WO2010014240A2 - Novel bioactive small molecules derived from sea sponges - Google Patents

Abstract

The invention encompasses a number of previously unknown latrunculol compounds that show significant anti-neoplastic activity.

Description

NOVEL BIOACTIVE SMALL MOLECULES DERIVED FROM SEA SPONGES

Government Funding

This invention was made partly using funds from National Institutes of Health grant number ROl CA 47135. The U.S. Federal Government has certain rights to this invention.

Relation to other applications

This application claims priority to and the benefit of US provisional Application No. 61/137,359, filed on 29 July 2008 and entitled "COMPOSITIONS COMPRISING AND METHODS OF USE OF LATRUNCULINS", the entire disclosure of which is hereby incorporated by reference for all purposes.

Field of the Invention

The invention provides new, previously unknown compounds with biological activity. The compounds are derived from sponges and are related to latrunculin compounds. Also provided are methods of using such compounds for the treatment of cancer and other diseases such as gout and various diseases in which actin is involved.

Background

The latrunculins are sponge-derived bioactive small molecules originally isolated from the Red Sea sponge Negombata magnified (old genus designation Latrunculia). Latrunculins are known to have certain bioactive properties. Summary of the Invention

The invention encompasses a number of previously unknown latrunculol compounds that include latrunculol A (7), latrunculol A acetonide (7a), latrunculol B (8), 18-epi- latrunculol A (9), latrunculol C (10), latrunculone A (I l), and latrunculone B (12). These latrunculols are believed to be novel, previously unknown compounds, and have been isolated and structurally characterized by the inventors. These novel compounds show significant anti-neoplastic activity.

Brief Description of the Figures and Tables

Figure 1 : Structure of compounds 7, 7a, 8, 9, 10, 11, 12 and 13.

Figure 2: A graph showing the cytotoxic properties of compounds 1-12.

Table 1 : Summary of latrunculin frameworks and their sources

Table 5*: Table showing biological activity with microfilament disruption date in far right column.

*Note there are no tables 2 and 3 and 4

Detailed description of the Invention

The inventors investigated various sponges and were able to isolate, purify and characterize a number of novel compounds with biological activity, including antineoplastic activity.

The latrunculins are sponge-derived bioactive small molecules whose properties have been studied for almost 30 years. Two interesting compounds of this type are latrunculin A (I) and latrunculin B (2), originally isolated from the Red Sea sponge Negombata magnified (old genus designation Latrunculia).

Their basic structural motif consists of a macrolide 1,3 fused to a tetrahydropyran containing a 2-thiazolidinone side chain. Latrunculin A (I) also shares a carbon skeleton with the anti-cancer active epothilone A, obtained from cultures of the terrestrial myxobacterium, Sorangium cellulosum. Latrunculins have a unique mixed biogenesis from polyketide/non-ribosomal peptide synthetases (PKS/NRPS), they are strong actin inhibitors, and they show cytotoxicity against cancer cell lines. Latrunculin A is the most widely used small molecule molecular probe. Its actin inhibition properties result from blocking the hyper-assembly of G-actin to F-actin by binding to a site in a cleft between subdomains II and IV.

The inventors have isolated and studied latrunculin analogues from two taxonomically unrelated sponges, Cacospongia mycofijiensis and Negombata magnified, and have discovered a number of novel biologically active molecules that may be used in therapy.

The details, materials and methods used in this investigation are disclosed in the paper titled "Interrogating the Bioactive Pharmacophore of the Latrunculin Chemotype by Investigating the Metabolites of Two Taxonomically Unrelated Sponges" J. Med. Chem., 2008, 51 (22), pp 7234-7242 which publication is incorporated by reference for all purposes. Extraction and isolation of various compounds was performed as described in the above-mentioned document(s) and the resultant CH₂Cl₂ extract was purified by reverse-phase HPLC.

The inventors initially studied compounds separated and isolated from the Red Sea sponge N. magnified, isolating three known compounds: latrunculin B (2), 16-epi- latrunculin B (3), and latrunculin C (4).

Additionally the inventors identified Latrunculenic acid (6) which is a previously unknown compound.

5: R = CH₃, 16R

Through their investigation, the inventors additionally isolated, purified, identified and characterized a number of previously unknown latrunculol compounds that include latrunculol A (7), latrunculol A acetonide (7a), latrunculol B (8), 18-epi- latrunculol A (9), latrunculol C (10), latrunculone A (I l), and latrunculone B (12). These latrunculols are believed to be novel, previously unknown compounds, and have been isolated and structurally characterized by the inventors. These compounds are particularly interesting because they possess highly modified macrocyclic rings and show significant anti-neoplastic activity.

