Enantioselective synthesis of cryptophycin analogs, their use and methods of synthesis thereof

Abstract

The present invention provides illudin derivatives, intermediates, methods of preparation, pharmaceutical compositions, and uses thereof. Specific examples include novel synthetic routes for preparing illudin derivatives and therapeutically valuable illudin derivatives having positive optical rotation.

Description

TECHNICAL FIELD

[0001] The present invention relates to illudin derivatives or analogs, intermediates, methods of preparation, pharmaceutical compositions, and uses thereof. BACKGROUND

[0002] Illudins are a family of sesquiterpenes with antitumor antibiotic properties, typically produced by various mushrooms. In its isolated form, illudin exhibits selective toxicity against myeloid leukemia and other malignant cells. Omphalotus species such as O. olearius and O. illudens (Jack o' Lantern mushroom), and O. nidiformis (Australian Ghost fungus) all produce illudin in its natural form. Illudin is highly toxic and its natural form has little therapeutic value.

[0003] Methods of manufacturing illudin analogs typically require production of illudin S from liquid growth of O. illudens cell cultures. FIG. 1 (prior art) shows the current semi-synthetic route from illudin S to hydroxymethylacylfulvene (HMAF) and (+) hydroxyurea methylacylfulvene. Although cell lines of O. illudens have been developed that produce a higher ratio of illudin S to illudin M, production of this starting compound has been difficult in terms of expression yield, time required to begin harvesting illudin S (e.g., >4 weeks of culture), contamination with illudin M, and other complications including difficulties with reproducibility. The fermentation process can require production, handling, and purification of large quantities of illudin S, which is highly toxic and biohazardous in a production facility.

[0004] Accordingly, there is a need for improved illudin analogs and methods of synthesis thereof. SUMMARY

[0005] The present invention provides illudin derivatives, intermediates, methods of preparation, pharmaceutical compositions, and uses thereof. Specific examples include new synthetic routes for preparing illudin derivatives and illudin derivatives with positive optical rotation, which have therapeutic value. Illudin derivatives react with DNA, thereby preventing the transcription process, and thus can be effective in treating cancer and inflammatory diseases.

[0006] Another embodiment provides a synthetic route to acylfulvene, ilofenpin (6-hydroxymethyl acylfulvene), UMAF, and other analogs of Compound I or illudin. A method of synthesizing a compound as shown in Formula (I), wherein R1, R2, and R3 are independently (C1-C4)alkyl, methyl, or hydroxyl. In addition, specific embodiments include enantiomerically pure and racemic forms of derivatives of acylfulvene, ilofenpin, and UMAF. The racemic and (+) enantiomer of UMAF are novel compounds disclosed in the present invention. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 shows a semi-synthetic route from illudin S to hydroxymethyl acylfulvene (HMAF) and (+) hydroxyureamethyl acylfulvene in the prior art.

[0008] Figure 2 An embodiment of the present invention is shown that prepares a pivitol intermediate or tertiary alcohol.

[0009] Figure 3 Another embodiment is shown that includes methods of synthesizing compounds to (±)-acylfulvene by two exemplary strategies.

[0010] Figure 4 The (+) enantiomer is shown to be more toxic to the DU145 cell line.

[0011] Figure 5 The PC3 cell line is shown to have similar sensitivity to both enantiomeric forms of hydroxyureamethyl acylfulvene,

[0012] Figure 6 The (+) enantiomer is shown to be more toxic to the OVCAR3 cell line,

[0013] Figure 7 The (+) enantiomer is shown to be more toxic to the SK-OV3 cell line.

[0014] Figure 8 The HCC827 cell line is shown to have similar sensitivity to both enantiomeric forms of hydroxyureamethyl acylfulvene, and

[0015] Figure 9 The (+) enantiomer is shown to be more toxic to the H1975 cell line.

[0016] DEFINITIONS

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. As used herein, the following definitions are provided for ease in understanding the present invention.

[0018] As used herein, the terms "patient," "subject," "individual," and "host" refer to either a human or non-human animal that has or is suspected of having a disease or disorder associated with abnormal biological or cellular growth activity.

[0019] The terms "treat" and "treatment" of such a disease or disorder refer to the improvement of at least one symptom of the disease or disorder. These terms, when used in relation to a disease such as cancer, refer to one or more of the following: arresting the growth of a cancer, reducing the size or volume of a cancer, prolonging the expected survival time of a patient, inhibiting the growth of a tumor, reducing the weight of a tumor, reducing the size or number of metastatic lesions, inhibiting the development of new metastatic lesions, prolonging survival, prolonging progression-free survival, prolonging time to progression, and / or improving quality of life.

[0020] The term "prevent" when used in relation to a disorder or disease such as cancer, refers to reducing the frequency or delaying the onset of symptoms of the disorder or disease. Thus, the prevention of cancer includes, for example, reducing the number of detectable cancerous growths in a population of patients receiving prophylactic treatment relative to a control population that is not treated, and / or delaying the appearance of detectable cancerous growths in a population of treated patients relative to a control population that is not treated, e.g., in a statistically and / or clinically significant amount.

[0021] The term "pharmaceutically acceptable" means useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable for use in humans and animals.

