WO2013142326A1

WO2013142326A1 - 5-carbonylamino-/(sulfonamido-) substituted benz imidazoles and use thereof treatment of tuberculosis

Info

Publication number: WO2013142326A1
Application number: PCT/US2013/032083
Authority: WO
Inventors: Iwao Ojima
Original assignee: The Research Foundation Of State University Of New York
Priority date: 2012-03-23
Filing date: 2013-03-15
Publication date: 2013-09-26

Abstract

The present invention discloses novel 5-/6- or 5-/7- substituded benzimidazoles and pharmaceutically acceptable salts thereof. Another aspect of the invention relates to their use in treating a patient infected by Mycobacterium tuberculosis or Francisella tularensis.

Description

5 - CARBONYLAMINO - / ( SULFONAMIDO - ) SUBSTITUTED BENZ IMIDAZOLES AND USE THEREOF IN

TREATMENT OF TUBERCULOSIS

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

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Nos. 61/614,898, filed March 23, 2012; 61/614,908, filed March 23, 2012; 61/691,009, filed August 20, 2012; and 61/767,891, filed February 22, 2013, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

There is a need for additional ways of treating infectious diseases, especially those associated with, for example, Mycobacterium tuberculosis or Francisella tularensis.

Tuberculosis (TB) was one of the first infectious diseases to be identified as associated with the aforesaid bacteria. 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 A. Krunner et al, "Drug resistant tuberculosis in Estonia", Int. J. Tuberc. Lung Dis. (1998) Vol. 2, pages 130-133. 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 the 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 Francisella tularensis, the bacteria which causes tularemia. Tularemia is primarily enzootic; however, in humans, it causes lesions and flu-like symptoms. Finding new methods of treating F. tularensis is of great importance because it is one of the most pathogenic microorganisms presently known. As such, it is currently listed as a category A select agent by the Centers for Disease Control and

Prevention because of its potential as a bioterrorism agent.

There is also need for novel Filamenting temperature sensitive mutant Z ("FtsZ") inhibitors as well as antibacterial agents that have efficacy for the treatment of infectious disorders such as, for example, tuberculosis.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a compound having the formula:







or a pharmaceutically acceptable salt thereof. In another embodiment, the invention realtes to a compound of formula I:

wherein:

R represents

R represent

R represents

or a pharmaceutically acceptable salt thereof. In yet another embodiment, the invention realtes to a compound of formula II:

wherein:

R⁴ represents

5 represents

6 represents

or a pharmaceutically acceptable salt thereof. In another embodiment, the invention relates to a benzimidazole intermediate having the formula:

or a pharmaceutically acceptable salt thereof.

The invention also relates to methods of treating a patient infected by Mycobacterium tuberculosis or Francisella tularensis by administering to the patient a benzimidazole of the invention or a pharmaceutically acceptable salt thereof.

DETAILED DESCRIPTION

The invention relates to the novel benzimidazole derivatives shown in Table I and their intermediates shown in Table II, and the benzimidazole derivatives of formula I and II. These benzimidazole derivatives or pharmaceutically acceptable salts thereof can be used to treat a pateint infected by Mycobacterium tuberculosis or Francis ella tularensis. In fact, the benzimidazole derivates in Table I have unexpectedly high activities against Mycobacterium tuberculosis and/or Francisella tularensis.

The benzimidazole intermediates in Table II are also novel.

Table II

Formula I is shown below:

R² represents

R³ represents

Formula II is show below:

4 represents

R⁵ represents

R represents

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. 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 other group.

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.

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 the invention. Scheme 1 shows an example of a synthesis that yields 2,5,6-trisubstituted benzimidazoles of the invention. For example, the compounds of the invention may be made using polymer- assisted solution-phase (PASP) synthesis. PASP is a parallel synthesis method for creation of a trisubstituted benzimidazoles (BAZ-1) library using 2,4-dinitro-5-fluoroaniline (1) as the starting material.

