WO2015142683A1 - Functionalized tyrosine kinase inhibitors modified with precious metal electrophiles and methods associated therewith - Google Patents

Abstract

Newly synthesized thiourea-modified 3-chloro-4-fluoroanilio-quinazoline derivatives have been studied, as terminal carrier ligands in linear gold(I) complexes. The molecules mimic the tyrosine kinase inhibitor gefitinib (by computational docking experiments). Thiourea groups were either directly attached to quinazoline-C6 or linked to this position via a flexible ethylarmino chain. One compound tested acts as a thiourea-S/quinazoline-Nl mixed-donor ligand, giving an unusual dinuclear complex as determined by X-ray crystallography and/or electrospray mass spectrometry. One compound formed the desired stable linear complex. The biological activity of the carrier ligands and corresponding gold(I) complexes was studied in NCI-H460 and NCI-H1975 lung cancer cells. One compound that was tested partially overcomes resistance to gefitmib in NCI-H1975 (with IC₅₀ values of 1.7 and 30 μΜ, respectively), and the corresponding gold complex (13) maintains activity in the low-micromolar concentration range.

Description

and Methods Associated Therewith

The present invention claims priority under 35 USC 119(e) Co US Provisional

Application No. 61/953,7 1 filed March 15, 2014, the entire contents of which is

incorporated by reference in its entirety.

Field of t he I ntion

The present invention relates to thiowea/amidrae iimetionaiized tyrosine kinase inhibitors modified with go!d, platinum, and other precious metal compounds. The

Chiourea ariudine-modified 3-chloro-4-fluoroaniI.ino-q«ina .oime derivatives were studied as carrier ligands in linear go (l) or square planar platmurn(ii) complexes. ^"The molecules mimic the tyrosine kinase inhibitor gefitinib (by computational docking experiments).

Thiourea or amidine groups were either direciiy attached to qiiinazoiine-C'6 or linked to this position via a flexible alkylamino chain. The approach allows the selective targeting of clinically relevant tyrosine kinases with classical anticancer metals as a means of overcoming drug resistance in intractable cancer. The metals introduced may act as electrophiles and form bonds with amino acid residues in the kinases' active sites, such as cysteine, methionine, histidine, and lysine.

Background of the invention

Mutational! - activated tyrosine kinases (TK) of the epidermal growth factor receptor (EOFR) family are considered a major driver of cancer cell survival and aggressive tumor, growth. In non-small cell lung cancer, expression levels of EGFR are inversely correlated with survival of the disease. Somatic (activating) EOFR mutations sensitize cancer cells to small-molecule tyrosine kinase inhibitor's (T is), which target the enzyme's ATP binding site and show potent antiproliferative properties. Gefitinib is a qumazoime-based TKl indicated against cancers harboring aberrant EGFR, in particular lung carcinomas. Unfortunately, the efficacy of this drug is limited b the emergence of acquired resistance as a consequence of a secondary mutation within the ATP binding pocket. One currently pursued approach to combatting this form of resistance observed for mutant EGFR and other clinicall relevant kinases is to turn the reversible TKIs into irreversible inhibitors (ITKIs). These molecules contain a strategically positioned reactive electrophilie group (usually a Michael acceptor), which is able to form a covalent bond with accessible cysteine residues in the active sites of the tareetabte enzymes.

i Brief summary o f the fo vea lion

hi ail embodiment, the present invention relates to introducing a gold(I) or platmum(Il) based electrophile into a TKI structure tha can be targeted to the solvent- accessible cysteine-7 7 near the entrance to the ATP binding pocket of EGFR-T . These phannacophores would combine the high sulfur affinity of gold(I) or platinura(O) with the selectivity and nanomolar binding affinity of an EGFR-TK-targeted ligand. The use of gold(I) or platimim(ll) as e^'lectrophilic moieties in the proposed structures in place of fee Michael acceptor was inspired by the mechanisms of action of goid(l) drugs, which react with (seleno)cysteine residues in proteolytic and redox-active enzymes and the reactivity of platinum drugs with cysteine containing species demonstrated in the inventors' previous research. While these interactions are generally unselective, targeting of specific cysteine residues by goid(I) and platinutn(If) complexes containing target-selective carriers may occur.

Thus, in an embodiment the present invention relates to introducing a precious metal electrophile into a TKI structure that: can be targeted to the solvent-accessible cysteine-797 near the entrance to the ATP binding pocket of EGFR-TK. The desired pharmacophore combines the high sulfur affinity of goki(I) and platinum(ll) with the selectivity and

nanomolar binding affinity of an EGFR-TK-targeted ligand,

in an embodiment, the present invention relates to novel thiourea and araidtne- modified TKls derived from a. quinazo!me scaffold. The present invention also relates to the ability of novel fuiictionali zed TKls to serve as potential carrier l igands in (pseudo)1iali d.e and phosphine substituted goid(I) and mixed-Hgand platinumCH) complexes. At least one TKI derivative was identified that partially overcomes resistance to gefitinib in NSCLC ceil lines harboring wild-type and mutated EGFR kinase domains. Additionally, two piatinom(Ii) derivatives, P3-T2 and P9-T2, displayed low nanomolar binding affinities to wild-type

EGFR, similar to gefitinib Accordingly, in an embodiment, the present invention relates to generating unique types of hybrid pharmacophores-, which, lend themselves to modular high- throughput screening and extended structure-activity relationship studies. Libraries can be assembled using simple ligand substitution chemistry (see for example Scheme 1 ) and metal - facilitated armiie-to-nitri!e addition chemistry (see for example Schemes 4-6).

in an embodiment, although the relati vely high IC₅₀ of one deri ative can be explained by its aqueous reactivity, which leads to an uodesired, deactivating auto-cheiation, which does not allow for nucleophile binding and possibly prevents receptor binding, other compounds of the present invention have lower IC^'so values. The prototypes P3-T3 and P3- T4 are expected to have tower IC»> values, in one variation, the chemical strategy used to generate these unique types of hybrid pharmacophores bod themselves to modular screening and extended structure-activity relationship studies.

Thus, the compounds, compositions and methods of the present invention are likely to add to the useful platinum-based drugs that are currently being used worldwide against genitourinal (bladder, ovaries, testes) cancers and carcinomas of the head and neck, as well as colon cancers. The present compounds, compositions and methods show an altered spectrum of activity compared to the currently available drugs nd show excellent activity in cancers insensitive to the available clinical platinums, as well as the traditional kinase inhibitors.

