Patent Application
20090170803
The present invention provides a method for treating inflammation caused by bacterial, fungal or viral infections and the inflammation caused by the treatment of these infections, e.g., by the death of the bacterial or viral cells.
Bacterial, fungal and viral pathogens can cause infections which can lead to severe illness and even death. For example, the spore-forming Gram-positive rod Bacillus anthracis causes anthrax, a worldwide disease primarily affecting herbivores. Human infections occur sporadically from contact with infected animals or contaminated animal products. The disease is a constant threat in endemic regions because spores can persist for years in the soil. Recent events in the United States underscore the potential of anthrax as a bioterrorism agent.
The pathogenisis of lethal infections is complex, requiring germination of the spore inoculum, systemic invasion, multiplication, and toxin production leading to death. Often the symptoms of infections can include development of fatal inflammatory (septic) shock. Thus, adjunctive therapies to minimize the detrimental effects of inflammatory shock are under active investigation.
Inflammatory shock can be caused by the pathogens directly or by the death of the pathogens after treating the patient with a drug that kills the pathogen. Often, the sudden development of fatal inflammatory (septic) shock and the progression of the disease, despite the availability of a bacteriological or antiviral cure, account for the high mortality from these pathogens.
The inflammatory shock can be caused by the bacteria, fungal or viral pathogens directly or from the treatment thereof, i.e., the death of the pathogens due to treatment with antibacterial, antifungal or antiviral agents. Agonists of A2A adenosine receptors inhibit inflammation caused by dying pathogens. Accordingly, there is a need for selective, potent, and specific A2AAR agonists for use in adjunctive therapy for treating inflammatory bacterial, fungal and viral infections.
In accordance with the present invention, selective, potent, and specific A2AAR agonists have utility as a potential adjunct in therapy for treatment in combination with other agents that kill bacterial, fungal and viral infections such as, for example, anthrax, tularemia, escherichia coli and plague.
There is currently a need for pharmaceutical agents that are useful to reduce an inflammatory response due to the invasion of bacteria, funguses, or viruses or to reduce the inflammatory response due to toxins released by the bacteria, funguses, or viruses while alive or after they are killed using antibacterial agents, anti fungal agents or antiviral agents.
The present invention provides a therapeutic method for treating biological diseases that includes the administration of an effective amount of a suitable antibiotic agent, antifungal agent or antiviral agent in conjunction with an A2A adenosine receptor agonist. If no anti-pathogenic agent is known the A2A agonist can be used alone to reduce inflammation, as may occur during infection with antibiotic resistant bacteria, or certain viruses such as those that cause SARS or Ebola. Optionally, the method includes administration of a type IV PDE inhibitor. The A2A adenosine receptor agonist can provide adjunctive therapy for treatment conditions such as, the inflammation, caused by sepsis, for example, human uremic syndrome when administered with antibiotics in the treatment of bio-terrorism weapons, such as anthrax, tularemia, Escherichia coli, plague and the like. The present invention also provides adjunctive therapy for treatment of lethal bacterial, fungal and viral infections such as anthrax, tularemia, escherichia and plague comprising administration of an antibacterial agent, an antifungal agent or an antiviral agent in conjunction with selective, A2A adenosine receptor agonists.
The present invention provides a therapeutic method for treating biological diseases that provoke inflammation either alone or in combination with a disease killing medicine. These include bacteria in combination with antibiotics, including but not limited to bacteria that cause anthrax, tularemia, plague, lyme disease and anthrax. Also included are viruses including but not limited to those that cause RSV, severe acute respiratory syndrome (SARS), influenza and Ebola with or without anti-viral therapy. Also included are yeast and fungal infections with or without anti-yeast or anti-fungal agents.
The antibacterial agent, antifungal agent or antiviral agent can be co-administered (e.g., simultaneously) with the A2A adenosine receptor agonist or they can be can be administered either simultaneously or as a mixture or they can be administered subsequently. The subsequent administration of the A2A adenosine receptor agonists can be prior to the agent, within minutes or up to about 48 hours after the administration of the agent. Preferably the administration of the A2A adenosine receptor agonists will be within about 24 hours and more preferably within about 12 hours.
The method of the invention will also be useful for treating patients with sepsis, severe sepsis, and potentially, the systemic inflammatory response syndrome, in addition to septic shock. The A2AAR agonists exert multiple anti-inflammatory effects early in the inflammatory cascade, and thus a short course of an A2AAR agonists could produce profound benefit in serious, life-threatening infectious and inflammatory disorders of humans, including inhalational anthrax, tularemia, escherichia and plague.
The anti-inflammatory effect of A2AAR agonists has been documented in vivo, in experimental models of meningitis, peritonitis and arthritis. The potentially fatal syndrome of bacterial sepsis is an increasingly common problem in acute care units. Sepsis and septic shock, now the eleventh leading cause of death in the United States, are increasing in frequency. Current estimates indicate that about 900,000 new cases of sepsis (approximately 60% Gram negative) occur in the United States annually with an estimated crude mortality rate of 35%. Furthermore, the mortality rate, as assessed in recent clinical trials, is approximately 25%, while approximately 10% of patients die from their underlying disease. Shock develops in approximately 200,000 cases annually with an attributable mortality rate of 46% (92,000 deaths). Sepsis accounts for an estimated $ 5-10 billion annually in health care expenditures. It is now widely appreciated that among hospitalized patients in non-coronary intensive care units, sepsis is the most common cause of death. Sepsis syndrome is a public health problem of major importance. A2AAR agonists are anticipated to have use as a new and unique adjunctive therapeutic approach to reduce morbidity and mortality. It is believed that this treatment will improve the outcome in systemic anthrax, tularemia, escherichia and plague.
The agonists of A2A adenosine receptors of the invention can inhibit neutrophil, macrophage and T cell activation and thereby reduce inflammation caused by bacterial and viral infections. The compounds, in conjunction with antibiotics or antiviral agents can prevent or reduce mortality caused by sepsis or hemolytic uremic syndrome or other inflammatory conditions. The effects of adenosine A2A agonists are enhanced by type IV phosphodiesterase inhibitors such as rolipram.
The invention also provides a compound of formula I for use in medical therapy (e.g., for use as an adjunct in the treatment of potentially lethal bacterial infections, such as, anthrax, tularemia, Escherichia, plague, or other bacterial or viral infections, and treatment of systemic intoxification caused by bacterial and/or viral infections, as well as the use of a compound of formula I for the manufacture of a medicament for reducing inflammation caused by the bacteria or virus or the treatment thereof in a mammal, such as a human. The compounds of the invention are also useful for treatment of treating systemic intoxification wherein the bacterial or viral agents cause inflammation either directly or as a result of treatment, e.g., with an antibiotic or antiviral agent.
Sepsis is a severe illness caused by overwhelming infection of the bloodstream by toxin-producing bacteria or viruses. The infection, which can manifest as inflammation, can be caused by the bacteria or virus pathogens directly or from the treatment thereof, i.e., the death of the pathogens due to treatment with antibacterial or antiviral agents. Sepsis can be also be viewed as the body's response to an infection. The infection can be caused by micro-organisms or “germs” (usually bacteria) invade the body, can be limited to a particular body region (e.g., a tooth abscess) or can be widespread in the bloodstream (often referred to as “septicemia” or “blood poisoning”)
The systemic intoxification or inflammatory shock is often referred to as Septic shock; Bacteremic shock; Endotoxic shock; Septicemic shock; or Warm shock.
Septic shock is a serious, abnormal condition that occurs when an overwhelming infection leads to low blood pressure and low blood flow. Vital organs, such as the brain, heart, kidneys, and liver may not function properly or may fail. Septic shock occurs most often in the very old and the very young. It also occurs in people with underlying illnesses. Any bacterial organism can cause septic shock. Fungi and viruses may also cause this condition. Toxins released by the bacteria, fungi or viruses may cause direct tissue damage, and may lead to low blood pressure and/or poor organ function. These toxins can also produce a vigorous inflammatory response from the body, which contributes to septic shock.
In another aspect, the present invention also provides a method to treat severe acute respiratory syndrome (SARS), comprising administering to a mammal in need of said therapy, an effective anti-inflammatory amount of an agonists of A2A adenosine receptor, optionally with a PDE-IV inhibitor, such as, rolipram.
The following definitions are used, unless otherwise described. Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl, alkylaryl, etc. denote both straight and branched alkyl groups; but reference to an individual radical such as “propyl” embraces only the straight chain radical, a branched chain isomer such as “isopropyl” being specifically referred to. Aryl includes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical attached via a ring carbon of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C1-C4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
It will be appreciated by those skilled in the art that the compounds of formulas (I), (II), (III), and (IV) have more than one chiral center and may be isolated in optically active and racemic forms. Preferably, the riboside moiety of the compounds is derived from D-ribose, i.e., the 3′,4′-hydroxyl groups are alpha to the sugar ring and the 2′ and 5′ groups is beta (3R,4S,2R,5S). When the two groups on the cyclohexyl group are in the 1- and 4-position, they are preferably trans. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, or enzymatic techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase) and how to determine adenosine agonist activity using the tests described herein, or using other similar tests which are well known in the art.
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
Specifically, (C1-C8)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl or octyl. As used herein, the term “cycloalkyl” encompasses bicycloalkyl (norbornyl, 2.2.2-bicyclooctyl, etc.) and tricycloalkyl (adamantyl, etc.), optionally comprising 1-2 N, O or S. Cycloalkyl also encompasses (cycloalkyl)alkyl. Thus, (C3-C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like. (C1-C8)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl; (C1-C6)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1-C6)alkoxycarbonyl (CO2R2) can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio, (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, puridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).
Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl denotes a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1, 2, 3, or 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C1-C8)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
The term “heterocycle” generally represents a non aromatic heterocyclic group, having from 3 to about 10 ring atoms, which can be saturated or partially unsaturated, containing at least one heteroatom (e.g., 1, 2, or 3) selected from the group consisting of oxygen, nitrogen, and sulfur. Specific, “heterocycle” groups include monocyclic, bicyclic, or tricyclic groups containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur. A “heterocycle” group also can include one or more oxo groups (═O) attached to a ring atom. Non-limiting examples of heterocycle groups include 1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline, 4H-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl, isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine, piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine, pyrroline, quinuelidine, thiomorpholine, and the like.
The term “alkylene” refers to a divalent straight or branched hydrocarbon chain (e.g. ethylene —CH2CH2—).
The term “aryl(C1-C8)alkylene” for example includes benzyl, phenethyl, 3-phenylpropyl, naphthylmethyl and the like.
The terms “systemic intoxification” or “inflammatory shock” refer to the build-up of toxins or an intense inflammatory response in the body due to the invasion and/or treatment of bacteria, fungi or viruses.
As used herein “anti-pathogenic agent” refers to compounds that have anti-bacterial, anti-fungal or antiviral activity.
As used herein the term “in conjunction with” refers to co-administration of an antibacterial agent, an antifungal agent or an antiviral agent with the A2A adenosine receptor agonist. The agents and the A2A adenosine receptor agonists can be administered either simultaneously or as a mixture or they can be administered subsequently. The subsequent administration of the A2A adenosine receptor agonists can be prior to the agent, within minutes or up to about 48 hours after the administration of the agent. Preferably the administration of the A2A adenosine receptor agonists will be within about 24 hours and more preferably within about 12 hours.
The carbon atom content of various hydrocarbon-containing moieties is indicated by a prefix designating the minimum and maximum number of carbon atoms in the moiety, i.e., the prefix Ci-Cj indicates a moiety of the integer “i” to the integer “j” carbon atoms, inclusive. Thus, for example, (C1-C8)alkyl refers to alkyl of one to eight carbon atoms, inclusive.
The compounds of the present invention are generally named according to the IUPAC or CAS nomenclature system. Abbreviations which are well known to one of ordinary skill in the art may be used (e.g., “Ph” for phenyl, “Me” for methyl, “Et” for ethyl, “h” for hour or hours and “rt” for room temperature).
