Patent Grant
8202910
The present invention relates to methods and compositions for treating infectious disease and disease caused by microorganisms, particularly tuberculosis. The present invention also relates to methods and compositions having improved anti-mycobacterial activity, namely compositions comprising novel substituted ethylene diamine compounds. In certain embodiments, the present invention comprises compositions comprising novel substituted ethylene diamine compounds further comprising antitubercular agents such as rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol.
Mycobacterial infections often manifest as diseases such as tuberculosis. Human infections caused by mycobacteria have been widespread since ancient times, and tuberculosis remains a leading cause of death today. Although the incidence of the disease declined, in parallel with advancing standards of living, since the mid-nineteenth century, mycobacterial diseases still constitute a leading cause of morbidity and mortality in countries with limited medical resources. Additionally, mycobacterial diseases can cause overwhelming, disseminated disease in immunocompromised patients. In spite of the efforts of numerous health organizations worldwide, the eradication of mycobacterial diseases has never been achieved, nor is eradication imminent. Nearly one third of the world's population is infected with mycobacterium tuberculosis complex, commonly referred to as tuberculosis (TB), with approximately 8 million new cases, and two to three million deaths attributable to TB yearly. Tuberculosis (TB) is the cause of the largest number of human deaths attributable to a single etiologic agent (see Dye et al., J. Am. Med. Association, 282, 677-686, (1999); and 2000 WHO/OMS Press Release).
After decades of decline, TB is now on the rise. In the United States, up to 10 million individuals are believed to be infected. Almost 28,000 new cases were reported in 1990, constituting a 9.4 percent increase over 1989. A sixteen percent increase in TB cases was observed from 1985 to 1990. Overcrowded living conditions and shared air spaces are especially conducive to the spread of TB, contributing to the increase in instances that have been observed among prison inmates, and among the homeless in larger U.S. cities. Approximately half of all patients with “Acquired Immune Deficiency Syndrome” (AIDS) will acquire a mycobacterial infection, with TB being an especially devastating complication. AIDS patients are at higher risks of developing clinical TB, and anti-TB treatment seems to be less effective than in non-AIDS patients. Consequently, the infection often progresses to a fatal disseminated disease.
Mycobacteria other than M. tuberculosis are increasingly found in opportunistic infections that plague the AIDS patient. Organisms from the M. avium-intracellulare complex (MAC), especially serotypes four and eight, account for 68% of the mycobacterial isolates from AIDS patients. Enormous numbers of MAC are found (up to 1010 acid-fast bacilli per gram of tissue), and consequently, the prognosis for the infected AIDS patient is poor.
The World Health Organization (WHO) continues to encourage the battle against TB, recommending prevention initiatives such as the “Expanded Program on Immunization” (EPI), and therapeutic compliance initiatives such as “Directly Observed Treatment Short-Course” (DOTS). For the eradication of TB, diagnosis, treatment, and prevention are equally important. Rapid detection of active TB patients will lead to early treatment by which about 90% cure is expected. Therefore, early diagnosis is critical for the battle against TB. In addition, therapeutic compliance will ensure not only elimination of infection, but also reduction in the emergence of drug-resistance strains.
The emergence of drug-resistant M. tuberculosis is an extremely disturbing phenomenon. The rate of new TB cases proven resistant to at least one standard drug increased from 10 percent in the early 1980's to 23 percent in 1991. Compliance with therapeutic regimens, therefore, is also a crucial component in efforts to eliminate TB and prevent the emergence of drug resistant strains. Equally important is the development of new therapeutic agents that are effective as vaccines, and as treatments, for disease caused by drug resistant strains of mycobacteria.
Multidrug-resistant tuberculosis (MDR TB) is a form of tuberculosis that is resistant to two or more of the primary drugs used for the treatment of tuberculosis. Resistance to one or several forms of treatment occurs when bacteria develop the ability to withstand antibiotic attack and relay that ability to their progeny. Since an entire strain of bacteria inherit this capacity to resist the effects of various treatments, resistance can spread from one person to another.
The World Health Organization estimates that up to 50 million persons worldwide may be infected with drug resistant strains of tuberculosis. Also, 300,000 new cases of MDR-TB are diagnosed around the world each year and 79 percent of the MDR-TB cases now show resistance to three or more drugs routinely used to treat tuberculosis.
In 2003, the Centre for Disease control (CDC) reported that 7.7 percent of tuberculosis cases in the U.S. were resistant to isoniazid, a first line drug used to treat Tuberculosis. The CDC also reported that 1.3 percent of tuberculosis cases in the U.S. were resistant to both isoniazid and rifampin. Rifampin is the drug most commonly used with isoniazid.
Clearly, the possibility of drug resistant strains of tuberculosis that develop during or before treatment are a major concern to health organizations and heath care practitioners. Drugs used in the treatment of tuberculosis include, but are not limited to, Ethambutol, Pyrazinamide, Streptomycin, Isoniazid, Moxifloxacin and Rifampin. The exact course and duration of treatment can be tailored to a specific individual, however several strategies are well known to those skilled in the art.
Although over 37 species of mycobacteria have been identified, more than 95% of all human infections are caused by six species of mycobacteria: M. tuberculosis, M. avium intracellulare, M. kansasii, M. fortuitum, M. chelonae, and M. leprae. The most prevalent mycobacterial disease in humans is tuberculosis (TB) which is predominantly caused by mycobacterial species comprising M. tuberculosis, M. bovis, or M. africanum (Merck Manual 1992). Infection is typically initiated by the inhalation of infectious particles which are able to reach the terminal pathways in lungs. Following engulfment by alveolar macrophages, the bacilli are able to replicate freely, with eventual destruction of the phagocytic cells. A cascade effect ensues wherein destruction of the phagocytic cells causes additional macrophages and lymphocytes to migrate to the site of infection, where they too are ultimately eliminated. The disease is further disseminated during the initial stages by the infected macrophages which travel to local lymph nodes, as well as into the blood stream and other tissues such as the bone marrow, spleen, kidneys, bone and central nervous system. (See Murray et al. Medical Microbiology, The C.V. Mosby Company 219-230 (1990)).
There is still no clear understanding of the factors which contribute to the virulence of mycobacteria. Many investigators have implicated lipids of the cell wall and bacterial surface as contributors to colony morphology and virulence. Evidence suggests that C-mycosides, on the surface of certain mycobacterial cells, are important in facilitating survival of the organism within macrophages. Trehalose 6,6′ dimycolate, a cord factor, has been implicated for other mycobacteria.
The interrelationship of colony morphology and virulence is particularly pronounced in M. avium. M. avium bacilli occur in several distinct colony forms. Bacilli which grow as transparent, or rough, colonies on conventional laboratory media are multiplicable within macrophages in tissue culture, are virulent when injected into susceptible mice, and are resistant to antibiotics. Rough or transparent bacilli, which are maintained on laboratory culture media, often spontaneously assume an opaque R colony morphology, at which time they are not multiplicable in macrophages, are avirulent in mice, and are highly susceptible to antibiotics. The differences in colony morphology between the transparent, rough and opaque strains of M. avium are almost certainly due to the presence of a glycolipid coating on the surface of transparent and rough organisms which acts as a protective capsule. This capsule, or coating, is composed primarily of C-mycosides which apparently shield the virulent M. avium organisms from lysosomal enzymes and antibiotics. By contrast, the non-virulent opaque forms of M. avium have very little C-mycoside on their surface. Both the resistance to antibiotics and the resistance to killing by macrophages have been attributed to the glycolipid barrier on the surface of M. avium.
Diagnosis of mycobacterial infection is confirmed by the isolation and identification of the pathogen, although conventional diagnosis is based on sputum smears, chest X-ray examination (CXR), and clinical symptoms. Isolation of mycobacteria on a medium takes as long as four to eight weeks. Species identification takes a further two weeks. There are several other techniques for detecting mycobacteria such as the polymerase chain reaction (PCR), mycobacterium tuberculosis direct test, or amplified mycobacterium tuberculosis direct test (MTD), and detection assays that utilize radioactive labels.
One diagnostic test that is widely used for detecting infections caused by M. tuberculosis is the tuberculin skin test. Although numerous versions of the skin test are available, typically one of two preparations of tuberculin antigens are used: old tuberculin (OT), or purified protein derivative (PPD). The antigen preparation is either injected into the skin intradermally, or is topically applied and is then invasively transported into the skin with the use of a multiprong inoculator (Tine test). Several problems exist with the skin test diagnosis method. For example, the Tine test is not generally recommended because the amount of antigen injected into the intradermal layer cannot be accurately controlled. (See Murray et al. Medical Microbiology, The C.V. Mosby Company 219-230 (1990)).
Although the tuberculin skin tests are widely used, they typically require two to three days to generate results, and many times, the results are inaccurate since false positives are sometimes seen in subjects who have been exposed to mycobacteria, but are healthy. In addition, instances of mis-diagnosis are frequent since a positive result is observed not only in active TB patients, but also in persons vaccinated with Bacille Calmette-Guerin (BCG), and those who had been infected with mycobacteria, but have not developed the disease. It is hard therefore, to distinguish active TB patients from the others, such as household TB contacts, by the tuberculin skin test. Additionally, the tuberculin test often produces a cross-reaction in those individuals who were infected with mycobacteria other than M. tuberculosis (MOTT). Therefore, diagnosis using the skin tests currently available is frequently subject to error and inaccuracies.
The standard treatment for tuberculosis caused by drug-sensitive organisms is a six-month regimen consisting of four drugs given for two months, followed by two drugs given for four months. The two most important drugs, given throughout the six-month course of therapy, are isoniazid and rifampin. Although the regimen is relatively simple, its administration is quite complicated. Daily ingestion of eight or nine pills is often required during the first phase of therapy; a daunting and confusing prospect. Even severely ill patients are often symptom free within a few weeks, and nearly all appear to be cured within a few months. If the treatment is not continued to completion, however, the patient may experience a relapse, and the relapse rate for patients who do not continue treatment to completion is high. A variety of forms of patient-centered care are used to promote adherence with therapy. The most effective way of ensuring that patients are taking their medication is to use directly observed therapy, which involves having a member of the health care team observe the patient take each dose of each drug. Directly observed therapy can be provided in the clinic, the patient's residence, or any mutually agreed upon site. Nearly all patients who have tuberculosis caused by drug-sensitive organisms, and who complete therapy will be cured, and the risk of relapse is very low (“Ending Neglect: The Elimination of Tuberculosis in the United States” ed. L. Geiter Committee on the Elimination of Tuberculosis in the United States Division of Health Promotion and Disease Prevention, Institute of Medicine. Unpublished.)
Although the FDA approved a medication that combines the three main drugs (isoniazid, rifampin, and pyrazinamide) used to treat tuberculosis into one pill. Thereby reducing the number of pills a patient has to take each day and making it impossible for the patient to take only one of the three medications, a common path to the development of MDR-TB, there is still a need in the art to treat tuberculosis, especially in those cases wherein the tuberculosis strain is drug resistant.
What is needed are effective therapeutic regimens that include improved vaccination and treatment protocols. Currently available therapeutics are no longer consistently effective as a result of the problems with treatment compliance, and these compliance problems contribute to the development of drug resistant mycobacterial strains.
Furthermore, there is a need in the art for novel compositions and methods that are effective against infectious disease. More particularly, there is a need for novel compositions and methods for the effective treatment of Mycobacterial disease.
Ethambutol (EMB) is a widely used antibiotic for the treatment of TB, with over 300 million doses delivered for tuberculosis therapy in 1988.
Ethambutol, developed by Lederle Laboratories in the 1950s, has low toxicity and is a good pharmacokinetic. However, ethambutol has a relatively high Minimum Inhibition Concentration (MIC) of about 5 μg/ml, and can cause optic neuritis. Thus, there is an increasing need for new, and more effective, therapeutic compositions (See for example, U.S. Pat. Nos. 3,176,040, 4,262,122; 4,006,234; 3,931,157; 3,931,152; U.S. Re. 29,358; and Häusler et al., Bioorganic & Medicinal Chemistry Letters 11 (2001) 1679-1681). In the decoder years since the discovery of the beneficial effects of ethambutol, few pharmacological advances in TB treatment have been developed. Moreover, with the combined emergence of drug resistant strains, and the more prevalent spread of mycobacterial disease, it is becoming seriously apparent that new therapeutic compositions are crucial in the fight against tuberculosis.
Clearly effective therapeutic regimens that include improved vaccination and treatment protocols are needed. A therapeutic vaccine that would prevent the onset of tuberculosis, and therefore eliminate the need for therapy is desirable. Although currently available therapeutics such as ethambutol are effective, the emergence of drug resistant strains has necessitated new formulations and compositions that are more versatile than ethambutol. Currently available therapeutics are no longer consistently effective as a result of the problems with treatment compliance, lending to the development of drug resistant mycobacterial strains. What is needed are new anti-tubercular drugs that provide highly effective treatment, and shorten or simplify tuberculosis chemotherapy.
The present invention comprises methods and compositions comprising ethylene diamine compounds effective for the treatment of infectious disease. The present invention also provides methods and compositions comprising substituted ethylene diamines having improved anti-mycobacterial activity, including substituted ethylene diamines having improved anti-tuberculosis activity.
The present invention contemplates substituted ethylene diamines, which can derive from a variety of amine compounds. In the present invention, the substituted ethylene diamines are based on the following structure.
The substituted ethylene diamine compounds described herein are synthesized and screened for activity as follows. A chemical library of substituted ethylene diamines is prepared on a solid polystyrene support using split and pool technologies. This technique allows for the synthesis of a diverse set of substituted ethylene diamines. These diamines are screened for anti-TB activity using in vitro, biological assays, including a High-Throughput Screening (HTS) assay, based on the recently completed genomic sequence of M. tuberculosis, and a Minimum Inhibition Concentration (MIC) assay.
The methods and compositions described herein comprise substituted ethylene diamines that are effective against disease caused by infectious organisms, including, but not limited to, bacteria and viruses. One embodiment of the invention provides methods and compositions comprising substituted ethylene diamines that are effective against mycobacterial disease. Another embodiment of the invention provides methods and compositions comprising substituted ethylene diamines that have MIC of 50 μM or lower for mycobacterial disease. Another embodiment of the present invention comprises substituted ethylene diamines that have an MIC of 25 μM or lower for mycobacterial disease. Yet another embodiment of the present invention comprises substituted ethylene diamines that have an MIC of 12.5 μM or lower for mycobacterial disease. Another embodiment of the present invention comprises substituted ethylene diamines that have an MIC of 5 μM or lower for mycobacterial disease In another embodiment of the present invention, the methods and compositions comprise substituted ethylene diamines with HTS Luc activity of 10% or greater. In yet another embodiment of the present invention, the methods and compositions comprise substituted ethylene diamines, wherein one amine group is derived from a primary amine, and wherein the other amine group is derived from a primary or secondary amine. In another embodiment of the present invention, the methods and compositions comprise substituted ethylene diamines, wherein one amine is derived from cis-(−)myrtanylamine, cyclooctylamine, 2,2-diphenylethylamine, 3,3-diphenylpropylamine, (+)-bornylamine, 1-adamantanemethylamine, (+)-isopinocampheylamine; or (−)-isopinocampheylaminc.
The present invention contemplates various salt complexes and other substituted derivatives of the substituted ethylene diamines. The present invention also contemplates enantiomers and other stereoisomers of the substituted ethylene diamines and their substituted derivatives. The present invention further contemplates treatment for animals, including, but not limited to, humans.
In addition the present invention contemplates compositions comprising novel substituted ethylene diamine compounds further comprising antitubercular agents including, but not limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof.
Accordingly, it is an object of the present invention to provide methods and compositions for the treatment and prevention of diseases caused by microorganisms
Accordingly, it is an object of the present invention to provide methods and compositions for the treatment and prevention of infectious diseases.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of mycobacterial disease, including but not limited to, tuberculosis.
Yet another object of the present invention is to provide methods and compositions for the treatment and prevention of infectious diseases using compositions comprising substituted ethylene diamines.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of mycobacterial disease using compositions comprising substituted ethylene diamines.
Still another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein the diamine has an MIC of 50 μM, or less.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein the diamine has an MIC of 25 μM, or less.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein the diamine has an MIC of 12.5 μM, or less.
Yet another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein the diamine has an MIC of 5 μM, or less.
Yet another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein the diamine has HTS/Luc activity of 10% or greater.
Another object of the present invention is to provide methods and compositions for the treatment and prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein one amine group is derived from a primary amine, and the other amine group is derived from a primary or secondary amine.
Yet another object of the present invention is to provide methods and compositions for the treatment and/or prevention of tuberculosis using compositions comprising substituted ethylene diamines, wherein one amine is derived from cis-(−)myrtanylamine, cyclooctylamine, 2,2-diphenylethylamine, 3,3-diphenylpropylamine, (+)-bornylamine, 1-adamantanemethylamine, (+)-isopinocampheylamine; or (−)-isopinocampheylamine.
Yet another object of the present invention is to provide composition for the therapeutic formulation for the treatment and prevention of mycobacterial disease.
Another object of the present invention is to provide compositions for therapeutic formulations for the treatment and prevention of mycobacterial disease caused by mycobacterial species comprising M. tuberculosis complex, M. avium intracellulare, M. kansarii, M. fortuitum, M. chelonoe, M. leprae, M. africanum, M. microti, M. bovis BCG or M. bovis.
Still another object of the present invention is to provide compositions and methods for the treatment or prevention of infectious disease caused by Mycobacterium-fortuitum, Mycobacterium marinum, Helicobacter pylori, Streptococcus pneumoniae and Candida albicans.
Another object of the instant invention is to provide one or more novel compounds in a combination therapy to provide a synergistic effect that is active against mycobacterial disease.
A further object of the instant invention is to provide compositions comprising novel substituted ethylene diamine compounds further comprising antitubercular agents including, but not limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof.