[0022] The term "stereoisomers" refers to any enantiomers, diastereomers, or geometric isomers of the compounds of Formula (I) as long as they are chiral or carry one or more double bonds. When the compounds of Formula (I) and related formulas are chiral, they can exist in racemic or optically active forms. Since the pharmaceutical activity of the racemates or stereoisomers of the compounds of the present application can be different, it can be desirable to use the enantiomers. In these cases, the final product or even the intermediates can be separated into the enantiomeric compounds by known or employed chemical or physical measures.

[0023] The term "therapeutic effect" refers to a beneficial local or systemic effect at the site of administration or elsewhere in the body of the animal, particularly a mammal, more particularly a human, resulting from the administration of a compound or composition of the present application. The phrase "therapeutically effective amount" refers to the amount of a compound or composition of the present application that is effective, at a reasonable benefit / risk ratio, in the treatment of a disease or disorder resulting from abnormal biological activity.

[0024] A therapeutically effective amount of such a substance will vary depending on the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the mode of administration, and the like, and can be readily determined by a person skilled in the art. DETAILED DESCRIPTION

[0025] One embodiment of the present application provides for derivatives of illudin, intermediates, methods of preparation, pharmaceutical compositions, and uses thereof. Specific examples include novel synthetic routes for preparing illudin derivatives and illudin derivatives having positive optical rotation, which have therapeutic value. The illudin derivatives react with DNA, thereby preventing the transcription process, and thus can be effective in treating cancer and inflammatory diseases.

[0026] One exemplary embodiment of the present application provides for a compound having the formula I:

[0027]

[0028] R1, R2, and R3 are independently (C1-C4)alkyl, methyl, or hydroxyl.

[0029] Another exemplary embodiment includes hydroxymethylacylfulvene (HMAF, Irofulven) having the following formula II

[0030]

[0031] Another illustrative embodiment includes (-)-hydroxyureamethylacylfulvene (UMAF) having the following formula III

[0032]

[0033] One exemplary embodiment features UMAF as an enantiomer that exhibits positive optical rotation in dichloromethane or methanol at room temperature. UMAF has positive optical rotation, or can be part of a racemic mixture of UMAF structures. The mixture can include the following structures:

[0034]

[0035] The present application provides for a method of synthesizing a compound as shown in formula (IV):

[0036]

[0037] Another embodiment provides for a synthetic route to acylfulvene, irofulven (6-hydroxymethylacylfulvene), UMAF, and other analogs of Compound I or illudin. A method of synthesizing a compound as shown in formula (I), wherein R1, R2, and R3 are independently (C1-C4)alkyl, methyl, or hydroxyl. In addition, specific embodiments include enantiomerically pure and racemic forms of derivatives of acylfulvene, irofulven, and UMAF. The racemic and positive (+) enantiomers of UMAF are novel compounds disclosed in the present application.

[0038] Referring now to Figure 2 One embodiment includes the following steps: (1) conversion of 2-furfural to an alcohol of the following formula using a Grignard reaction, wherein R1is a hydrogen atom, a methyl group, an alkyl group, an allyl group, or an a-methylallyl group.

[0039]

[0040] (2) Piancatelli rearrangement to a racemic cyclopentenone, and (3) protection of the hydroxyl group to provide 9. In the production of alcohols from carbonyl compounds via Grignard reactions, it is common practice to prepare the Grignard reagent and then react it with the carbonyl compound. The choice of appropriate protecting group can be readily determined by one skilled in the art - exemplary protecting groups include, but are not limited to, silyl [trimethylsilyl (TMS), t-butyl dimethylsilyl (TBS), acetyl (Acetate (Ac) and benzoyl (Bz)] or benzyl [benzyl (Bn, p-methoxybenzyl (PMB)]. Utilizing a carbonyl- lactone dipolar cycloaddition between cyclopentenone 9 and diazoketone 10 provides cycloadduct 11, which is converted to 12 via base-mediated elimination. Selective alkylation of the ketone on the enone provides a pivitol intermediate: tertiary alcohol 13. Removal of the protecting group from the molecule (e.g., via base-mediated elimination). Selective alkylation of the ketone on the enone provides a tertiary alcohol.

[0041] Another embodiment provides a method of synthesizing acylfulvene (3), irofulvene (4), and hydroxyl urea methyl acylfulvene (5) from a tertiary alcohol. The tertiary alcohol can be in racemic or enantiopure form, first alkylating 2-furfural (6) with a methyl Grignard reagent, followed by Piancatelli rearrangement to a racemic cyclopentenone (±)-8, which is then protected to provide 9 (Scheme 2).

[0042] Referring now to Figure 3 Another embodiment includes a method of synthesizing compound 13 to (±)-acylfulvene (3) by two exemplary strategies (Scheme 3). In one exemplary method, Lewis acid-mediated elimination provides dienone 14, which upon reduction and elimination of the ketone moiety, yields diol (±)-16. Oxidation of compound (±)-16 can then yield (±)-acylfulvene (3). In another exemplary method, reduction of compound 13 yields alcohol 15, which upon treatment under Lewis acidic conditions, yields diol (±)-16 and (±)-acylfulvene (3) in the same manner. In another example, (±)-iodo irofulvene (4) and (±)-UMAF (5) can be made by a similar sequence as described in Scheme 1. The disclosed conversion of 6 to acylfulvene (3), irofulvene (4), and UMAF (5) can be the shortest and most efficient synthesis of these compounds to date.

