Isobenzofuran analogs of sclerophytin A

Isobenzofuran analogs of sclerophytin A are prepared in a highly concise fashion via an aldol-cycloaldol sequence. The analogs exhibit IC50's as low as 1 μM in growth inhibitory studies against KB3 cells using an MTT assay. Preferred analogs have one of the following structural formulas, where R is hydrogen or a substituted or unsubstituted lower alkyl group and Ar is a substituted or unsubstituted aryl group.

FIELD OF THE INVENTION

The present invention relates to isobenzofurans, particularly compounds structurally related to sclerophytin A, having anti-cancer and/or antibiotic activities.

BACKGROUND OF THE INVENTION

The 2,11-cyclized cembranoids are a class of diterpenoids isolated from a variety of marine sources that display a range of biological activities.(1) Some 2,11-cyclized cembranoids reported to possess cytotoxic activity are shown below:

Sclerophytin A has been reported to exhibit growth inhibitory activity against the L1210 cell line with an IC50of 1.0 ng/mL (3 nM).(2-4) Eleutherobin(5,6) and sarcodictyins(7,8) A and B are reported to exhibit taxol-like anti-mitotic activity.(9,10) There have been several reports of structure/activity relationship studies of sarcodictyin (11,12) or eleutherobin(13-16) analogs. However, there are fewer biological studies of any analogs of the more common isobenzofuran-containing 2,11-cyclized cembranoids.(17,18)

Recently, synthetic approaches have been developed for making selected cyclized cembranoids in which the isobenzofuran bicycle is assembled in as few as three steps from commercially available (S)-(+)-carvone using a highly stereoselective aldol-cycloaldol sequence.(19) This approach is used herein to prepare structurally related isobenzofurans that are nominal analogs of sclerophytin A.

SUMMARY OF THE INVENTION

The present invention is directed to novel isobenzofuran analogs of sclerophytin A, which have anti-cancer and/or antibiotic properties. Some preferred compounds of the present invention have one of the following basic structural formulas:

In the formulas, R represents H, or a lower alkyl group, which is substituted or unsubstituted, and Ar represents a substituted or unsubstituted aryl group. A particularly preferred compound has formula 10, wherein Ar is 2-fluorophenyl (herein referred to as compound 10h). A compound of the present invention can have a structure indicated above, or can be provided as a pharmaceutically acceptable ester, salt, or prodrug that releases a compound of the invention when metabolized.

Also contemplated is a method of treating a patient suffering from cancer or a microbial infection comprising administering to the patient a therapeutically effective amount, i.e., one that inhibits proliferation of the cancer or microbial cells, of a compound of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A compound of the present invention has a chemical structure selected from the following formulas:

In the formulas, R represents H, a substituted lower alkyl group, or an unsubstituted lower alkyl group. Ar represents a substituted or unsubstituted aryl group. Pharmaceutically acceptable esters, salts, and prodrugs of such compounds are also contemplated within the scope of the invention.

Preferably, in the formulas R=methyl, ethyl, cyclopropylmethyl or cyclopentylmethyl. It is also preferred that Ar=2-Br-phenyl, 3-Br-phenyl, 2,3-di-Cl-phenyl, 2,4-di-Cl-phenyl, 1-naphthyl, 2-pyridyl, 2-furyl, or 2-fluoropheynyl. A particularly preferred compound has structure 10, wherein Ar=2-fluorophenyl.

A method of synthesizing a compound of the present invention comprises converting (S)-(+)-carvone to an aryl glycolate derivative thereof. The conversion comprises reacting (S)-(+)-carvone with an arylaldehyde in an aldol condensation reaction to afford an aryl anti-alcohol, and etherifying the aryl anti-alcohol to afford the aryl glycolate derivative. The aryl glycolate derivative can then be cyclized to afford an isobenzofuran having structural formula 5. The isobenzofuran can then be converted by oxidative rearrangement to an enone having structural formula 6. Alternatively, the aryl glycolate derivative can be converted by β-lactonization-decarboxylation to a diene having structural formula 10.

Synthesis of isobenzofurans of the present invention by cycloaldolization is illustrated in Scheme 1. Intermolecular aldol reaction of (S)-(+)-carvone (1) and aryl aldehydes 2a-f gives anti-aldol adducts 3a-f in moderate to excellent yields and with diastereomer ratios from 1-20:1. Etherification of alcohols 3a-f proceeds in moderate to high yields to give glycolate esters 4a-f. Cycloaldolization under the influence of KHMDS afforded isobenzofurans 5a-f as single diastereomers based on1H NMR analysis.

Sclerophytin A and all other 2,11-cyclized cembranoids possess a C10 stereocenter in the alkane, rather than alcohol, oxidation state. The reduction of alcohols like 5a-d in a 3-step process via the corresponding enone have been previously reported.(19) Hence, enones 6a-d were prepared by oxidative rearrangement of the corresponding 3° alcohols.(20)

Reduction of the C10 stereocenter is illustrated by the conversion of enone 6a to tosyl hydrazone 7, which is followed by reductive transposition to afford cis-fused isobenzofuran 8 (Scheme 2).

A series of acyl derivatives (shown below) of ester 5a were prepared to evaluate the effect of the carboxyl substituent on activity. Esters 5a were prepared by transesterification in the presence of excess alcohol and Bu3SnOAc catalyst, while the corresponding carboxylic acid 9 was prepared by saponification of ester 5a.

