# Patent

## Title
Pyrimidine classical cannabinoid compounds and related methods of use

## Abstract

Disclosed are compounds of the formula I:




wherein R1, R2, V, W, X, Y and Z are as defined herein. The compounds can be used in the treatment of disorders mediated by the cannabinoid receptors, such as, for example, inflammation.

## Background
This application is a divisional of and claims priority benefit from application Ser. No. 12/468,777 filed May 19, 2009, now issued as U.S. Pat. No. 8,022,078, which claimed priority benefit from application Ser. No. 61/128,171 filed May 19, 2008, each of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
The classical cannabinoid, delta-9-tetrahydrocannabinol (Δ9-THC), is the major active constituent extracted from Cannabis sativa. The effects of cannabinoids are due to an interaction with specific high-affinity receptors. Presently, two cannabinoid receptors have been characterized: CB-1, a central receptor found in the mammalian brain and a number of other sites in the peripheral tissues; and CB-2, a peripheral receptor found principally in cells related to the immune system. In addition, it has recently been reported that the GPR35 and GPR55 orphan receptors bind cannabinoid type ligands and have been proposed as a third receptor subtype. The CB-1 receptor is believed to mediate the psychoactive properties associated with classical cannabinoids. Characterization of these receptors has been made possible by the development of specific synthetic ligands such as the agonists WIN 55212-2 (D'Ambra et al., J. Med. Chem. 35:124 (1992)) and CP 55,940 (Melvin et al., Med. Chem. 27:67 (1984)).
Pharmacologically, cannabinoids can be used to affect a variety of targets such as the central nervous system, the cardiovascular system, the immune system and/or endocrine system. More particularly, compounds possessing an affinity for either the CB-1 or the CB-2 receptors and potentially the GPR35 and GPR55 receptors are useful as anticancer agents, antiobesity agents, analgesics, myorelaxation agents and antiglaucoma agents. Such compounds can also be used for the treatment of thymic disorders, vomiting; various types of neuropathy, memory disorders, dyskinesia, migraine, multiple sclerosis; asthma, epilepsy, ischemia, angor, orthostatic hypotension, osteoporosis, liver fibrosis, inflammation and irritable bowel disease, and cardiac insufficiency.
However, certain cannabinoids such as Δ9-THC also affect cellular membranes, producing undesirable side effects such as drowsiness, impairment of monoamine oxidase function, and impairment of non-receptor mediated brain function. The addictive and psychotropic properties of some cannabinoids tend to limit their therapeutic value.
A number of structurally distinct classical cannabinoids are described in U.S. Pat. No. 7,169,942. The activity of these compounds addresses the ongoing need in the art for compounds, whether classical or non-classical cannabinoid analogs, that can be used for therapeutic purposes to affect treatment of conditions or disorders that are mediated by the CB-1 receptor and/or the CB-2 receptor.
SUMMARY OF THE INVENTION
In light of the foregoing, it is an object of the present invention to provide a range of heterocyclic cannabinoid analog compounds, compositions and/or related methods, thereby overcoming various deficiencies and shortcomings of the prior art, including those outlined above. It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the following objects can be viewed in the alternative with respect to any one aspect of this invention.
It can be an object of the present invention to identify one or more classes of cannabinoid compounds exhibiting affinity for cannabinoid and related receptors found in human cells and tissues.
It can also be an object of this invention to provide one or more pyrimidine classical cannabinoid receptor ligands, such compounds as can comprise a pyrimidine system substitution in the A-ring, and comprising all known and inferred C3 side chain substitutions and a hexahydro-, tetrahydro- or non-pyrane ring system.
It can be another object of the present invention to identify such compounds exhibiting cannabinoid receptor selectivity, for directed therapeutic use.
Other objects, features, benefits and advantages of the present invention will be apparent from this summary and the following descriptions of certain embodiments, and will be readily apparent to those skilled in the art having knowledge of various cannabinoid compound and related therapeutic methods. Such objects, features, benefits and advantages will be apparent from the above as taken into conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom, alone or with consideration of the references incorporated herein.
In part, the present invention can be directed to a cannabinoid analog compound selected from compounds of a formula I below




wherein the C-ring can comprise or be selected from hexahydro, 6a,10a-dehydro, 8,9-dehydro, and 9,10-dehydro structures; Y can be selected from S, O, CH2, CH(CH3), CH(OH), C(CH3)(OH), C(CH3)2, C(—V(CH2)nV—), C(O), NH, S(O), and S(O)2; V can be selected from CH2, S and O; n can be an integer≧1, and preferably from 1 to 6; Z can be selected from H, substituted and unsubstituted alkyl, substituted and unsubstituted cycloalkyl, substituted and unsubstituted aryl, substituted and unsubstituted heteroaryl, substituted and unsubstituted heterocycloalkyl, and arylalkyl, cycloalkylalkyl, heteralkylalkyl and heteroarylalkyl, wherein each alkyl portion can be optionally substituted up to three times and the ring portion of each can be optionally substituted with one, two, three, four or five substituents; and R1 can be selected from H and unsubstituted or substituted alkyl, including but not limited to aminoalkyl, morpholinoalkyl, and hemisuccinate moieties.

