1 H) 3. 2-cyclohexyl-6-ethoxy-3H-benzo[</]imidazol-5-amine (4a)

A solution of 3a (1.0 g, 2.97 mmol), tin (II) chloride dihydeate (0.47 g, 7.0 eq.) in 150 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 5 hours. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted ethyl acetate and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 4a as a brownish solid (0.64 g, 83 % yield); HPLC purity % 303 nm: 94.6, 254 nm: 89.9

4. 5-butoxy-2,4-dinitrobenzenamine (2b)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (2.0 g, 9.95 mmol) in 45 mL of acetone, 1.2 equiv of 1-butanol was added and then 1 M KOH solution was slowly added. The reaction mixture was stirred mechanically at room temperature for 6 hours. The desired product was washed with water three times and then diluted with

dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 2b as a yellow solid (1.94 g, 76 % yield): 1H NMR (300 MHz, CDCI3) δ 0.99 (t, 3 H, /= 7.2 Hz), δ 1.55 (q, 2 H), δ 1.85 (q, 2 H), δ 4.10 (t, 3 H, /= 6.3 Hz), δ 6.23 (s, 1 H), δ 8.96 (s, 1 H) 5. N-(5-butoxy-2,4-dinitrophenyl)cyclohexanecarboxamide (3b)

To a solution of 2b (1.0 g, 3.92 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (0.64 mL, 1.3 eq.) was added and magnetically stirred. The mixture was carried in the microwave at 100 °C for 1.5 hour. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 3b as a yellow solid as a yellow solid (1.44 g, crude yield): 1H NMR (300 MHz, CDC1₃) δ 0.99 (t, 3 H, /= 7.2) δ 1.28 (m, 4 H), δ 1.44 (m, 2 H), δ 1.55 (q, 2 H), δ 1.82 (m, 2 H), δ 1.85 (q, 2 H), δ 1.95 (m, 2 H), δ 8.82 (s, 1 H), δ 8.96 (s, 1 H), δ 10.99 (s, 1 H)

6. 6-butoxy-2-cyclohexyl-lH-benzo[rf]imidazol-5-amine (4b)

A solution of 3b (1.39 g, 3.81 mmol), tin (II) chloride dihydeate (6.01 g, 7.0 eq.) in 150 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 3.5 hours. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted dichloromethane and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 4b as a pale beige color solid (0.42 g, 38 % yield): 1H NMR (300 MHz, CDC1₃) δ 0.84 (q, 3 H), δ 0.96 (m, 5 H), δ 1.52 (m, 5 H), δ 1.62 (q, 2 H), δ 2.08 (m, 2 H), δ 3.95 (t, 2 H, /= 6.3 Ηζ)δ 6.77 (s, 1 H), δ 6.98 (s, 1 H) 7. 2,4-dinitro-5-phenoxybenzenamine (2c)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (1.03 g, 5.12 mmol/ 1.74 g, 8.67 mmol) in 20 mL/ 45 mL of acetone, 1.2 equiv of phenols and 2.0 equiv of anhydrous K₂CO₃ were added. The reaction mixture was stirred mechanically at room temperature for 9.5 /14 h until the total disappearance of starting material as detected by mass analysis was confirmed. Enough water (100 mL) was added to give the desired compound 2c as a yellow precipitate. The desired product was washed with water three times and then diluted with dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 2c as a yellow solid

(0.94 g, 68 % yield/ 1.95 g, 82 % yield): 1H NMR (300 MHz, CDC1₃) δ 6.03 (s, 1 H), δ 7.14 (t, 2 H, /= 5.7 Hz), δ 7.35 (m, 1 H), δ 7.48 (t, 2 H, /= 7.5 Hz), δ 9.07 (s, 1 H)

8. N-(2,4-dinitro-5-phenoxyphenyl)cyclohexanecarboxamide (3c)

To a solution of 2c (1.01 g, 3.67 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (0.64 mL, 1.3 eq.) was added and magnetically stirred and refluxed at 120 °C for 18 hours. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 3c as a yellow solid as a yellow solid (1.09 g, 77 % yield): 1H NMR

