Patent Abstract:
n1 -- 2 -- hydrazine - 1 - carbothioamide and its cu 2 + , pd 2 + and pt 2 + complexes are effective anti - tumor agents . also disclosed are compositions comprising and its cu 2 + , pd 2 + and pt 2 + complexes , and the use of the compositions in the treatment of malignant tumors .

Detailed Description:
methyl hydrazinecarbodithioate . potassium hydroxide ( 13 . 2 g , 0 . 2 mol ) is dissolved in 15 ml water and 12 ml 2 - propanol . the solution was cooled to 5 ° c . hydrazine hydrate ( 10 g , 0 . 2 mol ) was added slowly under stirring . carbon disulfide ( 15 . 2 g , 0 . 2 mol ) was added drop - wise , and the solution stirred for 120 min at 5 ° c . methyl iodide ( 28 . 3 g , 0 . 2 mol ) was added slowly . after the addition stirring was continued for 2 hrs . the precipitate was filtered off and dried . yield 6 . 8 g , 37 %. m . p . 81 - 83 ° c . 1 h nmr ( cdcl 3 ): 2 . 4 ppm ( 3h , s ), 4 . 0 ppm ( 2h , broad ), 8 . 7 ppm ( 1h , broad ). methyl 2 -( 2 - pyridylmethylene )- hydrazinecarbodithioate . methyl hydrazinecarbodithioate ( 2 . 2 g , 18 . 3 mmol ) was dissolved in 2 - propanol ( 10 ml ). after adding 2 - pyridine aldehyde ( 2 . 0 g , 18 . 6 mmol ) drop - wise to the solution stirring was continued for 90 min . the reaction mixture was stored in a refrigerator overnight , and the precipitate filtered off and dried . yield 2 . 2 g , 55 %. m . p . 171 - 173 ° c . 1 h nmr ( dmso - d 6 ): 2 . 6 ppm ( 3h , s ), 7 . 4 ppm ( 1h dd ), 7 . 9 ppm ( 1 + 1h dd , d ), 8 . 2 ppm ( 1h , s ), 8 . 6 ppm ( 1h , dd ). n1 -( 3 - methoxypropyl )- 2 -( pyridylmethylidene )- hydrazine - 1 - carbothioamide ( vlx50 ). methyl 2 -( 2 - pyridinylmethylene )- hydrazinecarbodithioate ( 0 . 5 g , 2 . 4 mmol ) and 1 - amino - 3 - methoxypropane ( 0 . 25 g , 2 . 8 mmol ) was dissolved in dry methanol and refluxed for 12 hrs . the precipitate was filtered off and recrystallized from ethyl acetate . yield 0 . 18 g , 30 %. m . p . 108 - 110 ° c . 1 h nmr ( cdcl 3 ): 2 . 0 ppm ( 2h , tt ), 3 . 4 ppm ( 3h , s ), 3 . 6 ppm ( 2h , t ), 3 . 8 ppm ( 2h , t ) 7 . 3 ppm ( 1h , dd ), 7 . 7 ppm ( 1h , t ), 7 . 8 ppm ( 1h , s ), 7 . 9 ppm ( 1h , d ), 8 . 2 ppm ( 1h , broad singlett ) 8 . 6 ppm ( 1h , d ), 9 . 0 ppm ( 1h , s ). vlx50 is a known compound commercially obtainable from maybridge plc ( fischer scientific ). cu 2 + complex of n1 -( 3 - methoxypropyl )- 2 -( pyridylmethylidene )- hydrazine - 1 - carbothioamide , ( vlx60 ). to a solution of vlx50 ( 41 mg ) in 5 ml of ethanol is added 60 mg of cucl 2 in 2 ml of ethanol . the mixture is stirred for 3 hrs at room temperature . the green precipitate is filtered off and washed with ethanol . yield 74 mg . 1 h nmr ( dmso - d 6 ): could not be recorded due to cu 2 + being paramagnetic . pd 2 + complex of n1 -( 3 - methoxypropyl )- 2 -( pyridylmethylidene )- hydrazine - 1 - carbothioamide , ( vlx61 ). to a solution of vlx50 ( 41 mg ) in 5 ml of ethanol is added 42 mg of pdcl 2 in 37 ml of ethanol . the mixture is stirred for 20 hrs at room temperature . the yellow precipitate is filtered off and washed with ethanol . yield 35 mg . 1 h nmr ( dmso - d 6 ): 1 . 8 ppm ( 2h , t ), 3 . 4 ppm ( 3h , s ), 3 . 5 ppm ( 4h , t ), 8 . 0 ppm ( 1h , s ), 8 . 3 ppm ( 1h , t ), 8 . 5 ppm ( 1h , d ). pt 2 + complex of n1 -( 3 - methoxypropyl )- 2 -( pyridylmethylidene )- hydrazine - 1 - carbothioamide , ( vlx62 ). to a solution of vlx50 ( 41 mg ) in 0 . 5 ml of ethanol is added 42 mg of pdcl 2 in 0 . 5 ml of ethanol . the mixture is stirred for 20 hrs at reflux temperature . the red - brown precipitate is filtered off and washed with ethanol . yield 65 mg . 1 h nmr ( dmso - d 6 ): 1 . 8 ppm ( 2h , t ), 3 . 6 ppm ( 7h , broad ), 7 . 8 ppm ( 2h , m ), 8 . 5 ppm ( 1h , s ), 8 . 8 ppm ( 1h , d ). pharmaceutical compositions . the following illustrate representative pharmaceutical dosage forms containing the compound of the invention , for therapeutic use in humans . ( a ) tablet . compound of formula i ( 2 . 0 mg ), lactose ( 76 . 0 mg ), povidone ( 14 . 0 mg ) croscarmellose sodium ( 12 . 0 ), microcrystalline cellulose 90 . 0 , magnesium stearate ( 3 . 0 mg ). ( b ) tablet . compound of formula ii , iii or iv ( 1 . 0 mg ), microcrystalline cellulose ( 400 mg ), starch ( 50 . 0 mg ), sodium starch glycolate ( 14 . 0 ), magnesium stearate ( 5 . 0 mg ). ( c ) hard gelatin capsule . compound of formula i ( 10 . 0 mg ), colloidal silicon dioxide ( 1 . 