Anti-migration and anti-invasion thiazole analogs for treatment of cellular proliferative disease

Abstract

Thiazole analog compounds and their pharmaceutically acceptable salts are disclosed, including pharmaceutical compositions comprising the thiazole analog compounds, either alone or in combination with at least one additional therapeutic agent, and/or with a pharmaceutically acceptable carrier. Methods of using the thiazole analog compounds, either alone or in combination with at least one additional therapeutic agent, in the prophylaxis or treatment of cellular proliferative diseases, such as cancer, are also disclosed.

Claims

1. A compound of Formula (I) ##STR00019## wherein R.sup.1 is phenyl; R.sup.2 is absent; R.sup.3 is ethyl, n-propyl, n-butyl, n-hexyl, n-dodecyl, allyl, or propynyl; R.sup.4 is hydrogen; R.sup.5 is C(O); R.sup.6 is phenyl or xylyl; and the dashed lines represent optional double bonds; and wherein the optional double bond between the amino nitrogen and position 2 of the thiazole ring is present; or a salt thereof; or a compound of formula 5k: ##STR00020## or a salt thereof.

2. The compound of claim 1, wherein said compound is formula 5l: ##STR00021## or a salt thereof.

3. The compound of claim 1, wherein said compound is formula 5m: ##STR00022## or a salt thereof.

4. The compound of claim 1, wherein said compound is formula 5n: ##STR00023## or a salt thereof.

5. The compound of claim 1, wherein said compound is formula 5o: ##STR00024## or a salt thereof.

6. The compound of claim 1, wherein said compound is formula 5p: ##STR00025## or a salt thereof.

7. The compound of claim 1, wherein said compound is formula 5q: ##STR00026## or a salt thereof.

8. The compound of claim 1, wherein said compound is formula 5r: ##STR00027## or a salt thereof.

9. The compound of claim 1, wherein said compound is formula 7b: ##STR00028## or a salt thereof.

10. The compound of claim 1, wherein said compound is formula 7c: ##STR00029## or a salt thereof.

11. The compound of claim 1, wherein said compound is formula 7d: ##STR00030## or a salt thereof.

12. The compound of claim 1, wherein said compound is formula 8a: ##STR00031## or a salt thereof.

13. A method of treating a cancer selected from the group consisting of metastatic cancer, breast cancer and non-small cell lung cancer, in a mammal in need thereof, the method comprising administering to the mammal a composition comprising the compound of claim 1.

14. A method of inhibiting cell migration in a mammal with metastatic cancer, breast cancer or non-small cell lung cancer, the method comprising administering to the mammal a composition comprising the compound of claim 1.

15. A method of inhibiting cell invasion in a mammal with metastatic cancer, breast cancer or non-small cell lung cancer, the method comprising administering to the mammal a composition comprising the compound of claim 1.

16. A composition comprising the compound of claim 1, wherein said composition is in a form of a product for oral delivery, said product form being selected from the group consisting of a concentrate, dried powder, liquid, capsule, pellet, and pill.

17. A composition comprising the compound of claim 1, wherein said composition is in a form of a product for parenteral, intravenous, intradermal, intramuscular, or subcutaneous administration.

18. A composition comprising the compound of claim 1, further comprising at least one carrier, binder, diluent, or excipient.

19. A composition comprising the compound of claim 1, further comprising a chemotherapeutic agent.

20. The method of claim 13, wherein said compound is administered at from about 0.01 to about 40 mg/kg/day.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a further understanding of the nature, objects, and advantages of the present disclosure, reference should be had to the following detailed description, read in conjunction with the following drawings, wherein like reference numerals denote like elements.

(2) FIG. 1 shows the X-ray crystal structures of 4e (FIG. 1A) and 5j (FIG. 1B).

(3) FIG. 2 shows the synthesis of novel anti-migration and anti-invasion thiazole analogs.

(4) FIG. 3 shows that the thiazole analogs inhibit migration of MDA-MB-231 breast cancer cells.

(5) FIG. 4 shows clonogenic assay results demonstrating no cytotoxicity of the thiazole analogs that potently inhibit migration of MDA-MB-231 cells in four out of five selected compounds. Images of colony formation are shown for cells treated with 1) DMSO, 2) 2b, 3) 5j, 4) 3n, 5) 4e, and 6) 5k.

(6) FIG. 5 shows the effect of selected thiazole analogs on the migration and proliferation of HeLa cells.

(7) FIG. 6 shows that analog 5k strongly inhibits migration of A549, a metastatic non-small cell lung cancer cell line.

(8) FIG. 7 shows the effect of selected thiazole analogs on the invasion of MDA-MB-231 breast cancer cells.

(9) FIG. 8 shows the possible mechanism of action of 5k in blocking cell migration and invasion. Analog 5k strongly suppresses actin-rich membrane protrusions in MDA-MB-231 cells. F-actin staining in (A) Control cells (DMSO-treated); or (B) Cells treated with 5k; and (C). Quantitation of-actin intensity in vehicle and 5k-treated cells.

(10) FIG. 9 shows the immunofluorescence microscopic images of localization of fascin (green) and f-actin (magenta) in MDA-MB-231 cells treated with DMSO (vehicle) or synthetic analog 5k. Co-localization of f-actin and fascin appears as white pixels.

(11) FIG. 10 shows a reaction scheme for synthesis of the analogs of lead compounds. NaH is sodium hydride; THF is tetrahydrofuran; rt is room temperature; RBr indicates a brominated R group that provides the R.sup.2 and R.sup.3 substituents of compounds 5-8, as shown; PTC is phase transfer catalyst; DMAP is 4-dimethylaminopyridine; and MeI is methyl iodide.

DETAILED DESCRIPTION

(12) Before the subject disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments of the disclosure described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present disclosure will be established by the appended claims.

(13) In this specification and the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.

(14) Detailed descriptions of one or more preferred embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.

(15) One aspect of the present invention describes the synthesis of novel thiazole analogs. Another embodiment of the invention describes the potent anti-migration and anti-invasion effects on metastatic cancer cells exhibited by the disclosed thiazole analogs.

(16) To determine if these analogs have anti-migration and anti-invasion activities in different cancer cell lines, transwell migration assays were performed using MDA-MB-231 breast cancer cells (FIG. 3) and HeLa cells (FIG. 5) for all synthetic analogs. The most potent analog, 5k, was also tested in a non-small cell lung cancer cell line, A549 (FIG. 6). Next, clonogenic assays were performed on the breast cancer cells treated with the compounds to rule out any indirect effect on cell migration due to cytotoxicity (FIG. 4). MDA-MB-231 cells were allowed to grow for 14 days in six-well plates in the presence or absence of the synthetic compounds at 10 M.

(17) As used herein, the term metastatic cancer refers to any cancer having invasive and metastatic potential. The terms minimize or reduce, or derivatives thereof, include a complete or partial inhibition of a specified biological effect (which is apparent to one of ordinary skill from the context in which the terms minimize or reduce are used).

(18) Methods of Administration

(19) The compounds of the invention are useful in vitro or in vivo in inhibiting the growth of cancer cells. The compounds may be used alone or in compositions together with a pharmaceutically acceptable carrier or excipient. Suitable pharmaceutically acceptable carriers or excipients include, for example, processing agents and drug delivery modifiers and enhancers, such as, for example: calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl--cyclodextrin, polyvinyl-pyrrolidinone, low melting waxes, ion exchange resins, and the like, as well as combinations of any two or more thereof. Other suitable pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences, Mack Pub. Co., New Jersey, 1991, incorporated herein by reference.

(20) Effective amounts of the compounds of the invention generally include any amount sufficient to: (1) detectably limit actin polymerization in a cell of interest; (2) detectably inhibit cancer presentations, such as metastasis; or (3) alleviate symptoms of cancer in a patient or animal treated with a thiazole analog described herein.

(21) The amount of thiazole analog active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific thiazole analog employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease being treated.