(300 MHz, CDCI3) δ 1.28 (m, 4 H), δ 1.44 (m, 2 H), δ 1.82 (m, 2 H), δ 1.95 (m, 2 H), δ 7.16 (m, 2 H), δ 7.36 (m, 1 H), 8 7.51 (d of t, 2 H), δ 8.53 (s, 1 H), 9.06 (s, 1 H), δ 10.81 (s, 1 H) 9. 2-cyclohexyl-6-phenoxy-lH-benzo[</]imidazol-5-amine (4c)

A solution of 3c (1.48 g, 3.85 mmol), tin (II) chloride dihydeate (6.08 g, 7.0 eq.) in 150 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 3 hours. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted dichloromethane and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 4c as a pale beige color solid (0.55 g, 46 % yield) and get darker color with 0.44 g with 37 % yield: 1H NMR (300 MHz, CDC1₃) δ 6.95 (t, 2 H, /= 6.9 Hz), δ 6.98 (d, 2 H, /= 1.2 Hz), δ 7.03 (s, 1 H), δ 7.28 (d, 2 H, /= 1.2 Hz), δ 7.31 (s, 1 H), δ 2.12 (m, 5 H), δ 1.88 (m, 5 H); HPLC purity % 303 nm: 98.1, 254 nm: 96.3

5-(4-fluorophenoxy)-2,4-dinitrobenzenamine (2d)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (1.0 g, 4.97 mmol) in 20 mL of acetone, 1.2 equiv of 4-fluorophenols and 2.0 equiv of anhydrous K₂C0₃ were added. The reaction mixture was stirred mechanically at room temperature for 16 hours until the total disappearance of starting material as detected by mass analysis was confirmed. Enough water (100 mL) was added to give the desired compound 2d as a yellow precipitate. The desired product was washed with water three times and then diluted with dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 2d as a yellow solid (1.33 g, 91 % yield): 1H NMR (300 MHz, CDC1₃) δ 6.0 (s, 1 H), δ 7.14 (m, 4 H), δ 9.08 (s, 1 H) 11. N-(5-(4-fluorophenoxy)-2,4-dinitrophenyl)cyclohexanecarboxamide (3d)

To a solution of 2d (1.30 g, 4.44 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (0.77 mL, 1.3 eq.) was added and magnetically stirred and refluxed at 120 °C for 18 hours. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 3d as a yellow solid as a yellow solid (1.47 g, 82 % yield): 1H NMR (300 MHz, CDC1₃) δ 1.28 (m, 4 H), δ 1.44 (m, 2 H), δ 1.82 (m, 2 H), δ 1.95 (m, 2 H), δ 7.17 (m, 4 H), δ 8.53 (s, 1 H), δ 9.06 (s, 1 H), δ 10.8 (s, 1 H)

12. 2-cyclohexyl-6-(4-fluorophenoxy)-2,3-dihydro-lH-benzo[</]imidazol-5-amine

(4d)

A solution of 3d (1.46 g, 3.61 mmol), tin (II) chloride dihydeate (5.71 g, 7.0 eq.) in 150 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 1 hours. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted dichloromethane and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 4d as a pale beige color solid (0.81 g, 69 % yield): 1H NMR (300 MHz, CDC1₃) δ 1.34 (m, 5 H), δ 1.60 (m, 5 H), δ 6.87 (m, 3 H), δ 7.07 (m, 2 H); HPLC purity % 303 nm: 93.5, 254 nm: 86.8 13. 5-(Butylthio)-2,4-dinitrobenzenamine (6a)

A solution of 1 M KOH was added dropwise to a magnetically stirred solution of 2,4- dinitro-5-fluoroaniline (1.0 g, 4.97 mmol) and /i-butanethiol (1.5 eq. 0.8 mL ) in 25 mL of THF until color of the reaction mixture was changed from yellow to red. The solvent was evaporated on a rotary evaporator. The residue was washed with water and ethyl acetate three times. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 5-(butylthio)-2,4-dinitrobenzenamine 6a as a yellow solid (1.3 g, quant, yield): mp 148- 149 °C; 1H NMR (300 MHz, CDC13) δ 0.95 (m, 3 H), 1.38 (m, 2 H), 1.77 (m, 2 H), 6.55 (s, 2 H), 2.53 (s, 1 H), 9.18 (s, 1 H); LCMS (FIA) found 272.0 (M+l)