Brief description of the several views of the drawing

Figure 1 shows the structure of geflti.oib

Figure 2 (a) shows the structure of { Au(7)) 2 jC!^DMF ( l2a*DMF) in the solid state with selected atoms labeled. Counter ions and crystal solvent, have been omitted for clarity. fb) shows electrospray ionization mass spectrum recorded in positive-ion mode of dinuclear complex 12a. Characteristic ions: raft 420.3 |M(7H-HJ\ 518.3 MU2a -Aii÷H f \ 616.3 [M(] 2a}~2CS ^: 1035.5 [M(12a)~Au ^'.

Detailed description of the invention

In an embodiment, the present invention relates to a new class of chemotherapy compositions, compounds and methods aimed towards treating cancer caused by the

overexpression and/or mutation of the epidermal growth factor receptor (EGFR), specifically non-small cell lung cancer (NSCLC). In an embodiment, the present invention relates to synthetic routes capabl of utilizing EGFR tyrosine kinase inhibitors (TKis) as scaffolds for precious metal conjugation, leading to .i reversible inhibition of wildtype and .mutant EGF . in cancerous models via a solvent-accessible reactive cvsteme residue in the ATP-binding pocket of the enzyme. The chemical compos itions of these metal -T I hybrid agents are distinct .from those of current reversible and irreversible TKis currently in the clinic and on the market. These new metal-TKl hybrid agents (e.g., gold and platinum) possess the ability to result in decreased systemic toxicity when compared to classical DNA-targeted raetal- based chemotherapy (e.g. dspiathx) b driving the new agents towards proteins i volved in cancer proliferation and survival rather tha towards genomic DMA .

Thus, in an embodiment, the present invention may be useful for first- and/or second- line treatment option for cancers that are inherently resistant, or, have become resistant to clinical therapies, especially classical platinum drugs. The present invention may meet the urgent need (hat exists tor mechanistically novel drugs for the life-prolonging/curative treatment of NSCLC.

Thus, in an embodiment, the present invention relates to new gold(i) and platihum(.il)~ TK1 hybrid agents capable of utilizing the inherent nanomolar binding affinity of the TKI to create metal -induced irreversible enzyme inhibition, which was only previousl availabie via Mi chael acceptor/donor interaction s - la an embodiment_., the present invention also relates to synthetic methodologies capable of producing precious metal-TKl hybrid agents that are able to irreversibly inhibit a wide array of structurally diverse tyrosine kinases with solvent-accessible cysteine or methionine residues in the ATP binding pocket with great specificity.

Moreover, in an embodiment, the present invention relates to the design and synthesis of structurally new and previously undi sclosed go!dd) and piaiimun(JI)-TKJ

compounds/compositions displaying promising kinase selectivity and biological activity when compared to FDA-approved and widely used gefitinib.

In a variation, the present .invention also relates to ne thiourea-contajning kinase inhibitors (TI ) that are capable of overcoming the intrinsic resistance caused by the

secondary T790M EGFR. mutation to classical T Is (gefitinib).

In an embodiment, the present compounds/compositions containing P3-T2 and/or P - T2 show wiidtype EGFR dissociation constants comparable to gefitinib.

in an embodiment, the present invention relates to a new use for anti-cancer metals, allowing them to be repurposed for selective molecularly targeted therapies for the

management of resistant forms of cancer.

in an embodiment, the present invention relates to a new, selective target for platinom-based chemotherapy.

In an embodiment, the present invention uses the platinum and gold-based compounds to target several kinases for cancer chemotherapy, including the ErbB family of enzymes, VEGFR, and FGFR, among others. Small molecule kinase inhibitors often lose their effectiveness in tumors over time as a consequence of secondary mutations that significantly decrease the drag-enzyme binding affinity. The present invention attempts to overcome this type of acquired tumor resistance b introducing irreversible inhibitors thai covalently attach to the ATP binding pocket. The componnds/composuions and methods will serve as kinase inhibitors containing cystetne-binditig metal-based electrophi!es. The compounds feature organic ATP-mimicking scaffolds that bind to the target kinase active site with high affinity and selectivity while the eiectrophilic metal induces a permanent coordinative bond with the amino acid residue to achieve irreversible inhibition. Accordingly, in one embodiment, the present: invention presents structure-guided desi n and synthesis of these classes of molecules.

Several of the synthesized target molecules showed binding constants ( d) in the low- nanomolar range in competition binding assays and high selectivity for the targeted kinase domain in a panel of 145 wild-type and clinically relevant mutated kinases (KinoraeScan, DiscoverX, Fremont, CA). ^"The ability of the electrophilic agents to induce irreversible adducts was studied by LC ESI-MS/MS in pepsin digests of kinase incubated with inhibitor. Moreover, the in vitro cytotoxicity of selected derivatives was studied in solid tumor cell lines. The data generated suggest that the new pharmacophores will, likely have applications as therapeutics capable of overcoming acquired tumor resistance by irreversibly inhibiting deregulated kinases.

To generate a gold-modified. TKI., an N-heterocycHc quinazolme scaffold was used and the N~(3~chloro~4^«fluorophenyl) group from gefitinib (Figure 1 ), which target the adenine bindin site and an adjacent hydrophobic pocket of EGF -TK, respectively. The 6-position of the qutnay.oline C6 ring was chosen as attachment point for a side chain carrying a cysteine-affinic gold(!) moiety, similar in design to relevant Michael acceptor-based ITKls. The goal, of this design was to favor binding of the metal with cysteine- 797 proximal to the kinase's catalytic cleft without compromising the ligand's interactions with the binding pocket. The linkage between the eiectrophile and the TKI may be achieved via formation of a strong An(i)-S bond wit a thiourea residue; a thiol-like donor group previously explored in biologically relevant carrier Iigands of this metal.

A docking study was performed to examine the conformational space and receptor binding of thionrea-modified. TKIs. Structures of the gold-free Iigands were geometry optimized using DFT calculations and studied in complex with the EGFR kinase domain

(L858R/T790M mutant) (PDB-iD; 2JIV). hi these experiments, a scaffold was identified that produces energetically favorable binding geometries in which the thiourea sulfur of the TKI is positioned in close proximity (4-4.5 A) to the sulfur atom of cysteine-797. This orientation was considered compatible with electrophilic attack of the metal in a

corresponding gold(I)-rnodif?ed TKI on the protein thiol.