Another object of the present invention is to provide combination therapy for infectious disease comprising substituted ethylene diamines and antitubercular agents.
An additional object of the present invention is to provide combination therapy for mycobacterial disease comprising substituted ethylene diamines and antitubercular agents including, but not limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof either singularly or in combination.
Yet another object of the present invention is to provide combination therapy for mycobacterial disease such as tuberculosis comprising substituted ethylene diamines and antitubercular agents including, but not limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof either singularly or in combination.
Another object of the present invention is to provide combination therapy for mycobacterial disease such as tuberculosis comprising substituted ethylene diamines such as SQ 109, SQ 73 or SQ 609 and antitubercular agents including, but not limited to, rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof either singularly or in combination.
Another object of the present invention is to provide combination therapy for mycobacterial disease such as tuberculosis comprising substituted ethylene diamines such as SQ 109, SQ 73 or SQ 609 and antitubercular agents including, but not limited to, rifampicin, and rifampicin analogues such as rifapentine, rifalazil and rifabutin.
Yet another object of the instant invention is to provide one or more novel compounds in combination with a drug to provide a synergistic effect active against mycobacterial disease.
Another object of the instant invention is to provide one or more novel compounds in combination with a standard tuberculosis drug to provide a synergistic effect active against mycobacterial disease.
It is a further objective that the instant invention provide novel methods of treatment wherein one or more novel compounds are used in combination with at least one known drug to provide a synergistic effect, by which to treat or prevent infectious disease.
These and other objects, features and advantages of the present invention will become apparent after a review of the following detailed description of the disclosed embodiments and the appended claims.
a)-2(ac) provide chemical structures of a variety of primary amines.
a)-3(f) provide chemical structures of a variety of acyclic secondary amines.
a)-4(i) provide chemical structures of a variety of cyclic secondary amines.
The present invention may be understood more readily by reference to the following detailed description of the specific embodiments included herein. However, although the present invention has been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention. The entire text of the references mentioned herein are hereby incorporated in their entireties by reference including U.S. patent application Ser. No. 11/145,499, filed Jun. 3, 2005, U.S. patent application Ser. No. 10/147,587 filed May 17, 2002, and U.S. Provisional Patent Application Ser. No. 60/381,220 filed May 17, 2002.
Mycobacterial infections, such as those causing tuberculosis, once thought to be declining in occurrence, have rebounded, and again constitute a serious health threat. Tuberculosis (TB) is the cause of the largest number of human deaths attributed to a single etiologic agent with two to three million people infected with tuberculosis dying each year. Areas where humans are crowded together, or living in substandard housing, are increasingly found to have persons affected with mycobacteria. Individuals who are immunocompromised are at great risk of being infected with mycobacteria and dying from such infection. In addition, the emergence of drug-resistant strains of mycobacteria has led to treatment problems of such infected persons
Many people who are infected with mycobacteria are poor, or live in areas with inadequate healthcare facilities. As a result of various obstacles (economical, education levels, etc.), many of these individuals are unable to comply with the prescribed therapeutic regimens. Ultimately, persistent non-compliance by these and other individuals results in the prevalence of disease. This noncompliance is frequently compounded by the emergence of drug-resistant strains of mycobacteria. Effective compositions and vaccines that target various strains of mycobacteria are necessary to bring the increasing number of tuberculosis cases under control.
Chemotherapy is a standard treatment for tuberculosis. Some current chemotherapy treatments require the use of three or four drugs, in combination, administered daily for two months, or administered biweekly for four to twelve months. Table 1 lists several treatment schedules for standard tuberculosis drug regimens.
Decades of misuse of existing antibiotics and poor compliance with prolong and complex therapeutic regimens has led to mutations of the mycobacterium tuberculosis and has created an epidemic of drug resistance that threatens tuberculosis control world wide. The vast majority of currently prescribed drugs, including the front line drugs, such as isoniazid, rifampin, pyrazinamide, ethambutol and streptomycin were developed from the 1950s to the 1970s. Thus, this earlier development of tuberculosis chemotherapy did not have at its disposal the implications of the genome sequence of Mycobacterium tuberculosis, the revolution in pharmaceutical drug discovery of the last decades, and the use of national drug testing and combinational chemistry.
Consequently, the treatments of drug-resistant M. tuberculosis strains, and latent tuberculosis infections, require new anti-tuberculosis drugs that provide highly effective treatments, and shortened and simplified tuberculosis chemotherapies. Moreover, it is desirable that these drugs be prepared by a low-cost synthesis, since the demographics of the disease dictate that cost is a significant factor.
The present invention provides methods and compositions comprising a class of substituted ethylene diamine compounds effective in treatment and prevention of disease caused by microorganisms including, but not limited to, bacteria. In particular, the methods and compositions of the present invention are effective in inhibiting the growth of the microorganism, M. tuberculosis. The methods and compositions of the present invention are intended for the treatment of mycobacteria infections in human, as well as other animals. For example, the present invention may be particularly useful for the treatment of cows infected by M. bovis.
As used herein, the term “tuberculosis” comprises disease states usually associated with infections caused by mycobacteria species comprising M. tuberculosis complex. The term “tuberculosis” is also associated with mycobacterial infections caused by mycobacteria other than M. tuberculosis (MOTT). Other mycobacterial species include M. avium-intracellulare, M. kansarii, M. fortuitum, M. chelonae, M. leprae, M. africanum, and M. microti, M. avium paratuberculosis, M. intracellulare, M. scrofulaceum, M. xenopi, M. marinum, M. ulcerans.
The present invention further comprises methods and compositions effective for the treatment of infectious disease, including but not limited to those caused by bacterial, mycological, parasitic, and viral agents. Examples of such infectious agents include the following: staphylococcus, streptococcaceae, neisseriaaceae, cocci, enterobacteriaceae, pseudomonadaceae, vibrionaceae, campylobacter, pasteurellaceae, bordetella, francisella, brucella, legionellaceae, bacteroidaceae, gram-negative bacilli, clostridium, corynebacterium, propionibacterium, gram-positive bacilli, anthrax, actinomyces, nocardia, mycobacterium, Helicobacter pylori, Streptococcus pneumoniae, Candida albicans, treponema, borrelia, leptospira, mycoplasma, ureaplasma, rickettsia, chlamydiae, systemic mycoses, opportunistic mycoses, protozoa, nematodes, trematodes, cestodes, adenoviruses, herpesviruses, poxviruses, papovaviruses, hepatitis viruses, orthomyxoviruses, paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, bunyaviridae, rhabdoviruses, human immunodeficiency virus and retroviruses.
The present invention further provides methods and compositions useful for the treatment of infectious disease, including by not limited to, tuberculosis, leprosy, Crohn's Disease, acquired immunodeficiency syndrome, lyme disease, cat-scratch disease, Rocky Mountain Spotted Fever and influenza.
The anti-infective methods and compositions of the present invention contain one or more substituted ethylene diamine compounds. In particular, these compounds encompass a wide range of substituted ethylene diamine compounds having the following general formula:
where “R1NH” is typically derived from a primary amine, and “R2R3N” is typically derived from a primary or secondary amine. The ethylene diamines of the present invention are prepared by a modular approach using primary ard secondary amines as building blocks, and coupling the amine moieties with an ethylene linker building block. Representative primary amines, acyclic secondary amines, and cyclic secondary amines are shown in
Generally, chemical moieties R1, R2, and R3 of the ethylene diamine compounds of the present invention are independently selected from H, alkyl; aryl; alkenyl; alkynyl; aralkyl; aralkenyl; aralkynyl; cycloalkyl; cycloalkenyl; heteroalkyl; heteroaryl; halide; alkoxy; aryloxy; alkylthio; arylthio; silyl; siloxy; a disulfide group; a urea group; amino; and the like, including straight or branched chain derivatives thereof, cyclic derivatives thereof, substituted derivatives thereof, heteroatom derivatives thereof, heterocyclic derivatives thereof, functionalized derivatives thereof, salts thereof, such salts including, but not limited to hydrochlorides and acetates, isomers thereof, or combinations thereof. For example, nitrogen-containing heterocyclic moieties include, but are not limited to, groups such as pyridinyl (derived from pyridine, and bonded through a ring carbon), piperidinyl (derived from piperidine and bonded through the ring nitrogen atom or a ring carbon), and pyrrolidinyl (derived from pyrrolidine and bonded through the ring nitrogen atom or a ring carbon). Examples of substituted, or functionalized, derivatives of R1, R2, and R3 include, but are not limited to, moieties containing substituents such as acyl, formyl, hydroxy, acyl halide, amide, amino, azido, acid, alkoxy, aryloxy, halide, carbonyl, ether, ester, thioether, thioester, nitrile, alkylthio, arythio, sulfonic acid and salts thereof, thiol, alkenyl, alkynyl, nitro, imine, imide, alkyl, aryl, combinations thereof, and the like. Moreover, in the case of alkylated derivatives of the recited moieties, the alkyl substituent may be pendant to the recited chemical moiety, or used for bonding to the amine nitrogen through the alkyl substituent.
Examples of chemical moieties R1, R2, and R3 of the present invention include, but are not limited to: H; methyl; ethyl; propyl; butyl; pentyl; hexyl; heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl; propynyl; butynyl; cyclopropyl; cyclobutyl; cyclopentyl; cyclohexyl; cyclooctyl cyclobutenyl; cyclopentenyl; cyclohexenyl; phenyl; tolyl; xylyl; benzyl; naphthyl; pyridinyl; furanyl; tetrahydro-1-napthyl; piperidinyl; indolyl; indolinyl; pyrrolidinyl; 2-(methoxymethyl)pyrrolidinyl; piperazinyl; quinolinyl; quinolyl; alkylated-1,3-dioxolane; triazinyl; morpholinyl; phenyl pyrazolyl; indanyl; indonyl; pyrazolyl; thiadiazolyl; rhodaninyl; thiolactonyl; dibenzofuranyl; benzothiazolyl; homopiperidinyl; thiazolyl; quinonuclidinyl; isoxazolidinonyl; any isomers, derivatives, or substituted analogs thereof; or any substituted or unsubstituted chemical species such as alcohol, ether, thiol, thioether, tertiary amine, secondary amine, primary amine, ester, thioester, carboxylic acid, diol, diester, acrylic acid, acrylic ester, methionine ethyl ester, benzyl-1-cysteine ethyl ester, imine, aldehyde, ketone, amide, or diene. Further examples of chemical moieties R1, R2, and R3 of the present invention include, but are not limited to, the following species or substituted or alkylated derivatives of the following species, covalently bonded to the amine nitrogen: furan; tetrahydrofuran; indole; piperazine; pyrrolidine; pyrrolidinone; pyridine; quinoline; anthracene; tetrahydroquinoline; naphthalene; pyrazole; imidazolc; thiophene; pyrrolidine; morpholine; and the like. One feature of the recited species or substituted or alkylated derivatives of these species, is that they may be covalently bonded to the amine nitrogen in any fashion, including through the pendant substituent or alkyl group, through the heteroatom as appropriate, or through a ring atom as appropriate, as understood by one of ordinary skill in the art.
The chemical moieties R1, R2, and R3 of the present invention also include, but are not limited to, cyclic alkanes and cyclic alkenes, and include bridged and non-bridged rings. Examples of bridged rings include, but are not limited to, the following groups: isopinocamphenyl; bornyl; norbornyl; adamantanetetyl; cis-(−)myrtanyl; adamantyl; noradamantyl; 6-azabicyclo[3.2.1]octane; exo-norbornane; and the like.
In one embodiment of the present invention, NR2R3 is derived from a cyclic secondary amine. Examples of a cyclic chemical moiety, NR2R3, of the present invention include, but are not limited to, 4-benzyl-piperidine; 3-piperidinemethanol; piperidine; tryptamine; moropholine; 4-piperidinopiperidine; ethyl 1-piperazine carboxylate; 1-(2-amino-ethyl)-piperazine; decahydroquinoline; 1,2,3,4-tetrahydro-pyridoindole (reaction at either amine); 3-amino-5-phenyl pyrazole; 3-aminopyrazole; 1-(2-fluorophenyl)piperazine; 1-proline methyl ester; histidinol; 1-piperonyl-piperazine; hexamethyleimine; 4-hydroxypiperidine; 2-piperidinemethanol; 1,3,3-trimethyl-6-azabicyclo[3.2.1]octane; 3-pyrrolidinol; 1-methylpiperazine; (S)-(+)-(2-pyrrolidinylmethyl)pyrrolidine; 1-methylhomopiperazine; 2-ethyl-piperidine; 1,2,3,4-tetrahydroisoquinoline; 1-(4-fluorophenyl)piperazine; d,l-tryptophan methyl ester; tert-butyl (15, 45)-(−)-2,5-diazabiclyclo[2.2.1]heptane-2-carboxylate; isonipecotamide; heptamethyleneimine; alpha-methyltryptamine; 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline; 3-aminopyrrolidine; 3,5-dimethylpiperidine; 2,6-dimethylmorpholine; 1,4-dioxo-8-azaspiro[4.5]decane; 1-methol-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; 1,3,4,6,7,8-hexahydro-2H-pyrido(1,2-A)pyrimidine; 1,2,3,4-tetrahydroquinoline; 1-(2-methoxyphenyl)piperazine; 1-(2-(2-hydroxyethoxy)ethyl)piperazine; (S)-(+)-2-(aminomethyl)pyrroli-dine; (3S(3a, 4Ab), 8Ab)-N-t-butyl-D-ecahydro-3-isoquino-linecarboxamide; (R)-cycloserine; homopiperazine; 2,6-dimethylpiperazine (reaction at either amine); iminodibenzyl; 5-methoxytryptamine; 4,4′-bipiperidine; 1-(2-hydroxyethyl)piperazine; 4-methylpiperidine; 1-histidine methyl ester; or methyl pipecoliate.
The R1HN substituent is derived from a primary amine. The R2R3N substituent is typically derived from a primary or secondary amine, but may also arise from an amino acid, or an amino acid precursor. The amino acid can transform into an amino alcohol. When an amino acid is employed as the source of the R2R3N moiety, the precursor compound may be selected from, among others, the following compounds and their derivatives: d,l-tryptophan methyl ester; 1-methionine ethyl ester; 1-lysine methyl ester (via reaction at either primary amine); (S)-benzyl-1-cysteine ethyl ester; 1-arginine methyl ester (via reaction at either primary amine); 1-glutamic acid ethyl ester; 1-histidine methyl ester; or (3S (3a, 4Ab), 8A b)-N-t-butyl-D-ecahydro-3-iso-quino linecarboxamide.
The R4 moiety of the substituted ethylene diamine compounds of the present invention is typically selected from H, alkyl or aryl, but R4 can also constitute alkenyl, alkynyl, aralkyl, aralkenyl, aralkynyl, cycloalkyl, cycloalkenyl, and the like. Examples of the R4 chemical moiety include, but are not limited to: H; methyl; ethyl; propyl; butyl; pentyl; hexyl, heptyl; octyl; ethenyl; propenyl; butenyl; ethynyl; propynyl; butynyl; cyclobutyl; cyclopentyl; cyclohexyl; cyclobutenyl; cyclopentenyl; cyclohexenyl; phenyl; tolyl; xylyl; benzyl; naphthyl; straight or branched chain derivatives thereof; cyclic derivatives thereof; substituted, functionalized, and heteroatom derivatives thereof; and heterocyclic derivatives thereof, and the like. Typically, R4 is selected from H, methyl, ethyl, butyl or phenyl. However, when R4 is “H” the ethylene diamine does not contain ethambutol.
A majority of the ethylene diamine compounds described herein are preferably prepared using a solid support synthesis, as set forth in one of the representative reaction schemes shown in
The preparation of the ethylene diamines is preferably accomplished in six steps, using a rink-acid resin. The first step of the synthesis is converting the rink-acid resin to rink-chloride by treatment with triphenylphosphine and hexachloroethane in tetrahydrofuran (THF). This step is followed by addition of the primary amine in the presence of Hunig's base (EtN(i-Pr)2) in dichloroethane. The third step is the acylation of the resin-attached amine using either one of the two acylation routes shown in
Introduction of the second nitrogen moiety is preferably achieved in the presence of Hunig's base in dimethylformamide (DMF). Reduction of the intermediate amine-amide is carried out using Red-Al (3.4M solution of sodium his (2-methoxyethoxy) aluminum hydride in toluene). The final product is cleaved from the resin support using a 10% solution (by volume) of trifluoroacetic acid (TFA) in dichloromethane (DCM). The solvent is evaporated, and the TFA salts of the final diamine products are analyzed by mass spec, and screened against M. tuberculosis for effectiveness. Some of the substituted ethylene diamines, prepared using the above-described solid-support synthesis, are also prepared using a solution phase synthesis described below.
Formation of the Substituted Ethylene Diamine Library
The solid support syntheses, shown in
Prior to the solid support synthesis, each amine, within numbers 1 to 288, as shown in
Screening Against M. tuberculosis
An entire library of synthesized substituted ethylene diamines (targeted number of compounds about 100,000), prepared as described above, was screened, in vitro, against M. tuberculosis in ethambutol (EMB) sensitive Luc-assay. The MIC (Minimum Inhibition Concentration) was also determined. The MIC is the minimum concentration of a growth inhibitor, here the substituted ethylene diamine, where there is no multiplication of the microorganism under examination. Screening was done using a High-Throughout Screening (HTS) Luc assay with recombinant mycobacteria containing a promoter fusion of a luciferase to the EB-inducible gene (Luc assay). The Luc-assay and MIC assay are described in detail below. These assays are well known to those skilled in the art. Based on this initial screening, 300+ compound mixtures showed anti-TB activity.