(22) Accordingly, the novel thiazole analogs of the present invention can be provided in vivo to a mammal in need thereof, in any manner, for treatment of metastatic cancer for example, with the desired outcome of blocking or delaying the onset of metastasis. The thiazole analogs of the present invention can be provided by any route acceptable for administration, and at any dose acceptable for a non-cytotoxic therapeutic agent.

(23) For example, the thiazole analogs of the present invention can be provided to an animal at about 0.01, about 0.1, about 1, about 5, about 10, about 20, about 30, or about 40 mg per kilogram per animal per day. The thiazole analogs of the present invention can be provided at from about 0.01 to about 40, from about 0.1 to about 40, from about 1 to about 40, from about 5 to about 40, from about 10 to about 40, from about 20 to about 40, from about 30 to about 40, from about 0.01 to about 30, from about 0.01 to about 20, from about 0.01 to about 10, from about 0.01 to about 5, and from about 0.01 to about 1 mg per kilogram per day. A particular embodiment of the present invention comprises administration of the thiazole compound 5k at at least one of the aforementioned dosages.

(24) As aforementioned, the thiazole analogs of the present invention can be administered at from about 1 mg to about 40 mg/kg of body weight daily, in a human or animal, being so treated. Further, a therapeutically effective dosage of a thiazole compound or composition comprising such a compound, may include a total daily dose administration of for example, from about 0.001 to 1000 mg/kg of body weight daily, or from about 0.01 to 100 mg/kg of body weight daily, or from about 0.1 to 10 mg/kg of body weight daily. Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dosages.

(25) The thiazole compounds of the present invention may be administered orally, parenterally, sublingually, by aerosolization or inhalation of a spray, rectally, or topically, in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches.

(26) Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using skiable dispersing or wetting agents and suspending agents.

(27) Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active thiazole analog may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents.

(28) While various preferred embodiments of the invention have been disclosed above, it will be appreciated that changes can be made to these embodiments without departing from the spirit and scope of the invention.

EXAMPLE 1

Synthesis of Thiazole Analogs

(29) Thiazole derivatives were designed by varying the substituent groups R.sup.6, R.sup.1, R.sup.2, and R.sup.3 either individually or in combination on the basic structures of 2, 3, 4, and 5 as illustrated in FIG. 2.

(30) To synthesize 2-Amino-4-phenylthiazole (2a), a mixture of 2-bromo-1-phenylethanone (19.9 g, 0.1 mol) and thiourea (8.4 g, 0.11 mol) in anhydrous EtOH (200 mL) was heated at reflux for 1 h. After that, the solvent was removed in vacuo, and saturated aqueous NaHCO.sub.3 was added to make the mixture basic (pH=8-9). Then, the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic phases were washed with brine and dried with MgSO.sub.4. After removal of the solvent, the residue was stirred for 20 min with petroleum ether and filtered to afford 2a (17.1 g, 97%) as a solid. H-NMR (CDCl.sub.3): 7.78-7.76 (2H, m), 7.40-7.36 (2H, m), 7.31-7.27 (1H, m), 6.72 (1H, s), 5.13 (2H, s, br). MS-EI: 176 (M.sup.+). HRMS (ESI(+)): Calcd. for C.sub.9H.sub.9N.sub.2S (M+H): 177.0486. Found: 177.0477.

(31) To synthesize 2-Amino-4-(2,4-dimethylphenyl)thiazole (2b), a mixture of 2-bromo-1-(2,4-dimethyl phenyl)ethanone (22.7 g, 0.1 mol) and thiourea (8.4 g, 0.11 mol) in anhydrous EtOH (200 mL) was heated at reflux for 1 h. After that, the solvent was removed in vacuo, and saturated aqueous NaHCO.sub.3 was added to make the mixture basic (pH=8-9). Then, the mixture was extracted with CH.sub.2Cl.sub.2. The combined organic phases were washed with brine and dried with MgSO.sub.4. After removal of the solvent, the residue was stirred for 20 min with petroleum ether and filtered to afford 2b (24.6 g, 100%) as a solid. .sup.1H-NMR (CD.sub.3OD): 7.27 (1H, J=7.6 Hz, d), 7.19 (1H, s), 7.13 (1H, J=0.4 and 7.6 Hz, dd), 6.74 (1H, s), 2.35 (3H, s), 2.34 (3H, s). MS-EI: 204 (M.sup.+). HRMS (ESI(+)): Calcd. for C.sub.13H.sub.13N.sub.2S (M+H): 205.0799. Found: 205.0798.

(32) To synthesize analogs 3a, 3b, 3c, 3d, 3e, and 3f, a solution of 2 (0.01 mol) and R.sup.1CO.sub.2H (0.015 mol) was prepared in anhydrous dichloromethane, to which were added dicyclohexylcarbodimide (0.02 mol) and DMAP (0.61 g, 0.005 mol). After stirring overnight at room temperature under nitrogen atmosphere, petroleum ether was added to the reaction mixture to facilitate precipitates, and then the solution was filtered, and concentrated in vacuo. The residue was purified by flash chromatography to give the corresponding 3 analogs as solid.

(33) To synthesize analogs 3g, 3h, 3i, 3j, 3k, 3l, 3m, 3n, and 3o, a mixture of R.sup.1CO.sub.2H (0.015 mol) and thionyl chloride (7.12 g, 4.4 mL, 0.06 mol) was refluxed for 2 h followed by removal of excess thionyl chloride in vacuo. To a solution of 2 (0.01 mol) and DMAP (1.24 g, 0.01 mol) in anhydrous dichloromethane was added the above acyl chloride or commercially available acyl chloride in dichloromethane dropwise at 0 C. After stirring at room temperature for 2 h under nitrogen atmosphere, the reaction mixture was concentrated in vacuo. The saturated Na.sub.2CO.sub.3 solution was added to quench the reaction, and the solution was extracted with ethyl acetate, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by flash chromatography to give the desired 3 analogs as a solid.

(34) To synthesize analogs 4 and 5, a cooled mixture of NaH (0.26 g, 60% in oil, 6.5 mmol) in THF (20 mL) was prepared, to which was added a solution of various 3 analogs (5 mmol) in THF (10 mL) dropwise. The mixture was warmed up to room temperature and stirred for 20 min. After that, the mixture was cooled to 0 C. again and MeI or EtBr (6.5 mmol) was added dropwise. The mixture was then warmed up to room temperature and stirred for 2 h. Water (5 mL) was added to quench the reaction and the mixture was further diluted with water (50 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic phase was dried with anhydrous MgSO.sub.4. After removal of all the solvent, the residue was purified by silica gel chromatography (hexane/EtOAc=4:1) to afford product 4 and 5 (more polar) as solid.

EXAMPLE 2

Thiazole Analogs Inhibit Migration of MDA-MB-231 Breast Cancer Cells

(35) To determine the effects of the synthesized mackroketone analogs on cancer cell migration, we performed transwell migration assay on each compound using an invasive and metastatic breast cancer cell line, MDA-MB-231. When cells were seeded at a density of 2.510.sup.4 in media free of serum in the upper chamber but containing 5% FBS in the lower chamber, their ability to migrate in the presence and absence of 10 M analogs were measured by counting the total number of cells in the lower chamber after 24 hrs. As shown in FIG. 3, of the 37 synthetic analogs, most displayed moderate or potent anti-migration activity, 19 analogs showed greater than 50% inhibition, and three most potent analogs (3g, 5j and 5k) blocked cell migration by over 80%. These results demonstrate that the synthetic analogs are effective migration inhibitors.