From, the modeling studies, two structures, 4 and 5 (see Scheme 1), emerged, which were considered viable candidates for the desired application.. The two derivatives (and all other deri vatives reported) were synthesized from common intermediate 3, which was generated by installing a 3-chioro-4-fluoroanilino group at the 4-positton of the quinazoline ring in precursor .1 and subsequent reduction of the 6-nitro group in 2 to a 6-araino group. Reaction of 3 with (he appropriate isothiocyanates afforded the ,Ν'-disubstituted iMourea derivatives 4 and 5.

Scheme 1 Synthesis of quina¾olitie derivatives. Reagents and conditions: a) 3~chloro~4~ fiuoroaniline, i-PrOH, ri_? overnight; b) Fe, AcOH, NaOAc, MeOH, reflux, 3 h; c) RN^'CS, EtOH, rt, 3 h; d) paraformaldehyde, NaOMe, N&BH4, MeOH, reft ax, 4 fi; e) MeNCS, DMAP, EtOH, reflux, 30 h; f) tert-butylmethyl(2-oxoeihyt)c-ar aniate, aCNBH3, AcOH, MeOH, rt, 2 h; g) 4 M HCL reflux, 2 h; h) MeNCS, EtOH, rt, I h.

Attempts to introduce gold(l) e!ectrophiies such as [AoX} (where X ^:::: CI~ or SCN-) into these structures using the common precursor j AiiXiiht}] (tht ^:::: tetrahydrothiophene), led to a product that was not isotatable (Scheme 2). Electrospray ionization raass spectra (E SIMS) of these reaction mixtures showed little sign of the desired ligand exchange to produce complexes | A.uX{4,5}], but were consistent with decomposition of 4 and 5 resulting in the analogous eyanamides (loss of H₂S, based on (M~34-f-H fragment ions) and gold(i) sulfide. Desulluraiion of {aromatic) N,N ^'-disubsiituted thioureas has previously been observed in the presence of heavy metals.

To circumvent the problem of !igaud desutfuration, an Ν,Ν,Ν '-trisubstituied derivative ? was synthesized from the Nfi-methyiqninazo!ine derivative 6, which was generated from intermediate 3 via reductive animation, in feactioos with [AuX(tht)], compound 7 showed greatly improved Ugand properties and was able to act as a stable sulfur- donor. X-ray cr siaUographic and mass spectrometfie analysis of the product isolated .from die reaction mixtures indicated that compound 7 does not act as a terminal S -donor !igand m the desired complexes [AiiX(7)j. instead, 7 produces dinuclear complexes f {Au(7}};? jX_;; in which 7 acts as a bridging Itgand (see 1.2a and 1.2b in Scheme 2).

?

not iso!abte

13

Scheme 2 Synthesis of gold(!) complexes. Reagents and conditions: a) ( AuX(tht)] (10) tht === tetrahydrottuophene, CH₂C , rt, 30 min; b) (AuCI(PEC₃)) (1 1 ), eOH/THF, AgNO¾, tt.

in the solid state structure of J 2a, the Ati(l) centers are coordinated by a thiourea-S atom of one Ugand and endocyclic qvdnazoline-N 1 of another ligaad. This produces a unusual arrangement in which the aromatic moieties of the two bridging ligands are mutually stacked at. van der Waals distance (see Figure 2a). Proton NM^'R chemical shift anomalies in conjunction with electrospray mass spectra recorded in positive-ion mode (see Figure 2b) confirm that the dinuclear caiionic structures persist in solution and are not solely a

consequence of packing effects in the solid state. Importantly, no reversal of the dinuclear structure was observed, when 12a was incubated under physiologically relevant

concentrations in phosphate-buffered saline (containing -140 m.M chloride) for several days ((ddaattaa nnoott sshhoowwnn)).. T Thhiiss oobbsseerrvvaattiioonn ssuuggggeessttss t thhaatt 1122aa ccaannnnoott bbee ccoonnssiiddeerreedd aa cclleeaavvaabbllee •p prreeccuurrssoorr ooff tthhee ccoorrrreessppoonnddiinngg mmoonnoonnuucclleeaarr ccoommpplleexx,, jj AAuuCCli((77))||..

AAtttteemmppttss ttoo iinnssttaallll aa mmoorree iinneerrtt pphhoosspphhiinnee l liiggaanndd iinn ppllaaccee ooff XX vviiaa r reeaaccttiioonn ooff^™ 7 w wiitthh jj AAuuCCII((PPEEt. )HH 1155]] ttoo aavvooiidd ssuubbssttiittuuttiioonn ooff tthhee l liiggaanndd ttrraannss ttoo ssuullffuurr b byy qqiittiinnaazzoolliinnee n niittrrooggeenn 55 wweerree u unnssuucccceessssffuull ((nnoo ssuubbssttii t tuuttiioonn o off c chhlloorriiddee bbyy tthhiioouurreeaa wwaass oobbsseerrvveedd)).. TThhee llaatttteerr rreeaaccttiioonn ssuucccceeeeddeedd wwhheenn t thhee ttrriissuubbssttiittuutteedd tthhiioouurereaa m mooiieettyy wwaass iinnccoorrppoorraatteedd iinnttoo aann eexxtteennddeedd ssiiddee cchhaaiinn aatt t thhee i 6m-ppoossiittiioonn ooff tthhee qquuiionaazzooiiiinnee ssccaaffffoolldd,. Innt trroodduuccttiioonn ooff tthhee ((22--mmeemmyyllaammiinnoo))eetthhyyll ggrroouupp iinnvvoollvveedd rreedduuccttiivvee a ammiinnaattiioonn ooff 33 ttoo ggiivvee 88,, wwhhiicchh w waass fi finnaallllyy ccoonnvveerrtteedd ttoo tthhiioouurreeaa 99 wwiitthh iissootthhiiooccyyaannaattee.. WWhhiillee 99 ddiidd nnoott yyiieelldd tthhee ddeessiirreedd ggoolldd --mmooddiif liieedd m moonnoonnuucclleeaarr ddeerriivvaattiivvee 1100 wwhheenn rreeaacctteedd wwiitthh ff AAuuXX((tthhtt))]],, i itt. pprroodduucceedd tthhee ccaattiioonnkk ccoommpplleexx | [AAuu((PPEEit^;¾»)X(99))]|{(NN0O₃)