[0043] If enantiopure acylfulvene (3), irofulvene (4) or UMAF (5) is desired, the mixture or racemic intermediate (±)-16, which can produce both (+)-(16) and (-)-16, can be purified by preparative chiral chromatography or other methods known to those skilled in the art, can be used to synthesize acylfulvene (3), irofulvene (4) or UMAF (5) (Scheme 4). This represents a process for the enantioselective synthesis of these compounds.

[0044]

[0045] Scheme 4. New route to (+)-acylfulvene (3) and (-)-acylfulvene (3) using chiral resolution of (±)-16.

[0046] As shown below, either enantiomer of acylfulvene (3), irofulvene (4) or UMAF (5) can also be produced by known enzymatic resolution of racemic (±)-9 to either (+)-9 or (-)-9, which can be converted to any of acylfulvene (3), irofulvene (4) or UMAF (5) by the steps described in Schemes 2 and 3 (Scheme 5). 1

[0047]

[0048] Scheme 5. New route to (+)-acylfulvene (3) and (-)-acylfulvene (3) using enzymatic resolution of (±)-8.

[0049] Specific embodiments also have pharmaceutical compositions containing a pharmaceutically acceptable carrier and any of the compounds of Formula (I), (II), (III) and other compounds described above.

[0050] Specific embodiments also have compositions and compounds, such as UMAF, that reduce toxicity and side effects, including those related to the eye. Other embodiments allow for therapeutic methods that take advantage of these properties.

[0051] Pharmaceutically acceptable salts of these compounds are also contemplated for use in the uses described herein. "Pharmaceutically acceptable salt" refers to any salt of a compound of the application that retains its biological properties and is not toxic or otherwise unacceptably deleterious to pharmaceutical use. Pharmaceutically acceptable salts can be derived from many organic and inorganic counterions well known in the art, including. Such salts include: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethanedisulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-methylbenzenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-l-carboxylic, gluheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinine, muconic acid, and the like; or (2) salts formed when acidic protons present in the parent compound are replaced by a metal ion, e.g., sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and mercury ion, or by a base equivalent, e.g., ammonium, phosphonium, and alkali or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide, or with the quaternary ammonium salts, such as ammonium, choline, and the like. Pharmaceutically acceptable salts further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, when the compound contains a basic functionality, and salts of the acid form with organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, besylate, acetate, maleate, oxalate and the like.

[0052] The pharmaceutical compositions of the present application comprise one or more compounds of the present application and one or more physiologically or pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and the subject matter and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) other nontoxic compatible substances used in pharmaceutical formulations.

[0053] The compositions of the present application can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term "parenterally" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions of the present application are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions of this application can be aqueous or oleaginous suspensions. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium.

[0054] To this end, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents, which are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans, and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purposes of formulation.

[0055] The pharmaceutically acceptable compositions of this application can be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents can also be added.

[0056] Alternatively, the pharmaceutically acceptable compositions of this application can be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

[0057] The pharmaceutically acceptable compositions of this application can also be administered topically, especially when the target of treatment includes areas or organs readily accessible by such administration. Suitable formulations for this purpose can include those suitable for transdermal, buccal, or sublingual administration. Transdermal patches have the added advantage of providing controlled delivery of a compound of the present application at a predetermined rate and time. For topical administrations, the pharmaceutically acceptable compositions can be formulated in the form of ointments, creams, salves, and the like for application directly to the skin of the patient. Such dosage forms contain the active compound in an amount varying from 0.1 to 5% by weight.

[0058] The compounds of the application can be combined with a carrier material to produce a single dosage form composition in an amount that will vary depending upon the host treated, the particular mode of administration. Formulation of the composition should set the dose of the inhibitor between 0.01-100 mg / kg body weight / day, which can be taken by a patient receiving these compositions.

[0059] Dosage, toxicity and therapy according to the application of the compounds, including pharmaceutically acceptable salts and deuterated variants, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. LD 50 is the dose lethal to 50% of the population. ED 50This is the dose that has a therapeutic effect on 50% of the population. The dose-to-total-to-toxicity ratio (LD50) is... 50 / ED 50 The therapeutic index is the highest possible value. Compounds exhibiting a high therapeutic index are preferred. Although compounds exhibiting toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the affected tissue site to minimize potential damage to uninfected cells, thereby reducing side effects. Additionally, a second therapeutic agent can be administered to mitigate the toxic side effects of the first therapeutic agent.

[0060] Data obtained from cell culture experiments and animal studies can be used to determine dosage ranges for human use. Dosages of these compounds can range from ED to [specific dosage range]. 50 The toxicity is minimal or non-toxic within the range of circulating concentrations. Dosage can vary within this range depending on the dosage form and route of administration. For any compound, the therapeutically effective dose can be initially estimated from cell culture assays. Doses can be formulated in animal models to achieve the range of circulating plasma concentrations, which includes the IC50 determined in cell culture. 50 (That is, the concentration of the test compound that achieves half of the maximum symptom suppression). This type of information can be used to more accurately determine the dosage that is useful to humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0061] It should also be understood that the specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular compound used, age, weight, general health condition, sex, diet, timing of administration, excretion rate, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of the compound of the present invention in the composition will also depend on the specific compound in the composition. Detailed Implementation

[0063] Example

[0064] In the following embodiments, the following abbreviations are used.

