Preparation of Phantasmidine and Analogues

Thereof

RELATED APPLICATIONS

This application claims the benefit of priority to United States Provisional Patent

Application serial number 61/421,697, filed December 10, 2010, the contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Amphibians, in general, and poison frogs, in particular, have been a source of many biologically active natural products. A number of frog-skin alkaloids have been shown to have activity at nicotinic acetylcholine receptors (nAChRs). The ability of poison frogs to sequester alkaloids from their diet results in a unique complexity, and to date over 800 alkaloids in more than 20 structural classes have been characterized.

Nicotinic acetylcholine receptors are broadly distributed in both the peripheral and central nervous systems; activation of these receptors in the brain results in enhanced release of various key neurotransmitters. Dysfunction of these receptors is associated with a variety of neurological diseases, including nicotine and alcohol addiction. Nicotinic agonists enhance action at nicotinic acetylcholine receptors and have been shown to possess potential clinical utility in many of these diseases, although development is hindered by the existence of a large number of receptor subtypes with highly variable properties. Nicotinic agonists may ultimately play a role in the treatment of Alzheimer's disease, attention deficit hyperactivity disorder (ADHD), or schizophrenia.

In 1992 epibatidine (2) was isolated and characterized from the frog, Epipedobates anthonyi. See Figure 1. The compound has become one of the most well-studied frog alkaloids due to its potent analgesic activity resulting from its ability to activate nicotinic receptors; unfortunately, it is also extremely toxic.

The chemical complexity of E. anthonyi extract (over 80 alkaloids) has prompted the investigation of other constituent alkaloids, including epiquinamide. Although the activity initially ascribed to this molecule was later found to be due to a cross- contamination artifact, other compounds within the extract were also found to have nicotinic activity. One was the known N-methylepibatidine. Another, a condensed tetracyclic alkaloid, phantasmidine (1), is distinct from the epibatidine skeleton and represents a new structural class. See Figure 1. Initial studies suggest that this novel rigid nicotinic ligand has selectivity for ^-containing nicotinic receptors, distinct from the activity of epibatidine. As such, phantasmidine may serve as a useful pharmacological probe and potential lead compound for the development of selective nicotinic receptor therapeutics. However, phantasmidine was obtained in only microgram amounts, insufficient for full pharmacological characterization. Consequently, there exists a need for an efficient synthesis of phantasmidine and analogs thereof.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to a method of preparing a product, comprising the step depicted in Scheme I:

Scheme I

wherein

A represents an optionally-substituted aromatic ring;

R ¹ represents H or lower alkyl;

Y represents C(R ¹ ) ₂ , O or NR ¹ ; and

n is 1, 2, 3, or 4.

In certain embodiments, the invention relates to a method of preparing a product, comprising the step depicted in Scheme II:

Scheme II.

In certain embodiments, the invention relates to a method of preparing a product, comprising the steps depicted in Scheme III:

Scheme III

wherein

A represents an optionally-substituted aromatic ring;

R ¹ represents H or lower alkyl;

Y represents O or NR ¹ ;

n is 1, 2, 3, or 4;

"Step a" comprises a first reagent, a first acid, and a first solvent, at a first temperature, for a first period of time;

"Step b" comprises a base, and a second solvent, at a second temperature, for a second period of time;

"Step c" comprises a second reagent, and a third solvent, at a third temperature, for a third period of time;

"Step d" comprises a third reagent, at a fourth temperature; and

"Step e" comprises a fourth reagent, and a fourth solvent, at a fifth temperature, for a fourth time. In certain embodiments, the invention relates to a method of preparing a product, comprising the steps depicted in Scheme IV:

a) BH ₃ «THF, THF,

0 to 25 °C, 30 h;

2 M aq. KOH, b) MeOH, 0-25 °C; f-BuOH, 25 °C c) piperazine, MeOH

15 min, degassed reflux, 3 h

Scheme IV.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts a retrosynthetic analysis of ±-phantasmidine (1), and the structure of epibatidine (2)

Figure 2 depicts a synthetic scheme that failed to produce ±-phantasmidine (1). Figure 3 depicts a synthetic scheme that produced ±-phantasmidine (1).

Figure 4 depicts the x-ray structure of lactam 3.

Figure 5 depicts the circular dichroism (CD) spectra of the purified enantiomers of phantasmidine.

Figure 6 depicts the structures and observed ratios of various conformers of derivatives of phantasmidine.

Figure 7 depicts (a) the structure of the major conformer 19ma (MMX energy =

46.44) of the more active (+)-2ai?-enantiomer(5)-Mosher amide showing shielding (δ 0.68 and 0.87) of the cyclobutane methylene group, and (b) the structure of the minor conformer 19mi (MMX energy = 47.22) of the more active (+)-2ai?-enantiomer(S)-Mosher amide showing shielding (δ 6.41 and 6.56) of the pyridine hydrogens. DETAILED DESCRIPTION OF THE INVENTION

Overview of a Synthesis of Phantasmidine

We speculated that phantasmidine (1) could be prepared by reduction of lactam 3 (Figure 1). In the key step of the proposed synthesis we planned to prepare lactam 3 from keto amide 5 by a novel, tandem intramolecular aldol reaction-intramolecular nucleophilic aromatic substitution sequence. Addition of the amide enolate of 5 to the cyclobutanone carbonyl group would provide alkoxide 4, which would undergo a nucleophilic aromatic substitution reaction at the activated 2-halopyridine to form the furan ring of 3. Although two stereoisomeric aldol products could be formed, the alkoxide can only displace the halide in 4. Moreover, the aldol reaction is reversible, so the two isomers would be in equilibrium with the reaction driven to completion by the cyclization of the desired stereoisomer 4 to give lactam 3. Keto amide 5 was expected to be readily available by the reaction of primary amide 6 with l,2-bis(trimethylsilyloxy)cyclobutene (7), the readily available acyloin formed from succinate esters. We began with the more readily available, but less reactive, dichloro amide 6a. Schlosser has shown that nucleophilic substitution of a 2-fluoropyridine is -320 times faster than substitution of a 2-chloropyridine. The use of fluoro chloro amide 6b, therefore, offered an attractive alternative if nucleophilic aromatic substitution of dichloropyridine 4a failed to form the furan ring of 3a.

Commercially available 2,6-dichloropyridine-3-carboxyaldehyde (8a) was reduced with NaBH ₄ in MeOH at 0 °C for 30 minutes to give primary alcohol 9a in 99% yield (see Figure 2). Reaction of the alcohol with thionyl chloride and catalytic DMF in CH ₂ CI ₂ at 25 °C for 6 hours gave chloride 10a, which was treated with NaCN in aqueous EtOH at reflux for 4 hours to give nitrile 11a in 64% yield from alcohol 9a. Hydrolysis of nitrile 11a with methanesulfonic acid and alumina at 120 °C for 15 minutes afforded the requisite dichloro amide 6a in 70% yield. Reaction of primary amide 6a with 1,2- bis(trimethylsilyloxy)cyclobutene (7) in ether saturated with hydrogen chloride in a sealed tube at 80 °C for 2 h afforded cyclobutanone amide 5a in 78% yield.

Unfortunately, our attempts to convert keto amide 5a to tetracyclic lactam 3 were unsuccessful. For example, treatment of 5a with aqueous potassium hydroxide in t-BuOH gave a complex mixture of products. Based on limited literature precedent, we thought that acylation of the amide would not only remove the acidic NH proton, but would also facilitate enolization and the intramolecular aldol reaction because the a-protons of an imide are more acidic than those of an amide. Treatment of 5a with (Boc) ₂ 0, Et ₃ N, and DMAP in CH ₂ CI ₂ at 25 °C acylated the amide to give imide 12, which underwent the desired aldol reaction to give alkoxide 13. Unfortunately, nucleophilic aromatic substitution with displacement of the chloride was slow and only the unstable α,β-unsaturated lactam 14 resulting from dehydration of the aldol product was isolated in 61% yield. Because we were unable to form the furan ring of phantasmidine from dichloropyridine 5a, we turned our attention to the preparation of more-reactive chlorofluoropyridine 5b.

Metalation of 2-chloro-6-fluoropyrdine (15) adjacent to the fluoride with LDA in THF at -78 °C followed by trapping with DMF afforded aldehyde 8b in 90% yield (Figure 3). The reactive fluoride was sensitive to basic hydrolysis to give the pyridine, so the reaction was quenched by addition of saturated hydrogen chloride in ether at -78 °C. Reduction of aldehyde 8b with NaBH ₄ in MeOH at -10 °C gave alcohol 9b in 90% yield. Conversion of the alcohol to the chloride with thionyl chloride and catalytic DMF in CH ₂ C1 ₂ proceeded to give 10b because the reaction conditions are acidic. Substitution of the benzylic chloride without hydrolysis of the 2-fluoropyridine was best accomplished by reaction with sodium cyanide in DMSO at 25 °C for 1 hour to give nitrile lib in 63% yield from alcohol 9b. Hydrolysis of the nitrile to the amide with methanesulfonic acid and alumina at 120 °C for 15 minutes afforded fluoro chloro amide 6b in 61% yield, which was converted to keto amide 5b in 85% yield by reaction with 7 in ether saturated with hydrogen chloride for 2 h at 80 °C in a sealed tube.

Remarkably, the increased reactivity of the fluoride of keto amide 5b was sufficient to facilitate formation of the furan ring of lactam 3. Treatment of a degassed solution of 5b in t-BuOH with degassed 2 M aqueous KOH resulted in aldol cyclization to give alkoxide 4b, which underwent the desired nucleophilic aromatic substitution reaction to form furan 3 in 46%o yield. The structure of 3 was readily assigned by analysis of the NMR spectra and was confirmed by X-ray crystal structure determination. No other products were isolated, suggesting that the aldol reaction to give 4b is stereospecific or the aldol reaction is reversible and the undesired stereoisomer 16 reverts to 5b, which then cyclizes again to give 4b and then 3.

