Nitro-[2,1-b]imidazopyran compounds and antibacterial uses thereof

Methods, chemicals and compositions are provided for inhibiting the growth of pathogenic microbes in vitro and of treatment of pathogenic bacterial diseases, including mycobacterial, Clostridium, Cryptosporidium and Helicobacter infections, in vivo using bicyclic nitroimidazole compounds of the formula (II): ##STR1## wherein R.sub.1 is hydrogen, halogen, loweralkyl, haloloweralkyl, cycloalkyl, heterocycle, substituted heterocycle and heterocyclicalkyl; X is oxygen, sulfur or NR.sub.2, where R.sub.2 is hydrogen, loweralkyl, aryl, cycloalkyl, heterocycle, substituted heterocycle, heterocyclicalkyl, COR.sub.3 or SO.sub.2 R.sub.4 CONR.sub.4 R.sub.5, where R.sub.3, R.sub.4 and R.sub.5 are independently chosen from hydrogen, loweralkyl, aryl, alkylaryl, alkoxyalkyl, alkoxyaryl, alloxyalkoxyaryl, alkylheterocycle, and alkoxyheterocycle; n is 1, 2 or 3; Y and Y are independently selected from oxygen, CH.sub.2, CO, CR.sub.4 R.sub.5 or NR.sub.4, where R.sub.4 and R.sub.5 are as defined previously; therefore when n is 3 or 2, the compounds of formula II may be additionally substituted as follows: ##STR2## wherein R.sub.6, R.sub.7, R.sub.8 and R.sub.9 are independently chosen from hydrogen, loweralkyl, aryl, alkylaryl, alkoxyalkyl, alkoxyalkylaryl, alkoxyalkylheterocycle, alkylary2alkylaryl, alkylarylaryl, alkylcycloalkyl, alkoxyaryl, alkylheterocycle, and alkoxyheterocycle; and the pharmaceutically acceptable salts thereof.

 

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BACKGROUND

 

 
After a decline in rates of infection over several decades, a troubling gain in the prevalence of tuberculosis (TB) is happening. Since TB is highly infectious it poses a profound threat to public health. TB bacteria can easily be passedfrom person to person in airborne droplets formed when a individual who has active TB sneezes or coughs.
 

 
Much more alarming has been the rise of multidrug-resistant tuberculosis (MDRTB). Before 1984, about 10% of TB bacteria isolated from patients in the United States were resistant to a single antibacterial drug. In 1984, 52% of patientswere infected with Mycobacterium tuberculosis (also referred to as tubercle bacilli) resistant to one drug, and 32% were resistant to one or more drugs. Outbreaks of MDRTB have been reported in 13 states. Ten percent of the listed MDRTBcases to date have occurred in previously healthy individuals whose mortality rate–70 to 90%–has been almost the same as that of immunosuppressed individuals with MDRTB (Snider and Roper, 1992).
 

 
The United States Centers for Disease Control (CDC) has released preliminary results of a joint research with the New York State Health Department demonstrating that instances of drug-resistant TB have more than doubled since 1984. CDC data in the firstquarter of 1991 show that several of these drug-resistant breeds are resistant to both of the frontline TB medications, rifampin and isoniazid. Outbreaks of MDRTB have occurred in hospitals in Miami and New York City, in Addition to in the New York State prisonsystem. In one hospital in nyc, the median interval between identification of MDRTB and death was only four weeks. Further clusters of MDRTB were reported to the CDC in 1990 and 1991 in Mississippi, Missouri, and Michigan.
 

 
There are five frontline drugs known to be highly effective against Mycobacterium tuberculosis and five second-line drugs which may be used when resistance to one or more of the frontline drugs is detected. Paradoxically, in the USA, untilApril 1992, there were deficits of antituberculosis drugs, some of which are needed when resistance to the frontline drugs rifampin and isoniazid is present. These shortages had happened because several pharmaceutical companies had ceasedproduction of those drugs.
 