[0065] AcOH (acetic acid)

[0066] DIBAL-H diisobutylaluminum hydroxide

[0067] DIPEA (diisopropylethylamine)

[0068] DMSO (dimethyl sulfoxide)

[0069] IBX 2-iodobenzoic acid

[0070] L rise

[0071] M Moore

[0072] MeOAc methyl acetate

[0073] MeOH (methanol)

[0074] Mmol

[0075] MTBE (methyl tert-butyl ether)

[0076] NMR (Nuclear Magnetic Resonance)

[0077] TFA (trifluoroacetic acid)

[0078] THF Tetrahydrofuran

[0079] TMS Tetramethylsilane

[0080] TMSOTf Trimethylsilyl Trifluoromethanesulfonate

[0081] Unless otherwise specified, solvents and reagents can be used without purification. CH2Cl2 and MeCN are stored in [location missing]. Molecular sieves. Volatile solvents were removed under reduced pressure using a Buchi rotary evaporator. Thin-layer chromatography (TLC) was performed on pre-coated silica gel plates (0.25 mm thick 60F254) with a glass backing and visualized using one or more of the following methods: UV light (254 nm), silica impregnated with I2, KMnO4, or cerium ammonium molybdate (CAM) staining. Rapid column chromatography was performed using Biotage Isolera One columns, using pre-packed Silicycle 25g high-performance (14–40 μM) columns. Unless otherwise specified, 1 1H nuclear magnetic resonance (NMR) spectra were obtained at 400 MHz using a solution of CDCl3 containing 0.05% v / v tetramethylsilane (TMS). Deuterated solvents were obtained at 100 MHz as shown. 13 C-NMR. Chemical shifts are expressed in parts per million (ppm, δ) with TMS as a reference, and coupling constants are expressed in Hertz (Hz). Spectral splitting modes are designated as s, singlet; d, doublet; t, triplet; q, quartet; quintet; sextet; sept, septet; m, multiplet; comp, multiplet of overlapping magnetically inequivalent protons; br, broad peak; and app, sharp peak.

[0082]

[0083] 1 -acetylcyclopropanecarboxylic acid. Tert-butyl 3-oxobutanoate (339 g, 350 mL, 2.146 mol) was added to a flask of a vigorously stirred mixture of K2CO3(1186 g, 8.58 mol) and DMSO (3.5 L). The mixture was stirred for 10 minutes, then neat 1,2-dibromoethane (806 g, 370 mL, 4.29 mol) was added and the mixture was stirred overnight. The reaction mixture was then diluted with H2O (2 L) and the mixture was extracted with MTBE (3 x 500 mL). The combined organic phases were washed with a 10% aqueous salt solution (4 x 200 mL), the organic layer was dried over Na2SO4and concentrated under reduced pressure to an oil. Neat TFA (486 g, 328 mL, 4.29 mol) was added to the oil and the mixture was stirred at room temperature overnight. The TFA was removed under reduced pressure and a 20% (w / w) aqueous NaOH solution was added until the pH of the aqueous layer was < 11. The mixture was then washed with MTBE (3 x 200 mL) and the pH of the aqueous layer was adjusted to > 2 with a 20% (w / w) aqueous H2SO4solution and the mixture was extracted with CH2Cl2(3 x 200 mL) and the combined organic layers were dried over Na2SO4and concentrated under reduced pressure to give 182 g (66%) of crude 1 -acetylcyclopropanecarboxylic acid as an orange oil.

[0084]

[0085] 1 -(Furan-2-yl)ethan-1 -ol. A three necked round bottom flask (22 L) was washed with anhydrous THF (100 mL), evacuated and filled with nitrogen (3x). The flask was then charged with THF (1500 mL) and furfural (600.0 g, 547.2 mL, 6.244 mol) and the solution was cooled to 0 °C with an ice bath and kept under a nitrogen atmosphere. A solution of methyl magnesium chloride (2289 mL, 3M, 6.838 mol) in THF was added slowly over a period of about 180 minutes using a cannula with nitrogen pressure, carefully keeping the reaction temperature less than 10 °C. The mixture was allowed to cool to 0 °C and stirred for another 30 minutes before carefully quenching with the addition of 1 N aqueous HCl (1000 mL) during which time a large amount of solid was formed and the reaction mixture solidified. Water (2000 mL) was then added and an additional 1 N aqueous HCl (5000 mL) was added before the solid was broken up with mechanical stirring while carefully keeping the internal temperature < 10 °C. The mixture was extracted with MTBE (3 x 1500 mL), the organic phases were combined, washed with water (1000 mL), brine (1500 mL) and then dried over sodium sulfate. The organic layer was then concentrated under reduced pressure by a rotary evaporator to give 678 g (96%) of crude 1 -(furan-2-yl)ethan-1 -ol as a ruby red liquid which was used directly in the next step.

[0086]

[0087] (±)-4-hydroxy-5-methylcyclopent-2-en-l-one. (8) In a 2000 mL round bottom flask equipped with a reflux condenser and magnetic stir bar was added deionized water (1600 mL) and l-(2-furyl)ethanol (7) (130 g, 1159 mmol). The reaction mixture was purged with nitrogen and heated to reflux with vigorous stirring for 120 minutes. The reaction mixture was then cooled to ambient temperature and the aqueous solution was decanted from the brown, oily resin. The aqueous layer was then washed with a mixture of MTBE and hexanes (1 : 1, 3 x 250 mL) and the organic extracts were discarded. Sodium chloride (250 g) was then added to the aqueous solution and extracted with EtOAc (3 x 250 mL). The combined organic extracts were dried over sodium sulfate, filtered, and concentrated on a rotary evaporator under reduced pressure to yield 8.0 g (6%) of (±)-4-hydroxy-5-methylcyclopent-2-en-l-one (8) as a light yellow oil.