Reduction of lactam 3 with borane in THF afforded the borane complex of phantasmidine, which was decomplexed by treatment with piperazine to give racemic phantasmidine (1) in 67% yield. Addition of one equivalent of TFA to a CD ₃ OD solution of phantasmidine afforded the monocation, with an 1H NMR spectrum identical to that reported for natural phantasmidine ^» DCl. Pyrrolidine 1 was treated with AcCl and NEt ₃ in CH ₂ C1 ₂ at -15 to 25 °C to give acetamide 17 in 67% yield. The 1H and ¹³ C NMR spectra of 17 (mixture of rotamers) are identical to those reported for the acetamide of phantasmidine. The GC retention times of synthetic and natural phantasmidine are identical, as are the mass spectra of synthetic and natural phantasmidine and phantasmidine acetamide. Therefore, the remarkable synthesis of 1 validates the structure of phantasmidine assigned based on incomplete data obtained from the limited amount (~20 μg) of natural material available.

The synthetic method leads to racemic phantasmidine. The coupling of 6b and 7 affords racemic keto amide 5b. However, even if we were able to prepare 5b in enantiomerically pure form, it might be racemized under the basic reaction conditions of the tandem intramolecular aldol reaction-intramolecular nucleophilic aromatic substitution sequence. Therefore, we resolved the enantiomers of phantasmidine by chiral HPLC on a Chiralcel OJ-H column (retention times of 27 and 40 minutes, respectively). Consequently, both pure enantiomers are available for full biological investigation.

In general, starting materials for the processes described in the present patent application are known or can be prepared by known processes from commercially available materials. The products of the reactions described herein are isolated by conventional means such as extraction, crystallization, distillation, chromatography, and the like.

In conclusion, the invention relates to a short, efficient synthesis (8 steps, 8% overall yield) of racemic phantasmidine that confirms the structure assigned from the limited amount of available natural material. In certain embodiments, the invention relates to a method of producing phantasmidine for further biological evaluation. In certain embodiments, the key step, a novel tandem intramolecular aldol reaction-intramolecular nucleophilic aromatic substitution, should be broadly useful for making phantasmidine analogues.

It will be appreciated by those skilled in the art that the compounds made by the methods of the present invention contain several chiral centers and that such compounds exist in the form of isomers (e.g., enantiomers). The invention includes all such isomers and any mixtures thereof including racemic mixtures.

Racemic forms can be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Another method for resolving racemates into optical antipodes is based upon chromatography on an optically active matrix. Racemic compounds of the present invention can thus be resolved into their optical antipodes, e.g., by fractional crystallization of D- or L-(tartrate, mandelate, or camphorsulphonate) salts, for example. The compounds of the present invention may also be resolved by the formation of diastereomeric amides by reaction of the compounds of the present invention with an optically active activated carboxylic acid such as that derived from (+) or (-) phenylalanine, (+) or (-) phenylglycine, (+) or (-) camphanic acid or by the formation of diastereomeric carbamates by reaction of the compounds of the present invention with an optically active chloroformate or the like.

Additional methods for the resolution of optical isomers, known to those skilled in the art may be used, and will be apparent to the average worker skilled in the art. Such methods include those discussed by J. Jaques, A. Collet, and S. Wilen in "Enantiomers, Racemates, and Resolutions", John Wiley and Sons, New York (1981).

Another option for resolution of optical isomers is preparative high-performance liquid chromatography (HPLC).

In the above-outlined synthetic scheme, for example, stereoisomers of compounds 5b, 3, or 1 may be resolved. In certain embodiments, stereoisomers of compound 3 are resolved. In certain embodiments, stereoisomers of compound 1 are resolved.

Optically active compounds can also be prepared from optically active starting materials.

Biological Activity of Phantasmidine and its Analogues

The effect of drug abuse in the US is devastating. Abuse of illicit drugs and alcohol resulted in over 102,000 deaths in just the year 2000. Abuse of tobacco products alone is linked to 435,000 deaths in that same year. The combined medical, economical, criminal, and societal impact of abuse of alcohol, nicotine, and illicit drugs is estimated to cost upwards of half-a-trillion dollars per year in the US. Drug addiction has been viewed primarily as a social problem, not a health problem. This stance has resulted in a paucity of medical approaches available to physicians for treating chemical dependency. Drug addiction is now recognized to be a chronic brain disease. There is no reliable cure for drug addiction, and the rate of relapse can be as high as 60%, which correlates with other chronic diseases. Therefore, an urgent need exists to address directly this dearth of treatment options by developing novel small molecule agents (such as nicotinic desensitizers) for the treatment of drug abuse.

Drugs of abuse have differing mechanisms of action, but share a common pathway toward physical dependency. The mesolimbic dopamine pathway is widely accepted as a central pathway in producing the rewarding effects of addictive drugs. This pathway includes the dopaminergic neurons in the ventral tegmental area (VTA) of the midbrain and their targets in the limbic forebrain, especially the nucleus accumbens (NAc). All drugs of abuse, regardless of their mechanisms of actions, converge on the VTA-NAc pathway. Acute exposure to addictive drugs results in the elevation of dopamine levels, a reward signaling event, which promotes repeated drug intake. Addiction is then reinforced by the drugs producing a negative emotional symptom when the drug is removed, developing a period of sensitization, and associative learning toward drug-related environmental cues.

The nAChRs may be an important target for the treatment of multiple addictions, not just nicotine addiction. Activation of the central nAChRs has also been shown to mediate the reinforcing effects of other drugs of abuse, including alcohol and cocaine. Behavior sensitization induced by nicotine, amphetamine, or cocaine was shown to be associated with an increase in electrically evoked release of [ ³ H] dopamine in nucleus accumbens slices, suggesting a related pathway for these three different drugs.

The nAChR α4β2 subtype is implicated in the addictive effects of nicotine. In general, the nAChRs are integral membrane proteins of approximately 290 kDa and members of the Cys-loop superfamily of receptor-coupled ion channels. These receptors are ligand-gated ion channels that are permeable to cations, particularly Na+, K+, and Ca++. The neuronal nAChRs are pentameric membrane proteins composed of five subunits. To date, nine a subunits (a2 - a 10) and three β subunits (β2 - β4) have been found in vertebrates. Different combinations of these a and β subunits define the various nAChR subtypes. Although the theoretical number of subtypes is very large, a much smaller number of native nAChR subtypes represents the majority of neuronal nAChRs, including two heteromeric subtypes, α4β2 and α3β4, and one homomeric subtype, a7. In most areas of mammalian brain subtype α4β2 represents the predominant population of nAChRs.

The nAChRs are allosteric proteins that respond to the action of ACh at the binding site by changing the status of the channel gate to carry out the function of the nAChR. The receptors have at least three discrete conformational states: a resting state (closed), an open state (opened) and a desensitized state (closed). A particular nicotinic ligand, such as ACh, has a certain affinity for each of the three states. In the absence of bound ligand, nAChRs fluctuate among all three conformational states, but most of the time they are in the resting state. The binding of a ligand to a certain state of the receptor increases the probability of the receptor to be in that state. For example, an agonist binds with a reasonably high affinity to the open state of a receptor, and thus increases the probability of it being in the open (active) conformational state. For a population of receptors, the overall initial effect of an agonist is to shift a certain subpopulation of receptors from the resting state to the open state. In the open state, cations flow through the channel. However, agonists have an even higher affinity for the desensitized state of the receptor; therefore, the eventual effect of an agonist is to "drive" the receptor population from the resting and open states to the desensitized state, in which receptors remain closed. The kinetic rates for transitions between states vary greatly among different nACfiR subtypes, which contributes to the great functional diversity of neuronal nACfiRs.

In addition to ACh and nicotine, many other natural products and synthetic compounds act on nACfiRs. Nicotinic ligands belong to the following four major classes, defined classically, according to their actions.

(1) Agonists. Nicotinic agonists, such as ACh or nicotine, activate nACfiRs leading to the opening of their channels, which allows cations to cross the membrane; but prolonged presence of agonists desensitizes the receptors. The actions can be explained by the three-state model described above. Agonists have low binding affinity to the resting state of nACfiRs, higher affinity to the open state, and highest binding affinity to the desensitized state. After an agonist binds, the transition from the resting state to the open state is fast, but the transition from open state to desensitized state is slow. Therefore, agonists can activate receptors to open their channels initially, but if present for an extended period agonists desensitize receptors to close the channels.

(2) Competitive Antagonists. A competitive antagonist, such as dihydro-β- erythroidine (ϋΗβΕ), does not activate nACfiRs but prevents agonists from activating the receptor by occupying the ACh binding site. A possible mechanism is that competitive antagonists have higher binding affinity at the resting state of receptors than at the open state; therefore, they do not increase the probability of the open state but can prevent agonists from binding to the ACh site.

(3) Noncompetitive Antagonists. A noncompetitive antagonist, such as mecamylamine, does not activate nACfiRs but prevents an agonist from activating nACfiRs by binding to a site different from the ACh site. For example, the binding site for mecamylamine is in the central pore of the receptors, and so it blocks the pathway for ions, preventing the functional activity of an agonist. (4) Allosteric Modulators. An allosteric modulator, such as progesterone, does not bind to the acetylcholine binding site (orthosteric site) but modulates nAChR signaling through its binding to an allosteric site. There are positive and negative allosteric modulators of nAChRs and some of them show selectivity among nAChR subtypes.