Deconvolution of the Reactive Wells
The M. tuberculosis screening revealed approximately 300 active compounds mixtures that were selected for deconvolution. In particular, wells possessing activity of approximately <12.5 μM in the HTS Luc assay, and/or an MIC of approximately <12.5 μM, were selected for a total of 336 wells.
Deconvolutions were performed by discrete re-synthesis of each substituted ethylene diamine compound in each active compound pool. The pooled compounds in each active well were individually synthesized, and screened. Syntheses of the targeted diamine compounds in each active pool were done in the 96-well plates using stored archived α-haloacetyl amides (resin attached haloacetyl amides), according to the previously described reaction steps (the addition of the second amine, the reduction with Red-Al, and the cleavage from the solid support). The archived resins were stored as individual compounds at 4° C. The 96-well plates were used for the remaining synthesis steps as previously described.
The same screening tests, MIC and HTS Luc assay, were performed on each deconvoluted compound. Representative Luminescence data for deconvoluted compounds are shown in
Summary of Screening Results
Overall, the deconvolution screening results revealed about 2,000 ethylene diamine compounds with inhibitory activity against M. tuberculosis. More than 150 of these compounds exhibited MICs equal to or lower than approximately 12.5 μM.
The total frequency of the top thirteen amines that contributed to the activity of the substituted ethylene diamines are shown in
#11 2,3-Dimethylcylochexy amines
#18 3,3-Diphenylpropylamine
#44 1-Adamantanemethylamine
#47 2,2-Diphenylethylaminne
#63 (S)-2-Amino-1-butanol
#74.1 (−)-cis-Myrtanylamine
#77.1 Cyclooctylamine
#78.1 2-Adamantamine
#105a (1R,2R,3R,5S)-(−)-Isopinocampheylamine
#231 2-Methoxyphenethylamine
#255 (S)-Cylcohexylethylamine
#266 Undecylamine
#272 Geranylamine
Other amines that contributed to the activity of the substituted ethylene diamines are shown in Table 2. The compounds in Table 2 are sorted by their MIC results. Some compounds, synthesized in larger quantities (2-60 mg) on the Quest® Synthesizer, and purified by HPLC using semi-preparative C18-column, are shown in Table 3. Generally, the final purity of each compound in Table 3 was at least 90%.
In one embodiment, the present invention comprises a composition comprising compound 109 and compound 73.
In another embodiment, the present invention comprises a composition comprising compound 109 and compound 73 and a standard tuberculosis drug.
In yet another embodiment, the present invention comprises a method of treating disease caused by an infectious agent comprising administering an effective amount of compound 109.
Still a further embodiment, the present invention comprises a method of treating disease caused by an infectious agent comprising administering an effective amount of compound 109 and compound 73.
In yet another embodiment, the present invention comprises a method of treating disease caused by an infectious agent comprising administering an effective amount of compound 109 and compound 73 and a standard tuberculosis drug. In another embodiment, the present invention comprises a method of treating disease caused by an infectious agent comprising administering an effective amount of compound 109 and compound 73 and a pharmaceutical carrier.
The present invention is also directed to a new library of diamine compounds useful against infectious disease. To further enhance the structural diversity of prior diamine compounds, a synthetic scheme to incorporate amino acids into a bridging linker between the two amine components has been developed. The use of amino acids allowed for diverse linker elements, as well as chirality see
The reaction scheme followed is shown in
Solid phase syntheses using Rink resin. Twenty one 96-well plates have been prepared. Six-step synthetic route starting from the Rink resin similar to what that had been used to create our first 100,000 compound library (Scheme 1,
Attachment of the first amine to the support was done according to the Garigipati protocol. Rink acid resin (Novabiochem) was converted into the Rink-chloride upon treatment with triphenylphosphine and dichloroethane in THF. This activated resin was then loaded by addition of an amine N1 in presence of Hunig's base in dichloroethane. The amine N1 includes, but is not limited to, alkyl and aryl primary amines. Out of 177 primary amines that had been previously used as N1 for 100,000 library preparation, only 30 were selected in this Scheme, based upon in vitro activity data of their ethylenediamine derivatives (from the previous ˜100K library) as well as structural diversity (
On the next step, the acylation reaction was accomplished via peptide coupling with FMOC protected amino acids in presence of HATU (O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate) and EtN(iso-Pr)2 in DCM/DMF mixture at room temperature. The reaction was done twice to improve product yields. The list of the amino acids used to create this library is shown in the Table 26 (
Deprotection (removal of the FMOC group) was carried out by reaction with piperidine at room temperature. Derivatization of the amino group was achieved by reductive alkylation with various carbonyl compounds, such as aldehydes, ketones, and carboxylic acids, in the presence of NaBCNH3 at room temperature for 72-96 h. The selection of the carbonyl compounds was made so that the final diamine products would carry the same or similar types of substituents that had been observed in the hit compounds generated from the previous library of ethambutol analogs, as well as structural diversity (
Reduction of the aminoethyleneamides into corresponding diamines was carried out using the soluble reducing reagent 65+w % Red-Al at room temperature. Cleavage of the products from the resin was achieved with a 10% solution of trifluoroacetic acid in dichloromethane resulting in the formation of TFA salts of the diamines.
For library production the first three steps of the synthetic scheme (resin activation, amine loading, and acylation) were carried out using a Quest 210 Synthesizer on scale of 0.1-0.15 g of resin per tube. Following the acylation, formed resins were thoroughly washed, dried, and then groups of ten resins were pooled together. A small amount of each resin (˜0.05 g) was archived prior to pooling to facilitate re-synthesis and deconvolution of actives.
Deprotection of the FMOC group, addition of the carbonyl component, reduction, and cleavage were carried out in 96-well reaction blocks using the Combiclamps system by Whatman Polyfiltronics or the FlexChem system by Robbins Scientific. A suspension of the pooled resins in 2:1 mixture of DCM/THF was evenly distributed into one reaction plate resulting in approximately 10 mg of the resin per well. The 96 diverse carbonyl compounds were arrayed in one 96-well plate template and added, one carbonyl compound per well, to each individual pool of ten resins, resulting in an anticipated 960 diamines produced per plate. Reduction was carried out in the same format and cleavage and filtering into storage plates was followed by evaporation of the TFA prior to biological assay.
Quality assessment of the prepared compounds was done by Electrospray Ionization mass spectrometry using two randomly selected rows (16 samples) per plate, 17% of the total number. Successful production of a compound was based on an appearance of a molecular ion of the calculated mass. Depending on the amino acid that had been used for the synthesis, the percentage of the predicted ions were observed, and therefore the predicted compounds were formed, varied from 5-60% (Table 25,
As discussed herein, there is a need in the art for novel compounds and methods that are effective against infectious disease. More particularly, there is a need for novel compounds and methods for the effective treatment of Mycobacterial disease. The instant invention satisfies the long felt need of the prior art by providing novel compositions and methods that are effective in the treatment of infectious disease, including but not limited to, tuberculosis.
In one embodiment the instant invention comprises at least two novel compounds from Table 3 (compounds I-165) for the treatment of infectious disease.
In another embodiment, the instant invention comprises one or more novel compounds of Table 3 (compounds I-165) in combination with one or more drugs for the treatment of infectious disease.
In another embodiment, a composition comprising at least one of compound I-165 is combined with one or more drugs for the treatment of M. tuberculosis. In a further embodiment, a composition comprising one or more novel compounds selected from the group consisting of compounds I-165 is combined with one or more drug to provide a synergistic effect that is active as a method of treating Mycobacterial disease.
In one embodiment the instant invention comprises a composition comprising one or more compounds of Table 3 in combination with at least one known standard tuberculosis drug.
In yet another embodiment a method of treating infectious disease comprises one or more compounds of Table 3 in combination with at least one known standard tuberculosis drug. While not wishing to be bound by the following theory it is believed that the combination of a standard tuberculosis drug with at least one or more of compounds comprising compounds I-165 produces a synergistic effect resulting in the treatment or prevention of infectious disease, including but not limited to, tuberculosis.
The bactericidal activity of streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide alone and in combination against Mycobacterium Tuberculosis is discussed by Dickinson et al. (Am Rev Respir Dis 116(4): 627-35): Log-phase cultures of Mycobacterium tuberculosis in Tween-albumin medium were exposed to streptomycin, isoniazid, rifampin, ethambutol, and pyrazinamide in concentrations in the range likely to be present in serum during treatment of patients. The bactericidal activity of the drugs was measured as the decrease in viable counts at 4 and 7 days. The activity of single drugs was highest for streptomycin and next highest for rifampin and isoniazid, but ethambutol only started to kill after 4 days. When exposed to 2 drugs, bactericidal synergism was found with streptomycin/isoniazid and isoniazid/ethambutol; additivity, with streptomycin/rifampin; indifference, with isoniazid rifampin and streptomycin/ethambutol; and antagonism, with rifampin/ethambutol and isoniazid/pyrazinamide. When cultures were exposed to the 3 drugs, isoniazid, rifampin, and ethambutol, marked antagonism was found between isoniazid and rifampin, whereas the addition of isoniazid or an increase in its concentration increased the bactericidal activity. Combination therapy including the novel ethylene diamine compositions as described herein have not been identified prior to the present invention. The present invention contemplates combination therapy comprising novel ethylene diamine compositions as presently described together with one or more antitubercular drugs, including but not limited to rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol. Also included herein are analogues and chemical equivalents and substitutes of such antitubercular agents. For example, the present inventors contemplate the use of rifampicin as well as its analogues, including but not limited to, rifapentine, rifalazil and rifabutin.
In another embodiment, the present invention comprises a composition effective against Mycobacterium-fortuitum, Mycobacterium marinum, Helicobacter pylori, Streptococcus pneumoniae and Candida albicans comprising at least one compound selected from the group consisting of compounds 1-165.
In another embodiment, the present invention comprises a composition effective against Mycobacterium-fortuitum, Mycobacterium marinum, Helicobacter pylori, Streptococcus pneumoniae and Candida albicans comprising at least one compound selected from the group consisting of compounds I-165, alternatively combined with one or more antitubercular agents wherein the antiburcular agents, include but are not limited to rifampicin, isoniazid, pyrazinamide, moxifloxacin and ethambutol and analogues thereof.
Formulations
Therapeutics, including compositions containing the substituted ethylene diamine compounds of the present invention, can be prepared in physiologically acceptable formulations, such as in pharmaceutically acceptable carriers, using known techniques. For example, a substituted ethylene diamine compound is combined with a pharmaceutically acceptable excipient to form a therapeutic composition.
The compositions of the present invention may be administered in the form of a solid, liquid or aerosol. Examples of solid compositions include pills, creams, soaps and implantable dosage units. Pills may be administered orally. Therapeutic creams and anti-mycobacteria soaps may be administered topically. Implantable dosage units may be administered locally, for example, in the lungs, or may be implanted for systematic release of the therapeutic composition, for example, subcutaneously. Examples of liquid compositions include formulations adapted for injection intramuscularly, subcutaneously, intravenously, intraarterially, and formulations for topical and intraocular administration. Examples of aerosol formulations include inhaler formulations for administration to the lungs.
A sustained release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis, or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained release matrix is chosen desirably from biocompatible materials, including, but not limited to, liposomes, polylactides, polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipds, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide.
The dosage of the composition will depend on the condition being treated, the particular composition used, and other clinical factors, such as weight and condition of the patient, and the route of administration. A suitable dosage may range from 100 to 0.1 mg/kg. A more preferred dosage may range from 50 to 0.2 mg/kg. A more preferred dosage may range from 25 to 0.5 mg/kg. Tablets or other forms of media may contain from 1 to 1000 mg of the substituted ethylene diamine. Dosage ranges and schedules of administration similar to ethambutol or other anti-tuberculosis drugs may be used.
The composition may be administered in combination with other compositions and procedures for the treatment of other disorders occurring in combination with mycobacterial disease. For example, tuberculosis frequently occurs as a secondary complication associated with acquired immunodeficiency syndrome (AIDS). Patients undergoing AIDS treatment, which includes procedures such as surgery, radiation or chemotherapy, may benefit from the therapeutic methods and compositions described herein.
The following specific examples will illustrate the invention as it applies to the particular synthesis of the substituted ethylene diamine compounds, and the in vitro and in vivo suppression of the growth of colonies of M. tuberculosis. In addition, the teachings of R. Lee et al. J. Comb. Chem. 2003, 5, 172-187 are hereby incorporated by reference in their entirety. It will be appreciated that other examples, including minor variations in chemical procedures, will be apparent to those skilled in the art, and that the invention is not limited to these specific illustrated examples.
The Rink-acid resin was obtained from NOVABIOCHEM® Inc., San Diego, Calif. Solvents: acetonitrile, dichloromethane, dimethylformamide, ethylenedichloride, methanol and tetrahydrofuran were purchased from ALDRICH®, Milwaukee, Wis., and used as received. All other reagents were purchased from SIGMA-ALDRICH®, West Monroe Highland, Ill. Solid phase syntheses were performed on a QUEST® 210 Synthesizer, from ARGONAUT TECHNOLOGIES®, Foster City, Calif., with the aid of combinatorial chemistry equipment, from WHATMAN® POLYFILTRONICS® (Kent, England; Rockland, Mass.) and ROBBINS SCIENTIFIC®, Sunnyvale, Calif. Evaporation of solvents was done using SPEEDVAC® AES, from SAVANT®, Holbrook, N.Y. All necessary chromatographic separations were performed on a WATERS' ALLIANCE HT SYSTEM®, Milford, Mass. Analytical thin-layer chromatography was performed on MERCK® silica gel 60F254 plates, purchased from SIGMA-ALDRICH®, West Monroe Highland, Ill.
The activation of the Rink-acid resin, the addition of the first amine, and the acylation step were carried out in 10 ml tubes using the QUEST® 210 Synthesizer. The addition of the second amine, the reduction with Red-AL, and the cleavage from the solid support were carried out in 96-deep (2 ml) well, chemically resistant plates.
A. Activation of the Rink-Acid Resin
The Rink-acid resin had a coverage of 0.43-0.63 mmol of linker per gram resin. Four to five grams of this resin were suspended in 80 ml of a 2:1 mixture of dichloromethane and tetrahydrofuran (THF), and distributed into ten, 10 ml tubes, with 8 ml of resin suspension per tube. Each suspension was filtered and washed twice with THF. A solution of triphenylphosphine (3.80 g, 14.5 mmol) in 30 ml of THF was prepared, and 3 ml of this solution was added to each tube, followed by the addition of 3 ml of a solution of hexachloroethane in THF (3.39 g/14.3 mmol hexachloroethane in 30 ml THF). After agitation for six hours at room temperature, each activated resin was washed twice with THF and twice with dichloromethane.
B. Addition of the First Amine
Each tube, containing the activated rink resin, was charged with 3 ml of dichloroethane, 0.3 ml (1.74 mmol) N1N-diisopropylethylamine (EtN(iPr)2) and the corresponding amine (around 1 mmol). If the selected amine was a solid at room temperature, it was added as a solution, or a suspension in DMF. Enough dichloroethane was added to each tube for a final volume of 8 ml. The reaction mixture was heated at 45° C. for 6-8 hours. The resins were filtered, washed with a 2:1 mixture of dichloromethane and methanol (1×8 ml), then with methanol (2×8 ml), and then dried under argon for 10 minutes.
C. Acylation with the Halo-Acylchloride
a. Acylation with Chloroacetyl Chloride. Each resin was prewashed with THF (2×8 ml), and then charged with THF (8 ml), pyridine (0.3 ml, 3.67 mmole) and chloroacetyl chloride (0.25 ml, 2.5 mmole). The reaction mixture was stirred for 8 hours at 45° C., and then for 6-8 hours at room temperature. Each resin was filtered, washed with a 2:1 mixture of dichloromethane/methanol (1×8 ml), methanol (2×8 ml) and THF (2×8 ml). The acylation was repeated using the same loading of reagents, but a shorter reaction time of 4 hours at 45° C., and 2 hours at room temperature. Each resin was then filtered, washed with a 2:1 mixture of dichloromethane and methanol (1×8 ml), and then with methanol (3×8 ml). Each resin was dried under argon for 10 minutes. Each resin was then transferred into a vial and dried in a desiccator under vacuum for 1 hour.
b. Acylation with α-Phenyl-α-Chloroaceryl Chloride. The same procedure set out for the acylation with chloroacetyl chloride was used. A 2.5 mmol excess of α-phenyl-α-chloroacetyl chloride, relative to mmol amount of linker in the rink-acid resin, was used.
C. Acylation with α-Halo-α-Methyl; α-Halo-α-Ethyl and α-Halo-α-Butylacetyl Bromide. A 1:1:1 mixture (by volume) of the α-bromoproponyl bromide (R4=Me), α-bromobutyryl bromide (R4=Et), and α-bromohexanoyl bromide (R4=Bu) was used to give a molar ratio of 0.52:0.56:0.42 (in mmols). This resulted in a molar excess of 1.65, 1.75 and 1.31, respectively, if the original coverage of the resin was 0.63 mmol/g (0.5 g resin per tube), and 2.4, 2.6 and 1.9 if the original coverage of the resins was 0.43 mmol/g (0.5 g resin per tube).
d. Acylation with α-Chloro-α-Methyl Acetic acid. Each resin was prewashed with dichloromethane. Each tube was charged with 3 ml of a solution of PyBrop (0.29 g, 0.62 mmole) in dichloromethane, a solution of the α-chloro-α-methylacetic acid (0.095 g, 0.62 mmole) in 3 ml of DMF, and EtN(iPr)2 (0.2 ml, 1.2 mmole). Each reaction mixture was allowed to react for 16-18 hours at room temperature. Each resin was then filtered, washed with dichloromethane (2×8 ml) and methanol (2×8 ml), and the acylation was repeated. Each resin was then filtered, washed with dichloromethane (2×8 ml), methanol (3×8 ml), and dried under argon for about 10 minutes. Each resin was transferred into a vial, and dried in a desiccator under vacuum for one hour.