Scheme 1

The first step involves the nucleophilic substitution of compound 1 with a secondary amine in the presence of Ν,Ν-diisopropylethylamine. The reaction produces compound 2 in high yields and purity at room temperature.

Then the acylation of the free amino group of compound 2 with an acyl or aroyl chloride takes place. This reaction occurs under reflux conditions using pyridine as the solvent.

Subsequently, reduction of the aromatic m-dinitro groups of compound 3 using HCOO^" NH₄ ⁺ and Pd-C generates diamine compound 4. The benzimidazole ring is formed through acid- catalyzed dehydration.

The free aromatic amino group of compound 5 is modified in different ways. To introduce diversity at the -C(X)-R⁵ position, anhydride, acyl chloride, sulfonyl chloride, and isocyanate are used as modifying agents. The modification of the aromatic amine moiety takes place smoothly in dry dichloromethane and all excess acylating reagents are scavenged by commercially available aminomethylated polystyrene resin (from nova-biochem) to give the desired product 6 in 80-95% yield.

The synthesis of specific compounds of the invention is shown below in Scheme 2.

Scheme 2

Scheme 3 shows the synthesis of 2,5,7-trisubstituted benzimidazoles of the invention.

Scheme 3

Commercially available 5-amino-2,4-dinitrobenzamide (6) was hydrolyzed to give 4- amino-3,5-dinitrobenzoic acid, which was converted to acyl chloride 7. The reaction of 7 with sodium azide afforded the corresponding acyl azide, which was subjected to the Curtius rearrangement to give the corresponding isocyanate. The isocyanate was treated with corresponding alcohol to form intermediate 8a-8b as bright yellow solid in 85 % yield. The reduction of 8a-8b followed by cyclocondensation with the bisulfite salts of different aldehydes afforded 7-aminobenzimidazoles 9a-9e. The derivatization of the 7-aminobenzimidazole using various acyl chloride, chloroformates or the corresponding N-hydroxysuccinimide esters generated the desired hit compounds lOa-lOi in analytically pure form in 41-53% yields.

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 non-toxic 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.

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 Table 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 Table I or a pharmaceutically acceptable salt thereof as used herein is any amount effective to treat a patient infected by Mtb, Mycobacterium avium complex, or Francisella tularensis. 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 is not to be in any way limited by the examples set forth herein.

EXAMPLE 1

5-Butoxycarbonylamino-2-cvclohexyl-6 -N, N-dimethylamino- lH-benzo \d] imidazole (SB - P17G-C2)

A solution of 5-amino-2-cyclohexyl-6-(N,N-dimethylamino)-lH-benzo[d]imidazole (100 mg, 0.39 mmol) and Ι,Γ-carbonyldiimidazole (69 mg, 0.43 mmol) in dichloromethane (2 mL) was stirred under reflux conditions for 4 hours. After all the starting material had disappeared, n- butanol (71 uL, 0.77 mmol) was added and the reaction was refluxed for additional 12 hours. After the completion of the reaction, the reaction mixture was concentrated. The crude was purified by flash chromatography on silica gel (gradient: 30-70% ethyl acetate /hexanes) to give 5-butoxycarbonylamino-2-cyclohexyl-6-N,N-dimethylamino-lH-benzo[d]imidazole as a white solid (111 mg, 79 % yield); 1H NMR (300 MHz, CDC13) δ 0.97 (t, 3 H, / = 7.2 Hz), 1.25- 1.48 (m, 5 H), 1.56-1.76 (m, 5 H), 1.85 (m, 2 H, / = 9.6 Hz), 2.09 (m, 2 H, /= 12.9 Hz), 2.64 (s, 6 H), 2.83 (m, 1H), 4.20 (t, 2 H, / = 6.9 Hz), 7.53 (s, 1 H), 8.20 (s, 2 H), 9.17 (s, 1 H).