((ccoommppoouunndd 1133)) ((sseeee SScchheemmee 22)),, wwhhiicchh pprroovveedd ttoo bbee ssttaabbllee wwiitthh rreessppeecctt ttoo ddeessiittiififiiirraattiioonn aanndd ddiinnuucclleeaattiioonn ((bbaasseedd oonn E ESSII--MMSS aanndd NNMMRR ddaattaa)).. C Chhaarraacctteeririssttiicc vvaarriiaattiioonnss iinn ¹¹HH NNMMRR cchheemmiiccaall sshhiiftftss oobbsseerrvveedd ffoorr ccoommpplleexx 1133 rreellaattiivvee t too lliiggaanndd 99 ccoonnffiirrmm sseelleeccttiivvee bbiinnddiinngg ooff tthhee {{AAuu((PPEE ))FF mmooiieettyy ttoo tthhiioouurreeaa ssuullffuurr..

Cell proliferation assays in two NSCLC cell lines were performed to assess the cell growth inhibitory effects of the newly synthesized Τ Ϊ derivatives 7 and 9 alone and as ligands in complexes 12a, 12b, and 13. For comparison the clinical drug gefitinib was included in the screening. The ceil Ike NCI-H460 is a model of a large-cell carcinoma and i 20 characterized by wild-type EGFR-TK. By contrast, NCl-Hl 975 adenocarcinoma cells harbor a point mutation (L858 ) in exon 21 as well as a secondary mutation CT790M) at the bottom of the hydrophobic ATP binding pocket. Whi l e the former somatic (activating) mutation sensitizes cancer cells to EGFR-TKIs (including gefitinib), the latter causes resistance to these therapies.

25 The resul s of the cell proliferation assays are summarized in Table 1. Both NCI-H46Q

(wild-type) and CI-IT1975 (mutated) show the expected resistance to gefitinib with inhibitory concentrations hi the high rnicromolar range, in agreement with previously reported dat (typically, ¾β > it⁾ p [2b]), NCTH46 cells were found to be more sensitive to all of the analogues tested than NCI-Hi 975, except for compound 9. The new analogue 7

30 was the least acti ve compound tested with high- icrornokr IC50 value in a range previously observed for structurally related thionrea-modilled TKls. By contrast, extension of the side chain on carbon-6 of the quinazoiine ring to generate 9 led to a pronounced increase in potency, in particular in NCl-Hl 975, where a cytotoxic enhancement of 30-fold is observed. Most notably, compound 9 partially overcomes the acquired resistance against gefitinib

S observed in this cancer model. The low-micromolar inhibitory concentration detemiined for compound 9 in. resistant NCI-HI.975 compares favorably with tbe bjgh-nanomolar/low- micromolar activity typically observed for gefiiinib in. sensitive NSCLC models.

Modification of compounds 7 and 9 with { AuX} and {Au(PEt;)F groups, respectively, had only a minor effect on the inhibitory concentrations, with compound 13 maintaining low- rnicromolar activity in both cell Hues.

Tabie 1. Summary of Cytotoxicity Oata MC.v Vaiues}- "

Compd NCI-H460 NCI-H1975

7 36.0 ± 4.4 51.7 ± 1.1

9 4.2 0.4 1.7 + 0.1

12a 19.1 ± 2.0 39.7 ± 1.7

12b 20.1 ± 0.2 31 .9 ± 0.4

13 1.9 ± 0.1 2.5 ± 0.1 gefiiinib 14.2 ± 0.5 30.0 ± 1.3 !C$o values ± S.D. (piVI) were extracted from drug-response curves for two experiments performed in triplicate for each concentration.

Using simple !igand substitution chemistry, which has been previously developed to generate mixed ihiourea-(pseudo)halldo and iiiiourea-phosphme goid(I) complexes, a sulfur- modified TKJ derived from gefiiinib was introduced as a ligand. Compound 9 alone showed significantly better activity than gefiiinib in a T Jkesistant cancer cell line. This observation suggests thai the newl introduced thiourea-containing side chain may enhance the binding affinity of the classical TKI structure with the enzyme's active site. In the lowest-energy model generated for compound 9 in complex with EGFR-TK (L858 /T7 0M mutant) the tluourea-modifted side chain is located in the hydrophihe region of the protein cleft produced by the bilobal kinase Ibid, (see Supplementary Information). This orientation, positions the i ourea-NH group in close hydrogen-bonding distance to residues Asn-842 and Asp-855, which may promote strong binding to the EGFR-TK domain.

The goal of turning compound 9 into a goid(I)-modified TKI was to further enhance the biological activ ity of the TKI by promoting reaction of cysteine- 797 of the EGFR-TK with a targeted metal-based electrophile. Potential binding mechanisms of such a

pharamcophore may involve cross-link formation between the protein and the ligand in the ATP binding pocket of the TKI, or complete transfer of the gold moiety from the TKI to tire amino-acid sulfur (Scheme 3). Both types of irreversible modification have the potential to help overcome resistance mediated b reduced TKI binding affinity in the T790M mutant as a consequence of secondary mutations in the active site of the kinase. Unfortunately, unlike thiourea sulfur in compound 7, the corresponding side chain in 9, while confcrcnationaliy more flexible, appears to adopt an unfavorable orientation for this application (S_t¾0,_iK;i-— Scy* * 9 A). The ability of compound 9 to act as a targeted carrier of ( Au(PEt¾)] ^r would also depend on the stability of the mixed thiourea-phosphine coordination in compound 13 in the presence of competing nucleophiies. These include sulfur-containing proteins in circulation and in the eytosol Thiourea as a donor was chosen .for its relative ease of synthesis and for its ihioi-like properties and ability to compete with thiolate sulfur in gold(J) coordination. Alternati ve gold-affniic iigands remain to be tested as part of a broader effort to establish structure-activity relationships in gold-modified EGFR-TKis.

Scheme 3. Potential reactions of go!d(I) with cysteine sulfur hi the ATP binding site of EGFR-TK via gold(i) transfer (left side pathway) of g ki(i)-raediated cross-link formation (right side pathway). The same concept applies to derivatives containing plaiinum(ll) and other metals.