D. Addition of the Second Amine
Ten, or thirty prepared α-haloacetyl amide resins from the first three steps were pooled together, leaving 0.05-0.10 gram of each individual resin for necessary deconvolutions. A suspension of the pooled resin mixture in 100 ml of a 2:1 mixture of dichloromethane and THF was distributed into one, two or three, 96-well reaction plates. For one reaction plate, 1.7 to 2.0 grams of resin were used. For two reaction plates, 3.0 to 3.3 grams of resin were used, and for three reaction plates, 4.7 to 5.0 grams of resin were used. The distributed suspension was then filtered using a filtration manifold, a small lightweight manifold that is generally used for drawing solvents and reagents from the chambers of the 96-well reaction plates. The reaction plates were transferred into COMBICLAMPS® (Huntington, W. Va.), and 10% EtN(iPr)2 in DMF was added at 0.2 ml per well (0.21 mmole of EtN(iPr)2 per well), followed by the addition of a 1.0M solution of the appropriate amine from the corresponding master plate, 0.1 ml per well (0.1 mmole amine per well). The COMBICLAMPS® are used to accommodate 96-well reaction plates during synthesis, allowing for the addition of reagents into the plates, and a proper sealing that maintains reagents and solvents for hours at elevated temperatures. These clamps consist of a top and bottom cover provided with changeable, chemically resistant sealing gaskets. They are designed to accommodate 96-well reaction plates between the top and bottom covers. The reaction plates were sealed and kept in an oven at 70-75° C. for 16 hours. After cooling to room temperature, the resins were filtered, washed with a 1:1 mixture of DCM/methanol (1×1 ml), methanol (2×1 ml), and then dried in a desiccator under vacuum for 2 hours.
E. Reduction with Red-Al
The reaction plates were placed into COMBICLAMPS®. A 1:6 mixture of Red-Al (65+w % in toluene) and THF was added, at 0.6 ml per well (0.28 mmole of Red-Al per well), and allowed to react for 4 hours. Each resin was then filtered, washed with THF (2×1 ml), and methanol (3×1 ml). The addition of methanol should proceed with caution. Each resin was then dried under vacuum.
F. Cleavage of Final Ethylene Diamine Compound
This step was carried out using a cleavage manifold, a Teflon coated aluminum, filter/collection vacuum manifold, designed for recovering cleavage products from the reaction plates into collection plates. The manifold is designed to ensure that the filtrate from each well is directed to a corresponding well in a receiving 96-well collection plate. The reaction plates (placed on the top of the collection plates in this manifold) were charged with a 10:85:5 mixture of TFA, dichloromethane, and methanol (0.5 ml of mixture per well). After fifteen minutes, the solutions were filtered and collected into proper wells on the collection plates. The procedure was repeated. Solvents were evaporated on a SPEED VAC®, Holbrook, N.Y., and the residual samples (TFA salts) were tested without further purification.
Deconvolution of the active wells was performed by re-synthesis of discrete compounds, from the archived α-haloacetyl amide resins (10 resins, 0.05-0.10 g each), which were set aside at the end of the acylation step before the pooling. Each resin was assigned a discrete column (1, or 2, or 3, etc., see the template) in a 96 well filterplate, and was divided between X rows (A, B, C, etc), where X is the number of hits discovered in the original screening plate. To each well, in a row, a selected N2 (R3R2NH) hit amine (0.1 mmol), DMF (180 ml) and EtNiPr2 (20 ml) were added: the first selected amine was added to the resins in the row “A”, the second amine—to the resins in the row “B”, the third amine—to the resins in the row “C”, etc. A lay-out of a representative 96-well filter plate is shown in Table 4.
Deconvolution of the active wells was performed by re-synthesis of discrete compounds, from the archived α-haloacetyl amide resins (10 resins, 0.05-0.10 g each), which were set aside at the end of the acylation step before the pooling. Each resin was assigned a discrete column (1, or 2, or 3, etc., see the template) in a 96 well filterplate, and was divided between X rows (A, B, C, etc), where X is the number of hits discovered in the original screening plate. To each well, in a row, a selected N2 (R3R2NH) hit amine (0.1 mmol), DMF (180 ml) and EtNiPr2 (20 ml) were added: the first selected amine was added to the resins in the row “A”, the second amine—to the resins in the row “B”, the third amine—to the resins in the row “C”, etc. A lay-out of a representative 96-well filter plate is shown in Table 4.
The reaction plates were sealed and kept in an oven at 70-75° C. for 16 hours. After cooling to room temperature, the resins were filtered, washed with a 1:1 mixture of DCM and methanol (1×1 ml), methanol (2×1 ml), and dried in desiccator under vacuum for 2 h. Reduction and cleavage were performed according to steps 5 and 6 in the original synthetic protocol. The product wells from the cleavage were analyzed by ESI-MS (Electro Spray Ionization Mass Spectroscopy) to ensure the identity of the actives, and were tested in the same Luc and MIC assays.
The solid-phase protocol described above in Example I was applied to the scaled-up synthesis of the selected substituted ethylene diamine compounds. Here, all reaction steps, from the activation of the Rink-acid resin to the cleavage of the final product, were carried out using the QUEST® instrument only, which allowed for the simultaneous syntheses of twenty parallel reactions. Purification of all crude samples was done by HPLC to yield desirable products in purity greater than 90%. Table 3 lists the scale-ups of substituted ethylene diamines. Here, the synthesis of one of the active compounds, N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine is described below as an example.
The Preparation of N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine (compound 109) is set forth in
Representative Solution Phase Synthesis of the Active Compounds Preparation of N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−) -isopinocampheylethane-1,2-diamine as hydrochloride (compound 59) is set forth in
Bromocyclooctylacetylamide. To a mixture of cyclooctylamine (3.3 g, 0.026 mol) and pyridine (2.42 g, 0.031 mmol) in anhydrous THF (80 ml) at 0° C. was added dropwise, via syringe, bromoacetylbromide (5.78 g, 0.029 mol). The reaction temperature was maintained by an ice bath. The reaction mixture was allowed gradually to warm up to room temperature, and was stirred at room temperature for 1 hour. The precipitate was removed by filtration, washed with ethyl ether (1×30 ml), and the filtrate was concentrated to dryness on a rotory evaporator. Bromocyclooctylacetylamide was forwarded to the second step without additional purification.
N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheyl-1-carbonylethane-1,2-diamine. To a solution of the bromocyclooctylacetylamide in DMF (60 ml) were added Hunig's base (4.64 g, 0.036 mol) and (1R,2R,3R,5S)-(−) -isopinocampheylamine (4.5 g, 0.029 mol), and the reaction mixture was stirred at 80° C. for 16 hours. After cooling off to the room temperature, the reaction mixture was diluted with 150 ml of ethyl ether, and washed with 1M NaOH solution (2×50 ml). The organic layer was washed with brine (1×50 ml), dried over MgSO4, and concentrated to dryness on the rotory evaporator. The residue (11.04 g) as brown oil was purified on COMBIFLASK® (Isco, Lincoln, Nebr., USA), using Silicagel cartridges commercially available from BIOTAGE® (Biotage, Inc. of Dyax Corp, Va., USA), and the following mobile phase gradient: 30 min run, starting with DCM, 100%, and finishing up with a mixture DCM:MeOH:N4OH (600:400:10). The final product (7.29 g) was obtained as a brown oil; 76% yield, purity 90%.
N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine. To a solution of the amide, from previous step, in anhydrous THF (160 ml), was added dropwise via syringe commercially available (SIGMA-ALDRICH®) Red-Al, as 65 wt % solution in THF (28 ml, 0.09 mol). The reaction mixture was stirred at reflux for 20 hours. After cooling down to the room temperature, the reaction mixture was poured into 1.5M NaOH (200 ml), and extracted with ethyl ether (2×100 ml). The organic layer was washed with brine (1×100 ml), dried over MgSO4, and evaporated to dryness on the rotory evaporator to yield 7.2 g of a crude product, as a brown oil. Chromatographic purification of the crude using the same equipment and conditions as for the previous step, gave 3.5 g of the diamine. The diamine was treated with 2.0M solution of HCl in ethyl ether (25 ml), and kept in a refrigerator overnight. A dark yellow solid (4.2 g) formed, and was filtered off, and recrystallized from MeOH and ethyl ether to yield 1.5 g of the diamine as an HCl salt (of purity greater than 98%, NMR and MS are available), 19% overall yield.
Mass spectra data were obtained by Elecrospray Ionization technique on a PERKIN ELMER®/SCIEX®, API-300, TQMS with an autosampler, manufactured by SCIEX®, Toronto, Canada.
A. Library of Substituted Ethylenediamines
Mass spectroscopy served as a means for monitoring the reaction results of the library of ethylenediamines. Mass spectroscopy was done on two randomly selected rows (24 samples) per reaction plate, for roughly 28,000 compounds in pool of 10 or 30 compounds per well. Thus, if ten compounds per well were synthesized, the mass spectra for each well should contain ten signals, correlating with the proper molecular ions for each compound. The presence or absence of a particular signal indicated the feasibility of the particular synthesis. Based on the mass spectral data, and on a general analysis of the reactivity of the various amines, it is estimated that 67,000 compounds were formed out of 112,000 compounds.
1H NMR Spectroscopy
Proton NMR data was recorded on a VARIAN® Nuclear Magnetic Resonance Spectrometer (Palto Alto, Calif.) at 500 MHz.
Representative substituted ethylene diamines were purified by HPLC, and analyzed by proton NMR. A representative proton NMR profiles is shown in
NMR and MS data for some representative hit compounds are shown below.
Compound 6.
N2-(1-Adamantylmethyl)-N1-(3,3-diphenylpropyl)propane-1,2-diamine. 55 mg, 36% yield. 1H NMR: δ 7.28-7.15 (m, 5H), 3.95 (t, J=7.9 Hz, 1H), 2.94 (br s 4H), 2.71 (dd, J=7.6, 9.8 Hz, 2H), 2.41 (s, 2H), 2.32 (dd, J=7.6, 7.9 Hz, 2H), 2.16 (s), 2.08-1.98 (m, 4H), 1.72 (m, 6H), 1.62 (m, 6H), 1.51 (d, J=2.4 Hz, 3H). Mass spectrum (ESI) m/z (MH)+417.
Compound 7.
N-(3,3-Diphenylpropyl)-N′-(1-adamanthylmethyl)ethane-1,2-diamine. 28 mg, 22% yield. 1H NMR (500 MHz) δ 7.30-7.12 (m, 10H); 3.95 (t, J=7.6 Hz, 1H); 2.91 (d, J=1.2 Hz, 4H); 2.70 (dd, J=7.6 and 1.2 Hz, 2H); 2.40 (d, J=1.3 Hz, 2H); 2.32 (q, J=8.0 Hz, 2H); 1.98 (br d, J=1.7 Hz, 4H); 1.72 (d, J=12.2 Hz, 4H); 1.62 (d, m? J=12.2 Hz, 4H); 1.51 (br s, 6H). Mass spectrum (ESI) m/z (MH)+403.6.
Compound 10.
N-(−)-cis-Myrtanyl-N′-(3,3-diphenylpropyl)ethane-1,2-diamine. 14 mg, 11% yield. 1H NMR (500 MHz) δ 7.30-7.10 (m, 10H); 3.95 (m, 1H); 2.92-2.83 (m, 4H); AB: 2.80 (d, J=7 Hz, 1H); 2.76 (d, J=8 Hz, 1H); 2.65 (dd, J=9.6 and 7.6 Hz, 2H); 2.42-2.20 (m, 4H), 2.29 (d, J=8 Hz, 2H), 1.90 (m, 8H); 1.42 (m, 1H); 1.19 (m, 2H); 1.17 (s, 3H); 0.95 (s, 3H); 1.00-0.8 (m, 2H). Mass spectrum (ESI) m/z (MH)+391.3.
Compound 14.
N-(3,3-Diphenylpropyl)-N′-exo-(2-norborny)ethane-1,2-diamine. 17 mg, 16% yield. 1H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 3.95 (t, J=7.9 Hz, 1H); 2.86 (dd, J=11.5 and 1.5 Hz, 4H); 2.73 (dd, J=8.0 and 3.3 Hz, 1H); 2.64 (t, J=7.6 Hz, 2H); 2.29 (t, J=7.5 Hz, 2H), 2.31-2.26 (m, 2H) 2.30 1.96 (s, 3H); 1.63 (ddd, J=13.1, 7.9 and 2.5 Hz, 1H); 1.60-1.50 (m, 1H); 1.50-1.43 (m, 2H); 1.30 (dq, J=4.0 and 13.5 Hz, 1H), (1H, m), 1.20 (dd, J=10.4 and 1.1 Hz, 1H), 1.11 (dd, J=2.0, and 8.5 Hz, 1H), 1.08 (dd, J=2.5, and 8.5 Hz, 1H), 1.10 (dq, J=8.3 and 2.1, 2H). Mass spectrum (ESI) m/z (MH)+349.1.
Compound 21.
N-(3,3-Diphenylpropyl)-N′-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine. 5 mg, 4% yield. Mass spectrum (ESI) m/z (MH)+365.5.
Compound 32.
N-(2,2-Diphenylethyl)-N′-®-(+)-bornylethane-1,2-diamine. 58 mg, 48% yield. 1H NMR (500 MHz): δ 7.30-7.10 (m, 10H); 4.18 (t, J=6.8 Hz, 1H); 3.34 (d, J=7.6 Hz, 2H); 3.02 (m, 4H); 2.95-2.90 (m, 1H); 2.15-2.08 (m, 1H); 1.94 (m, 1H); 1.72-1.65 (m, 2H); 1.48-1.30 (m, 2H); 1.27-1.10 (m, 2H); 1.06 (dd, J=13.6 and 4.1 Hz, 1H); 0.82 (s, 3H); 0.81 (s, 3H); 0.78 (s, 3H). Mass spectrum (ESI) m/z (MH)+377.2
Compound 34.
N-(2,2-Diphenylethyl)-N′-(1-adamanthylmethyl)ethane-1,2-diamine. 6.8 mg, 6% yield. 1H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 4.15 (t, J=7.6 Hz, 1H); 3.24 (dd, J=7.9 and 1.2 Hz, 2H); 2.79 (t, J=6.5 Hz, 2H); 2.74 (t, J=6.0 Hz,m, 2H); 1.95 (m, 8H); 1.69 (d, J=12.5 Hz, 4H); 1.59 (d, J=11.9 Hz, 4H); 1.40 and 1.39 (br s, 3H); Mass spectrum (ESI) m/z (MH)+389.0.
Compound 37.
N-(2,2-Diphenylethyl)-N′-(−)-cis-myrtanylethane-1,2-diamine. 54 mg, 38% yield. 1H NMR: δ 7.31-7.18 (m, 10H), 4.13 (t, J=7.6 Hz, 1H), 3.26 (d, J=7.6 Hz, 2H), 2.86 (dd, J=4.3, 8.0 Hz, 4H), 2.76 (dd, J=7.6, 12.2 Hz, 2H), 2.37 (ddd, J=1.8, 9.0, 12.5 Hz, 1H), 2.12 (dq, J=1.8, 7.6 Hz, 1H), 1.98 (br s, 2H), 1.98-1.84 (m, 4H), 1.39 (ddd, J=2.4, 4.0, 6.1 Hz, 1H), 1.18 (s, 3H), 0.95 (s, 3H), 0.91 (d, J=10.0 Hz, 1H) Mass spectrum (ESI) m/z (MH)+377.2.
Compound 38.
N-(−)-cis-Myrtanyl-N′-(2,2-diphenylethyl)propane-1,2-diamine. 39 mg, 30% yield. 1H NMR (500 MHz) δ 7.30-7.15 (m, 10H); 4.13 (t, J=8.0 Hz, 1H); AB: 3.28 (d, J=7.5 Hz, 1H); 3.24 (d, J=7.5 Hz, 1H), 3.26 (d, J=6.1 Hz, 2H); 2.96 (m, 1H); 2.88-2.75 (m, 2H); 2.71 (ddd, J=4.5, 9.0, 13.0 Hz, 1H), 2.58 (ddd, J=7.0, 10.0, 14.0 Hz, 1H); 2.35 (m, 1H); 2.21 (m, 1H); 2.00-1.80 (m, 6H); 1.40-1.20 (m, 1H); 1.17 (s, 3H); 0.93 (s, 3H); 0.89 (dd, J=9.7 and 4.2 Hz, 1H). Mass spectrum (ESI) m/z (MH)+391.0.
Compound 40.
N-(2,2-Diphenylethyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine. 33 mg, 23% yield. 1H NMR: δ 7.31-7.18 (m, 10H), 4.13 (t, J=7.5 Hz, 1H), 3.27 (d, J=8.0 Hz, 2H), 3.14 (dt, J=6.0, 10 Hz, 1H), (4H), 2.36 (qd, J=2.0, 6.0 Hz, 1H), 2.34 (dt, J=2.0, 10 Hz, 1H), 2.07-1.96 (m, 3H), 1.82 (dt, J=2.0, 6.0 Hz, 1H), 1.71 (ddd, J=2.5, 5.5, 13.5 Hz, 1H), 1.22 (s, 3H), 1.09 (d, J=7.0 Hz, 3H), 0.96 (d, J=10.5 Hz, 1H), 0.91 (s, 3H). Mass spectrum (ESI) m/z (MH)+377.3.
Compound 47.