In one embodiment, agonists of A2A adenosine receptors that are useful in the practice of the present invention include compounds having the formula (I):
wherein
Z is CR3R4R5 or NR4R5; each R1 is independently hydrogen, halo, —ORa, —SRa, (C1-C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3-C8)cycloalkyl, heterocycle, heterocycle(C1-C8)alkylene-, aryl, aryl(C1-C8)alkylene-, heteroaryl, heteroaryl(C1-C8)alkylene-, —CO2Ra, RaC(═O)O—, RaC(═O)—, —OCO2Ra, RbRcNC(═O)O—, RaOC(═O)N(Rb)—, RbRcN—, RbRcNC(═O)—, RaC(═O)N(Rb), RbRcNC(═O)N(Rb)—, RbRcNC(═S)N(Rb)—, —OPO3Ra, RaOC(═S)—, RaC(═S)—, —SSRa, RaS(═O)—, RaS(═O)2—, —N═NRb, or —OPO2Ra;
each R2 is independently hydrogen, halo, (C1-C8)alkyl, (C3-C8)cycloalkyl, heterocycle, heterocycle(C1-C8)alkylene-, aryl, aryl(C1-C8)alkylene-, heteroaryl, or heteroaryl(C1-C8)alkylene-; or
R1 and R2 and the atom to which they are attached is C═O, C═S or C═NRd,
R4 and R5 together with the atoms to which they are attached form a saturated or partially unsaturated, mono-, bicyclic- or aromatic ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms optionally comprising 1, 2, 3, or 4 heteroatoms selected from non-peroxide oxy (—O—), thio (—S—), sulfinyl (—SO—), sulfonyl (—S(O)2—) or amine (—NRb—) in the ring;
wherein any ring comprising R4 and R5 is substituted with from 1 to 14 R6 groups; wherein each R6 is independently halo, —ORa, —SRa, (C1-C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C1-C8)cycloalkyl, (C6-C12)bicycloalkyl, heterocycle or heterocycle (C1-C8)alkylene-, aryl, aryl (C1-C8)alkylene-, heteroaryl, heteroaryl(C1-C8)alkylene-, —CO2Ra, RaC(═O)O—, RaC(═O)—, —OCO2Ra, RbRcNC(═O)O—, RaOC(═O)N(Rb)—, RbRcN—, RbRcNC(═O)—, RaC(═O)N(Rb)—, RbRcNC(═O)N(Rb)—, RbRcNC(═S)N(Rb)—, —OPO3Ra, RaOC(═S)—, RaC(═S)—, —SSRa, RaS(═O)—, —NNRb, —OPO2Ra, or two R6 groups and the atom to which they are attached is C═O, C═S or; two R6 groups together with the atom or atoms to which they are attached can form a carbocyclic or heterocyclic ring;
R3 is hydrogen, halo, —ORa, —SRa, (C1-C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3-C8)cycloalkyl, heterocycle, heterocycle(C1-C8)alkylene-, aryl, aryl(C1-C8)alkylene-, heteroaryl, heteroaryl(C1-C8)alkylene-, —CO2Ra, RaC(═O)O—, RaC(═O)—, —OCO2Ra, RbRcNC(═O)O—, RaOC(═O)N(Rb)—, RbRcN—, RbRcNC(═O)—, RaC(═O)N(Rb)—, RbRcNC(═O)N(Rb)—, RbRcNC(═S)N(Rb)—, —OPO3Ra, RaOC(═S)—, RaC(═S)—, —SSRa, RaS(═O)—, RaS(═O)2—, —NNRb, —OPO2Ra; or if the ring formed from CR4R5 is aryl or heteroaryl or partially unsaturated then R3 can be absent;
each R7 is independently hydrogen, (C1-C8)alkyl, (C3-C8)cycloalkyl, aryl or aryl(C1-C8)alkylene, heteroaryl, heteroaryl(C1-C8)alkylene-;
X is —CH2ORa, —CO2Ra, OC(O)Ra, —CH2OC(O)Ra, —C(O)NRbRb, —CH2SRa, —C(S)ORa, —OC(S)Ra, —CH2OC(S)Ra or —C(S)NRbRc or —CH2N(Rb)(Rc);
wherein any of the alkyl, cycloalkyl, heterocycle, aryl, or heteroaryl, groups of R1, R2, R3, R6 and R7 is optionally substituted on carbon with one or more (e.g. 1, 2, 3, or 4) substituents selected from the group consisting of halo, —ORa, —SRa, (C1-C8)alkyl, cyano, nitro, trifluoromethyl, trifluoromethoxy, (C3-C8)cycloalkyl, (C6-C12)bicycloalkyl, heterocycle or heterocycle(C1-C8)-alkylene-, aryl, aryloxy, aryl(C1-C8)alkylene-, heteroaryl, heteroaryl(C1-C8)-alkylene-, —CO2Ra, RaC(═O)O—, RaC(═O)—, —OCO2Ra, RbRcNC(═O)O—, RaOC(═O)N(Rb)—, RbRcN—, RbRcNC(═O)—, RaC(═O)N(Rb)—, RbRcNC(═O)N(Rb)—, RbRcNC(═S)N(Rb)—, —OPO3Ra, RaOC(═S)—, RaC(═S)—, —SSRa, RaS(═O)p—, RbRcNS(O)p—, N═NRb, and —OPO2Ra;
wherein any (C1-C8)alkyl, (C3-C8)cycloalkyl, (C6-C12)bicycloalkyl, (C1-C8)alkoxy, (C1-C8)alkanoyl, (C1-C8)alkylene, or heterocycle, is optionally partially unsaturated;
each Ra, Rb and Rc is independently hydrogen, (C1-C8)alkyl, or (C1-C8)alkyl substituted with 1-3 (C1-C8)alkoxy, (C3-C8)cycloalkyl, (C1-C8)alkylthio, amino acid, aryl, aryl(C1-C8)alkylene, heteroaryl, or heteroaryl(C1-C8)alkylene; or Rb and Rc, together with the nitrogen to which they are attached, form a pyrrolidino, piperidino, morpholino, or thiomorpholino ring; and Rd is hydrogen or (C1-C6)alkyl; m is 0 to about 8 and p is 0 to 2; or a pharmaceutically acceptable salt thereof.
In another embodiment, the invention includes the use of compounds of formula (I) provided that when CR4R5 is a carbocyclic ring then at least one of R1, R2, or R3 is a group other than hydrogen or at least one R6 group is a group other than —CH2OH, —CO2Ra, RaC(═O)O—, RaC(═O)OCH2— or RbRcNC(═O)—; and provided that m is at least 1 when Z is NR4R5.
Specific and preferred values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.
A specific value for R1 is hydrogen, —OH, —CH2OH, —OMe, —OAc, —NH2, —NHMe, —NMe2 or —NHAc.
Another specific value for R1 is hydrogen, —OH, —OMe, —OAc, —NH2, —NHMe, —NMe2 or —NHAc.
Another specific value for R1 is hydrogen, —OH, —OMe, or —NH2.
Another specific value for R1 is hydrogen, —OH, or —NH2.
A more specific value for R1 is hydrogen or —OH.
A specific value for R1, R2 and the carbon atom to which they are attached is carbonyl (C═O).
A specific value for R2 is hydrogen or (C1-C8)alkyl, cyclopropyl, cyclohexyl or benzyl.
Another specific value for R2 is hydrogen, methyl, ethyl or propyl.
Another specific value for R2 is hydrogen or methyl.
A more specific value for R2 is hydrogen
A specific value for R3 is hydrogen, OH, OMe, OAc, NH2, NHMe, NMe2 or NHAc.
Another specific value for R3 is hydrogen, OH, OMe, or NH2.
Another specific value for R3 is hydrogen, OH, or NH2.
A more specific value for R3 is hydrogen or OH.
A specific value for the ring comprising R4, R5 and the atom to which they are connected is cyclopentane, cyclohexane, piperidine, dihydro-pyridine, tetrahydro-pyridine, pyridine, piperazine, decaline, tetrahydro-pyrazine, dihydro-pyrazine, pyrazine, dihydro-pyrimidine, tetrahydro-pyrimidine, hexahydro-pyrimidine, pyrazine, imidazole, dihydro-imidazole, imidazolidine, pyrazole, dihydro-pyrazole, and. pyrazolidine.
A more specific value for the ring comprising R4 and R5 and the atom to which they are connected is, cyclohexane, piperidine or piperazine.
A specific value for R6 is (C1-C8)alkyl, or substituted (C1-C8)alkyl, —ORa, —CO2Ra, RaC(═O)—, RaC(═O)O—, RbRcN—, RbRcNC(═O)—, or aryl.
Another specific value for R6 is (C1-C8)alkyl, —ORa, —CO2Ra, RaC(═O)—, RaC(═O)O—, RbRcN—, RbRcNC(═O)—, or aryl.
Another specific value for R6 is methyl, ethyl, butyl, OH, ORa, —CO2Ra, RaC(═O)—, OC(═O)CH2CH3, —CONRbRc, —NRbRcC or phenyl.
Another specific value for R6 is OH, OMe, methyl, ethyl, t-butyl, —CO2Ra, —C(═O)NRbRc, —OAc, —NH2, —NHMe, —NMe2, —NHEt or —N(Et)2.
Another specific value for R6 is —(CH2)1-2ORa, —(CH2)1-2C(═O)ORa, —(CH2)1-2C(═O)Ra, —CH2)1-2C(═O)Ra, —(CH2)1-2OCO2Ra, —(CH2)1-2NHRa, —(CH2)1-2NRbRc, —(CH2)1-2C(═O)NHRa, or —(CH2)1-2C(═O)NRbRc.
Another specific value for R6 is —CH2OH, —CH2OAc, CH2OCH3, —CH2C(═O)OCH3, —CH2C(═O)CH3, CH2C(═O)CH3, —CH2OCO2CH3, —CH2NH(CH3), or (CH2)1-2N(CH3)2.
Another specific value for R6 is methyl, ethyl, t-butyl, phenyl, —CO2Ra, —CONRbRc, or RaC(═O)—.
Another specific value for R6 is —CH2OH, —CH2OAc, —C(═O)OCH3, —C(═O)CH3, OCO2CH3—OCO2CH3, —CH2NH(CH3), or —(CH2)1-2N(CH3)2.
A more specific value for R6 is methyl, ethyl, —CO2Ra—CONRbRc, or RaC(═O).
A specific number of R6 groups substituted on the R4R5 ring is from 1 to about 4.
Specific values for Ra and Rb are independently hydrogen, (C1-C4)alkyl, aryl or aryl(C1-C8)alkylene.
More specific values for Ra and Rb are independently hydrogen, methyl, ethyl, phenyl or benzyl.
A more specific value for Ra is (C1-C8)alkyl.
Another specific value for R1 is methyl, ethyl, propyl or butyl.
A more specific value for Ra is methyl, ethyl, i-propyl, i-butyl or tert-butyl.
Another specific value for Rb and Rc is a ring
A specific value for R7 is hydrogen, alkyl, aryl or aryl(C1-C8)alkylene.
Another specific value for R7 is hydrogen, methyl or ethyl, phenyl or benzyl.
A more specific value for R7 is H, or methyl.
A specific value for —N(R7)2 is amino, methylamino, dimethylamino, ethylamino, pentylamino, diphenylethylamino, pyridylmethylamino, diethylamino or benzylamino.
A specific value for —N(R7)2 is amino, methylamino, dimethylamino, ethylamino, diethylamino diphenylethylamino, pentylamino or benzylamino.
A specific value for N(R7)2 is amino, or methylamino.
A specific value for X is —CH2ORa, —CO2Ra, OC(O)Ra, —CH2OC(O)Ra, —C(O)NRbRc.
Another specific value for X is —CH2ORa or —C(O)NRbRc.
A more specific value for X is —CH2OH or —C(O)NHCH2CH3.
A specific value for m is 0, 1, or 2.
A more specific value for m is 0, or 1.
Specific examples of rings comprising R4, R5 and the atom to which they are connected include:
where q is from 0 to 14 and Rd is hydrogen, provided that when q is zero then Rd is not hydrogen.
More specific examples of rings comprising R4, R5 and the atom to which they are connected include:
Specific values for the ring comprising R4, R5 and the atom to which they are connected are 2-methyl cyclohexane, 2,2-dimethylcyclohexane, 2-phenylcyclohexane, 2-ethylcyclohexane, 2,2-diethylcyclohexane, 2-tert-butyl cyclohexane, 3-methyl cyclohexane, 3,3-dimethylcyclohexane, 4-methyl cyclohexane, 4-ethylcyclohexane, 4-phenyl cyclohexane, 4-tert-butyl cyclohexane, 4-carboxymethyl cyclohexane, 4-carboxyethyl cyclohexane, 3,3,5,5-tetramethyl cyclohexane, 2,4-dimethyl cyclopentane. 4-cyclohexanecarboxylc acid, 4-cyclohexanecarboxylc acid esters, or 4-methyloxyalkanoyl-cyclohexane.
More specific values for the ring comprising R4, R5 and the atom to which they are connected are 4-piperidine, 4-piperidene-1-carboxylic acid, 4-piperidine-1-carboxylic acid methyl ester, 4-piperidine-1-carboxylic acid ethyl ester, 4-piperidine-1-carboxylic acid propyl ester, 4-piperidine-1-carboxylic acid tert-butyl ester, 1-piperidine, 1-piperidine-4-carboxylic acid methyl ester, 1-piperidine-4-carboxylic acid ethyl ester, 1-piperidine-4-carboxylic acid propyl ester, 1-piperidine-4-carboxylic acid tert-butyl ester, 1-piperidine-4-carboxylic acid methyl ester, 3-piperidine, 3-piperidene-1-carboxylic acid, 3-piperidine-1-carboxylic acid methyl ester, 3-piperidine-1-carboxylic acid tert-butyl ester, 1,4-piperazine, 4-piperazine-1-carboxylic acid, 4-piperazine-1-carboxylic acid methyl ester, 4-piperazine-1-carboxylic acid ethyl ester, 4-piperazine-1-carboxylic acid propyl ester, 4-piperazine-1-carboxylic acid tert-butylester, 1,3-piperazine, 3-piperazine-1-carboxylic acid, 3-piperazine-1-carboxylic acid methyl ester, 3-piperazine-1-carboxylic acid ethyl ester, 3-piperazine-1-carboxylic acid propyl ester, 3-piperidine-1-carboxylic acid tert-butylester, 1-piperidine-3-carboxylic acid methyl ester, 1-piperidine-3-carboxylic acid ethyl ester, 1-piperidine-3-carboxylic acid propyl ester or 1-piperidine-3-carboxylic acid tert-butyl ester.