EXAMPLE 3

Clonogenic Assay of MDA-MB-231 Cells Treated with Analogs

(36) To rule out any indirect effect on cell migration due to cytotoxicity, clonogenic assays were performed on the breast cancer cells treated with the compounds. MDA-MB-231 cells were allowed to grow for 14 days in six-well plates in the presence or absence of the synthetic compounds at 10 M. FIG. 4 shows the proliferation and colony formation of five anti-migration analog-treated colony images compared to DMSO treated control cells. The five compounds, 2b, 5j, 3n, 4e, and 5k all inhibited cell migration by over 50% (55% to 86%), yet no apparent cell toxicity was observed when cells were treated with the analogs at the same dose of 10 M for two weeks, with the exception of 5j, which significantly inhibited colony formation of the breast cancer cells by 75%. A few other anti-migration analogs also exhibit moderate level of inhibition of the breast cancer cell proliferation. For example, analog 3g blocked both cell migration (81.4%) and colony formation (42.7%). For these analogs cytotoxity may have partially contributed to their overall anti-migration effect. The cell toxicity data for all analogs are listed in Table 1 along with anti-migration data for comparison.

(37) TABLE-US-00001 TABLE 1 List of all synthetic analogs with migration inhibition and colony formation data when MDA-MB-231 breast cancer cells were treated with 10 M analogs Effect on cell proliferation Migration Inhibition (colony formation) Analogs (% of vehicle control) (% of vehicle control) DMSO 100.0% 100.0% 2a 86.0% 103.6% 2b 45.5% 110.9% 3a 34.5% 85.9% 3b 88.0% 98.8% 3c 78.0% 95.0% 3d 103.0% 98.6% 3e 46.5% 80.9% 3f 40.9% 67.5% 3g 18.6% 57.3% 3h 80.8% 102.8% 3i 32.3% 95.5% 3j 31.3% 105.2% 3k 68.2% 99.8% 3l 92.7% 103.4% 3m 75.2% 90.7% 3n 28.3% 71.5% 3o 33.7% 85.0% 4a 78.2% 107.7% 4b 72.2% 97.7% 4c 43.5% 97.1% 4d 54.5% 96.5% 4e 39.2% 123.4% 4f 54.3% 132.4% 4g 72.7% 97.5% 4h 71.4% 127.5% 4i 80.0% 111.1% 5a 43.3% 85.0% 5b 77.6% 107.3% 5c 28.1% 98.2% 5d 59.7% 63.8% 5e 26.5% 75.4% 5f 37.1% 102.3% 5g 35.9% 40.9% 5h 62.0% 111.5% 5i 32.1% 97.7% 5j 13.0% 25.2% 5k 14.3% 120.9%

EXAMPLE 4

Anti-Migration Activities and Effect on Colony Formation in Metastatic HeLa Cells

(38) Based on the potent effects of synthetic thiazole analogs in blocking migration of MDA-MB-231 cells, ten analogs with high anti-migration activity but low or negligible cytotoxicity were selected to test their anti-migration activity in another metastatic cell line, HeLa. For comparison of possible cancer cell specific mode of action we also included 5j, a potent anti-migration analog that also inhibited the proliferation of the triple negative breast cancer cells. Results are summarized in FIG. 4. The analogs exhibited excellent anti-migration activity in HeLa cells, as evidenced by the dramatically reduced transwell migration (60-86% inhibition) when treated with 10 M analogs. While these analogs demonstrated similar anti-migration efficacies in MDA-MB-231 and HeLa cells, some differences were noted. For example, the analog 3n showed 30% inhibition of colony formation in MDA-MB-231 cells but had no apparent cytotoxicity in HeLa cells. On the other hand, the analog 5i was not toxic to 231 cells, but it suppressed the clonogenic capability of HeLa cells by 45%. Interestingly, the analog 5j was a strong inhibitor of cell proliferation for both HeLa and MDA-MB-231 cells. The analog that emerged as the most potent anti-migration agent with no apparent cytotoxity in both metastatic breast cancer and cervical cancer cell lines was 5k, achieving over 85% inhibition of transwell migration in both cell lines at the dose of 10 M. Thus 5k may be further evaluated for its potential as an anti-migration and anti-metastatic agent.

EXAMPLE 5

Antimigration Activity of Thiazole Analog 5k in A549 Non Small Cell Lung Cancer Cells

(39) Additional migration assay of a non small cell lung cancer cell line, A549 also demonstrated that in the presence of 10 M 5k, the metastatic lung cancer cells lost nearly 80% of migratory capacity as indicated in FIG. 6.

EXAMPLE 6

Thiazole Analogs Show Low IC50 Concentration in MDA-MB-231 Cells

(40) Given the potent anti-migration efficacy demonstrated by most of the thiazole analogs, we decided to study the dose response of the most potent analogs to obtain their IC50 values in suppressing the transwell migration of the MDA-MB-231 cells. As shown in Table 2, the IC50 values for the 10 selected analogs varied from 2.87 M to 0.176 M.

(41) TABLE-US-00002 TABLE 2 IC.sub.50 values for 10 most potent anti-migration compounds in MDA-MB-231 breast cancer cells Compound IC.sub.50 (M) 3a 2.49 3i 2.87 3j 1.29 3n 1.01 3o 0.839 4e 0.366 5c 1.12 5i 2.08 5j 0.189 5k 0.176

EXAMPLE 7

Effect of Selected Thiazole Analogs on the Invasion of MDA-MB-231 Breast Cancer Cells

(42) To determine if the synthetic thiazole analogs block invasion of metastatic cancer cells we performed matrigel invasion assays of MDA-MB-231 cells treated with 10 selected analogs.

(43) As shown in FIG. 7, all 10 analogs exhibited marked inhibition of matrigel invasion of the breast cancer cells, with percent invasion reduced to approximately 40-60% of the control.

(44) The analog 5k, the most active anti-migration agent without any apparent cytotoxity, also appears to be the most potent compound in blocking cell invasion.

EXAMPLE 8

Analog 5k Strongly Suppresses Actin-Rich Membrane Protrusions in MDA-MB-231 Breast Cancer Cells

(45) An essential component of migration is protrusion of the cell membrane, which is driven by actin polymerization. The related compound known as migrastatin has been shown to block actin bundling by binding to the actin regulatory protein fascin, which is linked to migration in cell culture systems, and metastasis in vivo. See Chen, L.; Yang. S.; Jakoncic, J.; Zhang, J. J.; Huang, X. Y. Migrastatin analogues target fascin to block tumour metastasis. Nature 2010; 464:1062-1066. Erratum in: Nature. 2011; 476:240; Hashimoto, Y., D. J. Kim, and J. C. Adams. The roles of fascins in health and disease. J. Pathol. 2011; 224:289-300; and Jayo, A., and M. Parsons. Fascin: a key regulator of cytoskeletal dynamics. Int. J. Biochem. Cell Biol. 2010; 42:1614-1617. While the previous study by Chen et al. showed that migrastatin blocked the actin bundling activity of fascin using purified proteins in vitro, the effects on actin structures in cells were not tested. See Chen, 2010. Thus, we tested the hypothesis that compound 5k interferes with f-actin in membrane protrusions associated with cell motility. MDA-MB-231 cells were serum starved /+10 M 5k, then stimulated with serum for 2 hours to induce actin-rich membrane protrusions, which were analyzed by fluorescent microscopy. FIG. 8 shows that control cells (DMSO-treated) had robust actin-rich membrane protrusions, which were significantly reduced in cells treated with 5k (55% reduction in f-actin intensity; p<0.01).

EXAMPLE 9

Compound 5k Significantly Blocks f-Actin and is Correlated with the Absence of Fascin in Membrane Protrusions

(46) To further probe for a possible role of fascin in the reduction of actin-rich membrane protrusions, we determined the localization of fascin by immunofluorescence microscopy. FIG. 9 shows that in control cells, a pool of fascin is localized within the zone of f-actin in the protrusions. This pool of fascin was notably missing from the actin-rich membrane regions in the cells treated with 5k (indicated by arrowheads). Thus, the results demonstrate that compound 5k significantly blocks f-actin and is correlated with the absence of fascin in the membrane protrusions, suggesting that its mechanism of action is to perturb the actin dynamics required for tumor cell migration.