 
Because of its persistence in the body, the tubercle bacillus is a notoriously tough pathogen to control. Though bacille Calmette-Guerin (BCG) vaccine protects against acute tuberculosis meningitis and disseminated TB in children, itsefficacy against pulmonary TB in adults has varied widely in different parts of the world. Treatment of conventional TB is effective, but expensive, requiring daily therapy with a number of drugs for no less than six months. There’s a frequent tendencyamong TB patients to stop taking their medication once the drugs start to have their favorable impact or to choose the medications only intermittently. While this occurs, relapses are frequent and quite often are caused by drug-resistant tubercle bacilli thathave endured the initial course of treatment. The emergence of drug-resistant M. tuberculosis is in many ways an index of individual compliance with antituberculosis chemotherapy and of the inability of the health care infrastructure to guarantee adequatetreatment. Many public health agencies that once can play key roles in this process have had their budgets cut radically in the past several years and hence are unable to perform this crucial service.
 

 
MDRTB is extremely difficult to treat, and also the vast majority of patients don’t respond to treatment. Overall treatment costs for an individual with MDRTB can be as much as 10 times the price of traditional treatment; the cost of the treatment drugsalone can be as much as 21 times as great.
 

 
The favored treatment for classical TB consists of isoniazid, rifampin, and pyrazinamide. For individuals whose tubercle bacilli are thought to be resistant to isoniazid, a fourth drug, ethambutol, is commonly added to the regimen until drugsusceptibility outcomes are understood. Isolates of tubercle bacilli resistant to both isoniazid and rifampin, now representing about 20 percent in some cities, demand specialized treatment with additional drugs, which might consist of streptomycin andciprofloxacin for two years.
 

 
The tubercle bacillus is a slow-growing organism. Three to six weeks are required to increase the bacteria in the clinical laboratory, and an additional three to six weeks are required to monitor for antibiotic resistance. Such extended laboratoryprocedures can result in a delay in diagnosis, which means that patients with unrecognized drug-resistant TB may be treated ineffectively and stay contagious for a longer period. In HIV-positive individuals, MDRTB usually causes death within 4 to 16weeks later being diagnosed, and this can be before laboratory tests on drug susceptibility and resistance can be finished.
 

 
There is no evidence that mutation rates in M. tuberculosis organisms have improved or that increased virulence would be to blame for the recent deadly outbreaks of TB. It is likely that drug-resistant kinds of tuberculosis arose because of patientnoncompliance with the 6- to 12-month regimen of antibiotics necessary to treat TB. Ineffective therapy regimens also play a part in the increasing incidence of TB. To address noncompliance, some states with high TB rates are considering approaches tooutreach, like expanding directly observed therapy (DOT); others may reestablish inpatient centers similar to the TB sanatoria of the first half of this century. Standard treatment regimens for TB also have been updated. Rather than carrying two orthree antibiotics, TB patients take four. Still, as noted earlier, the present shortages of antituberculosis medications in america have made even standard treatment tough.
 