14. N-(5-(Butylthio)-2,4-dinitrophenyl)cyclohexanecarboxamide (7a)

To a solution of 6a (1.0 g, 3.92 mmol) in 10 mL of acetonitrile and 1 mL of pyridine, cyclohexanecarbonyl chloride (0.7 mL, 1.3eq.) was added and magnetically stirred. The mixture was carried in the microwave at 100 °C for 2 h. The reaction mixture was diluted with ethyl acetate and washed with water three times. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product compound 7a (1.97 g >100 yield) 15. 6-(Butylthio)-2-cyclohexyl-lH-benzo[d]imidazol-5-amine (8a)

A solution of 7a (1.97 g, 5.16 mmol), tin (II) chloride dihydrate (7 eq. 15.5 g), and 12 M HC1 (80 mL) in 200 mL of EtOH was magnetically stirred and refluxed for 4 hours. The reaction mixture was cooled and quenched with 1M NaOH until ~ pH 10. Tin chloride precipitated in solution upon addition of 1 M NaOH. The solution was filtered from the tin chloride solid layer. The solution was washed with water and ethyl acetate three times. The organic layers were dried with magnesium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 8a as a solid (0.413g, 26% yield): 1H NMR (300 MHz, CDC1₃) δ 0.86 (t, 3 H, /= 7.5 Hz), 1.37 (m, 5 H), 1.55 (m, 5 H), 1.82 (m, 2 H), 2.05 (m, 5 H), 2.71 (t, /= 7.2 Hz), 6.85 (s, 1 H), 7.66 (s, 1 H); ¹³C NMR (400 MHz, CDC1₃) δ 13.567, 21.778, 25.777, 25.974, 31.560, 31.825, 35.437, 38.553, 98.180, 114.427, 122.957, 133.543, 138.748, 143.909, 158.873; LCMS (FIA) found 304.1(M+1); HPLC purity % 303 nm: 94.8, 254 nm: 88.0

2,4-Dinitro-5-(phenylthio)benzenamine (6b)

A solution of 1 M KOH was added dropwise to a magnetically stirred solution of 2,4- dinitro-5-fluoroaniline (2.0 g, 9.95 mmol)) and benzenethiol (1.2 eq.) in 45 mL of THF until a yellow precipitate appeared and then let the reaction mixture stir for another 30 min. The solvent was evaporated on a rotary evaporator. The residue was washed with water and ethyl acetate three times. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 5-butoxy-2,4-dinitrobenzenamine

6b as a yellow solid (2.91 g, quant, yield): mp 214-217 °C; 1H NMR (300 MHz, CDC1₃) δ 5.96 (s, 1 H), 7.56 (m, 3 H), 7.59 (m, 2 H), 9.02 (s, 1 H) 17. N-(2,4-dinitro-5-(phenylthio)phenyl)cyclohexanecarboxamide (7b)

To a solution of 6b (1.0 g, 3.4 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (0.7 mL, 1.3 eq.) was added and magnetically stirred. The mixture was carried in the microwave at 100 °C for 3 h. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 7b as light yellow solid (1.09 g , 79 % yield): 1H NMR (300 MHz, CDC1₃); δ 7.61 (m, 5 H), δ 8.47 (s, 1 H), δ 9.23 (s, 1 H), δ 10.63 (s, 1 H)

18. 2-cyclohexyl-6-(phenylthio)-lH-benzo[d]imidazol-5-amine (8b)

A solution of 7b (1.0 g, 2.5 mmol), tin (II) chloride dihydeate (7.0 eq. 5.62 g), and 12 M HC1 (40 mL) in 80 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen. After for 6 hours TLC showed the starting material had been left. To the reaction mixture, added 3.0 equivalents more of tin (II) chloride dihydeate and refluxed 2 more hours. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted ethyl acetate and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 8b as a brownish solid (0.46g, 57 % yield): 1H NMR (300 MHz, CDC1₃); δ 6.87 (s, 1 H), δ 7.07 (m, 3 H), δ 7.15 (m, 2 H), δ 7.71 (s, 1 H); LCMS (FIA) found 324.1 (M+l); HPLC purity % 303 nm: 96.1, 254 nm: 94.2