EXAMPLE 10

Synthesis of Additional Thiazole Analogues

(47) As shown in FIG. 10, the compounds 5, 7 and their isomers 6 and 8 were respectively obtained from N-alkylation of the amides 3 and 4 which were prepared following the literature procedure. See Zheng, S.; Zhong, Q.; Jiang, Q.; Mottamal, M.; Zhang, Q.; Zhu, N.; Burow, M. E.; Worthylake, R. A.; Wang, G. Discovery of a series of thiazole derivatives as novel inhibitors of metastatic cancer cell migration and invasion. ACS Med. Chem. Lett. 2013; 4:191-196. 4-(2,4-Dimethylphenyl)thiazol-2-amine (9) was treated with benzenesulfonic chloride to give 10, which was transferred to the analog 11 and its isomer 12 by the N-methylation reaction in THF. The acylation of the 2-aminothiazoles 13 by acyl chloride provided the amides 14 at room temperature in dichloromethane, and further methylation of 14 led to the desired analogs 15 and their corresponding isomers 16.

(48) All reagents and solvents were purchased from AK Scientific, Sigma-Aldrich Chemical Co., Fisher Scientific, ACROS and Pharmco-AAPER and were used as received. Aldrich Chemical Co. (WI, USA) or Acros organics (NY, USA) and were used as received. All organic solvents (Pharmco-AAPER) used were of reagent grade quality and were used without further purification. NMR spectra were recorded on a Bruker Fourier-300 spectrometer (Bruker Inc., Billerica, Mass.) in ppm. Melting points were determined with a Mel-temp II point apparatus and are uncorrected. Crude synthetic products were purified by the following methods: chromatography on Silica Gel (60-100 mesh, Fisher Scientific) column. Analytical thin layer chromatography (TLC) was performed on 250 fluorescent plates (Agela Tech., DE, USA) and visualized by using UV light. For all products, the purity was ascertained to be greater than 95% by the HPLC method using a Shimadzu (Columbia, Md.) 2010 HPLC-UV/MS system with a C-18 reverse phase column and by GC-MS analyses using an Agilent Technologies 5975C inert MSD mass spectrometer.

(49) General procedure for N-alkylation of 3 and 4. To a cooled mixture of NaH (0.26 g, 60% in oil, 6.5 mmol) in THF (20 mL) was added a solution of compound 3 or 4 (5 mmol) in THF (10 mL) dropwise. The mixture was warmed up to room temperature and stirred for 20 min. After that, the mixture was cooled to 0 C. again and MeI or RBr (6.5 mmol) was added dropwise. The mixture was then warmed up to room temperature and stirred for 2 h. Water (5 mL) was added to quench the reaction and the mixture was further diluted with water (50 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic phase was dried with anhydrous MgSO.sub.4. After removal of all the solvent, the residue was purified by silica gel chromatography (hexane/EtOAc=4:1) to afford product 5 (more polar) and 6 or 7 (more polar) and 8 as solid.

(50) 5l: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (dd, J=1.8 and 8.1 Hz, 2H), 7.49-7.42 (m, 3H), 7.18-7.10 (m, 3H), 6.45 (s, 1H), 4.35 (m, 1H), 3.82 (m, 1H), 1.24 (t, J=6.9 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 167.8, 140.1, 137.82, 137.75, 137.2, 131.3, 131.2, 130.6, 129.2, 128.0, 127.2, 126.8, 106.8, 42.1, 21.3, 19.6, 13.8. GC-MS: 336 (M.sup.+).

(51) 5m: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (m, 2H), 7.52-7.42 (m, 3H), 7.17-7.09 (m, 3H), 6.45 (s, 1H), 4.28 (m, 1H), 3.71 (m, 1H), 2.41 (s, 3H), 2.13 (s, 3H), 1.70 (m, 2H), 0.81 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.0, 168.1, 140.0, 138.2, 137.7, 137.2, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8, 106.8, 48.5, 21.8, 21.3, 19.6, 11.2. GC-MS: 350 (M.sup.+).

(52) 5n: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (m, 2H), 7.52-7.42 (m, 3H), 7.17-7.09 (m, 3H), 6.45 (s, 1H), 4.33 (m, 1H), 3.73 (m, 1H), 2.40 (s, 3H), 2.14 (s, 3H), 1.64 (m, 2H), 1.22 (m, 2H), 0.81 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 168.1, 140.1, 138.2, 137.7, 137.2, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8, 106.9, 46.7, 30.4, 21.3, 19.9, 19.6, 13.6. GC-MS: 364 (M.sup.+).

(53) 5o: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (dd, J=1.5 and 7.8 Hz, 2H), 7.52-7.42 (m, 3H), 7.17-7.09 (m, 3H), 6.45 (s, 1H), 4.33 (m, 1H), 3.72 (m, 1H), 2.41 (s, 3H), 2.14 (s, 3H), 1.61 (m, 2H), 1.23-1.17 (m, 6H), 0.81 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 168.0, 140.1, 138.1, 137.7, 137.2, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8, 106.9, 46.9, 31.1, 28.2, 26.2, 22.4, 21.3, 19.6, 13.9. GC-MS: 392 (M.sup.+).

(54) 5p: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.35 (m, 2H), 7.49-7.26 (m, 3H), 7.17-7.09 (m, 3H), 6.44 (s, 1H), 4.32 (m, 1H), 3.73 (m, 1H), 2.40 (s, 3H), 2.13 (s, 3H), 1.59-1.50 (m, 4H), 1.28-1.17 (m, 16H), 0.88 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 168.1, 140.0, 138.1, 137.7, 137.3, 131.3, 131.2, 130.7, 129.2, 128.0, 127.3, 126.8, 106.9, 46.9, 32.8, 31.9, 29.7, 29.6, 29.5, 29.4, 29.3, 28.9, 28.2, 26.5, 25.8, 22.7, 21.3, 19.6, 14.1. MS (ESI): 477 (M+H).

(55) 5q: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.35 (m, 2H), 7.51-7.41 (m, 3H), 7.14-7.07 (m, 3H), 6.46 (s, 1H), 5.87 (m, 1H), 5.10 (dd, J=1.2 and 10.2 Hz, 1H), 4.98 (dd, J=1.2 and 17.1 Hz, 2H), 4.42 (m, 1H), 2.40 (s, 3H), 2.13 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.2, 168.1, 140.1, 137.8, 137.1, 131.3, 131.1, 130.8, 129.2, 128.0, 127.0, 126.6, 118.4, 106.8, 49.1, 21.3, 19.7. GC-MS: 348 (M.sup.+).

(56) 5r: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.40 (m, 2H), 7.52-7.42 (m, 3H), 7.23-7.11 (m, 3H), 6.47 (s, 1H), 4.80 (m, 2H), 2.40 (s, 3H), 2.21 (m, 1H), 2.19 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.4, 168.1, 140.4, 138.1, 137.1, 136.9, 131.6, 131.4, 130.8, 129.4, 128.1, 126.9, 126.4, 106.9, 77.3, 72.4, 36.0, 21.4, 19.9. MS (ESI): 347 (M+H).

(57) 6: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (dd, J=1.5 and 8.1 Hz, 2H), 7.52-7.43 (m, 4H), 7.20 (s, 1H), 7.18 (s, 2H), 4.75 (m, 2H), 2.42 (s, 3H), 2.22 (m, 1H), 2.21 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.7, 167.3, 141.0, 138.5, 136.2, 135.9, 131.9, 131.6, 130.5, 129.5, 128.1, 127.3, 124.8, 98.0, 76.97, 72.7, 37.1, 21.5, 19.6. MS (ESI): 347 (M+H).

(58) 7a: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.92 (m, 2H), 7.55-7.48 (m, 5H), 7.42 (m, 2H), 7.34 (m, 1H), 7.25 (s, 1H), 4.27 (q, J=7.5 Hz, 2H), 1.38 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.6, 159.0, 149.5, 135.2, 134.8, 130.4, 128.68, 128.65, 127.8, 126.7, 126.0, 109.2, 45.2, 14.1. GC-MS: 308 (M.sup.+).