Another group of specific values for the ring comprising R4 and R5 are 2-methyl cyclohexane, 2,2-dimethylcyclohexane, 2-phenyl cyclohexane, 2-ethylcyclohexane, 2,2-diethylcyclohexane, 2-tert-butyl cyclohexane, 3-methyl cyclohexane, 3,3-dimethylcyclohexane, 4-methyl cyclohexane, 4-ethylcyclohexane, 4-phenyl cyclohexane, 4-tert-butyl cyclohexane, 4-carboxymethyl cyclohexane, 4-carboxyethyl cyclohexane, 3,3,5,5-tetramethyl cyclohexane, 2,4-dimethyl cyclopentane, 4-piperidine-1-carboxylic acid methyl ester, 4-piperidine-1-carboxylic acid tert-butyl ester 4-piperidine, 4-piperazine-1-carboxylic acid methyl ester, 4-piperidine-1-carboxylic acid tert-butylester, 1-piperidine-4-carboxylic acid methyl ester, 1-piperidine-4-carboxylic acid tert-butyl ester, tert-butylester, 1-piperidine-4-carboxylic acid methyl ester, or 1-piperidine-4-carboxylic acid tert-butyl ester, 3-piperidine-1-carboxylic acid methyl ester, 3-piperidine-1-carboxylic acid tert-butyl ester, 3-piperidine, 3-piperazine-1-carboxylic acid methyl ester, 3-piperidine-1-carboxylic acid tert-butylester, 1-piperidine-3-carboxylic acid methyl ester, 1-piperidine-3-carboxylic acid tert-butyl ester
Specific compounds of formula (I) are those wherein each R7 is H, X is ethylaminocarbonyl and
R1 is hydroxy, R2 is hydrogen, and Z is 4-carboxycyclohexyl, wherein Ra is hydrogen, 4; Z is 4-methoxycarbonylcyclohexylmethyl, Ra is methyl, 5; R1 and R2 together are oxo, Z is a 4-carbonylcyclohexyl group, wherein Ra is methyl, methoxy, ethyl, ethoxy, propyl, isopropoxy, -isobutyl, tert-butyl, amine, methylamine or dimethylamine, 6.
Another group of specific compounds of formula (I) are those wherein each R7 is H, X is ethylaminocarbonyl, R1 is hydroxy, R2 is hydrogen, and Z is a substituted 4-(methyleneoxycarbonyl)cyclohexyl group, wherein Ra is methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, methylamine or dimethylamine, 7; or R1 and R2 together are oxo, and Z is a substituted-(methyleneoxycarbonyl)cyclohexyl group, wherein Ra is methyl, ethyl, propyl, tert-butyl, methoxy, ethoxy, methylamine or dimethylamine, 8.
Another group of specific compounds of formula (I) are those wherein each R7 is H, X is ethylaminocarbonyl, and R1 and R2 are each hydrogen, and Z is a 1-piperidyl-4-carboxylic acid or ester group, wherein Ra is hydrogen, methyl, ethyl, propyl, isopropyl, or t-butyl, 9; R1 and R2 together are oxo, and Z is a 1-piperidyl-4-carboxylic acid or ester group, wherein Ra is hydrogen, methyl, ethyl, propyl, isopropyl, or t-butyl, 10; R1 and R2 are each hydrogen and Z is a 4-(methyleneoxycarbonyl)piperidin-4-yl group wherein Ra is methyl, ethyl, propyl or t-butyl, amine, methylamine, dimethylamine, 11; or R1 and R2 together are oxo, and Z is a 4-(methyleneoxycarbonyl)piperidin-4-yl wherein Ra is methyl, ethyl, propyl or t-butyl, amine, methylamine, dimethylamine, 12; R1 and R2 are each hydrogen and Z is a 4-(methyleneoxycarbonyl)piperidin-4-yl-oxy wherein Ra is hydrogen, methyl, ethyl, propyl isopropyl, isobutyl, or t-butyl, 13 or R1 and R2 together are oxo, Z is a 4-(methyleneoxycarbonyl)piperidin-4-yl-oxy wherein Ra is hydrogen, methyl, ethyl, propyl, isopropyl, isobutyl, or t-butyl, 14.
Another group of specific compounds of formula (I) are those wherein each R7 is H, X is ethylaminocarbonyl, R1 and R2 are each hydrogen, and Z is a 4-piperidyl-1-carboxylic acid or ester group, wherein Ra is methyl, ethyl, propyl, isopropyl, isobutyl, or t-butyl, 15, R1 is hydroxy, R2 is hydrogen, and Z is a 4-piperidyl-1-carboxylic acid or ester group, wherein Ra is methyl, ethyl, propyl, isopropyl, isobutyl, or t-butyl, 16; or R1 and R2 together are oxo, and Z is a 4-piperidyl-1-carboxylic acid or ester group, wherein Ra is methyl, ethyl, propyl, isopropyl, isobutyl, or t-butyl, 17.
Another group of specific compounds of formula (I) are those wherein each R7 is H, X is ethylaminocarbonyl, R1 and R2 are each hydrogen, Z is a 4-piperazine-1-carboxylic acid or ester group wherein Ra is methyl, ethyl, isopropyl, isobutyl, or t-butyl, 18; or R1 and R2 together are oxo, Z is a 4-piperazine-1-carboxylic acid or ester group wherein Ra is methyl, ethyl, isopropyl, isobutyl, or t-butyl, 19.
Additional compounds useful to practice the invention are depicted in tables 1, 2, 3, 4, 5, 6 and 7 below:
Examples of anti-bacterial agents suitable for use in the present invention include, but are not limited to, acediasulfone, acetosulfone, amikacin, amoxicillin, amphotericin B, ampicillin, apramycin, arbekacin, aspoxicillin, aztreonam, brodimoprim, butirosin, capreomycin, carumonam, cefadroxil, cefatrizine, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefmenoxime, cefminox, cefodizime, ceforanide, cefotaxime, cefotiam, cefozopran, cefpirome, cefprozil, cefroxadine, ceftazidime, cefteram, ceftibuten, ceftriaxone, cefuzonam, cephalexin, cephaloglycin, cephalosporin C, cephradine, ciprofloxacin, clinafloxacin, colistin, cyclacillin, dapsone, diathymosulfone, dibekacinm, enviomycimm, epicillin, fortimicin(s), gentamicin(s), gramicidin S, isepamicin, kanamycin(s), lucensomycin, lymecycline, micronomicin, natamycin, neomycin, netilmicin, paromomycin, pazufloxacin, penicillin N, peplomycin, perimycin A, polymyxin, p-sulfanilylbenzylamine, ribostamycin, ristocetin, sisomicin, sparfloxacin, succisulfone, 2-p-sulfanilylanilinoethanol, 4,4′-sulfinyldianiline, sulfachrysoidine, sulfamidochrysoidine, sulfanilic acid, sulfoxone, teicoplanin, tetroxoprim, thiazolsulfone, tigemonam, tobramycin, tosufloxacin, trimethoprim, trovafloxacin, tuberactinomycin, vancomycin and the like. Preferred antibiotic agents are ciprofloxacin and ceftriaxone.
Examples of anti-fungal (anti-yeast) agents suitable for use in the present invention include, but are not limited to amphotericin B, azaserine, candicidin(s), lucensomycin, mepartricin, natamycin, nystatin, tubercidin and the like.
Examples of antiviral agents suitable for use in the present invention include, but are not limited to abacavir, acyclovir, amantadine, famciclovir, foscavir, ganciclovir, indinavir, lamivudine, lopinavir, ritonavir and the like.
In another embodiment, agonists of A2A adenosine receptors that are useful in the practice of the present invention include compounds having the formula (II):
wherein Z is CR3R4R5; each R1, R2 and R3 is hydrogen; R4 and R5 together with the carbon atom to which they are attached form a cycloalkyl ring having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms; and
wherein the ring comprising R4 and R5 is substituted with (CH2)0-6—Y; where Y is —CH2ORa, —CO2Ra, —OC(O)Ra, CH2OC(O)Ra, C(O)NRbRc, —CH2SRa, —C(S)ORa, —OC(S)Ra, —CH2OC(S)Ra or C(S)NRbRcC or —CH2N(Rb)(Rc);
each R7 is independently hydrogen, (C1-C8)alkyl, (C3-C8)cycloalkyl, aryl or aryl(C1-C8)alkylene;
X is —CH2ORa, —CO2Ra, —OC(O)Ra, —CH2OC(O)Ra, —C(O)NRbRc, —CH2SRa, —C(S)ORa, —OC(S)Ra, —CH2OC(S)Ra or C(S)NRbRc or —CH2N(Rb)(Rc);
each Ra, Rb and Rc is independently hydrogen, (C1-C8)alkyl, or (C1-C8)alkyl substituted with 1-3 (C1-C8)alkoxy, (C3-C8)cycloalkyl, (C1-C8)alkylthio, amino acid, aryl, aryl(C1-C8)alkylene, heteroaryl, or heteroaryl(C1-C8)alkylene; or Rb and Rc, together with the nitrogen to which they are attached, form a pyrrolidino, piperidino, morpholino, or thiomorpholino ring; and m is 0 to about 6; or a pharmaceutically acceptable salt thereof.
A specific value for —N(R7)2 is amino, monomethylamino or cyclopropylamino.
A specific value for Z is carboxy- or —(C1-C4)alkoxycarbonyl-cyclohexyl(C1-C4)alkyl.
A specific value for Ra is H or (C1-C4)alkyl, i.e., methyl or ethyl.
A specific value for Rb is H, methyl or phenyl.
A specific value for Rc is H, methyl or phenyl.
A specific value for —(CR1R2)m— is —CH2— or —CH2—CH2—.
A specific value for X is CO2Ra, (C2-C5)alkanoylmethyl or amido.
A specific value for Y is CO2Ra, (C2-C5)alkanoylmethyl or amido.
A specific value for m is 1.
Specific A2A adenosine receptor agonists suitable for use with the present invention having formula (II) include those described in U.S. Pat. No. 6,232,297. Preferred compounds of formula (II) are those wherein each R7 is H, X is ethylaminocarbonyl and Z is 4-carboxycyclohexylmethyl (DWH-146a), Z is 4-methoxycarbonylcyclohexylmethyl (DWH-146e), Z is 4-isopropylcarbonylcyclohexylmethyl (AB-1), Z is 4-acetoxymethyl-cyclohexylmethyl (JMR-193) or Z is 4-pyrrolidine-1-carbonylcyclohexylmethyl (AB-3). These compounds are depicted below.
The specific A2A adenosine receptor agonists suitable for use with the present invention having formula (II) include those described in U.S. Pat. No. 6,232,297. These compounds, having formula (II), can be prepared according to the methods described therein.
Another specific group of agonists of A2A adenosine receptors that are useful in the practice of the present invention include compounds having the general formula (III):
wherein Z2 is a group selected from the group consisting of —OR12, —NR13R14, a —C≡C-Z3, and —NH—N═R17;
each Y2 is individually H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl or phenyl C1-C3 alkyl;
R12 is
one of R13 and R14 has the same meaning as R12 and the other is hydrogen; and
R17 is a group having the formula (I)
wherein each of R15 and R16 independently may be hydrogen, (C3-C7)cycloalkyl or any of the meanings of R12, provided that R15 and R16 are not both hydrogen;
X2 is CH2OH, CH3, CO2R20 or C(═O)NR21R22 wherein R20 has the same meaning as R13 and wherein R21 and R22 have the same meanings as R15 and R16 or R21 and R22 are both H;
Z3 has one of the following meanings:
or a pharmaceutically acceptable salt thereof.
Specific C6-10-aryl groups include phenyl and naphthyl.
Preferably, in the compound of formula (I), Z2 is a group of the formula (iii)
—O—(CH2)n—Ar (iii)
wherein n is an integer from 1-4, preferably 2, and Ar is a phenyl group, tolyl group, naphthyl group, xylyl group or mesityl group. Most preferably Ar is a para-tolyl group and n=2.
Preferably, in the compound of formula (II), Z2 is a group of the formula (iv)
—NH—N═CHCy (iv)
wherein Cy is a C3-7-cycloalkyl group, preferably cyclohexyl or a C1-4 alkyl group, preferably isopropyl.
Preferably, in the compound of formula (II), Z2 is a group of the formula (vii)
—C≡C-Z3 (v)
wherein Z3 is C3-C16 alkyl, hydroxy C2-C6 alkyl or (phenyl) (hydroxymethyl).
Specific examples of such compounds of formula (I) include WRC-0470, WRC-0474 [SHA 211], WRC-0090 and WRC-0018, shown below:
wherein the H on CH2OH can optionally be replaced by ethylaminocarbonyl. Of these specific examples, WRC-0474[SHA 211] and WRC-0470 are particularly preferred.