7: R = H. 18R 10 8: R = CH₃, 18R 9: R = H, 18S

11 12

Compounds 8, 9, and 10 were close in structure to 7. Compound 8 has the formula C₂₃H₃₅NO₇S; Compound 9, C₂₂H₃₃NO₇S; Compound 10, (C₂₂H₃₃NO₇S); Compound 11 of C₂₂H₃iNO₇S and Compound 12, C₂₂H_{3 I}NO₆S.

Latrunculol A (7) was found (by HRESIMS) to have the formula C₂₂H₃₃NO₇S. Compared to Latrunculin A (1) there are two less Hydrogen atoms, two additional Oxygen atoms and one less unsaturation equivalent. The diene chromophore of Latrunculin A is present as a monoene diol in Latrunculol A. This was substantiated using NMR. The ¹H and ¹³C NMR spectra of Latrunculol A revealed that, vs Latrunculin A, signals of the Δ⁶ double bond of Latrunculin A were replaced by two oximethine signals for a vicinal diol. Though the ³J6,₇ was not detectable, mutual gCOSY correlations were observed. As expected, the remaining functional groups included (1) a thiazolidinone carbonyl [δ 173.9 (C-20)], (2) an ester carbonyl [δ 166.6 (C-I)], (3) a disubstituted double bond [δ 5.64 (H-8), 5.05 (H-9), δ 132.3 (C-8), 136.5 (C-9)], (4) a trisubstituted double bond [δ 5.54 (H-2), δ 1 18.7 (C-2), 158.3 (C-3)], and (5) a hemiacetal quaternary carbon [δ 97.6 (C-17)]; all were consistent with the type Ia system. Finally, the COSY and HMBC NMR data further substantiated the proposed planar structure of Latrunculol A.

Additionally, the inventors also discovered a new Latrunculin A analogue: 15, 17, dimethoxy latrunculin A (13). In the new analogue has a double bond on the five membered thiazolidinone moeity and a ring opened macrolide structure, as below.

15, 17, dimethoxy latrunculin A (13)

Cytotoxicity assays were carried out using the compounds of the invention. Significant cytotoxicity against cancer cells was shown by compounds No. 1, 2, 7, 7a, 8, 9 and 12 against both human and murine cancer cell lines, and the IC50 value range was 0.5-10 μM.

Compounds 1, 2, 3, 7, 7a, 8, 10 and 12 exhibited microfilament-disrupting effects. The other five compounds were inactive.

18-epi-latrunculol A (No.9) is of particular interest and exhibited a powerful cytotoxic effect at micromolar concentrations but was shown to have a reduced anti-actin cytotoxic effect when compared with other latrunculin and latrunculol compounds. 18-epi-latrunculol A appears to have a strongly selective anti-tumor activity and is more specifically cytotoxic to cancerous cells than other related compounds. The graph of Figure 2 shows that 18-epi-latrunculol A is almost twice as cytotoxic to cancer cells as to non-cancer cells. Obviously such specific anti-cancer toxicity may provide a desirable clinical property. Anti-neoplastic cytotoxic bioactivity. A mini-library of 13 compounds was obtained in sufficient quantity to engage in bioactivity assessment. This included 10 metabolites (1-4, 7-12) isolated in this study, two latrunculin analogues (5, 6) from our pure compound repository, and the synthetic derivative 7a. Each was evaluated in the disk diffusion soft agar cell-based assay against murine cell lines colon 38, Ll 210, and CFU-GM (see graph, "Table 2"). Compounds were adsorbed onto the disk at similar concentrations, and a zone of inhibition (Z) in mm was recorded. Somewhat surprisingly, one of the new compounds, 7, showed the best cytotoxic effect against colon 38 (Zc38 ⁼ 23.5 mm at 1.1 μM), which was 2.6 and 1.8 times greater than 1 (Z_C38 = 9.0 mm at 1.9 μM) and 2 (Zc₃₈ = 13.0 mm at 5.1 μM), respectively. Four other compounds were almost as effective against colon 38, and these included acetonide 7a (Z_C38 = 21.0 mm at 4.0 μM), 8 and 12 (each Z_C38 = 19.5 mm at 1.1 μM), followed by 9 (Zc₃₈ ⁼ 17.0 mm at 3.5 μM). Examining the relative sensitivity of the two other cell lines to these and the other compounds provided an assessment of their relative solid tumour selective cytotoxicity. Four compounds fit this designation and are flagged by a an asterisk (*) in the bar graph plotting zone of inhibition (mm) vs Concentration (Micromolar) (See graph, "Table 2"). These include 1, 3, 7, and 9 with colon 38 selective in their cytotoxicity effect as follows. The magnitude of the differential selective cytotoxicity for 7 was identical to that of 1 (Zc3₈ ~ Z_CFU-_GM ⁼ 9.0 mm). Interestingly, compound 9 (Zc₃₈ - ZCFU-G_M ⁼ 15.0 mm), the 185 epimer of 7, exhibited the greatest overall differential selective cytotoxicity. A similar enhancement of relative solid tumor selectivity was also seen when comparing the epimers 3 (Zc₃₈ - ZL_I21₀ ⁼ 9.0 mm) and the 16S isomer 2 (Zc₃₈ - Z_LI_∑I_O = 1 5 mm). The next steps in this evaluation involved expanding the bioassay results by including additional cancer cell lines.