(59) 7b: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (dd, J=1.2 and 7.5 Hz, 2H), 7.50-7.39 (m, 8H), 6.52 (s, 1H), 4.19 (q, J=7.5 Hz, 2H), 1.76 (m, 2H), 0.83 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 168.5, 139.3, 137.2, 131.3, 130.9, 129.6, 129.5, 129.2, 128.8, 128.7, 128.0, 126.0, 107.2, 48.8, 21.9, 11.1. GC-MS: 322 (M.sup.+).

(60) 7c: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.35 (dd, J=1.8 and 6.3 Hz, 2H), 7.49-7.43 (m, 8H), 6.55 (s, 1H), 5.98 (m, 1H), 5.19 (d, J=10.2 Hz, 1H), 4.98 (d, J=17.4 Hz, 1H), 4.82 (d, J=5.1 Hz, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.3, 168.5, 139.3, 137.0, 131.9, 131.4, 130.6, 129.7, 129.6, 129.2, 128.7, 128.0, 118.0, 107.1, 49.6. GC-MS: 320 (M.sup.+).

(61) 7d: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.40 (d, J=6.6 Hz, 2H), 7.57-7.44 (m, 8H), 6.56 (s, 1H), 4.92 (d, J=2.1 Hz, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.4, 168.5, 138.6, 136.8, 131.6, 130.1, 129.9, 129.44, 129.37, 129.0, 128.1, 77.8, 72.8, 36.9. LC-MS (ESI): 319 (M+H).

(62) 8a: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (dd, J=1.5 and 7.5 Hz, 2H), 7.51-7.40 (m, 8H), 6.52 (s, 1H), 4.26 (q, J=6.9 Hz, 2H), 1.34 (t, J=6.9 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.2, 168.1, 139.1, 137.1, 130.9, 129.6, 129.4, 129.2, 128.9, 128.0, 107.2, 42.5, 14.0. GC-MS: 308 (M.sup.+).

(63) 8b: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.91 (dd, J=1.5 and 6.9 Hz, 2H), 7.53-7.32 (m, 8H), 7.24 (s, 1H), 4.18 (q, J=7.5 Hz, 2H), 1.83 (m, 2H), 0.83 (t, J=7.5 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.8, 159.3, 149.5, 135.2, 134.8, 130.4, 128.68, 128.66, 128.5, 127.9, 127.0, 126.0, 109.2, 51.6, 21.9, 11.2. GC-MS: 322 (M.sup.+).

(64) Procedure for Preparation of 10. To a solution of 9 (0.61 g, 3.0 mmol) and pyridine (0.96 mL, 12 mmol) in anhydrous dichloromethane was added benzenesulfonic chloride (0.76 mL, 6 mmol) in dichloromethane dropwise at 0 C. After stirring at room temperature for 2 h under nitrogen atmosphere, the reaction mixture was concentrated in vacuo. The saturated Na.sub.2CO.sub.3 solution was added to quench the reaction, and the solution was extracted with ethyl acetate, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by flash chromatography to give product 10 (0.57 g, 55% yield) as a solid. .sup.1H-NMR (300 MHz, CDCl.sub.3): 9.85 (bs, 1H), 7.93 (d, J=7.8 Hz, 2H), 7.53-7.43 (m, 3H), 7.16 (d, J=7.8 Hz, 1H), 7.08-7.04 (m, 3H), 6.26 (s, 1H), 2.34 and 2.32 (ds, 6H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 168.7, 141.9, 140.1, 136.3, 136.1, 132.2, 132.0, 128.9, 128.7, 127.2, 126.5, 125.7, 103.6, 21.2, 20.4. GC-MS: 344 (M.sup.+).

(65) Methylation of 10 to afford 11 and 12. To a cooled mixture of NaH (0.07 g, 60% in oil, 1.6 mmol) in THF (5 mL) was added a solution of compound 10 (0.34 g, 1 mmol) in THF (5 mL) dropwise. The mixture was warmed up to room temperature and stirred for 20 min. After that, the mixture was cooled to 0 C. again and MeI (0.25 mL, 4.0 mmol) was added dropwise. The mixture was then warmed up to room temperature and stirred for 2 h. Water (3 mL) was added to quench the reaction and the mixture was further diluted with water (10 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (310 mL). The combined organic phase was dried with anhydrous MgSO.sub.4. After removal of all the solvent, the residue was purified by silica gel chromatography (hexane/EtOAc=4:1) to afford product 11 (more polar) (0.27 g, 75% yield) and 12 (0.034 g, 9% yield) as solid.

(66) 11: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.05-8.02 (m, 2H), 7.58-7.43 (m, 3H), 7.12-7.01 (m, 3H), 6.27 (s, 1H), 3.19 (s, 3H), 2.36 (s, 3H), 2.11 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 166.8, 142.3, 140.6, 139.1, 137.6, 131.9, 131.4, 130.5, 128.6, 127.1, 126.6, 126.4, 103.5, 33.6, 21.3, 19.5. GC-MS: 358 (M.sup.+).

(67) 12: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.83 (d, J=7.2 Hz, 2H), 7.58-7.38 (m, 4H), 7.02-6.98 (m, 2H), 6.88 (s, 1H), 3.45 (s, 3H), 2.32 and 2.30 (ds, 6H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 160.0, 151.2, 137.9, 136.8, 135.9, 133.8, 131.8, 131.4, 129.4, 129.3, 127.4, 126.6, 112.0, 36.7, 21.2, 21.1. GC-MS: 358 (M.sup.+).

(68) General Procedure for the Acylation of 13 (see FIG. 10) to afford 14 (see FIG. 10). To a solution of 13 (0.01 mol) and DMAP (1.24 g, 0.01 mol) in anhydrous dichloromethane was added the acyl chloride in dichloromethane dropwise at 0 C. After stirring at room temperature for 2 h under nitrogen atmosphere, the reaction mixture was concentrated in vacuo. The saturated Na.sub.2CO.sub.3 solution was added to quench the reaction, and the solution was extracted with ethyl acetate, dried over MgSO.sub.4, and concentrated in vacuo. The residue was purified by flash chromatography to give product 14 as a solid.

(69) 14a: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.99 (dd, J=1.5 and 8.4 Hz, 2H), 7.52-7.47 (m, 3H), 6.55 (d, J=0.9 Hz, 1H), 2.11 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 172.0, 165.3, 159.3, 146.8, 133.1, 132.8, 132.3, 130.0, 128.9, 128.3, 127.8, 108.4, 16.4. GC-MS: 218 (M.sup.+).

(70) 14b: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.5 (bs, 1H), 7.75 (dd, J=1.2 and 8.4 Hz, 2H), 7.63 (dd, J=2.1 and 7.2 Hz, 1H), 7.51-7.45 (m, 2H), 7.38-7.28 (m, 3H), 7.19-7.08 (m, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 165.3, 158.5, 146.3, 132.8, 132.7, 132.0, 131.7, 130.8, 130.4, 128.9, 128.7, 127.4, 126.8, 113.2. GC-MS: 314 (M.sup.+).

(71) 14c: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.68 (bs, 1H), 8.17-7.15 (m, 10H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 171.3, 165.3, 160.1, 148.3, 135.9, 133.5, 133.1, 133.0, 131.7, 131.1, 130.3, 130.2, 130.1, 129.5, 129.3, 128.9, 128.4, 127.8, 124.9, 122.9, 109.3. GC-MS: 360, 358 (M.sup.+).

(72) 14d: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.34 (bs, 1H), 7.90 (m, 2H), 7.67-7.41 (m, 7H), 7.20 (s, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.9, 158.7, 149.1, 133.1, 133.0, 131.83, 131.79, 129.0, 127.6, 127.4, 122.1, 108.5. GC-MS: 358, 360 (M.sup.+).

(73) 14e: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.19 (bs, 1H), 7.90 (m, 2H), 7.77 (t, J=1.8 Hz, 1H), 7.66-7.56 (m, 2H), 7.49-7.44 (m, 1H), 7.32-7.26 (m, 2H), 7.20 (s, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.8, 158.5, 148.7, 135.9, 134.7, 133.0, 131.8, 130.0, 129.0, 128.0, 127.4, 126.2, 124.1, 109.1. GC-MS: 314 (M.sup.+).