Such compounds may be synthesized as described in: Olsson et al. (U.S. Pat. Nos. 5,140,015 and 5,278,150); Cristalli (U.S. Pat. No. 5,593,975); Miyasaka et al. (U.S. Pat. No. 4,956,345); Hutchinson, A. J. et al., J. Pharmacol. Exp. Ther., 251, 47 (1989); Olsson, R. A. et al., J. Med. Chem., 29, 1683 (1986); Bridges, A. J. et al., J. Med. Chem., 31, 1282 (1988); Hutchinson, A. J. et al., J. Med. Chem., 33, 1919 (1990); Ukeeda, M. et al., J. Med. Chem., 34, 1334 (1991); Francis, J. E. et al., J. Med. Chem., 34, 2570 (1991); Yoneyama, F. et al., Eur. J. Pharmacol., 213, 199-204 (1992); Peet, N. P. et al., J. Med. Chem., 35, 3263 (1992); and Cristalli, G. et al., J. Med. Chem., 35, 2363 (1992); all of which are incorporated herein by reference.
Another embodiment includes compounds having formula (III) where Z2 is a group having formula (vi):
wherein R34 and R35 are independently H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl, phenyl C1-C3 alkyl or R34 and R35 taken together with the nitrogen atom are a 5- or 6-membered heterocyclic ring containing 1-2 heteroatoms selected from nonperoxide oxygen, nitrogen (N(R13)) or sulphur atoms. Preferably one of R34 and R35 is hydrogen and the other is ethyl, methyl or propyl. More preferably one of R34 and R35 is hydrogen and the other is ethyl or methyl.
The 2-(pyrazol-1-yl)adenosine compounds of the invention, wherein Z2 is a group having formula (vi), can be prepared by reacting a 2-chloro- or 2-iodo adenosine derivative with an 1H-pyrazole-4-carboxamides compound having formula (vii):
where R34 and R35 are as described above, wherein selective protection/deprotection of the amido group is used as needed. A preferred pyrazole derivative useful in practicing this invention is a compound having the formula:
The 1H-pyrazole-4-carboxamides can be prepared starting with 1H-pyrazole-4-carboxylic acid, available from Aldrich Chemical Co. In the first step, the acid is converted to an ester, e.g., a methyl or ethyl ester. The ester converted to the amide via aminolysis, e.g., with methylamine to form the methyl amide. The pyrazole-4-carboxamide will react with the 2-halopurines in the presence of a strong base to provide the 2-(pyrazol-1-yl)adenosine compounds having formula (III).
Another specific group of agonists of A2A adenosine receptors that are useful in the practice of the present invention include compounds having the general formula (IV):
wherein Z4 is —NR28R29;
R28 is hydrogen or (C1-C4) alkyl; and R29 is
wherein each Y4 is individually H, (C1-C6)alkyl, (C3-C7)cycloalkyl, phenyl or phenyl(C1-C3)alkyl; and X4 is —C(═O)NR31R32, —COOR30, or —CH2OR30;
wherein each of R31 and R32 are independently; hydrogen; C3-7-cycloalkyl; (C1-C4)alkyl; (C1-C4)alkyl substituted with one or more (C1-C4)alkoxy, halogen, hydroxy, —COOR33, amino, mono((C1-C4)alkyl)amino, di((C1-C4)alkyl)amino or (C6-C10)aryl wherein aryl is optionally substituted with one or more halogen, (C1-C4)alkyl, hydroxy, amino, mono((C1-C4) alkyl)amino or di((C1-C4) alkyl)amino; (C6-C10)aryl; or (C6-C10)aryl substituted with one or more halogen, hydroxy, amino, mono((C1-C4)alkyl)amino, di((C1-C4)alkyl)amino or (C1-C4)alkyl;
R26 and R27 independently represent hydrogen, lower alkanoyl, lower alkoxy-lower alkanoyl, aroyl, carbamoyl or mono- or di-lower alkylcarbamoyl; and R30 and R33 are independently hydrogen, (C1-C4)alkyl, (C6-C10)aryl or (C6-C10)aryl((C1-C4)alkyl); or a pharmaceutically acceptable salt thereof.
In one embodiment of formula (IV), at least one of R28 and R29 is (C1-C4)alkyl substituted with one or more (C1-C4)alkoxy, halogen, hydroxy, amino, mono((C1-C4)alkyl)amino, di((C1-C4)alkyl)amino or (C6-C10)aryl wherein aryl is optionally substituted with one or more halogen, hydroxy, amino, (C1-C4)alkyl, R30OOC—(C1-C4)alkyl, mono((C1-C4)alkyl)amino or di((C1-C4)alkyl)amino.
In another embodiment, at least one of R31 and R32 is C1-4-alkyl substituted with one or more (C1-C4)alkoxy, halogen, hydroxy, amino, mono((C1-C4)alkyl)amino, di((C1-C4)alkyl)amino or C6-10-aryl wherein aryl is optionally substituted with one or more halogen, hydroxy, amino, (C1-C4)alkyl, R30OOC—(C1-C4)alkylene-, mono((C1-C4)alkyl)amino or di((C1-C4)alkyl)amino.
In another embodiment, at least one of R28 and R29 is C6-10-aryl substituted with one or more halogen, hydroxy, amino, mono((C1-C4)alkyl)amino, di((C1-C4)alkyl)amino or (C1-C4)alkyl.
In another embodiment, at least one of R31 and R32 is C6-10-aryl substituted with one or more halogen, hydroxy, amino, mono((C1-C4)alkyl)-amino, di((C1-C4)alkyl)amino or (C1-C4)alkyl.
In a preferred combination, R31 is hydrogen and R32 is (C1-C4)alkyl, cyclopropyl or hydroxy-(C2-C4)alkyl. A preferred R28 group is (C1-C4)alkyl substituted with (C6-C10)aryl, that is in turn substituted with R30O(O)C—(C1-C4)alkyline-.
A preferred compound having formula (IV) is:
wherein R30 is hydrogen, methyl, ethyl, n-propyl or isopropyl. More preferred is a compound wherein the R30 group is methyl or ethyl. The most preferred R30 group is methyl.
Two compounds that are particularly useful in practicing the present invention have the formula:
wherein R30 is hydrogen (acid, CGS21680) and where R30 is methyl (ester, JR2171).
The compounds of the invention having formula (IV) may be synthesized as described in: U.S. Pat. No. 4,968,697 or J. Med. Chem., 33, 1919-1924, (1990)
Specifically, the invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating systemic intoxification in a mammal (e.g. a human),
Specifically, the invention also provides the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof to prepare a medicament for treating inflammation caused by bacterial, fungal or viral infections and the inflammation caused by the treatment of these infections, e.g., by the death of the bacterial or viral cells in a mammal (e.g. a human).
The present method also includes the administration of a Type IV phosphodiesterase (PDE) inhibitor in combination with compounds having formulae (I), (II), (III), and (IV). The combination of the compounds of the invention with type IV phosphodiesterase inhibitor provides synergistic decreases in the inflammatory response of immune cells. Examples of Type IV phosphodiesterase (PDE) inhibitors include those disclosed in U.S. Pat. No. 4,193,926, and WO 92-079778, and Molnar-Kimber, K. L. et al., J. Immunol., 150, 295A (1993), all of which are incorporated herein by reference.
Suitable Type IV phosphodiesterase (PDE) inhibitors include racemic and optically active 4-(polyalkoxyphenyl)-2-pyrrolidones of general formula (VI):
(disclosed and described in U.S. Pat. No. 4,193,926) wherein R18 and R19 are independently the same or different and are hydrocarbon radicals having up to 18 carbon atoms with at least one being other than methyl, a heterocyclic ring, or alkyl of 1-5 carbon atoms which is substituted by one or more of halogen atoms, hydroxy, carboxy, alkoxy, alkoxycarbonyl or an amino group or amino.
Examples of hydrocarbon R18 and R19 groups are saturated and unsaturated, straight-chain and branched alkyl of 1-18, preferably 1-5, carbon atoms, cycloalkyl and cycloalkylalkyl, preferably 3-7 carbon atoms, and aryl and aralkyl, preferably of 6-10 carbon atoms, especially monocyclic.
Rolipram is an example of a suitable Type IV phosphodiesterase or PDE inhibitor included within the above formula. Rolipram has the following formula:
In cases where compounds are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.
Compounds of the present invention can conveniently be administered in a pharmaceutical composition containing the compound in combination with a suitable excipient. Such pharmaceutical compositions can be prepared by methods and contain excipients which are well known in the art. A generally recognized compendium of such methods and ingredients is Remington's Pharmaceutical Sciences by E. W. Martin (Mark Publ. Co., 15th Ed., 1975). The compounds and compositions of the present invention can be administered parenterally (for example, by intravenous, intraperitoneal or intramuscular injection), topically, orally, or rectally.
For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.
The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.
The compounds or compositions can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
Pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.
Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.
Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.
The compound is conveniently administered in unit dosage form; for example, containing about 0.05 mg to about 500 mg, conveniently about 0.1 mg to about 250 mg, most conveniently, about 1 mg to about 150 mg of active ingredient per unit dosage form. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations.
The compositions can conveniently be administered orally, sublingually, transdermally, or parenterally at dose levels of about 0.01 to about 150 μg/kg, preferably about 0.1 to about 50 μg/kg, and more preferably about 0.1 to about 10 μg/kg of mammal body weight.
For parenteral administration the compounds are presented in aqueous solution in a concentration of from about 0.1 to about 10%, more preferably about 0.1 to about 7%. The solution may contain other ingredients, such as emulsifiers, antioxidants or buffers.
The exact regimen for administration of the compounds and compositions disclosed herein will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment and, of course, the judgment of the attending practitioner.
The preparation of compounds useful in practicing the present invention are disclosed in U.S. patent application Ser. No. 10/236,379, filed Oct. 1, 2002, and can generally be prepared as illustrated in Schemes 1A and 1B below. Starting materials can be prepared by procedures described in these schemes, procedures described in the General methods below or by procedures that would be well known to one of ordinary skill in organic chemistry. The variables used in Schemes 1A and Scheme 1B are as defined herein or as in the claims.
The preparation of alkynyl cycloalkanols is illustrated in Scheme 1A. A solution of an appropriate cycloalkanone (where j is from 0-5) is prepared in a solvent such as THF. A solution of a suitable ethynylmagnesium halide compound in a solvent is added to the cycloalkanone. After addition, the solution is allowed to stir at about 20 C. for about 20 hours. The reaction is monitored via TLC until the starting material is consumed. The reaction is quenched with water, filtered over a plug of sand and silica, washed with a solvent, such as EtOAc, and evaporated to provide the product. Typically, two products are formed, the isomers formed by the axial/equatorial addition of the alkyne (where m is as defined above, and the sum of m1 and m2 is from 0 to about 7) to the ketone. The compounds are purified via flash chromatography using EtOAc/Hexanes to provide the product.
In accordance with one embodiment of the present invention a composition comprising an agonist of A2AAR is administered to a patient to treat septic shock and systemic inflammatory response syndrome. As used herein the term “treating” includes prophylaxis of the specific disorder or condition, or alleviation of the symptoms associated with a specific disorder or condition and/or preventing or eliminating said symptoms. In one embodiment a method for treating septic shock or systemic inflammatory response syndrome is provided wherein an agonist of A2AARs is administered to a patient to reduce inflammation and improve survival in a patient suffering from septic shock or systemic inflammatory response syndrome. In one embodiment the A2AAR agonist is selected from the group consisting of ATL146e, AB-1, AB-3 and JR-3213.
The preparation of 2-alkynyladenosines is illustrated in Scheme 1B. A flame-dried round bottom under nitrogen is charged with 5-(6-Amino-2-iodo-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-carboxylic acid ethylamide (NECA 2-Iodoadenosine) and a solvent such as DMF. The appropriate alkyne is added followed by acetonitrile and TEA. (The solvents are degassed.) The appropriate alkyne is added in acetonitrile, followed by TEA, 5 mole % Pd(PPh3)4, and CuI. The solution is allowed to stir for about 24 hours at room temperature, and monitored until complete by HPLC. If the reaction is not complete after this time, additional catalyst, CuI, and TEA are added. After the reaction is complete, the solvents are removed under high-vacuum and the residue taken up in a small amount of DMF. This product is isolated using preparative silica TLC. The product is purified by RP-HPLC.
The following abbreviations have been used herein:
All melting points were determined with a Thomas Hoover capillary melting point apparatus and are uncorrected. Nuclear magnetic resonance spectra for proton (1H NMR) were recorded on a 300 MHz GE spectrophotometer. The chemical shift values are expressed in ppm (parts per million) relative to tetramethylsilane. For data reporting, s=singlet, d=doublet, t=triplet, q=quartet, and m=multiplet. Mass spectra were measured on a Finnigan LcQ Classic. High resolution mass spectrometry (HRMS) data was provided by the Nebraska Center for Mass Spectrometry. Analytical HPLC was done on a Waters 2690 Separation Module with a Waters Symmetry C8 (2.1×150 mm) column operated at room temperature. Compounds were eluted at 200 μL/min with 70:30 acetonitrile:water, containing 0.5% acetic acid, with UV detection at 214 nm using a Waters 486 Tunable Detector. Preparative HPLC was performed on a Shimadzu Discovery HPLC with a Shim-pack VP-ODS C18 (20×100 mm) column operated at room temperature. Compounds were eluted at 30 mL/min with a gradient 20-80% of water (containing 0.1% TFA) to methanol over 15 minutes with UV detection at 214 nm using a SPD10A VP Tunable detector. All final compounds presented here were determined to be greater than 98% pure by HPLC. Flash chromatography was performed on Silicyle 60A gel (230-400 mesh) or using reusable chromatography columns and system from RT Scientific, Manchester N.H. Analytical thin-layer chromatography was done on Merck Kieselgel 60 F254 aluminum sheets. Preparative thin-layer chromatography was done using 1000 micron Analtech Uniplate with silica gel. All reactions were done under a nitrogen atmosphere in flame-dried glassware unless otherwise stated.
To a solution of about 10 mmol of the appropriate cyclohexanone in about 50 mL of THF is added to about 60 mL (30 mmol) of 0.5 M ethynylmagnesium bromide in THF. The solution is allowed to stir at about 20° C. for about 20 hours. After the starting material had been consumed, monitored by TLC, the reaction is quenched with about 5 mL of water, filtered over a plug of sand and silica, washed with EtOAc, and evaporated to yield a yellow oil. Usually the oil contained two spots on TLC with 20% EtOAc/Hexanes, which are visualized with Vanillin. Usually these two products are the different isomers formed by the axial/equatorial addition of the alkyne to the ketone. The compounds are purified via flash chromatography using 10% EtOAc/Hexanes to provide clear oils or white solids in a yield of about 50-80%.
To a solution of the appropriate piperazine/piperadine (about 10.0 mmol), in about 20 mL acetonitrile, is added about 12.0 mmol of propargyl bromide (80% stabilized in toluene) and about 50.0 mmol of anhydrous potassium carbonate. The reaction mixture is filtered, and evaporated to dryness. The residue is taken up in about 50 mL of dichloromethane/water and the organic layers removed. The aqueous layer is washed with an additional 3×25 mL dichloromethane. The organic layer is dried using anhydrous sodium sulfate, filtered, and concentrated to provide the crude product, which is purified using column chromatography.
To about 100 mg of the appropriate Boc-protected piperazine/piperadine is added 2-4 mL of neat TFA. The solution is allowed to stir for 6 hours. The TFA is removed under reduced pressure to yield a yellow oil. This oil is taken up in about 10 mL of dichloromethane to which is added 10-fold excess of TEA and 3 equivalents of the appropriate acyl chloride. The yellow solution is allowed to stir at room temperature for about 12 hours, after which time the solvents are removed and the product purified using a 1.1×30 cm 14 g column from Robert Thompson Scientific with a 5%-30% gradient of ethyl acetate/hexanes.
General Method 4: Preparation of 2-AAs (2-alkynyladenosines).
A flame-dried 25 mL round bottom under nitrogen is charged with 5-(6-amino-2-iodo-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-carboxylic acid ethylamide (2-Iodoadenosine) (about 40 mg) (X═CH3CH2NHC(O)—) and dissolved in about 2 mL of DMF. The appropriate alkyne (approx. 0.1 mL) is then added followed by about 4 mL of acetonitrile and about 0.1 mL of TEA. All three solvents had been degassed with nitrogen for at least 24 hours. To this solution is added 5 mole percent Pd(PPh3)4 and 6 mole % copper iodide. The yellowish solution is allowed to stir for 24 hours at room temperature, or until complete by HPLC. If the reaction is not complete at this time, additional catalyst, CuI, and TEA are added. After the reaction is complete, the solvents are removed under high-vacuum and the red/black residue taken back up in a small amount of DMF. This solution is added to a preparative silica TLC plate (Analtech 1000 microns, 20 cm×20 cm) and eluted first with 120 mL of 40% Hexanes/CH2Cl2, and then again after addition of 40 mL of MeOH. The UV active band (usually yellow in color) in the middle of the plate is collected, slowly washed with 4×25 mL 20% MeOH/CH2Cl2, and concentrated. This product is then purified by RP-HPLC.
A suspension of 113 g (0.4 mol) of dry guanosine (6.1), acetic anhydride (240 mL, 2.5 mol), dry pyridine (120 mL) and dry DMF (320 mL) was heated for 3.75 hours at 75° C. without allowing the temperature to exceed 80° C. The clear solution was then transferred to a 3 L Erlenmyer flask and filled with 2-propanol. Upon cooling the solution to room temperature crystallization was initiated and allowed to proceed at 4° C. overnight. The white solid filtrate was filtered, washed with 2-propanol and recrystallized from 2-propanol to provide 6.2 (96%). 1H NMR (300 Mhz, CDCl3) 8.20 (s, 1H, H-8), 6.17 (d, J=5.41 Hz, 1H, H-1) 5.75 (t, J=5.39 Hz, 1H, H-2), 5.56 (t, J=5.0, H-3), 4.41 (m, 3H, H-4.5), 2.14 (s, 3H, Ac), 2.11 (s, 3H, Ac), 2.10 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) 171.0, 170.3, 1702, 157.7, 154.8, 152.4, 136.7, 117.7, 85.5, 80.4, 73.0, 71.3, 64.0, 31.3, 21.2, 21.0.
To a 1 L flask was added 80 g (0.195 mol) [(2R,3R,4R,5R)-3-4-diacetyloxy-5-(2-amino-6-oxohyropurin-9-yl)oxolan-2-yl]m ethyl acetate (6.2), tetramethylammonium chloride (44 g, 0.4 mol), anhydrous acetonitrile (400 mL) and N,N-dimethylaniline (25 mL). The flask was placed in an ice salt bath and cooled to 2° C. To this solution was added dropwise POCl3 (107 mL 1.15 mol) at a rate that maintained the temperature below 5° C. (45 minutes). The flask was then removed from the ice bath, outfitted with a condenser, placed in an oil bath and allowed to reflux for 10 minutes. The solution changed to a red/brown color. The solvent was removed under reduced pressure to yield an oily residue which was transferred to a beaker containing 1000 g of ice and 400 mL of CHCl3 and allowed to stir for 1.5 hours to decompose any remaining POCl3. The organic phase was removed and the aqueous phase extracted with 3×50 mL of CHCl3 and pooled with the organic phase. The pooled organic layers were back extracted with 50 mL of water followed by stirring with 200 mL of saturated NaHCO3. The organic layer was further extracted with NaHCO3 until the aqueous extract was neutral (2×). The organic layer was finally extracted with brine and dried over MgSO4 for 16 hours. To the solution was added 800 mL of 2-propanol after which the solution was concentrated under reduced pressure. To the oily solid was added 200 mL of 2-propanol and the solution was refrigerated overnight. The crystalline product was filtered, washed, and allowed to dry overnight to give 6.3 (77%). 1H NMR (300 MHz, CD3OD) 8.31 (s, 1H, H-8), 7.00 (s, 2H, NH2) 6.06 (d, J=5.8 Hz, 1H, H-1), 5.83 (t, J=6.16 Hz, 1H, H-2), 5.67 (m, 1H, H-3), 4.29 (m, 3H, H-4.5), 2.07 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.98 (s, 3H, Ac). 13C NMR (300 MHz, CD3OD) 171.0, 170.4, 170.2, 160.8, 154.6, 150.8, 142.2, 124.5, 85.8, 80.6, 72.8, 71.2, 63.9, 21.4, 21.3, 21.1.
Isoamyl nitrite (5 mL, 37 mmol) was added to a mixture of 5.12 g (12 mmol) [(2R,3R,4R,5R)-3-,4-diacetyloxy-5-(2-amino-6-chloropurin-9-yl)oxolan-2-yl]methyl acetate (6.3), 12 (3.04 g, 12 mmol), CH2I2 (10 mL, 124 mmol), and CuI (2.4 g, 12.6 mmol) in THF (60 mL). The mixture was heated under reflux for 45 minutes and then allowed to cool to room temperature. To this solution was added 100 ml of saturated Na2S2O3. This step removed the reddish color. The aqueous layer was extracted 3× with chloroform, which was pooled, dried over MgSO4, and concentrated under reduced pressure. The product was then purified over a silica gel column using CHCl3-MeOH (98:2) to collect [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]meth yl acetate (6.4) (80% crystallized from EtOH). 1H NMR (300 MHz, CDCl3) 8.20 (s, 1H H-8), 6.17 (d, J=5.41 Hz, 1H, H-1), 5.75 (t, J=5.39 Hz, 1H, H-2), 5.56 (t, J=5.40 Hz, 1H, H-3), 4.38 (m, 3H, H-4.5), 2.14 (s, 1H, Ac), 2.11 (s, 1H, Ac), 2.10 (s, 1H, Ac).
To a flask containing 6.0 g (11.1 mmol) [(2R,3R,4R,5R)-3,4-diacetyloxy-5-(6-chloro-2-iodopurin-9-yl)oxolan-2-yl]methyl acetate (6.4) was added 100 ml of liquid NH3 at −78° C. and the solution was allowed to stir for 6 hours. After which time it was allowed to come to room temperature overnight with concurrent evaporation of the NH3 to yield a brown oil. The product was crystallized from hot isopropanol to provide 6.5 (80%), m.p. 143-145° C., r.f.=0.6 in 20% MeOH/CHCl3. 1H NMR (300 MHz, DMSO-d6) 8.24 (s, 1H), 7.68 (s, 2H), 5.75 (d, J=6.16, 1H), 5.42 (d, J=5.40 Hz, 1H), 5.16 (d, J=4.62 Hz, 1H), 4.99 (t, J=5.39 Hz, 1H), 4.67 (d, J=4.81 Hz, 1H), 4.06 (d, J=3.37 Hz, 1H), 3.89 (m, 1H), 3.54 (m, 2H).
To a solution of 2.0 g (5.08 mmol) (4S,2R,3R,5R)-2-(6-amino-2-iodopurin-9-yl)-5(hydroxymethyl)oxolane-3,4-diol (6.6) in 100 mL acetone was added 9.6 g of p-toluenesulfonic acid and 5 ml of dimethoxypropane. The reaction was stirred at room temperature for 1 hour. Solid NaHCO3, 15 g, was added to the solution. The slurry was stirred for an additional 3 hours. The residue was filtered and washed 2× with EtOAc. The filtrate was then concentrated under reduced pressure. The residue was chromatographed on a silica gel column with MeOH—CHCl3 (1:99) to give 6.6 (72%) as a solid, m.p. 185-187° C. 1H NMR (300 MHz, DMSO-d6) 8.22 (s, 1H, H-8), 7.69 (s, 2H), NH2), 6.00 (d, J=2.70 Hz, 1H, H-1), 5.21 (m, 1H, H-2), 5.07 (bs, 1H, OH), 4.88 (m, 1H, H-3), 4.13 (m, 1H, H-4), 3.47 (m, 2H, H-5), 1.49 and 1.28 (s, 3H, C(CH3)2).
To a stirred solution of 1.6 g (3.7 mmol) of [(1R,2R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7-7-dimethyl-3,6,8-trioxabicyclo[3.3.0]oct-2-yl]methan-1-ol (6.6) in 200 mL of H2O was added 0.60 g of KOH and, dropwise, a solution of 1.70 g (10.8 mml) of KMnO4 in 50 mL of H2O. The mixture was placed in the dark at room temperature for 2-4 days. The reaction mixture was then cooled to 5-10° C. and decolorized by a solution of 4 mL of 30% H2O2 in 16 mL of water, while the temperature was maintained below 10° C. using an ice-salt bath. The mixture was filtered through Celite and the filtrate was concentrated under reduced pressure to about 10 mL and then acidified to pH 4 with 2N HCl. The resulting precipitate was filtered off and washed with ether to yield 6.7 (70%) after drying as a white solid, m.p. 187-190 C. 1H NMR (300 MHz, DMSO-d6) 8.11 (s, 1H, H-8), 7.62 (s, 2H, NH2), 7.46 (s, 1H, COOH), 6.22 (s, 1H, H-1), 5.42 (d, J=5.71 Hz, 1H, H-2), 5.34 (d, J=6.16 Hz, 1H, H-3), 4.63 (s, 1H, H-4), 1.46 and 1.30 (s, 3H, C(CH3)2).
A solution of 1.72 g (3.85 mmol) of (2S,1R,4R,5R)-4-(6-amino-2-iodopurin-9-yl)-7,7-dimethyl-3,6,8-trioxabicyclo[3.3.0]octane-2-carboxylic acid (6.7) in 80 mL of 50% HCOOH was stirred at 80° C. for 1.5 hours. The reaction mixture was evaporated under reduced pressure, dissolved in H2O, and the solvent was evaporated again. This process was repeated until there was no odor of formic acid in the residue. Recrystallization from water provided 1.33 g (85%) 6.8 as a white solid, m.p. 221-223° C., dec. 1H NMR (300 MHz, DMSO-d6) 8.31 (s, 1H, H-8), 7.68 (s, 2H, NH2), 5.90 (d, J=6.55 Hz, 1H, H-1), 4.42 (m, 1H, H-2), 4.35 (d, J=2.31 Hz, 1H, H-4), 4.22 (m, 1H, H-3).
To a cooled (5° C.) and stirred solution of 1.29 g (3.17 mmol) of (2S,3S,4R,5R)-5-(6-amino-2-iodopurin-9-yl)-3,4-dihydroxyoxolane-2-carboxylic acid (6.8) in 150 mL of absolute ethanol was added dropwise 1.15 mL of ice-cooled SOCl2. The mixture was stirred at room temperature overnight and then brought to pH 8 with saturated aqueous NaHCO3. The mixture was filtered, and then the filtrate was concentrated under reduced pressure to yield a white solid which was dried and then redissolved in 20 mL of dry ethylamine at −20° C. for 3 hours and then at room temperature overnight. The reaction mixture was diluted with absolute ethanol, and the precipitated product was filtered off and washed with dry ether to provide 530 mg (72%) of 6.9 as a pure solid, m.p. 232-234° C. 1H NMR (300 MHz, DMSO-d6) 8.34 (s, 1H, H-8), 8.12 (t, 1H, NH), 7.73 (s, 2H, NH2), 5.85, (d, J=6.93 Hz, 1H, H-1), 4.54 (m, 1H, H-2), 4.25 (d, J=1.92 Hz, 1H, H-4), 4.13 (m, 1H, H-3), 3.28 (m, 2H, CH2CH3), 1.00 (t, J=7.2 Hz, 3H, CH2CH3).
To a 100 mL-flask containing 79 (4.0 g, 27.8 mmol) in DMF (40 mL) was added TBDMSCl (3.56 g, 23.6 mmol) and imidazole (3.79 g, 55.6 mmol). The reaction was allowed to stir at 25° C. for 16 hours after which time saturated aqueous LiBr (50 mL) was added and the reaction extracted with ether (2×50 mL). The ether layers were pooled and extracted again with LiBr (2×35 mL). The ether layer became clear. The ether layer was then concentrated in vacuo and the product purified by flash chromatography, on a silica gel column, eluting with 1:2 ether/petroleum ether to yield 83 (3.80 g, 62%) as a homogenous oil. 1H NMR (CDCl3) δ 3.46 (d, J=6.2 Hz, 2H), 3.39 (d, J=6.2 Hz, 2H), 1.95-1.72 (m, 4H), 1.65 (m, 1H), 1.40 (m, 1H), 1.03-0.89 (m, 4H), 0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (CDCl3) δ69.2, 69.1, 41.2, 41.1, 29.5, 26.5, 18.9, −4.8; APCI m/z (rel intensity) 259 (MH+, 100).
To a 100 mL-flask containing 83 (3.4 g, 13.2 mmol) in CHCl3 (30 mL) was added tosyl chloride (3.26 g, 17.1 mmol) and pyridine (3.2 mL, 39.6 mmol). The reaction was allowed to stir at 25° C. for 14 hours after which time the reaction was concentrated in vacuo to yield a wet white solid. To this solid was added ether (50 mL) and the solid was filtered and subsequently washed with additional ether (2×50 mL). The ether layers were pooled, concentrated in vacuo to yield a clear oil which was purified by flash chromatography, on a silica gel column, eluting with 1:4 ether/petroleum ether to yield 84 (4.5 g, 83%) as a white solid. 1H NMR (CDCl3) δ 7.78 (d, J=7.7, 2H), 7.33 (d, J=7.7 Hz, 2H), 3.81 (d, J=6.2 Hz, 2H), 3.37 (d, J=6.2, 2H), 2.44 (s, 3H), 1.95-1.72 (m, 4H), 1.65 (m, 1H), 1.40 (m, 1H), 1.03-0.89 (m, 4H), 0.88 (s, 9H), 0.04 (s, 6H); 13C NMR (CDCl3) δ 145.1, 133.7, 130.3, 128.4, 75.8, 68.9, 40.7, 38.0, 29.1, 26.5, 22.1, 18.9, −4.9; APCI m/z (rel intensity) 413 (MH+, 100).
A 3-neck 250 mL-flask equipped with a gas inlet tube and dry-ice condenser was cooled to −78° C. and charged with liquid ammonia (40 mL). To the reaction mixture was added lithium wire (600 mg, 86.4 mmol) generating a deep blue solution. The mixture was allowed to stir for 1 hour. Acetylene, passed through a charcoal drying tube, was added to the ammonia until all the lithium had reacted and the solution turned colorless, at which time the flow of acetylene was stopped, the acetylene-inlet tube and condenser removed and the flask outfitted with a thermometer. DMSO (20 mL) was added and the ammonia evaporated with a warm water bath until the mixture reached a temperature of 30° C. The solution was stirred at this temperature for 2 hours until the solution stopped bubbling. The mixture was cooled to 5° C. and compound 84 (11.25 g, 27.3 mmol), in DMSO (10 mL), was added. The temperature was maintained at 5° C. The mixture was allowed to stir at 5° C. for 0.5 hours. Then the solution was gradually warmed to room temperature and stirred for an additional 18 hours. The brown/black reaction mixture was poured slowly over ice (300 g) and extracted with ether (4×100 mL), dried with anhydrous sodium sulfate, and concentrated in vacuo to yield a yellow oil. The oil was subsequently dissolved in THF (200 mL) and changed to a brownish color upon addition of TBAF hydrate (11.20 g, 35.5 mmol). The solution was allowed to stir for 24 hours under N2 atmosphere. After stirring, the reaction was quenched with water (200 mL) and extracted with ether (3×100 mL). The ether extracts were combined and concentrated in vacuo. The crude product was purified by chromatography, on a silica gel column, eluting with 1:1 ether/petroleum ether to yield 86 (3.91 g, 93%) as a yellow oil. 1H NMR (CDCl3) δ 3.45 (d, J=6.2, 2H), 2.10 (d, J=6.2, 2H), 1.9 (s, 1H), 1.94-1.69 (m, 4H), 1.52-1.34 (m, 2H), 1.16-0.83 (m, 4H); 13C NMR (CDCl3) δ 83.8, 69.5, 69.0, 40.8, 37.7, 32.3, 29.7, 26.5.
To a solution of 960 mg (6.31 mmol) of 86 in 6 mL DMF was added 0.62 mL (7.57 mmol) pyridine and 0.78 mL (8.27 mmol) acetic anhydride. The reaction was allowed to stir overnight at room temperature. After 16 hours, starting material still remained. The reaction mixture was heated at 75° C. for 3 hours. The solvent was removed under reduced pressure to yield a yellow oil which was purified by flash chromatography, on silica gel, eluting with 1:3 ether/petroleum ether to yield 1.12 g (91%) of 87 as an oil. 1H NMR (CDCl3) δ3.87 (d, J=6.2 Hz, 2H), 2.06 (d, J=4.3 Hz, 2H), 2.03 (s, 3H), 1.98-1.93 (m, 1H), 1.92-1.83 (m, 2H), 1.83-1.74 (m, 2H), 1.63-1.36 (m, 2H), 1.12-0.90 (m, 4H); 13C NMR (CDCl3) δ 171.7, 83.7, 69.9, 69.6, 37.4, 37.3, 32.1, 29.7, 26.5, 21.4; APCI m/z (rel intensity) 195 (M+, 30), 153 (M+, 70), 135 (M+, 100).
A solution of chromium trioxide (600 mg, 6.0 mmol) in 1.5 M H2SO4 (2.6 mL, 150 mmol) was cooled to 5° C. and added to a solution of 86 (280 mg, 1.84 mmol) in acetone (15 mL). The mixture was allowed to warm to room temperature and allowed to stir overnight. Isopropanol (4 mL) was added to the green/black solution, which turned light blue after 1 hr. After adding water (15 mL), the solution was extracted with CHCl3 (6×25 mL). The organic layers were pooled and concentrated in vacuo to yield a white solid. The solid was dissolved in ether (50 mL) and extracted with 1 M NaOH (2×30 mL). The basic extracts were pooled, acidified w/10% HCl, and re-extracted with ether (3×30 mL). The ether layers were combined, dried with sodium sulfate and concentrated in vacuo to yield a white solid. The product was recrystallized from acetone/water to yield 88 (222 mg, 73%) as white needles: mp 84-85° C.; 1H NMR (CDCl3) δ 2.30-2.23 (m, 1H), 2.17-2.11 (m, 2H), 2.07-2.03 (m, 2H), 1.97-1.91 (m, 3H), 1.51-1.39 (m, 3H), 1.13-1.01 (m, 2H); 13C NMR (CDCl3) δ 182.5, 83.8, 69.6, 40.7, 37.7, 32.3, 29.6, 26.5; APCI m/z (rel intensity) 165 (M−, 100).
To a solution of 88 (240 mg, 1.45 mmol) in 7:3 CH2Cl2:MeOH (10 mL) was added TMS Diazomethane (2.0 M in hexanes) (0.9 mL, 1.8 mmol) in 0.2 ml aliquots until the color remained yellow. The reaction was allowed to stir for an additional 0.25 hours at room temperature. After stirring, glacial acetic acid was added dropwise until the solution became colorless. The reaction was concentrated in vacuo to an oil which was purified by flash chromatography on silica gel using ether:petroleum ether (1:9) to yield 89 (210 mg, 80%) as a clear oil. 1H NMR (CDCl3) δ 3.60 (s, 3H), 2.25-2.13 (m, 1H), 2.08-1.94 (m, 3H), 1.95-1.90 (m, 2H), 1.49-1.31 (m, 3H), 1.10-0.93 (m, 2H); 13C NMR (CDCl3) δ 176.7, 83.3, 69.8, 51.9, 43.4, 36.7, 31.9, 29.2, 26.3; APCI m/z (rel intensity) 181 (MH+, 100).
Yield: 345 mg, 81%. 1H NMR (CDCl3) δ 0.98-1.07, 1.40-1.52, 1.57-1.70, 1.78-1.93 (4×m, 10H, cyclohexyl), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, —C6H10CH2CCH), 3.78 (s, 3H, —OCH3), 3.96 (d, —C6H10CH2O—).
Yield: 433 mg, 83%. 1H NMR (CDCl3) δ 0.95 (d, 4H, —OCH2CH(CH3)2), 0.98-1.09, 1.40-1.51, 1.57-1.70, 1.78-1.93 (4×m, 10H, cyclohexyl), 1.94-2.04 (m, 1H, —OCH2CH(CH3)2), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, —C6H10CH2CCH), 3.91, 3.95 (2×d, 4H, —OCH2CH(CH3)2, —C6H10CH2O—).
Yield: 340 mg, 69%. 1H NMR (CDCl3) δ 0.97-1.08, 1.40-1.49, 1.55-1.69, 1.77-1.93 (4×m, 10H, cyclohexyl), 1.96 (t, 1H, acetylene), 2.10 (dd, 2H, —C6H10CH2CCH), 3.98 (d, —C6H10CH2O—), 5.15 (s, 2H, —OCH2Ph), 7.33-7.40 (m, 5H, Ar).
A solution of N-Boc-4-piperidinemethanol, 5.0 g (23.2 mmol) in chloroform, 50 mL, was prepared. Toluene sulfonyl chloride, 5.75 g (30.2 mmol), in 5.6 mL of pyridine (69.6 mmol) was added. The solution was stirred under nitrogen allowed to stir for 24 hours. Standard workup and chromatographic purification provided the title compound. Yield 6.0 g
To a solution of 1.0 g (8.9 mmol) (R)-(+)-3-methyl-cyclohexanone in 50 mL of THF was added 54 mL (26.7 mmol) of 0.5 M ethynylmagnesium bromide in THF. The solution was allowed to stir at 20° C. for 20 hours. Analysis by TLC indicated that the starting material had been consumed. The reaction was quenched with 5 mL of water, filtered over a plug of sand and silica, washed with EtOAc, and evaporated to yield 1.15 g of a yellow oil containing two spots (r.f.'s 0.33 (minor, JR3217A) and 0.25 (major, JR3217B), 20% EtOAc/Hexanes) which were visualized with Vanillin. The compound was purified via flash chromatography using 10% EtOAc/Hexanes (225 mL silica) to provide JR3217A and JR3217B.
The title compound was prepared starting with 4.0 g (22.3 mmol) of methylpipecolinate hydrochloride according to general method 2.
To a solution of methyl isonipecotate 3.5 g (24.4 mmol, 3.30 mL) in 100 mL dichloromethane was added TEA (1.5 eq, 36.6 mmol, 5.1 mL), propargyl bromide (3.0 eq, 73.2 mmol, 6.5 ml), at room temperature for 36 hrs. The reaction was quenched with 35 mL water to yield to provide a clear solution. The solution was extracted with dichloromethane 2×25 mL, dried with Na2SO4, and the solvent evaporated to provide a yellow oil. r.f. (40% EtOAc/Hexanes) 0.26 stains faint white with Vanillin, starting material r.f. 0.05 stains yellow with Vanillin. The product appeared pure after extraction.
The title compound was prepared starting with 2.0 g (12.7 mmol) of ethyl isonipecotate according to general method 2.
To a solution of 10.0 g (54.8 mmol) of tert-butyl-1-piperazine carboxylate in 60 mL acetonitrile was added 5.20 mL (60.4 mmol) propargyl bromide and 37.9 g (274 mmol) anhydrous potassium carbonate. Additional propargy bromide, 1.5 mL, was added after stirring for 36 hours at room temperature. The residue was evaporated to dryness. Dichloromethane, 50 mL, and water, 50 mL, were added. The reaction mixture was extracted with CH2Cl2, 4×40 mL, dried over magnesium sulfate, and evaporate to provide a brown oil. The oil was dissolved in dichloromethane and purify with a RT Scientific system using hexane/ethyl acetate gradient to yield 5.5 g (46%) of yellow oil, which ultimately crystallized upon standing.
To a solution of 3 g (19.0 mmol) of ethyl N-piperazinecarboxylate in 25 mL of CH3CN was added 1.57 g (1.32 mL 20.1 mmol) of 2-chloroacetonitrile and 15.6 g (95 mmol) K2CO3.1½H2O. The suspension was stirred at room temperature for 16 hours. The reaction was analyzed using TLC (35% Ethyl acetate/Hexanes, product r.f. 0.38 vs. sm r.f. of 0.02). The analysis indicated the reaction was complete. The golden yellow solution was evaporated to dryness. The residue was extracted with CH2Cl2/H2O, dried with MgSO4, and concentrated.
The title compound was prepared starting with 500 mg (2.52 mmol) of 2,5-Diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester according to general method 2.
The title compound was prepared starting with 3 g (17.9 mmol) of 1-cyclohexylpiperazine according to general method 2
To a flame-dried 25 mL round bottom flask under nitrogen was added 2.1 g of 4-Prop-2-ynyl-piperazine-1-carboxylic acid tert-butyl ester. To this solid was added 5 mL of 98% TFA in 1 mL portions. The solution turned wine red, bubbled and smoked. The additional portions of TFA were added when this activity subsided. After the third portion of TFA had been added only minimal bubbling occurred. The solution was allowed to stir under nitrogen at room temperature for an additional hour and evaporated under reduced pressure to yield the product as a thick red syrup. Assumed quantitative yield of 1.16 g. The residue was suspended in 20 mL dichloromethane and used immediately without further purification for the preparation of compounds JR4031, JR4033, and JR4035.
The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using methylchloroformate according to general method 3.
The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using isobutylchloroformate according to general method 3.
The title compound was prepared starting with tert-butyl ester (JR3257) and using tert-butylacetylchloride according to general method 3.
The title compound was prepared starting with 385 mg (3.1 mmol) of JR4029 and using acetyl chloride according to general method 3.
To a solution of piperidine-4-carboxylic acid (10 g, 77.5 mmol) and potassium carbonate (21.4 g, 155 mmol) in 150 mL of water was prepared. A solution of di-tert-butyl dicarbonate (16.9 g, 77.5 mmol) in 40 mL of THF was added dropwise via addition funnel at 0° C. The reaction was allowed to warm to room temperature gradually over 30 minutes and stirred for an additional 4 hours. The THF was removed under reduced pressure and the aqueous phase extracted with 50 mL of ether. The aqueous phase was then adjusted to pH 2 with 10% HCl and extracted with EtOAc, 4×50 mL. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to yield 17.2 g (97%) of JR3183 as a white solid. Rf=0.2 (35% EtOAc/Hexanes stained w/vanillin). 1H NMR (CDCl3) δ 11.83 (s, 1H), 3.98 (d, J=11.8 Hz, 2H), 2.83 (t, J=11.8, 2H), 2.46 (m, 1H), 1.88 (d, J=12.9 hz, 2H), 1.2 (m, 2H), 1.42 (s, 9H). 13C NMR (CDCl3) δ 180.0, 154.8, 79.8, 42.9, 40.8, 28.3, 27.7. APCI m/z (rel intensity) M−228.2 (100).
The following intermediate compounds are prepared using the general method 1 described herein and the appropriate starting materials.
The reaction of 110 with five equivalents of LiOH in THF/water for 6 hours gave 109 (7 mg, 72%) as a white solid which was crystallized from MeOH/H2O (0.1% TFA) after purification by reverse phase HPLC. 1H NMR (DMSO-d6) δ 8.70 (s, 1H), 8.41 (s, 1H), 7.62 (s, 2H), 5.89 (d, J=7.25 Hz, 1H), 4.53 (m, 1H), 4.27 (s, 1H), 4.08 (d, J=3.6 Hz, 1H), 2.29 (d, J=6.4 Hz, 2H), 2.15-1.99 (m, 1H), 1.92-1.76 (m, 4H), 1.52-1.38 (m, 1H), 1.38-1.19 (m, 2H), 1.02 (t, J=6.3 Hz 3H); 13C NMR (DMSO-d6) 176.7, 169.2, 155.6, 148.9, 145.2, 141.6, 119.0, 87.7, 85.0, 84.6, 81.6, 73.1, 71.9, 43.2, 35.9, 33.3, 31.2, 28.3, 25.6, 15.0. HRMS (FAB) m/z 474.2196 [(M+H)+ cacld for C22H29N6O6 474.2182].
The reaction of 89 with 2-IodoNECA under the general conditions described above provided 110 (74 mg, 60%) as a white solid. 1H NMR (CD3OD) δ 8.23 (s, 1H), 5.92 (d, J=7.7 Hz, 1H), 4.69-4.65 (dd, J=7.7 Hz, 4.6 Hz, 1H), 4.40 (s, 1H), 4.24 (d, J=4.6 Hz, 1H), 3.59 (s, 3H), 3.49-3.31 (m, 2H), 2.31 (d, J=6.6 Hz, 2H), 2.10-2.09 (m, 1H), 2.01-1.89 (m, 4H), 1.61-1.32 (m, 5H), 1.13 (t, J=7.3 Hz, 3H); 13C NMR (CD3OD) δ 177.1, 171.1, 156.3, 149.3, 146.7, 142.4, 119.7 89.6, 86.0, 85.5, 81.6, 74.0, 72.2, 51.2, 43.2, 36.8, 34.2, 31.8, 28.9, 26.2, 14.4; HRMS (FAB) m/z 487.2325 [(M+H)+ cacld for C23H31N6O6 487.2305].
The reaction of 87 with 2-IodoNECA under the general conditions described above gave 111 (78 mg, 62%) as a white solid. 1H NMR (CD3OD) δ 8.22 (s, 1H), 5.92 (d, J=8.1 Hz, 1H), 4.70-4.66 (dd, J=8.1 Hz, 4.6 Hz, 1H), 4.40 (d, J=1.2 Hz, 1H), 4.25-4.23 (dd, J=4.6 Hz, 1.2 Hz, 1H), 3.83 (d, J=6.5, 2H), 3.53-3.31 (m, 2H), 2.29 (d, J=6.5 Hz, 2H), 1.97 (s, 3H), 1.93-1.89 (m, 2H), 1.79-1.75 (m, 2H), 1.64-1.42 (m, 2H), 1.12 (t, J=7.3 Hz, 3H), 1.09-0.91 (m, 4H); 13C NMR (CD3OD) δ 172.0, 171.2, 156.2, 149.3, 146.7, 142.5, 119.7, 89.6, 86.3, 85.5, 81.5, 74.0, 72.2, 69.6, 37.4, 37.2, 34.2, 32.1, 29.4, 26.4, 19.9, 14.5; HRMS (FAB) m/z 501.2469 [(M+H)+ cacld for C24H33N6O6 501.2462].
The reaction of 86 (30 mg, 0.2 mmol) with 2-IodoNECA (28 mg, 0.07 mmol) under the general conditions described above gave 112 (7 mg, 24%) as a white solid. 1H NMR (CD3OD) δ 8.22 (s, 1H), 5.92 (d, J=7.7 Hz, 1H), 4.70-4.66 (dd, J=7.7 Hz, 4.8 Hz, 1H), 4.40 (d, J=1.2 Hz, 1H), 4.25-4.23 (dd, J=4.8 Hz, 1.2 Hz, 1H), 3.51-3.37 (m, 2H), 3.31 (d, J=6 Hz, 2H), 2.30 (d, J=6.8 Hz, 2H), 1.94-1.89 (m, 2H), 1.83-1.78 (m, 2H), 1.64-1.42 (m, 2H), 1.12 (t, J=7.3 Hz, 3H), 1.09-0.91 (m, 4H); 13C NMR (CD3OD) δ 170.3, 155.4, 148.5, 146.0, 141.6, 118.8, 88.7, 85.5, 84.6, 80.6, 73.1, 71.3, 66.8, 39.6, 36.9, 33.3, 31.5, 28.6, 25.6, 13.5; HRMS (FAB) m/z 459.2373 [(M+H)+ cacld for C22H31N6O5 459.2356].
To a sealed tube containing 5 mL of freshly distilled ethylamine was added 10 mg (0.02 mmol) of ATL146e. The flask was sealed and allowed to stir at 60° C. for 80 hours. After this time the reaction was only about 50% complete by HPLC. The vessel was cooled to 0° C., opened, and the ethylamine was removed in vacuo to yield 4.5 mg (73%) of JR3037 as a white solid and the recovery of 4.0 mg of starting material after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ. 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 500.8 (MH+, 100), 327.4 (3).
To a sealed tube containing 10 mL of saturated MeOH/NH3 solution was added 5 mg (0.01 mmol) of ATL146e. The flask was sealed and allowed to stir at 25° C. for 48 hours. The vessel was cooled to 0° C., opened, and the ammonia removed by bubbling N2 for 1 hour. The remaining solvent was then removed in vacuo to yield 4.0 mg (83%) of JR3055 as a white solid after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ 8.41 (s, 1H), 5.98 (d, J=7.2 Hz, 1H), 4.65 (dd, J=7.3 Hz, 4.8 Hz, 1H), 4.41 (d, J=2.0 Hz, 1H), 4.28 (dd, J=4.6 Hz, 2.0 Hz, 1H), 3.35 (m, 2H), 2.37 (d, J=6.4 Hz, 2H) 2.10 (m, 1H), 1.90 (m, _H), 1.53 (m, _H_), 1.23 (m, _H), 1.12 (t, J=7.3 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 472.3 (MH+, 100), 299.4 (10).
To a sealed tube containing 10 mL 2.0 M methylamine in methanol was added 16.5 mg (0.03 mmol) of ATL146e. The flask was sealed and allowed to stir at 70° C. for 120 hours. The vessel was cooled to 0° C., opened, and the solvent was removed in vacuo to yield 8.0 mg (48%) of JR3065 as a white solid after the residue was purified by RP-HPLC. 1H NMR (CD3OD-d4) δ. 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 486.3 (MH+, 100), 313.4 (35).
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.48 (s, 1H), 6.04 (d, J=6.9 Hz, 1H), 4.72 (dd, J=6.9 Hz, J=4.4 Hz, 1H), 4.46 (d, J=2.3 Hz, 1H), 4.33 (dd, J=4.6 Hz, J=1.9 Hz, 1H), 3.42 (m, 2H), 2.04 (m, 4H), 1.83, (m, 4H), 1.16 (t, J=7.3 Hz, 3H). 13C NMR (CD3OD-d4) δ 171.9, 155.3, 150.0, 144.3, 120.6, 95.4, 90.6, 89.5, 86.2, 79.9, 74.9, 74.0, 70.5, 42.9, 35.3, 24.4, 15.3. APCI m/z (rel intensity) 417.2 (MH+, 100), 399.4 (85), 244.3 (15), 26.5 (25). HRMS M+ actual 417.18864, observed 417.18880.
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.57 (s, 1H), 6.09 (d, J=6.6 Hz, 1H), 4.77 (dd, J=6.7, Hz, J=4.8 Hz, 1H), 4.46 (d, J=2.3 Hz, 1H), 4.37 (dd, J=4.6 Hz, J=2.3 Hz, 1H), 3.42 (m, 2H) 1.80 (m, 13H), 1.28 (m, 9H), 1.13 (t, J=7.3 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 527.3 (MH+, 60), 509.5 (100), 354.4 (5), 336.5 (5), 279.5 (8). HRMS M+actual 527.29819, observed 527.29830
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.51 (s, 1H), 6.06 (d, J=7.0 Hz, 1H), 4.75 (dd, J=6.4 Hz, J=4.9 Hz, 1H), 4.46 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.9 Hz, J=2.1 Hz, 1H), 3.42 (m, 2H), 2.12 (d, J=11.9 Hz, 2H), 1.80 (d, J=11.9 Hz, 2H), 1.58 (t, J=12.1 Hz, 2H), 1.28 (m, 4H), 1.15 (t, J=7.1 Hz, 3H), 0.91 (t, J=7.1 Hz, 3H). 13C NMR (CD3OD-d4) δ 171.9, 155.4, 150.0, 144.2, 143.8, 120.6, 94.5, 90.5, 86.1, 81.8, 74.9, 74.1, 70.3, 40.5, 39.8, 35.3, 31.0, 30.2, 15.2, 12.0. APCI m/z (rel intensity) 459.4 (MH+, 100), 441.4 (60), 268.4 (10). HRMS M+ actual 459.23559, observed 459.23550.