The same procedure was followed for the synthesis of SB-P17G-C4, SB-P17G-C8, SB-P1G- C12, SB-P3G2-OL1, SB-P6B-C12, and SB-OL1-C8.

EXAMPLE 2

5-Benzyloxycarbonylamino-2-cvclohexyl-6-N,N-dimethylamino-lH-benzord1imidazole (SB-

P17G-C4)

White solid, 82% yield; 1H NMR (300 MHz, CDC13) δ 1.26 (m, 3 H), 1.51-1.76 (m, 5 H), 1.99 - 2.03 (m, 2 H, / = 12 Hz), 2.14 (s, 1 H), 2.58 (s, 6 H), 2.84 (m, 1 H), 5.22 (s, 2 H), 7.33 -7.49 (m, 6 H), 8.28 ( 1 H), 10.25 (s, 1 H)

EXAMPLE 3

2-Cvclohexyl-5-ethoxyethoxycarbonylamino-6-N,N-dimethylamino-lH-benzord1imidazole (SB-P17G-C8)

White solid, 86% yield; 1HNMR (300 MHz, CDC1₃) δ 1.22 - 1.39 (m, 8 H), 1.61 -1.85 (m, 5 H), 2.10 (m, 2 H, / = 12 Hz ), 2.60 (s, 6 H), 2.88 (m, 1 H), 3.57 (q, 2 H, / = 6.9 Hz), 3.71 (t, 2 H, / = 4.5 Hz), 4.35 (t, 2 H, / = 4.5 Hz), 7.50 ( s, 1 H), 8.23 (s, 1 H), 8.32 (s, 1 H).

EXAMPLE 4

2-Cvclohexyl-6-N,N-dimethylamino-5-(4-dimethoxy)benzamido-lH-benzord1imidazole (SB- P17G-A16)

To a solution of 5-amino-2-cyclohexyl-6-(N,N-dimethylamino)-lH-benzo[d]imidazole (100 mg, 0.39 mmol) in dichloromethane (2 niL) was added triethylamine (60 μί, 0.43 mmol). To this, 4- methoxybenzoyl chloride (53.3 μί, 0.39 mmol) was added dropwise at 0 °C and reacted at room temperature. After completion of the reaction, the reaction mixture was diluted with ethyl acetate and transferred to a separatory funnel. The organic layer was washed with saturated sodium bicarbonate solution, followed by water and finally brine. The organic layer was dried over magnesium sulfate. The crude product was purified by flash chromatography on silica gel (gradient: 30-70% ethyl acetate /hexanes) to obtain the product which was then treated with activated charcoal to yield the product as a white solid (130 mg, 86%). 1H NMR (300 MHz, CDC13) δ 1.17 (m, 3 H), 1.57-1.75 (m, 5 H), 2.01 (m, 2 H, / = 12.9 Hz), 2.73-2.77 (s+m, 7 H), 3.90 (s, 3 H), 7.04(d, 1 H, / = 9 Hz), 7.60 ( s, 1H), 7.93 (d, 2 H, / = 9 Hz), 8.86 (s, 1 H), 9.82 (s, 1 H).

EXAMPLE 5

5-Benzamido-2-cvclohexyl-6-N, -dimethylamino-lH-benzord1imidazole (SB-P17G-A15)

To a solution of 5-amino-2-cyclohexyl-6-(N,N-dimethylamino)-lH-benzo[d]imidazole (70 mg, 0.27 mmoles) in 2 mL of dichloromethane was added l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (57.5 mg, 0.3 mmol) (EDC) and benzoic acid (36.4 mg, 0.30 mmol). 4- Dimethylaminopyridine (DMAP) (36.4, 0.30 mmol) was added to the above reaction mixture and the reaction was refluxed overnight. After completion of the reaction as per TLC, the reaction mixture was diluted with dichloromethane followed by the addition of triethylamine. The reaction mixture was transferred to a separatory funnel and the organic layer was washed with water, sodium bicarbonate and finally brine. The organic layer was dried over magnesium sulfate and concentrated using rotary evaporator. The crude product was purified by flash chromatography (column was packed with hexane, 30% ethyl acetate was used with gradual increase to 50% ethyl acetate) to obtain the product as a white solid (83 mg, 85% yield). 1H