 
A series of nitroimidazo[2,1-b]oxazole derivates was clarified in Sehgal, K. et al.,”Novel Nitroimidazo[2,1-b]oxazole Formation from Reaction of 2,4(5)-Dinitroimidazole with Oxiranes (1),” J. Heterocyuclic Chem. 16:1499-1500 (1979). Compoundsof this type have the following general formula (I): ##STR3## These compounds were described as potential radiosensitizing agents for use in the radiotherapy of cancer (Agrawal, K. et al.,”Possible Radiosensitizing Agents. Dinitroimidazole;” J. Med.Chem. 22(5):583-586 (1979); Sehgal, R. et al,”Possible Radiosensitizing Agents. 2. Synthesis and Biological Activity of Derivatives of Dinitroimidazole with Oxiranes,” J. Med. Chem. 24:601-604 (1981). More recently, certain nitroimidazole compoundswere reported to exhibit antifungal properties, such as antitubercular action (see, e.g., Nagarajan, K. et al.,”Nitroimidazoles XXI. 2,3-dihydro-6-nitroimidazo [2.1-b] oxazoles with antitubercular action,” Eur. J. Med. Chem. 24:631-633(1989). In addition, the compound of formula (I) in which R is ethyl (2-ethyl-5-nitro-2,3-dihydro[2,1-b]imidazo-oxazole, also known as Ceiby-Geigy CGI 17341) has recently been demonstrated to show activity against Mycobacterium tuberculosis (Ashtekar, D. etal.,”In Vitro and In Vivo Activities of the Nitroimidazole CGI 17341 against Mycobacterium tuberculosis,” Antimicrobial Agents and Chemotherapy, 37(2):183-186 (1993).
 

 
Pseudomembranous colitis (PMC) is a severe autoimmune disorder marked by acute colonic inflammation, nausea, stomach cramps, and mucosal plaques or pseudomembranes. PMC results from the over production of toxigenic Clostridium difficile inthe gut. C. difficile is a spore-forming anaerobe and is the significant nosocomial pathogen of PMC. The greater growth of C. difficile takes place when the bacterial flora of the GI tract has been modified because of extensive use of wide spectrum antibiotics. Twotoxins, A and B, are made by C. difficile. The toxins attack membranes or microfilaments of colon cells producing inflammation and necrosis. Toxin A triggers intestinal hemorrhage and fluid secretion whilst toxin B is cytotoxic.
 

 

PMC for a subclass of diarrheal disease has become a frequent complication

 

 
Of antibiotic use. PMC generally appears 5-10 days following onset of antibiotic treatment. A watery diarrhea is the most frequent symptom, occurring in 90-95percent of PMC cases (Aronsson, B. et al., J. Infect. Dis. 151:476-481 (1985)). Intense casesof PMC can cause high fever, leukocytosis, dehydration, electrolyte imbalance, and death (see Clostridium difficle: Its function in Intestinal Disease. R. D. Rolfe and S. M. Finegold, Ed., Academic Press Inc., New York (1988), and R. Fekety,OAntibiotic-Associated Colitis. Mediguide to Infectious DiseaseO Vol. 4, pp. 1-7 (1984)).
 

 
Patients at greatest risk include the elderly, debilitated cancer patients, and patients undergoing abdominal surgery. Untreated C. difficile generates 10-20percent mortality in older or chronically debilitated patients (Dosik, G. M. et al., Am. J.Med. 67:646-656 (1979)). Worldwide incidence of PMC is unknown due to the lack of proper studies. However, in industrialized countries, C. difficile is rapidly becoming the most popular enteric bacterial pathogen following Campylobacter and Salmonella(Bartlett, J., visit Clostridium difficle: Its function in Intestinal Illness. R. D. Rolfe and S. M. Finegold, Ed., Academic Press Inc., New York, pp. 1-13 (1988)).
 

 
Chemicals most frequently used to take care of PMC include vancomycin, metronidazole, and bacitracin. Vancomycin is a really expensive treatment, $100-$400 to get a ten day course. Relapse rate after vancomycin therapy was shown in experimentalanimals (Swannson, B. et. al., Antimicrobial Agents and Chemotherapy, 35:1108-1111 (1991) and Bartlett, J. G. et al., Clin. Infect. Dis. (S4) S265-72 (1994)). Due to the gain of vancomycin resistant germs, the use of vancomycin for C. difficileinfections may be on the decline. Metronidazole is significantly less powerful than vancomycin, however, it’s also less expensive. Metronidazole is absorbed and may expose patients to potential side effects that are associated with the drug (PHYSICIANS DESKREFERENCE, 48TH EDITION, 1994, pp. 1704-1706). Metronidazole has a relapse rate similar to vancomycin. Bacitracin is an antibiotic polypeptide and is commercially available as a combination of nine peptides. It’s also expensive and no suitable oraldosage form can be obtained.
 