In part, the present invention can be directed to a salt of a compound in accordance herewith.
In part, the present invention can be directed to a pro-drug of a compound in accordance herewith.
In part, the present invention can also be directed to a pharmaceutical composition comprising a compound of the sort described herein, a salt and/or a pro-drug thereof; and a pharmaceutically acceptable carrier component.
In part, the present invention can be directed to a method of modifying the activity of a cannabinoid receptor. Such a method can comprise providing a compound, salt and/or pro-drug of the present invention or any other compound disclosed herein that has activity at a cannabinoid or related receptor, a salt and/or pro-drug thereof; and contacting a cell and/or cannabinoid receptor of a cell with such a compound. As illustrated below, such contact can be at least partially sufficient to at least partially modify activity of such a cannabinoid receptor.
In part, the present invention can also be directed to a method of treating a cannabinoid receptor-mediated condition. Such a method can comprise providing a compound in accordance herewith or any other compound disclosed herein that has activity at a cannabinoid receptor, a salt and/or pro-drug thereof; and administering to a patient an amount of such a compound, salt and/or pro-drug, that is at least partially effective to treat a cannabinoid receptor-mediated condition. This aspect of the invention can relate to the use of agonists of a CB-1 or a related receptor, antagonists of a CB-1 or related receptor, agonists of a CB-2 or related receptor, and/or antagonists of a CB-2 or related receptor to treat or prevent disease conditions mediated by hyperactivity of CB-1 and/or CB-2 (or related) receptors or either inactivity or hypoactivity of the CB-1 and/or CB-2 (or related) receptors.
In part, the present invention can also be directed to a compound selected from compounds of a formula




wherein the C-ring can comprise or be selected from hexahydro-, 6a,10a-dehydro-, 8,9-dehydro- and 9,10-dehydro-structures; R1 can be selected from H, alkyl, aminoalkyl, morpholinoalkyl, and hemisuccinate moieties; Y can be selected from carbonyl, dimethylmethylene and hydroxymethylene moieties; and Z can be selected from substituted and unsubstituted alkyl, phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl, thiophenyl and substituted thiophenyl moieties, such substituents as would be understood by those skilled in the art made aware of this invention, including but not limited to those described elsewhere herein. In certain embodiments, Z can be an alkyl, phenyl, thiophenyl or a cycloalkyl moiety and, optionally, Y can be a dimethylmethylene or carbonyl moiety. Regardless, such a compound can be selected from salts and/or pro-drugs of such a compound.

Without limitation, this invention can also be directed to a method of cancer treatment. Such a method can comprise providing a cancer cell comprising a cannabinoid receptor, such a cell of a growth of cancer cells; and contacting such a growth with a cannabinoid compound selected from compounds of a formula




wherein the C-ring, R1, Y and Z are as described above. In an embodiment, R1 can be selected from H, alkyl, alkylamino, alkylmorpholino and hemisuccinate moieties; Y can be selected from carbonyl, dimethylmethylene and hydroxymethylene moieties; and Z can be selected from substituted and unsubstituted alkyl, phenyl, substituted phenyl, cycloalkyl, substituted cycloalkyl, thiophenyl and substituted thiophenyl moieties, with such substituents as would be understood by those skilled in the art made aware of this invention, including but not limited to those described elsewhere herein, and salts and pro-drugs of said compounds, and combinations thereof, such compound(s) in an amount at least partially sufficient to induce death of a cell of such a growth. In certain other embodiments, Z can be selected from substituted alkyl, unsubstituted alkyl, substituted cycloalkyl, unsubstituted cycloalkyl, phenyl, substituted phenyl, thiophenyl and substituted thiophenyl moieties, with such substituents as can be selected from chloro, hydroxy and methoxy moieties. In certain such embodiments, R1 can be selected from H and methyl moieties. Regardless, without limitation and as illustrated elsewhere herein, Y can be dimethylmethylene or carbonyl.




BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the functional activity of compound 6b at the CB-1 receptor.
FIG. 2 shows the secretion profiles of G-CSF by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α at 4 and 18 hour intervals.
FIG. 3 shows the secretion profiles of IL-1β by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α at 4 and 18 hour intervals.
FIG. 4 shows the secretion profiles of IL-6 by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 18 hour interval.
FIG. 5 shows the secretion profiles of IL-6 by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 4 hour interval.
FIG. 6 shows the secretion profiles of IL-8 by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 18 hour interval.
FIG. 7 shows the secretion profiles of IL-8 by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 4 hour interval.
FIG. 8 shows the secretion profiles of MCP-1 by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α at 4 and 18 hour intervals.
FIG. 9 shows the secretion profiles of MIF by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 4 hour interval.
FIG. 10 shows the secretion profiles of MIF by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 18 hour interval.
FIG. 11 shows the secretion profiles of RANTES by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 18 hour interval.
FIG. 12 shows the secretion profiles of RANTES by A549 cells exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α scaled to show the levels at the 4 hour interval.



DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
The novel compounds encompassed by the instant invention are those described by the general Formula I set forth above, and the pharmaceutically acceptable salts and prodrugs thereof.
By “alkyl” in the present invention is meant straight or branched chain alkyl radicals having from 1-20 carbon atoms. Optionally, an alkyl group of the instant invention can contain one or more double bonds and/or one or more triple bonds.
By “cycloalkyl” is meant a carbocyclic radical having from three to twelve carbon atoms. The cycloalkyl can be monocyclic or a polycyclic fused system. Optionally, a cycloalkyl group of the instant invention can contain one or more double bonds and/or one or more triple bonds.
The term “heterocyclyl” refers to one or more carbocyclic ring systems of 4-, 5-, 6- or 7-membered rings which includes fused ring systems and contains at least one and up to four heteratoms selected from nitrogen, oxygen or sulfur and combinations thereof.
By “aryl” is meant an aromatic carbocyclic ring system having a single ring, multiple rings or multiple condensed rings in which at least one ring is aromatic.
The term “heteroaryl” refers to one or more aromatic ring systems having from three to twelve atoms which includes fused ring systems and contains at least one and up to four heteroatoms selected from nitrogen, oxygen or sulfur and combinations thereof.
By “arylalkyl” is meant an alkyl radical substituted with an aryl, with the point of attachment is a carbon of the alkyl chain.
As used herein, “substituted” refers to those substituents as would be understood by those skilled in the art. At least one and as many as five substituents can exist on a single group. Examples of such substituents include, but are not limited to, halo, alkyl, alkoxy, hydroxyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cyano, nitro, amino, alkylamino, dialkylamino, thiol, alkylthiol, haloalkyl (e.g. trifluoromethyl), carboxy, alkylcarboxy, carbamoyl and the like.
According to one approach, representative, non-limiting pyrimidine analogs can be prepared by reacting an intermediate pyrimidine II with the appropriate terpine to generate the described C-rings according to the Schemes shown below. The hexahydro is synthesized utilizing a modification of the tandem cyclization method described by Tietz, (Chem. Rev. 1996, 96, 115-136 and the references cited therein, all incorporated herein by reference in their entirety.) The ring formations are accomplished under microwaves conditions with the appropriately substituted pyrimidine and citronellal, either racemic or optically active, as depicted in Scheme 1.