Responses of two human solid cancer cell lines, HCT-116 and MDA-MB-435, were measured by Trypane blue and the SRB method, respectively, for all 13 compounds, and the IC₅₀ values are summarized in Table 5 Collectively these data showed significant inhibition for 7 of the 13 compounds and the trends against the HCT-116 cell line were somewhat similar to that observed in the disk diffusion assay. Overall, there were seven very active compounds led by 7 with the smallest IC₅₀ = 0.48 μM against HCT-116. Other similarly active compounds (with HCT-1 16 IC₅₀ values in μM) included 12 (0.92), 1 (1.1), 8 (2.1), 7a (5.1), 9 (5.5), and 2 (7.1). The activity pattern against the MDA-MB-435 cell line was, with the exception of the response by 9 (IC50 > 50), almost parallel to that observed against HCT-116. The most active compound was 12 with IC₅₀ = 1.0 μM. The remaining order of activity was (with IC₅₀ in μM) 7 (2.1), 1 (2.8), 2 (4.8), 8 (4.0), and 7a (7.9). While these responses are appealing, there is another important issue. Effective cytotoxins such as 1 and 2 that are powerful anti-actin agents are usually considered to be unsuitable for therapeutic development because of their unselective profile. The IC₅₀ ratios tabulated in the third column of Table 5 for the last six compounds could be interpreted as consistent with this view. Alternatively, the very different ratio computed for 9 could foreshadow a special circumstance. This possibility was investigated next.

Microfϊlament-disrupting activity. The mini-library was further assessed in a microfilament-disrupting assay using AlO cells. Figure 3 (in J. Med. Chem., 2008, 57 (22), pp 7234-7242) shows the results of the phenotypic assay used, and the last column of Table 5 summarizes the level of anti-actin action at 5 μM for each of the compounds. Results are consistent with previous studies that show, relative to the control, the expected microfilament-modulating effects exhibited by 1 and 2. The inventors also observed significant disruption action by 3 and 7. Analogous perturbation effects were observed (tabulated in Table 5) for 7a, 8, 10, 12 at 5 μM. By contrast, the response for 9, while not identical to the control (Figure 3), represents the pattern expected for a compound devoid of significant microfilament-disrupting action. Similar negative responses were observed for four other compounds, 4, 5, 6, and 11. Overall, these results when evaluated side-by-side with the cytotoxicity data indicate that six of the seven very active compounds (1, 2, 7, 7a, 8, 12) are aggressive actin disruptors. The different profile for 9 is intriguing, and it is reasoned that a variation in the 18i? to \8S configuration of the thiazolidinone ring (7 vs 9) diminishes the anti-actin effect without eliminating the cytototxicity properties. This pattern does not appear to hold for the latrunculin B series, as the microfilament-disrupting activities were similar for 2 vs 3, in which the configuration at 16 changes from R to S.

It should be noted that more than 50% of the latrunuculin analogues (seven compounds) evaluated in this cell based assay study were significantly active (IC₅₀ ⁼ 0.5-10 μM) against one or both cell lines. All of these compounds possess the type Ia framework. The remaining six compounds had modest to inactive IC₅₀ values, and this group included all four of the type 2 framework compounds plus two with a type

1 a framework. The conformation of the 16-membered macrolide ring appears to be critical for activity as judged by the significant activity of 1, 7, 7a, 8, and 12 vs the poor responses for 10 and 1 1. Not to be ignored is the requirement for the R configuration of the thiazolidinone ring for maximizing IC₅₀ against human tumour cell lines.

The present findings have increased the understanding about the latrunculin family chemotypes and their cytotoxicity properties. The inventors have obtained both 1 and

2 from a Fijian C. mycofijiensis, which represents the first example of types 1 and 2 structures co-occurring in Cacospongia. The isolation and characterization of 7-11 possessing unprecedented new macrolide oxygenation patterns serves to further extend the record of structural information. The discovery that there are two distinct cytotoxicity modes of action operating for the latrunculin family, illustrated by the data in Table 5 for 1, 7, 12 vs 9, provide useful therapeutic advantages for these compounds. The properties of 9 are especially important because they are similar to those of oxolatrunculin B, as each may inhibit cancer cell line growth by an actin independent pathway.