 
Organisms of the genus Cryptosporidium are little obligate intracellular coccidian parasites that infect the microvilli epithelial lining of the digestive tract and rarely the lymph nodes. Cryptosporidium parvum, which is the very commonmember of the genus, is the causative agent of Cryptosporidiosis. These organisms are at precisely the same sequence as the Plasmodium (the Malaria parasite), but the developmental life cycle, transmissibility and ailments are very different. Although recognizedand identified as a parasite for quite a while, the very first cases of individual Cryptosporidiosis were reported in 1976. Cryptosporidium is effective at infecting a variety of types of farm and domestic animals too. This parasite is recognized worldwide as acausative agent of nausea. The source of human infections is supposed to be through zoonotic transmission (mainly calves, but other animals such as rodents, puppies, and kittens) and through person-to-person contact. However, this mode of transmissionalone does not account for the transmission that is senile, and epidemiological studies have demonstrated that Cryptosporidium parvum is a water-borne pathogen. At the Spring of 1993, a huge outbreak of Cryptosporidiosis occurred in the Metropolitan Milwaukeearea afflicting an estimated 400,000 men (the largest single documented epidemic of an infectious disease in North America). This epidemic was linked to the town’s water supply.
 

 
The most common clinical indications of Cryptosporidium infections are frequent watery diarrhea and low grade fever. Other symptoms include: cramps, nausea, vomiting and weight loss. Both the length of symptoms and severity of disorder andoutcome vary based on age and immune status of the patient.
 

 
In immuno-competent individuals, the disease causes watery diarrhea of a median period of 10 days (range 1-20), with varying levels of occurrence of different symptoms. The disease is considered self-limiting, but in children and infants, it hasbeen associated with causing malnutrition, severe morbidity and spread to cause large outbreaks.
 

 
In immuno-compromized persons, the duration, severity and effect of disease depend on the severity and cause of immune deficiency. For example, in certain patients with AIDS, infections with Cryptosporidium causes acute, prolonged diarrhealillness with malnutrition and dehydration and may be a significant factor resulting in death due to excessive loss of water. Involvement of lymph and lymph trees can also happen and complicates disease further. For different individuals (e.g., persons onsteroid treatment ), the infection may be cleared upon termination of their immuno-suppressive agent.
 

 
The infection begins with the organism colonizing the ileum and jejunum causing impaired digestion and malabsorption as a result of parasite-induced damage to the villi. The secretory (Cholera-like) nausea suggests that a toxin-mediated outpouring offluids into the gut, but no toxins are recorded as yet. Cryptosporidium is associated with diarrheal illness in all areas of the world. It is projected that the overall prevalence of Cryptosporidium in people with nausea is 2-2.5percent forpersons living in industrialized countries and 7-8.5percent for men living in developing countries. The overall prevalence rate reported in various studies in North America has ranged between 0.6%-4.3percent (2% in AIDS patients).
 

 
There’s currently no standard effective treatment for Cryptosporidiosis. As a diarrheal disease, treatment for Cryptosporidiosis is based on relieving symptoms as well as specific treatment, with anti-cryptosporidial drugs and hyper immune globulin.Current therapy in ordinary hosts is symptomatic. Fluid and electrolyte replacement is the primary importance in management. Non-specific anti-diarrheal agents like Kaopectate, Loperamide (Immodium), Phenoxylate (Lomotil), and Pepto-Bismol are notconsistently powerful. So far, specific treatment of Cryptosporidium immune-deficient men has been also unsuccessful. A number of drugs have been evaluated using animal models and not have shown good promise in removing the infection.Immunotherapy using Bovine dialyzable Leukocyte extracts and Passive lacteal immunity using Compounds in hyper immune Bovine colustrum have revealed different results.
 