The synthesis of the 9,10- and 8,9-dehydro analogs is accomplished under the same reaction conditions as the hexahydro, however, citronellal is substituted with TBS protected 3-hydroxyl citronellal (Scheme 2). The synthesis of this compound has previously been reported by Kesenheimer and Groth, Org. Lett., 8:2507 (2006), incorporated herein by reference in its entirety.




Synthesis of the 6a,10a-dehydro is accomplished using a method of Adams (U.S. Pat. No. 2,419,934 as incorporated herein by reference in its entirety) utilizing racemic or optically active pulegone and the appropriately substituted pyridine analog (Scheme 3).




The corresponding pyrimidines are prepared by reacting dimethyl-, diethyl-, or bis(trichlorophenyl)-malonates with the appropriately substituted amidines in a Traube-type synthesis. As seen in Scheme 4, the amidines are prepared from aromatic, heteroaromatic, and aliphatic nitriles using established chemistry including methanol/HCl followed by NH3 or by direct conversion using amino-chloro-methyl-aluminum (See, e.g., Moss, Tet. Lett. 36:8761 incorporated herein by reference in its entirety).




The amidines and malonates so prepared are cyclized in the presence of base, e.g. sodium ethoxide, to form the substituted pyrimidines (Scheme 5).




Derivatives containing a gem-dialkyl, heterocyclic, or carbocyclic substituent at Y, where commercial compounds are not available, are prepared either by direct alkylation of the methylene nitrile (See, U.S. Pat. No. 7,057,076 to Makriyannis and Pub. No. 2004/087590, each of which is incorporated herein by reference in its entirety) or from the appropriately substituted aryl-, heteroaryl-halogen-, and isopropyl-nitrile (See, U.S. Pub. No. 2005/0065033 filed Aug. 21, 2003, the entirety of which is incorporated herein by reference). Schemes 6 and 7 are representative of, but not limited to, the scope of the chemistry.








Derivatives containing a keto, hydroxyl, or alkylhydroxyl substituent at Y can be prepared by direct oxidation of compounds bearing a Y═CH2 or from the C2-aldehyde pyrimidine, prepared from 2,2-bis-ethylsulfanyl-acetamidine and the appropriately malonic acid ester (Scheme 7) using chemistry previously reported (See, U.S. Pat. No. 7,169,942, the entirety of which is incorporated herein by reference in its entirety).




While syntheses of several representative, non-limiting compounds are described herein, it will be understood by those skilled in the art that various other compounds can be prepared using similar such procedures and/or straight-forward modifications thereof. Accordingly, the identities of moieties R1, Y and Z are limited only by the respective reagents, starting materials, intermediates and chemistry thereon. Various other such moieties and/or substituents thereof include but are not limited to those described in the aforementioned co-pending application.
Likewise, the present invention contemplates, more broadly, various other such compounds, salts and/or pro-drugs thereof, together with corresponding pharmaceutical compositions thereof, as also described in the aforementioned co-pending application. Such compounds, salts, pro-drugs and/or pharmaceutical compositions can be used as described therein. For instance, the present invention can be used to modify the activity of one or both of the CB-1 and CB-2 receptors. Such a method can be carried out by contacting a cell and/or cannabinoid receptor thereof with a compound of the present invention, such contact at least partially sufficient to at least partially modify the activity of such a cannabinoid receptor, whether ex vivo or in vivo.
More generally, various physiological and/or therapeutic advantages of the present compounds and/or compositions can be realized with consideration of the authorities cited in the aforementioned co-pending application. The inventive analogs, as described herein, can be administered in therapeutically-effective amounts to treat a wide range of indications. Without limitation, various such conditions and/or disease states are described in paragraph 0067 of co-pending application Ser. No. 12/074,342, filed Mar. 3, 2008 and entitled “Tri-Aryl/Heteroaromatic Cannabinoids and Use Thereof,” the entirety of which is incorporated herein by reference.
Accordingly, this invention can be directed to a method comprising providing a compound of the sort described herein, such a compound exhibiting activity at a cannabinoid receptor; and contacting a cell comprising a cannabinoid receptor with such a compound and/or administering such a compound to a patient, such a compound in an amount at least partially effective to treat a cannabinoid receptor/mediated condition. Such a cannabinoid receptor can be a receptor described herein or as would otherwise be understood or realized by those skilled in the art made aware of this invention.
The activity of cannabinoid and related receptors can be affected, mediated and/or modified by contacting such a receptor with an effective amount of one or more of the present compounds as can be present in or as part of a pharmaceutical composition or treatment, or by contacting a cell comprising such a receptor with an effective amount of one or more such compounds, so as to contact such a receptor in the cell therewith. Contacting may be in vitro or in vivo. Accordingly, as would be understood by those skilled in the art, “contact” means that a cannabinoid and/or related receptor and one or more compounds are brought together for such a compound to bind to or otherwise affect or modify receptor activity. Amounts of one or more such compounds effective to modify and/or affect receptor activity can be determined empirically and making such a determination is within the skill in the art.
Without limitation, analog compounds of this invention can be used ex vivo in receptor binding assays of the sort described in Example 2 of the aforementioned co-pending '342 application. In vitro activity of the present analog compounds can be demonstrated in a manner similar to that described in Example 3 of the co-pending application. Alternatively, in vivo activity can be demonstrated using the protocols described in Examples 4 and 6, thereof. More specifically, anti-cancer activity of various representative compounds of this invention can be shown against human lung, prostate, colorectal and pancreatic cancer cell lines using the methodologies described in Example 9 of the aforementioned co-pending '342 application. Extending such a methodology, the present invention can also be used to treat cancer growth of the central nervous system and/or induce cellular death within such growth. In accordance with this invention, various cannabinoid compounds of the sort described herein, including but not limited to those discussed above, can also be used in conjunction with a method to treat human glioma and/or brain cancers. Illustrating such embodiments, one or more compounds of the present invention can be provided and used, as described in the co-pending application, to contact and/or treat human brain cancers, such contact and/or treatment as can be confirmed by cell death and/or related effects.
EXAMPLES OF THE INVENTION
The following non-limiting examples and data illustrate various aspects and features relating to the compounds, compositions and/or methods of the present invention, including the synthesis of pyrimidine classical cannabinoid receptor ligands and/or compounds, as are available though the methodologies described herein. In comparison with the prior art, the present compounds and methods provide results and data which are surprising, unexpected and contrary thereto. While the utility of this invention is illustrated through the preparation and use of several compounds, moieties and/or substituents thereof, it will be understood by those skilled in the art that comparable results are obtainable with various other compounds, moieties and/or substituents, as are commensurate with the scope of this invention. All compounds are named using ChemBioDraw Ultra Version 11.0.01.
Example 1a