Description of Various Embodiments

The invention relates to a novel latrunculin-related compounds. These compounds include latrunculol A (7), latrunculol B (8), 18-epi-latrunculol A (9), latrunculol C (10), latrunculone A (1 1), latrunculone B (12), and latrunculol A acetonide (7a), and to derivatives, metabolites, analogues, variants, congeners, or isomeric forms thereof.

A generic structure incoφorating all variations of structures 7 to 12 can be represented as shown below, wherein R groups (R1-R7) specify R or S stereochemistry and functional groups, either H, Me, OMe or O so as to provide the formulae 7-12, above.

Rl may be CH3

R2 may be O or OH (R or S)

R3 may be H (R or S), OH, OCH3

R4 may be H or O

R5 may be CH3

R6 may be OH or H

R7 may be H (R or S) In another embodiment

Rl may be CH3 or H

R2 may be O or OH (R or S) or H

R3 may be H (R or S), OH, OCH3

R4 may be H or O or H

R5 may be CH3 or H

R6 may be OH or H

R7 may be H (R or S)

The invention encompasses variants, derivatives and analogues of the above structures, and in alternative embodiments the composition of the invention may include structures wherein any one or more of Rl - R7 can alternatively be H, OH, CH3.

In other embodiments, any one or more of Rl - R7 may be derivatized by any method with the addition of any group such that at least a portion of the biological activity of the molecule is retained.

The invention includes novel compounds and mixtures of compounds as described above that specifically exclude one or more of structures 1, 2, 3, 4 and 5.

The invention includes novel compounds defined by the generic formula above, wherein

Rl is CH3 or H

R2 is O or OH (R or S) or H

R3 is H (R or S), OH, OCH3

R4 is H or O or H

R5 is CH3 or H

R6 is OH or H

R7 is H (R or S)

And wherein the compound is not latrunculin A (1) or latrunculin B (2) or 16-epi- latrunculin B (3) or latrunculin C (4). Positions occupied by hydrogen in the groups described herein can be further substituted with substituents exemplified by, but not limited to, hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, fluoro, chioro, bromo, iodo, methyl, ethyl, propyl, butyl, alkyl, substituted alkyl, thio, thioalkyl, acyl, carboxyl, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylaniino, sulfonamide, substituted sulfonamide, cyano, amino, substituted amino, acylarnino, trifluoromethyl, trifluoromethoxy, phenyl, aryl, substituted aryl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, substituted cycloalkyl, pyrrolidinyl, piperidinyl, morpholjno, and hetero cycle; and preferred hetero atoms are oxygen, nitrogen, and sulfur. It is understood that where open valences exist on these substituents they can be further substituted with alkyl, cycloalkyl, aryl, heteroaryl, and/or heterocycle groups, and where multiple such open valences exist, these groups can be joined to form a ring, either by direct formation of a bond or by formation of bonds to a new heteroatom, preferably oxygen, nitrogen, or sulfur. It is further understood that the above subtitutions can be made provided that replacing the hydrogen with the sub stituent does not introduce unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable.

The invention encompasses derivatives, metabolites, analogues, variants, congeners, or isomeric forms of any of the disclosed compounds. In another aspect, the invention relates to a compound comprising any of the aforementioned latrunculin compounds or fragments thereof.

The novel molecule may be an isolated and/or purified molecule, removed and separated from its natural cellular environment, substantially or completely free of other cellular components. The molecule may be natural or synthetic. The latrunculin compound may be isolated from a natural source such as a sponge (e.g., Cacospongia mycofϊjiensis). Alternatively, the latrunculin compound can be made synthetically (10) or it can be isolated from an organism genetically engineered to produce the molecules of the invention. The molecule may be present on its own or mixed with one or more other latrunculin molecules such as those named herein. The molecule may be mixed or formulated with any pharmaceutical excipient. In yet another aspect, the invention relates to a method of preventing or treating a disease, such as, but not limited to cancer, the method comprising administering a therapeutically effective amount of a latrunculin-containing composition to a subject in need thereof. This invention also relates to methods of using pharmaceutical formulations of the invention in the prevention or treatment of diseases or disorders that are affected by modification of the integrity of the actin cytoskeleton, for example, treatment of disorders in which intraocular pressure is elevated, such as primary open-angle glaucoma and ocular neuroprotection in humans and other mammals. Other specific conditions that the method of the invention can be used to treat include Gout and any other actin related disease. The invention also may be used to provide a method of reducing intraocular pressure, a method of treating glaucoma, a method of inhibiting wound healing after trabeculectomy, and a method of inhibiting angiogenesis.