While the intention of the current design was to hijack gold into the ATP-binding pocket of the tyrosine kinase, a potential mechanism by which complex 13 disassembles into a gokl~phosphhie species and analogue 9 in the presence of reactive bio iigands prior to reaching the enzyme active site has also to be considered. Gold(I>-phosphmes directly target the mitochondria to trigger oxidative stress which leads to apoptosis. Aberrant EGFR expression is associated with defective mitochondrial apoptotic signaling., which renders affected cancer cells insensitive to conventional chemotherapy. Thus, a dual mechanism promoted by the individual components that involves inhibition of EGFR signaling and cell, death triggered by mitochondrial toxicity may help overcome tumor resistance.

Derivative 9 showed promising activity in the ceil proliferation assay, but attachment of the gold electrophiie in .13 did not result in the desired enhancement in cell kill. One possible explanation for this observation would be that, the [Au(PEt, 3⁺ group, while not compromising the binding affinity of the T l moiety with the receptor, may be positioned unfavorably for reaction with the sulfur atom of cysteine-797. It is also possible that cytotoxic gold-phospbine species generated from complex 13 may not significantly

contribute to the cell kill in the two cancer models, which, ma be dominated by TK

inhibition.

Hydrogen ietraehloroaurate trihydrate was purchased from Aha Aesar, The tetnihydrothionplienego!d(i) complexes 1 Oa and. 1 Ob. I i 1 chlorotriethylphosphinegoldii) 1 1 ,( 15] compound L[ 12] and tert-but.yim.etliyi(2-o oethyl.)carbamate| l j were synthesized. using published procedures. For the preparation of biological buffers, biochemical grade chemicals (Fisher/Acros) were used. HPLC-grade solvents were used for ail HPLC and mass spectrometry experiments. Reagents and chemicals were acquired from common vendors and used without further purification. Reactions involving gold were performed and solutions stored in the dark.

Ή M.R spectra of the target compounds and intermediates were recorded on Bruker

Advance 300 and D.RX-50G instruments. 'C MR spectra were recorded on a Bruker DRX~ 500 instrument operating 125.8 MHz. Chemical shifts (o) are reported m parts per million (ppm) relative to terramethylsilane (TMS). Electrospray ionization mass spectra (ESI- S) were recorded, on an Agilent J. iOOLC/M.SD trap instrument. Ion evaporation, was assisted by a flow of j drying gas (300-350 °C) at a pressure o 40-50 psi and a flow rate of 1 1 L min. Mass spectra, were typically recorded with a capillary voltage of 2800 V over a mass-to- charge (m/z) scan range of 200-2200. The purity and stability of the target, compounds was analyzed by reverse-phase high-performance liquid chromatography (HPLC) using the LC module of the Agilent Technologies 1 1 0 LC/MSD trap system equipped with a .nrulti- wavelength diode-array detector. Separations were accomplished with a 4.6 mm ^x 150 mm reverse-phase Agilent. ZORBA SB-C.18 (5 um) analytical column, at 25 ^,;:C. Separations were accomplished with the following solvent, system: solvent A-optima water/0.1 % formic acid; solvent B-methanol J % formic acid; solvent C --acetonitrile/0. 1. % .formic acid.

Separations were performed at a flow rate of 0.5 mL/min and a gradient of 50 % A / 50 % 8 to 5 % A 95 % 8 over 20 mm (for 7), and at a flow rate of 0.5 mL min and a gradient of 95 % A / 5 % C to 5 % A / 95 % C over 20 rain (for 9). HPLC traces were recorded over a wavelength range of 363 -463 ran. High-resohrtion mass spectrometry (HRMS) was performed on a Thermo Scientific LTQ Orbitrap XL equipped with an electrospray ionizaiion source. Single crystals of 7 were grown from a saturated solution in methanol, while crystals of 12 were grown by slow diffusion of diethyl ether into a concentrated DMF solution. HPLC peaks were integrated with MestReNova 8.1. For all organic Hgaads studied, in cancer cells, an analytical purity of > 95 % was confirmed by reversed-phase HPLC. The purity of compounds 12 and 1 3 was determined by CHN elemental analysis (intertek Pharmaceutical Services, Whitehouse, NJ). Single-crystal X-ray data acquired for compounds 7 and 12a and experimental details of the structure solution and refinement have been deposited with ihe Cambridge Crysialiographie Data Centre (Cambridge, U.K.) under deposition codes

CCDC9766U and CC.DC976612, respectively.

Computational studies

The inhibitor structures were built in GaussView 4,0 (Seroichera Inc., Shawnee

Mission, KS, 2009), Structures were optimized at the ti>3!yp level of theory using the 6- 31 1 ** basis set in Gaussian 03 (Gaussian, Inc., Pittsburgh PA, 2003). [22] Prior to docking studies, structures were checked in AutoDockTools 1.5.4 (The Scripps Research institute, La Joila, CA) for possible bond torsion errors, and appropriate Gasteiger charges were calculated and assigned as necessary. The crystal structure of EGFR (L858R T790M mutant) from the Brookhaven Protein Data Bank (PDB ID: 2J1V) was used as the 3-D receptor structure for docking studies, and the active site coordination ranges were defined using the grid box in AutoDockTools. The inhibitor structures were then docked into the active site using

uioDock Vina, 231 Conformational searches were performed with an exhausfiveness setting of '9\ Results were evaluated based on relative docking energies.

Tested Coinputtnds

New thiourea and aniine-derivatives, T1 and T2 (see below for structures), have been synthesized and fully characterized. New metal-TKl derivatives GT-Tl , P3-T2, and P9-T2 have been synthesized and fully characterized (see below for structures).

Compounds 'Π, Gl-Tl, P3-T2, and Ρ -Γ2 (see Scheme 4) have been tested in vivo in both CI-H460 (wildtype EGFR) and NCI-H1 75 (double-mutaai EGFR) for biological activiiy, EGFR dissociation constants for P3-T2 and P9-T2 have been, determined utilizing the KmomeScan at Discove Corporation (see Tables 2 and 3 below for IC50 and K4 values). Along with EGFR dissociation constants, a kinase selectivity assay was performed for P3-T2 by DiscoveRx utilizing 145 distinct kinases, which resulted in multiple selectivity

^■Mis", mostly representing wildtype and mutant EGFR proteins.

