Targeting Estrogen Receptor Sites in Human Breast Cancer Cell Line T47D With Copper Conjugates of Nonsteriodal Anti-inflammatory Drug Derivatives: Antiproliferative Activity of Ketoprofen Derivative and its Copper Complex

A square planar copper complex of derivatized NSAID drug (Ketoprofen thiosemicarbazone [3-benzoyl-α-methyl benzene acetic acid thiosemicarbazone]), is characterized by elemental analysis, spectroscopy, electrochemistry and magnetic susceptibility studies which exhibits dose-dependent and enhanced antiproliferative effects on human breast cancer cell line T47D rich in progesterone receptors.


Introduction
The pharmacology of nonsteroidal anti-inflammatory drugs (NSAIDs) and roles of cycloo.genase enzymes, viz. COX-1 and COX-2, have now been integated into a unified model suggesting that inhibition of both COX isoforms by NSAIDs leads to prevention of cancers i-. 4animal model through induction of apoptosis and stimulation of immune serveillance. The actual mechanism of this action, however, is not known. Ketoprofen ) is a widely used NSAID in clinical practice possessing a variety of biological activities which has close structural resemblance to non-steroidal antiestrogens viz. Trioxifene (II) and Keoxifene (Ill). Since II and HI have been shown to compete with estradiol for estrogen recept.
(ER) binding site and thereby prevent the growth of malignancies in breast cancers, I may also be looked upon as a potential antacancer agent. In order to exploit ER binding affinities of I for the purposes of evolving compounds which can deliver cytotoxic drugs to the hormone responsive cancer cells we decided to modify ketoprofen moiety with thiosemicarbazone functionality which has been known to attribu.. antiproliferative properties to several organic compounds of clinical significance. Combination of clinically used NSAIDs with cytotoxac drugs has also been shown to exert synerg, i[c effects on the cytotoxicities of many anticcer drugs which is of potential clinical significance in the treatment of certain cancers. Additionally appendage by the thiosemicarbazone pharmacophore allows for metal conjugation especially With copper leading_ to jancement in selective binding to the estrogen receptors as shown by Predki and Sarkar. In the present communication we describe the synthesis and characterization of ketoprofen thiosemicarbazone (KFTSC, IV) and its Cu (II) conjugate and evaluation of their antiproliferative activities against human bret cancer cells T47D, which is a progesterone receptor rich, and ER sensitive cell line.

Material and method
All chemicals used in the syntheses of lig_and and copper conjugates were of AR grade while solvents were distilled prior to their use. Ketoprofen (Aldrich), thiosemicarbazide (Sisco Chem. Pvt. Ltd.) and CuCI.2HO (Qualigens)were used-as supplied. Synthesis.. ofli.and Kdtolrofen thiosemicarbazone (IV) was prepared by reacting I (0.254g, 0.001mole) and thiosemicarbazide (0.091g, 0.001mole) both dissolfed in methanol (10 ml) with one drop of concentrated hydrochloric acid and bringing the reaction mixare to a reflux on the water batfi for two hours. A white colored microc_rystallirie product separated out when the mixture was allowed to cool. It was washed with ether and dried in vacuum.
T.rtiate..d thymdne uptake assay T47D cells were incubated over a peri.od of 48 hours with v_aring concentrations of the test compounds in 48 well plates. At 48 hours, ['HI thymidine was added to the cells and incubated at 37C for 2/3 hours. After removing the medium, cells were precipitated with ice cold 10% Trichloro acetic acid (TCA) and kept at 4C for 1-2 hours. Finally, the cells were washed with ice cold Phosphate buffered saline (PBS), to remove unbound tritium while DNA was hydrolysed with 0.5M NaOH/0.1% Triton X-100. Contents of each well were mixed with Scintillation fluid and radioactivity was measured in Liquid Scintillation Counter. The reaction of KFTSC with CuC12.2H20 in methanol results in a neutral, green complex where metal coordination takes place through thione sulfur and imine mtrogen atom as shown in Figure 1.
The IR Spectrum of the liga_nd exhibits a broad ld around 3166 cm " which ,an be .a.scrib.ed to tlie H-bonded OH bf the carboxyl group.. The strongband at 1697 cm is attributed [o the C=O stretchin.g band of the carBoxylate group while_ the abso_rotion at 1654 cm" j# assigned to stretching frequency or 3-benzoyl carbonyl group respectively. Condensation with thiosemicarbazone moiety results in the loss of band at 1654 cm confirming successful derivatization of the benzoyl carbonyl grouo. Two additional bands can be seen at 3417 andl340 cm"which are due to asymmeiric and symmetri.c, stretches, of the amin_o_ gr9u_p_." T.hle i.m.iqe v(c=aqd th,i,arbo,ny],,y(c= tunctionalities are observed at 1595 and 924 cm whicn are slaltted to t3[i aria vu3cl respectively on copp.er conjugation indicating their involvement in metal .o0rdination.': The electronic spectra of IV in DMF shows a band at 31056 cm due to extended conjugation in the thosemicarbazone moiety while its copper conjugate exhibits a molerately intense.band at 27027 cm"due to Iigand to etal charge trans'er." Th.e broad band observed foctlae metal-based transition at 17153 cm is attributable to a combinatiarl of. th.e tra_nsitjons'Blg---,'.Eg an.d'B.lg "--t"Alg respectively in a. sg.u,ar,,e,,anar cqp.figumfiqn" wlaicla is turtlaer supported by the observed magnetic moment oi .vu tw IOr tins com.pex. The cyclic voltammograms for KF.TSC and its copper com.p.]ex in DMF solvent are.siaown in . Figure  (31xM) is lowered on complexation with copper (> 1 lxM) indicating that metal complextion with copper clearly offers an advantage in designing antiproliferative compounds especially against hormone responsive cancers. The selective affinity of copper ions towards the i-ntranuclear estrogen receptor binng sites and consequent inhibition of DNA dimerization noted by Predki and Sarkar. may be contributing to the observed enhancement in antiproliferative activities of these copper conjugates.