Another embodiment relates to a pharmaceutical composition comprising one or more of the aforementioned latrunculin-related compounds in which the latrunculin-related compound(s) is considered to be a "drug" and wherein the drug is mixed with a carrier to provide a formulation. Such formulations may contain one or more ingredients apart from the primary active ingredient or drug, such as excipients, fillers, carrier materials etc that may be used to modify or improve the drug release, improve its physical and/or chemical stability, dosage form performance, processing, manufacturing, etc. Excipients may include binding agents (e.g. methyl cellulose); fillers (e.g. corn starch, sucrose); lubricants (e.g. stearic acid, magnesium stearate, colloidal silica); and disintegrators (e.g. sodium starch glycolate, alginic acid. Further details on techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Other embodiments include formulations for use in therapy or diagnosis wherein, for example, the therapy or diagnosis involves delivering a therapeutically effective amount of a drug to a subject.

Other embodiments include the use of a pharmaceutical formulation in the manufacture of a medicament for the treatment of any of the diseases enumerated herein such as cancer, In still another aspect, a latrunculin may be administered to a subject to treat or prevent cancer, including, but not limited to, adenocarcinoma, sarcoma, melanoma, lymphoma, and leukemia; particularly, those cancers may include, but are not limited to, cancers of the pancreas, prostate, ovary, breast, lung, liver, colon, bladder, adrenal gland, heart, kidney, and brain. In a particular aspect, a latrunculin is used to treat a solid tumor, such as those of any of the aforementioned cancers.

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, ungulates, lagomorphs, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable excipient or carrier or adjuvant, for any of the therapeutic purpose. Such pharmaceutical compositions include at least one latrunculin-like compound described herein or a derivative, metabolite, analogue, variant, congener, or isomeric form thereof. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Adjuvants may also be used. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. The determination of an effective dose is well within the capability of those skilled in the art. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD5O/ED5O. In particular regard for the administration of latrunculins, minimization of the anti-actin cytotoxic effect of the latrunculin is desirable. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, every two weeks, every month or every three months, depending on half- life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Sustained release and controlled release formulations may also be

Materials and Methods

General Experimental Procedures. Optical rotations were obtained on a digital polarimeter. The NMR spectra were recorded in CDCI₃ and acetone-c?₆ at 500 and 600 MHz for ¹H and 125.6 and 150.0 MHz for ¹³C, respectively. Semipreparative HPLC was performed using a 5 μm Ci₈ ODS column by means of a single wavelength (λ = 230 nm) for compound detection. High resolution mass measurements were obtained from an ESI-TOF mass spectrometer. DFT calculations were performed by Spartan 06 with a basis set at the B3LYP/6-31G*//B3LYP/6-31G* level. The computer used for DFT calculations was equipped with a dual 2.80 GHz CPU and 4 GB DDR2 SDRAM at 667 MHz with 222 GB disk space.

Biological Material, Collection, and Identification. Specimens of N. magnifϊca(19) (collection, no. 01600) (0.62 kg wet weight) were collected using SCUBA in 2001 off the coast of Eilat, Israel, at depths of 15 m. Two separate specimens of C. mycofijiensis(32) (collection nos. OOIOO-I and 00100-11) (1.9 and 0.6 kg wet weight) were collected using SCUBA in 2000 from the Beqa Lagoon, Fiji, at depths of 15-20 m. Taxonomic identification was based on comparison of the biological features to other samples in our repository and physical features of those previously published.(19, 32) Voucher specimens and underwater photos are available. DFT Calculations for Carbon Chemical Shifts. The carbon chemical shifts predicted by Spartan 06 were corrected by the least-squares method. (33) Score was defined by the equation [(no. of carbon - points)/(no. of carbon)] x 100 (%). The points (0-3 points) are given on the basis of the absolute difference between predicted carbon and experimental carbon chemical shift: (1) 0 points (<5 ppm), (2) 1 point (6-10 ppm), (3) 2 points (1 1-20 ppm), and (4) 3 points (>21 ppm).

Biological Assays. The detailed methods of the disk diffusion soft agar colony formation assay, IC₅₀ determination for HCT-1 16 and MDA-MB-435, microfilament- disrupting assay were described previously.(34) The assay results appear in Figures 2 and 3 and Table 5.