Of the major nAChR subtypes, the α4β2 subtype stands out not only because of its prevalence in most of the brain, but also because its expression is increased by chronic administration of nicotine in rats and mice. Moreover, recent in vivo studies of imaged brain α4β2 nAChRs in human smokers indicate that these receptors are virtually saturated by the nicotine taken in during the smoking of a single cigarette. Therefore, it is likely that most of the α4β2 nAChRs in a smoker's brain are in a state of desensitization during the time the addicted individual is awake and smoking at a typical rate. In fact, as the nicotine concentration drops and the increased numbers of desensitized receptors begin to recover function, the resumption of endogenous acetylcholine signaling through even a relatively small percentage of these receptors, may provide the critical neurophysiological cues for the individual to smoke his/her next cigarette, silencing those cues by once again desensitizing the receptors.

There are significant differences in the pharmacological profile between β2- and β4- expressing cells. Much interest has been displayed in nicotinic agonists having selectivity for ?2-containing receptors (principally 4β2). These receptors have been implicated as targets for analgesia, cognition, and smoking cessation. A substantial number of ligands have been developed with ?2-selectivity.

Cytisine has been used extensively as a probe for 4β2 receptors in natural and radiolabeled forms for in vitro pharmacology, as well as clinically as a smoking cessation agent, where the drug varenicline is an analogue of cytisine.

However, at present there are few ^-selective nicotinic agonists.

Norchloroepibatidine and UB-165 display only a modest (3-10-fold) β4 preference over β2, while AR-R17779 is more selective (>20-fold), though the latter is considered a selective l agonist. Mice in which the ?4-subunit has been knocked out are resistant to nicotine- induced seizures and exhibited reduced nicotine withdrawal relative to their wild-type counterparts. Preliminary studies showed that phantasmidine was a ?4-selective agonist. If phantasmidine is, indeed, a ?4-selective agonist, it may provide a useful tool for probing subtype-selectivity in this receptor class, especially due to its highly rigid structure. Like many receptors, high-potency, subtype-selective nicotinic-receptor agonists are rare, whereas antagonists are more common. While antagonists need only bind to the receptor, the additional structural requirements for activation of receptors make the design of agonists more demanding. Derivatives of phantasmidine may serve as useful pharmacologic probes for /^-containing nicotinic receptors, much as the semirigid laburnum alkaloid cytisine and pyridohomotropane (a derivative of the dinoflagellate alkaloid anatoxin) have for /?2-containing receptors. Phantasmidine and its analogs also have the potential to be active at a7 and/or 5-HT3 receptors given its structural similarity to pyridofurans.

On the other hand, more recent experimental data suggest that phantasmidine shows better selectivity than epibatidine for /?2-containing receptors over /^-containing receptors. More studies must be completed in order to uncover the range of biological activity for phantasmidine and its analogs.

In certain embodiments, compounds made by methods of the present invention may be useful as agonists of nicotinic acetylcholine receptors. In certain embodiments, compounds made by methods of the present invention may be useful as agonists of β2- containing nicotinic acetylcholine receptors. In certain embodiments, compounds made by methods of the present invention may be useful as agonists of /^-containing nicotinic acetylcholine receptors.

Phantasmidine may fill a new niche for characterization of β2- or /^-containing nicotinic receptors.

Exemplary Methods of the Invention

In certain embodiments, the invention relates to a method of preparing a product, comprising the step depicted in Scheme I:

Scheme I

wherein

A represents an optionally-substituted aromatic

R ¹ represents H or lower alkyl;

Y represents C(R ¹ ) ₂ , O or NR ¹ ; and

n is 1, 2, 3, or 4.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally-substituted aromatic ring selected from the group consisting of benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, indole, quinoline, isoquinoline, benzothiophene, benzofuran, benzimidazole, benzothiazole, benzoxazole, and quinazoline.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally-substituted aromatic ring selected from the group consisting of benzene, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, indole, quinoline, isoquinoline, benzothiophene, and benzofuran.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally- substituted aromatic ring selected from the group consisting of pyridine, pyrazine, pyridazine, pyrimidine, indole, quinoline, isoquinoline, benzothiophene, and benzofuran.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein R ¹ is H, methyl, or ethyl.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein R ¹ is H.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein Y is O or NR ¹ .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein Y is NR ¹ .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein n is 1 or 2.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein n is 1.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the base is a carbonate, phosphate, oxide, hydroxide, alkoxide, aryloxide, or metal amide.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the base is sodium tert-butoxide, potassium hydroxide, cesium hydroxide, calcium hydroxide, or sodium hydroxide.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the period of time is about 3 minutes to about 60 minutes. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the period of time is about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the temperature is about 10 °C to about 40 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the temperature is about 15 °C, about 20 °C, about 25 °C, about 30 °C, or about 35 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of resolving the product, thereby producing a substantially enantiomerically pure product.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is substantially enantiomerically pure.

In certain embodiments, the invention relates to a method of preparing a product, comprising the step depicted in Scheme II:

Scheme II.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product is substantially enantiomerically pure.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of resolving the product, thereby producing a substantially enantiomerically pure product.

In certain embodiments, the invention relates to a method of preparing a product, comprising the steps depicted in Scheme III:

Scheme III

wherein

A represents an optionally-substituted aromatic ring;

R ¹ represents H or lower alkyl;

Y represents O or NR ¹ ;

n is 1, 2, 3, or 4;

"Step a" comprises a first reagent, a first acid, and a first solvent, at a first temperature, for a first period of time;

"Step b" comprises a base, and a second solvent, at a second temperature, for a second period of time;

"Step c" comprises a second reagent, and a third solvent, at a third temperature, for a third period of time;

"Step d" comprises a third reagent, at a fourth temperature; and

"Step e" comprises a fourth reagent, and a fourth solvent, at a fifth temperature, for a fourth time.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally- substituted aromatic ring selected from the group consisting of benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, indole, quinoline, isoquinoline, benzothiophene, benzofuran, benzimidazole, benzothiazole, benzoxazole, and quinazoline. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally-substituted aromatic ring selected from the group consisting of benzene, pyridine, pyrazine, pyridazine, pyrimidine, naphthalene, indole, quinoline, isoquinoline, benzothiophene, and benzofuran.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein A is an optionally-substituted aromatic ring selected from the group consisting of pyridine, pyrazine, pyridazine, pyrimidine, indole, quinoline, isoquinoline, benzothiophene, and benzofuran.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein R ¹ is H, methyl, or ethyl.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein R ¹ is H.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein Y is NR ¹ .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein n is 1 or 2.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein n is 1.

In certain embodiments, the invention relates to any one of the aforementioned

methods, wherein the first reagent is

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first acid is HI, HBr, HC10 ₄ , HC1, H ₂ S0 ₄ , HN0 ₃ , or HC10 ₃ .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first solvent is hexane, cyclohexane, toluene, 1,4-dioxane, chloroform, tetrahydrofuran, or diethyl ether.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first solvent is diethyl ether.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first temperature is about 60 °C to about 100 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first temperature is about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, or about 95 °C. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first period of time is about 20 min to about 5 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the first period of time is about 45 min, about 1 h, about 1.5 h, about 2 h, about 2.5 h, or about 3 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of purifying the product of "Step a."

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the base is a carbonate, phosphate, oxide, hydroxide, alkoxide, aryloxide, or metal amide.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the base is sodium tert-butoxide, potassium hydroxide, cesium hydroxide, calcium hydroxide, or sodium hydroxide.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second solvent is water.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second temperature is about 10 °C to about 40 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second temperature is about 15 °C, about 20 °C, about 25 °C, about 30 °C, or about 35 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second period of time is about 3 minutes to about 60 minutes.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second period of time is about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of purifying the product of "Step b."

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of resolving the product of "Step b."

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the second reagent is BH ₃ .

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third solvent is tetrahydrofuran or diethyl ether. In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third temperature is about 0 °C to about 40 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third period of time is about 10 h to about 50 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third period of time is about 15 h, about 20 h, about 25 h, about 30 h, about 35 h, or about 40 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the third reagent is methanol.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth temperature is about 0 °C to about 40 °C.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth reagent is piperazine.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth solvent is methanol, ethanol, water, or ethyl acetate.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth solvent is methanol.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fifth temperature is the refiux temperature of the fourth solvent.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth period of time is about 1 h to about 5 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the fourth period of time is about 2 h, about 2.5 h, about 3 h, about 3.5 h, or about 4 h.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of purifying the product of "Step e."

In certain embodiments, the invention relates to any one of the aforementioned methods, wherein the product of "Step e" is substantially enantiomerically pure.

In certain embodiments, the invention relates to any one of the aforementioned methods, further comprising the step of resolving the product of "Step e", thereby producing a substantially enantiomerically pure product. In certain embodiments, the invention relates to a method of preparing a product, comprising the steps depicted in Scheme IV:

a) BH ₃ «THF, THF,

0 to 25 °C, 30 h;

2 M aq. KOH, b) MeOH, 0-25 °C; f-BuOH, 25 °C c) piperazine, MeOH

15 min, degassed reflux, 3 h

Scheme IV.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

The term "nucleophile" is recognized in the art, and as used herein means a chemical moiety having a reactive pair of electrons. Examples of nucleophiles include uncharged compounds such as water, amines, mercaptans and alcohols, and charged moieties such as alkoxides, thiolates, carbanions, and a variety of organic and inorganic anions. Illustrative anionic nucleophiles include simple anions such as hydroxide, azide, cyanide, thiocyanate, acetate, formate or chloroformate, and bisulfite. Organometallic reagents such as organocuprates, organozincs, organolithiums, Grignard reagents, enolates, acetylides, and the like may, under appropriate reaction conditions, be suitable nucleophiles. Hydride may also be a suitable nucleophile when reduction of the substrate is desired.