P9-T2

Scheme 4

Compounds to be Tested

Compounds T3 (see Scheme 5) and T4 (see Scheme 6) and their platinum derivatives are bemg synthesized. Biological testing will be completed in NCI-H460 and NCI-H1975 cell lines. All P!aiinum-TKI derivatives will be tested in SKBR3 cells this spring to determine (heir uiiiity in Her2-positive breast caacer as irreversible Her2 inhibitors.

P3-T3 P3-T4

P3-T3

Scheme 5 Scheme 5a

Schemes 5 and 5a (top) show different schemes to make compounds wherein. X is cis to Rj. The bottom portion of scheme 5a shows how to make compounds wherein the linker that i s Rs-E in formulas S. and. la can be made any len¾th. in an embodiment, scheme 5b shows how one can varv- the structural diversity in chemical libraries by performing reactions at different temperatures. In. an embodiment, the ratio of nilrile substitution vs. nitriie addition can be varied by varying the temperature.

Scheme 5i>

P3-T4

Scheme 6 ίί should be noted that for the reactions in schemes 4-6, the outcome products of the reactions can be varied by var ing the temperatures with either the nitrite substitution product or the nitrite addition product as the major component (for an example, see scheme 5b).

In one embodiment, the design using metal-based electrophiles has a fundamental advantage over Michael-acceptor-based inhibitors (suc as afatmib): with the latter structures only active-site cysteine residues can be effectively targeted. In contrast, platinum forms stable bonds with many other ammo-acid residues, such as methionine, histidine, and lysine, as well as nitrogen of the peptide backbone, in addition, in one embodiment, the present design can be tailored for use with other kinases. This includes enzymes involved in the escape mechanisms that confer resistance to EGF (e.g. FGFRl) and kinases that cannot be targeted with current irreversible drugs because their active sites do not have targetable cysteines.

Preparation of compounds

-(3~Chloro-4-fiiKirophenyi}-7-ethoxy-6~nit (2). A mixture of I. (4.55 g, 18.0 rnmoi) in 40 niL ofdichloromethane (DCM) and 3-chIoro-4-tTuoroaniline (2.61 g, 18.0 mmo!) in 80 raL of isopropanol was stirred at room temperature overnight. The solid was collected and washed with small amount of DCM to give 6.6? g (93 %) of 2 as a bright yellow powder. Ή^" N R (300 MHz, DMSO-d6) δ i 1.69 (bs, 1 H), 9.59 (S, I E), 8,93 (s, iH), 8.07 _f J ==== 6.8, 2.6 Hz, i H), 7.77 (ddd, J - 9.0, 4.4, 2,6 Hz, I H), 7.63 (s, I H), 7.54 (L J - 9.1 Hz, IH), 7,27 (q, J - 8.8 Hz, 1 H), 4.39 (q, J - 6,9 Hz, 2H), ! .44 (t, S - 6.9 Hz, 3H); ^UC NMR (126 MHz, DMSO-46) δ 1.59.45, 156.29, 155.51, 154.99, 154,34, 139.92, 134.79, 126.18, 124.92, 123.34, 1 19.75, ί 17.54, 107.29, 105.92, 99.98, 66.76, 14.50; MS (ESI) mix

N⁴^3 ¾loro-4-flaomphenyl)-7^moxyquina lme-4,6-diam.ine (3). A mixture of 2 (2 g, 5 mmol), iron (1.68 g, 30.1 mmol), acetic acid ( 1,8 g, 30.3 mmoi), and sodium acetate (0.41 g, 5.0 nimol) in 80 .raL of methanol wa refl.ux.ed for 3 h . To this mixture were added 3 ml. of cone , ammonia. The mixture was filtered while hot, the solid, washed with hot.

methanol, and the solvent removed from the filtrate by rotary evaporation. The resulting solid was extracted four times with hot acetone. After the acetone was removed from the extracts, water was added to the residue and the slurry was stirred for 30 min. The yellow precipitate was filtered, washed with water, and dried in a vacuum at 60 °C to yield 1.42 g (85 %) of 3. ^!H MR (300 MHz, DMSOd6) δ 9.38 (s, IH), 8.38 (s, I H), 8.25 ~ 8.1 1 (m, IH). 7.90-7.73 (m, IH), 7.38 (m, 2H). 7.08 (s, IH), 5.33 (s, 2H), 4.22 (q, J === 7.2 Hz, 2H), 1.45 (l, J ~^: 7.0 Hz, 3H); ⁱ³C NMR ( 126 MHz, DMSO-d6) 6 154.90, 153.51 , 151.83, 151.58, 150.12, 144.70, 138,42, 137.46, 122.34, 121.34, 1 18.58, 1 16.38, 1 10.19, 106.24, 100.75, 63.77, 14.34; MS (ESI) τη.·7. 333. 1 j \HH } \

]-(4-((3~Chloro^-flnorophen.yl}amm^

(4) . A mixture of 0.1 g (0,3 mmol) of 3 and 0.033 g (0.45 mmol) of methylisothiocyanaie was stirred in 3 mL of ethanol at room temperature for 3 h. The solution was concentrated to half its volume and stored in die refrigerator overnight. The precipitate formed was collected, washed with ethanol to yield 47 rug (39 %) of 4. 1H NMR (50 MHz, DMSO-d6) § 9.71 (s, IE), 9.08 (s, 1 H), 8.61 (s, 1 M), 8.57 (s, IH), 8.20 (dd, J = 6.8, 2.6 Hz, IH), 7.84 (did, J = 9.2, 4.3, 2.7 Hz, 2H), 7.43 (t, J - 9.1 Hz, Hi), 7.26 is, IH), 4.24 (q, J ==== 7.0 Hz, 2H), 2.94 (d, J ==== 4.3 Hz, 3H), .1.41. (t, I - 6.9 Hz, 3H); ¾ NMR (126 MHz, DMSO-d6) 5 182.39, 157.41 , 157.17, 154.69, 154.52, 1 52.59, 1 50,42, 137.22, 128.17, 123,57, 122.48, 121.31 , 1 19.26, 1 17.07, 109.16, .108.22, 64.79, 31.94, 14.83; MS (ESI) ra/z 406.1 [M÷Bf.