19. 5-(4-fluorophenylthio)-2,4-dinitrobenzenamine (6c)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (2.0 g, 9.95 mmol) in 45 mL of acetone, 1.2 equiv of 4-fluorobenzenethiol and 2.0 equiv of anhydrous K₂CO₃ were added. The reaction mixture was stirred mechanically at from ice bath to room temperature for 14 hours until the total disappearance of starting material as detected by mass analysis was confirmed. Enough water (100 mL) was added to give the desired compound 6c as a yellow precipitate. The desired product was washed with water three times and then diluted with dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 6c as a yellow solid

(2.59 g, 84 % yield): 1H NMR (300 MHz, CDC1₃) δ 5.93(d, 1 H, /= 3.3 Hz), δ 7.25 (m, H), δ 7.61 (m, 2 H), δ 9.21 (d, 1 Η, /= 3 Hz)

20. N-(5-(4-fluorophenylthio)-2,4-dinitrophenyl)cyclohexanecarboxamide (7c)

To a solution of 6c (1.0 g, 3.24 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (0.56 mL, 1.3 eq.) was added and magnetically stirred and refluxed at 120 °C for 15 hours. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 7c as a yellow solid as a yellow solid (1.28 g, 94 % yield): 1H NMR (300 MHz, CDCI3) δ 1.28 (m, 4 H), δ 1.44 (m, 2 H), δ 1.82 (m, 2 H), δ 1.95 (m, 2 H), δ 7.43 (m, 2 H), δ 7.60 (m, 2 H), δ 8.47 (s, 1 H), δ 9.22 (s, 1 H), δ 10. 61 (s, 1 H)

21. 2-cyclohexyl-6-(4-fluorophenylthio)-lH-benzo[</]imidazol-5-amine (8c) A solution of 7c (1.20 g, 2.86 mmol), tin (II) chloride dihydeate (4.5 g, 7.0 eq.) in 125 mL of EtOH was magnetically stirred and refluxed at 90 °C under nitrogen for 2 hours. However, there is no reaction so added 60 mL of 4 M of HCl. The reaction mixture was stirred for another 6 hours and then cooled and basified with 30 % KOH until ~ pH 13. The solution was diluted dichloromethane and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 8c as a pale beige color solid (0.60 g, 61 % yield): 1H NMR (300 MHz, CDC1₃) δ 5.30 (s, 1 H), δ 6.88 (m, 3 H), δ 7.04 (m, 2 H), δ 7.70 (s, 1 H); HPLC purity % 303 nm: 97.7, 254 nm: 95.3

22. 5-(benzylthio)-2,4-dinitrobenzenamine (6d)

To a magnetically stirred solution of 2,4-dinitro-5-fluoroaniline (2.02 g, 10.04 mmol) in 45 mL of THF, 1.2 equiv of benzyl mercaptan and followed by 1 M of KOH solution were slowly added. The reaction mixture was stirred mechanically at room temperature for 5 h until the total disappearance of starting material as detected by TLC. The organic layer were evaporated to give the desired compound 6d as a yellow precipitate. The desired product was washed with water three times and then diluted with

dichloromethane. The organic layers were dried with magnesium sulfate, filtered, and concentrated to afford the product 6d as a yellow solid (3.49 g, crude): 1H NMR (300 MHz, CD₃OD) δ 3.31 (s, 2 H), δ 7.01 (s, 1 H), δ 7.30 (m, 3 H), δ 7.34 (m, 2 H), δ 9.01 (s, 1 H)

23. N-(5-(benzylthio)-2,4-dinitrophenyl)cyclohexanecarboxamide (7d)

To a solution of 6d (2.01 g, 6.56 mmol) in 15 mL of pyridine, cyclohexanecarbonyl chloride (1.14 mL, 1.3 eq.) was added and magnetically stirred and refluxed at 120 °C for 16 hours. The reaction mixture was evaporated with toluene and then washed with CuS0₄ solution twice to get rid of left over pyridine. The reaction mixture washed with brine and then diluted with ethyl acetate and methylene chloride and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated to afford the product compound 7d as a yellow solid as a yellow solid (2.43 g, 89 % yield): 1H NMR

(300 MHz, CD₃OD) δ 3.31 (s, 2 H), δ 7.01 (s, 1 H), δ 7.30 (m, 3 H), δ 7.34 (m, 2 H), δ 9.01 (s, 1 H)