The term "electrophile" is art-recognized and refers to chemical moieties which can accept a pair of electrons from a nucleophile as defined above. Electrophiles useful in the method of the present invention include cyclic compounds such as epoxides, aziridines, episulfides, cyclic sulfates, carbonates, lactones, lactams and the like. Non-cyclic electrophiles include sulfates, sulfonates (e.g., tosylates), chlorides, bromides, iodides, and the like

The terms "electrophilic atom", "electrophilic center" and "reactive center" as used herein refer to the atom of the substrate which is attacked by, and forms a new bond to, the nucleophile. In most (but not all) cases, this will also be the atom from which the leaving group departs.

The term "electron-withdrawing group" is recognized in the art and as used herein means a functionality which draws electrons to itself more than a hydrogen atom would at the same position. Exemplary electron-withdrawing groups include nitro, ketone, aldehyde, sulfonyl, trifluoromethyl, -CN, chloride, and the like. The term "electron-donating group", as used herein, means a functionality which draws electrons to itself less than a hydrogen atom would at the same position. Exemplary electron-donating groups include amino, methoxy, and the like.

The term "meso compound" is recognized in the art and means a chemical compound which has at least two chiral centers but is achiral due to an internal plane or point of symmetry.

The term "chiral" refers to molecules which have the property of non- superimposability on their mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner. A "prochiral molecule" is an achiral molecule which has the potential to be converted to a chiral molecule in a particular process.

The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of their atoms or groups in space. In particular, the term "enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another. The term "diastereomers", on the other hand, refers to the relationship between a pair of stereoisomers that comprise two or more asymmetric centers and are not mirror images of one another.

Furthermore, a "stereoselective process" is one which produces a particular stereoisomer of a reaction product in preference to other possible stereoisomers of that product. An "enantioselective process" is one which favors production of one of the two possible enantiomers of a reaction product. The subject method is said to produce a "stereoselectively-enriched" product (e.g., enantioselectively-enriched or diastereoselectively-enriched) when the yield of a particular stereoisomer of the product is greater by a statistically significant amount relative to the yield of that stereoisomer resulting from the same reaction run in the absence of a chiral catalyst. For example, an enantioselective reaction catalyzed by one of the subject chiral catalysts will yield an e.e. for a particular enantiomer that is larger than the e.e. of the reaction lacking the chiral catalyst.

The term "regioisomers" refers to compounds which have the same molecular formula but differ in the connectivity of the atoms. Accordingly, a "regioselective process" is one which favors the production of a particular regioisomer over others, e.g., the reaction produces a statistically significant preponderence of a certain regioisomer.

The term "reaction product" means a compound which results from the reaction of a nucleophile and a substrate. In general, the term "reaction product" will be used herein to refer to a stable, isolable compound, and not to unstable intermediates or transition states.

The term "substrate" is intended to mean a chemical compound which can react with a nucleophile, or with a ring-expansion reagent, according to the present invention, to yield at least one product having a stereogenic center.

The term "catalytic amount" is recognized in the art and means a substoichiometric amount relative to a reactant.

As discussed more fully below, the reactions contemplated in the present invention include reactions which are enantioselective, diastereoselective, and/or regioselective. An enantioselective reaction is a reaction which converts an achiral reactant to a chiral product enriched in one enantiomer. Enantioselectivity is generally quantified as "enantiomeric excess" (ee) defined as follows:

% Enantiomeric Excess A (ee) = (% Enantiomer A) - (% Enantiomer B) where A and B are the enantiomers formed. Additional terms that are used in conjunction with enatioselectivity include "optical purity" or "optical activity". An enantioselective reaction yields a product with an e.e. greater than zero. Preferred enantioselective reactions yield a product with an e.e. greater than 20%, more preferably greater than 50%, even more preferably greater than 70%, and most preferably greater than 80%.

A diastereoselective reaction converts a chiral reactant (which may be racemic or enantiomerically pure) to a product enriched in one diastereomer. If the chiral reactant is racemic, in the presence of a chiral non-racemic reagent or catalyst, one reactant enantiomer may react more slowly than the other. This class of reaction is termed a kinetic resolution, wherein the reactant enantiomers are resolved by differential reaction rate to yield both enantiomerically-enriched product and enantimerically-enriched unreacted substrate. Kinetic resolution is usually achieved by the use of sufficient reagent to react with only one reactant enantiomer (i.e., one-half mole of reagent per mole of racemic substrate). Examples of catalytic reactions which have been used for kinetic resolution of racemic reactants include the Sharpless epoxidation and the Noyori hydrogenation.

A regioselective reaction is a reaction which occurs preferentially at one reactive center rather than another non-identical reactive center. For example, a regioselective reaction of an unsymmetrically substituted epoxide substrate would involve preferential reaction at one of the two epoxide ring carbons.

The term "non-racemic" with respect to the chiral catalyst, means a preparation of catalyst having greater than 50% of a given enantiomer, more preferably at least 75%. "Substantially non-racemic" refers to preparations of the catalyst which have greater than 90% ee for a given enantiomer of the catalyst, more preferably greater than 95% ee.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C30 for straight chain, C3-C30 for branched chain), and more preferably 20 of fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths.

The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one double or triple carbon-carbon bond, respectively.

The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiet that can be represented by the general formula: wherein R9, Ri g and R' lO each independently represent a group permitted by the rules of valence.

The term "acylamino" is art-recognized and refers to a moiety that can be represented by the general formula:

wherein R ₉ is as defined above, and R'i j represents a hydrogen, an alkyl, an alkenyl or -(CH2) _m -R8, where m and Rg are as defined above.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

wherein R9, Rj Q are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2) _m -Rg, wherein m and Rg are defined above. Representative alkylthio groups include methylthio, ethyl thio, and the like.

The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula:

O O II— x _{Rl 1} , or _ _x _U_ _Rlii

wherein X is a bond or represents an oxygen or a sulfur, and R\ \ represents a hydrogen, an alkyl, an alkenyl, -(CH2) _m -Rg or a pharmaceutically acceptable salt, R'j \ represents a hydrogen, an alkyl, an alkenyl or -(CH2) _m -Rg, where m and Rg are as defined above. Where X is an oxygen and R _j \ or R' _j \ is not hydrogen, the formula represents an "ester". Where X is an oxygen, and Rj \ is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Ri j is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'j \ is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and Rj \ or R'j \ is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and R^ \ is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and Ri 1 ' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and Rj \ is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and \ \ is hydrogen, the above formula represents an "aldehyde" group.

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -0-(CH2) _m -Rg, where m and Rg are described above.

The term "sulfonate" is art recognized and includes a moiety that can be represented by the general formula:

O II

S— OR ₄₁

II

o

in which R4\ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term "sulfonylamino" is art recognized and includes a moiety that can be represented by the general formula:

O II

N— S-R

I ^I o II

R

The term "sulfamoyl" is art-recognized and includes a moiety that can be represented by the general formula:

The term "sulfonyl", as used herein, refers to a moiety that can be represented by the general formula: O

_ll_

S R

II

o

in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

The term "sulfoxido" as used herein, refers to a moiety that can be represented by the general formula:

0

I I

— s-R ₄₄

in which R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

The term "sulfate", as used herein, means a sulfonyl group, as defined above, attached to two hydroxy or alkoxy groups. Thus, in a preferred embodiment, a sulfate has the structure:

in which R40 and R41 are independently absent, a hydrogen, an alkyl, or an aryl. Furthermore, R40 and R4 , taken together with the sulfonyl group and the oxygen atoms to which they are attached, may form a ring structure having from 5 to 10 members.

Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, alkenylamines, alkynylamines, alkenylamides, alkynylamides, alkenylimines, alkynylimines, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls, alkenoxyls, alkynoxyls, metalloalkenyls and metalloalkynyls.

The term "aryl" or "aromatic" as used herein includes 4-, 5-, 6- and 7-membered single-ring aromatic groups or multiple-ring aromatic groups, which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycle". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogens, alkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, or - (CH ₂ ) _m -R7, -CF ₃ , -CN, or the like.

The terms "heterocycle" or "heterocyclic group" refer to 4 to 10-membered ring structures, more preferably 5 to 7 membered rings, which ring structures include one to four heteroatoms. Heterocyclic groups include pyrrolidine, oxolane, thiolane, imidazole, oxazole, piperidine, piperazine, morpholine. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogens, alkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, or -(CH2) _m -Ry, -CF3, -CN, or the like.

The terms "polycycle" or "polycyclic group" refer to two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocycles) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogens, alkyls, alkenyls, alkynyls, hydroxyl, amino, nitro, thiol, amines, imines, amides, phosphonates, phosphines, carbonyls, carboxyls, silyls, ethers, thioethers, sulfonyls, selenoethers, ketones, aldehydes, esters, or -(CH2) _m -R7, -CF3, -CN, or the like.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur, phosphorus and selenium.

For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, /?-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, /?-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, /?-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.

The terms ortho, meta and para apply to 1,2-, 1,3- and 1 ,4-disubstituted benzenes, respectively. For example, the names 1 ,2-dimethylbenzene and ortAo-dimethylbenzene are synonymous.

The phrase "protecting group" as used herein means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations. Examples of such protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively. The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 ^nd ed.; Wiley: New York, 1991).

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described hereinabove. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

EXEMPLIFICATION

The following examples are provided to illustrate the invention. It will be understood, however, that the specific details given in each example have been selected for purpose of illustration and are not to be construed as limiting the scope of the invention. Generally, the experiments were conducted under similar conditions unless noted.