Extraction and Isolation. Samples were preserved in the field according to our standard laboratory procedures and stored in a cold room until extraction was performed. The sponges were extracted 3 * with methanol, and then the resultant oil was partitioned using a modified Kupchan-type solvent partition scheme. (35) The CH₂Cl₂ extract (FD, 180 mg) of the sponge, N. magnified (collection no. 01600) was fractionated using repeated semipreparative reverse phase gradient HPLC (80:20 CH₃CΝ/H₂O up to 100% over 40 min) to give six fractions. Fraction H3 (7.8 mg) was then further purified using the above conditions to yield 4 (3.3 mg). Fractions H4 (51.2 mg) and H5 (1 1.3 mg) afforded 2 and 3. The CH₂Cl₂ extract (FD, 75 mg) of the sponge C. mycofijiensis (collection no. OOIOO-I) was also purified using repeated semipreparative reversed-phase gradient HPLC (30:70 CH₃CNZH₂O up to 80:20 over 50 min) to give nine fractions. The resultant fractions H1-H9 eluted in the following order and afforded 10 (1.6 mg), 7 (8.3 mg), 9 (2.5 mg), 1 1 (2.6 mg), 8 (3.1 mg), 12 (2.8 mg), 1 (25.3 mg). Fractions H5 and H9 were not fully pursued because of limited sample size and purity. A separate purification of the lesser CH₂CI₂ extract (00100-11 FD, 75 mg) was made using the same HPLC conditions and generated 1 1 fractions. Additional amounts of compounds 1 and 7-12 were obtained along with 2 (2.2 mg).(22).

Latrunculin compounds were provided as follows: Latrunculin A (1): white powder; [α]_D ²³ +144.4 (c 1.0, CHCl₃); ¹H and ¹³C NMR data in Table Sl (61/137,359, filed on 29 July 2008); HRESITOFMS mlz 444.1827 [M + Na]⁺ (calcd for C₂₂H_3INO₅SNa 444.1815). This compound was identified by comparison of spectral data with those of the literature values.(22)

Latrunculin B (2): white powder; [α]_D ²³ +120.1 (c 4.0, CHCl₃); ¹H and ¹³C NMR data in Table S2; HRESITOFMS mlz 418.1653 [M + Na]⁺ (calcd for C₂oH₂₉NOsSNa 418.1659). This compound was identified by comparison of spectral data with those of the literature values.(22)

16-epi-Latrunculin B (3): white powder; [α]_D ²³ + 81.2 (c 1.0, CHCl₃); ¹H and ¹³C NMR data in Table S3; HRESITOFMS mlz 418.1649 [M + Na]⁺ (calcd for C₂oH₂₉N0₅SNa 418.1659). This compound was identified by comparison of spectral data with those of the literature values.(8)

Latrunculin C (4): white powder; ¹H and ¹³C NMR in Table S4; HRESITOFMS mlz 420.1815 [M + Na]⁺ (calcd for C₂₀H₃₁NO₅SNa 420.1798). This compound was identified by comparison of spectral data with those of the literature values. (18)

Library Compounds: 15-Methoxylatrunculin B (5) and latrunculeic acid (6) were used for the disk diffusion cell-based assay. The purity (>95%) for 5 and 6 was confirmed by LCMS and ¹H NMR analysis.(19, 36)

Latrunculol A (7): white powder; [α]_D ²⁶ +64.8 (c 5.6, MeOH); UV (MeOH) λ_max (logε) 216 nm (4.07); ¹H and ¹³C NMR data in Tables 2, 3, and S5; HRESITOFMS mlz 478.1864 [M + Na]⁺ (calcd for C₂₂H₃₃NO₇SNa 478.1870).

Formation of Acetonide 7a from 7

To a solution of 7 (5.0 mg) in CH₂Cl₂ (0.5 mL) were added 2,2-dimethoxypropane (0.75 mL) and pyridinium toluene /(-sulfonate (9.0 mg). After stirring of the mixture at room temperature for 2 h, the solvent was evaporated under reduced pressure. The residue was purified by HPLC using acetonitrile-water (1 :1 to 1 :0) as the eluent to afford acetonide 7a (2.5 mg) as a colorless powder.