2-Methyl-2-thiophen-2-yl-propanenitrile—To a solution of 2-(thiophen-2-yl)acetonitrile (1 g, 8.13 mmol) in 4 ml anhydrous THF, KHMDS (24.4 mmol, 48.9 ml, 0.5M in toluene) was added at 0° C. The mixture was allowed to stir for 3 minutes, after which a solution of 16.26 mmol iodomethane (1.13 ml in 26 ml anhydrous THF) was added slowly over a period of 10 minutes. The mixture was stirred for 5 minutes and monitored by TLC. Upon completion, the reaction was quenched with aqueous ammonium chloride. The organic phase was separated with ethyl acetate and dried over sodium sulfate. The product was purified via vacuum distillation. (bp 42° C. at 1 torr) Yield: 89%. 1H NMR (500 MHz, CDCl3): δ (ppm) 7.4 ppm (d, 1H), 7.2 ppm (t, 1H), 7.0 ppm (d, 1H), 1.9 ppm (s, 6H).
Example 1b
In a similar fashion the following compound was synthesized.




2,2-Dimethyloctanenitrile—Purified via vacuum distillation (bp 50-55° C. at 1.1 torr). Yield: 84% I.R. (neat) nitrile 2230 cm−1, 1H NMR (500 MHz, CDCl3): δ (ppm) 1.5 ppm (m, 4H). 1.4-1.3 ppm (m, 12H), 0.9 ppm (s, 3H).
Example 2a




2-Methyl-2-phenylpropionitrile—To a solution of fluorobenzene (5.85 mL, 62.4 mmol) in 100 mL of anhydrous toluene was added isobutyronitrile (22.5 mL, 250 mmol) followed by 200 mL (100 mmol) of a 0.5 M solution of KHMDS in toluene. The reaction was stirred at 80° C. for 24 hours. The reaction was then allowed to cool to room temperature, diluted with diethyl ether, and washed with water and brine. The organic fraction was then dried over sodium sulfate and concentrated under reduced pressure. The product was purified by flash chromatography using an ethyl acetate/hexanes gradient to yield 4.57 g (50%) of the objective compound as a brown oil. MS: (ESI, Pos) m/z 168.0 (M+23) 1H NMR (500 MHz, CDCl3): δ (ppm) 7.48 (d, 2H), 7.39 (t, 2H), 7.31 (t, 1H), 1.73 (s, 6H).
Example 2b
In a similar fashion the following compounds were synthesized.




2-Cyclohexyl-2-methylpropanenitrile—Purified in a manner similar to 2-methyl-2-phenylpropanenitrile using 2-bromopyridine as the starting material to yield a brown oil. MS: (ESI, Pos) m/z 168.9 (M+23).
Example 2c




2-Methyl-2-(pyridin-2-yl)propanenitrile—Purified in a manner similar to 2-methyl-2-phenylpropanenitrile using 2-bromopyridine as the starting material to yield a brown oil. MS: (ESI, Pos) m/z 168.9 (M+23).
Example 3a




2-Phenylacetamidine—Ammonium chloride (2.9 g, 54 mmol) was suspended in 20 mL of anhydrous toluene, stirred under argon, and cooled to 0° C. To this suspension was added 25 ml (50 mmol) of a 2M solution of trimethylaluminum in toluene. The reaction was allowed to warm to room temperature and stirring was continued until the evolution of methane ceased ( ̃1 hour). Then, 3.46 mL (30 mmol) of benzyl cyanide in 10 mL of anhydrous toluene was added and the reaction was heated to 80° C. for 18 hours. The reaction was then cooled to room temperature and slowly poured into a slurry of 15 g of silica gel in 50 mL of chloroform and stirred for 5 minutes. The silica was filtered and washed with methanol. The filtrate and washings were combined and reduced to a volume of  ̃15 mL and refiltered. Then, 18 mL (54 mmol) of a 3N solution of methanolic HCl was added to the filtrate followed by 400 mL of diethyl ether. After 16 hours of stirring at room temperature, a white precipitate formed was subsequently filtered. This crude solid was then added to 150 mL of a 4:1 mixture of isopropanol-acetone and stirred for an additional 16 hours at room temperature. Undissolved ammonium chloride was then removed by filtration and the filtrate reduced to  ̃15 mL. After the addition of 300 mL of diethyl ether the mixture was stirred for  ̃1 hour. The white precipitate was then filtered and dried to yield 3.7 g (72%) of the objective compound as the hydrochloride salt. MS: (ESI, Pos) m/z 157.9 (M+23) 1H NMR (500 MHz, DMSO-d6): δ (ppm) 9.06 (br.d, 3H), 7.49 (d, 2H), 7.36 (t, 2H), 7.31 (t, 1H), 3.74 (s, 2H).
Example 3b
In a similar fashion the following amidines were synthesized.