(74) 14f: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.08 (bs, 1H), 8.24 (s, 2H), 7.93 (m, 2H), 7.77 (s, 1H), 7.60 (m, 1H), 7.51-7.45 (m, 2H), 7.39 (s, 1H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 165.0, 159.1, 147.3, 136.3, 133.4, 132.5, 132.0, 131.7, 129.3, 127.6, 126.2, 125.3, 121.7, 110.9. GC-MS: 416 (M.sup.+).

(75) 14g: 1.9 g, Yield: 51%. .sup.1H-NMR (300 Hz, CDCl.sub.3): 10.10 (1H, bs), 7.93 (2H, J=7.2 Hz, d), 7.59 (1H, m), 7.52-7.47 (2H, m), 7.13 (1H, s), 7.04 (2H, s), 3.92 (6H, s), 3.87 (3H, s). .sup.13C-NMR (75 Hz, CDCl.sub.3): 164.6, 158.1, 153.4, 150.0, 138.1, 133.0, 131.8, 130.0, 129.0, 127.3, 107.6, 103.3, 61.0, 56.2. MS-EI: 370 (M.sup.+). HRMS (ESI(+)): Calcd. for C.sub.19H.sub.19N.sub.2O.sub.4S (M+H): 371.1066. Found: 371.1052.

(76) 14h: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.20 (d, J=6.9 Hz, 2H), 8.09 (d, J=7.2 Hz, 2H), 7.62-7.43 (m, 6H), 2.51 (s, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 171.3, 165.2, 157.1, 144.4, 133.7, 133.3, 132.8, 131.9, 130.3, 130.1, 128.8, 128.4, 128.3, 128.1, 127.9, 122.0, 12.0. GC-MS: 294 (M.sup.+).

(77) 14i: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.6 (bs, 1H), 7.82 (dd, J=1.2 and 8.1 Hz, 2H), 7.53-7.41 (m, 5H), 7.01 (m, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.7, 155.1, 144.2, 132.7, 132.0, 130.7, 130.0, 129.9, 128.8, 127.4, 122.2, 115.4, 115.2, 12.1. GC-MS: 312 (M.sup.+).

(78) 14j: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.01 (bs, 1H), 7.92 (d, J=7.5 Hz, 2H), 7.69 (d, J=8.1 Hz, 2H), 7.57-7.45 (m, 3H), 7.19 (d, J=8.1 Hz, 2H), 7.14 (s, 1H), 2.37 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 165.2, 158.9, 150.3, 137.9, 132.7, 131.9, 131.4, 129.4, 128.7, 127.4, 126.0, 107.3, 21.2. GC-MS: 294 (M.sup.+).

(79) 14k: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.92 (bs, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.10 (s, 1H), 6.94 (s, 2H), 6.84 (d, J=8.7 Hz, 2H), 3.86 (s, 6H), 3.84 (s, 3H), 3.83 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.6, 163.1, 159.1, 153.2, 149.9, 137.9, 130.0, 129.4, 124.0, 114.0, 107.5, 103.3, 60.8, 56.0, 55.4.

(80) 14l: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.43 (bs, 1H), 7.73 (m, 2H), 7.30 (m, 2H), 7.12 (s, 1H), 6.88 (s, 2H), 3.85 (s, 6H), 3.83 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.4, 159.0, 153.3, 150.1, 139.1, 138.0, 130.3, 129.7, 129.0, 128.8, 107.9, 103.3, 60.8, 56.0. GC-MS: 404 (M.sup.+).

(81) 14m: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.07 (bs, 1H), 7.44 (dd, J=1.8 and 8.1 Hz, 1H), 7.39 (s, 1H), 7.11 (s, 1H), 6.92 (s, 2H), 6.78 (d, J=8.4 Hz, 1H), 3.92 (s, 3H), 3.86 (s, 6H), 3.83 (s, 3H), 3.82 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.8, 159.2, 153.3, 152.8, 150.1, 148.9, 138.0, 129.9, 124.2, 120.8, 110.4, 110.3, 107.6, 103.3, 60.9, 56.0, 55.8. GC-MS: 430 (M.sup.+).

(82) 14n: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.03 (bs, 1H), 8.28 (d, J=9.0 Hz, 1H), 7.10 (s, 2H), 7.09 (s, 1H), 6.68 (dd, J=2.1 and 9.0 Hz, 1H), 6.56 (d, J=2.1 Hz, 1H), 4.11 (s, 3H), 3.96 (s, 6H), 3.90 (s, 3H), 3.89 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.8, 162.6, 159.2, 158.1, 153.4, 149.9, 138.0, 134.5, 130.4, 112.0, 107.5, 106.1, 103.4, 98.7, 60.9, 56.4, 56.2, 55.7.

(83) 14o: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.50 (bs, 1H), 7.13 (d, J=0.3 Hz, 1H), 7.06 (s, 2H), 6.90 (s, 2H), 3.87 (s, 3H), 3.84 (s, 6H), 3.82 (s, 3H), 3.78 (s, 6H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 165.1, 159.3, 153.3, 153.1, 150.2, 141.9, 138.1, 129.6, 126.8, 107.8, 104.8, 103.2, 60.9, 60.8, 56.1, 56.0.

(84) 14p: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.38 (bs, 1H), 7.84 (dd, J=1.5 and 7.8 Hz, 1H), 7.24-7.15 (m, 3H), 7.12 (s, 2H), 4.11 (s, 3H), 3.96 (s, 6H), 3.92 (s, 3H), 3.89 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 162.6, 157.5, 153.4, 152.6, 150.1, 148.0, 138.1, 130.3, 124.8, 124.0, 123.0, 117.0, 107.6, 103.4, 61.9, 60.9, 56.2.

(85) 14q: .sup.1H-NMR (300 MHz, CDCl.sub.3): 11.19 (bs, 1H), 8.33 (dd, J=1.5 and 7.8 Hz, 1H), 7.57 (dt, J=1.5 and Hz, 1H), 7.19-7.07 (m, 5H), 4.14 (s, 3H), 3.96 (s, 6H), 3.89 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 162.7, 157.8, 157.7, 153.4, 150.0, 138.0, 134.5, 132.6, 130.3, 121.7, 119.0, 111.6, 107.7, 103.4, 60.9, 56.4, 56.2.

(86) 14r: .sup.1H-NMR (300 MHz, CDCl.sub.3): 10.75 (bs, 1H), 7.41-7.39 (m, 2H), 7.31 (m, 1H), 7.12 (s, 1H), 7.05 (m, 1H), 6.96 (s, 2H), 3.88 (s, 6H), 3.84 (s, 3H), 3.77 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 164.8, 159.8, 158.5, 153.3, 150.1, 138.0, 133.1, 129.9, 129.8, 119.4, 119.2, 112.2, 107.6, 103.3, 60.9, 56.0, 55.3.

(87) General procedure for methylation of 14 (see FIG. 10). To a cooled mixture of NaH (0.26 g, 60% in oil, 6.5 mmol) in THF (20 mL) was added a solution of compound 14 (5 mmol) in THF (10 mL) dropwise. The mixture was warmed up to room temperature and stirred for 20 min. After that, the mixture was cooled to 0 C. again and MeI (6.5 mmol) was added dropwise. The mixture was then warmed up to room temperature and stirred for 2 h. Water (5 mL) was added to quench the reaction and the mixture was further diluted with water (50 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (350 mL). The combined organic phase was dried with anhydrous MgSO.sub.4. After removal of all the solvent, the residue was purified by silica gel chromatography (hexane/EtOAc=4:1) to afford product 15 (more polar) and 16 as solid.

(88) 15a: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.83 (dd, J=1.8 and 7.8 Hz, 2H), 7.47-7.43 (m, 3H), 6.29 (d, J=1.2 Hz, 1H), 3.79 (s, 3H), 2.31 (d, J=1.2 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.0, 169.0, 137.1, 134.4, 131.3, 129.2, 128.0, 104.3, 32.9, 14.4. GC-MS: 232 (M.sup.+).