Acetonide 7a: colorless powder; [α]_D ²⁸ +21.7 (c 1.7, MeOH); ¹H NMR δ ppm (CDCl₃) 5.70 (IH, d, J= 1.4 Hz, H-2), 5.64 (IH, br s, NH), 5.44 (IH, dd, J = 10.7 and 9.6 Hz, H-9), 5.39 (IH, dd, J= 10.8 and 8.7 Hz, H-8), 5.38 (IH, m, H-15), 4.45 (IH, t, J= 8.5 Hz, H-7), 4.05 (I H, br t, 9.8 Hz, H-13), 3.83 (IH, ddd, J = 8.8, 5.6, and 1.0 Hz, H-18), 3.77 (IH, ddd, J= 8.6, 5.9 and 4.3 Hz, H-6), 3.50 (IH, dd, J= 11.6, 8.9 Hz, H-19b), 3.41 (IH, dd, J= 11.6 and 6.0 Hz, H-19a), 2.55-2.63 (3H, m, H₂-4 and H-IO), 2.08 (IH, dt, J = 14.6 and 2.2 Hz, H-16b), 1.95 (3H, s, H₃-21), 1.90 (IH, br d, J = 14.6 Hz, H-16a), 1.67-1.76 (5H, m, H₂-5, H-I Ib and H-14b), 1.40-1.56 (3H, m, H₂- 12, H- 14a), 1.44 (3H, s, H₃-acetonide), 1.43 (3H, s, H₃-acetonide), 1.20 (IH, m, H- 1 Ia), 1.02 (3H, d, J= 5.5 Hz, H₃-22); NOESY ID (mixing time = 0.5 ms) H-8/H-6, H-7/H-10; HRESIMS mlz 518.2178 [M + Na]⁺ (calcd for 518.2183, C₂₅H₃₇NO₇SNa).

Latrunculol B (8): white powder; [α]_D ²⁷ +47.5 (c 1.6, MeOH); UV (MeOH) X_n13x (logε) 216 nm (3.95); ¹H and ¹³C NMR data in Tables 2, 3, and S7. HRESITOFMS mlz 492.2025 [M + Na]⁺ (calcd for C₂₃H₃₅NO₇SNa 492.2027).

18-epi-Latrunculol A (9): white powder; [α]_D ²⁷ +55.2 (c 4.4, MeOH); UV (MeOH) λ_maX (logε) 216 nm (4.06); ¹H and ¹³C NMR data in Figures S15 and S16, and Table S8; HRESITOFMS mlz 478.1866 [M + Na]⁺ (calcd for C₂₂H₃₃NO₇SNa 478.1870).

Latrunculol C (10): white powder; [α]_D ²⁸ +56.1 (c 1.3, MeOH); UV (MeOH) λ_max (logε) 216 nm (4.50); ¹H and ¹³C NMR data in Tables 2, 3, and S9; HRESITOFMS mlz 478.1878 [M + Na]⁺ (calcd for C₂₂H₃₃NO₇SNa 478.1870).

Latrunculone A (11): white powder; [α]_D ²⁷ +5.8 (c 1.2, MeOH); UV (MeOH) λ_π,_ax (logε) 216 nm (4.23); ¹H and ¹³C NMR data in Tables 2, 3, and SlO; HRESITOFMS mlz 476.1707 [M + Na]⁺ (calcd for C₂₂H₃₁NO₇SNa 476.1714).

Latrunculone B (12): white powder; [α]_D ²⁹ +129.2 (c 0.1, MeOH); ¹H and ¹³C NMR data in Tables 2, 3, and Sl 1 ; HRESITOFMS mlz 460.1764 [M + Na]⁺ (calcd for C₂₂H₃,NO₆SNa 460.1735).

Abbreviations and Definitions

The following definitions are used: DFT, density functional theory; PKS/NRPS, polyketide synthase / nonribosomal peptide synthetase; MAE, mean absolute error; colon 38, murine colon adenocarcinoma; Ll 210, murine lymphocytic leukemia; CFU-GM, murine bone marrow; HCT-1 16, human colorectal carcinoma; MDA-MB-435, human breast cancer; SRB, sulforhodamine B; AlO, rat smooth muscle; DTP, developmental therapeutics program.

Latrunculin: The term "latrunculin" (interchangeably used with "latrunculin molecule" or "latrunculin compound") is used to describe a compound that is derived from or related to latrunculin A or latrunculin B. The Latrunculin-like compounds described herein are compounds that share gross structural similarity with the Latrunculin A molecule, but include analogues, derivatives, metabolites, variants, congeners, or isomeric forms thereof including any of the structures 6-12 and including the generic structure as shown herein in any of the variations described herein.

"Purified" and "Isolated" when used to describe a latrunculin-like compound of the invention means that the compound is substantially free of the cellular contents that would be present in its natural physiological state within the organism from which it is derived.

"Cancer" refers to any neoplasm or neoplastically transformed cell.

"Administration of a drug/administering a drug" refers to providing a drug and contacting the drug with a subject.

The terms "drug", "therapeutic", "therapeutic agent" or "drug" mean any substance which is biologically, physiologically, or pharmacologically active, in a human being or other mammal, locally and/or systemically, and includes diagnostic agents.