2-Methyl-2-phenylpropanimidamide hydrochloride—Yield: 29% MS: (ESI, Pos) m/z 163.0 (M+1) 1H NMR (500 MHz, DMSO-d6): δ (ppm) 9.27 (br.s, 2H), 8.71 (br.s, 2H), 7.41 (m, 2H), 7.36 (m, 2H), 7.32 (m, 1H), 1.60 (s, 6H) 13C NMR (500 MHz, DMSO-d6): δ (ppm) 175.91, 143.27, 128.71, 127.42, 125.65, 44.06, 26.31.
Example 3c




2-Methyl-2-(thiophen-2-yl)propanimidamide—Yield: 36% MS: (ESI, Pos) m/z 169.0 (M+1) 1H NMR (500 MHz, DMSO-d6): δ (ppm) 8.8 (br.d, 3H), 7.53 (dd, 1H), 7.13 (dd, 1H), 7.04 (dd, 1H), 1.70 (s, 1H) 13C NMR (500 MHz, DMSO-d6): δ (ppm) 174.75, 147.04, 127.18, 125.72, 125.48, 41.92, 27.20.
Example 3d




2-Methyl-2-pyridin-2-yl-propanenitrile—prepared from 2-bromopyridine as the starting material to yield a brown oil. MS: (ESI, Pos) m/z 168.9 (M+23).
Example 3e




Hexanamidine—Yield: 84 MS: (ESI, Pos) m/z 115.1 (M+1) 1H NMR (500 MHz, DMSO-d6): δ (ppm) 9.11 (s, 2H), 8.78 (s, 2H), 2.37 (t, 2H), 1.60 (p, 2H), 1.27 (m, 4H), 0.86 (t, 3H) 13C NMR (500 MHz, DMSO-d6): δ (ppm) 171.15, 31.58, 30.33, 25.96, 21.59, 13.77.
Example 3f
Various other amidines can be prepared from the respective nitriles, using synthetic procedures, comparable to those described above en route to the Y- and/or Z-substituted pyrimidine intermediates, as illustrated below.
Example 4




2,2-Bis(ethylthio)acetimidamide—5.16 mmol of trimethyl aluminum (2.6 ml, 2M in toluene) was slowly added to a suspension of ammonium chloride (5.58 mmol, 300 mg) in 2 ml of anhydrous toluene at 5° C. The mixture was brought to room temperature and allowed to stir until gas evolution of methane had ceased. 3.1 mmol (500 mg) of 2,2-bis(ethylthio)/acetimidamide in 1 ml anhydrous toluene was added. The mixture was refluxed at 80° C. for 18 hours, TLC indicated the nitrile had been consumed. The mixture was poured into a mixture of 1.55 g silica gel in 5 ml chloroform and stirred for 5 minutes. The silica was filtered off and washed with methanol. The filtrate was stripped to a volume of 2 ml and filtered to remove NH4Cl. 2 ml of 3N methanolic HCl (6 mmol) was added followed by 31 ml ethyl ether and the mixture was stirred overnight. The resulting crude white precipitate was filtered and then combined with 4:1 v/v isopropanol-acetone and stirred at room temperature overnight. Undissolved ammonium chloride was removed by filtration, the filtrate was again stripped to a volume of 2 ml and combined with 31 ml of ether. The white precipitate was filtered and identified as the desired product. Product was confirmed via mass spectroscopy and NMR in DMSO. +MS=179 m/z, 1H NMR (500 MHz, DMSO-d6): 9.35 ppm (d 3H, split). 5.1 ppm (s, 1H). 2.77 ppm (q, 4H). 1.27 ppm (t, 6H).
Example 5a




2-Benzylpyrimidine-4,6-diol—Sodium metal (168 mg, 7.3 mmol) was dissolved in 8 mL of anhydrous ethanol. To this was added 500 mg (2.93 mmol) of 2-phenylacetimidamide hydrochloride 3a and the reaction was allowed to stir at room temperature for 5 minutes. Diethyl malonate (2.9 mmol) was then added and the reaction was heated to reflux overnight. The reaction was then cooled to room temperature and filtered. The solid was washed several times with absolute ethanol. The filtrate and washings were combined and diluted with 2 volumes of water and acidified with 5N HCl to a pH of  ̃2 and the product precipitated. The mixture was chilled and then filtered. The solid was washed several times with diethyl ether and then dried to yield 262 mg (44%) of a peach colored solid. MS: (ESI, Neg) m/z 200.8 (M−1) 1H NMR (500 MHz, DMSO-d6): δ (ppm) 11.80 (br.s, 2H), 7.32 (m, 4H), 7.25 (m, 1H), 5.09 (s, 1H), 3.80 (s, 2H).
Example 5b
In a similar fashion the following compounds were synthesized.