Example 1 - General Procedures

Reactions were conducted in flame- or oven-dried glassware under a nitrogen atmosphere and were stirred magnetically. The phrase "concentrated" refers to removal of solvents by means of a rotary evaporator attached to a diaphragm pump (15-60 Torr) followed by removal of residual solvents at < 1 Torr with a vacuum pump. Flash chromatography was performed on silica gel 60 (230-400 mesh). Analytical thin layer chromatography (TLC) was performed using silica gel 60 F-254 pre-coated glass plates (0.25 mm). TLC Plates were analyzed by short wave UV illumination, or by dipping in vanillin stain (27 g of vanillin in 380 mL of EtOH, 50 mL of water and 20 mL of concentrated sulfuric acid) and heating on a hot plate or by spray with permanganate spray (5 g of KMn0 ₄ in 495 mL of water). THF and ether were dried and purified by distillation from sodium/benzophenone. DIPEA, Et ₃ N, MeOH, and benzene were distilled from Ca¾. 1H and ¹³ C NMR spectra were obtained on a 400 MHz spectrometer in CDCI ₃ with tetramethylsilane as internal standard unless otherwise indicated. Chemical shifts are reported in δ (ppm downfield from tetramethylsilane). Coupling constants are reported in Hz with multiplicities denoted as s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), m (multiplet) and br (broad). COSY spectra were recorded for all compounds and used to assign 1H NMR spectra. IR spectra were acquired on an FT-IR spectrometer and are reported in wave numbers (cm ¹ ). High resolution mass spectra were obtained using the following ionization techniques: chemical ionization (CI), electron impact (EI), electrospray ionization analyzed by quadrupole time of flight (QTOF).

Example 2 - Synthesis of Phantasmidine (Figure 2)

2,6-Dichloropyridine-3-pyridinemethanol (9a). A solution of 2,6- dichloropyridine-3-carboxaldehyde (8a) (950 mg, 5.40 mmol) in MeOH (10 mL) was treated with NaBH ₄ (205 mg, 5.40 mmol) in one portion at 0 °C. The reaction was stirred at 0 °C for 30 min. 10% aqueous hydrochloric acid was added dropwise to the reaction until pH 1 was reached and MeOH was removed under vacuum. The residue was extracted with CH ₂ CI ₂ (50 mL x 3). The combined CH ₂ CI ₂ layers were washed with H ₂ 0 (50 mL) and brine (50 mL), dried (Na ₂ S0 ₄ ), and concentrated to give 952 mg (99%) of 9a as a white solid, which was used directly without further purification. A small portion of crude 9a was recrystallized to give an analytical sample: mp 73-74 °C (lit. 62-64 °C); 1H NMR 7.88 (d, 1, J = 8.0), 7.33 (d, 1, J = 8.0), 4.78 (br s, 2), 2.21 (br s, 1, w _m = 16, OH); ¹³ C NMR 148.8, 147.8, 139.1, 133.8, 123.2, 60.9; IR 3369. The data are identical to those previously reported.

2,6-Dichloro-3-pyridineacetonitrile (11a). A solution of 9a (848 mg, 4.76 mmol) in 5 mL of CH ₂ C1 ₂ was treated with 5 mL of SOCl ₂ and 3 drops of DMF at 0 °C. The resulting solution was stirred at 0 °C for 1 h and then warmed to 25 °C and stirred for another 5 h. The reaction was concentrated. The residue was dissolved in 50 mL of CH ₂ CI ₂ and the solution was washed with saturated NaHC0 ₃ and brine, dried (Na ₂ S0 ₄ ), and concentrated to give 1.02 g of crude 2,6-dichloro-3-chloromethylpyridine (10a), which was used without further purification. A small portion of crude 10a was recrystallized to give an analytical sample: mp 83-84 °C; 1H NMR 7.82 (d, 1, J = 8.0), 7.33 (d, 1, J = 8.0), 4.66 (s, 2); ¹³ C NMR 150.1, 149.5, 141.2, 130.6, 123.5, 41.8; IR 1580, 1552, 1426, 1353. The data are identical to those previously reported.

A solution of crude 10a dissolved in 20 mL 9: 1 EtOH/H ₂ 0 was treated with NaCN (466 mg, 9.52 mmol). The resulting solution was heated at 80 °C for 4 h. The reaction was cooled and diluted with H ₂ 0 (50 mL), and extracted with CH ₂ C1 ₂ (3 x 50 mL). The combined organic layers were washed with H ₂ 0 and brine and dried (Na ₂ S0 ₄ ). Concentration gave 952 mg of crude 11a. Flash chromatography (5: 1 hexanes/EtOAc) gave 572 mg (64%) of pure 11a: mp 90-91 °C (lit. 80-82 °C, lit. 89-91 °C ); 1H NMR 7.86 (d, 1, J = 8.0), 7.38 (d, 1, J = 8.0), 3.85 (s, 2); ¹³ C NMR 150.4, 149.3, 140.2, 124.0, 123.7, 115.4, 21.4; IR 2265. The 1H NMR data are identical to those previously reported.

2,6-Dichloro-3-pyridineacetamide (6a). A mixture of 11a (410 mg, 2.19 mmol) and alumina (447 mg, 4.38 mmol) in MsOH (5 mL) was heated at 120 °C for 15 min. The reaction was cooled and H ₂ 0 (30 mL) was added. The mixture was extracted with CH ₂ C1 ₂ (30 mL x 5). The combined CH ₂ C1 ₂ layers were washed with brine (30 mL), dried (MgS0 ₄ ), and concentrated to give 370 mg of 6a as a white solid. Crude 6a was washed with cold ether to give 315 mg (70%) of pure 6a that was used directly for the next step: mp 175-176 °C; 1H NMR (CD ₃ OD) 7.79 (d, 1, J = 8.0), 7.40 (d, 1, J = 8.0), 3.71 (s, 2); ¹³ C NMR (CD ₃ OD) 174.0, 151.7, 149.8, 144.8, 131.2, 124.6, 39.6; IR 3408, 3205, 1667.

2,6-Dichloro- V-(2-oxocyclobutyl)-3-pyridineacetamide (5a). Primary amide 6a (250 mg, 1.22 mmol) and l,2-bis(trimethylsilyloxy)cyclobutene (7, 350 mg, 1.52 mmol) in 5 mL of saturated HC1 in anhydrous ether was sealed in a resealable tube. The reaction was heated at 80 °C for 2 h and cooled to 25 °C. The solvent was removed and the residue was dissolved in 30 mL of CH ₂ C1 ₂ . The solution was washed with saturated NaHC0 ₃ to remove the side product 2,6-dichloro-3-pyridineacetic acid, brine, and dried (Na ₂ S0 ₄ ). Concentration gave 309 mg of light-yellow gummy material. Flash chromatography (25: 1 CH ₂ Cl ₂ /MeOH) gave 259 mg (78%) of pure 5a as a white solid: R _f = 0.2 (20:1 CH ₂ Cl ₂ /MeOH); 1H NMR 7.70 (d, 1, J = 8.0), 7.29 (d, 1, J = 8.0), 6.45 (br d, 1, J = 8.4, NH), 4.91 (ddd, 1, J = 10.0, 8.4, 8.0), 3.65 (s, 2), 2.96 (apparent dd, 2, J = 8.4, 8.4, CH ₂ group adjacent to ketone), 2.44 (dddd, 1, J = 12.0,10.0, 8.4, 8.4), 2.09 (dddd, 1, J = 12.0, 8.4, 8.4, 8.4); C NMR 205.0, 168.0, 150.1, 149.2, 142.4, 128.2, 123.4, 64.2, 42.1, 39.0, 19.3; IR 3299, 1786, 1654; HRMS (ESI+) calcd for C ₁₁ H ₁₁ N ₂ O ₂ CI ₂ (MH ⁺ ) 273.0198, found 273.0198.

2-(i-Butoxycarbonyl)-4-(2,6-dichloro-3-pyridinyl)-2-azabicyc lo[3.2.0]hept-4-en- 3-one (14). A solution of secondary amide 5a (14 mg, 51 mmol) in 3 mL of anhydrous CH ₂ CI ₂ was treated with NEt ₃ (35 mL, 0.25 mmol) and DMAP (2 mg) at 0 °C. (Boc) ₂ 0 (46 mL, 0.2 mmol) was then added. The reaction was stirred at 0 °C for 15 min and 25 °C for another 45 min. H ₂ O was added and the resulting mixture was extracted with CH ₂ CI ₂ (10 mL x 3). The combined CH ₂ CI ₂ layer was washed with H ₂ 0 and brine and dried (Na ₂ S0 ₄ ). Concentration gave 37 mg of crude 14 contaminated with (Boc^O. Flash chromatography (5: 1 hexanes/EtOAc) gave 11 mg (61%) of pure 14: 1H NMR 8.29 (d, 1, J = 8.4), 7.34 (d, 1, J= 8.4), 5.03 (ddd, 1, J= 8.4, 6.4, 2), 3.33 (ddd, 1, J = 14, 9.2, 9.2), 2.95 (dddd, 1, J = 14, 7.2, 3, 2), 2.72 (dddd, 1, J= 9.6, 9.2, 8.4, 3), 1.57 (s, 9), 1.52 (dddd, 1, J = 9.6, 9.2, 7.2, 6.4); ¹³ C NMR 169.6, 161.8, 149.32, 149.27, 148.6, 140.7, 124.9, 123.0, 119.3, 83.4, 63.1, 34.9, 28.1, 25.3.