24. 6-(benzylthio)-2-cyclohexyl-lH-benzo[d]imidazol-5-amine (8d)

A solution of 7d (2.03 g, 4.82 mmol), tin (II) chloride dihydeate (7.61 g, 7.0 eq.) in 200 mL of EtOH and 100 mL of HC1 was magnetically stirred and refluxed at 90 °C under nitrogen for 6 hours. Even after 6 hours the TLC indicated that the reaction was not completed yet. Therefore, tin (II) chloride dihydeate (7.61 g, 7.0 eq.) was more added and then refluxed another 3 hours at 90 °C under nitrogen. The reaction mixture was cooled and quenched with 30 % KOH until ~ pH 13. The solution was diluted

dichloromethane and washed with water three times. The organic layers were dried with sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography to afford compound 8d as a pale beige color solid (0.90 g, 56 % yield): 1H NMR (300 MHz, CD₃OD) δ 1.33 (m, 6 H), δ 1.86 (m, 2 H), δ 2.12 (m, 2 H), δ 3.91 (s, 3 H), 8 7.11 (s, 1 H), 8 7.49 (s, 1 H) EXAMPLE II. Screening of Compounds

A. The libraries of 2,5,6- trisubstituted benzimidazoles 5 and 9 (376 compounds) were screened for their activity against Mtb H37Rv using the "Microplate Alamar Blue Assay (MABA)" [Collins, L.; Franzblau, S. G., Microplate Alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against

Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemother. 1997, 41, (5), 1004-1009] in a 96-well format at 5 μg/mL concentration (single point assay in triplicates). Then, the growth inhibition was measured in percentage. The results are shown below.