NMR (300 MHz, CDC13) δ 1.25 (m, 3 H), 1.61-1.71 (m, 5 H), 2.03 (m, 2 H, / = 12.9 Hz), 2.74 (s, 6 H), 2.85 (m, 1 H),7.58 (m, 3 H), 7.67 (s, 1 H), 7.95 (d , 1H, / = 6.6 Hz), 8.91 (s, 1 H), 9.88 (s, 1 H).

The same procedure was followed for the synthesis of SB-PIG-Al 1, SB-P1G-A12, and SB-P1G- A10.

EXAMPLE 6

2-Cvclohexyl-6-N,N-diethylamino-5-trifluoroethoxycarbonylamino-lH-benzord1imidazole (SB-PI G-C12)

White solid, 42 % yield; 1HNMR (300 MHz, CDC1₃) δ 0.89 (t, 6 H, / = 6.9 Hz), 1.21-1.28 (m, 4 H), 1.59-1.65 (m, 4 H), 2.08 (m, 2 H), 2.93 (m, 5H), 2.82 (m, 1 H), 4.56 (m, 2 H), 7.58 (s, 1 H), 8.31 (s, 1 H), 8.78 (s, 1 H).

EXAMPLE 7

5-Butoxycarbonylamino-2-cvclohexyl-6-N,N-dipropylamino-lH-benzord1imidazole (SB- P3G2-OL1)

White solid, 33 % yield; 1H NMR (300 MHz, CDC13) δ 0.86 (t, 6 H, / = 7.2 Hz), 0.96 (t, 3 H, / = 7.5 Hz), 1.34-1.46 (m, 7 H), 1.57-1.72 (m, 7 H), 1.81 (m, 2 H, / = 10 Hz), 2.07 (m, 2 H, / = 10 Hz), 2.83 (m, 5 H), 4.17 (t, 2 H, / = 6.5 Hz), 7.57 (s, 1 H), 8.19 (s, 1 H), 8.51 (s, 1 H) EXAMPLE 8

2-Cvclohexyl-6-N,N-diethylamino-5-(4-N,N-dimethylamino)benzamido-lH- benzordlimidazole (SB-PI G-

Yellow solid, 65 % yield; 1H NMR (300 MHz, CDC13) δ 0.94 (t, 6 H, / = 7.2 Hz), 1.12-1.28 (m, 3 H), 1.54-1.71 (m, 5 H), 2.07 (m, 2 H, / = 10 Hz), 3.25 (m, 5 H), 3.00 (s, 6 H), 6.80 (d, 2 H, / = 9 Hz), 7.75 (s, 1 H), 7.85 (d, 2 H, / = 9 Hz), 9.01 (s, 1 H), 10.2 (s, 1 H).

EXAMPLE 9

2-Cvclohexyl-6-pyrrolidino-5-trifluoroethoxycarbonylamino-lH-benzord1imidazole (SB- P6B-C12)

White solid, 69 % yield; 1HNMR (500 MHz, CDC1₃) δ 1.25-1.39 (m, 3 H), 1.65-1.84 (m, 5 H), 1.97 (m, 4 H), 2.13 (s, 2 H), 2.98 (m, 5H), 4.58 (m, 2 H, J = 1.4 Hz), 7.51 (s, 1 H), 8.19 (s, 1 H).