Example 3 - Synthesis of Phantasmidine (Figure 3)

6-Chloro-2-fluoropyridine-3-carboxaldehyde (8b). A solution of diisopropylamine (5.80 mL, 41.5 mmol) in anhydrous THF (100 mL) was treated with n- BuLi (2.1 M in hexanes, 18.0 mL, 37.8 mmol) at -78 °C. The cold bath was removed and the resulting solution was stirred at 0 °C for 30 min. The light yellow solution was re- cooled to -78 °C and 2-chloro-6-fluoropyridine (15, 3.80 g, 29.0 mmol) in anhydrous THF (20 mL) was added dropwise. The reaction mixture was stirred at -78 °C for 1 h. Dimethylformamide (4.49 mL, 58.0 mmol) was added dropwise and the mixture was stirred an additional 1 h and saturated HCl in ether was added at -78 °C slowly to the mixture until pH 1 was reached. The reaction was warmed to 25 °C and H ₂ 0 (50 mL) was added. The layers were separated. The aqueous layer was extracted with EtOAc (50 mL x 2). The combined organic layers were washed with H ₂ 0 (50 mL) and brine (50 mL), dried (MgS0 ₄ ), and concentrated to give crystalline crude 8b. The crude needles were recrystallized from hexanes to give 4.14 g (90%) of 8b: R _f = 0.27 (10: 1 hexanes/EtOAc); mp 49-50 °C; 1H NMR 10.27 (s, 1), 8.27 (dd, 1, J = 8.8, 8.0), 7.41 (d, J = 8.0); ¹³ C NMR 185.0, 162.6 (d, J = 254), 154.8 (d, J = 15), 141.3, 123.0 (d, J = 5.3), 116.9 (d, J

1697, 1595, 1562. The 1H NMR spectrum is identical to that previously reported.

6-Chloro-2-fluoro-3-pyridinemethanol (9b). A solution of 8b (2.00 g, 12.6 mmol) in MeOH (20 mL) was treated with NaBH ₄ (479 mg, 12.6 mmol) in portions over 5 min at - -10 °C. The reaction was stirred at -10 °C for 30 min. 10% aqueous hydrochloric acid was added dropwise to the reaction until pH 1 was reached and MeOH was removed under vacuum. The residue was extracted with CH ₂ CI ₂ (50 mL x 3). The combined CH ₂ CI ₂ layers were washed with H ₂ 0 (50 mL) and brine (50 mL), dried (Na ₂ S0 ₄ ), and concentrated to give 1.99 g of 9b as a white solid. Recrystallization from hexanes/EtOAc gave 1.84 g

(91%) of pure 9b: R _f = 0.24 (3:1 hexanes/EtOAc); mp 60-61 °C; 1H NMR 7.91 (dd, 1, J = 8.8, 8.0), 7.26 (d, 1, J = 8.0), 4.75 (d, 2, J = 4.8), 2.80 (t, 1, J = 4.8, OH); ¹³ C NMR 159.1 (d, J = 244), 147.1 (d, J = 14), 141.6 (d, J = 6), 121.8 (d, J = 5.3), 121.4 (d, J = 27), 57.7; IR 3230; HRMS (EI) calcd for C ₆ H ₅ NC1F0 (M ⁺ ) 161.0044, found 161.0052.

6-Chloro-2-fluoro-3-pyridineacetonitrile (lib). A solution of 9b (1.66 g, 10.3 mmol) in 10 mL of CH ₂ C1 ₂ was treated with 10 mL of SOCl ₂ and 5 drops of DMF at 0 °C. The resulting solution was stirred at 0 °C for 1 h and then warmed to 25 °C and stirred for another 5 h. The reaction was concentrated. The residue was dissolved in 50 mL of CH ₂ CI ₂ and the solution was washed with saturated NaHC0 ₃ and brine, dried (Na ₂ S0 ₄ ), and concentrated to give 1.93 g of crude 6-chloro-3-(chloromethyl)-2-fluoropyridine (10b), which was used without further purification. A small portion of crude 10b was recrystallized to give an analytical sample: 1H NMR 7.85 (dd, 1, J = 9.2, 8.0), 7.28 (d, 1, J = 8.0), 4.59 (s, 2); ¹³ C NMR 159.4 (d, J = 246), 148.7 (d, J = 14), 143.2 (d, J = 4.5), 122.2 (d, J = 5.1), 118.2 (d, J = 28), 37.9; IR 1603, 1573, 1437, 1398; HRMS (EI) calcd for C ₆ H ₄ NC1 ₂ F (M ⁺ ) 178.9705, found 178.9710. Powdered NaCN (1.10 g, 22.4 mmol) was added to 20 mL of DMSO. The mixture was stirred at 25 °C for 20 min. A solution of crude 10b in 5 mL of DMSO was added to the NaCN suspension dropwise over 10 min. The NaCN fully dissolved after addition. The resulting solution was stirred for 1 h at 25 °C and cooled to 0 °C. Water (100 mL) was added slowly to the reaction and the mixture was extracted with CH ₂ CI ₂ (50 mL x 3). The combined organic layers were washed with H ₂ 0 and brine, dried (Na ₂ S0 ₄ ), and concentrated to give 1.50 g of crude lib. Flash chromatography (6: 1 hexanes/EtOAc to 3: 1 hexanes/EtOAc) gave 1.23 g of pure lib. Recrystallization gave 1.10 g (63%) of analytically pure lib: R _f = 0.37 (3: 1 hexanes/EtOAc); mp 47-48 °C; 1H NMR 7.89 (dd, 1, J = 9.2, 8.0), 7.33 (d, 1, J = 8.0), 3.78 (s, 2); ¹³ C NMR 159.2 (d, J = 246), 148.9 (d, J = 14), 142.3 (d, J = 4.1), 122.4 (d, J = 5.3), 115.4, 111.2 (d, J = 29), 17.2; IR 2261; HRMS (EI) calcd for C ₇ H ₄ N ₂ C1F (M ⁺ ) 170.0047, found 170.0055.

6-Chloro-2-fluoro-3-pyridineacetamide (6b). A mixture of lib (300 mg, 1.76 mmol) and alumina (570 mg, 5.59 mmol) in MsOH (5 mL) was heated at 120 °C for 15 min. The reaction was cooled and H ₂ 0 (30 mL) was added. The mixture was extracted with CH ₂ C1 ₂ (30 mL x 5). The combined CH ₂ C1 ₂ layers were washed with brine (30 mL), dried (MgS0 ₄ ), and concentrated to give 285 mg of 6b as a white solid. Crude 6b was dissolved in the minimum amount of MeOH at 60 °C and the solution was cooled and hexanes were added to facilitate recrystallization. Needles crystallized slowly from the interface to give 201 mg (61%) of pure 6b: mp 179-180 °C; 1H NMR (CD ₃ OD) 7.85 (dd, 1, J = 9.2, 8.0), 7.34 (d, 1, J = 8.0), 3.60 (s, 2); ¹³ C NMR (CD ₃ OD) 174.2, 161.8 (d, J = 242), 148.0 (d, J = 14), 146.5 (d, J = 5.3), 123.1 (d, J = 5.3), 118.2 (d, J = 29), 35.3; IR 3310, 3205, 1671; HRMS (ESI+) calcd for C ₇ H ₇ N ₂ 0C1F (MH ⁺ ) 189.0231, found 189.0233.

6-Chloro-2-fluoro- V-(2-oxocyclobutyl)-3-pyridineacetamide (5b). Primary amide 6b (161 mg, 0.86 mmol) and l,2-bis(trimethylsiloxy)cyclobutene (7, 256 mg, 1.11 mmol) in 5 mL of saturated HC1 in anhydrous ether was sealed in a resealable tube. The reaction was heated at 80 °C for 2 h and cooled to 25 °C. The solvent was removed and the residue was dissolved in 30 mL of CH ₂ CI ₂ . The solution was washed with saturated NaHC0 ₃ to remove the side product, 6-chloro-2-fluoro-3-pyridineacetic acid, and then brine, and dried (Na ₂ S0 ₄ ). Concentration gave 219 mg of a light brown solid. The crude product was washed with 5 mL of cold ether to give 186 mg (85%) of pure 5b that was used directly for the next step: R _f = 0.23 (20: 1 CH ₂ Cl ₂ /MeOH); 1H NMR 7.77 (dd, 1, J= 9.2, 8.0), 7.24 (d, 1, J = 8.0), 6.28 (br d, 1, J = 8.4, NH), 4.93 (ddd, 1, J = 10.0, 8.4, 8.4), 3.54 (s, 2), 2.96 (apparent dd, 2, J = 8.4, 8.4, CH ₂ group adjacent to ketone), 2.45 (dddd, 1, J = 12.0, 10.0, 8.4, 8.4), 2.07 (dddd, 1, J = 12.0, 8.4, 8.4, 8.4); ¹³ C NMR 204.8, 168.0, 160.0 (d, J = 243), 147.6 (d, J = 14), 144.3 (d, J = 4.7), 122.1 (d, J = 5.1), 115.0 (d, J = 29), 64.2, 42.2, 34.7, 19.5; IR 3307, 1782, 1650; HRMS (ESI+) calcd for C ₁₁ H ₁₁ N ₂ O ₂ CIF (MH ⁺ ) 257.0493, found 257.0492.