2-(2-Phenylpropan-2-yl)pyrimidine-4,6-diol—MS: (ESI, Neg) m/z 228.9 (M−1); 1H NMR (500 MHz, DMSO-d6): δ (ppm) 11.35 (br.s, 2H), 7.33 (t, 2H), 7.23 (m, 3H), 5.19 (s, 1H), 1.62 (s, 6H).
Example 5c




2-(2-(thiophen-2-yl)propan-2-yl)pyrimidine-4,6-diol—MS: (ESI, Neg) m/z 235.1 (M−1); (500 MHz, DMSO-d6): δ (ppm) 11.39 (br.s, 2H), 7.41 (d, 1H), 6.99 (d, 1H), 6.97 (t, 1H), 5.2 (s, 1H), 1.72 (s, 6H).
Example 5d




2-(2-Cyclohexylpropan-2-yl)pyrimidine-4,6-diol—MS: (ESI, Neg) m/z 235.1 (M−1); (500 MHz, DMSO-d6): δ (ppm) 11.41 (br.s, 2H), 5.12 (s, 1H), 1.80 (t, 1H), 1.69 (d, 2H), 1.38 (s, 1H), 1.72 (d, 2H), 1.19-1.02 (m, 4H), 1.15 (s, 6H), 0.94 (dd, 2H).
Example 5e




2-(2-Methyloctan-2-yl)pyrimidine-4,6-diol—MS: (ESI, Neg) m/z 236.8 (M−1); (500 MHz, DMSO-d6): δ (ppm) 11.33 (br.s, 2H), 5.13 (s, 1H), 1.62 (m, 2H), 1.22 (s, 6H), 1.20 (m, 6H), 1.04 (m, 2H), 0.83 (t, 3H).
Example 6a




3-Benzyl-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6a)—2-benzylpyrimidine-4,6-diol (5a, 100 mg, 0.49 mmol) was added to 3 mL of absolute ethanol in a 10 mL microwave reaction vessel. To this was also added 66 μL of pyridine and 3 μL of piperidine followed by 266 μL (1.47 mmol) of (R)-(+)-citronellal. The reaction vessel was then sealed and irradiated at 200 watts to 130° C. for 1 hour. The solvent was then removed by rotary evaporation and the product purified by flash chromatography using a methanol/methylene chloride gradient to yield 180 mg (69%) of the objective product as a light brown resin. MS: (ESI, Neg) m/z 337.0 (M−1). 1H NMR (500 MHz, CDCl3): δ (ppm) 13.01 (br.s, 1H), 7.49 (d, 2H), 7.30 (t, 3H), 7.25 (m, 1H), 3.92 (s, 2H), 3.28 (d, 1H), 2.37 (m, 1H), 1.84 (m, 2H), 1.69 (m, 1H), 1.46 (s, 3H), 1.38 (m, 1H), 1.19 (s, 3H), 1.05 (m, 2H), 0.92 (d, 3H), 0.66 (q, 1H).
Example 6b
In a similar fashion the following compounds were synthesized.




(6aS,9R,10aR)-6,6,9-Trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6b)—yield: 31% MS: (ESI, Neg) m/z 373.1 (M−1) 1H NMR (500 MHz, DMSO-d6): δ 11.57 (s, 1H), 3.10 (d, 1H), 2.16 (m, 1H), 1.75 (m, 2H), 1.56 (m, 6H), 1.34 (s, 3H), 1.18 (m, 12H), 1.02 (m, 5H), 0.89 (d, 3H), 0.82 (t, 3H), 0.48 (q, 1H).
Example 6c




(6aS,9R,10aR)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6c)—yield: 60.4% MS: (ESI, Neg) m/z 365.0 (M−1) 1H NMR (500 MHz, DMSO-d6): δ 11.43 (s, 1H), 7.32 (q, 2H), 7.21 (m, 3H), 3.06 (d, 1H), 2.16 (m, 1H), 1.76 (m, 2H), 1.56 (m, 11H), 1.37 (s, 3H), 1.01 (m, 2H), 0.87 (d, 3H), 0.48 (q, 1H).
Example 6d




(6aS,9R,10aR)-3-(2-Cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6d)—yield: 36% MS: (ESI, Neg) m/z 371.0 (M−1) 1H NMR (500 MHz, DMSO-d6): δ 11.52 (s, 1H), 3.10 (s, 1H), 2.16 (m, 1H), 1.73 (m, 5H), 1.52 (m, 2H), 1.34 (s, 3H), 1.26 (m, 4), 1.10 (m, 10H), 1.05 (s, 3H), 0.96 (m, 2H), 0.88 (d, 3H), 0.48 (q, 1H).
Example 6e




(6aS,9R,10aR)-6,6,9-Trimethyl-3-(2-(thiophen-2-yl)propan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6e)—yield: 46% MS: (ESI, Neg) m/z 371.0 (M−1) 1H NMR (500 MHz, DMSO-d6): δ 11.52 (s, 1H), 7.40 (d, 1H), 6.97 (m, 2H), 3.06 (d, 1H), 2.16 (m, 1H), 1.70 (m, 8H), 1.50 (m, 2H), 1.36 (s, 3H), 1.06 (m, 5H), 0.87 (d, 3H), 0.47 (q, 1H).
Example 6f




(6aR,9S,10aS)-6,6,9-Trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (6f)—yield: 9% MS: (ESI, Neg) m/z 365.1 (M−1) 1H NMR (500 MHz, methanol-d4): δ 7.31 (m, 2H), 7.22 (m, 3H), 2.93 (m, 1H), 2.30 (m, 1H), 1.72 (m, 2H), 1.64 (m, 8H), 1.58 (s, 3H), 1.45 (s, 3H), 1.09 (m, 2H), 0.90 (d, 3H), 0.56 (q, 1H).
Example 7




3-Benzyl-6,6,9-trimethyl-tetrahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (7)—The material was synthesized from 2-benzylpyrimidine-4,6-diol 5a and TBS protected 3-hydroxyl citronella (Kesenheimer and Groth, Org. Lett., 8:2507 (2006)) as described for the previous compound. The TBS protecting group was removed using TBAF and the compound was dehydrated using a general acid to yield a mixture of separable isomers.
Example 8