Plate 23

At 5 μg/mL concentration

SB-P23E2 52 Cyclohexyl PhCO BuS

SB-P23G8 52 Cyclohexyl 4-MeC₆H₄CO 4-FC₆H₄S

SB-P23B9 51 Cyclohexyl 4-MeOC₆H₄CH₂CO BuO

SB-P23E7 51 Cyclohexyl 4-PrC₆H₄CO BuS

SB-P23A7 49 Cyclohexyl 4-PrC₆H₄CO EtO

SB-P23D2 46 Cyclohexyl PhCO 4-FC₆H₄0

SB-P23E10 46 Cyclohexyl 4-CNC₆H₄CO BuS

SB-P23F8 45 Cyclohexyl 4-MeC₆H₄CO PhS

SB-P23A3 42 Cyclohexyl W-C5H11CO EtO

SB-P23D7 42 Cyclohexyl 4-PrC₆H₄CO 4-FC₆H₄0

SB-P23D10 39 Cyclohexyl 4-CNC₆H₄CO 4-FC₆H₄0

SB-P23D9 37 Cyclohexyl 4-MeOC₆H₄CH₂CO 4-FC₆H₄0

SB-P23D8 36 Cyclohexyl 4-MeC₆H₄CO 4-FC₆H₄0

SB-P23C9 34 Cyclohexyl 4-MeOC₆H₄CH₂CO PhO

SB-P23D6 32 Cyclohexyl 4-FC₆H₄CO 4-FC₆H₄0

SB-P23C2 22 Cyclohexyl PhCO PhO

Plate 24

At 5 μg/mL concentration

SB-P24A10 36 Cyclohexyl Cl₃CCO EtO

SB-P24F5 32 Cyclohexyl Et₂NCO PhS

Plate 25

At 5 μg/mL concentration

Plate 26

At 5 μg/mL concentration

SB-P26D9 65 Cyclohexyl 4-MeC₆H₄CO 4-FC₆H₄0

SB-P26E9 65 Cyclohexyl 4-MeC₆H₄CO BuS

SB-P26C11 63 Cyclohexyl Ph(CH₂)₂CO PhO

SB-P26D10 63 Cyclohexyl CH₂=CH(CH₂)₂CO 4-FC₆H₄0

SB-P26D8 62 Cyclohexyl 4-i-BuC₆H₄CO 4-FC₆H₄0

SB-P26H4 62 Cyclohexyl CH₃(CH₂)₂OCO PhCH₂S

SB-P26D5 62 Cyclohexyl PhCH₂OCO 4-FC₆H₄0

SB-P26G9 61 Cyclohexyl 4-MeC₆H₄CO 4-FC₆H₄S

SB-P26B11 57 Cyclohexyl Ph(CH₂)₂CO BuO

SB-P26A8 56 Cyclohexyl 4-i-BuC₆H₄CO EtO

SB-P26A9 56 Cyclohexyl 4-MeC₆H₄CO EtO

SB-P26C4 56 Cyclohexyl CH₃(CH₂)₂OCO PhO

SB-P26D4 55 Cyclohexyl CH₃(CH₂)₂OCO 4-FC₆H₄0

SB-P26G6 55 Cyclohexyl PhS0₂ 4-FC₆H₄S

SB-P26G8 55 Cyclohexyl 4-i-BuC₆H₄CO 4-FC₆H₄S

SB-P26C6 54 Cyclohexyl PhS0₂ PhO

SB-P26H10 54 Cyclohexyl CH₂=CH(CH₂)₂CO PhCH₂S

SB-P26H11 54 Cyclohexyl Ph(CH₂)₂CO PhCH₂S

SB-P26B4 53 Cyclohexyl CH₃(CH₂)₂OCO BuO

SB-P26B8 52 Cyclohexyl 4-i-BuC₆H₄CO BuO

SB-P26D7 52 Cyclohexyl 4-ClC₆H₄CO 4-FC₆H₄0

SB-P26E10 51 Cyclohexyl CH₂=CH(CH₂)₂CO BuS

SB-P26C8 51 Cyclohexyl 4-i-BuC₆H₄CO PhO

SB-P26A10 51 Cyclohexyl CH₂=CH(CH₂)₂CO EtO

SB-P26B9 50 Cyclohexyl 4-MeC₆H₄CO BuO

SB-P26D6 50 Cyclohexyl PhS0₂ 4-FC₆H₄0

SB-P26B6 49 Cyclohexyl PhS0₂ BuO

SB-P26E11 49 Cyclohexyl Ph(CH₂)₂CO BuS

SB-P26H6 49 Cyclohexyl PhS0₂ PhCH₂S

SB-P26C5 48 Cyclohexyl PhCH₂OCO PhO

SB-P26C10 47 Cyclohexyl CH₂=CH(CH₂)₂CO PhO

SB-P26H9 47 Cyclohexyl 4-MeC₆H₄CO PhCH₂S

SB-P26B10 46 Cyclohexyl CH₂=CH(CH₂)₂CO BuO

SB-P26G12 46 Cyclohexyl PhNHCO 4-FC₆H₄S

SB-P26H8 45 Cyclohexyl 4-i-BuC₆H₄CO PhCH₂S

SB-P26H5 43 Cyclohexyl PhCH₂OCO PhCH₂S

SB-P26E12 43 Cyclohexyl PhNHCO BuS

SB-P26A4 42 Cyclohexyl CH₃(CH₂)₂OCO EtO

SB-P26E8 42 Cyclohexyl 4-i-BuC₆H₄CO BuS

SB-P26E5 42 Cyclohexyl PhCH₂OCO BuS

SB-P26E4 37 Cyclohexyl CH₃(CH₂)₂OCO BuS SB-P26C12 34 Cyclohexyl PhNHCO PhO

SB-P26D12 30 Cyclohexyl PhNHCO 4-FC₆H₄0

SB-P26E6 30 Cyclohexyl PhS0₂ BuS

B. Certain 2,5,6- trisubstituted benzimidazoles were screened for their activity against Mtb H37Rv using the "Microplate Alamar Blue Assay (MABA)" in a 96-well format at 1 μg/mL concentration and 0.5 μg/mL concentration (single point assay in triplicates). Then, the growth inhibition was measured in percentage. The results are shown below.

At 0.5 μg/mL concentration

At 1.0 μg/mL concentration

C. MIC₅₀ values against Mtb were determined for several compounds using the microplate Alamar Blue assay (MABA). Stock solutions of the compounds were prepared in DMSO and were serially diluted 2-fold in 96-well microtiter plates, and each Mtb strain was added to each well to an OD600 of 0.005. Plates were incubated for 6 days at 37 °C. Alamar Blue (Invitrogen) was added to the plates, and the plates were incubated for an additional 24 h at 37 °C. Plates were monitored for color change, and MIC₅₀ was determined in triplicate. The results are shown below.