 
Several treatment modalities have been attempted either in individual cases or at limited scale controlled studies, and also have demonstrated various levels of success. Examples include: Diloxamide furoate and furazolidone (DNA damaging agents Nitrofurananalog anti-Giardia drug), Quinine and Clindamycin, oral Spiramycin (a macrolide), alpha-difluoromethylornithine (active against other ailments and P. carinii), and Interleukin-2.
 

 
As mentioned above, effective therapy of Cryptosporidium infections is missing. Normally, this has not been a major problem in healthy persons because nausea usually lasts for less than 20 days and clinical signs are usually resolvedspontaneously. However, recent resurgence of big outbreaks have shown a connection between this infection and disease, and therapy may be warranted. If a safe and effective therapy were available, most clinicians would tend to take care of theinfection, whatever the immune status of the patient (this could be done to avoid progression to more serious disease, and also to block transmission to other susceptible hosts). Because most immuno-compromized patients often develop a prolonged, lifethreatening disease, a successful therapy is required for this particular patient population.
 

 
Helicobacter pylori causes chronic gastritis in humans and has been implicated as a pathogenic element in gastric and duodenal ulcers, gastric carcinoma and non-ulcer dyspepsia. All these are important diseases because of their incidence, theirimpact on morbidity and mortality and because of their cost to the health system. Diseases related to H. pylori infection are mostly chronic conditions with multifactorial causes, although goods that successfully eradicate H. pyloriinfection should significantly lower the incidence and prevalence of the diseases.
 

 
Worldwide sales of anti-ulcer drugs exceed $6.5 billion. H. pylori-associated diseases generate enormous amounts of earnings for pharmaceutical companies. The market for GI medication is currently dominated by histamine H2 receptor antagonists.Consequently, there exists a medical need for novel anti-H. pylori agents. The major thrust amongst pharmaceutical companies has been to evaluate existing products. Only two new antibiotics are in evolution: Abbott’s Biaxin that was recently approvedfor the treatment of H. pylori infections and Azithromycin, a related macrolide in Pfizer, has shown promise.
 

 
From an economic perspective, antibiotics signify the treatment of choice for treatment of duodenal ulcers. Compared with different choices (intermittent or maintenance treatment with H2 antagonists, highly selective vagatomy), antibiotics arerelatively cheap and offer the least time spent with an active ulcer.
 

 
The significant barrier to successful eradication of H. pylori is gaining entry to the organism. H. pylori is comparatively easy to kill in vitro. It’s vulnerable to acids, bismuth and several antibiotics, but none of these are powerful when used formonotherapy in vivo. Eradication rates with monotherapies have rarely exceeded 10%. Successful treatment of H. pylori demands an understanding of the structure of the gastrointestinal websites where the disease resides and also the pharmacokinetic natureof the representatives used. The germs live under and within gut mucus, in adrenal glands and intracellular spaces, and at the duodenal mucosa. These varied sites mean that successful delivery of antimicrobial agents by either local or systemic isdifficult to realize. Levels of amoxycillin, bismuth and imipenem/cilastatin from the human gastric mucosa after oral treatment have all been demonstrated to exceed the in vitro MIC for the organism, although none of the agents have demonstrated efficacyin vivo. Reasons for this include failure of these medication to permeate into all websites of H. pylori colonization and inability to maintain adequate bactericidal levels in the mucosa. Failure of drugs like clindamycin, erythromycin and the quinolones may bedue to the effect of intragastric pH. In addition, evolution of immunity happens quickly in H. pylori and continues to be documented for fluoroquinolones, nitroimidazoles and macrolides.
 

 
A requirement continues in the art, however, for enhanced agents that exhibit antimicrobial activity against pathogenic mycobacteria, Clostridium, Cryptosporidium and Helicobacter, and more particularly for agents and their derivatives which may behighly helpful in the treatment of MDRTB.
 

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