In the course of optimizing the cycloaldol reaction of glycolate 4a to ester 5a, it was found that diene product 10 was formed in significant amounts (ca. 15%) when the reaction mixture is maintained at −78° C. for longer periods of time prior to quenching with HOAc (Scheme 3). For methyl glycolates 4g and 4h, the diene product was the only cyclized product isolated from the reaction mixture. The dienes are presumably formed by lactonization of the aldol intermediates to form unstable β-lactones 11, which upon loss of CO2afford dienes 10.(21)

Biological Assays

The human KB-3 carcinoma cell line was used to perform the MTT colorimetric assay of cell viability.(22-24) Cells were treated with increasing concentrations of compounds to assess their growth inhibitory properties. IC50values (concentration of the drug required to reduce cell viability by 50%) ranged from 1 to >100 μM (Table 1). The concentration curve for 5c, which had an IC50of 5 μM, is shown inFIG. 1, Panel B. Compounds 5a (FIG. 1, Panel A) and 6a (FIG. 1, Panel C) had higher IC50in the range of 20 and 70 μM, respectively. Several other compounds, e.g., 8 (FIG. 1, Panel D), showed no evident growth inhibitory activity at the highest concentration tested (100 μM).

TABLE 1Inhibition of KB-3 cell survival assessed by MTT viability assay.EntryCompoundIC50(μM)15a2025a.i535a.ii5045a.iii>10055b365c575d385e1095f100105i>100116a70126b30136c5146d3158>100169>1001710a41810e>1001910g22010h1
Discussion

Several structure-activity trends become apparent upon examination of the assay data. Firstly, for the 2-Br ester compounds 5a (entries 1-6), the smaller the alcohol moiety of the ester, the lower the IC50, i.e. Me<Et<cyclopropylmethyl<cyclopentylmethyl˜neryl. Secondly, the ethyl ester alcohols 5a-d as a group were more active than the ethyl ester enones 6a-d as a group (entries 1, 5-7 vs. 11-14), with individual differences in the IC50values in the groups varying by factors of 1 to 23.

The essentially equal potency of hydroxy esters 5b-d (entries 5-7) and dienes 10a, g, h (entries 17, 19, 20) is intriguing and suggests that the esters may undergo lactonization and decarboxylation to the dienes in vivo (cf. Scheme 3). This notion is supported by the substantial difference in activity between β-OH ester 5a, which can undergo lactonization, and the corresponding C10 reduction product 8, which cannot (entries 1 and 15, respectively). On the other hand, the 1-napthyl methyl ester 5e (entry 8) is at least 10-fold more active than the corresponding diene 10e (entry 18). Furthermore, enones 6a-d (entries 11-14) exhibit essentially equal potency to the β-OH methyl and ethyl esters, and also cannot undergo β-lactone formation. Further studies would be needed to determine whether β-hydroxy esters 5, enones 6, and dienes 10 are inhibiting growth by the same or different mechanisms of action.

The most active representatives of the compound classes (alcohol 5d, enone 6d and diene 10h) were submitted to NCI's Developmental Therapeutics program for 60-cell line screening. Single dose assays revealed no significant activity for alcohol 5d. Enone 6d exhibited significant differential activity against the RPMI-8226 leukemia and the PC-3 prostate cancer cell lines. Diene 10h was the most active compound tested, possessing significant differential activity against the entire leukemia panel and the NCI-H522 non-small cell lung cancer cell line. Subsequent 5-dose testing of 10h revealed a GI50=0.148 μM and LC50=9.36 μM for the RPMI-8226 leukemia cell line, and a GI50=0.552 μM and LC50=26.8 μM for the HOP-92 non-small cell lung cancer cell line.

CONCLUSION

A structurally novel set of analogs has been developed based on the isobenzofuran bicycle common to most of the 2,11-cyclized cembranoids that exhibit IC50's as low as 1 μM for growth inhibition against KB3 cells. Analog 10h possesses sub-micromolar growth inhibitory activity against the RPMI-8226 leukemia and HOP-92 non-small cell lung cancer cell lines.

The present invention is now described with reference to certain examples, which explain but do not limit it.

EXAMPLES

Cell Proliferation Studies

Inhibition of cell proliferation was assessed by the MTT assay. This assay is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and form a dark blue formazan crystal product which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells.(22) Solubilization of the cells by the addition of solvent results in the liberation of the crystals which are solubilized. The level of the formazan product created is directly proportional to the number of living cells.(23,24)

All stock solutions of compounds were made at 10 mM in dimethyl sulfoxide (DMSO). DMSO and MTT were from Sigma Chemical Co. (St. Louis, Mo.). Cell culture reagents were obtained from Life Technologies (Carlsbad, Calif.). The KB-3 human carcinoma cell line was maintained in monolayer culture at 37° C. and 5% CO2in Dulbecco's Modified Eagle's Medium, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 units/mL penicillin, and 50 μg/mL streptomycin.

KB-3 cells (2000/well) were plated in 96-well dishes, and after 24 h were treated with 0.1 nM-100 μM of the compound in question. The final concentration of DMSO did not exceed 1%, and controls received vehicle alone. After 96 h, cells were incubated with 50 μg MTT/well/0.2 mL for 4 h at 37° C., the media was removed, and the formazan crystals were dissolved in 0.15 mL of DMSO. Absorbance at 570 nm was measured with an ELx800™ Microplate Reader (Bio Tek Instruments, Inc., Winooski, Vt.). All treatments were performed in triplicate. Results are shown inFIGS. 1A-Das a percentage of control samples (mean±SD). IC50is the concentration that reduced survival to 50% of the control (no drug).

The present invention has been described hereinabove with reference to particular examples for purposes of clarity and understanding rather than by way of limitation. It should be appreciated that certain improvements and modifications can be practiced within the scope of the appended claims.

REFERENCES