N-(−)-cis-Myrtanyl-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine. 42 mg, 33% yield. 1H NMR: 33.35-3.20 (m, 6H), 2.93 (dd, J=4.6, 2.0 Hz, 2H), 2.45-2.33 (m, 4H), 2.17 (s, 3H), 2.06 (quint, J=7.0 Hz, 1H), 2.0-1.9 (m, 6H), 1.90 (dd, J=2.1, 5.2 Hz, 1H), 1.87 (dt, J=1.8, 4.6 Hz, 1H), 1.51 (ddd, J=4.6, 10.0, 13.0 Hz, 1H), 1.23 (s, 3H), 1.19 (s, 3H), 1.12 (d, J=8 Hz, 3H), 1.03 (d, J=10.3 Hz, 1H), 0.98 (s, 3H), 0.94 (d, J=9.8 Hz, 1H), 0.94 (s, 3H). Mass spectrum (ESI) m/z (MH)+333.6.
Compound 52.
N-(3,3-Diphenylpropyl)-N′-cyclooctylethane-1,2-diamine. 20 mg, 18% yield. 1H NMR (500 MHz): δ 7.30-7.10 (m, 10H); 3.96 (t, J=7.9 Hz, 1H); 3.00 (m, 1H); 2.90 (dd, J1=J2=5.5 Hz, 2H); 2.84 (dd, J1=J2=5.0 Hz, 2H); 2.61 (t, J=7.3 Hz, 2H), 2.27 (q, J=7.6 Hz, 2H); 1.83 (m, 2H); 1.74 (m, 2H); 1.65-1.40 (m, 10H).
Compound 55.
N-(1-Adamantylmethyl)-N′-cyclooctylethane-1,2-diamine. 6.7 mg, 6% yield. 1H NMR (500 MHz): δ 3.08-3.02 (m, 1H), 3.02-2.98 (m, 2H); 2.97-2.92 (m, 2H); 2.36 (s, 2H); 1.98 (m, 2H); 1.93-1.86 (m, 2H); 1.80-1.50 (m, 19H).
Compound 57.
N-(−)-cis-Myrtanyl-N′-(cyclooctyl)ethane-1,2-diamine. 18 mg, 18% yield. 1H NMR (500 MHz) δ 3.05-2.95 (m, 4H); AB: 2.76 (d, J=7.5 Hz, 1H), 2.23 (d, J=8.0 Hz, 1H); 2.76 (dd, J=11.6 and 7.3 Hz, 1H); 2.73 (dd, J=11.9 and 8.2 Hz, 1H); 2.40-2.34 (m, 1H); 2.28 (quintet, J=8.0 Hz, 1H); 1.97 (s, 3H); 2.00-1.84 (m, 6H); 1.80-1.70 (m, 2H); 1.68-1.38 (m, 1H); 1.18 (s, 3H); 0.97 (s, 3H); 0.92 (d, J=9.8 Hz, 1H). Mass spectrum (ESI) m/z (MH)+307.5.
Compound 58.
N-(2-Adamantyl)-N′-cyclooctylethane-1,2-diamine. 25 mg, 23% yield. 1H NMR: δ 3.06 (m, 1H), 3.00 (t, J=6.1 Hz, 2H), 2.93 (t, J=5.5 Hz, 2H), 2.83 (br s, 1H), 1.96 (s, 3H), 1.92-1.80 (m, 10H), 1.80-1.50 (m, 20H). Mass spectrum (ESI) m/z (MH)+305.1.
Compound 59.
N-(Cyclooctyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine. 15 mg, 14% yield. 1H NMR (400 MHz): δ 3.47 (dt, J=6.0, 10.0 Hz, 1H), 3.40-3.28 (m, 7H), 2.44 (tq, J=2.0, 10.0 Hz, 1H), 2.36 (dtd, J=2.0, 6.0, 10.0 Hz, 1H), 2.09 (dq, J=2.0, 7.2 Hz, 1H), 2.00-1.90 (m, 3H), 1.88-1.78 (m, 2H), 1.78-1.63 (m, 4H), 1.65-1.30 (m, 8H), 1.18 (d, J=6.0 Hz, 3H), 1.16 (s, 3H), 1.17 (d, J=7.2 Hz, 1H), 0.90 (s, 3H). Mass spectrum (ESI) m/z (MH)+307.4.
Compound 62.
N-(−)-cis-Myrtanyl-N′-(1S)-(1-ethylcyclohexane)ethane-1,2-diamine. 48 mg, 46% yield. 1H NMR (500 MHz): δ 3.06-3.00 (m, 1H); 2.98-2.95 (m, 2H); 2.92-2.84 (m, 1H); 2.79 (dd, J=11.9 and 7.0 Hz, 1H); 2.75 (dd, J=11.9 and 7.9 Hz, 1H); 2.73 (m, 1H); 2.39 (m, 1H); 2.28 (quintet, J=8.5 Hz, 1H); 2.00-1.86 (m, 6H); 1.82-1.76 (m, 2H); 1.68 (m, 2H); 1.54-1.42 (m, 2H); 1.32-1.10 (m, 6H); 1.19 (s, 3H); 1.13 (d, J=6.7 Hz, 3H); 1.07 (dd, J=12 and 3 Hz, 2H); 1.02 (dd, J=12 and 3 Hz, 2H); 0.98 (s, 3H); 0.93 (d, J=9.7 Hz, 1H). Mass spectrum (ESI) m/z (MH)+306.9.
Compound 65.
N-trans-(2-phenylcyclopropyl)-N′-(1-adamanthyl)ethane-1,2-diamine. 18 mg, 16% yield. Mass spectrum (ESI) m/z (MH)+311.3.
Compound 66.
N-(3,3-Diphenylpropyl)-N′-(1R,2R,3R,5S)-(−)-isopinocampheylethane-1,2-diamine. 2 mg, 2% yield. 1H NMR (500 MHz) δ 7.26 (m, 10H); 3.96 (t, J=7.6 Hz, 1H); 3.09 (m, 1H); 2.92 (m, 1H); 2.84 (m, 2H); 2.62 (m, 2H); 2.35 (m, 4H); 1.97 (s, 3H); 1.82 (m, 1H); 1.68 (m, 1H); 1.21 (s, 3H); 1.12 (d, J=7.3 Hz; 3H); 1.01 (m, 1H); 0.92 (s, 3H). Mass spectrum (ESI) m/z (MH)+391.4.
Compound 73.
N-(2-Adamantyl)-N′-[2-(2-methoxyphenyl)ethyl]ethane-1,2-diamine. 21 mg, 19% yield. 1H NMR: δ 7.22 (dd, J=8.2, 7.3 Hz, 1H), 7.14 (d, J=7.3 Hz, 1H), 6.89 (d, J=7.1, Hz, 1H), 6.87 (d, J=8.2, Hz, 1H), 3.81 (s, 3H), 3.06 (t, J=7.1 Hz, 2H), 3.06 (m, 2H), 3.01 (m, 2H), 2.93 (t, J=7.1, 2H), 1.95 (br s, 2H), 1.90-1.80 (m, 7H), 1.78-1.66 (m, 6H), 1.59 (d, J=2.5 Hz, 2H). Mass spectrum (ESI) m/z (MH)+329.4.
Compound 78.
N-2-Adamantyl-N′-2,3-dihydro-1H-inden-2-yl-ethane-1,2-diamine. 4.3 mg, 3% yield. 1H NMR: δ 7.20 (dd, J=4.9, 8.5 Hz, 2H), 7.14 (dd, J=5.5, 2.1 Hz, 2H), 3.71 (quint, J=6.1 Hz, 2H), 3.19 (dd, J=5.8, 15.9 Hz, 2H), 3.13 (br.s, 1H), 3.05 (m, 4H), 2.86 (dd, J=4.8, 15.8 Hz, 2H), 2.08 (m, 2H), 2.00 (m, 6H), 1.96-1.88 (m, 4H), 1.88-1.80 (m, 3H). 1.74 (m, 4H), 1.68-1.60 (m, 2H). Mass spectrum (ESI) m/z (MH)+303.4.
Compound 109.
N-Geranyl-N′-(2-adamanthyl)ethane-1,2-diamine. 27 mg, 24% yield. 1H NMR (400 MHz): δ 5.40 (t, J−7.2 Hz, 1H), 4.78 (br s, 2H), 3.64 (d, J=7.6 Hz, 2H), 3.34 (m, 2H), 2.07 (m, 2H), 2.08-1.95 (m, 4H), 1.95-1.85 (m, 4H), 1.82 (m, 2H), 1.88-1.70 (m, 4H), 1.70-1.62 (m, 3H), 1.67 (s, 3H), 1.56 (s, 3H), 1.50 (s, 3H). Mass spectrum (ESI) m/z (MH)+307.4.
Compound 111.
N-Geranyl-N′-(2-ethylpiperidine)ethane-1,2-diamine. 44 mg, 42% yield. 1H NMR (500 MHz): δ 5.22 (t, J=6.1 Hz, 1H); 5.04 (m, 1H), 3.52 (d, J=7.3 Hz, 2H); 3.05-2.85 (m, 4H); 2.66 (m, 1H); 2.44 (m, 2H); 2.08 (m, 4H); 1.80-1.50 (m, 2H); 1.70 (s, 3H); 1.65 (s, 3H); 1.58 (s, 3H); 1.50-1.35 (m, 2H), 0.89 (t, J=7.3, 3H). Mass spectrum (ESI) m/z (MH)+293.4
Compound 116.
N-Geranyl-N′-allyl-N′-(cyclopentyl)ethane-1,2-diamine. 45 mg, 42% yield. 1H NMR: δ 5.86 (ddd, J=10.0, 16.1, 6.7 Hz, 1H), 5.28 (d, J=15.9 Hz, 1H), 5.25 (d, J=8.7 Hz, 1H), 5.23 (t, J=7.3 Hz, 1H), 5.30 (m, 1H), 3.59 (d, J=7.3 Hz, 2H), 3.28 (br d, J=6.4 Hz, 2H), 3.16 (quintet, J=8.2 Hz, 1H), 3.02 (m, 2H), 2.95-2.86 (m, 2H), 1.88-1.80 (m, 4H), 1.70 (s, 3H), 1.74-1.66 (m, 3H), 1.65 (s, 3H), 1.58 (s, 3H), 1.56-1.50 (2H), 1.50-1.40 (m, 2H). Mass spectrum (ESI) m/z (MH)+305.3.
Compound 117.
N-Geranyl-N′-diphenylmethylethane-1,2-diamine. 24 mg, 20% yield. 1H NMR (500 MHz): δ 7.40 (d, J=7.2 Hz, 4H); 7.29 (t, J=7.3 Hz, 4H); 7.21 (t, J=7.0 Hz, 2H); 5.15 (t, J=7.5, 1H); 5.01 (m, 1H); 4.89 (br s, 1H); 3.42 (d, J=7.0 Hz, 2H); 3.00-2.78 2.93 (m, 4H); 2.20-2.00 2.17 (m, 4H); 1.63 (s, 3H); 1.59 (s, 3H); 1.56 (s, 3H). Mass spectrum (ESI) m/z (MH)+363.3.
Compound 125.
N,N′-bis-(−)-cis-Myrtanylpropane-1,2-diamine. 82 mg, 70% yield. 1H NMR (500 MHz): δ 3.62 (m, 1H); 3.18 (dd, J=13.7 and 3.7 Hz, 1H); 3.05 (dt, J=11.5 and 7.5 Hz, 1H); 3.06-2.92 (m, 2H); 2.86 (dt, J=12.2 and 7.3 Hz, 1H); 2.40 (m, 4H); 2.06-1.84 (m, 10H); 1.56-1.46 (m, 2H); 1.37 and 1.36 (two d, J=6.7 and J=7.0 Hz, 3H); 1.20 (s, 3H); 1.19 (m, 3H), 0.99 and 0.98 (two s, 3H) Hz, H); 0.97 (s, 3H); 0.94 (two d, J=10.1 Hz, 2H). Mass spectrum (ESI) m/z (MH)+346.9.
Compound 151.
N-[2-(2-Methoxy)phenylethyl]-N′-(1R,2R,3R,5S)-(−)-isopinocampheyl-ethane-1,2-diamine. 67 mg, 60% yield. 1H NMR (500 MHz): δ 7.23 (t, J=5.8 Hz, 1H); 7.13 (dd, J=5.8 and 1.8 Hz, 1H); 6.88 (m, 2H); 3.81 (s, 3H); 3.13 (m, 1H); 3.1-3.0 (m, 3H); 3.01 (t, J=7.0 Hz, 2H); 2.89 (t, J=7.0 Hz, 2H); 2.42-2.35 (m, 2H); 2.00 (m, 3H); 1.82 (dt, J=6.0 and 2.0 Hz, 1H); 1.72 (ddd, J=2.5, 5.5, 13.5 Hz, 1H); 1.22 (s, 3H) 1.13 (d, J=7.3 Hz, 3H). 0.99 (d, J=10.1 Hz, 1H); 0.93 (s, 3H). Mass spectrum (ESI) m/z (MH)+331.5.
N-2-(2-Methoxyphenyl)ethyl-N′-allyl-N′-cyclopentyl-ethane-1,2-diamine. 8 mg, 7% yield. 1H NMR: δ 7.26 (dd, J=7.3, 8.5, 1H), 7.18 (d, J=7.2 Hz, 1H), 6.91 (m, 2H), 5.61 ddd, (J=6.7, 17.0, 9.4 Hz, 1H), 5.13 (d, J=15.3 Hz, 1H), 5.10 (d, J=9.2 Hz, 1H), 3.83 (s, 3H), 3.13 (dd, J=7.0, 6.7 Hz, 2H), 3.10 (d, J=6.7 Hz, 1H), 3.00 (d, J=7.3 Hz, 1H), 3.05-2.90 (m, 2H), 2.97 (dd, J=8.2, 6.1 Hz, 2H), 2.75 (t, J=6.1 Hz, 2H), 1.73 (m, 2H), 1.62 (m, 2H), 1.50 (m, 2H), 1.22 (m, 2H). Mass spectrum (ESI) m/z (MH)+311.4.
N2-(3-Phenylpropyl)-N′-[2-(4-fluorophenyl)ethyl]-1-phenylethane-1,2-diamine. 23 mg, 19% yield. 1H NMR: δ 7.35 (d, J=7.6 Hz, 2H), 7.34 (quart, J=7. Hz, 1H), 7.26 (d, J=6.4 Hz, 3H), 7.23 (d, J=7.6 Hz, 2H), 7.17 (dd, J=7.3, 6.4 Hz, 1H), 7.12 (d, J=7.0 Hz, 2H), 3.21 (m, 1H), 3.03 (ddd, J=4.2, 8.0, 12.8 Hz, 4H), 2.86 (t, J=8.0 Hz, 2H), 2.85-2.79 (m, J=12. Hz, 2H), 2.74-2.64 (m, 4H), 2.61 (t, J=7.7 Hz, 2H), 1.96 (quint, J=7.6 Hz, 2H). Mass spectrum (ESI) m/z (MH)+377.3.
Substituted ethylene diamines, as described herein, were tested on Mycobacterium tuberculosis using high-throughout screening assay with recombinant mycobacterial containing promoter fusion of luciferase to Rv0341EMB-inducible promoter. This assay quickly and reliably identifies antimycobacterial activity in compound mixtures and/or in individual compounds. In this assay, bioluminescence increases when the mycobacteria is tested against an active compound, or an active compound mixture. During this assay, a theoretical yield of 100% was assumed for every unpurified substituted ethylene diamine, and the activity of each sample was compared to commercially available ethambutol (99.0% purity). Results were reported in LCPS, and % Max. LCPS based on the activity of EMB at 3.1 μM.
The substituted ethylene diamines were analyzed according to the following procedure. The diamines were dried in a speed vacuum to an approximate concentration of 6.3 mmoles per well. Each diamine, or diamine mixture, was then resuspended or dissolved in 200 μl of methanol for a concentration of 31.5 mM diamine(s). The diamine(s) solution was diluted to a concentration of 200 μM in 7H9 broth medium (a 1:15.75 dilution of the 31.5 mM stock, followed by a 1:10 dilution; each dilution in 7H9 broth medium). Next, 50 μl of the diluted diamine(s) solution was added to the first well of a row of twelve in an opaque, 96-well plate. The 7H9 broth medium, 25 μl, was added to each of the remaining wells (#2-12) in the row. The diamine(s) solution in “well one” was serially diluted by transferring 25 μl from “well one” to “well two”, and repeating a 25 μl transfer from “well two” to “well three”, and so on, on through “well eleven”. In “well eleven”, the extra 25 μl of solution was discarded. “Well twelve” was used as a growth control to assess background activity of the reporter strain. The plate was then covered and incubated at 37° C. for 24 hours. Immediately prior to analysis, the following substrates were prepared: a buffer solution containing 50 mM HEPES at pH 7.0 and 0.4% Triton X-100. Then, 0.25 ml of 1M DTT, and 14 μl of 10 mg/ml luciferin in DMSO were added to 5 ml of the buffer solution. This final solution (50 μl) was added to each of the twelve wells, immediately after the incubation period had run. The luminescence from each well was measured 20 minutes after the luciferin substrate was added, using a TOPCOUNT® (Downers, Grove, Ill.) NXT luminometer (55/well).