[0088]

[0089] (±)-4-(methoxymethoxy)-5-methylcyclopent-2-en-l-one. (9) In a flask was added (±)-4-hydroxy-5-methylcyclopent-2-en-l-one (8) (1.5 g, 13.38 mmol), CH2Cl2(80 mL), and diisopropylethylamine (DIPEA) (5.19, 6.99 mL g, 40.14 mmol), the mixture was purged with nitrogen and cooled to 0 °C with an ice bath. Chloromethyl methyl ether (3.14 M in MeOAc; 8.51 mL) was added dropwise via syringe over 20 minutes. The reaction was stirred at 0 °C for an additional hour, then allowed to slowly warm to ambient temperature and stirred overnight. The reaction mixture was then diluted with CH2Cl2(50 mL), quenched with 10% aqueous NH4Cl (50 mL) and the organic phase separated. The aqueous layer was extracted with CH2Cl2, dried over sodium sulfate (50 mL), the organic extracts were combined, and concentrated under reduced pressure on a rotary evaporator. The crude residue was purified by silica gel flash chromatography (column: 40 g silica gel column; eluent: gradient elution CH2Cl2 0→ 100% hexanes) to yield 10.5 g (84%) of (±)-4-(methoxymethoxy)-5-methylcyclopent-2-en-l-one (9) as a light yellow oil.

[0090]

[0091] Diazoketone. (10) Oxalyl chloride (39.6 g, 26.4 mL, 312 mmol) was added to a solution of l-acetylcyclopropanecarboxylic acid (20 g, 156 mmol) and DMF (110 mg, 0.12 mL, 1.5 mmol) in CH2Cl2(200 mL) at room temperature and stirred for 2 h. The reaction mixture was concentrated under reduced pressure to an oil, dissolved in anhydrous CH2Cl2(300 mL) and cooled to -78 °C (dry ice / acetone bath). Pure 2,6-lutidine (19.3 g, 21.0 mL, 180 mmol) was added followed by TMSCHN2(2.0 M, 200 mL, 300 mmol) in hexanes. The cooling bath was removed and the mixture was allowed to warm to room temperature and stirred overnight. The mixture was concentrated under reduced pressure to an oil and stored in the freezer for 30 min before adding MTBE (200 mL). The mixture was filtered through celite (50 g), concentrated to an oil and purified on a silica gel column (350 g eluted with 1000 mL CH2Cl2) and the eluent concentrated to give 29.3 g of a dark red-brown oil containing 11.23 g of diazoketone (10) (49%) as determined by NMR using mesitylene as an internal standard. This material was used directly in the next step.

[0092]

[0093] Cycloadduct. (11) A stirred solution of diazoketone (10) (11.23 g, 73.8 mmol) and (±)-4-(methyloxy)-5-methylcyclopent-2-en-l-one (9) (5.76 g, 36.9 mmol) in CH2Cl2was evacuated and filled with nitrogen (3x), then solid Rh2(OAc)4(42 mg, 0.095 mmol) was added at room temperature and stirred overnight. The reaction was then concentrated under reduced pressure and purified by flash chromatography on silica gel (column: 120 g silica gel column; eluent: gradient EtOAc 0→80% hexanes) to give 10.8 g (96%) of cycloadduct (11) as a waxy orange solid as determined by NMR using mesitylene as an internal standard.

[0094] 1H-NMR (400 MHz) δ 4.96 (s, 1H), 4.80 (d, J = 7.2 Hz, 1H), 4.74 (d, J = 7.2 Hz, 1H), 3.96 (dd, J = 8.0, 11.2 Hz, 1H), 3.45 (s, 3H), 2.93 (t, J = 7.6 Hz, 1H), 2.67 (d, J = 6.8 Hz, 1H), 2.59 (dq, J = 6.4, 11.6 Hz, 1H), 1.30 (ddd, J = 4.0, 6.8, 9.6 Hz, 1H), 1.20 (s, 3H). 1.16 (ddd, J = 4.0, 6.4, 8.4 Hz, 1H). 1.12 (s, 3H), 1.07 (ddd, J = 4.4, 7.6, 9.6 Hz, 1H), 0.73 (ddd, J = 4.0, 7.2, 9.6 Hz, 1H); 13 C-NMR (100 MHz) δ 212.8, 211.8, 96.2, 87.4, 81.5, 79.9, 59.5, 50.2, 44.1, 39.0, 14.2, 13.8, 12.4, 11.2.

[0095] NMR assignment: 1 H-NMR (400 MHz) δ 4.96 (C9-H), 4.80 (C7-H), 4.74 (C7-H), 3.96 (C4-H), 3.45 (C8-H), 2.93 (C3-H), 2.67 (C2-H), 2.59 (C5-H), 1.30 (C14 or C15-H), 1.20 (C13-H). 1.16 (C14 or C15-H). 1.12 (C6-H), 1.07 (C14 or C15-H), 0.73 (C14 or C15-H); 13 C-NMR (100 MHz) δ 212.8 (C1), 211.8 (C10), 96.2 (C7), 87.4 (C12), 81.5 (C9), 79.9 (C4), 59.5 (C2), 50.2 (C8, C5), 44.1 (C3), 39.0 (C11), 14.2 (C14 or C15), 13.8 (C13), 12.4 (C14 or C15), 11.2 (C6).