D. MIC₉₉ values against M. Smegmatis are shown below. MIC values were determined by using the microplate Alamar Blue assay (MABA) and these values were compared to MIC values found in the literature for certain drugs.

MIC99 values against M. Smegmatis were determined by using the microplate Alamar Blue assay (MABA).

APPENDIX

The diagram for the library

10 11 12

 Structures of some compounds showing activity

41

42

Claims

CLAIMS We claim:

1. A molecule having formula I

I

wherein:

R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰, NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(0)R⁵, S(0)₂R⁵, CH(O), C(0)OR⁶, C(0)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo;

R represents alkyl, cycloalkyl, or aryl;

R represents H, alkyl, cycloalkyl, or aryl;

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰;

R⁵ represents R⁶ or OR⁶;

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl;

Y represents C(X²)R⁴, S(0)R⁵, or S(0)₂R⁵;

X¹ represents O or S;

X² represents O, S, NH, or NR⁶; R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a heterocyclic alkyl or a heterocyclic aryl;

R 7' and R 8° may be combined to represent a carbocyclic alkyl; alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain; cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings; aryl groups are carbocyclic or heterocyclic; carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings; heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings; halo substituents are fluoro, chloro, bromo, or iodo; each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or

substituted with one or more substituent at any position; alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁵, S(0)₂R⁵, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl; cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl; aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl; and heterocyclic alkyl and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen and sulfur; and pharmaceutically acceptable salts thereof.

2. A molecule according to claim 1, wherein R¹ is alkyl, cycloalkyl, or aryl.

3. A molecule according to claim A 1, wherein R is cyclohexyl.

4. A molecule according to claim A, wherein X¹ is O.

5. A molecule according to claim A3, wherein R is alkyl.

6. A molecule according to claim A3, wherein R is aryl.

7. A molecule according to claim A, wherein R²X^X is BuS, PhS, 4-FC₆H₄S, PhCH₂S, 4-FC₆H₄0, EtO, BuO, or PhO.

8. A molecule according to claim A, wherein R is H.

9. A molecule according to claim A, wherein Y is C(X )R .

10. A molecule according to claim A7, wherein X¹ is O and R⁴ is alkyl, aryl, or OR⁶.

11. A molecule according to claim A, wherein Y is S(0)₂Ph.

12. A molecule according to claim A, wherein R is H and Y is 4-MeC₆H₄CO, 4- CNC₆H₄CO, PhCO, 4-PrC₆H₄CO, 4-MeOC₆H₄CH₂CO, rj-C₅HnCO, C₆H₅CH₂CO, 4- FC₆H₄CO, Cl₃CCO, C1CH₂C0, 2,4-F₂C₆H₃CO, CH₂=CHCH₂CO, 4-N0₂C₆H₄CO, Et₂NCO, CH₂=CHCH₂NHCO, Me₂NCS, N-i-BuNHCO, N-i-PrNHCO, N-(2,6- Me₂C₆H₃)NHCO, PhNHCO, CH₂=CH(CH₂)₂CO, PhCH₂OCO, 4-i-BuC₆H₄CO, PhS0₂, 4-ClC₆Fi CO, or Ph(CH₂)₂CO.

13. A method of treating a patient infected with Mycobacterium tuberculosis, the method comprising administering to the patient a compound of formula I

I

wherein:

R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰, NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(0)R⁵, S(0)₂R⁵, CH(O), C(0)OR⁶, C(0)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo;

R represents alkyl, cycloalkyl, or aryl;

R represents H, alkyl, cycloalkyl, or aryl;

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰;

R⁵ represents R⁶ or OR⁶;

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl;

Y represents C(X²)R⁴, S(0)R⁵, or S(0)₂R⁵;

X¹ represents O or S;

X² represents O, S, NH, or NR⁶;

R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a

heterocyclic alkyl or a heterocyclic aryl; R 7' and R 8° may be combined to represent a carbocyclic alkyl; alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain; cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings; aryl groups are carbocyclic or heterocyclic; carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings; heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings; halo substituents are fluoro, chloro, bromo, or iodo; each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or

substituted with one or more substituent at any position; alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁵, S(0)₂R⁵, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl; cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl; aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl; and heterocyclic alkyl and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen and sulfur; and pharmaceutically acceptable salts thereof.