5 mg ), lactose 430 mg , pregelatinized starch ( 120 mg ), magnesium stearate ( 3 . 0 mg ). ( d ) solution for injection . compound of formula i ( 5 . 0 mg ), sodium dihydrogen phosphate ( 10 . 0 mg ), disodium hydrogen phosphate ( 5 . 7 mg ), sodium chloride ( 4 . 5 mg ), 01 . 0 n sodium hydroxide solution q . s . ( ph adjustment to 7 . 0 - 7 . 5 ), water for injection q . s . ad 1 ml . ( e ) solution for injection . compound of formula ii , iii or iv ( 0 . 5 mg / ml ), sodium dihydrogen phosphate ( 1 . 3 mg ), disodium hydrogen phosphate ( 0 . 6 mg ), polyethylene glycol 400 ( 200 . 0 mg ), 0 . 1 n sodium hydroxide solution q . s . ( ph adjustment to 7 . 0 - 7 . 5 ), water for injection q . s . ad 1 ml . cell culture . patient tumor samples ( 98 ) and preparations ( 4 ) of normal peripheral blood mononuclear cells ( pbmc ), detailed in table 1 , were used to determine the activity of vlx50 and , for comparison , six other cytotoxic drugs chosen to represent different mechanistic classes . the tumor samples were obtained by bone marrow / peripheral blood sampling , routine surgery or diagnostic biopsy . leukemic cells and pbmcs were isolated by 1 . 077 g ml - 1 ficoll - paque centrifugation ( larsson et al ., 1992 ). tumor tissue from solid tumor samples was minced into small pieces and tumor cells were isolated by collagenase dispersion followed by percoll density gradient centrifugation ( csoka et al ., 1994 ). the sampling of primary tumor cells was approved by the local ethics committee at uppsala university hospital . cell viability was determined by trypan blue exclusion test and the proportion of tumor cells in the preparation was judged by inspection of may - grunvald - giemsa stained cytospin slides ( fig1 a ). all samples used in this study contained more than 70 % tumor cells . the cell lines used in this study were breast cancer mcf7 and htert - rpe ( normal epithelial cell line ) obtained from american type culture collection ( atcc ) and clontech ( palo alto , calif . ), respectively . the remaining cell line panel used has been described in detail previously ( dhar et al ., 1996 ) and consists of the parental cell lines rpm ; 8226 ( myeloma ), ccrf - cem ( leukemia ), nci - h69 ( small cell lung cancer ), u - 937 gtb ( lymphoma ), achn ( renal cell carcinoma ) and the drug - resistant sub - lines 8226 / dox40 , 8226 / lr5 , cem / vm - 1 , u - 937 vcr , and h69ar . the sub - line 8226 / dox40 was exposed to 0 . 24 μg / ml doxorubicin once a month and over - expresses pgp / mdr1 / abcb1 ( dalton et al ., 1986 ). the 8226 / lr5 sub - line was exposed to 1 . 53 μg / ml of melphalan at each change of medium ; resistance is suggested to be associated with increased levels of glutathione as well as genes involved in cell cycle and dna - repair ( mulcahy et al ., 1994 ). u937 vcr was continuously cultured in the presence of 10 ng / ml vincristine and the resistance is proposed to be tubulin associated ( botling et al ., 1994 ). h69ar was alternately fed with drug - free medium and medium containing 0 . 46 doxorubicin and over - expresses mrp1 / abcc1 cole ( cole et al ., 1992 ). cem / vm - 1 was cultured in drug - free medium and could be grown for 3 - 4 months without loss of resistance against teniposide which is proposed to be topoisomerase ii associated ( bugg et al ., 1991 ; danks et al ., 1988 ). the resistant phenotypes were stable for more than three months . normal epithelial htert - rpe cells were cultured in in modified eagles medium nutrient mixture f - 12 ham . the htert - rpe cell culture were supplemented with 10 % heat - inactivated fetal calf serum , 2 mm glutamine , 100 μg / ml streptomycin and 100 u / ml penicillin ( all from sigma aldrich co , st louis , mo .) at 37 ° c . in humidified air containing 5 % co 2 . the remaining cell lines cells were grown in culture medium rpmi - 1640 supplemented with 10 % heat - inactivated fetal calf serum , 2 mm glutamine , 100 μg / ml streptomycin and 100 u / ml penicillin ( sigma ) at the same conditions . the resistant cell lines were tested regularly for maintained resistance to the selected drugs . growth and morphology of all cell lines were monitored on a weekly basis . fluorometric microculture cytotoxicity assay . the fluorometric microculture cytotoxicity assay , fmca , described in detail previously ( lindhagen et al ., 2008 ), is based on measurement of fluorescence generated from hydrolysis of fluorescein diacetate ( fda ) to fluorescein by cells with intact plasma membranes . cells were seeded in the drug - prepared 384 - well plates using the pipetting robot precision 2000 ( bio - tek instruments inc ., winooski , vt .). the number of cells per well were 2500 - 5000 . two columns without drugs served as controls and one column with medium only served as blank . the plates were incubated for 72 h and then transferred to an integrated hts saigan core system consisting of an orca robot ( beckman coulter ) with co2 incubator ( cytomat 2c , kendro , sollentuna , sweden ), dispensor module ( multidrop 384 , titertek , huntsville , ala . ), washer module ( elx 405 , bio - tek instruments inc ), delidding station , plate hotels , barcode reader ( beckman coulter ), liquid handler ( biomek 2000 , beckman coulter ) and a multipurpose reader ( fluostar optima , bmg labtech gmbh , offenburg , germany ) for automated fmca . quality criteria for a successful assay included a mean coefficient of variation of less than 30 % in the control and a fluorescence signal in control wells of more than 5 times the blank . multiparametric high content screening assays . to study the cell death characteristics a multi - parametric high content screening ( hcs ) assay was used ( cellomics cytotoxicity hitkit ™) and an hcs assay for measurement of apoptosis , which has been described in detail previously ( lovborg et al ., 2004 ). the cells ( 1500 cells / well ) were seeded into flat - bottomed 96 - well plates ( perkin elmer inc ., wellesley , mass .) and were left to attach before addition of drugs . for the cytotoxicity assay the cytotoxicity hitkit ™ reagents ( cellomics inc ., pittsburgh , pa ., usa ) was used according to the manufacturer &# 39 ; s instructions . multi - parameter cytotoxicity hitkit ™ contains a nuclear dye , a cell permeability dye , and a lysosomal mass / ph indicator . in the apoptosis assay fam - devd - fmk ( part of the caspatag kit , chemicon , temecula , calif .) at a final concentration of 20 μm was added one hour before the end of the drug exposure to stain activated caspase - 3 and partly caspase - 7 . the staining solution was removed and the plates were washed twice with pbs followed by a 30 min fixation in 3 . 7 % formaldehyde and nuclear staining with 10 μm hoechst 33342 ( sigma ). plates were then washed twice . the plates were centrifuged before each aspiration to avoid loss of cells detached due to toxic stimuli . processed plates were kept at + 4 ° c . for up to 24 h before analysis . plates were analyzed using the arrayscan ™ hcs software ( cellomics inc ). the system is a computerized automated fluorescence - imaging microscope that automatically identifies stained cells and reports the intensity and distribution of fluorescence in individual cells . images were acquired for each fluorescence channel , using suitable filters with 20 × objective . in each well at least 800 cells were analyzed . images and data were stored in a microsoft sql database . phase contrast microscopy . time lapse phase contrast microscopy was performed using an automated incucyte phase contrast microscope . mcf - 7 cells ( 10 , 000 / well ) were plated on 24 - well imagelock plates ( essen instruments , ann arbor , mich .) and cultured in rpmi 1640 media containing 10 % fetal bovine serum and antibiotics . the plates were immediately placed into incucyte imaging system ( essen instruments ). the chamber is designed to fit into a standard , humidified , co 2 incubator in an atmosphere of 5 % co 2 , and a moving objective allows the cell culture to be stationary while images are captured at different positions from well to well . images were collected at hourly intervals starting 30 minutes after addition of the plate to the incucyte - flr chamber . drug treatment was performed 24 hours after the plates were placed in the incucyte . cell density was calculated using the incucyte software . movies were generated using incucyte software ( essen instruments ) at three frames / second , which is equivalent to 30 minutes of culture / second . measurement of intracellular iron . the fluorescent membrane permeable fe sensor , phen green ( invitrogen ab , göteborg , sweden ) was used to measure changes in intracellular free iron concentration . fluorescence of the phengreen indicators is quenched upon binding fe 2 + and fe 3 + . the emission intensity of the phengreen fl indicator depends on both the metal ion &# 39 ; s concentration and the indicator &# 39 ; s concentration . mcf - 7 cells were harvested and diluted to 500 , 000 per ml in complete medium , loaded with 10 μm phengreen diacetate for 45 minutes and plated to flat - bottomed 96w microtiter plates . the plates were washed × 2 with pbs , resuspended in 180 μl pbs per well and subsequently analyzed by a plate reading fluorometer fluostar optima which is programmed to scan fluorescence once a minute for 20 minutes with a break after 5 minutes , during which 20 μl pbs , or vlx50 ( 100 μm or 500 μm ) was added . in vivo studies . cells from two ovarian carcinoma patients and the cell line cem were cultured inside semi - permeable polyvinylidene fluoride fibers and assessed by the hollow fiber assay ( friberg et al ., 2005 ; jonsson et al ., 2000 ). the fibers were implanted subcutaneously into nmri male mice ( scanbur , sollentuna sweden ), which were treated with a single dose ( 0 . 76 mg / mouse ) of vlx50 subcutaneously , or vehicle only ( n = animals / group ). fibers were retrieved after 6 days and cell density evaluated ( fig3 a ) using the mtt ( 3 -[ 4 , 5 - dimethylthiazol - 2 - yl ]- 2 , 5 - diphenyltetrazolium bromide )- assay ( alley et al . 1988 ). the method is based on the conversion of mtt to blue formazan crystals by living cells . the formazan was extracted by dmso as previously described ( jonsson et al . 2000 ), and optical density ( od ) read at 570 nm . cell density for each fiber on retrieval day was expressed as net growth , defined as ( od retrieval day — od implantation day )/ od implantation day , i . e . the percent change in cell density in the fibers during the 6 days of in vivo experiment . the animals were observed regarding behavior and weight gain throughout the experiment . blood samples ( 200 μl ) were obtained through the orbital plexus after anesthetization with isofluran just before euthanasia , and analyzed for hematological parameters . four animals were caged per cage and fed a commercial diet ( lactamin ab , sweden ), water being given ad libitum . data analysis and statistics . small laboratory information and management system ( kelley et al ., 2004 ) was used for screening data management and analysis . raw fluorescence data files were loaded into the slims software which calculates percent inhibition according to the formula : percent inhibition = 100 ×( x - negative control / positive control - negative control ) − 1 , where x denotes fluorescence from experimental wells . slims also identifies and corrects systematic spatial errors . more than or equal to 50 % mean inhibition in ovca phtc was set as the criteria for qualifying as hit compound . structural similarity to other compounds in the library was calculated based on a structural fingerprint consisting of binary vectors representing structures located within the compound and which are automatically computed for each compound loaded into the program . the z ′- value was calculated to evaluate the quality and usefulness of the assay in the screening setting using the equation : z ′= 1 −[( 3sdposcontrol + 3sdnegcontrol )/( meanposcontrol − meannegcontrol )] where sd and mean are the standard deviation and mean values of screening raw data from wells with untreated cells ( positive control ) and blank wells ( negative control ), respectively ( zhang et al ., 1999 ). dose - response data were analyzed using calculated survival index values and the software program graphpadprism4 ( graphpad software inc ., san diego , calif ., usa ). data was processed using non - linear regression to a standard sigmoidal dose - response model to obtain ic50 - values ( inhibitory concentration 50 %). response rate was defined as the fraction of samples having a survival index below the median at the concentration from the dose - response curves showing the largest standard deviation . for vlx50 this concentration was 4 μm ( cf , fig3 b , 5 a ). the relative effect of a drug on solid and hematological tumors was indicated by the s / h ratio , defined as the ratio between the total response rates for the solid and the hematological samples ( cf , fig2 b ). diagnosis - specific activity ex vivo . to examine the effect of vlx50 in a setting close to the clinic , its anti - tumor activity was studied in 98 tumor samples from patients with a variety of solid and hematological cancer diagnoses as well as in four pbmc . the ic50 - values ranged from