EXAMPLE 10

2-Cvclohexyl-6-N,N-dipropylamino-5-ethoxyethoxycarbonylamino-lH-benzord1imidazole (SB-OL1-C8)

White solid, 71 % yield; 1HNMR (300 MHz, CDC1₃) δ 0.83 (t, 6H, J = 12 Hz), 1.23 (t, 3H, / = 6.9 Hz), 1.30-1.43 (m, 7 H), 1.61-1.81 (m, 5 H), 2.81 (m, 5 H), 3.55 (m, 2H, / = 6.9 Hz), 3.70 (t, 2 H, / = 4.5 Hz), 4.34 (t, 2 H, / = 4.5 Hz), 7.53 ( s, 1 H), 8.18 (s, 1 H), 8.63 (s, 1 H).

EXAMPLE 11 2-Cvclohexyl-6-N,N-diethylamino-5-(pyrazine-2 arboxamido)-lH-benzord1imidazole (SB-

P1G-A12)

White solid, 64 % yield; 1H NMR (300 MHz, CDC13) δ 0.94 (t, 6 H, / = 7.2 Hz), 1.16-1.39 (m, 3 H), 1.61-1.82 (m, 5 H), 2.11 (m, 2 H, / = 10 Hz), 3.00 (m, 5 H), 7.62 (s, 1 H), 8.66 (d, 1 Η, / = 5.1 Hz), 8.79 (d, 1 H, / = 5.1 Hz), 8.91 (s, 1 H), 9.51 (s, 1 H), 11.54 (s, 1 H).

EXAMPLE 12

2-Cvclohexyl-6-N,N-diethylamino-5-(3,5-dimethoxy)benzamido-lH-benzord1imidazole (SB- P1G-A10)

White solid, 72 % yield; 1H NMR (300 MHz, CDC13) δ 0.94 (t, 6 H, / = 7.2 Hz), 1.18-1.27 (m, 3 H), 1.6-1.74 (m, 5 H), 2.07 (m, 2 H, / = 10 Hz), 2.77 (m, 1 H), 3.00 (q, 4 H, / = 7.2 Hz), 3.87 (s, 6 H), 6.65 (s, 1 H), 7.07 (d, 2 H), 7.60 (s, 1 H), 9.93 (s, 1 H), 10.3 (s, 1 H). EXAMPLE 13

Procedure for the determination of the minimum inhibitory concentration (MIC):

MIC values were determined using the microplate dilution method reported by R. A. Slayden et al, "The role ofKasA and KasB in the biosynthesis of meromy colic acids and isoniazid resistance in Mycobacterium tuberculosis", Tuberculosis (Edinburgh, 2002) Vol. 82, pagesl49-60.

Bacteria were cultivated in liquid medium to an optical density of -0.4 at 600 nm. The bacterial cultures were then prepared for testing by diluting 1: 100 in liquid medium. A total of 50 μL· of each culture was added to each well of a 96-well optical plate. Analogs were prepared at 60 μΜ in 100% DMSO. Compound stock solutions were diluted 1:2 in liquid medium and then distributed in the plate as 2-fold serial dilutions to achieve a concentration range of 200-0.2 mg/mL in a total final volume of 100 μL· The plates were incubated at 37 °C and evaluated for the presence of bacterial growth or non-growth by optical density using an inverted plate reading method. The MIC99 was determined to be the lowest concentration of compound that inhibited bacterial growth. MIC values shown below in Table III represent measurements performed independently in triplicate.

MIC₅₀ values indicated below:

EXAMPLE 14

Compounds with hits against Mtb at the 5 μg/mL cut-off:

EXAMPLE 15

Antibacterial activity against F. tularensis F. tularensis LVS provided by Dr. J. Petersen (Centers for Disease Control, Fort Collins,