14. A method of treating a patient infected with Mycobacterium avium compli method comprising administering to the patient a compound of formula I

I

wherein:

R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰, NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(0)R⁵, S(0)₂R⁵, CH(O), C(0)OR⁶, C(0)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo;

R represents alkyl, cycloalkyl, or aryl;

R represents H, alkyl, cycloalkyl, or aryl;

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰;

R⁵ represents R⁶ or OR⁶;

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl;

Y represents C(X²)R⁴, S(0)R⁵, or S(0)₂R⁵;

X¹ represents O or S;

X² represents O, S, NH, or NR⁶; R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a heterocyclic alkyl or a heterocyclic aryl;

R 7' and R 8° may be combined to represent a carbocyclic alkyl; alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain; cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings; aryl groups are carbocyclic or heterocyclic; carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings; heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings; halo substituents are fluoro, chloro, bromo, or iodo; each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or

substituted with one or more substituent at any position; alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁵, S(0)₂R⁵, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl; cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl; aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl; and heterocyclic alkyl and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen and sulfur; and pharmaceutically acceptable salts thereof.

15. A method of treating a patient infected with Mycobacterium avium, the method comprising administering to the patient a compound of formula I

I

wherein:

R¹ represents NH₂, NHR⁶, NR⁹R¹⁰, NR⁶C(0)NR⁹R¹⁰, NR⁶C(S)NR⁹R¹⁰, OH, OR⁶, SH, SR⁶, S(0)R⁵, S(0)₂R⁵, CH(O), C(0)OR⁶, C(0)R⁶, CH₂OH, CR⁷R⁸OH, CH₂OR⁶, CR⁷R⁸OR⁶, CH₂NH₂, CR⁷R⁸NH₂, CR⁷R⁸NR⁹R¹⁰, alkyl, cycloalkyl, aryl, or halo;

R represents alkyl, cycloalkyl, or aryl;

R represents H, alkyl, cycloalkyl, or aryl;

R⁴ represents H, R⁶, OR⁶, SR⁶, NH₂, NHR⁶, or NR⁹R¹⁰;

R⁵ represents R⁶ or OR⁶;

R⁶, R⁷, R⁸, R⁹, and R¹⁰ independently represent alkyl, cycloalkyl, or aryl;

Y represents C(X²)R⁴, S(0)R⁵, or S(0)₂R⁵;

X¹ represents O or S;

X² represents O, S, NH, or NR⁶; R³ and Y, and R⁹ and R¹⁰ independently, may be combined to represent a heterocyclic alkyl or a heterocyclic aryl;

R 7' and R 8° may be combined to represent a carbocyclic alkyl; alkyl groups are branched or unbranched, saturated or unsaturated, and have 1-18 carbon atoms in their longest chain; cycloalkyl groups are carbocyclic or heterocyclic, fused or unfused, non-aromatic ring systems having a total of 5-16 ring members including substituent rings; aryl groups are carbocyclic or heterocyclic; carbocyclic aryl groups are fused or unfused ring systems having a total of 6-16 ring members including substituent rings; heterocyclic aryl groups are fused or unfused ring systems having a total of 5-16 ring members including substituent rings; halo substituents are fluoro, chloro, bromo, or iodo; each alkyl, cycloalkyl, and aryl, independently, may be unsubstituted or

substituted with one or more substituent at any position; alkyl substituents are halo, hydroxyl, OR⁶, SR⁶, S(0)R⁵, S(0)₂R⁵, NH₂, NHR⁶, NR⁹R¹⁰, cycloalkyl, or aryl; cycloalkyl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, alkyl, cycloalkyl, or aryl; aryl substituents are halo, hydroxyl, OR⁶, SR⁶, NH₂, NHR⁶, NR⁹R¹⁰, CN, alkyl, cycloalkyl, aryl, nitro, or carboxyl; and heterocyclic alkyl and heterocyclic aryl have at least one heteroatom selected from oxygen, nitrogen and sulfur; and pharmaceutically acceptable salts thereof.

16. A benzimidazole having the formula: