Patent Application
20130197025
This invention relates to A3 adenosine receptor (A3AR) allosteric modulators and uses thereof.
The following is a list of prior art which is considered to be pertinent for describing the state of the art in the field of the invention. Acknowledgement of these references herein will at times be made by indicating their number within brackets from the list below.
G protein-coupled receptors (GPCRs) class is the largest family of cell-surface receptors which plays a crucial role in intracellular signal transduction. Adenosine receptors are part of the GPCR class, which belongs to the Class A or rhodopsin-like subfamily of GPCRs. Adenosine, a purine nucleoside, produces numerous physiological actions via cell surface adenosine receptors. These receptors are widely distributed throughout the body and are divided into four subclasses, A1, A2A, A2B and A3 receptors, the latter being the most recently identified receptor.
The A3 adenosine receptor (A3AR) is involved in a variety of physiological processes. The receptor is highly expressed in various tumor cell types while expression in adjacent normal tissues is relatively low. Activation of the receptor by a specific synthetic agonist induces modulation of downstream signal transduction pathways which include the Wnt and the NF-kB, resulting in tumor growth inhibition (1-5).
In vivo studies have shown that A3AR agonists inhibit the development of colon, prostate and pancreatic carcinomas as well as melanoma and hepatoma. A3AR agonists were also been shown to act as anti-inflammatory agents by ameliorating the inflammatory process in different experimental autoimmune models such as rheumatoid arthritis, Crohn's disease and multiple sclerosis (6-10). It was proposed also that the A2A and A3 receptors mediate the anti-inflammatory effects of methotrexate (11).
A3 adenosine receptor (A3AR) expression levels are elevated in cancer cells as compared to normal cells (12). Thus, the A3AR expression level has been described as a means for the diagnosis of cancer (13). In addition, A3AR expression levels have also been described to be elevated in peripheral blood cells of patients with colorectal cancer (14).
Several members of the GPCR class of receptors have been reported to be modulated allosterically (15), i.e. these receptors have additional binding site(s) on a receptor that are distinct from the agonist binding site (orthosteric site, orthosterically modulated receptors), but that can modulate receptor activity.
Allosteric modulation of GPCRs has been characterized most extensively for muscarinic receptors (16), and it has been suggested that allosteric modulators may provide therapeutic advantages over orthosteric agonists. Such advantages may include greater subtype selectivity and fewer side effects (15).
The adenosine receptors are natural allosteric proteins because agonist-mediated signaling by GPCRs requires a conformational change in the receptor protein transmitted between two topographically distinct binding sites, one for the agonist and another for the G protein. Allosteric sites on GPCRs represent novel drug targets because allosteric modulators possess a number of advantages over classic orthosteric ligands, such as a ceiling level to the allosteric effect and a potential for greater GPCR subtype-selectivity.
Allosteric modulation of A1 adenosine receptors was reported (17). A number of aminobenzoylthiophenes, including PD81723, were allosteric modulators of the A1 adenosine receptor (19). These compounds were shown to be highly subtype-selective enhancers for A1 adenosine receptors (19) and were less likely to cause desensitization and down-regulation of receptors than selective A1 adenosine receptor agonists.
Some 1H-imidazo-[4,5-c]quinoline derivatives were described as selective allosteric enhancers of human A3 adenosine receptors (20). Specifically, the derivatives were shown to potentiate the potency and maximal efficacy of agonist-induced responses while decreasing the dissociation of the agonist N6-(4-amino-3-[125I]iodobenzyl)-5′-N-methylcarboxamidoadenosine from human A3 adenosine receptors.
In accordance with the first aspect, the present invention provides the use of an A3 adenosine receptor allosteric modulator (A3RM) for the preparation of a pharmaceutical composition for the preparation of a pharmaceutical composition for the treatment of a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR), wherein said A3RM has the following general formula (I):
wherein:
and any pharmaceutically acceptable salt thereof.
In accordance with a further aspect, there is also provide a method of modulating A3 adenosine receptor (A3AR) in a subject, the method comprises administering to said subject an amount of an A3 adenosine receptor allosteric modulator (A3RM), the amount being effective to modulate A3AR activity,
In accordance with yet a further aspect, there is provided a method of treating a subject for a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR), the method comprises administering to said subject an amount of an A3 adenosine receptor allosteric modulator (A3RM) having the general formula as defined above, the amount of the A3RM being effective to modulate A3AR activity.
The invention also provides a pharmaceutical composition for the treatment of a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR), the composition comprises as active ingredient an A3 adenosine receptor allosteric modulator (A3RM), having the general formula (I) as defined above.
A preferred composition in accordance with the invention is in the form suitable for oral administration.
The A3RM is preferably utilized in accordance with the invention for enhancing the A3AR.
The present invention also provides imidazoquinoline derivatives having the general formula I as defined above, for use as an A3AR allosteric enhancer.
The invention also provides imidazoquinoline derivatives selected from:
These imidazoquinoline derivatives are also some preferred A3RM in accordance with the invention. A specifically preferred imidazoquinoline derivative in accordance with the invention is N-(3,4-Dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (45)
In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
The present invention concerns allosteric modulation of the A3 adenosine receptor (A3AR) by the use of imidazoquinoline derivatives. It has been found that imidazoquinoline derivatives may effectively increase the efficacy of the receptor, upon binding to thereto.
As appreciated, while the invention is described in the following detailed description with reference to therapeutic methods comprising treatment with allosteric modulators of A3AR, it is to be understood that also encompassed within the present invention are pharmaceutical compositions comprising A3 adenosine receptor allosteric modulators, the uses of such A3 adenosine receptor allosteric modulators, as well as some novel imidazoquinoline derivatives found to be specifically effective as allosteric modulators of the receptor.
As used herein, the term “allosteric modulation” which may be used interchangeably with the term “allosteric regulation” denotes the regulation or modulation of an enzyme, receptor or other protein by binding of an effector molecule at the protein's allosteric site which is different from the binding site of the endogenous ligand.
Effectors that enhance the protein's activity are referred to as “allosteric activators” or “allosteric enhancers”, whereas those that decrease the protein's activation are called “allosteric inhibitors”.
Further, as used in the specification and claims, the forms “a”, “an” and “the” include singular as well as plural references unless the context clearly dictates otherwise. For example, the term “an imidazoquinoline derivative” denotes one or more compounds being the same or different chemical modifications of imidazoquinoline.
Further, as used herein, the term “comprising” is intended to mean that the methods and compositions of the invention may include the recited imidazoquinoline derivative but not excluding other substances. The term “consisting essentially of” is used to define methods and compositions that include the recited components but exclude other components that may have an essential significance on the biochemical response resulting from the binding of imidazoquinoline derivative to the receptor. For example, a composition consisting essentially of an imidazoquinoline derivative as the active ingredient and a pharmaceutically acceptable carrier will not include or include only insignificant amounts (amounts that will have an insignificant effect on the activity of the receptor) of other compounds capable of binding to the allosteric site or binding site of the receptor. “Consisting of” shall thus mean excluding more than trace elements of other components. Embodiments defined by each of these transition terms are within the scope of this invention.
Further, all numerical values, e.g. when referring the amounts or ranges of the components constituting the composition of the invention, are approximations which are varied (+) or (−) by up to 20%, at times by up to 10% of from the stated values. It is to be understood, even if not always explicitly stated that all numerical designations are preceded by the term “about”.
Thus, in accordance with the first aspect, the present invention provides the use of an A3 adenosine receptor allosteric modulator (A3RM) for the preparation of a pharmaceutical composition for the preparation of a pharmaceutical composition for the treatment of a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR), wherein said A3RM has the following general formula (I):
wherein:
and any pharmaceutically acceptable salt thereof, for the preparation of a pharmaceutical composition for the treatment of a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR).
Preferably, the composition is for enhancing A3AR activity.
The term “alkyl” is used herein to refer to a linear or branched hydrocarbon chain having from 1 to 10 carbon atoms and more preferably 1 to 6 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-heptyl, octyl and the like.
Similarly, the terms “alkenyl” and “alkynyl” denote a linear or branched hydrocarbon chain having, respectively, from 2 to 10, or from 3 to 10 carbon atoms and more preferably 2 to 6 or 3 to 6 carbon atoms, the alkenyl or alkynyl having at least one unsaturated bond.
The alkyl, alkenyl or alkynyl substituents may be substituted with a heteroatom containing group. Thus, it should be understood that while not explicitly stated, any of the alkyl modifications defined hereinabove and below, such as alkylthio, alkoxy, akanol, alkylamine etc, also include the corresponding alkenyl or alkynyl modifications, such as, akenylthio, akenyloxy, alkenol, alkenylamine, or respectively, akynylthio, alkynyloxy, alkynol, alkynylamine.
The term “aryl” denotes an unsaturated aromatic carbocyclic group of from 5 to 14 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Preferred aryls include phenyl, indanyl, benzimidazole.
The term “alkaryl” refers to -alkylene-aryl groups preferably having from 1 to 10 carbon atoms in the alkylene moiety and from 6 to 14 carbon atoms in the aryl moiety. Such alkaryl groups are exemplified by benzyl, phenethyl and the like.
The term “Substituted aryl” refers to an aromatic moiety which is substituted with from 1 to 3 substituents as defined above. A variety of substituents are possible, as appreciated by those versed in the art. Nonetheless, some preferred substituents include, without being limited thereto, halogen, (substituted) amino, nitro, cyano, alkyl, alkoxy, acyloxy or alkanol, sulphonyl, sulphynyl.
The term “Halo” or “halogen” refers to fluoro, chloro, bromo and iodo, preferably to chloro.
The term “acyl” refers to the groups H—C(O)— as well as alkyl-C(O)—.
The term “alkanol” refers to the group —COH as well as alk-OH, “alk” denoting an alkylene, alkenylene or alkynylene chain.
The term “alkoxy” is used herein to mean —O-alkyl, including, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy and the like.
The term “alkylthio” is used herein to mean —S-alkyl, including, but not limited to, methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio and the like.
The term “alkoxyalkyl” is used herein to mean -alkyl-O-alkyl, including, but not limited to, methoxymethyl, ethoxymethyl, n-propoxymethyl, isopropoxymethyl, n-butoxymethyl, isobutoxymethyl, t-butoxymethyl and the like.
The term “cycloalkyl” is used herein to mean cyclic hydrocarbon radicals including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
The term “alkoxycarbonyl” is used herein to mean —C(O)O-alkyl, including, but not limited to, methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl and the like.
The term “fused cycloalkyl” is used herein to mean any compound or substituent comprising at least two aliphatic rings which are connected at a single atom (to form a spirocyclic moiety), at two mutually bonded atoms or across a sequence of atoms (bridgehead). The fused rings may include any bicyclic, tricyclic as well as polycyclic moieties. Bicyclic substituents are preferred in accordance with this embodiment of the invention.
The term “haloform” as used herein is meant to encompass a —CX3 radical, wherein X is any of the halogen atom, namely, Cl, Br, F and I. Specifically, the halogen atom is F.
The term “heterocycloalkyl” as used herein is meant to encompass a cyclic hydrocarbon having from 4 to 10 carbon atoms and at least one heteroatom selected from S, O, NH and NR, wherein R is a substituent as defined hereinabove. It should be noted that the heteroatom may be located at any position on the heterocycloalkyl ring. A non-limiting list of heterocycloaklyl moieties include: tetrahydro-pyranyl, tetrahydro-thiopyranyl, pyperidinyl and the like. The term “heterocycloalkenyl” as used herein is meant to encompass a cyclic hydrocarbon having from 4 to 10 carbon atoms, at least one double bond and at least one heteroatom selected from S, O, NH and NR, wherein R is a substituent as defined hereinabove. It should be noted that the heteroatom may be located at any position on the heterocycloalkylene ring.
The term “isoindole-1,3-dione” as used herein is meant to encompass a radical having the following formula:
According to a more specific and preferred embodiment of the invention, the R1 substituent in A3RM has the following general formula (II):
wherein n is 0 or an integer selected from 1-5; preferably, n is 0, 1 or 2; and
In yet a further preferred embodiment, R1 in said A3RM is a substituent having the above formula (II), wherein X1 or X2, which may be the same or different, are selected from hydrogen, chloro, methoxy, methanol or a substituent having the formulae (IIIa) or (IIIb):
wherein Y is selected from N or CH.
In further embodiment R2 in said A3RM is selected from H, C1-10 alkyl, C4-10 cycloalkyl, the alkyl chain may be a straight or branched or form a four to seven membered cycloalkyl ring.
In yet a further embodiment, R2 in said A3RM is selected from a five to seven membered heterocyclic aromatic ring.
More preferred R2 substituents are selected from H, n-pentyl, or a five membered heterocyclic aromatic ring having the following formula (IV):
wherein Z is selected from O, S or NH, preferably O.
In accordance with another embodiment of the invention R2 comprises fused rings, particularly so as to form bicyclic substituents. Non-limiting examples of bicyclic compounds which may be used to form the substituents in the context of the invention comprise bicyclo[2.2.1]heptane, bicyclo[4.1.0]heptane, bicyclo[4.1.0]heptan-3-carboxylic acid, bicyclo[3.1.0]hexan-3-carboxylic acid, bicyclo[4.1.0]heptan-2-carboxylic acid, bicyclo[3.1.0]hexan-2-carboxylic acid, and bicyclo[2.2.1]heptan-2-carboxylic acid.
In accordance with yet another embodiment, R2 may be selected from 2-cyclohexene and 3-cyclohexene.
Specific imidazoquinoline derivatives in accordance with the invention are listed below:
In general, the novel derivatives 31-47 were synthesized as shown in the scheme depicted in
It was surprisingly found that modifying the Van Galen procedure by using microwave irradiation (a short period of about 40 min max) in the last reaction step resulted in a significant reduction in the required reaction time as well as an easier and more straightforward purification of the end products. This was unexpected since microwave irradiation has not been thought to be relevant for this particular reaction.
It is noted that the above specific imidazoquinoline derivatives are novel per se and all have been shown to modulate the response obtained by their allosteric binding to A3AR.
The imidazoquinoline derivatives of the invention where shown to have, on the one hand, reduced affinity, if any, to the orthosteric binding sites of the A1 and A2A, A2B adenosine receptors and reduced affinity to the orthosteric binding site of the A3 adenosine receptor, and on the other hand, high affinity to the allosteric site of the A3 adenosine receptor. This finding was unexpected in light of the non-specific affinity of known imidazoquinoline derivatives, such as N-phenyl-2-cyclopentyl-1H-imidazo[4,5-c]quinolin-4-amine (compound 30) to the orthosteric as well as allosteric binding sites of these receptors. The selective affinity of the derivatives disclosed herein is particularly evident with respect to compounds 33, 42, 44 and 45 in Tables 1 and 2.
As further shown in Table 2 hereinafter, the specific imidazoquinoline derivatives of the invention were shown to increase the activity of the A3AR. Thus, in accordance with a further preferred embodiment of the invention modulation comprises enhancement of A3AR activity. In this context, the A3AM are considered as A3AR activators.
The term “enhancement” denotes the effect of the imidazoquinoline derivative on the receptor exhibited by an increase of at least 15% in the efficacy of the A3 adenosine receptor by binding of the imidazoquinoline to the allosteric site of the receptor and/or by a decrease in dissociation rate of adenosine or an A3AR agonist to the orthosteric binding site.
Thus, in accordance with another of its aspects, the present invention provides a method of modulating A3 adenosine receptor (A3AR) in a subject, the method comprises administering to said subject an amount of an A3 adenosine receptor allosteric modulator (A3RM), the amount being effective to modulate A3AR, wherein said A3RM has the general formula (I) as defined above.
The invention also provides a method of treating a subject for a condition which requires for said treatment modulation of A3 adenosine receptor (A3AR), the method comprises administering to said subject an amount of the A3RM as defined above, the amount being effective to modulate A3AR activity.
The term treatment “treatment” as used herein refers to the administering of a therapeutic amount of the imidazoquinoline derivative as defined herein, alone or in combination with a ligand to the A3AR orthosteric binding site which is effective to achieve a therapeutic effect, the therapeutic effect being selected from one or more of the following: amelioration of undesired symptoms associated with a disease, prevention of the manifestation of such symptoms before they occur, slowing down a progression of the disease, slowing down any deterioration of symptoms, enhancement of onset of a remission period, slowing down of any irreversible damage caused in a progressive chronic stage of the disease, delaying of the onset of said progressive stage, lessening of the severity or cure of a disease, improving survival rate or more rapid recovery from a disease, preventing the disease form occurring or a combination of two or more of the above.
A variety of conditions may be treated by the modulation of the A3AR depending on the specific effect the imidazoquinoline has on the receptor.
When said modulation comprises inhibition of or decrease in efficacy of the receptor, the condition may be any condition which may also be treated by the binding of an A3 adenosine receptor antagonist. Such conditions comprise, without being limited thereto, certain malignancies, certain immuno-compromised afflictions, and high intraocular pressure.
When said modulation comprises enhancement or increase in efficacy of the receptor, the condition may be any condition which may also be treated by the binding of an A3 adenosine receptor agonist. Such conditions comprise, without being limited thereto, hyperproliferative disorders, and in particular all types of solid tumors; skin proliferative diseases (e.g. psoriasis); a variety of benign hyperplasic disorders; inflammatory diseases; ischemic conditions, such as myocardial or renal ischemia.
The term “solid tumors” refers to carcinomas, sarcomas, adenomas, and cancers of neuronal origin and if fact to any type of cancer which does not originate from the hematopoeitic cells and in particular concerns: carcinoma, sarcoma, adenoma, hepatocellular carcinoma, hepatocellularcarcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, cohndrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing's tumor, leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hematoma, bile duct carcinoma, melanoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma, ependynoma, pinealoma, retinoblastoma, multiple myeloma, rectal carcinoma, thyroid cancer, head and neck cancer, brain cancer, cancer of the peripherial nervous system, cancer of the central nervous system, neuroblastoma, cancer of the endometrium, as well as metastasis of all the above. It has been shown in accordance with the invention that increased expression of A3AR can be found not only in the primary tumor site but also in metastases thereof.
Benign hyperplasic disorders include, without being limited thereto, benign prostate hyperplasia (BPH), non-tumorigenic polyps in the digestive tract, in the uterus and others.
Inflammatory diseases include, without being limited thereto, rheumatoid arthritis, Crohn's disease, multiple sclerosis and others.
It should be appreciated that the method of the invention may also comprise the combined administration of the A3RM in combination with a ligand to the orthosteric binding site. When modulation involves enhancement of the receptor, the A3RM may be administered in combination with adenosine or an A3AR agonist.
The term “combination” includes a schedule of treatment that involves administration of at least the A3RM and the ligand to the orthosteric site. The schedule of treatment may comprise simultaneous or co-administration of the two active principles, or with an interval between administrations.
According to a preferred embodiment, the A3RM is administered to the subject orally, although other administration routes are applicable, including parenteral (intravenous, intramuscular, intra-arterial, subcutaneous, intranasal, via the lungs (inhalation)).
The A3RM is preferably used and administered in combination with a physiologically acceptable carrier to form a pharmaceutical composition, the latter also forming part of the invention.
The term “physiologically acceptable excipient” denotes any excipient that is useful in preparing a pharmaceutical composition or formulation that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and includes a excipient that is acceptable for veterinary use as well as human pharmaceutical use.
In making the compositions of this invention, the imidazoquinoline derivative is usually mixed with the excipient, diluted by an excipient or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the imidazoquinoline derivative. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
The effective amount of imidazoquinoline derivative in the pharmaceutical composition and unit dosage form thereof may be varied or adjusted widely depending upon the particular application, the manner or introduction, the potency of the particular compound, and the desired concentration. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the affinity of the imidazoquinoline derivative to the allosteric binding site, its distribution profile within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender, etc.
The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. The amount of therapeutically active compound in such a unit dosage form may vary from about 0.5 mg to 500 mg.
In this case, the composition of the invention will typically be administered over an extended period of time in a single daily dose, in several doses a day, as a single dose and in several days, etc. The treatment period will generally have a length proportional to the length of the disease process and the specific imidazoquinoline derivative effectiveness and the patient species being treated.
Microwave-assisted chemistry was performed on an Emrys™ Optimizer with Emrys™ Optimizer software. For the reactions round-bottom vials with a volume of 2-5 mL were used.
1H-NMR spectra were measured at 200 MHz with a Bruker AC 200 or Bruker DMX 600 spectrometer. 13C-NMR spectra were measured at 50 or 150 MHz. Chemical shifts for 1H and 13C are given in ppm (δ) relative to tetramethylsilane (TMS) as internal standard, coupling constants are given in Hz. Melting points were determined with a Büchi capillary melting point apparatus and are uncorrected. Combustion analyses of new target compounds were performed by the analytical department of the Gorlaeus Laboratories, Leiden University (The Netherlands) and are within 0.4% of theoretical values unless otherwise specified.
Compound 3 was prepared as described elsewhere [Bachman, G. B. et al. Quinoline Derivatives from 3-Nitro-4-hydroxyquinoline, J. Am. Chem. Soc., 1947, 69, 365-371]. In brief, a solution of NaOH (13.4 g) in water (26.8 ml) was cooled and stirred. Nitromethane (6.7 g, 5.9 ml, 110 mmol) was added dropwise, keeping the temperature at 25-30° C. The mixture was then warmed to 40° C. and again cooled and stirred while another portion of nitromethane (6.7 g, 5.9 ml, 110 mmol) was added slowly at 40-45° C. This temperature was maintained until all solid was dissolved and a clear red solution was obtained. The solution was then heated to 50-55° C. for two to five minutes and finally cooled to 30° C., poured on chipped ice (30 g) and acidified with concentrated HCl (30 ml). The resultant solution of methazonic acid 2 was immediately added to a solution of anthranilic acid 1 (13.7 g, 100 mmol) and concentrated HCl (9.2 ml) in water (200 ml). Yellow precipitate formed, which was left to stand at room temperature for 12-18 h. The precipitate was filtered off and washed with water. The cake was sliced into thin flakes and dried. Yield: 18.40 g (89%). Mp.: 196°-197° C. 1H NMR (DMSO-d6): δ 6.76 (d, 2H, J=6.6 Hz, CH2); 7.24 (t, 1H, J=6.6 Hz, N═CH); 7.54-7.82 (m, 2H, Ar); 8.02-8.12 (m, 2H, Ar); 12.09 (s, 1H, COOH).
Compound 5 was prepared as described elsewhere [Bachman, G. B. et al. Quinoline Derivatives from 3-Nitro-4-hydroxyquinoline, J. Am. Chem. Soc., 1947, 69, 365-371]. In brief, a mixture of 2-β-nitroethylideaminobenzoic acid 3 (10.4 g, 50 mmol) and technical acetic anhydride (50 ml) was placed in a 3-necked flask of at least 100 mL capacity fitted with thermometer, magnetic stirrer and reflux condenser. It was stirred and heated to 100-105° C. until a clear solution was obtained. Heating was then discontinued and anhydrous potassium acetate (5.0 g, 51 mmol) was added rapidly with stirring. The temperature rose spontaneously to 134-138° C. When it began to fall (five to ten minutes) external heating was applied and the mixture was refluxed for fifteen minutes with vigorous stirring, then allowed to cool slowly to room temperature. The product filtered off and washed with glacial acetic acid until the washings were colorless. Then suspended in water (50 ml) and dried in vacuum at 80° C. Yield: 6.93 g (49%). Mp.: >300° C. 1H NMR (DMSO-d6): δ 7.54 (t, 1H, J=7.3 Hz, Ar); 7.76-7.86 (m, 2H, Ar); 8.27 (d, 1H, J=8.0 Hz, Ar); 9.21 (s, 1H, Ar).
Compound 6 was prepared as described elsewhere [Van Galen, P. J. M. et. al. 1-H-imidazo[4,5-c]quinolin-4-amines: Novel Non-Xanthine Adenosine Antagonists. J. Med. Chem. 1991, 34, 1202-1206]. In brief, 3-Nitro-4-hydroxyquinoline 5 (5.7 g, 30 mmol) was added to phosphorus oxychloride (70.0 g, 41.2 ml, 450 mmol) with stirring. The mixture was refluxed for 30 minutes. After cooling the solvent was poured over crushed ice while stirring. After 1 hour the solid that was formed was filtered off, washed with cold water and dissolved in ethyl acetate. The solution was extracted with ice-cold NaOH (1 M) till pH=8-9 and dried over MgSO4. The solvent was evaporated and the residue was dried. Yield: 5.05 g (81%). Mp.: 118°-119° C. 1H NMR (DMSO-d6): δ 7.94-8.11 (m, 2H, Ar); 8.25 (d, 1H, J=8.0 Hz, Ar); 8.47 (d, 1H, J=9.5 Hz, Ar); 9.42 (s, 1H, Ar).
Compound 7 was prepared as described elsewhere [Van Galen, P. J. M. et. al. (1991) ibid.]. In brief, ammonia gas was passed, while stirring, through a solution of 3-nitro-4-chloroquinoline 6 (7.0 g, 30 mmol) in toluene (95 ml) and propanol (15 ml) till the product was formed. During the course of the reaction, the temperature was gradually raised till 70° C. After cooling, the solid was separated by filtration and washed successfully with toluene/2-propanol (70:30), ether and cold water until Cl− could no longer be detected. The solid was filtered off and dried at 80° C. Yield: 6.1 g (95%). Mp.: 255-257° C. 1H NMR (DMSO-d6): δ 7.50-7.66 (m, 1H, Ar); 7.81-7.92 (m, 2H, Ar); 8.59 (d, 1H, J=8.0 Hz, Ar); 9.03 (broad s, 2H, NH2); 9.18 (s, 1H, Ar)
Compound 8 was prepared as described elsewhere [Van Galen, P. J. M. et. al. (1991) ibid.]. In brief, to a mixture of 3-nitro-4-aminoquinoline 7 (3.2 g, 20 mmol) in absolute ethanol (60 ml) was added 10% palladium on charcoal (0.17 g). The mixture was hydrogenated under 2.5-3.5 atm pressure till the product was formed and subsequently filtered over Hyflo. The filtrate was evaporated and the residue was gradually solidified and dried in vacuum. Yield: 2.66 g (98%). Mp.: 183-185° C. MS (ESI) m/z 161.0 (M+1)+. 1H NMR (DMSO-d6): δ 4.73 (broad s, 2H, NH2); 5.88 (s, 2H, NH2); 7.16-7.37 (m, 2H, Ar); 7.64-7.70 (m, 1H, Ar); 7.98-8.03 (m, 1H, Ar); 8.22 (s, 1H, Ar).
1H-Imidazo[4,5-c]quinolines were prepared as described elsewhere [Young, R. C. et al. Purine Derivatives as Competitive Inhibitors of Human Erythrocyte Membrane Phosphatidylinositol 4-Kinase. J. Med. Chem. 1990, 33, 2073-2080]. In brief, polyphosphoric acid (1.3 ml/mmol) was added to 3,4-diaminoquinoline 8 and the appropriate carboxylic acid (1.2 eq.). The mixture was stirred at 100° C. for 5 hours. Then cooled to 0° C. and NH4OH was added slowly till pH=8-9. The mixture was extracted with ethyl acetate (3 times 15 ml), then washed with water, brine and again water and dried over MgSO4. The solvent was evaporated and the residue was dried.
Compound 9 was prepared as described elsewhere [Van Galen, P. J. M. et. al. (1991) ibid.]. In brief, compound 8 (1.4 g, 8.71 mmol) was heated under stirring with trimethyl orthoformate (23.8 ml). To a clear solution was cautiously added formic acid (0.45 ml) whereupon solid precipitated. The mixture was refluxed for 1 hour. After cooling to 40° C., ether (3.1 ml) and absolute ethanol (0.36 ml) were added and the mixture was cooled on ice for 1 hour. The solid that was formed was filtered off, washed with ether and subsequently with ethyl acetate and dried. Yield: 0.44 g (81%). Mp.: 263-265° C. 1H NMR (DMSO-d6): δ 7.67-7.72 (m, 2H, Ar); 8.18 (s, 1H, Ar); 8.29-8.34 (m, 1H, Ar); 8.61-8.65 (m, 1H, Ar); 8.93 (s, 1H, Ar).
Scale: 6.2 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=1:1. Yield: 0.88 g (63%). Mp.: 191-192° C. MS (ESI) m/z 223.6 (M+1)+. 1H NMR (CD3OD): δ 1.95-2.32 (m, 2H, CH2); 2.44-2.66 (m, 4H, 2CH2); 3.82-4.01 (m, 1H, CH); 7.58-7.71 (m, 2H, Ar); 8.01-8.11 (m, 1H, Ar); 8.32-8.38 (m, 1H, Ar); 9.01 (s, 1H, Ar).
Scale: 4.0 mmol Eluent for column chromatography was 5% methanol in dichloromethane. Yield: 0.77 g (81%). Mp.: 192° C. 1H NMR (CDCl3): δ 1.70-1.88 (m, 6H, 3CH2); 2.03-2.22 (m, 2H, CH2); 3.39-3.56 (m, 1H, CH); 7.27 (s, 1H, Ar); 7.53-7.66 (m, 2H, Ar); 8.21 (d, 2H, J=8.0 Hz, Ar); 9.18 (s, 1H, NH).
Scale: 6.3 mmol Eluent for column chromatography was 1-5% methanol in dichloromethane. Yield: 0.60 g (38%). Mp.: 205-206° C. MS (ESI) m/z 251.7 (M+1)+. 1H NMR (CDCl3): δ 1.12-1.39 (m, 4H, 2CH2); 1.66-1.77 (m, 4H, 2CH2); 2.16-2.21 (m, 2H, CH2); 3.01-3.13 (m, 1H, CH); 7.41-7.60 (m, 2H, Ar); 8.19 (d, 1H, J=8.8 Hz, Ar); 8.31 (d, 1H, J=8.0 Hz, Ar); 9.16 (s, 1H, Ar).
Scale: 6.3 mmol Eluent for column chromatography was 1-5% methanol in dichloromethane. Yield: 0.45 g (27%). Mp.: 225-226° C. MS (ESI) m/z 266.3 (M+1)+. 1H NMR (CDCl3): δ 1.25-1.24 (m, 12H, 6CH2); 3.20-3.32 (m, 1H, CH); 7.42-7.62 (m, 2H, Ar); 8.20 (d, 1H, J=8.0 Hz, Ar); 8.33 (d, 1H, J=8.0 Hz, Ar); 9.19 (s, 1H, Ar).
2-Furoyl chloride (1.1 g, 0.8 ml, 8.1 mmol) in dry dichloromethane (15 ml) was added dropwise to a solution of to 3,4-diaminoquinoline 8 (1.0 g, 6.0 mmol) in dry pyridine (6.2 ml) under nitrogen. The solution was stirred for 2 hours at room temperature. Water (15 ml) was added to quench the reaction and the solvent was evaporated under reduced pressure to afford an orange solid. This crude solid in 2N NaOH (15 ml) was refluxed for 2 hours. After cooling on ice the pH was adjusted to 7 using concentrated HCl. The solid that precipitated was filtered off and washed with water and ether. Then extracted with ethyl acetate (3 times 15 ml) and washed with water (3 time 15 ml) and dried over MgSO4. The solvent was evaporated and the residue was dried. Eluent for column chromatography was 1-5% methanol in dichloromethane. Yield: 0.62 g (44%). Mp.: 236-238° C. 1H NMR (DMSO-d6): δ 6.74 (s, 1H, Ar); 7.35 (d, 1H, J=3.7 Hz, Ar); 7.69-7.73 (m, 2H, Ar); 7.83 (s, 1H, Ar); 8.09 (s, 1H, Ar); 8.46 (s, 1H, Ar); 9.09 (s, 1H, Ar). 13C NMR (DMSO-d6): δ 111.4, 112.6, 120.7, 121.8, 126.5, 127.2, 129.5, 138.2, 143.6, 144.3, 145.1, 147.2, 155.4.
Prepared as described for the furyl compound (14) using hexanoyl chloride (1.75 g, 13 mmol). Eluent for column chromatography was ethyl acetate:petroleum ether 1:4 to 4:1. Yield: 0.85 g (41%): mp 142-143° C. 1H NMR (DMSO-d6): δ 0.91 (t, 3H, J=6, CH3), 1.37-1.39 (m, 4H, 2×CH2), 1.83-1.91 (m, 2H, CH2), 1.93-2.30 (t, 2H, J=8, CH2), 7.64-7.69 (m, 2H, Ar); 8.08-8.13 (m, 1H, Ar); 8.28-8.42 (m, 1H, Ar); 9.12 (s, 1H, Ar).
Staring material (9-15) was almost completely dissolved (with heating) in chloroform (2.5 ml/mmol), dichloromethane (2.5 ml/mmol) and methanol (0.25 ml/mmol). 3-Chloroperoxybenzoic acid (2.5 eq.) was added and the solution was reflux. After 30 minutes Na2CO3 (0.04 g/mmol) was added and the mixture was refluxed for one more hour. The reaction mixture was cooled on an ice-bath and the solvent was evaporated. Column chromatography was needed for purification and removal of 3-chloroperoxybenzoic acid.
Scale: 8.3 mmol Eluent for column chromatography was 2% methanol in dichloromethane. Yield: 0.55 g (36%). Mp.: 290-295° C. 1H NMR (CD3OD): δ 7.41-7.61 (m, 1H, Ar); 7.87-7.96 (m, 2H, Ar); 8.45-8.53 (m, 1H, Ar); 8.75-8.80 (m, 1H, Ar); 9.16 (s, 1H, Ar).
Scale: 1.8 mmol Eluent for column chromatography was 2% (increased to 6%) methanol in dichloromethane. Yield: 0.18 g (42%). Mp.: 123-130° C. 1H NMR (DMSO-d6): δ 1.95-2.22 (m, 2H, CH2); 2.38-2.59 (m, 4H, 2CH2); 3.41-3.93 (m, 1H, CH); 7.72-7.93 (m, 2H, Ar); 8.39-8.41 (m, 1H, Ar); 8.65-8.99 (m, 1H, Ar); 11.52 (s, 1H, Ar).
Scale: 0.8 mmol Eluent for column chromatography was 5% methanol in dichloromethane. Yield: 0.19 g (95%). Mp.: 155-157° C. 1H NMR (CD3OD): δ 1.74-2.13 (m, 6H, 3CH2); 2.18-2.39 (m, 2H, CH2); 3.38-3.54 (m, 1H, CH); 7.29-7.42 (m, 2H, Ar); 7.83-7.92 (m, 2H, Ar); 9.03 (s, 1H, Ar).
Scale: 2.4 mmol Eluent for column chromatography was 5-10% methanol in dichloromethane. Yield: 0.59 g (92%). Mp.: 160-165° C. 1H NMR (CD3OD): δ 1.17-2.19 (m, 10H, 5CH2); 2.97-3.12 (m, 1H, CH); 7.79-7.93 (m, 2H, Ar); 8.42-8.46 (m, 1H, Ar); 8.69-8.77 (m, 1H, Ar); 9.03 (s, 1H, Ar). 13C NMR (CD3OD): δ 26.6, 26.8, 32.5, 39.7, 120.8, 123.1, 128.6, 129.9, 130.1, 130.3, 132.5, 138.0, 142.6, 164.1, 166.0, 170.9, 173.8.
Scale: 2.0 mmol Eluent for column chromatography was 3-8% methanol in dichloromethane. Yield: 0.22 g (39%). Mp.: 115-120° C. 1H NMR (CD3OD): δ 1.67-2.11 (m, 12H, 6CH2); 3.05-3.31 (m, 1H, CH); 7.78-7.90 (m, 2H, Ar); 8.28-8.48 (m, 1H, Ar); 8.62-8.73 (m, 1H, Ar); 9.00 (s, 1H, Ar). 13C NMR (CD3OD): δ 25.0, 26.4, 32.1, 39.2, 117.4, 118.2, 120.6, 126.2, 127.4, 127.8, 128.2, 130.0, 130.6, 132.3, 132.6, 135.2, 162.7, 166.5.
Scale: 2.2 mmol Eluent for column chromatography was 1-10% methanol in dichloromethane. Yield: 0.36 g (66%). Mp.: >280° C. 1H NMR (CD3OD): δ 6.74-6.77 (m, 1H, Ar); 7.37-7.47 (m, 2H, Ar); 7.87-7.96 (m, 2H, Ar); 8.55-8.60 (m, 1H, Ar); 8.74-8.79 (m, 1H, Ar); 9.07 (s, 1H, Ar).
Scale: 3.34 mmol Eluent for column chromatography was 1-5% methanol in dichloromethane. Yield: 0.12 g (14%): 1H NMR (CD3OD): δ 0.88-0.94 (t, 3H, CH3, J=6 Hz); 1.26-1.39 (m, 4H, 2×CH2); 1.81-1.88 (m, 2H, CH2), 2.88-2.95 (t, 2H, CH2, J=8 Hz); 7.44-7.58 (m, 2H, Ar); 7.90-8.09 (m, 2H, Ar); 9.03 (s, 1H, Ar).
A mixture of toluene (0.45 ml/mmol) and dimethylformamide (0.90 ml/mmol) was cooled in an ice-bath and phosphorus oxychloride (2.6 eq.) was added. After 10 minutes the appropriate 1H-imidazo[4,5-c]quinolin-5-oxide was added and the solution was stirred at room temperature for 10 minutes. Subsequently the solution was heated to 100° C. on a steam bath for 30 minutes. Upon cooling, the solvent was evaporated and the resulting syrup was poured over crushed ice while stirring. The mixture was then warmed to room temperature and carefully adjusted to pH 6-7 with solid NaHCO3. After 2 hours, the solid that was formed was filtered off, washed with water and diisopropylether and subsequently dried.
Scale: 3.5 mmol Eluent for column chromatography was 2% methanol in dichloromethane. Yield: 0.31 g (44%). Mp.: 257-258° C. MS (ESI) m/z 203.8 (M+1)+, 206.0 (M+3)+. 1H NMR (CDCl3): δ 8.46-8.56 (m, 2H, Ar); 8.82-8.87 (m, 1H, Ar); 9.14-9.19 (m, 1H, Ar); 9.35 (s, 1H, Ar); 10.16 (broad s, 1H, NH).
Scale: 0.6 mmol Yield: 0.16 g (99%). Mp.: 142-145° C. 1H NMR (CDCl3): δ 2.06-2.24 (m, 2H, CH2); 2.52-2.66 (m, 4H, 2CH2); 3.87-4.00 (m, 1H, CH); 7.56-7.68 (m, 2H, Ar); 8.02-8.06 (m, 1H, Ar); 8.30-8.32 (m, 1H, Ar).
Scale: 12.7 mmol Yield: 2.65 g (75%). Mp.: >265° C. 1H NMR (CD3OD): δ 1.81-2.22 (m, 6H, 3CH2); 2.25-2.31 (m, 2H, CH2); 3.40-3.51 (m, 1H, CH); 7.63-7.74 (m, 2H, Ar); 8.00-8.03 (m, 1H, Ar); 8.33-8.38 (m, 1H, Ar).
Scale: 2.2 mmol Yield: 0.65 g (97%). Mp.: 245-250° C. MS (ESI) m/z 285.9 (M+1)+, 288.0 (M+3)+. 1H NMR (CD3OD): δ 1.37-2.17 (m, 10H, 5CH2); 3.03-3.18 (m, 1H, CH); 7.68-7.71 (m, 2H, Ar); 8.00-8.08 (m, 1H, Ar); 8.32-8.40 (m, 1H, Ar).
Scale: 0.8 mmol Yield: 0.38 g (86%). Mp.: 195-200° C. MS (ESI) m/z 299.9 (M+1)+, 301.7 (M+3)+. 1H NMR (CD3OD): δ 1.73-2.13 (m, 12H, 6CH2); 3.21-3.40 (m, 1H, CH); 7.63-7.79 (m, 2H, Ar); 7.97-8.05 (m, 1H, Ar); 8.29-8.38 (m, 1H, Ar).
Scale: 1.4 mmol Eluent for column chromatography was petroleum ether:ethyl acetate: =75:25. Yield: 0.1 g (26%). Mp.: 235-238° C. MS (ESI) m/z 269.8 (M+1)+, 272.1 (M+3)+. 1H NMR (CD3OD): 6.72-6.75 (m, 1H, Ar); 7.40-7.43 (m, 1H, Ar); 7.63-7.75 (m, 2H, Ar); 7.83-7.88 (m, 1H, Ar); 7.98-8.01 (m, 1H, Ar); 8.36-8.40 (m, 1H, Ar).
Scale: 0.45 mmol Yield: 0.052 g (41%): mp 236-237° C.; MS (ESI) m/z 273.9 (M+1)+, 1H NMR (CDCl3): δ 0.65-0.72 (t, 3H, CH3, J=6 Hz); 1.14-1.21 (m, 4H, 2×□CH2); 1.61-1.72 (m, 2H, CH2); 2.76-2.84 (t, 2H, CH2, J=8 Hz); 7.33-7.47 (m, 2H, Ar); 7.76-7.80 (m, 1H, Ar); 7.95-8.15 (m, 1H, Ar).
These compounds were prepared by means of microwave-assisted chemistry. Absolute ethanol (2.5-3.0 ml) was added to the appropriate 4-chloro-1H-imidazo[4,5-c]quinoline and appropriate aniline (2-3 eq.). Conditions: pre stirring 60 seconds, temperature 120° C., time 2400 seconds, normal sample absorption, fixed hold time. After the reaction was completed the solvent was evaporated and the remaining product was purified by column chromatography and recrystallized.
Compound 30 was prepared as described elsewhere [Van Galen P. J. M. et al. (1991) ibid.].
Scale: 0.8 mmol Eluent for column chromatography was 2.5-10% methanol in dichloromethane. Yield: 40 mg (15%). The product was recrystallized from methanol. Mp.: 155-157° C. MS (ESI) m/z 328.9 (M+1)+. 1H NMR (CDCl3): δ 1.80-2.27 (m, 8H, 4CH2); 3.31-3.55 (m, 1H, CH); 7.00-7.09 (m, 1H, Ar); 7.15-7.29 (m, 4H, Ar); 7.49-7.56 (m, 1H, Ar); 7.78 (d, 1H, J=6.6 Hz, Ar); 7.96-8.09 (m, 2H, Ar). 13C NMR 750 MHz (CDCl3): δ 24.9, 31.7, 38.9, 114.7, 117.9, 118.1, 118.3, 119.2, 119.8, 120.9, 121.9, 123.6, 124.1, 126.2, 126.4, 126.5, 126.7, 128.1, 128.2, 128.9, 133.5, 140.5, 143.3, 146.6, 156.3, 159.1. Anal. (C21H20N4.0.7H2O) C, H, N.
Scale: 0.7 mmol Eluent for column chromatography was 1% methanol in dichloromethane. Yield: 80 mg (35%). The product was recrystallized from methanol. Mp.: 125-126° C. MS (ESI) m/z 342.7 (M+1)+, 343.8 (M+2)+. 1H NMR (CDCl3): δ 1.55-2.15 (m, 8H, 4CH2); 2.24 (s, 3H, CH3); 3.21-3.33 (m, 1H, CH); 7.07 (d, 2H, J=8.0 Hz, Ar); 7.21-7.28 (m, 1H, Ar); 7.46 (t, 1H, J=7.3 Hz, Ar); 7.74-7.87 (m, 4H, Ar). 13C NMR 400 MHz (CDCl3): δ 20.7, 24.4, 29.7, 32.4, 39.6, 62.5, 115.8, 120.7, 122.6, 126.7, 127.3, 129.4, 132.4, 137.1, 143.8, 147.5, 157.1. Anal. (C22H22N4.1.7H2O) C, H, N.
Scale: 0.7 mmol Eluent for column chromatography was 1% methanol in dichloromethane. Yield: 90 mg (38%). The product was recrystallized from methanol. Mp.: 106-107° C.; MS (ESI) m/z 358.8 (M+1)+. 1H NMR (CDCl3): δ 1.71-2.29 (m, 8H, 4CH2); 3.22-3.38 (m, 1H, CH); 3.65 (s, 3H, OCH3); 6.80-6.84 (m, 2H, Ar); 7.26 (t, 1H, J=8.0 Hz, Ar); 7.46 (t, 1H, J=7.3 Hz, Ar); 7.76-7.93 (m, 4H, Ar). 13C NMR 400 MHz (CDCl3): δ 25.5, 32.5, 39.6, 53.4, 55.4, 114.2, 114.8, 116.5, 120.4, 122.1, 122.4, 127.0, 127.2, 133.1, 144.0, 147.8, 155.4, 156.7. Anal. (C22H22N4O.CH3OH) C, H, N.
Scale: 0.7 mmol Eluent for column chromatography was dichloromethane. Polarity was slowly increased with methanol till 2%. Yield: 150 mg (54%). The product was recrystallized from methanol. Mp.: 114-115° C. MS (ESI) m/z 396.5 (M+1)+, 398.5 (M+2)+, 400.5 (M+3)+. 1H NMR (CDCl3): δ 1.68-2.29 (m, 8H, 4CH2); 3.29-3.43 (m, 1H, CH); 7.33 (t, 2H, J=8.8 Hz, Ar); 7.54 (t, 1H, J=8.0 Hz, Ar); 7.78 (t, 2H, J=8.0 Hz, Ar); 7.99 (d, 1H, J=8.0 Hz, Ar); 8.44 (s, 1H, Ar); 9.62 (broad s, 1H, NH). Anal. (C21H18Cl2N4.2.2H2O) C, H, N.
Scale: 1.0 mmol Eluent for column chromatography was 0.5-2% methanol in dichloromethane. Yield: 120 mg (33%). The product was recrystallized from methanol. Mp.: 189-190° C. MS (ESI) m/z 362.7 (M+1)+, 365.0 (M+3)+. 1H NMR (CDCl3): δ 1.67-2.25 (m, 8H, 4CH2); 3.28-3.43 (m, 1H, CH); 7.27-7.39 (m, 3H, Ar); 7.49-7.59 (m, 1H, Ar); 7.71-7.79 (m, 1H, Ar); 7.97 (t, 3H, J=8.0 Hz, Ar); 8.46 (broad s, 1H, NH). Anal. (C21H19ClN4.1.5H2O) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=50:50, later increased to 70:30 respectively. Yield: 33 mg (25%). The product was recrystallized from methanol giving off-white crystals. Mp.: 228-230° C. MS (ESI) m/z 359.0 (M+1)+. 1H NMR (CDCl3/CD3OD): δ 1.63-2.16 (m, 8H, 4CH2); 3.23-3.24 (m, 1H, CH); 3.95-4.05 (q, 2H, J=7.3 Hz, CH2); 6.93 (d, 1H, J=8.0 Hz, Ar); 7.18-7.28 (m, 3H, Ar); 7.39 (t, 1H, J=7.3 Hz, Ar); 7.72-7.92 (m, 3H, Ar, OH). Anal. (C22H22N4O.0.5CH3OH) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was 1% methanol in dichloromethane. Yield: 120 mg (81%). The product was recrystallized from methanol giving light brown crystals. Mp.: 142-145° C. MS (ESI) m/z 369.0 (M+1)+. 1H NMR (CDCl3): δ 1.15-1.34 (m, 2H, CH2); 1.65-2.19 (m, 8H, 4CH2); 2.76 (t, 4H, J=7.3 Hz, 2 CH2); 3.24-3.27 (m, 1H, CH); 7.06 (d, 1H, J=8.0 Hz, Ar); 7.22-7.29 (m, 1H, Ar); 7.40-7.49 (m, 2H, Ar); 7.73 (s, 1H, Ar); 7.85-7.96 (m, 2H, Ar). Anal. (C24H24N4.1.2H2O) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was ethylacetate:petroleumether=80:20. Yield: 110 mg (74%). The product was recrystallized from ethyl acetate:petroleum ether=50:50 giving off-white crystals. Mp.: 235-236° C. MS (ESI) m/z 368.8 (M+1)+. 1H NMR (CD3OD): δ 1.70-2.38 (m, 8H, 4CH2); 3.23-3.50 (m, 1H, CH); 7.31-7.38 (m, 1H, Ar); 7.47-7.60 (m, 2H, Ar); 7.71-7.75 (m, 1H, Ar); 7.82-7.86 (m, 1H, Ar); 8.01-8.08 (m, 2H, Ar); 8.68 (s, 1H, Ar). Anal. (C22H20N6.1.5H2O) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was diisopropylether. Yield: 33 mg (22%). The product was recrystallized from methanol giving white crystals. Mp.: 245-247° C. MS (ESI) m/z 372.9 (M+1)+. 1H NMR (CD3OD): δ 1.72-2.30 (m, 8H, 4CH2); 3.26-3.41 (m, 1H, CH); 3.77 (s, 3H, OCH3); 4.76 (s, 2H, CH2); 6.87-6.92 (m, 2H, Ar); 7.23-7.49 (m, 4H, Ar); 7.78 (d, 1H, J=8.8 Hz, Ar); 8.00 (d, 1H, J=7.3 Hz, Ar). Anal. (C23H24N4O) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=20:80, increased to 30:70. Yield: 30 mg (20%). The product was recrystallized from methanol giving light grey crystals. Mp.: 260-261° C. MS (ESI) m/z 368.0 (M+1)+. 1H NMR (CD3OD): δ 1.79-2.29 (m, 8H, 4CH2); 3.30-3.46 (m, 1H, CH); 6.45 (d, 1H, J=2.9 Hz, Ar); 7.23-7.50 (m, 5H, Ar); 7.77 (d, 1H, J=7.8 Hz, Ar); 8.01 (d, 1H, J=8.0 Hz, Ar); 8.20 (s, 1H, Ar). Anal. (C23H21N5) C, H, N.
Scale: 0.8 mmol Eluent for column chromatography was 2% methanol in dichloromethane. Yield: 0.07 mg (25%). The product was oily. 1H NMR (CDCl3): δ 1.50-2.11 (m, 8H, 4CH2); 3.14-3.31 (m, 1H, CH); 4.85 (s, 2H, CH2); 6.25 (broad s, 2H, 2 NH); 7.08-7.20 (m, 6H, Ar); 7.38 (t, 1H, J=8.8 Hz, Ar); 7.87 (d, 1H, J=8.8 Hz, Ar); 8.09 (d, 1H, J=8.0 Hz, Ar). 13C NMR (CDCl3): δ 25.3, 32.3, 39.7, 45.4, 116.1, 121.3, 122.2, 125.3, 127.0, 127.4, 128.1, 138.1, 143.3, 144.0, 149.7, 157.4, 160.6. Anal. (C22H22N4) C, H, N.
Scale: 0.8 mmol Eluent for column chromatography was 1% methanol (increased to 4%) in dichloromethane. Yield: 160 mg (56%). The product was product was recrystallized from absolute methanol giving off-white crystals: mp 95-97° C.; MS (ESI) m/z 356.9 (M+1)+, 357.7 (M+2)+, 359.0 (M+3)+. 1H NMR (CD3OD): δ 1.75-2.21 (m, 8H, 4CH2); 3.02 (t, 2H, J=8.0 Hz, CH2); 3.30-3.38 (m, 1H, CH); 3.89 (t, 2H, J=8.0 Hz, CH2); 7.15-7.45 (m, 7H, Ar); 7.76 (d, 1H, J=8.8 Hz, Ar); 7.97 (d, 1H, J=6.3 Hz, Ar). 13C NMR (CD3OD): δ 27.6, 29.0, 34.9, 41.6, 116.8, 119.1, 121.1, 121.6, 124.2, 125.1, 128.2, 128.3, 131.2, 133.3, 141.7, 144.4, 147.3, 160.1. Anal. (C23H24N4) C, H, N.
Scale: 0.8 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=70:90, increased to 100% ethyl acetate. Yield: 164 mg (50%). The product was recrystallized from methanol giving off-white crystals. Mp.: 236-238° C. MS (ESI) m/z 424.9 (M+1)+, 426.8 (M+2)+, 428.1 (M+3)+. 1H NMR (CDCl3/CD3OD 9/1, v/v): δ 1.62-2.00 (m, 10H, 5CH2); 2.17-2.24 (m, 2H, CH2); 3.09-3.23 (m, 1H, CH); 7.32-7.39 (m, 2H, Ar); 7.50-7.58 (m, 1H, Ar); 7.71-7.85 (m, 2H, Ar); 7.98 (d, 1H, J=8.0 Hz, Ar); 8.41 (s, 1H, Ar). 13C NMR (DMSO-d6): δ 26.2, 27.7, 33.4, 40.2, 115.2, 119.3, 120.2, 121.0, 122.0, 122.9, 127.0, 128.3, 130.1, 130.6, 134.2, 141.6, 142.7, 146.8, 158.6. Anal. (C23H22Cl2N4.1.3H2O) C, H, N.
Scale: 0.4 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=20:80. Yield: 96 mg (64%). The product was recrystallized from methanol giving orange crystals. Mp.: 135-138° C. MS (ESI) m/z 394.7 (M+1)+, 396.7 (M+3)+. 1H NMR (CD3OD): δ 6.64-6.67 (m, 1H, Ar); 7.10 (d, 1H, J=3.7 Hz, Ar); 7.21-7.44 (m, 4H, Ar); 7.65-7.72 (m, 2H, Ar); 7.95 (d, 1H, J=8.0 Hz, Ar); 8.43-8.44 (m, 1H, Ar). 13C NMR (DMSO-d6): δ 110.6, 112.6, 115.3, 119.5, 120.4, 121.5, 122.2, 123.2, 127.1, 127.6, 128.2, 130.1, 130.3, 130.6, 134.6, 141.5, 142.3, 143.2, 144.7, 145.1, 146.9. Anal. (C20H12Cl2N40.0.5H2O) C, H, N.
Scale: 0.7 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=20:80. Yield: 121 mg (48%). The product was recrystallized from methanol:ethyl acetate=20:80 giving off-white crystals. Mp.: 131-134° C. MS (ESI) m/z 382.8 (M+1)+. 1H NMR (CD3OD): δ 2.00-2.22 (m, 2H, CH2); 2.42-2.56 (m, 4H, 2CH2); 3.72-3.89 (m, 1H, CH); 7.26-7.33 (m, 2H, Ar); 7.47-7.54 (m, 1H, Ar); 7.64-7.69 (m, 1H, Ar); 7.88-7.96 (m, 2H, Ar); 8.30 (s, 1H, Ar). Anal. (C20H16Cl2N4.1.0 MeOH) C, H, N.
Scale: 0.9 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=15:85, increased to 30:70. Yield: 160 mg (44%). The product was recrystallized from methanol:ethyl acetate=1:99 giving off-white crystals. Mp.: 237-240° C. MS (ESI) m/z 410.8 (M+1)+, 412.7 (M+2)+, 414.7 (M+3)+. 1H NMR (CD3OD): δ 1.40-2.00 (m, 8H, 4CH2); 2.15-2.28 (m, 2H, CH2); 2.95-3.07 (m, 1H, CH); 7.34-7.56 (m, 4H, Ar); 7.77-7.88 (m, 2H, Ar); 8.52 (s, 1H, Ar). 13C NMR (DMSO-d6): δ 25.6, 31.6, 38.2, 115.5, 119.2, 120.1, 121.1, 122.0, 123.0, 127.0, 130.1, 130.6, 134.1, 141.6, 142.7, 146.7, 153.2, 157.5. Anal. (C22H20Cl2N4.2.1H2O) C, H, N.
Scale: 1.0 mmol Eluent for column chromatography was ethyl acetate:petroleum ether=20:80. Yield: 160 mg (50%). The product was recrystallized from ethyl acetate:petroleum ether=30:70 giving white crystals. Mp.: 167-172° C. MS (ESI) m/z 328.9 (M+1)+, 331.0 (M+2)+, 332.0 (M+3)+. 1H NMR (CDCl3): δ 7.23-7.36 (m, 2H, Ar); 7.39-7.43 (m, 1H, Ar); 7.62-7.72 (m, 1H, Ar); 7.65-7.95 (m, 3H, Ar); 8.32 (s, 1H, Ar). Anal. (C16H10Cl2N4.1.1H2O) C, H, N.
Scale: 0.2 mmol Eluent for column chromatography was dichloromethane. Yield: 44 mg (73%). The product was recrystallized from ethyl acetate:petroleum ether=50:50 giving white crystals. Mp.: 195-200° C. MS (ESI) m/z 398.9 (M+1)+, 400.8 (M+3)+. 1H NMR (CDCl3): δ 0.82-0.97 (m, 3H, CH3); 1.39-1.51 (m, 2H, CH2); 1.81 (m, 4H, 2CH2); 2.98 (t, 2H, J=8.0 Hz, CH2); 7.33-7.41 (m, 2H, Ar); 7.52-7.60 (m, 1H, Ar); 7.74-7.81 (m, 2H, Ar); 7.99 (d, 1H, J=8.0 Hz, Ar); 8.48 (s, 1H, Ar). Anal. (C21H20Cl2N4) C, H, N.
Table 1 summarizes the chemical structures and physico-chemical characteristics of the imidazoquinoline derivatives prepared as described above.
[125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (I-AB-MECA; 2000 Ci/mmol), [3H]R-PIA (R—N6-[phenylisopropyl]adenosine, 34 Ci/mmol), [3H] CGS21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamido-adenosine, 47 Ci/mmol) and [3H]cyclic AMP (40 Ci/mmol) were from Amersham Pharmacia Biotech (Buckinghamshire, UK).
CHO (Chinese hamster ovary) cells expressing the recombinant human ARs (HEK-293 cells were used for the human A2AAR) were cultured in DMEM and F12 (1:1) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 ng/ml streptomycin and 2 μmol/ml glutamine. Cells were harvested by trypsinization. After homogenization and suspension, cells were centrifuged at 500 g for 10 min, and the pellet was re-suspended in 50 mM Tris.HCl buffer (pH 7.4) containing 10 mM MgCl2. The suspension was homogenized with an electric homogenizer for 10 sec, and was then re-centrifuged at 20,000 g for 20 min at 4° C. The resultant pellets were resuspended in buffer in the presence of 3 Units/mL adenosine deaminase, and the suspension was stored at −80° C. until the binding experiments. The protein concentration was measured using the Bradford assay.
For binding to the human A1AR, [3H]R-PIA (2 nM) was incubated with membranes (40 μg/tube) from CHO cells stably expressing the human A1AR at 25° C. for 60 min in 50 mM Tris.HCl buffer (pH 7.4; MgCl2, 10 mM) in a total assay volume of 200 μL. Nonspecific binding was determined using 10 μM of N6-cyclopentyladenosine. For human A2A AR binding, membranes (20 μg/tube) from HEK-293 cells stably expressing the human A2A AR were incubated with 15 nM [3H]CGS21680 at 25° C. for 60 min in 200 μL of 50 mM Tris.HCl, pH 7.4, containing 10 mM MgCl2. N-5′-ethyluronamidoadenosine (10 μM) was used to define nonspecific binding. Reaction was terminated by filtration with GF/B filters.
Each tube in the competitive binding assay contained 100 μl membrane suspension (20 μg protein), 50 μl [125I]-AB-MECA (0.5 nM), and 50 μl of increasing concentrations of the test ligands in Tris.HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2, 1 mM EDTA. Nonspecific binding was determined using 10 μM of 5′-N-ethylcarboxamidoadenosine in the buffer. The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburgh, Md., USA). Filters were washed three times with 9 mL ice-cold buffer. Radioactivity was determined in a Beckman 5500B γ-counter.
Dissociation Kinetics of [125I]I-AB-MECA from Human A3ARs:
The dissociation of [125I]I-AB-MECA was measured as follows. Membranes (20 μg) were preincubated at 25° C. with 0.5 nM [125I]I-AB-MECA, in a total volume of 100 μl of Tris-HCl buffer (50 mM, pH 8.0) containing 10 mM MgCl2, and 1 mM EDTA for 60 min. The dissociation was then initiated by the addition of 3 μM Cl-IB-MECA with or without allosteric modulators. The time course of dissociation of total binding was measured by rapid filtration at appropriate time intervals. Nonspecific binding was measured after 60-min incubation in the presence of 3 μM Cl-IB-MECA. Further assay was as described above.
Intracellular cyclic AMP levels were measured with a competitive protein binding method (Nordstedt and Fredholm, 1990). CHO cells that expressed recombinant human A3ARs were harvested by trypsinization. After centrifugation and resuspension in medium, cells were plated in 24-well plates in 0.5 ml medium. After 24 hr, the medium was removed and cells were washed three times with 1 ml DMEM, containing 50 mM HEPES, pH 7.4. Cells were then treated with agonists and/or test compounds in the presence of rolipram (10 μM) and adenosine deaminase (3 units/ml). After 45 min forskolin (10 μM) was added to the medium, and incubation was continued an additional 15 min. The reaction was terminated by removing the supernatant, and cells were lysed upon the addition of 200 μL of 0.1 M ice-cold HCl. The cell lysate was resuspended and stored at −20° C. For determination of cyclic AMP production, protein kinase A (PKA) was incubated with [3H]cyclic AMP (2 nM) in K2HPO4/EDTA buffer (K2HPO4, 150 mM; EDTA, 10 mM), 20 μL of the cell lysate, and 30 μL 0.1 M HCl or 50 μL of cyclic AMP solution (0-16 pmol/200 μL for standard curve). Bound radioactivity was separated by rapid filtration through Whatman GF/C filters and washed once with cold buffer. Bound radioactivity was measured by liquid scintillation spectrometry.
Binding and functional parameters were calculated using Prism 5.0 software (GraphPAD, San Diego, Calif., USA). IC50 values obtained from competition curves were converted to Ki values using the Cheng-Prusoff equation. Data were expressed as mean±standard error.
aAll experiments were performed using adherent CHO (A1 and A3) and HEK293 (A2A) cells stably transfected with cDNA encoding the human ARs. Binding at human A1, A2A and A3 ARs in this study was carried out as described in Methods using [3H]R-PIA (2.0 nM), [3H]CGS 21680 (15 nM) or [125I]I-AB-MECA (0.5 nM) as a radioligand. Values from the present study are expressed as mean ± s.e.m., n = 3-5. Percentage inhibition at A1, A2A, or A3 receptors is expressed as the mean value from 2-4 separate experiments with similar results performed in duplicate.
bA2B receptor: effect of compounds at 10 μM on NECA (150 nM)-induced cyclic AMP accumulation from one experiment performed in triplicate; the inhibition of CGS15943 (10 μM) was expressed as 100%.
cdissociation: % decrease of dissociation at 30 min; the residual binding of [125I]AB-MECA after 30 min of dissociation was expressed as 100%.
dincrease of efficacy: compared to maximal effect by 2-Cl-IB-MECA alone (control = 100%)
Further, several additional 1H-imidazo-[4,5-c]quinolin-4-amine derivatives were synthesized in accordance with the procedure described above. the potency of these derivatives is provided in Tables 3 and 4.
aAll experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human ARs. Binding at human A1, A2A and A3ARs in this study was carried out as described in Experimental Procedures using [3H]R-PIA, [3H]CGS 21680 or [125I]I-AB-MECA as a radioligand. Values from the present study are expressed as mean ± s.e.m., n = 3-5. Percentage inhibition at A1, A2A, or A3 receptors is expressed as the mean value from 2-4 separate experiments with similar results performed in duplicate.
bValues from Göblyös et al.
cdissociation: % decrease of [125I]I-AB-MECA dissociation at 30 min (control = 100%)
dincrease of efficacy; compared to maximal effect by 2-Cl-IB-MECA alone (control)
e10 μM Cl-IB-MECA alone = 100%
aAll experiments were performed using adherent CHO cells stably transfected with cDNA encoding the human ARs. Binding at human A1, A2A and A3ARs in this study was carried out as described in Experimental Procedures using [3H]R-PIA, [3H]CGS 21680 or [125I]I-AB-MECA as a radioligand. Values from the present study are expressed as mean ± s.e.m., n = 3-5. Percentage inhibition at A1, A2A, or A3 receptors is expressed as the mean value from 2-4 separate experiments with similar results performed in duplicate.
bA2BAR: effect of compounds at 10 μM on NECA (150 nM)-induced cyclic AMP accumulation from one experiment performed in triplicate, CGS15943 (10 μM) = 100%.
cdissociation: % decrease of [125I]I-AB-MECA dissociation at 30 min (control = 100%)
dincrease of efficacy: compared to maximal effect by 2-Cl-IB-MECA alone (control)
emethylenedioxo substituent at the 3 and 4 poisitions of the phenyl ring, namely, a 5 memebered diether ring substituent
In Tables 2 to 4 the effects of the imidazoquinoline derivatives at the orthosteric sites of all four adenosine receptor subtypes were listed, together with their effects on the allosteric site on the human adenosine A3 receptor. Many compounds display little if any affinity for the orthosteric binding sites, particularly on the adenosine A1, A2A and A2B receptor, especially when R1=3,4-Cl2-phenyl (namely a 3,4-dichlororo phenyl substituent). Also the orthosteric binding site on the adenosine A3 receptor accommodates the imidazoquinoline derivatives rather poorly. The best separation between orthosteric and allosteric recognition was found with compound 45. Thus, this compound was selected for further in vivo assays.
A stock solution of imidazoquinoline derivative 45 was prepared by dissolving the same in DMSO. Further dilutions for treatment were performed in PBS. As a control, an equivalent amount of DMSO in PBS was used.
To test the effect of imidazoquinoline derivative 45 on the myeloid system, the compound was orally administered at a dose of 100 μg/kg, thrice daily to ICR mice, for two consecutive days. Blood samples were withdrawn 24 and 48 hours after the last drug administration. A differential blood count was performed.
The results presented in
Adjuvant induced arthritis model (model for rheumatoid arthritis (RA)) was established in rats by immunizing the animals with an emulsion contacting Mycobacterium tuberculosis in Freund's adjuvant (Animal Models for Autoimmune and Inflammatory disease:Adjuvant Arthritis in the Rat. Current Protocols in Immunology, Supplement 19, page 15.4.2.). Treatment with imidazoquinoline derivative 45 was initiated upon onset of disease. The imidazoquinoline derivative 45 was administered orally twice daily at a dose of 100 μg/kg.
Evaluation of disease development was carried out daily, starting upon onset of disease until the end of the study. Intensity of arthritis was scored by grading each paw from 0-4 on erythema, swelling and deformity of the joint, every day: 0=no erythema or swelling; 1=slight erythema or swelling of one of the toes or fingers; 2=erythema and swelling of more than one toe or finger; 3=erythema and swelling of the ankle or wrist; 4=complete erythema and swelling of toes or fingers and ankles or wrist, and inability to bend the ankle or wrist. All scores of the four legs from each rat are added to give up to a maximum arthritic score of 16. The results are presented in
The results presented above suggest that the imidazoquinoline derivative and in the particular example imidazoquinoline derivative 45 may have a dual effect, on the one hand of enhancing the myeloid system and on the other hand, as a remedy for conditions such as RA.
| Number | Date | Country | |
|---|---|---|---|
| 60762141 | Jan 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12219772 | Jul 2008 | US |
| Child | 13800560 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2007/001930 | Jan 2007 | US |
| Child | 12219772 | US |