3-Benzyl-6,6,9-trimethyl-7,8,9,10-tetrahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol (8)—The material was synthesized from 2-benzylpyrimidine-4,6-diol 5a and pulegone according to the method of Adams (U.S. Pat. No. 2,419,934). In general, 5a (1 eq), pulegone (1.5 eq), POCl3 (0.3 eq) and dry benzene are refluxed for 4 hours. The excess POCl3 was decomposed in aqueous sodium bicarbonate and the mixture was extracted with Et2O, dried, and evaporated. The product was purified by flash chromatography using a methanol/methylene chloride gradient.
Example 9
While several compounds with tetrahydro and dehydro C-ring structures are shown, other such compounds can be prepared to provide a range of Y and/or Z moieties, such compounds limited only by commercial or synthetic availability of the corresponding pyrimidine and terpine intermediates. Likewise, R1 can be varied depending upon choice of starting material or subsequent chemistry on the resulting cannabinoid compound.
Example 10




2-(Bis(ethylthio)methyl)pyrimidine-4,6-diol (10)—Prepared in a manner similar to 5a using 2,2-Bis(ethylthio)acetamidine hydrochloride 4 and diethyl malonate as the starting materials to yield a dark yellow waxy solid. MS: (ESI, Neg) m/z 245.3 (M−1). H1 NMR: 11.7 ppm (d) 2H, 5.2 ppm (s). 69H, 4.8 ppm (s) 1H, 2.65 ppm (m) 4H. 1.17 ppm (t) 6H. Subsequent sulfanyl oxidation can be used to generate various Y moieties, C(O), etc., as described above.
Receptor Binding Assays
Cell membranes from HEK293 cells transfected with the human CB-1 receptor and membranes from CHO-K1 cells transfected with the human CB-2 receptor were prepared. [3H]CP 55,940 having a specific activity of 120 Ci/mmol was obtained from Perkin-Elmer Life Sciences, Inc. All other chemicals and reagents were obtained from Sigma-Aldrich. The assays were carried out in 96 well plates obtained from Millipore, Inc. fitted with glass fiber filters (hydrophilic, GFC filters) having a pore size of 1.2μ. The filters were soaked with 0.05% polyethyleneimine solution and washed 5× with deionized water prior to carrying out the assays. The filtrations were carried out on a 96 well vacuum manifold (Millipore Inc.), the filters punched out with a pipette tip directly into scintillation vials at the end of the experiment, and the vials filled with 5 ml scintillation cocktail Ecolite (+) (Fisher Scientific). Counting was carried out on a Beckmann Scintillation Counter model LS6500. Drug solutions were prepared in DMSO and the radioligand was dissolved in ethanol.
Incubation buffer: 50 mM TRIS-HCl, 5 mM MgCl2, 2.5 mM EDTA, 0.5 mg/ml fatty acid free bovine serum albumin, pH 7.4.
Binding protocol for the CB-1 receptor: 8 μg of membranes (20 μl of a 1:8 dilution in incubation buffer) was incubated with 5 μl of drug solution (10−4M to 10−12M) and 5 μl of 5.4 nM [3H]CP 55,940 in a total volume of 200 μl for 90 mins at 30° C. Non-specific binding was determined using 10 μM WIN55,212-2 (Ki=4.4 nM). The membranes were filtered and the filters washed 7× with 0.2 ml ice-cold incubation buffer and allowed to air dry under vacuum.
Binding protocol for the CB-2 receptor: 15.3 μg of membranes (20 μl of a 1:20 dilution in incubation buffer) was incubated with 5 μl of drug solution (10−4M to 10−12M) and 5 μl of 10 nM [3H]CP 55,940 in a total volume of 200 μl for 90 minutes at 30° C. Non-specific binding was determined using 10 μM WIN55, 212-2 (Ki=4.4 nM). The membranes were filtered and the filters washed 7× with 0.2 ml ice-cold incubation buffer and allowed to air dry under vacuum.
Data accumulation and statistical analysis: Varying concentrations of drug ranging from 10−4M to 10−12M were added in triplicate for each experiment and the individual molar IC50 values were determined using GraphPad Prism. The corresponding Ki values for each drug were determined utilizing the Cheng and Prusoff equation and final data was presented as Ki±S.E.M. of n≧2 experiments.
Functional assays: HEK-293 cell lines stably transfected with a cyclic nucleotide-gated channel and either human CB-1 or CB-2 receptors (BD Biosciences, San Jose, Calif. USA) were seeded in poly-D-lysine coated 96-well plates at a density of 70,000 cells per well. Plates were incubated at 37° C. in 5% CO2 overnight prior to assay. Plates were then removed from the incubator and the complete growth medium (DMEM, 10% FBS, 250 μg/ml G418 and 1 μg/ml puromycin) was replaced with 100 μL DMEM containing 0.25% BSA. Next, 100 μL membrane potential dye loading buffer (Molecular Devices, Sunnyvale, Calif. USA) was prepared according to the manufacturer. The plates were placed back into the incubator for 30 minutes and then the baseline fluorescence was read on a BioTek Synergy 2 multi-mode microplate reader (BioTek Instruments, Winooski, Vt. USA) with 540 nm excitation and 590 nm emission filters prior to drug addition. Drugs were added in 50 μL DPBS containing 2.5% DMSO, 1.25 μM 5′-(N-ethylcarboxamido) adenosine and 125 μM Ro 20-1724. Plates were then incubated at room temperature for 25 minutes and fluorescence measured again at 540 nm excitation and 590 nm emission.
FIG. 1 depicts the functional activity of compound 6b at the CB-1 receptor.
Cytoxocity assay: Cells were seeded on a 96 well polystyrene plate in full serum media at a density of 75,000 cells per milliliter, 100 μL per well. Plates were incubated at 37° C. and 5% CO2 for 24 hours to allow cell attachment. Drug solutions were prepared in DMSO at 100× concentration and mixed 1:100 in 1% FBS media to yield the desired concentration. Drug-media mixtures were vortexed immediately prior to administration to cells. Full serum media was removed and replaced with drug-media mixtures and incubated for 18 hours. 10 μL of Cell Counting Kit 8 (CCK8, Dojindo# CK04-11) was added to each well to colormetrically assess viability. After 2-4 hours of incubation with the CCK8 dye, absorbance was read at 450 nm by a BioTek Synergy 2 plate reader.
The cytotoxicity of selected compounds against the glioblastoma brain cancer cell line LN-229 is depicted in Table 1.