The Minimum Inhibition Concentration (MIC) is the concentration of the growth inhibitor, here a substituted ethylene diamine, at which there is no multiplication of seeded cells. A microdilution method was used to determine the MIC of the substituted ethylene diamines, capable of inhibiting the growth of Mycobacterium tuberculosis in vitro. In a representative MIC experiment, bacteria, the H37Rv strain of Mycobacterium tuberculosis (M.tb), was cultivated in 7H9 medium to a density of 0.2 OD (optical density) at 600 nm. The bacterial culture was then diluted 1:100 in 7H9 broth medium. Stock solutions of isoniazid and ethambutol were each prepared at 32 μg/ml in 7H9 medium. A 3.2 mg/ml solution of isonizid and ethambutol were each prepared in water. The solutions were then filtered, and diluted 1:100 in 7H9 medium. Each drug, purchased from Sigma, was “laboratory use only” grade. A 10 mM solution of each substituted ethylene diamine was prepared in methanol. Next, 100 μl of the 7H9 medium was added to each well in a 96-well plate (rows (A through H)×columns (1 through 12)). To the first wells in rows C through H was added an additional 80 μl of the 7H9 medium. The isoniazid solution, 100 μl, was added to well A1, and the ethambutol solution, 100 μl, was added to well B1. Six substituted ethylene diamines, 20±1 each, were added to wells C1 through H1 (column 1), respectively. A serial dilution of each substituted ethylene diamine and the isoniazid and ethambutol controls, was performed across each row. For example, a serial dilution across row C1-C12 was done by mixing and transferring 100 μl of the previous well to the next consecutive well. In each well in “column 12,” 100 μl of the final dilution was discarded. Next, 100 μl of the diluted H37Rv strain of M.tb was added to each well. The 96-well plate was then covered and incubated at 37° C. for 10 days. The plate was read for bacterial growth, or non-growth, using an inverted plate reader. The MIC was determined to be the lowest concentration of substituted ethylene diamine that inhibited visible growth of the M. tuberculosis.
A representative plate layout, listing concentration in each well, is shown in Table 9. Table 10 lists MIC and LD50 data for selected compounds. The LD50 is the concentration of the substituted ethylene diamine at which 50% of the cells (H37Rv strain of M.tb) are killed. Table 11 lists MIC data for purified substituted ethylene diamines in comparison to ethambutol (EMB).
The above procedure was also used to examine batched compounds (10 compounds per well). Synthesized batches of substituted ethylene diamines were dried in speed vacuum and then resuspended in DMSO or sterile water to a concentration of 2.5 mg/ml.
Secondary screening was performed on some of the substituted ethylene diamine compounds to examine their activity against three clinically resistant MDR patient isolates. MDR Strain TN576 is classified as a W1 strain (STPR, INHR, RIFR, EMBR, ETHR, KANR, CAPR) strain TN587 is classified as a W strain (STPR, INHR, RIFR, EMBR, KANR), and the third strain TN3086 is classified as a W1 strain (STPR, INHR, RIFR, EMBR, KANR). Each MDR strain is highly resistant to ethambutol with MIC values exceeding 12.5-25 μM. The MICs for the following substituted ethylene diamines, MP 116, MP 117, RL 241, compounds #59 and #109, were determined for all three patient isolates.
The results from this study are shown in Tables 12-13. Table 14 characterizes each MDR strain according to its resistance.
Animal models were used in the final stages of the drug discovery cycle to assess the anti-microbial efficacy of some substituted ethylanediamine compounds in a representative system of human disease state. The in vivo testing approach involves the inoculation of four-six week old C57BL/6 mice via aerosol, containing approximately 200 colony forming units of M. tuberculosis H37Rv.
A. CFU Lung Study
Mice aerosolized with M. tuberculosis H37Rv were examined for 10 to 12 weeks following inoculation. Drugs (substituted ethylene diamines) were administered via the esophageal cannula (gavage) 7 days/week, starting at either 14 or 21 days post infection. Bacterial load in the lungs of five mice per group were determined at approximately one-week intervals by viable colony counts. The drugs tested were directly compared to the front line anti-tuberculosis drug isoniazid, and to the second line drug, ethambutol. Isoniazid and ethambutol were tested at 25 mg/kg and 100 mg/kg, respectively. The substituted ethylene diamines, compound 37, compound 59 and compound 109, were each tested at 1 mg/kg and 10 mg/kg.
B. Lesion Study
The ability of compound 59 and compound 109 to prevent the development of gross pathology due to bacterial burden was determined in conjunction with the CFU/Lung Study. The gross pathology was determined by visible quantitation of lesions on the surface of the lungs. Quantitation by inspection is a good surrogate for CFU determination, and directly correlates to the bacterial burden, as determined by the actual colony forming units. The lesions are first visibly examined, and then the lungs are processed and plated for CFU quantification. The lesion study demonstrates the ability of the drug to prevent the development of the disease pathology.
C. Toxicity Study
Toxicity was assessed using a dose escalation study. This study was performed with ten C57BL/6 mice per candidate. Every two days, the mice were administered an increased concentration of the drug, and monitored for detrimental effects. The administration scheme was 50, 100, 200, 400, 600, 800 and 1000 mg/kg. The maximum limit of 1000 kg/mg was based on the goal of dose escalation, and the solubility of the drugs in the delivery vehicle. Compound 37 was toxic in mice at 100 kg/mg. Compound 59 and compound 109 were tolerated in mice at 1000 mg/kg and 800 mg/kg, respectively.
It should be understood that the foregoing relates only to preferred embodiments of the present invention, and that numerous modifications, or alterations, may be made therein without departing from the spirit and scope of the invention. The entire text of each reference mentioned herein is hereby incorporated, in its entirety, by reference.
Twenty six compounds (including 37, 59 and 109) were tested in an in vitro model of toxicity using monkey kidney cells (Vero) and human cervical cancer cells (HeLa) using methods well known to those skilled in the art. The data from this toxicity testing and the MIC data were used to calculate a selectivity index (SI), the ratio of IC50:MIC (Table 15). Selectivity Indexes were ranging from 1.76 to 16.67. Compound 109 has the best selectivity index.
Compounds 58, 59, 73, 109, and 111 were selected for in vivo efficacy studies in a mouse model of TB. Compounds 58 and 59 share the same cyclooctyl fragment in their molecules; compounds 58, 73, and 109 share adamantly moiety, and 109 and 111—the geranyl fragment (
In these studies, 8-week old inbred female mice C57BL/6 were intravenously infected with M. tuberculosis. 3 weeks following infection drug treatment was initiated (detailed protocol is provided). The drugs were administered orally by gavage. Mice were sacrificed at three timepoints (15, 30, and 45 days post infection), and CFUs in spleen and lungs were determined (
Materials and Methods
Mice. Female C57BL/6 mice of 8 weeks old were purchased from Charles River (Raleigh, N.C.), housed in BSL-2 facility of BIOCAL, Inc. (Rockville, Md.), and were allowed to acclimate at least 4 days prior infection.
Mycobacteria. An example of frozen and thawed of M. tuberculosis H37Rv Pasteur was added to 5 ml 7H10 broth medium, with 0.5% BSA and 0.05% Tween 80, incubated 1 week at 37° C., and then 1 ml was added into 25 ml medium (2-d passage during 2 weeks). Culture was washed twice and resuspended in PBS with 0.5% BSA and 0.05% Tween 80, aliquoted and frozen at −80° C. To determined CFU of the culture aliquot was thawed, and 10-fold dilutions will be plated on agar 7H9 and CFU count will be calculated 20 days later.
Infection: Frozen sample of culture was thawed, and diluted for concentration about 106 CFU/ml. Mice were infected with M. tuberculosis H37Rv intravenously through lateral tail vein in corresponded dose in 0.2 ml of PBS.
Antimicrobial agents. INH, EMB, Ethambutol analogues.
Protocol of drug treatment: Treatment of mice with compounds was initiated 20 days following infection. Compounds were dissolved in 10% ethanol in water and administered by gavage (0.2 ml per mouse). Therapy was given 5 days per week and continued for four or six weeks. Two, four and six weeks following chemotherapy start mice (6 mice per group) were sacrificed, lungs and spleens were removed and homogenized in sterile in 2 ml PBS with 0.05% Tween-80. Homogenates were plated in serial dilutions on 7H10 agar dishes, and incubated at 37° C. CFU counts were calculated three weeks later.
Statistic analysis. To analyze results of CFUs in organs ANOVA test was performed; the significance of the differences was estimated by Student's test, p<0.05 was considered statistically significant.
Results
In vivo activities of new compounds. The activities of these compounds are presented in
Compounds 73 and 109 were also tested in shorter model with using higher dose of infection (
Testing of compounds 111 and 59 was performed in B6 mice infected with 5×105 CFU M. tuberculosis H37Rv and beginning chemotherapy 20 days following infection (
In all experiments, INH showed higher activity than EMB and other compounds decreasing load of bacteria in organs on 2-3 logs during 4-6 weeks of chemotherapy; new compounds similar to EMB (100 mg/kg) decreased load of bacteria on 1.0-2.0 logs. Among studied compounds 73 and 109 are the most preferable, because the highest capacity to decrease mycobacteria in organs and its parameters of toxicity and pharmacology kinetics.
Preliminary dose acceleration studies in mice have indicated that compound 109 can be well tolerated at doses up to 800 mg/kg and compound 59 up to 1000 mg/kg. Compound 37 was fatal at doses 100 mg/kg (Clif Barry, NIAID, unpublished results).
Compound 109 was mostly used in the form of dihydrochloride at five different doses, and 37—solely as hydrochloride salt at two doses.
Mice were given a one-time dose of the compounds at concentrations 100, 300 or 1000 mg/kg using the gavage method. Each dose of each compound consisted of one group of 3 mice. Monitoring of the mice was done twice a day for the duration of the experiment. Mice surviving one week post-drug administration were sacrificed; critical organs were aseptically removed and observed for abnormalities and evidence of drug toxicity. The MTD (mg/kg) is the highest dose that results in no lethality/tissue abnormality.
Methods:
These studies indicate that the maximum tolerated dose for the compound 109 is 600 mg/kg (Table 16). No visible changes in organs were observed. Dose 800 mg/kg was fatal: out of a group of 3 mice, two animals died within 3 days (Table 17). Compound 37 was well tolerated at doses 100 and 300 mg/kg. No visible changes in organs were observed. Additional experiments to evaluate maximum tolerated dose and in vivo efficacy for the compound 37 are being conducted.
Initially, analytical methods for determination of the compounds had been developed that allowed to carry out all the PK experiments, sec
Biostability studies of the compounds in plasma were carried out using concentrations 1 and 15 mg/ml. The compounds were incubated for 1, 2, 3 & 6 hr at 37° C. (Table 18). In addition, it was found that all tested compounds were stable in plasma at 24° C., pH 2 and 7.4 up to 24 hr.
Pilot PK study of the compounds 37, 59, and 109 in mice was conducted using a cassette dosing: all the three analogs were formulated together in saline at 1.5 mg/mL, and administered to mice simultaneously orally at 25 mg/kg, peritoneally at 6 mg/kg, and intravenously. It was found that doses 15 and 7.5 mg/kg caused death of mice, 3.75 mg/kg appeared lethargic immediately after dosing but then appeared normal appearance a few minutes later; 3 mg/kg displayed no adverse reactions and hence was used as intravenous dose. Obtained data are presented on
Conducted pharmacokinetic studies indicated that compound 59 (NSC 722040 by the NCI index) has relatively poor PK profiling (AUC, Cmax) and further testing of this compound was abandoned. Based on preliminary toxicity data compound 37 was also ruled out as possible candidate. Therefore, compound 109 (NSC 722041 by the NCI) was selected for further PK analyses.
It has been shown that compound SQ 109 reaches and exceeds its Minimum Bactericidal Concentration MBC (313 ng/ml) in plasma when administered either iv or intravenously orally (p.o.), has a half-life of 5.2 h, and has total clearance less than hepatic blood flow (
Its oral bioavailability is only 3.8% when administered p.o but this is explained by its unique tissue distribution pattern. Tissue distribution studies have demonstrated that SQ109 primarily distributes into the lungs and spleen (
By using an ultracentrifugation method, it was found that plasma protein binding of the compound 109 is concentration dependent and varies from 15% (20 ng/ml) to 74% (200 ng/ml) to 48% (2000 ng/ml). After i.v. dosing (3 mg/kg) the compound distributes between plasma and red blood cells in a ratio 70.6:29.4.
Little is known of the fate of the compound in the body, since the total amount of the compound after excretion (urine and feces) does not exceed 3% of the delivered dose (Table 2).
Initial attempts to identify metabolites of the compound 109 in urine, did not provide evidence of breakdown products,
In vitro Pharmacology and early ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) studies of the compound 109 were contracted out to CEREP (15318 NE 95th Street, Redmond, Wash. 98052, USA, www.cerep.com, tel 425 895 8666) under a Service Agreement and included testing against 30 standard receptors (see CEREP Tables 22 and 23, provided in
Compounds with the best Selectivity Indexes, such as 109, 58, 73, 78, (Table 15) and good in vivo data share the same adamantane fragment (
In the synthetic scheme used for preparation of the library Scheme 1 (
Compound SQBisAd can be prepared by “wet chemistry” using the same route, Scheme 3 (
General Methods All reagents were purchased from Sigma-Aldrich. Rink acid resin was purchased from NovaBiochem, Inc. Solvents acetonitrile, dichloromethane, dimethylformamide, ethylene dichloride, methanol, and tetrahydrofuran were purchased from Aldrich and used as received. Solid phase syntheses were performed on Quest 210 Synthesizer (Argonaut Technologies) and combinatorial chemistry equipment (Whatman Polyfiltronics and Robbins Scientific). Evaporation of the solvents was done using SpeedVac AES (Savant). Mass spectra data were obtained by Electrospray Ionization technique on Perkin Elmer/Sciex, API-300, TQMS with an autosampler.
The activation of the Rink-resin, the addition of the amine, and the acylation step were carried out in 10 ml tubes using the Quest 210 Synthesizer. Removal of the FMOC group, reductive alkylation reaction with carbonyl compounds, the reduction with Red-Al, and the cleavage from the solid support were carried out in 96-deep (2 ml) well, chemically resistant plates.
Step 1. Activation of the Rink-Acid Resin.
A suspension of the Rink-acid resin (coverage of 0.43-0.63 mmol/g), 6 g (up to 3.78 mmol), in 80 ml of 2:1 mixture of dichloromethane and THF was distributed into 20 tubes, 4 ml per tube, filtered and washed twice with THF. A solution of triphenylphosphine (5.7 g, 21.75 mmol) in 40 ml of THF was added, 2 ml/tube, followed by the addition of a solution of hexachloroethane (5.09 g, 21.45 mmol) in 20 ml of THF, 1 ml/tube. After 6 h the resins were washed with THF (2×4 ml) and dichloromethane (2×4 ml).
Step 2. Addition of the First Amine.
Each tube was charged with 3 ml of dichloroethane, EtNiPr2, (0.2 ml, 1.15 mmol), and the corresponding amine (1 mmol). (When a selected amine was a solid, it was added as a solution or a suspension in DMF). Dichloroethane was added to each tube to fill up the volume 4 ml. The reaction was carried for 8 h at 45° C. and 6-8 h at room temperature. The resins were filtered, washed with a 2:1 mixture of dichloromethane and methanol (1×4 ml), then with methanol (2×4 ml), and suck dry.
Step 3. Acylation with Fmoc Protected Amino Acid.
The resins were pre-washed with dichloromethane (2×4 ml). Each tube was charged with 2 ml of dichloromethane, HATU (2 mol excess to loaded resin, 0.14 g, 0.39 mmol, dissolved in 1 ml of DMF), and 0.47 mmol (2.5 mol excess to loaded resin) of amino acid dissolved in 1 ml of DMF, and allowed to stir for 8 h at 45° C. and 6-8 h at room temperature. After 16 h the resins were filtered, washed with 1:1 mixture of DMF and dichloromethane (1×3 ml), dichloromethane (1×3 ml) and acylation was repeated with the same amount of reagents. At the end, the resins were filtered, washed with 1:1 mixture of DMF and dichloromethane (1×3 ml), and methanol (3×3 ml), sucked dry (on Quest) for 30 min and transferred into vials (one resin per vial), and dried in a desiccator under vacuum for 1 h. After this step all resins were subjected for quality control using MS spectra.
Step 4. Alkylation of the Amino Group.
Deprotection. Ten prepared resins from the first three steps were pooled together, leaving approximately 0.05 g of each in the individual vials for all necessary deconvolutions. A suspension of the resin mixture (2.0-2.5 g) in 100 ml of a 2:1 mixture of dichloromethane and THF was distributed into two 96-well filterplates and filtered using a filtration manifold. The reaction plates were transferred into combiclamps, and 0.2 ml of 20% solution of piperidine in DMF was added to remove Fmoc protecting group and allowed to stay for 10 min. After 10 min plate was filtered, washed with 0.2 ml of DMF, and deprotection was repeated with 0.2 ml of 20% solution of piperidine in DMF and allowed to stay for 20 min. After that plate was filtered, washed with DMF (0.2 ml per well) and dichloromethane (2×0.5 ml per well).
Reaction with the carbonyl compounds. Each well in row A on the reaction plate was charged with 0.1 ml of dichloromethane, 0.08 ml of ˜1.0M solution of appropriate acid in DMF from master plate, 0.05 ml DMF solution of PyBrop, (0.015 g, 0.03 mmol, 2.5 mol excess to loaded resin) and 0.05 ml of EtNiPr2 in dichloromethane (0.022 ml, 0.13 mmol, 10 mol excess to loaded resin). Each well in rows B through H was charged with 0.1 ml of THF, 0.160 ml of 1.0 M solution of appropriate aldehyde or ketone in DMF from master plate and allowed to react for 30 min. After 30 min 0-075 ml (0.075 mmol) of 1.0 M solution of NaBCNH3 were added. The reaction plates were sealed and kept at RT for 72 h. At the end, the resins were filtered, washed with THF, DCM (1×1 ml), methanol (2×1 ml) and dried in desiccator under vacuum for 2 h.
Step 5. Reduction with Red-Al.
The reaction plates were placed into combiclamps. A 1:6 mixture of Red-Al (65+w % in toluene) and THF was added, 0.6 ml per well (0.28 mmol of Red-Al per well), and allowed to react for 4 h. After the reaction completion the resins were filtered, washed with THF (2×1 ml), methanol (3×1 ml) and dried in the filtration manifold.
Step 6. Cleavage.