The term "mammal" encompasses, but is not limited to, human beings and other mammals, and living tissue which is not part of a mammal.

The term "therapeutically effective amount" means an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent, effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the formulation to be administered, and a variety of other factors that are appreciated by those of ordinary skill in the art.

The term "diagnostic agent" means any chemical moiety that may be used for diagnosis or in a diagnostic test. For example, diagnostic agents include imaging agents containing radioisotopes, contrasting agents containing for example iodine, enzymes, fluorescent substances and the like.

The term "treatment" means the application of a process to an individual in order to alter a physiological state, whether or not the process includes a curative element.

"Controlled" release of a drug means release of the drug in a pre-determined or adjustable way such that the amount or rate or timing of release is pre-set or is altered in a desired way.

"Sustained" release of a drug means release over an extended period of time, for example minutes, hours or days, such that less than all the drug is released initially. A sustained release rate may provide, for example, a release of a certain specified amount of a drug from a dosage form, over a certain time period, under physiological conditions or in an in vitro test.

General Representations Concerning the Disclosure

In this specification, reference is made to particular features of the invention it is to be understood that the disclosure of the invention in this specification includes all appropriate combinations of such particular features. The embodiments disclosed in this specification are exemplary and do not limit the invention. As used in this specification, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a part" includes a plurality of such parts, and so forth. The term "comprises" and grammatical equivalents thereof are used in this specification to mean that, in addition to the features specifically identified, other features are optionally present. For example, a composition " comprising" (or "which comprises") ingredients A, B and C can contain only ingredients A, B and C, or can contain not only ingredients A, B and C but also one or more other ingredients. The term "consisting essentially of and grammatical equivalents thereof is used herein to mean that, in addition to the features specifically identified, other features may be present which do not materially alter the claimed invention. The term "at least" followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example "at least 1 " means 1 or more than 1, and "at least 80%" means 80% or more than 80%. The term "at most" followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, "at most 4" means 4 or less than 4, and "at most 40%" means 40% or less than 40 %. The terms "plural", "multiple", "plurality" and "multiplicity" are used herein to denote two or more than two features. Where reference is made in this specification to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility). Where reference is made herein to "a" or "an" feature, this includes the possibility that there are two or more such features (except where the context excludes that possibility). This specification incorporates by reference all documents referred to herein and all documents filed concurrently with this specification or filed previously in connection with this application, including but not limited to such documents which are open to public inspection with this specification including J. Med. Chem., 2008, 51 (22), pp 7234-7242 (Publication Date (Web): October 22, 2008) that formed part of US provisional Application No. 61/137,359, filed on 29 July 2009 the entire disclosure of which is hereby incorporated by reference for all purposes.

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Claims

Claims

1. A composition comprising one or more latrunculin-like compounds with the formula:

wherein

Rl is CH3 or H

R2 is O or OH (R or S) or H

R3 is H (R or S), OH , or OCH3

R4 is H or O or H

R5 is CH3 or H

R6 is OH or H

R7 is H (R or S)

2. The composition of claim A wherein the compound is not latrunculin A (1) or latrunculin B (2) or 16-epi-latrunculin B (3) or latrunculin C (4).

3. The composition of claim A wherein the compound is selected from the group consisting of: latrunculol A (7), latrunculol B (8), 18-epi-latrunculol A (9), latrunculol C (10), latrunculone A (I l), latrunculone B (12), latrunculol A acetonide (7a), and 15, 17, dimethoxy latrunculin A (13).

4. A pharmaceutical formulation comprising the composition of claim 1.

5. The pharmaceutical formulation of claim 4 wherein the compound exhibits cytotoxic activity against both human and murine cancer cell lines in vitro with an IC50 value range of between 0.5-10 μM.

6. The pharmaceutical formulation according to claims 4 or 5 wherein the compound exhibits significantly reduced microfϊlament-disrupting effects when compared with latrunculin A (I) when tested in an in vitro assay using AlO cells, wherein the latrunculin compound is present at a concentration of 5 μM.

7. The pharmaceutical formulation of claim 6 wherein the compound is 18-epi- latrunculol A (9).

8. The pharmaceutical formulation according to any one of claims 2, 3, 4, 5, 7 or 7 for use in the treatment of cancer.

9. A formulation comprising the composition of claim 1 for use in the treatment of gout, for reducing intraocular pressure, for treating glaucoma, for inhibiting wound healing after trabeculectomy, or for inhibiting angiogenesis.

10. A method for treating cancer comprising administering to a subject a formulation of any one of claims 2 to 9.

1 1. The method of claim 10 wherein the cancer is colon cancer, leukemia, breast cancer, lung cancer, or prostate cancer.