46% - 49%

(2aS,4aS,9aR)-i-^-7-Chloro-l,2,2a,3,4,4a-hexahydrobenzofuro[ 2,3- c]cyclobuta[6]pyrrol-4-one (3). In a 100 mL flask, a suspension of amide 5b (86 mg, 0.33 mmol) in 5 mL of t-BuOH was degassed using 3 freeze-thaw cycles. In another flask, 10 mL of 2 M aqueous KOH was treated with ultrasound under vacuum for 30 min. The degassed suspension of amide 5b was then treated with 3 mL of the degassed 2 M KOH solution 25 °C. The resulting reaction was stirred at 25 °C for 15 min, and 5% aqueous citric acid was then added until the pH reached 6. The mixture was extracted with CH ₂ CI ₂ (3 x 25 mL). The combined organic layers were washed with brine, dried (Na ₂ S0 ₄ ), and concentrated to give 82 mg of crude lactam 7. Flash chromatography (30: 1 CH ₂ Cl ₂ /MeOH) gave 37 mg (46%) of pure lactam 3: R _f = 0.33 (20: 1 CH ₂ Cl ₂ /MeOH); mp 208-209 °C (decomposition); 1H NMR 7.73 (dd, 1, J= 7.6, 1.3), 6.92 (d, 1, J= 7.6), 6.29 (br s, 1, w _m = 20, NH), 4.38 (dd, 1, J= 7, 6), 4.05 (s, 1), 2.81-2.68 (m, 1), 2.54-2.38 (m, 2), 1.72-1.60 (m, 1); ¹³ C NMR 175.5, 166.1, 149.9, 136.0, 117.3, 115.4, 89.0, 59.7, 52.1, 29.3, 24.8; IR 3380, 3271, 1690; HRMS (ESI+) calcd for C ₁₁ H ₁₀ N ₂ O ₂ CI (MH ⁺ ) 237.0431, found 237.0430. The structure of 3 was confirmed by X-ray crystal structure determination. X-Ray data collection, solution, and refinement for Lactam 3. All operations were performed on a Bruker-Nonius Kappa Apex2 diffractometer, using graphite- monochromated MoKa radiation. All diffractometer manipulations, including data collection, integration, scaling, and absorption corrections were carried out using the Bruker Apex2 software. Preliminary cell constants were obtained from three sets of 12 frames. Data collection was carried out at 120K, using a frame time of 10 sec and a detector distance of 60 mm. The optimized strategy used for data collection consisted of six phi and three omega scan sets, with 0.5° steps in phi or omega; completeness was 100.0%. A total of 3900 frames were collected. Final cell constants were obtained from the xyz centroids of 8153 reflections after integration.

From the systematic absences, the observed metric constants and intensity statistics, space group P2 n was chosen initially; subsequent solution and refinement confirmed the correctness of this choice. The structure was solved using SuperFlip, and refined (full- matrix-least squares) using the Oxford University Crystals for Windows program. All ordered non-hydrogen atoms were refined using anisotropic displacement parameters. Hydrogen atoms were refined using isotropic displacement parameters. The final least- squares refinement converged to Ri = 0.0306 (/ > 2σ(7), 2893 data) and wR ₂ = 0.0817 (F ² , 2992 data, 181 parameters). The relative stereochemistry of a single enantiomer (the crystal is a racemic mixture) is R, S, R for atoms C(6), C(7) and C(8), respectively. See Figure 4.

67%

(2aS,4aR,9aR)-i-^-7-Chloro-l,2,2a,3,4,4a-hexahydrobenzofuro[ 2,3- c]cyclobuta [b] yrrole (Phantasmidine, 1). A solution of lactam 3 (34 mg, 0.14 mmol) in 1 mL of anhydrous THF was treated with 3 mL of 1 M BH ₃ ^» THF in THF dropwise over 5 min at 0 °C. After addition, the reaction was slowly warmed to 25 °C and stirred at that temperature for 30 h. The reaction was cooled to 0 °C and 2 mL of MeOH was added dropwise over 5 min. The reaction was warmed to 25 °C and then concentrated. The residue was treated with 4 mL of MeOH and concentrated to give 48 mg of a mixture of 1, 1·ΒΗ ₃ and 1·2ΒΗ ₃ . The crude mixture was dissolved in 5 mL of MeOH and treated with 387 mg of piperazine. The resulting solution was heated at gentle reflux for 3 h and cooled. The reaction was concentrated and the residue was dissolved in 30 mL of CH ₂ CI ₂ . The solution was washed with H ₂ 0 (3 x 20 mL) and brine, dried (Na ₂ S0 ₄ ), and concentrated to give 32 mg of crude 1. Crude 1 was dissolved 10 mL of 4% aqueous HC1 and the resulting solution was washed with CH ₂ CI ₂ (2 x 5 mL). The aqueous layer was treated with 2 M aqueous Na ₂ C0 ₃ slowly until the pH reached 10 and extracted with CH ₂ CI ₂ (3 x 15 mL). The combined organic layers were washed with brine, dried (Na ₂ S0 ₄ ), and concentrated to give 22 mg (67%) of pure 1: R _f = 0.24 (20: 1 CH ₂ Cl ₂ /MeOH); 1H NMR (CD ₃ OD) 7.64 (dd, 1, J = 7.6, 1), 6.95 (d, 1, J = 7.6), 4.01 (dd, 1, J = 9, 7), 3.84 (br d, 1, J = 7.2), 3.67 (dd, 1, J = 12.4, 7.2), 3.45 (br d, 1, J = 12.4), 2.50 (ddd, 1, J = 12.4, 12, 9.5), 2.23 (ddd, 1, J = 12.4, 9.5, 3), 2.09 (dddd, 1, J = 12, 12, 9, 3), 1.58 (dddd, 1, J = 12, 9.5, 9.5, 7); ¹³ C NMR (CD ₃ OD) 168.2, 149.3, 137.8, 123.2, 118.1, 99.3, 67.4, 53.6, 51.4, 27.0, 17.8; IR 3287, 1593, 1598, 1416; HRMS (ESI+) calcd for CnHi ₂ N ₂ OCl (MH ⁺ ) 223.0638, found 223.0639.

A solution of 1 in CD ₃ OD was treated with a solution of TFA in CD ₃ OD. The 1H NMR spectrum of a sample containing 1 equivalent of TFA (monocation) matched that reported for phantasmidine: 1H NMR (CD ₃ OD) 7.70 (d, 1, J = 8.0), 7.01 (d, 1, J= 8.0), 4.24 (apparent br t, 1, J ~ 7), 4.07 (br d, 1, J = 6.8), 3.92 (br dd, 1, J = 12.4, 6.8), 3.69 (d, 1, J = 12.4), 2.63 (apparent q, 1, J ~ 11), 2.37 (apparent t, 1, J ~ 11), 2.22 (apparent qd, 1, J -11, 3), 1.74 (apparent qd, 1, J = 11, 7); ¹³ C NMR (CD ₃ OD) 167.9, 150.1, 138.2, 121.7, 118.7, 96.7, 65.8, 52.6, 50.3, 27.5, 16.3.

The enantiomers were separated by HPLC on an Agilent 1100 Series instrument equipped with a quaternary pump using a Daicel Chiralcel OJ-H column (250 x 4.6 mm) eluting with 80:20 hexane/isopropanol at a flow rate of 1 mL/min and monitoring at 230 nm. Fifteen runs injecting 0.15 mg of racemic phantasmidine gave a total of about 1 mg of each enantiomer. The faster eluting isomer eluted at 29 min, has a rotation [α] ²⁵ ο = +70 (c 0.17, CH ₂ CI ₂ ), and is (+)-phantasmidine. The slower eluting isomer eluted at 44 min, has a rotation [α] ²⁵ ο = -77 (c 0.17, CH ₂ CI ₂ ), and is (-)-phantasmidine. The two isomers also show the appropriate CD spectra (Figure 5).

l-[(2aS,4aR,9aR)-i-e/-7-chloro-2,2a,4,4a-tetrahydrobenzofuro [2,3- c]cyclobuta[6]pyrrol-3(lH)-yl]-ethanone ( V-Acetylphantasmidine, 17). A solution of phantasmidine (5 mg, 22 mmol) in 2 mL of anhydrous CH ₂ CI ₂ was treated with NEt ₃ (45 mL, 0.22 mmol) followed by AcCl (16 mL, 0.22 mmol) at -15 °C. The reaction was stirred at that temperature for 30 min. The reaction was warmed to 25 °C and stirred for another 30 min. The reaction was cooled to 0 °C and MeOH (0.5 mL) was added. The reaction was warmed to 25 °C and diluted with CH ₂ CI ₂ . The solution was washed with H ₂ 0 and brine and dried (Na ₂ S0 ₄ ). Concentration gave 7 mg of crude 17. Flash chromatography (40: 1 CH ₂ Cl ₂ /MeOH) gave 4 mg (67%) of pure 17 (a 2: 1 mixture of rotamers): 1H NMR (CD ₃ OD, major) 7.70 (dd, 1, J = 8.0, 1.2) 6.99 (d, 1, J = 8.0), 4.71 (dd, 1, J = 6.8, 6.8), 4.11-4.07 (m, 2), 4.03 (dd, 1, J = 11.6, 7.0), 2.56-2.34 (m, 3), 2.01 (s, 3), 1.74-1.66 (m, 1); 1H NMR (CD ₃ OD, minor) 7.73 (d, 1, J = 8.0, 1.2), 7.01 (d, 1, J = 8.0), 4.80 (dd, 1, J = 6.8, 6.8), 4.32 (dd, 1, J= 11.6, 8.4), 4.15 (br dd, 1, J= 8.4, 3.2), 3.97 (dd, 1, J= 11.6, 3.2), 2.56- 2.34 (m, 3), 2.00 (s, 3), 1.65-1.58 (m, 1); ¹³ C NMR (CD ₃ OD, major) 171.5 (C), 167.7 (C), 150.0 (C), 138.21 (CH), 123.0 (C), 118.6 (CH), 97.5 (C), 65.2 (CH), 53.4 (CH ₂ ), 48.6 (CH), 27.7 (CH ₂ ), 22.12 (CH ₃ ), 20.4 (CH ₂ ); ¹³ C NMR (CD ₃ OD, minor) 171.5 (C), 166.9 (C), 150.1 (C), 138.25 (CH), 123.0 (C), 118.6 (CH), 95.9 (C), 63.6 (CH), 55.0 (CH ₂ ), 50.0 (CH), 28.2 (CH ₂ ), 22.15 (CH ₃ ), 20.2 (CH ₂ ).