This step was carried out using a cleavage manifold. The reaction plates (placed on the top of the collection plates in this manifold) were charged with a 10:85:5 mixture of TFA, dichloromethane, and methanol, 0.5 ml per well. After 15 min, the solutions were filtered and collected into proper wells of the collection plates. The procedure was repeated. Solvents were evaporated on a speedvac, and the residual samples were ready for testing.
Deconvolution of the active wells was performed by re-synthesis of discrete compounds, from the archived FMOC-protected a-aminoacetamide resins (10 resins, 0.05-0.10 g each), which were set aside at the end of the acylation step before the pooling. Each resin was assigned a discrete column (1, or 2, or 3, etc.) in a 96-well filterplate, and was divided between X rows (A, B, C, etc), where X is the number of hits discovered in the original screening plate. To each well, in a row, a selected carbonyl compound (present in the hit) was added along with other required reagents: the first selected carbonyl compound was added to the resins in the row “A”, the second carbonyl compound—to the resins in the row “B”, the third carbonyl compound—to the resins in the row “C”, etc. A lay-out of a representative 96-well deconvolution plate is shown in Table 28,
The reaction plates were sealed and kept at RT for 72 h. At the end, the resins were filtered, washed with THF, DCM (1×1 ml), methanol (2×1 ml) and dried in desiccator under vacuum for 2 h. Reduction and cleavage were performed according to steps 5 and 6 of the synthetic protocol. The product wells from the cleavage were analyzed by ESI-MS (Electrospray Ionization Mass Spectroscopy) to ensure the identity of the actives, and were tested in the MIC assay. A summary of the ESI-MS data is provided below. A list of compound hits and structures is provided in Table 30,
4-methyl-N2-[(2E)-3-phenylprop-2-enyl]-N′-(3-phenylpropyl)pentane-1,2-diamine. Mass spectrum (ESI) m/z (MH)+351.2.
Compound 109 demonstrated potent in vitro and in vivo killing of M. tuberculosis as an individual compound. Herein, are examples providing a multi-drug regime to evaluate the effects of the compounds of table 3 on inhibition of bacterial growth in vitro and in mouse models of tuberculosis when used in combination with standard tuberculosis drugs. Rifampicin, compound 109 and isoniazid were selected for in vitro activity (
A rapid in vivo model of TB where infected animal weight loss is the indicator for tuberculosis disease progression was used to elevated novel compounds and combinational therapies. Rifampicin, INH, EMB, PZA, Moxi (standard tuberculosis drugs) and compound 109 were selected for in vivo studies in mice (
In this example Rifampicin and compound 109 were selected for in vivo studies in mice (
A combination therapy of SQ109 (compound 109) at 10 mg/kg and RIF at 2 mg/kg given orally was more efficacious in preventing body weight loss in infected mice than either single drug therapy given at the same dose (
The in vivo enhanced activity of combination treatment was confirmed through the use of a standard chronic mouse model of tuberculosis (
In vitro testing for safety pharmacology and early ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) studies of compound 109 were contracted out to CEREP (15318 NE 95th Street, Redmond, Wash., 98052, USA www.cerep.com) under a service agreement and included evaluation of inhibitory binding to a panel of 27 standard receptors and three transporters (See
The specific ligand binding to the receptors is defined as the difference between the total binding and the nonspecific binding determined in the presence of an excess of unlabelled ligand. Compound 109 (SQ109) was tested at 10 μM, and the results were expressed as a percent of control specific binding (Table 31) or as a percent of inhibition of control specific binding (Table 32) obtained in the presence of SQ109.
In summary it can be seen that greater than 50% inhibition was observed for D1 and D2S dopamine receptors (51% and 73%). Similarly, very significant inhibition was observed for melanocortin MC4 receptor (90%) which is known to exert a large influence on food intake. Furthermore, a very significant inhibition was observed for muscarinic M receptors (96%).
Opiate receptors which control pain, immune responses, and functions and are linked to effects of morphine and heroin were observed to display a 58% inhibition of control specific binding. Sigma receptors that have been shown to play an important role in antidepressive effects also demonstrated a high degree of inhibition (106%). Norepinephrine NE transporter plays an important role in the pathophysiology of depression and in the mechanism of action of antidepressant drugs was observed to have 87% inhibition of control specific binding. Another transporter, Dopamine DA transporter linked to substance abuse and attention deficit hyperactivity disorder (ADHD) was observed to display 63% inhibition. Serotonin 5-HT transporter implicated in the etiology of several disease states including, but not limited to, mental illnesses, for example, depression, anxiety, schizophrenia, eating disorders, migraines, obsessive compulsive disorder, and panic disorder were also observed to display a 95% inhibition of control specific binding. While not wishing to be bound by the following theory, it is believed that compounds comprising a Ph-ethyleneamine component, including but not limited to, compound 73 also share CNS activity.
Compound 109 was tested against a representative panel of commonly encountered clinical microorganisms comprising opportunistic pathogens, target pathogens and normal human flora. The spectrum of activity of compound 109 was evaluated by performing antimicrobial susceptibility testing against a collection of aerobic and anaerobic bacteria, fungi, and mycobacteria. MICs for all organisms were established using the appropriate NCCLS (National Committee for Clinical Laboratory Standards) recommended standard methods and quality control strains.
Compound 109 displayed activity against gram-positive aerobes. In particular, compound 109 (SQ-109) demonstrated the best activity against Streptococcus pneumoniae with MICs ranging from 4-8 ug/ml. MICs for all species of enterococci tested ranged from 16-64 ug/ml while MICs for all Staphylococci tested ranged from 16-32 ug/ml (
Compound 109 displayed activity against gram-negative aerobes. SQ-109 demonstrated limited activity against all enterobacteriaceae tested with MICs ranging from 32->64 ug/ml with slightly lower MICs seen among the non-enterobacteriaceae (16->64 ug/ml). MICs for three Haemophilus influenzae isolates tested ranged from 1-32 ug/ml. SQ-109 demonstrated the best activity when tested against Helicobacter pylori with an MIC of 4 ug/ml for all three strains tested (
Compound 109 displayed activity against anaerobes. SQ-109 MICs ranged from 16-64 ug/ml for anaerobes Propionibacterium acnes, Bacteroides fragilis, and Clostridium difficile (see
Compound 109 was tested against a representative panel of commonly encountered clinical microorganisms comprising opportunistic pathogens, target pathogens and normal human flora. The spectrum of activity of compound 109 was evaluated by performing recommended broth microdilution methods. NCCLS M27-A2, 2003 for yeast and NCCLS M38-A, 2003 for mold.
SQ-109 demonstrated good activity against Candida albicans with MICs ranging from 4-8 ug/ml. Additionally, Mold MICs for three isolates of Aspergillus fumigatus tested were 16 ug/ml (
Compound 109 was tested against a representative panel of commonly encountered clinical microorganisms comprising opportunistic pathogens, target pathogens and normal human flora. The spectrum of activity of compound 109 was performed by using the BACTEC 460 TB system (Becton Dickinson, Cockeysville, Md., USA) according to the manufacturers instructions. MICs for MOTT were determined suing the agar proportion method recommended for testing slow-growing mycobacterium species, while MICs for the rapid-grower mycobacteria were performed suing the recommended broth microdilution method (NCCLS M24-A, 2003).
SQ-109 demonstrated very good activity when tested against M. tuberculosis [MTB] (0.25-0.5 ug/ml), M. bovis (0.25 ug/ml), and M. bovis BCG (0.5 ug/ml). Among resistant MTB strains tested; SQ-109 had an MIC of 0.25 ug/ml for an INH-resistant MTB strain, and an MIC of 0.5 ug/ml against an EMB-resistant strain (Table 37).
SQ-109 showed less activity against Mycobacteria-other-than-TB (MOTT) with an MIC of 8 ug/ml for three M. marinum strains tested, an MIC of 16 ug/ml for three M. kansasii tested, and MICs ranging from 8-32 ug/ml for three M. avium complex (MAC) isolates examined (Table 38).
SQ-109 showed good activity against rapid-grower M. fortuitum with an MIC of 1 ug/ml for all strains tested and less activity against the more resistant members of the M. chelonae group (M. chelonae and M. abscessus) with an MIC of 16 ug/ml for three strains tested (
The results of examples XXV-XXVII demonstrate the best inhibitory activity against several species of mycobacterium in the MTB complex (M. tuberculosis, M. bovis, and M. bovis BCG), Mycobacterium-forluitum, Mycobacterium marinum, Helicobacter pylori, Streptococcus pneumoniae and Candida albicans. SQ-109 was also found to be equally active against susceptible and resistant strains of M. tuberculosis.
Summary
The purpose of this study was to determine interactions of SQ109 with existing antitubercular drugs in vitro and assess its potential to improve combination drug activities against Mycobacterium tuberculosis.
Two-drug combinations at various concentrations below their minimal inhibitory concentrations (MIC) were tested for growth inhibition of M. tuberculosis using the BACTEC 460 system in vitro. Drug interactions were evaluated based on the quotient values that were derived numerically from the growth indices of cultures treated with single antibiotics or combination treatment with two antibiotics.
SQ 109 at 0.5 its MIC demonstrated strong synergistic activity with 0.5 MIC isoniazid and as low as 0.1 MIC rifampicin in inhibition of M. tuberculosis growth. Additive effects were observed between SQ109 and streptomycin, but neither synergy nor additive effects were observed with the combination of SQ109 with ethambutol or pyrazinamide. The synergy between SQ109 and rifampicin was also demonstrated using rifampicin-resistant (RIFR) M. tuberculosis strains, SQ109 lowered the MIC of rifampicin against these drug-resistant strains.
SQ109 interacts synergistically with isoniazid and rifampicin, two of the most important front-line TB drugs.
Materials and Methods
Antimicrobial drugs isoniazid, rifampicin, streptomycin, and ethambutol were purchased from Sigma-Aldrich. St. Louis, Mo. Stock solutions of isoniazid, streptomycin, and ethambutol were prepared in distilled and deionized water at 10 mg/mL, sterilized by filtration, and stored frozen at −80° C. Stock solutions of rifampicin, at 1 or 10 mg/mL, were prepared in methanol and stored at −80° C. Pyrazinamide was purchased as the drug reconstituting kit from Becton Dickinson, Cockeysville, Md., and a stock solution was prepared following instructions by the manufacturer. Stock solutions of SQ109 were prepared in methanol at 1 mg/mL and stored at 80° C.
M. tuberculosis Strains:
M. tuberculosis strain H37Rv was the same strain used in previous studies documenting activity of chemical compounds in our library. The mono RIFR M. tuberculosis clinical isolates used in this study were obtained from the Department of Health and Mental Hygiene, Central Laboratory, State of Maryland. The drug susceptibility profiles of strains 3185 and 2482 were previously determined at the state laboratory and later confirmed at Sequella. Both strains were susceptible to 0.1 mg/L isoniazid, 2.5 mg/L ethambutol, and 2 mg/L streptomycin. Rifampicin MIC was 24 mg/L for strain 3185 and >100 mg/L for strain 2482. The nature of rpoB mutation associated with rifampicin resistance was not determined.
Media Middlebrook 7H11 agar (Difco, Becton Dickinson) supplemented with 10% OADC (Difco) was used to grow M. tuberculosis for BACTEC inocula. BACTEC 71112B medium (Becton Dickinson) was used for all drug combination experiments except for pyrazinamide+SQ109: this combination was evaluated in PZA Test Medium (Becton Dickinson).
Assessment of drug effects on M. tuberculosis growth BACTEC 460 system (Becton Dickinson) was used to determine MIC for each individual drug and to study the combined effects of drugs on M. tuberculosis growth in vitro. M. tuberculosis H37Rv or RIF R M. tuberculosis isolates were grown on 7H11 agar plates. Bacterial inocula were prepared from 4 to 6 week old plates by transferring loopfuls of bacteria into capped glass tubes containing dilution fluid and glass beads and vortexing to break the clumps. Suspensions of M. tuberculosis at 1 McFarland Standard, free of clumps, were inoculated into Middlebrook 7H12 medium vials containing various combinations of test drugs (SQ109, isoniazid, rifampicin, ethambutol, streptomycin). The growth of the bacilli, expressed as the growth index or GI, was monitored daily by measuring the release of 14CO2 after the bacteria consumed 14C labeled palmitic acid in the media. To determine the combined effects of pyrazinamide and SQ109, we used the special pyrazinamide test medium from Becton Dickinson. No-drug controls, including the undiluted and 1:100 diluted bacterial inocula, were included in each experiment to monitor bacterial growth. When the GI value of the 1:100 inoculum vial reached 30 or greater, ΔGI, the difference in GI values between the latest GI and the previous reading for all testing vials was derived. The MIC of a given drug was defined as the lowest concentration at which the ΔGI of the drug vial was less than the ΔGI of the 1:100 control. All the experiments were completed within 5-8 days.
Data Analysis: The effects of drugs in combination were evaluated based on GI values using the technique that was previously described.9 Synergy was defined as x/y<1/z, where x is the GI of the test vial with the combination of drugs, y is the lowest GI of the single drugs of the combination and z is the number of drugs combined. For a two-drug combination, a quotient of <0.5 was indicative of a synergistic effect, a quotient of 1 indicated no interaction, a quotient of >2 showed an antagonistic effect, and a quotient of <0.75 but >0.5 indicated an additive effect.
Results
Antimicrobial activity of SQ109 in vitro: We previously determined that SQ109 MIC is 0.2 μM (0.11 mg/L) by micro-broth dilution or 0.63CM (0.35 mg/L) by BACTEC for the laboratory strain H37Rv M. tuberculosis.3 Recently completed determination of SQ109 MIC on more than 30 M. tuberculosis clinical isolates (drug susceptible and drug resistant, including EMB R strains) found the susceptibility of these clinical strains to be indistinguishable from H37Rv (MIC range 0.16-0.64 mg/L). These in vitro activities of SQ109 suggest that it is equally active against drug-sensitive and drug-resistant M. tuberculosis, including strains resistant to the parent pharmacophore ethambutol.
Interaction of SQ109 with Isoniazid, Streptomycin, Ethambutol, and Pyrazinamide:
Using a checkerboard titration, in which series of dilutions of two antibiotics are studied for effects on bacterial growth inhibition at all possible concentrations, both alone and in combination, the nature of the interaction between the two antibiotics can be determined algebraically. The interaction between two antibiotics in combination can be described as synergistic, additive, no effect, or antagonistic. In order to translate the experimental data into the types of drug-drug interactions, the quotient x/y (where x is the data obtained when two antibiotics are in combination and y is the data with the lower value of the two agents when tested separately at the same concentration). If the x/y value equals 1, it is interpreted as one of the drugs in combination is inactive. If x/y is less than 0.5 for a two drug combination, it implies that the two drugs when used together are more effective than when they are used separately, suggesting synergistic effects. If x/y values fail between 0.5 to 0.75, an additive effect may exist between the two drugs, suggesting a weak enhancement between them. If x/y values are greater than 2, it suggests antagonistic interactions between the two drugs. The window between x/y greater than 1 and less than 2 is the transition area from no effect to antagonistic. By applying simple algebra as published and validated elsewhere, we can evaluate the potential outcome of two drugs in combination in an in vitro assay system.
To determine the optimal drug combination(s) that might include SQ109 in future human efficacy trials, we analyzed the interaction of SQ109 with antibiotics that are currently used to treat TB patients. The MIC of each drug is the lowest concentration that inhibits the growth of 99% of the bacterial inoculum, thus it is clear that synergistic, additive, or antagonistic effects of combination drugs must be evaluated at individual drug concentrations that are below the level of the MIC for effects to be observed. Table 40 (
Interaction of SQ109 with Rifampicin.
When SQ109 was used in combination with rifampicin, we observed marked synergy between the two drugs, as indicated by the average quotient values (Table 41,
SQ109 and rifampicin interaction in RIFR M. tuberculosis To examine whether the synergistic interaction between SQ109 and rifampicin also facilitated inhibition of drug resistant M. tuberculosis strains, we evaluated drug interactions with RIFR M. tuberculosis clinical isolates. The rifampicin MIC on RIFR M. tuberculosis isolates 3185 and 2482 was 24 mg/L and >100 mg/L, respectively, much higher than the average MIC of drug-susceptible M. tuberculosis H37Rv (0.17 mg/L, Table 41 legend). The RIFR phenotype did not affect the MIC of SQ109 on these strains: MIC on both was 0.32 mg/L, the same value as that determined on RIFs M. tuberculosis H37Rv.
Experiments with RIFR strain 3185 were repeated with a different RIFR M. tuberculosis, strain 2482, whose rifampicin resistance was even more profound: MIC>100 mg/L. As shown in
Discussion
Multi-drug therapy is essential to cure TB infections and avoid the emergence of drug-resistant bacteria. Any new anti-TB drug candidate needs to be evaluated for combination drug interactions to optimize individual drug activity and avoid drug antagonism. In this study we report our findings on the interaction of SQ109, a new diamine antitubercular drug candidate, with commonly used anti-TB drugs in vitro. The possible interactions that we could measure in these studies using growth inhibition included antagonistic, synergistic, additive or no effect at all. We found no antagonistic interactions between SQ109 and any of the five front-line TB drugs tested in this study. The combinations of [SQ109+ethambutol] or [SQ109+pyrazinamide] showed no interactions, positive or negative, and the effects on growth of M. tuberculosis of these combinations were indistinguishable from those obtained with single drug treatment. An additive interaction was observed between SQ109 and streptomycin at certain concentrations, but no synergy was observed. In contrast, SQ109 showed synergy with two different front-line drugs: isoniazid and rifampicin. The synergistic interactions between SQ109 and rifampicin were particularly interesting, and quite potent: greater than 99% inhibition of M. tuberculosis growth was achieved at very low concentrations of the individual drugs, 0.1 MIC rifampicin+0.5 MIC SQ109. The in vitro synergy results described in this paper were consistent with in vivo studies (Nikonenko, et al., in preparation) that demonstrated drug synergy in experimental animals infected with M. tuberculosis when SQ109 was combined with rifampicin and isoniazid. These in vivo results showed enhanced bactericidal activity and faster elimination of M. tuberculosis by SQ109-containing drug combinations compared to similar combinations containing ethambutol. Together, the in vitro and in vivo data presented in these two studies can guide us in achieving the most efficacious drug combination design in future SQ109 clinical trials for treatment of active TB disease.