TABLE 1







Compound 
EC50 (μM)









6b
27.3



6d
10.6



6e
30.1










Inflammation Studies

Differentiation of Monocytes: To THP-1 human leukemia monocytes (ATCC #TIB-202) in suspension was added phorbol 12-myristate 13-acetate (PMA Aldrich #P1585) and ionomycin (Aldrich #10634), 10 and 500 ng/ml respectively, to induce differentiation into macrophage-like cells. Cells were seeded at 30,000 cells/well and allowed to incubate at 37° C. in 5% CO2/95% air for 3-10 days to complete transformation. Media was refreshed as needed until assay.
Cytokine Assay: A549 (ATCC #CCL-185), HUV-EC-C (ATCC #CRL-1730), or differentiated THP-1 cells were seeded on 96-well polystyrene plates at a density of 300,000 cells/ml (100 μL per well) and incubated at 37° C. in 5% CO2/95% air for 24 hours to allow cell attachment. Drug solutions were prepared in DMSO at 100× concentration and mixed 1:100 in 1% FBS media to yield the desired concentration.
Plates were then removed from the incubator and the complete growth media was replaced with 50 μL media containing 1% FBS and lipopolysaccharide or peptidoglycan at 1 μg/ml (for differentiated THP-1), or TNF-α (10 ng/ml) or IL-1β (1 ng/ml) in the case of A549 and HUVEC or without stimulus in the case of control wells. Cells were returned to the incubator for 1 hour before drug treatments. Drug-media solutions were prepared at 2× desired final concentration in media containing 1% FBS and the appropriate stimulus at the previously mentioned concentration. Control media was also prepared which contained no drug. 50 μL of drug containing media or control was then added to appropriate wells and the plates returned to the incubator for 18 hours. Media supernatants were then removed from the wells and frozen at −80° C. until time of assay.
FIGS. 2-12 depict secretion profiles of various modulators by A549 exposed to compound 6b at the EC1 and EC10 in the presence and absence of TNF-α at 4 and 18 hour intervals. The graph legends for FIGS. 2-12 are as follows: pymC 1-4=compound 6b at 5.84 μM for 4 hours; pymC 2-4=compound 6b at 13.3 μM for 4 hours; pymC 1-18=compound 6b at 5.84 μM for 18 hours; pymC 2-18=compound 6b at 13.3 μM for 18 hours; TNF=TNF-α at 10 ng/ml.
The invention and the manner and process of making and using it are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as the invention, the following claims conclude this specification.

## Claims

1. A method of treating inflammation mediated by a cannabinoid receptor comprising contacting said receptor with a compound in an amount to treat said cannabinoid receptor-mediated disorder, wherein the compound is of the formula




wherein
----- represents an optional double bond wherein the ring that optionally contains it is selected from hexahydro, 6a,10a-dehydro, 8,9-dehydro, and 9,10-dehydro;
Y is selected from S, O, CH2, CH(CH3), CH(OH), C(CH3)(OH), C(CH3)2, C(—U(CH2)nU—), C(O), NH, S(O), and S(O)2;
n is an integer≧1;
U is selected from CH2, S, and O;
Z is selected from H, substituted and unsubstituted alkyl, and
cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocycloalkyl, and heterocycloalkylalkyl, wherein each alkyl portion is optionally substituted and the ring portion of each is optionally substituted with one, two, three, four or five substituents; and

R1 is selected from H and substituted and unsubstituted alkyl.

2. A method according to claim 1 wherein
R1 is selected from H and alkyl;
Y is selected from carbonyl, dimethylmethylene and hydroxymethylene; and
Z is selected from substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted phenyl, and substituted or unsubstituted thiophenyl.

3. A method according to claim 2 wherein Z is alkyl, cycloalkyl, phenyl or thiophenyl.

4. A method according to claim 3 wherein Z is hexyl, cyclohexyl, phenyl or thiophenyl.

5. A method according to claim 2 wherein Y is dimethylmethylene.

6. A method according to claim 2 wherein the ring having ----- is hexahydro or 6a,10a-dehydro.

7. A method according to claim 1 wherein the compound is selected from
a) 3-Benzyl-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
b) (6aS,9R,10aR)-6,6,9-Trimethyl-3-(2-methyloctan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
c) (6aS,9R,10aR)-6,6,9-trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
d) (6aS,9R,10aR)-3-(2-Cyclohexylpropan-2-yl)-6,6,9-trimethyl-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
e) (6aS,9R,10aR)-6,6,9-Trimethyl-3-(2-(thiophen-2-yl)propan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
f) (6aR,9S,10aS)-6,6,9-Trimethyl-3-(2-phenylpropan-2-yl)-6a,7,8,9,10,10a-hexahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol;
g) 3-Benzyl-6,6,9-trimethyl-tetrahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol; and
h) 3-Benzyl-6,6,9-trimethyl-7,8,9,10-tetrahydro-6H-isochromeno[3,4-d]pyrimidin-1-ol.

8. A method according to claim 1 wherein the cannabinoid receptor is selected from CB-1 and CB-2.