l~(4-((3-Chtoro-4-i1uoropl ⁱnyl)amino)-7-elhoxycpi.nazolin-6-y

(5) , This analogue was synthesized using the same procedure and stoichiometric amounts described for compound 4. Yield 35 mg (25 ¾). Ή NMR (300 MHz, DMSO-d6) δ 9.95 (s,

I B), 9.74 (s, 1 H), 9.36 (s, 1 H), 8,72 (s, l H), 8.57 (s, 11:1), 8,1 (dd, J ^::: 6,9, 2.7 Hz, IH), 7.83 (ddd, J - 9,4, 4.4, 2.7 Hz, IH), 7.56 ~ 7.30 (m, 5H), 7.27 (s, 1 H), 7. 16 (t, J - 7.3 Hz, IH), 4.26 (q, J = 6.9 Hz, 2H), 1.43 (t, J = 6.8 Hz, 3H): C NMR (126 MHz, DMSO-d6) § 180.38, 156,97, 156.69, 154.19, 1 54.17, 1 53.99, 152.06, 149.99, 139, 17, 136,66, 128.41 , 128.06, 124.72, 124.06, 1 23.12, 1 22.01 , 121 .16, 1 18.70, 116.50, 108.48, 107,54, 64.31 , .14.32; MS (ESI) m, z 468.2 [M+H] *^'.

N⁴-(3-Chloro-4-0iK}rophenyl)-7-etl½^ (6). To a solution of sodium methoxide in 30 mL of methanol (prepared from 0.35 g/15 mmol of sodium metal) were added 1 g (3 m ol) of 3 and 0.45 g (.15 mmol) of paraformaldehyde. The reaction mixture was refluxed for 2 h and subsequently cooled to 0 °C. After 0.57 g (15 mmol) of sodium teiral dro bo ate were added in small portions,, die orange mixture turned light yellow, and heating at reflux was continued, for another 2 h. Solvent was removed by rotary evaporation and the residue was washed with water. The solid, was then dissolved in a minimum amount of methanol^'DC ( 1: 1), and the solution was passed through a Celsie pad to remove a minor amount of a black solid. Solvent was removed to give 0,7 g (67 %) of 6 as a light yellow powder. *R R (300 MHz, DMSO~d6) § 9.31 is, I H), 8.37 (a, I H), 8.1 (dd, j - 6.9, 2.7 Hz, IH), 7.84 (ddd, J = 9.1 , 4.4, 2.6 Hz, IH), 7.42 (t, J - 9.1 Hz, IH), 7.13 (s, IH), 7.05 (s, IH), 5.71 (q, J === 5.1 Hz, I H), 4.23 (q, .1 - 6.9 Hz, 2H), 2.92 (d, J === 4. Hz, 3H), 1.45 0, j - 6.9 Hz, 3H); ^K5C NMR (75 MHz, DMSO-d6) <5 154.77. 154,31 , 151.69, 151.10, 149.94, 144.41, 139.89, 137.29, 122.83, 121.79, 1 38.67, 1 16.42, 1 10.11 105.56, 5.67, 63,86, 30,03, 14.33; MS (ESI.) m/z 347, 1 (^'M+Hf.

i-(4-((3~Chloro^- fluorophenyl}^

dimethylthionrea (7). A mixture of 2.5 g (7.2 mmol) of 6, 1 ,05 g (14.4 mmol) of

memylisothiocyanate, and 1.76 g (1 .4 mmol) of 4-dimethyIaminopyridine (DMAP) in 20 mL of ethanol was stirred at reflax for 30 h. Additional methylisothiocyanate was added after 6, 18, and 24 b (2 eq each time). Ethanol was removed and tbe resulting dark oil was applied io a triethylamine-treated silica gel column. DCM/methanol (50: 1) was used to remove non- polar impuri ties and the same sol vent at a 30: 1 rati was used to elute the product. The product fractions were combined, and solvent was removed by rotary evaporation. The residue was recrystallized iron^'s ethyl acetate to give 1.05 g (35 %) of 7 as orange crystalline solid; ^lH NMR (500 MHz, DMSO-d6) δ 9.72 (s, IH), 8.61 (s, IH), 8.48 {s, 1H), 8.23 (dd, J - 6.9, 2.7 Hz, 1H), 7.85 (ddd, j - 9.2, 4.3, 2.7 Hz, 1 H), 7.44 ft, J - 9.1 Hz, IH), 7,33 (s, H), 7.1 (s, IH), 4.24 (q, J = 6.9 Hz, 2H), 3.47 (s, 3H), 2.85 (d, J - 4.2 Hz, 3H), 1.36 (t, J = 6.9 Hz, 3H); ^UC NMR (126 MHz, 0MSO-d6) 5 183.41, 158.43, 157.37, 155.44, 154.59, 152.66, 151 .97, 1 37.07, 1.33, 10, 124.53, 123.48, 122.35, 1 19.33, 1 17.13, 109.82, 109.68, 64,81 , 56.49, 1.9.02, 14.8.1 ; HRMS m/z [M+Hf caicd for C_{f y}H ₀ClF>½OS: 420.1061 , found:

420.1032.

N4-(3-Chk}ro-4-i¾of0p

diamine (8). A mixture containing 2.0 g (6.0 mmol) of 3, 0.68 g (Ί 8 mmol) ofNaCNBHj, and 2.1 g (12 mmol) of tert-butylmethyl(2-oxoethyi)carbamate in 40 ni t of methanol was ^•prepared. Sufficient glacial acetic acid (-1.5 g) was added until the solid was completely dissolved, A yellow precipitate formed after the .reaction was stirred for 12 h at room temperature. Solvent was removed and the resulting oil was redissolved in. DCM, washed with saturated NaHCOs and brine, and dried over Na^SO*. DCM was removed and the residue was heated at reflux in 50 mL of 4 M HC1 for 2 h to remove die Boc protecting group. Acid was removed by rotary evaporation, and foe resulting oil. was allowed to solidify by stirring in ethanol at room temperature overnight. The solid was collected, washed with small amounts of ethanol, and partitioned between DCM and i M NaOH solution. The organic layer was recovered, washed with brine, and dried over ajSC . After the solvent was removed .1 .0 g (5 i %) of 8 was obtained as a white solid. ³H NMR (300 MHz, DMSO- d6) o 9. 1 (s, IH), 8.37 (s, !H), 8.15 (dd, J = 6.9, 2.6 Hz, ! H), 7.83 (ddd, J - 9.1 , 4.4, 2.7 Hz, HI). 7.42 (t, J ==== 9.1 Hz, IH), 7.24 (s, IH), 7.07 (s, 1H), 5.45 (t J === 5,4 Hz, 10), 4.2 (q, J === 6.9 Hz, 2H), 3,32 (q, J - 5.8 Hz, 2H), 2.S4 (f, J - 6.0 Hz, 2H), 2.35 is, 3H), 1.44 it, J - 6.9 Hz, 3H); ^lsC NMR (126 Mfite, DMS046) δ 154.82, 153.70, 151.77, 151.56, 150.16, 144.52, 138.77, 1.37.20, 122.93, 121.87, 1 18.63, 1 16.38, 1 10.04, .105.82, 96.42, 63.93, 49.75, 42.19, 35.81, 14.31 ; MS (ESI) ro/z 390.2 [M+H .