Interestingly, data in the present study also showed that SQ109 is fully active against RIFR M. tuberculosis clinical isolates. This is consistent with the recent results obtained from the study of over 30 drug-sensitive and drug-resistant M. tuberculosis, where the strains had the same SQ109 MIC (range 0.16-0.64 mg/L), suggesting that there is no pre-existing resistance to this compound. Synergy of SQ109 with rifampicin was also observed when tested against RIFR isolates. At 0.5 MIC, SQ109 was able to increase rifampicin activity against the resistant organisms in a dose-dependent manner. This activity was not observed with 0.5 MIC ethambutol, suggesting that a specific interaction between SQ109 and rifampicin is likely responsible for the observation. Although this observation may not have direct clinical relevance, it points out additional differences between SQ109 and its parent pharmacophore, ethambutol, and could be an interesting experimental model to tease out SQ109 actions on M. tuberculosis.
A definitive explanation for the profound synergy between rifampicin and SQ109 against M. tuberculosis is not yet available. Hypothetically, though not wishing to be bound by the following theory, the synergistic interaction could result from the differences in drug action. Although the precise target of SQ109 is unknown, it does affect mycobacterial cell wall synthesis. Perhaps the effect of SQ109 on the mycobacterial cell wall results in increasing permeability, allowing more rifampicin to enter the bacteria. Subinhibitory concentrations of cell wall inhibitors such as ethambutol were shown to increase bactericidal activity of clarithromycin for M. tuberculosis, presumably by decreasing the permeability barrier for drug entry. However, ethambutol, a cell-wall inhibitor, did not show any synergy when used in combination with rifampicin in our experiments with RIFS H37Rv or the RIF R M. tuberculosis clinical isolates (
It is also possible that the expression level of the currently unknown primary target of SQ109 is tightly regulated at the transcriptional level. Rifampicin is an inhibitor of DNA transcription. Since transcripts of mRNA of various genes differ in half-life, inhibition of transcription could have profound effects on those transcripts with shorter half-life than those with longer half life. Thus, rifampicin treatment, even at suboptimal concentrations, could exert a noticeable effect on the level of a short-lived mRNA target for SQ109 activity. However, this hypothesis is inconsistent with the observation that synergy between the two drugs was not affected by the RIFR phenotype of the M. tuberculosis strains. This suggests that other effects of rifampicin, rather than only its primary action as an antibiotic, contributed to its synergistic interaction with SQ109. That rifampicin antibiotic activity might certainly be a contributing factor, but perhaps not the only factor that determined the interesting interaction with SQ109, was suggested by data on the MIC of RIFR strains. As the MIC of rifampicin becomes greater in RIFR as compared with RIFs strains, the concentrations of rifampicin showing synergy with the same amount of SQ109 are also greater, even though the combinations expressed in terms of fractions of MIC were similar between the strains studied. To fully evaluate the effect(s) of rifampicin transcription inhibition on SQ109 activity requires the identification of the SQ109 target(s), which is ongoing work in our laboratory.
Another possible effect of rifampicin on the drug synergy interaction between rifampicin and SQ109 is the rifampicin action as an efficient inducer of cytochrome P450 (CYP). The CYP are a superfamily of heme-containing enzymes involved in a wide array of NADPH/NADH-dependent reactions, and they play pivotal roles in biosynthesis of compounds such as sterols, steroids, and fatty acids, as well as in detoxification of xenobiotics and chemicals. Rifampicin is a potent inducer of a variety of CYP in human hepatocytes, as well as in peripheral blood lymphocytes.
The complete genome of M. tuberculosis reveals at least 20 CYP, but precise functions for these genes remain to be elucidated. Like their mammalian counterparts, the mycobacterial CYP were induced by rifampicin. Ramachandran and Gurumurthy determined CYP activity present in bacterial membrane fractions extracted from rifampicin-treated M. smegmatis and M. tuberculosis. In both cases, the CYP activities in the treated fractions were elevated as compared to the untreated controls, and the increase in CYP activity was statistically significant. In parallel, they found that isoniazid did not induce CYP activity in its treated fractions. The ability of rifampicin to induce CYP in M. tuberculosis may contribute to its synergy with SQ109. In this case, instead of inactivating the active compounds, CYP may in fact activate SQ109 by producing oxidized metabolites of SQ109 within M. tuberculosis.
Recently obtained data on SQ109 metabolism showed that SQ109 was metabolized rapidly after incubation with human liver microsomes: only 8% SQ109 remained after 40 min incubation. Analysis of SQ109 metabolites produced by the action of the microsomes revealed 4 chemical groups based on molecular mass. Two of the groups contained oxidized metabolites. Furthermore, the study found that SQ109 was metabolized by human CYP2D6 and CYP2C19 to generate these metabolites. Based on the finding that SQ109 was metabolized rapidly by CYP, it is possible that its antimycobacterial activity may come from one of its metabolites. It is conceivable that SQ109 is a prodrug and requires activation by mycobacterial CYP. We speculate that when SQ109 is used in combination with rifampicin, the latter induces certain CYP activity within the Mycobacteria. Elevated CYP activity activates the SQ109 prodrug more efficiently, resulting in an apparent synergistic activity of the two drugs. In fact, the enhanced activity seen with the combination of SQ109+rifampicin may be the more efficient and effective activity of an SQ109 active metabolite, rather than enhanced activity of rifampicin itself.
SQ109 has a very narrow spectrum: it is active against M. tuberculosis and M. bovis BCG, but much less active against M. smegmatis and M. avium (unpublished data). By comparing the putative CYP open reading frames present in various mycobacterial genomes, Kelly et al showed there were several CYP that are unique in M. tuberculosis and M. bovis. These CYP are strong candidates for actors that might be responsible for converting SQ109 to an active metabolite(s) within M. tuberculosis, and they are the subjects of ongoing investigations.
The present experiment will investigate the efficacy of Isoniazid (INH)+Rifampin (RIF)+Pyrazinamide (PZA)+SQ109 (INH/RIF/PZA/SQ109) for elimination of M. tuberculosis in lung during intensive phase therapy in animal modeling experiments (see
The preferred Phase II Study will be a prospective, multi-center, double-blind, randomized, placebo-controlled, clinical study of SQ109 designed to:
An animal (mouse) experiment will be conducted to increase SQ109 dose while holding INH/RIF/PZA at the standard DOTS dose to mimic the design of the present study and predict potential human doses.
Preliminary INH, RIF & PZA drug interaction studies, including ADME in mice and in vitro drug interactions (microsomes), are also being conducted.
Alternate Multidose (5 Day) EBA Study
An alternative Phase II multidose study would be an ‘Early Bactericidal Assay (EBA)’ study as proposed by Dennis Mitchison for new TB drug development and as conducted by Stephen Gillespie (London) and. In these studies, antibiotic monotherapy is conducted for a short period of time while monitoring numbers of bacteria in sputum. These studies are capable of determining a minimum effective dose of a single antibiotic to reduce bacteria in sputum and can expand the safety profile of SQ109; however these studies are not capable of providing efficacy data for combination therapy. It is anticipated that tuberculosis will, for the foreseeable future, be treated with at least 3 drugs with different modes of action.
The alternative multidose (5 day) EBA study would be a randomized, open, clinical study of SQ109 designed to determine the clinical efficacy of SQ109 in patients with pulmonary tuberculosis based on EBA of SQ109 and to further evaluate its safety. EBA studies provide a fast and economic way to evaluate the clinical efficacy of potential agents for the treatment of tuberculosis in a brief period (3-14 days, usually 5 days) of study medication monotherapy.
Approximately one hundred patients (see criteria below) will be orally treated with either SQ109 once daily (n=40) or 6-mg/kg INH (n=10) for 5 days. The study treatment period will be followed by a standard therapeutic regimen for 6 months. The dose of SQ109 for this study will be determined on based on other studies, such as the highest amount of the drug that can be taken with good PK parameters and without harmful side effects, also taking into a consideration the therapeutic dose obtained in the efficacy studies with laboratory animals (30 mg/m2).
Enrollment Inclusion Criteria:
Adults, male or female, aged 18-60
Chest X-ray and clinical findings consistent with tuberculosis.
Sputum smear-positive patients will be eligible for enrollment. The diagnosis of tuberculosis must be also confirmed by culture. AFB smear positive patients found later not to have TB (i.e. those with non-tuberculous mycobacterial disease) and those without culture confirmation will be removed from the study.
Enrollment Exclusion Criteria:
Pregnant or breastfeeding
HIV-infected
History of prior tuberculosis or history of previous tuberculosis treatment
Cavitary tuberculosis on initial chest X-ray (taken within 14 days of study entry)
Exposure to person(s) with known drug resistant tuberculosis
Patients with drug resistant tuberculosis (resistance to INH, RIF, PZA or EMB)
Patients receiving chronic steroids or other immunosuppressive medications
Extra-pulmonary tuberculosis
Sputum and blood will be collected 2 days before treatment (baseline) and after 2 and 5 days of study treatment. Hematological and biochemistry parameters will be assessed simultaneously. The count of CFU per milliliter will be determined as described (Jindani A, Doré CJ, Mitchison DA, American Journal of Respiratory and Critical Care Medicine 2003 Vol 167. pp. 1348-1354). The EBA will be calculated for each patient by the formula (log CFU/mlday0−log CFU/mlday5)/5 and will be reported as the mean±standard deviation (SD). As recommended by several authors, we will define the EBA as the decrease in log10 CFU per milliliter of sputum per day during the first 5 days of treatment.
To determine the optimal drug combination that includes SQ109 for future efficacy trials in humans, we analyzed the interaction of SQ109 with antibiotics that are currently used to treat TB patients. The MIC of each drug is the lowest concentration that kills or inhibits the growth of 99 percent of the bacterial inoculum. It is clear that synergistic, additive, or antagonistic effects of combination drugs must be evaluated at individual drug concentrations that are below the level of the MIC for effects to be observed. Table 41 lists the MIC of each individual drug, as well as quotient values for combinations of SQ109 with INH, STR, EMB, and PZA at drug concentrations below their MIC values. The concentration of each of the drugs used in combination was expressed as the fraction of the MIC value. In general, synergy effects are observed when quotient values of a combination doesn't exceed 0.5, additive effects when the quotient values are within a range of 0.5-0.75.
The results of this synergy experiment care be summarized as follows:
When SQ109 was used in combination with RIF, we observed marked synergy between the two drugs, as indicated by the average quotient values (Table 41, and
To examine whether the synergistic interaction between SQ109 and RIF also facilitated killing of drug resistant M. tuberculosis strains, we evaluated drug interactions with RIFR M. tuberculosis clinical isolates. The RIF MIC on RIFR M. tuberculosis isolates 3185 and 2482 was 24 μg/ml and >100 μg/ml, respectively, much higher than the average MIC of drug-susceptible M. tuberculosis H37Rv (0.17 μg/ml). The RIFR phenotype did not affect the MIC of SQ109 on these strains: MIC on both was 0.32 μg/ml, the same value as that determined on RIFs M. tuberculosis H37Rv. We have found (
We have carried out in vivo studies where SQ109 was tested for its efficacy in combination with other anti-TB drugs: Rifampin, Isoniazid, Ethambutol, Pyrazinamide, Moxifloxacin. At first, effectiveness of the drug combinations was studied in a rapid mouse model (developed by Sequella' scientist Dr. Boris Nikonenko) that allows to predict quickly and with sufficient accuracy the drug's efficacy based on its ability to prevent body weight loss in the infected animals, one of the signs of TB severity.
Briefly, mice were inoculated iv with 106 CFU of virulent M. tuberculosis H37Rv to develop a rapid and progressive TB disease. Chemotherapy was initiated 7d after inoculation and continued for 10 days. Mice treated with a single drug (SQ109, Rif, INH, EMB, PZA, and Moxi), as well as uninfected animals and infected untreated placebo, were used as the controls. In this model, all standard drugs were used at doses below their most efficacious in order to see the effect (when used at their therapeutic doses, drug-treated mice did not loose weight): Rif was studied at 2 mg/kg, INH at 1 mg/kg, EMB at 10 mg/kg, Moxi at 10 mg/kg, PZA at 50 mg/kg. Body weights of mice in all groups were monitored starting from time 0. By 10d, infected placebo control mice started to lose weight; by 20d mice in this group lost more than 25% of their body weight.
The results of the Day 21 (
As part of our efforts to develop a new regimen for treatment of tuberculosis, we studied a combination of SQ109 (at 10 mg/kg) with Sequella's potential drug candidates, -dipiperidine SQ609 (at 10 mg/kg) and 1,2-ethylenediamine SQ73 (at 5 mg/kg),
The compounds were used in the following doses: SQ109 at 10 mg/kg; SQ609 at 10 mg/kg, SQ73 at 5 mg/kg, totaling overall dose of 25 mg/kg. Activity of this drug combination was compared to the efficacy of one of the most efficacious anti-TB drugs isoniazid (INH) which was used as control in this study at 25 mg/kg.
Mice were infected with low dose of M. tuberculosis H37Rv and chemotherapy was initiated 4 weeks following infection and continued for 2 weeks.
In this study, SQ609-SQ109-SQ73 combination demonstrated similar activity to INH at it's the most efficacious dose.
This application is a continuation of International Application No. PCT/US 2006/026078, filed 3 Jul. 2006, which claims priority to U.S. patent application Ser. No. 11/173,192, filed 1 Jul. 2005, both of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 2623878 | Isler et al. | Dec 1952 | A |
| 2709169 | Morren et al. | May 1955 | A |
| 3176040 | Wilkinson et al. | Mar 1965 | A |
| 3197468 | Sensi et al. | Jul 1965 | A |
| 3553257 | Halmos et al. | Jan 1971 | A |
| 3579586 | Zoja | May 1971 | A |
| 3579587 | Zoja | May 1971 | A |
| 3629333 | Boughton et al. | Dec 1971 | A |
| 3682922 | Klimstra et al. | Aug 1972 | A |
| 3718655 | Ferrer-Salat et al. | Feb 1973 | A |
| 3769347 | Kazan | Oct 1973 | A |
| 3789073 | Narayanan et al. | Jan 1974 | A |
| 3829493 | Butula et al. | Aug 1974 | A |
| 3847991 | Bernardi et al. | Nov 1974 | A |
| 3855300 | Takahashi et al. | Dec 1974 | A |
| 3876702 | Petersen et al. | Apr 1975 | A |
| 3878201 | Tomcufcik | Apr 1975 | A |
| 3931152 | Tomcufcik et al. | Jan 1976 | A |
| 3931157 | Child et al. | Jan 1976 | A |
| 3944608 | Singh | Mar 1976 | A |
| 3944616 | Kazan | Mar 1976 | A |
| 3944617 | Singh | Mar 1976 | A |
| 3944618 | Singh | Mar 1976 | A |
| 3944619 | Singh | Mar 1976 | A |
| 3953513 | Oppici | Apr 1976 | A |
| 3979457 | Fujii et al. | Sep 1976 | A |
| 4006234 | Child et al. | Feb 1977 | A |
| RE29358 | Tomcufcik | Aug 1977 | E |
| RE29588 | Halmos et al. | Mar 1978 | E |
| 4150030 | Singh | Apr 1979 | A |
| 4262122 | Lees et al. | Apr 1981 | A |
| 4450274 | Park | May 1984 | A |
| 4457931 | Milani et al. | Jul 1984 | A |
| 5104875 | Jurgen et al. | Apr 1992 | A |
| 5256391 | Chen et al. | Oct 1993 | A |
| 5439891 | Kapil et al. | Aug 1995 | A |
| 5864045 | Burkholder et al. | Jan 1999 | A |
| 5922282 | Ledley | Jul 1999 | A |
| 5985935 | Kharazmi et al. | Nov 1999 | A |
| 6300061 | Jacobs, Jr. et al. | Oct 2001 | B1 |
| 6660744 | Hirst et al. | Dec 2003 | B1 |
| 7456222 | Protopopova et al. | Nov 2008 | B2 |
| 20020007524 | Sorensen | Jan 2002 | A1 |
| 20030069204 | Inukai et al. | Apr 2003 | A1 |
| 20030171330 | Hotoda et al. | Sep 2003 | A1 |
| 20030236225 | Protopopova et al. | Dec 2003 | A1 |
| 20040033986 | Protopopova et al. | Feb 2004 | A1 |
| 20040058964 | Devadas et al. | Mar 2004 | A1 |
| 20040147591 | Kanie et al. | Jul 2004 | A1 |
| 20050014800 | Matsuoka et al. | Jan 2005 | A1 |
| 20050113574 | Bogatcheva et al. | May 2005 | A1 |
| Number | Date | Country |
|---|---|---|
| 669202 | Mar 1966 | BE |
| 2409741 | Oct 2002 | CA |
| 2007524 | Apr 1969 | FR |
| 729332 | May 1955 | GB |
| 961317 | Jun 1964 | GB |
| 1157143 | Jul 1969 | GB |
| 1234349 | Jun 1971 | GB |
| 2168986 | Jun 2001 | RU |
| 803348 | Sep 1981 | SU |
| 805605 | Sep 1981 | SU |
| WO 9110664 | Jul 1991 | WO |
| WO 9310073 | May 1993 | WO |
| WO 9428885 | Dec 1994 | WO |
| WO 9951213 | Oct 1999 | WO |
| WO 03068769 | Aug 2003 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20090192173 A1 | Jul 2009 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2006/026078 | Jul 2006 | US |
| Child | 12255976 | US |