Example 4 - Mosher Ester Analysis

Hoye prepared diastereomeric Mosher amides from (2i?,5i?)-2,5- dimethylpyrrolidine and showed that their NMR spectra are remarkably different. Conformational studies indicated that the dominant conformation about carbonyl-carbon bond has the trifluoromethyl group syn to the carbonyl group. This approach was used to assign the absolute stereochemistry of phantasmidine, although the analysis is complicated because the Mosher amides of phantasmidine will exist in two conformers about the carbonyl-nitrogen bond on the NMR time scale as does N-acetylphantasmidine.

MMX calculations by PCMODEL revealed that in the major conformations (more stable by 1 kcal/mol) of phantasmidine Mosher amides 18ma and 19ma (see Figure 6) the large Ph(OMe)CF ₃ C group is adjacent to the cyclobutane-substituted methine carbon rather than the methylene carbon as in 2-methylpyrrolidine Mosher amides. The methyl group of phantasmidine acetamide (17) also prefers to be adjacent to the cyclobutane-substituted methine carbon (see 17ma) by 1 kcal/mol. This suggests that the methylene group in a cyclobutane at C-2 of a pyrrolidine is smaller than a proton, whereas a C-2 methyl group of a pyrrolidine is obviously much larger than a proton. The -90° bond angles in the cyclobutane move the methylene carbon toward C-3 and away from the nitrogen so that it is effectively smaller than a proton.

Acylation of readily available synthetic (±)-phantasmidine with (i?)-Mosher acid chloride gave a mixture of diastereomers 18 and 19. The 1H NMR spectrum was complex with two rotamers for each diastereomer. We were able to correlate peaks in each of the four rotamers from the COSY spectrum. Acylation of 0.5 mg of (-)-phantasmidine and 0.5 mg of (+)-phantasmidine with R -Mosher acid chloride gave the corresponding (-)- phantasmidine S-Mosher amide 18 and (+)-phantasmidine S-Mosher amide 19, respectively. The crude products were washed with 5% aq. HC1 and used for spectroscopic analysis without further purification. The 1H NMR impurities peaks were excluded by comparison with the racemate spectra.

A 3 : 1 mixture of rotamers was observed for (-)-phantasmidine S-Mosher amide 18. The methine hydrogen adjacent to the phenyl group in the major rotamer 18ma (δ 4.30) is shifted upfield 0.74 ppm from the methine hydrogen in the minor rotamer 18mi (δ 5.04) as shown in Figure 6. The methylene hydrogen adjacent to the phenyl group in the minor rotamer 18mi (δ 3.04) is shifted upfield 0.91 ppm from the methylene hydrogen in the major rotamer 18ma (see Figure 7). These shifts allow us to assign the absolute stereochemistry and indicate that the methylene carbon is larger than the cyclobutane- substituted methine carbon, although we calculated an equilibrium constant of 7.4 (AG = - 1.2 kcal/mol) and the observed equilibrium constant was only 3.

On the other hand, a 4: 1 mixture of rotamers was observed for (+)-phantasmidine S- Mosher amide 19. The methylene hydrogens of cyclobutane adjacent to the phenyl group in the major rotamer 19ma (δ 0.68, δ 0.87) are shifted upfield 0.95 and 1.62 ppm from those in the minor rotamer 19mi. The methylene proton and two aryl protons that are adjacent to the phenyl group (see Figure 7) in the minor rotamer 19mi (δ 3.78, δ 6.41 , and δ 6.56) are also shifted upfield significantly (0.63, 1.07, and 0.35 ppm) compared those in the major rotamer. These shifts allow us to assign the absolute stereochemistry and indicate that the methylene carbon is larger than the cyclobutane-substituted methine carbon; we calculated an equilibrium constant of 4.5 (AG = -0.9) and the observed equilibrium constant was 4.

The exceptionally large Δδ values established the absolute configuration of (-)- phantasmidine as shown in Mosher amide 18 and (+)-phantasmidine as shown in Mosher amide 19. The agreement of experiment and theory which both show that the large Ph(OMe)CF ₃ C group is adjacent to the cyclobutane substituted methine carbon provide further support for this assignment.

Racemic Phantasmidine (S)-(-)-MPTA Amide. A mixture of (5)-(-)-MPTA-OH (8.5 mg, 36 μιηοΐ) in 1 mL of hexanes was treated with 2 μΐ _^ of DMF and then 10 (14.8 mg, 1 16 μιηοΐ) of oxalyl chloride at 25 °C. The reaction was kept at 25 °C for 30 min and filtered. The filtrate was concentrated to give crude (R)-(-)-MPTA-Cl as colorless oil, which was used directly without further purification.

A solution of racemic phantasmidine (4 mg, 18 μηιοΐ) and diisopropylethylamine (30 μΐ,, 170 μηιοΐ) in 0.2 mL of anhydrous CH ₂ C1 ₂ was treated with a solution of the crude (R)-(-)-MPTA-Cl in 0.5 mL of anhydrous CH ₂ C1 ₂ at 25 °C under N ₂ . The reaction was stirred at 25 °C for 1 h and quenched with 2 mL of 5% aqueous Na ₂ C0 ₃ . The resulting mixture was extracted with CH ₂ C1 ₂ (3 mL x 3) and the combined organic extracts were dried (MgS0 ₄ ) and concentrated. Flash chromatography of the residue on silica gel (CH ₂ C1 ₂ to 100:1 CH ₂ Cl ₂ /MeOH) gave 6.1 mg (77%) of a 1 : 1 mixture of (±)-phantasmidine S)- MPTA amide diastereomers. One diastereomer exists as a 3: 1 mixture of rotamers and the other as a 4: 1 mixture.

(S)-(-)-MPTA Amide of the More Biologically Active Phantasmidine Enantiomer (19). A solution of (+)-phantasmidine (-0.5 mg, 2.2 μιηοΐ) and dry pyridine (1 nL, 12.5 μπιοΐ) in anhydrous 100 μΐ, of CDC1 ₃ was treated with (R)-(-)-MPTA-Cl (6.4 μιηοΐ, prepared as described above from the (S)-(-)-acid) in 100 μΐ, of CDC1 ₃ at 25 °C under N ₂ . The reaction was kept at 25 °C for 1 h and diluted with 0.3 mL of dry CDC1 ₃ . The entire CDC1 ₃ solution was transferred to an NMR tube for spectroscopic analysis. The ¹ H NMR spectrum of this diastereomer showed a 4: 1 mixture of rotamers of the MPTA amide. The spectral data described below were obtained by analysis of the spectrum of this crude diastereomer and the spectrum of the purified mixture of both diastereomers described above: ¹ H NMR (major rotamer, Ph protons not reported): 7.48 (dd, 1 , J= 7.6, 1.2), 6.91 (d, 1, J = 7.6), 4.71 (dd, 1, J = 7.6, 7.2), 4.41 (dd, 1, J = 13.2, 1.7), 3.98 (dd, 1, J = 13.2, 8.0), 3.80 (br d, 1, J = 8.0), 3.34 (q, 3, J _H , _F =1.8, OMe), 2.23 (ddd, 1, J = 12.8, 11.2, 10.4), 2.02 (ddd, 1, J= 12.8, 9.2, 4), 0.91-0.83 (m, 1), 0.73-0.63 (m, 1); (minor rotamer, Ph protons not reported): 6.56 (d, 1, J = 7.6), 6.41 (dd, 1, J = 7.6, 1.2), 5.25 (dd, 1, J = 7.6, 7.2), 3.88 (dd, 1, J= 12.8, 7.6), 3.78 (br d, 1, J= 12.8), 3.77 (q, 3, J _H , _F = 1.8, OMe), 3.63 (br d, 1, J= 7.6), 2.66 (ddd, 1, J= 12.8, 11.2, 10.4), 2.54-2.45 (m, 1), 2.35-2.27 (m, 1), 1.67-1.59 (m, 1).

(S)-(-)-MPTA Amide of the Less Biologically Active Phantasmidine Enantiomer (18). The identical procedure using -0.5 mg of (-)-phantasmidine whose ¹ H NMR spectrum showed a 3: 1 mixture of rotamers of the MPTA amide. The spectral data described below were obtained by analysis of the spectrum of this crude diastereomer and the spectrum of the purified mixture of both diastereomers described above: ¹ H NMR (major rotamer, Ph protons not reported): 7.49 (dd, 1, J= 7.6, 1.2), 6.93 (d, 1, J= 7.6), 4.74 (dd, 1, J = 12.8, 1.2), 4.30 (dd, 1, J = 8.4, 6.8), 3.95 (dd, 1, J = 12.8, 7.2), 3.75 (br d, 1, J = 7.2), 3.59 (q, 3, J _H , _F = 1.8, OMe), 2.42-2.32 (m, 2), 2.21 (br dd, 1, J = 12, 10.4), 1.74-1.66 (m, 1); (minor rotamer, Ph protons not reported) 7.37 (presumed d, 1, assigned from COSY spectrum), 6.91 (d, 1, J = 7.6), 5.04 (dd, 1, J = 7.6, 7.6), 3.91 (dd, 1, J = 12.8, 2,1), 3.67 (br d, 1, J = 7.6), 3.28 (q, 3, J _H , _F = 1.8, OMe), 3.04 (dd, 1, J = 12.8, 7.6), 2.64 (br ddd, 1, J = 12, 10, 10), 2.54-2.46 (m, 1), 2.35-2.27 (m, 1), 1.44-1.36 (m, 1).

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.