(89) 15b: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.38 (dd, J=1.8 and 7.8 Hz, 2H), 7.54-7.39 (m, 7H), 6.60 (s, 1H), 3.63 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.3, 168.4, 136.9, 136.3, 134.8, 132.3, 131.53, 131.49, 130.0, 129.9, 129.2, 128.1, 127.3, 108.1, 34.0. GC-MS: 328 (M.sup.+).

(90) 15c: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.83 (dd, J=1.8 and 8.1 Hz, 2H), 7.64 (m, 1H), 7.59 (m, 1H), 7.51-7.36 (m, 6H), 6.60 (s, 1H), 3.75 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.4, 168.9, 137.8, 136.8, 132.8, 132.5, 132.2, 131.6, 130.5, 129.3, 128.1, 127.8, 123.0, 107.8, 34.9. GC-MS: 374, 372 (M.sup.+).

(91) 15d: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (dd, J=1.5 and 8.1 Hz, 2H), 7.65 (d, J=6.6 Hz, 2H), 7.51-7.43 (m, 4H), 7.28 (d, J=7.2 Hz, 1H), 6.58 (d, J=0.9 Hz, 1H), 3.74 (d, J=2.1 Hz, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.3, 168.9, 138.2, 136.9, 132.3, 131.9, 131.6, 130.8, 129.5, 128.1, 124.2, 107.4, 34.9. GC-MS: 372, 374 (M.sup.+).

(92) 15e: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (d, J=7.8 Hz, 2H), 7.51-7.43 (m, 6H), 7.32 (m, 1H), 6.59 (s, 1H), 3.75 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.4, 168.9, 137.9, 136.8, 135.0, 132.3, 131.6, 130.3, 129.9, 129.4, 129.3, 128.1, 127.4, 107.8, 34.9. GC-MS: 328 (M.sup.+).

(93) 15f: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (dd, J=1.5 and 8.1 Hz, 2H), 8.03 (s, 1H), 7.91 (s, 2H), 7.50-7.46 (m, 3H), 6.73 (s, 1H), 3.77 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.4, 168.9, 136.4, 136.0, 132.8, 132.7, 132.4, 131.7, 129.2, 128.0, 123.3, 120.9, 109.4, 34.8. GC-MS: 430 (M.sup.+).

(94) 15g: 0.69 g, Yield: 36%. .sup.1H-NMR (300 Hz, CDCl.sub.3): 8.37 (2H, J=7.8 Hz, d), 7.50-7.43 (3H, m), 6.60 (2H, s), 6.56 (1H, s), 3.92 (3H, s), 3.90 (6H, s), 3.76 (3H, s). .sup.13C-NMR (75 Hz, CDCl.sub.3): 174.3, 168.7, 153.5, 139.4, 139.1, 137.0, 131.5, 129.2, 128.1, 125.9, 106.7, 106.6, 61.0, 56.4, 35.0. MS-EI: 384 (M.sup.+). HRMS (ESI(+)): Calcd. for C.sub.20H.sub.21N.sub.2O.sub.4S (M+H): 385.1222. Found: 385.1214.

(95) 15h: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.37 (dd, J=1.8 and 8.1 Hz, 2H), 7.53-7.42 (m, 6H), 7.33-7.30 (m, 2H), 3.61 (s, 3H), 2.17 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.0, 167.0, 137.2, 134.1, 131.3, 130.4, 129.8, 129.5, 129.2, 129.1, 128.0, 117.8, 35.0. GC-MS: 308 (M.sup.+).

(96) 15i: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.36 (dd, J=0.9 and 7.8 Hz, 2H), 7.47-7.44 (m, 3H), 7.32-7.22 (m, 4H), 3.60 (s, 3H), 2.15 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.0, 167.0, 164.9, 161.6, 137.1, 133.0, 132.4, 132.2, 131.4, 129.2, 128.0, 125.74, 125.70, 118.2, 116.5, 116.2, 34.9, 12.2. GC-MS: 326 (M.sup.+).

(97) 15j: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.38 (dd, J=1.5 and 7.8 Hz, 2H), 7.48-7.44 (m, 3H), 7.30 (s, 4H), 6.53 (s, 1H), 3.74 (s, 3H), 2.44 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.2, 168.9, 139.8, 139.6, 137.1, 131.4, 129.6, 129.23, 129.17, 128.0, 127.7, 106.6, 34.9, 21.4. GC-MS: 308 (M.sup.+).

(98) 15k: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.33 (d, J=9.0 Hz, 2H), 6.95 (d, J=9.0 Hz, 2H), 6.60 (s, 2H), 6.53 (s, 1H), 3.92 (s, 3H), 3.90 (s, 6H), 3.88 (s, 3H), 3.74 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 173.9, 168.4, 162.4, 153.5, 139.2, 139.1, 131.1, 129.7, 126.0, 113.2, 106.6, 106.4, 61.0, 56.3, 55.3, 34.9, 29.7.

(99) 15l: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.30 (d, J=8.7 Hz, 2H), 7.42 (d, J=8.7 Hz, 2H), 6.60 (s, 2H), 6.58 (s, 1H), 3.93 (s, 3H), 3.90 (s, 6H), 3.75 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 173.2, 168.8, 153.6, 139.5, 139.3, 137.6, 135.5, 130.7, 128.3, 125.7, 106.8, 106.7, 61.0, 56.4, 35.0.

(100) 15m: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.00 (m, 1H), 7.91 (m, 1H), 7.37 (t, J=7.8 Hz, 1H), 7.05 (m, 1H), 6.60 (s, 2H), 6.57 (s, 1H), 3.93 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H), 3.76 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.1, 168.8, 159.5, 153.5, 139.4, 139.2, 138.5, 129.0, 125.8, 121.8, 118.0, 113.6, 106.7, 106.6, 61.0, 56.4, 55.4, 35.0.

(101) 15n: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.35 (dd, J=1.8 and 7.8 Hz, 2H), 7.49-7.41 (m, 3H), 6.23 (d, J=1.2 Hz, 1H), 3.95 (s, 3H), 1.75 (m, 1H), 1.02 (m, 2H), 0.73 (m, 2H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 174.0, 169.1, 140.6, 137.1, 131.3, 129.2, 128.0, 103.9, 33.2, 8.7, 5.9. GC-MS: 258 (M.sup.+).

(102) 15o: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.68 (s, 2H), 6.61 (s, 2H), 6.57 (s, 1H), 3.96 (s, 6H), 3.93 (s, 3H), 3.92 (s, 3H), 3.91 (s, 6H), 3.77 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 173.7, 168.8, 153.5, 152.7, 141.1, 139.4, 139.2, 132.3, 125.8, 106.7, 106.6, 106.4, 61.0, 60.9, 56.3, 56.1, 34.9.

(103) 16a: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.97 (dd, J=1.8 and 7.8 Hz, 1H), 7.60-7.46 (m, 7H), 7.37-7.23 (m, 2H), 3.74 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.4, 159.0, 146.0, 134.5, 133.5, 132.1, 131.3, 130.9, 130.6, 128.73, 128.66, 127.6, 126.9, 114.6, 38.5. GC-MS: 328 (M.sup.+).

(104) 16b: .sup.1H-NMR (300 MHz, CDCl.sub.3): 8.1 (s, 1H), 7.83 (d, J=7.8 Hz, 1H), 7.59-7.44 (m, 6H), 7.31-7.28 (m, 2H), 3.76 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.5, 160.2, 147.9, 136.7, 134.4, 131.0, 130.7, 130.2, 129.1, 128.7, 127.6, 124.5, 122.9, 110.2, 38.5. GC-MS: 374, 372 (M.sup.+).

(105) 16c: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.80 (d, J=8.7 Hz, 2H), 7.59-7.50 (m, 7H), 3.75 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.4, 160.2, 148.3, 134.5, 133.7, 131.8, 131.0, 128.7, 127.6, 121.8, 109.6, 38.5. GC-MS: 372, 374 (M.sup.+).