CO) was grown to an Οϋόοο of -0.6, frozen at -80°C in 10% glycerol and used as a standard bacterial stock for these studies. For each evaluation bacteria were prepared fresh by growth from the standard stocks on Cystine Heart Agar supplemented with 2% hemoglobin (BD, Franklin Lakes, NJ) grown at 37°C for 48-72 h. Bacteria recovered from the Cystine Heart Agar-Hemoglobin (CHAB) plates were used to inoculate 50 mL Mueller-Hinton broth (BD) supplemented with 0.025% ferric pyrophosphate (Sigma- Aldrich), 2% IsoVitaleX (BD), 0.1% glucose (Sigma- Aldrich). Broth cultures were then incubated for 18hrs at 37°C passed 1:20 and incubated for an additional 8 h at 37°C. Bacteria were then diluted to a concentration of lxlO⁷ colony forming units (CFU)/mL in modified Mueller-Hinton (MMH) broth and 50 μΐ_^ added to each well for each test plate. Trisubstituted benzimidazole compound library was screened in a percent inhibition high-throughput fashion against F. tularensis LVS in a 96-well plate format. All compounds were diluted in MMH broth to concentrations of 10, 2 and 0.4 μg/mL in a 50μί volume/well. For MIC determination, compounds were added to the 96-well plate starting at 512 μg/mL in the first column and serially diluted 1:2 to column 12 for a final concentration of 0.25 in MMH broth. MIC plates were incubated at 37°C for 18 h at which time 10 of Alamar Blue (Invitrogen, Carlsbad, California) was added to each well and plates were incubated for an additional 4 h. Reduction of Alamar Blue was determined by absorbance readings at wavelengths of 570 and 600 nm using a microplate reader (Biotek, Winooski, VT). Percent growth reduction was calculated and MIC₉₀ values were determined by plotting the percentage inhibition calculated from spectrophotometric readings over the drug concentration series.

Bacterial growth and MIC was confirmed by optical density. Non-linear regression analysis was performed on % growth inhibition curves to determine MIC₉₀ values. Structures of trisubstituted benzimidazoles against F. tularensis at 1 μg/ml with > 90% growth inhibition

EXAMPLE 16

Cytotoxicity Assay

The cytotoxicity of the compounds was tested against Vero cells. Epithelial cells from the kidneys of the African Green Monkey were used to start the Vero cell line. Vero cells were grown in L15 media without C0₂. Serial 2-fold dilutions of the drugs were prepared (200 μg/mL to 50 μg/mL) in triplicate using 96-well microtiter plates. The cells were added to the plates to a final concentration of 1.25 x 10⁵/well in media containing Resazurin for a final concentration 25 μg/mL. The plates were incubated for 3 days at 37 °C. The LD was the lowest drug concentration that inhibited cell growth and the resazurin was not reduced.

Claims

CLAIMS We claim:

1. A compound having the formula:

2. A molecule of formula I

wherein:

R represents

R re resents

R represents

or a pharmaceutically acceptable salt thereof.

3. A molecule according to claim 2, wherein R represents

4. A molecule according to claim 2, wherein R represents

5. A molecule having the formula II

wherein:

R⁴ represents

R⁵ represents

R⁶ represents

or a pharmaceutically acceptable salt thereof.

6. A benzimidazole intermediate having the formula:

or a pharmaceutically acceptable salt thereof.

7. A method of treating a patient infected with Mycobacterium tuberculosis, the method comprising administering to the patient a compound having the formula:

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

wherein:

R represents

R re resents

R represents

or a pharmaceutically acceptable salt thereof.

9. A method of treating a patient infected with Mycobacterium tuberculosis, the method comprising administering to the patient a compound having formula II:

wherein:

R⁴ represents

R⁵ represents

R⁶ represents

rmaceutically acceptable salt thereof.

10. A method of treating a patient infected with Francisella tularensis, the method com rising administering to the patient a compound having the formula:

11. A method of treating a patient infected with Francisella tularensis, the method comprising administering to the patient a com ound having formula I:

2 represents

R³ represents

or a pharmaceutically acceptable salt thereof.

12. A method of treating a patient infected with Francisella tularensis, the method comprising administering to the patient a compound having formula II:

wherein:

R⁴ represents

R⁵ represents

6 represents

or a pharmaceutically acceptable salt thereof.