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.45 (s, 1H), 7.26 (m, 4H), 7.14 (m, 1H), 6.05 (d, J=7.3 Hz, 1H), 4.80 (dd, J=7.3 Hz, J=4.8 Hz, 1H), 4.46 (d, J=1.6 Hz, 1H), 4.34 (dd, J=4.7 Hz, J=1.8 Hz, 1H), 3.44 (m, 2H), 2.58 (m, 1H), 2.23 (d, J=11.7H, 2H), 1.92 (m, 4H), 1.78, (m, 2H), 1.15 (t, J=7.2 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 507.3 (MH+, 100) 489.4 (70), 334.3 (5), 316.5 (8). HRMS M+ actual 507.23559, observed 507.23580.
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.54 (s, 1H), 6.04 (d, J=6.9 Hz, 1H), 4.74 (dd, J=6.9 Hz, J=5.0 Hz, 1H), 4.46 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.7 Hz, J=1.9 Hz, 1H), 3.44 (m, 2H), 1.74 (s, 4H), 1.13 (m, 17H). APCI m/z (rel intensity) 487.3 (MH+, 75), 469.4 (100), 296.4 (10).
The reaction of 1-Ethynyl-2-methyl-cyclohexanol (JR3169B) (100 mg, 0.72 mmol) with 2-iodo-NECA (25 mg, 0.06 mmol) under the general coupling conditions gave JR3177A (8.0 mg) and JR3177B (8.2 mg) (overall yield 65%) as white solids after purification by a silica plug and RP-HPLC. JR3177A: 1H NMR (CD3OD-d4) δ 8.47 (s, 1H), 6.05 (d, J=6.9 Hz, 1H), 4.77 (dd, J=6.9 Hz, J=4.9 Hz, 1H), 4.45 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.6 Hz, J=2.1 Hz, 1H), 3.41 (m, 2H), 2.13 (d, J=12.7 Hz, 2H), 1.65 (m, 5H), 1.32 (m, 2H), 1.14 (t, J=7.0 Hz, 3H), 1.13 (d, J=6.6 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.3 (MH+, 100), 427.4 (80), 254.4 (14). 1H NMR (CD3OD-d4) δ 8.49 (s, 1H), 6.05 (d, J=6.9 Hz, 1H), 4.78 (dd, J=6.4 Hz, J=4.9 Hz, 1H), 4.45 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.6 Hz, J=1.6 Hz, 1H), 3.42 (m, 2H), 2.12 (d, J=12.3 Hz, 2H), 1.65 (m, 4H), 1.35 (m, 4H), 1.14 (t, J=7.3 Hz, 3H), 1.12 (d, J=6.6 Hz, 3H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.7 (MH+, 100), 427.3 (35), 254.4 (3.5).
The reaction of 1-Ethynyl-3-methyl-cyclohexanol (JR3149B) (100 mg, 0.72 mmol) with 2-iodo-NECA (25 mg, 0.06 mmol) under the general coupling conditions gave JR3179 (15.0 mg, 59%) as a white solid after purification by a silica plug and RP-HPLC. 1H NMR (CD3OD-d4) δ 8.49 (s, 1H), 6.06 (d, J=6.9 Hz, 1H), 4.75 (dd, J=6.4 Hz, J=4.9 Hz, 1H), 4.46 (d, J=1.9 Hz, 1H), 4.34 (dd, J=4.9 Hz, J=2.1 Hz, 1H), 3.42 (m, 2H), 2.09 (d, J=12.3 Hz, 2H), 1.73 (m, 4H), 1.46 (m, 1H), 1.23 (m, 1H), 1.169 (t, J=7.1 Hz, 3H), 0.95 (d, J=6.2 Hz, 3H), 0.89 (m, 1H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 445.3 (MH+, 100), 427.4 (40), 254.4 (4).
The title compound was prepared using the appropriate starting materials and procedures described herein. The results are as follows:
1H NMR (CD3OD-d4) δ 8.48 (s, 1H), 6.00 (d, J=6.9 Hz, 1H), 4.67 (dd, J=6.5 Hz, J=5.0 Hz, 1H), 4.42 (d, J=1.9 Hz, 1H)), 4.39 (s, 2H), 4.35 (dd, J=4.7 Hz, J=1.9 Hz, 1H), 4.13 (q,) 3.42 (m, 2H). 13C NMR (CD3OD-d4) δ. APCI m/z (rel intensity) 503.4 (MH+, 100), 330.3 (6).
35 mg (0.081 mmol) IodoNECA (62 mg alkyne, 0.41 mmol), 2 ml DMF, 4 ml Acetonitrile, 0.2 ml TEA, d(PPH3)4, CuI. Stirred overnight at room temperature (Nov. 29, 2001). Rxn is tan w/brown precipitate. TLC (20% MeOH/CH2C12) indicates r×n complete (r.f. INECA=0.67, r.f. product=0.45). Filtered mixture through celite, washed with 3×2 mL DMF, and evaporated under vacuum to brown oil. (solid precipitates out upon the addition of MeOH, thus used DMF to load on prep plate).
The following compounds can be prepared by following the general method 4 described herein and the appropriate intermediate compounds described herein.
Yield 3.4 mg, 10%. 1H NMR (CD3OD) δ 1.18 (t, 3H, —NHCH2CH3), 1.03-1.20, 1.51-1.70, 1.79-1.85, 1.94-2.01 (4×m, 10H, cyclohexyl), 2.35 (d, 2H, —C6H10CH2CC—), 3.46 (m, 2H, —NHCH2CH3), 3.73 (s, 3H, —OCH3), 3.94 (d, 2H, —C6H10CH2O—), 4.29 (dd, 1H, 3′-H), 4.45 (d, 1H, 4′-H), 4.72 (dd, 1H, 2′-H), 5.97 (d, 1H, 1′-H), 8.27 (s, 1H, 8-H). APCI m/z 517.4 (M+H).
Yield 8.5 mg, 30%. 1H NMR (CD3OD) δ 0.94 (d, 4H, —OCH2CH(CH3)2), 1.18 (t, 3H, —NHCH2CH3), 1.04-1.24, 1.54-1.72, 1.79-2.03 (3×m, 11H, cyclohexyl, —OCH2CH(CH3)2), 2.38 (d, 2H, —C6H10CH2CC—), 3.43 (m, 2H, —NHCH2CH3), 3.89, 3.94 (2×d, 4H, —C6H10CH2O—, —OCH2CH(CH3)2), 4.30 (dd, 1H, 3′-H), 4.46 (d, 1H, 4′-H), 4.71 (dd, 1H, 2′-H), 6.00 (d, 1H, 1′-H), 8.37 (br s, 1H, 8-H). APCI m/z 559.5 (M+H+).
Yield 1.0 mg, 3%. 1H NMR (CD3OD) δ 1.17 (t, 3H, —NHCH2CH3), 1.03-1.23, 1.52-1.71, 1.78-1.86, 1.93-2.02 (4×m, 10H, cyclohexyl), 2.35 (d, 2H, —C6H10CH2CC—), 3.45 (m, 2H, —NHCH2CH3), 3.97 (d, 2H, —C6H10CH2O—), 4.29 (dd, 11H, 3′-H), 4.45 (d, 1H, 4′-H), 4.72 (dd, 1H, 2′-H), 5.13 (s, 2H, —OCH2Ph), 5.97 (d, 1H, 1′-H), 7.33-7.37 (m, 5H, Ar), 8.30 (br s, 1H, 8-H). APCI m/z 593.3 (M+H+).
The following compounds can be prepared by following the general methods described herein and the appropriate intermediate compounds:
The effects of A2AAR agonists, initially WRC-0470 and most recently ATL146e and ATL193, were studied on phagocytic cells in vitro and in animal models of acute inflammation. The results indicated that these compounds are potent agonists and provide anti-inflammatory responses, both in vitro and in vivo. The effect of these compounds on human PMNL (843 receptors per cell) was characterized and quantified A2AARs. It has been documented that A2AAR agonists increase PMNL intracellular cyclic AMP concentrations while decreasing TNF-enhanced adherence to a fibrinogen-coated surface. The A2AAR agonists reduce TNF-stimulated superoxide release from adherent PMNLs. The super oxide release is completely blocked by the A2AAR antagonist ZM 241385 (ZM). Further, A2AAR agonists reduced PMNL oxidative activity in whole blood assays and decreased degranulation of activated PMNLs adhering to a biological surface. Rolipram synergistically accentuates all the effects discussed above, on activation of PMNLs. Finally, the protein kinase A inhibitor H-89 completely reversed the inhibitory effect of A2AAR agonists on the PMNL oxidative burst. These findings indicate that A2AAR agonists will modulate inflammation in vivo through direct actions on phagocytic cells.
Activation of A2AARs on human monocytes was also shown to strongly inhibit TNF release. This illustrates the anti-inflammatory action of A2AAR agonists. ATL146e is more potent than CGS21680 in the inhibition of LPS-stimulated human monocyte TNF production, an effect reversed by the selective A2AAR antagonist ZM241385. These data show that A2AAR agonists exert A2AAR-mediated anti-inflammatory effect and reduce TNF production by monocytes.
In these experiments, (n=15-16 per group; LPS 12.5 mg/kg), control animals were compared to those treated with ATL146e. The agonist was dosed i.p. at 6-hour intervals for 24 hours, beginning simultaneously with the initial intraperitoneal dose of LPS. The initial dosage of ATL146e utilized was 5 μg/kg; the protection from death (p=0.0002) is illustrated in
At the highest dosages (of ATL146e), complete protection is achieved. All animals surviving to 4 days recover completely. At the highest doses (of ATL146e), survival at 4 days is 100% vs. mortality of 75% in mice receiving control vehicle (phosphate buffered saline i.p.). The effect of an A2AAR agonist on mortality in a septic shock model is orders of magnitude superior to other “anti-inflammatory” agents used in similar models of septic shock, including corticosteroids, anti-LPS monoclonal antibodies, anti-TNF monoclonal antibodies, soluble TNF receptors, and IL-1 receptor antagonists.
ATL146e reduced mortality in a murine model of endotoxin-induced septic shock even after a delay in the onset of therapy. In these experiments, (N=15-16 per group: LPS 12.5 mg/kg), ATL146e was administered at a dose of 5 μg/kg i.p. at six hour intervals at various times after LPS challenge for a total of four doses. The results are illustrated in
The protective effect of ATL146e on mortality in the murine model of endotoxin-induced septic shock is specific for the A2A receptor. Two experimental strategies were employed to investigate the specificity of the protective effect observed with ATL146e on mortality and endotoxin-induced shock through the A2A AR receptor. ZM 241385 (ZM), a specific, potent, and highly selective antagonist of the A2AAR is used. As illustrated in
Homozygous A2A-KO mice. A2A-KO mice have been generated from a heterozygous breeding pair. These mice lack A2A-ARs as confirmed by PCR and by localization studies of A2A-ARs in wild type and A2A-KO mouse brains using a selective A2A AR monoclonal antibody (
Mice were injected with live E. Coli. and treated with an antibiotic (ceftriaxone). The control group of mice were treated with antibiotic alone. All mice were injected with 20 million E. Coli IP at time 0. As indicated the mice were treated once at time 0 with ceftriaxone or with 50 μg/kg ATL146e 8 times at 6 hour intervals. The results are illustrated in
Male C57BL/6 mice were injected (i.p.) with E. coli LPS (60 ng) and purified E. coli Shiga toxin-2 (Stx2, 12 ng) at zero hour. ATL-146e or ATL-203 (both 50 μg/kg) was administered i.p. at zero hour. Animals were sacrificed at 6 hrs and the kidneys removed for processing and analysis. IL-6 protein was increased 45-fold by LPS/Stx2 at 6 hr in comparison to the saline control. Both ATL compounds sharply reduced the renal IL-6 levels to approximately 16% of those from mice exposed to LPS/Stx2 (
Mice received 2.4 ng purified Stx2, i.p. at zero hr. and were treated either with or without ATL-203 compound (I.p.) beginning at zero hr, and every 12 hrs thereafter. Fixed and paraffin-embedded renal samples cut to 3 μl thick sections were reacted with neutrophil-specific antibody, etc. prior to analysis. The results illustrated in
These data demonstrate that the adenosine A2A receptor agonist effectively reduces Stx2-dependent infiltration of neutrophils in kidneys of C57BL/6 mice (
All cited publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.
This application claims priority from U.S. provisional patent application Ser. No. 60/371,434, filed Apr. 10, 2002, and U.S. provisional patent application Ser. No. 60/387,184, filed Jun. 7, 2002, both of which are incorporated by reference herein.
The invention described herein was made with government support under Grant Number RO1 HL37942 awarded by the National Institute of Health. The United States Government has certain rights in the invention
| Number | Date | Country | |
|---|---|---|---|
| 60371434 | Apr 2002 | US | |
| 60387184 | Jun 2002 | US |