[0096]

[0097] Methyl adduct. The starting material (11) (5.6 g, 19.97 mmol) was dissolved in dry THF (80 mL) and cooled to -78 °C with a dry ice / acetone bath. The solution was placed under vacuum and backfilled with nitrogen (2x), then a MeMgCl solution (10 mL, 3M in THF, 30 mmol; 1.5 eq) was added dropwise and the reaction stirred at -78 °C for 3.5 h, then quenched with acetic acid (1.3 mL). The reaction was allowed to warm to room temperature, diluted with DCM (350 mL) and water (250 mL). The resulting mixture was separated. The aqueous phase was extracted with DCM (100 mL) and the combined organic extracts were washed with 20% NaCl (100 mL) in water and dried over Na2S04and concentrated to an oil. The oil was purified by silica gel column chromatography (column: 40 g silica gel column; eluent: gradient EtOAc 0→ 45% in hexanes) to give 778 mg (13.2%) of the methyl adduct target product as white crystals, along with 2.23 g (39.8%) of starting material, 762 mg (12.9%) of a second isomer, and 833 g (13.5%) of a bis-methyl adduct, for a total mass of 92.6%.

[0098]

[0099] Cyclopentenone. (13) Potassium carbonate (704 mg, 5.09 mmol; 1.0 eq) was added to a solution of the starting material (1.50 g, 5.09 mmol) in MeOH (60 mL) and the mixture was stirred at room temperature for 2 h, then quenched with 10% NH4Cl in water (10 mL) and diluted with 300 mL EtOAc. The resulting mixture was washed with 20% NaCl 100 mL. The organic layer was separated and washed with 20% NaCl (50 mL). The organic extracts were dried over Na2S04, concentrated to an oil, and purified by silica gel column chromatography (column: 25 g silica gel column; eluent: gradient EtOAc 0→ 65% in hexanes) to give 974 mg (82%) of the olefin cyclopentenone (13) as a pale yellow crystal.

[0100]

[0101] Diketone (14) A solution of pure TMSOTf (7.72 mL, 9.48 g, 42.68 mmol) was added to a solution of bicycle (13) (2.0 g, 8.54 mmol) and 2,6-lutidine (7.46 mL, 6.86 g, 64.05 mmol) in anhydrous CH2Cl2(100 mL) at 0 °C and stirred for 4 h under a nitrogen atmosphere, then quenched with MeOH (4 mL) and the reaction mixture was concentrated under reduced pressure to an orange-red oil. The crude oil was suspended in MeOH (50 mL) and NH4F (6.32 g, 170.8 mmol) and AcOH (8.55 mL, 8.97 g, 149.45 mmol) were added and the suspension was stirred at room temperature overnight. The reaction was then diluted with H2O (250 mL) and extracted with EtOAc (2 x 150 mL), the organic layer was washed with 5% aqueous sodium citrate (2 x 100 mL), dried over Na2SO4, concentrated to an oil and purified by column chromatography on silica gel (column: 25 g silica gel column; eluent: gradient EtOAc 0→55% in hexanes) to give 774 mg (39%) of the diketone product (14) as a light yellow solid.

[0102]

[0103] Dialcohol (16) A solution of the diketone (14) (770 mg, 3.28 mmol) in anhydrous CH2Cl2(50 mL) was cooled to -78 °C with a dry ice / acetone bath and then a solution of DIBAL-H (13.69 mL, 1.2 M in toluene, 16.43 mmol) in hexanes was added. The solution was stirred for 0.5 h, then the reaction was quenched with MeOH (4 mL) and the reaction mixture was concentrated under reduced pressure to an oil. In another flask, to a vigorously stirred suspension of silica (10 g) in CH2Cl2(50 mL) was added H2O (1 mL) followed by H3PO4(85% w / w in H2O, 0.35 mL) and the mixture was stirred at room temperature for 2 h. The solvent was then removed under reduced pressure until the silica was free flowing. The crude oil from the DIBAL-H reduction was dissolved in EtOAc (200 mL) and then TbPCE-doped hydrated silica (6.2 g) was added and the suspension was stirred vigorously for 3 h. The reaction was then quenched with TEA (430 μL, 312 mg, 3.08 mmol) and filtered. The resulting solution was concentrated to an oil and purified by column chromatography on silica gel (column: 4 g silica gel column; eluent: gradient EtOAc 0→50% in hexanes) to give 83 mg (12%) of the dialcohol (16) compound as a yellow solid.

[0104]

[0105] (-)-acylfulvene (3) diol (16) (38 mg, 0.174 mmol) was dissolved in toluene (5 mL) and azeotropically dried under reduced pressure, then dry DMSO (6 mL) was added, followed by IBX 45 wt.% (216 mg, 0.348 mmol), the suspension was stirred at room temperature for 2 h. The mixture was then diluted with H2O (15 mL), extracted with EtOAc (2 x 10 mL), dried over Na2SO4, concentrated to an oil, and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 0→ 50% in hexane) to give 37 mg (98%) of (-)-acylfulvene (3) as a yellow gum.