diagnoses with a median ic 50 of below 5 μm such as cll , all , ovarian cancer and lymphoma to the more resistant colon and renal cancer samples with ic50 above 40 μm ( table 1 ). cancers of the breast , lung , cml , aml and pbmc displayed intermediate sensitivity to vlx50 . in fig2 a the response rates for vlx50 at 4 μm for the patient samples are listed according to diagnoses . corroborating the ic50 patterns the lymphocytic malignancies showed the highest response rates followed by breast and ovarian cancer whereas pbmc , colon and renal cancer had the lowest response rates . lung cancer , aml and cml had intermediate response rates . the relative effect of vlx50 and six standard cytotoxic drugs , in solid and hematological tumor samples , expressed as the s / h ratio , is shown in fig2 b . vlx50 had a ratio of 0 . 73 indicating a relatively high activity against solid tumors , second only to cisplatin ( s / h ratio of 1 . 2 ). the remaining drugs had a s / h ratio below 0 . 5 . the results for the standard drugs are consistent with their main clinical use . to roughly estimate tumor cell specificity , drug effect in cells from cll and normal pbmc were compared ( fig2 c ), demonstrating a significantly higher activity against the malignant phenotype with a pbmc / cll median ic50 ratio of 7 . 6 . of the tested standard cytotoxic drugs only vincristine were significantly more active in cll than in pbmc . notably , both cytarabin and melphalan showed significantly higher activity in pbmc than in cll cells ( t - test , p & lt ; 0 . 05 ). no difference was observed for doxorubicin , etoposide and cisplatin . the effect of vlx50 on tumor cell survival in a panel of ten cell lines is shown in fig5 a . in vivo activity in phtc cultures of ovarian carcinoma . activity in vivo was determined in hollow fiber cultures of phtc from ovarian carcinoma patients subcutaneously implanted in mice ( fig3 a ; n = 8 each in test and control groups ). after a single dose of 760 μg / mouse significant growth inhibition compared to vehicle treatment were observed in the two phtc cultures ( p & lt ; 0 . 05 and p & lt ; 0 . 01 , respectively ). the difference between the vlx50 treated group and the control group did not reach statistical significance in the control cell line ccrf - cem ( p & gt ; 0 . 05 ). vlx50 induced a small but significant reduction in weight gain compared to the control group ( p & lt ; 0 . 05 ; fig3 b ). a significant decrease in platelet counts was also evident ( p & lt ; 0 . 05 ). there was no difference in wbc , rbc , and hemoglobin values ( fig3 c ). pharmacological profiling in a resistance based cell line panel . when comparing the log ic50 patterns with some commonly used cytotoxic agents , vlx50 showed low correlation ( r =− 0 . 24 − 0 . 19 ) indicating absence of cross resistance to these standard drugs . in response vlx50 , an increased sensitivity compared with parental cell lines was observed in the sub - lines with pgp - tubulin - gsh and topo ii - mediated drug resistance with resistance factors ranging from 0 . 13 - 0 . 62 , thus indicating collateral sensitivity . for ncl h69 and its resistant sub - line cem / vm - 1 the resistance factor was 3 . 55 suggesting the involvement of mrp in mediating vlx50 resistance ( fig5 d ). the resistance factors for some tested standard agents ( fig5 d ) confirmed the expected resistance pattern of the drug resistant sub - lines ( not shown ). mode of vlx50 induced cell death . vlx50 was profiled with respect to mode of action using time lapse phase contrast microscopy and multi - parameter analysis using arrayscan ii . the effect of vlx50 on growth and viability was delayed with little or no effect observable at 24 h ( fig4 a and 4 b ). at 48 - 72 h there was a gradual decrease in cell density and a parallel increase in caspase - 3 activity and dna fragmentation ( fig4 c ). phase contrast images of the cells at this time point revealed a typical apoptotic morphology with condensed nuclei surrounded by a bright halo ( fig4 a ). the increase in dna fragmentation and caspase activation preceded the increase in cell membrane permeability which is compatible with classical apoptosis . mechanism of action . mechanistic exploration was performed using gene expression analysis of drug treated tumor cell cultures to generate a drug specific signature . the breast cancer cell line mcf - 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