(106) 16d: 0.35 g, Yield: 18%. .sup.1H-NMR (CDCl.sub.3): 7.61-7.57 (2H, m), 7.55-7.50 (3H, m), 7.19 (1H, s), 7.15 (2H, s), 3.95 (6H, s), 3.89 (3H, s), 3.77 (3H, s). .sup.13C-NMR (CDCl.sub.3): 170.4, 160.0, 153.5, 149.5, 138.3, 134.5, 131.0, 130.5, 128.7, 127.6, 108.9, 103.4, 61.0, 56.2, 38.5. MS-EI: 384 (M.sup.+). HRMS (ESI(+)): Calcd. for C.sub.20H.sub.21N.sub.2O.sub.4S (M+H): 385.1222. Found: 385.1211.

(107) 16e: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.69-7.64 (m, 2H), 7.57-7.48 (m, 5H), 7.16-7.10 (m, 2H), 3.67 (s, 3H), 2.52 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.2, 163.7, 160.5, 156.2, 143.8, 134.6, 131.6, 130.8, 130.1, 130.0, 128.6, 127.6, 123.3, 115.4, 115.1, 37.9, 12.1. GC-MS: 326 (M.sup.+).

(108) 16f: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.82 (d, J=8.1 Hz, 2H), 7.59-7.49 (m, 5H), 7.24-7.19 (m, 3H), 3.76 (s, 3H), 2.39 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.4, 159.9, 149.5, 137.7, 134.7, 132.0, 130.8, 129.4, 128.6, 127.6, 126.0, 108.4, 38.4, 21.3. GC-MS: 308 (M.sup.+).

(109) 16g: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.58 (d, J=8.4 Hz, 2H), 7.16 (s, 1H), 7.16 (s, 2H), 7.00 (d, J=8.7 Hz, 2H), 3.95 (s, 6H), 3.89 (s, 6H), 3.81 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.2, 161.8, 160.4, 153.4, 149.2, 138.0, 130.6, 130.0, 126.4, 113.9, 108.7, 103.4, 60.9, 56.2, 55.4, 38.8, 29.7.

(110) 16h: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.23-7.17 (m, 3H), 7.16 (s, 2H), 6.95 (d, J=8.4 Hz, 1H), 3.96 (s, 3H), 3.95 (s, 6H), 3.93 (s, 3H), 3.88 (s, 3H), 3.82 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.1, 160.4, 153.4, 151.4, 149.3, 149.0, 138.1, 130.5, 126.5, 121.3, 111.3, 110.4, 108.7, 103.4, 61.0, 56.2, 56.0, 38.8, 29.7. GC-MS: 444 (M.sup.+).

(111) 16i: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.35 (d, J=8.4 Hz, 1H), 7.16 (s, 3H), 6.60 (dd, J=2.4 and 8.4 Hz, 1H), 6.52 (d, J=2.1 Hz, 1H), 3.94 (s, 6H), 3.88 (s, 3H), 3.87 (s, 3H), 3.84 (s, 3H), 3.64 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 168.9, 162.8, 159.9, 157.1, 153.4, 149.0, 138.0, 130.7, 129.9, 117.2, 108.5, 105.0, 103.4, 98.5, 60.9, 56.2, 55.6, 55.5, 36.7.

(112) 16j: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.19 (s, 1H), 7.16 (s, 2H), 6.82 (s, 2H), 3.95 (s, 6H), 3.92 (s, 3H), 3.90 (s, 6H), 3.89 (s, 3H), 3.80 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.1, 160.2, 153.5, 153.3, 149.4, 140.3, 138.2, 130.5, 129.5, 108.9, 105.1, 103.4, 61.01, 60.98, 56.3, 56.2, 38.7.

(113) 16k: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.20-7.18 (m, 2H), 7.15 (s, 2H), 7.05 (dd, J=1.5 and 8.1 Hz, 1H), 6.93 (dd, J=1.5 and 7.8 Hz, 1H), 3.95 (s, 6H), 3.93 (s, 3H), 3.88 (s, 3H), 3.86 (s, 3H), 3.63 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 168.4, 159.4, 153.4, 152.7, 149.1, 145.2, 138.0, 130.5, 130.0, 124.9, 119.0, 114.0, 108.7, 103.4, 61.7, 60.9, 56.2, 55.9, 36.7.

(114) 16l: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.47 (m, 1H), 7.38 (dd, J=1.8 and 7.5 Hz, 1H), 7.18 (s, 1H), 7.16 (s, 2H), 7.08 (dt, J=0.9 and 7.5 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H), 3.95 (s, 6H), 3.88 (s, 3H), 3.86 (s, 3H), 3.63 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 168.9, 155.5, 153.4, 149.1, 138.0, 131.7, 130.6, 128.3, 124.5, 121.1, 111.0, 108.7, 103.4, 60.9, 56.2, 55.6, 36.5.

(115) 16m: .sup.1H-NMR (300 MHz, CDCl.sub.3): 7.41 (m, 1H), 7.19 (s, 1H), 7.15 (s, 2H), 7.15-7.05 (m, 3H), 3.95 (s, 6H), 3.89 (s, 3H), 3.86 (s, 3H), 3.76 (s, 3H). .sup.13C-NMR (75 MHz, CDCl.sub.3): 170.2, 159.7, 153.4, 149.3, 138.1, 135.7, 130.5, 129.8, 119.5, 116.8, 112.8, 108.9, 103.4, 60.9, 56.2, 55.4, 38.4.

(116) The additional thiazole compounds were synthesized and tested for antimigration efficacy and cytotoxicity, with results shown in TABLE 3.

(117) TABLE-US-00003 TABLE 3 Cytotoxicity Thiazole IC.sub.50 (anti- (Survival at compound migration, M) 1 M) 5l 5.56 141.8% 5m 0.176 85.0% 5n 0.333 70.4% 5o 0.087 90.0% 5p 0.032 109.5% 5q 0.242 115.3% 5r 11.0 70.2% 6 0.000 106.4% 7a 0.565 68.5% 7b 0.432 100.0% 7c 0.0243 64.8% 7d 0.0647 63.0% 8a 0.883 128.9% 8b 0.0324 116.0% 10 11 137.4% 11 1.46 92.4% 12 >25 116.7% 14a 12 103.1% 14b 15 102.4% 14c 10 78.0% 14d 9.5 82.2% 14e 0.123 71.4% 14f 0.336 85.8% 14g >50 95.0% 14h >25 99.0% 14i 9.1 53.7% 14j 0.308 55.6% 14k >25 93.8% 14l >25 85.6% 14m >25 87.3% 14n >50 93.8% 14o 1.03 77.6% 14p 15 90.1% 14q 8.5 103.7% 14r 14 92.6% 15a 15 102.6% 15b 0.268 72.2% 15c 0.300 94.4% 15d 0.125 59.3% 15e 0.367 35.2% 15f 0.096 79.8% 15g 1.07 88.4% 15h 0.197 68.4% 15i 0.847 83.8% 15j >25 112.6% 15k 8.0 105.6% 15l 0.758 77.6% 15m >50 103.5% 15n 0.104 90.7% 15o 1.96 81.5% 16a 0.0416 88.9% 16b 0.437 104.4% 16c 0.317 65.4% 16d 10 96.4% 16e 1.62 107.6% 16f 1.09 59.3% 16g 0.312 100.0% 16h >25 101.9% 16i 5.04 118.5% 16j 0.470 114.8% 16k 0.108 117.6% 16l >25 109.3% 16m 0.646 88.9%

(118) All references cited in this specification are herein incorporated by reference as though each reference was specifically and individually indicated to be incorporated by reference. The citation of any reference is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such reference by virtue of prior invention.

(119) It will be understood that each of the elements described above, or two or more together may also find a useful application in other types of methods differing from the type described above. Without further analysis, the foregoing will so fully reveal the gist of the present disclosure that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this disclosure set forth in the appended claims. The foregoing embodiments are presented by way of example only; the scope of the present disclosure is to be limited only by the following claims.