[0106] 1 H-NMR (400 MHz) δ 7.16 (d, J = 0.8 Hz, 1H), 6.43 (quint, J = 1.6 Hz, 1H), 3.93 (bs, 1H), 2.15 (d, J = 1.2 Hz, 3H), 2.00 (s, 3H), 1.52 (ddd, J = 4.0, 6.4, 9.6 Hz, 1H), 1.38 (s, 3H), 1.29 (ddd, J = 4.8, 6.4, 9.6 Hz, 1H), 1.07 (ddd, J = 5.2, 7.2, 9.6 Hz, 1H), 0.71 (ddd, J = 4.0, 7.2, 9.6 Hz, 1H).

[0107] NMR assignment: 1 H-NMR (400 MHz) δ 7.16 (C4-H), 6.43 (C1-H), 3.93 (O-H), 2.15 (C6-H), 2.00 (C11-H), 1.52 (C12 or C13-H), 1.38 (C14-H), 1.29 (C12 or C13-H), 1.07 (C12 or C13-H), 0.71 (C12 or C13-H).

[0108]

[0109] (+)-acylfulvene (3) was prepared by dissolving in toluene (5 mL) and azeotropically drying under reduced pressure, then adding anhydrous DMSO (5 mL), then IBX 45 wt.% (171 mg, 0.275 mmol) and stirring the suspension at room temperature for 2 hours. The mixture was then diluted with H2O (15 mL), extracted with EtOAc (2 x 10 mL), dried over Na2SO4, concentrated to an oil and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 0→ 50% in hexane) to give 30 mg (100%) of (+)-acylfulvene as a yellow gum.

[0110] 1 H-NMR (400 MHz) δ 7.16 (d, J = 0.8 Hz, 1H), 6.43 (quint, J = 1.6 Hz, 1H), 3.93 (bs, 1H), 2.15 (d, J = 1.2 Hz, 3H), 2.00 (s, 3H), 1.52 (ddd, J = 4.0, 6.4, 9.6 Hz, 1H), 1.38 (s, 3H), 1.29 (ddd, J = 4.8, 6.4, 9.6 Hz, 1H), 1.07 (ddd, J = 5.2, 7.2, 9.6 Hz, 1H), 0.71 (ddd, J = 4.0, 7.2, 9.6 Hz, 1H).

[0111]

[0112] (-)-Irofulven (4) was prepared by heating a suspension of paraformaldehyde (231 mg, 7.7 mmol as monomer) in 2M aqueous H2SO4(4 mL) to 90 °C for 30 minutes and cooling to room temperature before adding acetone (4 mL) and a solution of (-)-acylfulvene (3) (37 mg, 0.171 mmol) in acetone (1 mL), the mixture was stirred at room temperature for 48 hours. The reaction was then diluted with H2O (25 mL), extracted with CH2Cl2(3 x 15 mL), the combined organic extracts were washed with aqueous NaHCO3(15 mL) then H2O (15 mL) to pH 7. The organic extracts were then dried over Na2SO4, concentrated to an oil and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 0→ 65% in hexane) to give 24.6 mg (58%) of (-)-Irofulven (4) as a yellow gum and 8.6 mg (23%) of (-)-acylfulvene.

[0113] 1H-NMR (400 MHz) δ 7.09 (s, 1H), 4.67 (d, J = 12.4 Hz, 1H), 4.63 (d, J = 12.4 Hz, 1H), 3.90 (bs, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 1.49 (ddd, 4.0, 6.0, 9.6 Hz, 1H), 1.38 (s, 3H), 1.36 (ddd, J = 5.2, 6.4, 9.6 Hz, 1H), 1.08 (ddd, J = 4.8, 7.2, 9.6 Hz, 1H), 0.72 (ddd, J = 4.0, 7.6, 10.0 Hz, 1H).

[0114] NMR assignment: 1 H-NMR (400 MHz) δ 7.09 (C4-H), 4.67 (C15-H), 4.63 (C15-H), 3.90 (O-H), 2.19 (C6-H), 2.15 (C11-H), 1.49 (C12 or C13-H), 1.38 (C14-H), 1.36 (C12 or C13-H), 1.08 (C12 or C13-H), 0.72 (C12 or C13-H).

[0115]

[0116] (+)-Irofulven. (4) A suspension of paraformaldehyde (186 mg, 6.21 mmol as monomer) in 1 M aqueous H2SO4(9 mL) was heated to 90 °C for 30 min and cooled to room temperature before adding acetone (9 mL) and a solution of (+)-acylfulvene (3) (30 mg, 0.138 mmol) in acetone (3 mL). The mixture was stirred at room temperature for 72 h. The reaction was then diluted with H2O (30 mL) and extracted with CH2Cl2(3 x 15 mL), and the combined organic extracts were washed with aqueous NaHCO3(15 mL) and then H2O (15 mL) to pH 7. The organic extracts were then dried over Na2SO4, concentrated to an oil, and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 0→ 65% in hexanes) to give 12.7 mg (37%) of (+)-irofulven as a yellow gum and 12.4 mg (41%) of (+)-acylfulvene (3).

[0117] 1H-NMR (400 MHz) δ 7.09 (s, 1H), 4.67 (d, J = 12.4 Hz, 1H), 4.63 (d, J = 12.4 Hz, 1H), 3.90 (bs, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 1.49 (ddd, J = 4.0, 6.0, 9.6 Hz, 1H), 1.38 (s, 3H), 1.36 (ddd, J = 5.2, 6.4, 9.6 Hz, 1H), 1.08 (ddd, J = 4.8, 7.2, 9.6 Hz, 1H), 0.72 (ddd, J = 4.0, 7.6, 10.0 Hz, 1H).

[0118] NMR assignment: 1 H-NMR (400 MHz) δ 7.09 (C4-H), 4.67 (C15-H), 4.63 (C15-H), 3.90 (O-H), 2.19 (C6-H), 2.15 (C11-H), 1.49 (C12 or C13-H), 1.38 (C14-H), 1.36 (C12 or C13-H), 1.08 (C12 or C13-H), 0.72 (C12 or C13-H).

[0119]

[0120] (-)-Hydroxyurea methylacyl fulvene. (5) Hydroxyurea (37 mg, 0.487 mmol) was added to a mixture of (-)-Irofulvene (4) (24 mg, 0.097 mmol) in acetone (1.5 mL) and 2 M H2SO4(1.5 mL) and the mixture was stirred at room temperature for 24 h, then diluted with H2O (15 mL) and EtOAc (15 mL), the organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic extracts were washed with 5% aqueous NaHCO3(10 mL), brine (10 mL). The organic extracts were then dried over Na2SO4, concentrated to an oil, and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 10→ hexane 95%) to give 18.9 mg (61%) of (-)-LP-184 (5) as an orange-yellow solid.

[0121] 1H-NMR (400 MHz) δ 7.03 (s, 1H), 6.86 (s, 1H), 5.37 (bs, 2H), 4.80 (d, J = 14.8 Hz, 1H), 4.52 (d, J = 14.5 Hz, 1H), 3.84 (bs, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 1.47 (ddd, J = 4.0, 6.4, 10.0 Hz, 1H), 1.35 (ddd, J = 5.2, 6.4, 9.6 Hz, 1H), 1.35 (s, 3H), 1.34 (ddd, J = 5.2, 7.6, 9.6 Hz, 1H), 0.68 (ddd, J = 4.0, 7.6, 10.0 Hz, 1H).

[0122]

[0123] (+)-hydroxyurea methylacyl fulvene. (5) Hydroxyurea (7.5 mg, 0.098 mmol) was added to a mixture of (-)-Irofulvene (4) (12 mg, 0.049 mmol) in acetone (1 mL) and 2 M H2SO4(1 mL) and the mixture was stirred at room temperature for 24 h, then diluted with H2O (15 mL) and EtOAc (15 mL), the organic layer was separated and the aqueous layer was extracted with EtOAc (2 x 10 mL). The combined organic extracts were washed with NaHCO3(aq) (10 mL), brine (10 mL). The organic extracts were then dried over Na2SO4, concentrated to an oil, and purified by silica gel column chromatography (column: 12 g silica gel column; eluent: gradient EtOAc 10→ 95% hexanes) to give 6.6 mg (44%) of (+)-LP-184 (5) as a yellow solid and 3.0 mg (25%) of (+)-Irofulvene (4).

[0124] 1 H-NMR (400 MHz) δ 7.03 (s, 1H), 6.86 (s, 1H), 5.37 (bs, 2H), 4.80 (d, J = 14.8 Hz, 1H), 4.52 (d, J = 14.5 Hz, 1H), 3.84 (bs, 1H), 2.17 (s, 3H), 2.08 (s, 3H), 1.47 (ddd, J = 4.0, 6.4, 10.0 Hz, 1H), 1.35 (ddd, J = 5.2, 6.4, 9.6 Hz, 1H), 1.35 (s, 3H), 1.34 (ddd, J = 5.2, 7.6, 9.6 Hz, 1H), 0.68 (ddd, J = 4.0, 7.6, 10.0 Hz, 1H).

[0125] Cytotoxicity. The growth inhibitory activity of the two purified UMAF (also referred to as UMAF in this example) optical enantiomers was investigated in standard 96-well cell assays using representative cell lines from prostate, ovarian and lung cancer. Luminescent cell viability assay reagent (CellTiter-Glo R , Promega Corporation) was used to determine the concentration (GI 50 ) that caused 50% inhibition of cell growth. Growth inhibition curves as a function of UMAF concentration are shown from representative assays. Figure 4 、 6 , 7 and 9 indicate that the (+) enantiomer was more toxic to the DU145, OVCAR3, SK-OV3 and H1975 cell lines, respectively. Figure 5 and 8 show that the PC3 and HCC827 cell lines were equally sensitive to both enantiomeric forms of the compounds. The GI 50 concentrations for the two forms of the six cell lines are shown below.

[0126]

Claims

1. A compound having a positive optical rotation angle and having the formula II ###0001### II 2. A method of synthesizing a hydroxy urea methyl acyl fulvalene (UMAF) having the formula (II), ###0002### II the method comprising: selecting a cyclopentenone having a hydroxyl group; protecting the hydroxyl group; reacting the protected cyclopentenone with a diazo ketone to form a cycloadduct; and reducing the cycloadduct; alkylating the ketone group of the cycloadduct; cleaving the oxo group of the cycloadduct; and oxidizing the cycloadduct.

3. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier.

4. The use of an effective amount of a compound of claim 1 for the manufacture of a medicament for the treatment of prostate cancer, ovarian cancer and lung cancer in a mammal. the mammal is a human.

5. Use according to claim 4, wherein, ​

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