1 -{2-{{4-((3-Chloro-4-i¾^

1 ,3-dimethylthiourea (9). A mixture of 0.14 g (1.92 moi) ofmelhyiisolhiocyanate and 0.5 g (1.28 ramol) of 8 in 10 mL of eihanol was stirred at room temperature for I h to produce a white solid precipitate, which was collected, washed with cold, eihanol, and dried to yield. 0.53 g (90 %) of 9. ^!H NMR ( 500 MHz, DMSO-d6) S 9.24 (s, 1 H), 8.39 (s, 1 H), 8.18 (dd, J = 6.9, 2.7 Hz, 1 H), 7.85 (ddd, J = 9.1, 4.0. 2.7 Hz, 1 H), 7.51 (q, J - 4.2 Hz, 1 H), 7.43 (t, J ^« 9.1 Hz, 1H), 7.25 (s, 1H), 7.06 (s, 1H), 5.85 (ΐ, J - 5.3 Hz, 1.H), 4.22 (q, 2H), 4.16 (t, J - 6.4 Hz, 2H), 3.47 <q, j - 6.0 Hz, 2K), 3, 10 (s, 3M), 2.94 (d, J - 4.1 Hz, 3H), 1.45 (t J = 6.9 Hz, 3H); "C NMR (126 MHz, DMSO-d6) 8 182.50, 155.32, 154.22, 152.29, 152.17, 150.70, 14$ M , 139.13, 137.76, 123.26, 122.19, 1 19.22, 116.99, i.10.57, 106,31 , 96.57, 64.51 , 51.89, 41.95, 37.90, 33.19, 14.97; HRMS m ¾. (M+.H. calcd for C₂i.¾₅ClFN₆OS: 463.1483, found: 463.1464.

|Au(7)} j]Clj (12a). A mixture of 0.10 g (0.24 ramol) of compound 7 and 76 mg (0.24 mmoi) of chlorotetrahydrot-iiophenegold(l) (10a) in 5 ml, of DCM and 1 mL of methanol and was stirred at room temperature for 30 mm. When the solution was concentrated to a volume of - 1 mL, compound 12a precipitated as an oil-white solid, which was collected, washed with DCM, and dried in a vacuum at 60 °C. Yield; 0.1 16 g (75 %}. Ή NMR (500 MHz, DMSO-46) δ 9.77 (s, 1H), 8.89 (s, IH), 8.63 (s, !H), 8.54 (s, IB), 8.23- 8.15 (ra, IH), 7.86-7.78 (m, IH), 7.46 (t, J - 9.1 Hz, 1H), 7.33 (s, 1H), 4.25 (s, 2H), 3.53 is, 3H), 3.1? (s, 3H), 1.45-1.30 (m_f 3H); ¾ NMR (126 MHz, DMSC 6) δ 175.87, 157.60, 157.44, 155.79, 1.54.75, 152,81 , 152.02, 136.88, 133.21 , 124.09, 123.79, 122.59, 119,37, 117.19, 109.68, 65.09, 55.37, 34.21, 14.88; HRMS m/z j j2+ calcd for

C.¾H₃sAu₂C}2F2 Ki02S₂: 16.0648, found: 616.0611; anal calcd for

C₃sH_3SAu₂Cl F₂N_Se0₂S₂CH₂CI₂: C 33.20, H 2.71, N 10.19, found: C 33.49, H 2.65, N 10.13. (The analogous ihiocyatiate salt 12b showed the same mass spectroscopic and NMR features; no other details are reported.)

|Au(9)PEtjK^'NO_s} (.! 3). To 0, 153 g (0,34 nunol) of 9 i a mixture of 10 mL of THF and 5 mL of methanol 0.122 g (0.34 ramol) of ch!orotriethylphosphinegold(l) was added. The mixture was stirred for 5 mm, and 5 μΤ of a Ig ml, aqueous solution of Ag Oj were added to exchange chloride with ni trate counter ions. Precipitated AgCl was filtered off and the filtrate was conceiiixated and added to 20 mi, of diethyl ether. Compound 13 precipi tates as a yellow solid, which was washed with diethyl ether and dried in a vacuum. Yield: 0.126 g (46 %). 'li NMR (500 MHz, DMSO-d6) δ 932 (s, 1 H), 8.41 (s, 1 H), 8.2? (q, J === 4.2 Hz, 1 li), 8.15 (dd, J - 6.8, 2.6 Hz, 1.H), 7.83 (ddd, J - 9.1 , 4.0, 2.7 Hz, 1H), 7.44 (id, J - J , 0.8 Hz, !H), 7.29 (s, !H), 7.10 (s, IH), 5.77 (t, J=5.35 Hz, f H), 4.24 (q, J = 6.9 Hz, 2H), 4.19 (t, J - 5.6 Hz, 2E), 3.57 (q, J - 6.1 Hz, 2H), 3.21 (s, 3H), 3.10 (d, J - 4.0 Hz, 3H), I .S9 (dq, J - 10.6, 7.6 Hz, 6H), 1.45 (t, J - 6.9 Hz, 3H), 1 .05 (dt, J - 19.3, 7.7 Hz, 9H); ⁿC NMR (126 MHz, DMSO-d6) o 175.45, 1 54.87, 153.77, 151.84, 151.76, 150.27, 144.14, 138.31 , 137.01 , 122,84, 121 .78, 1 18.70, 1 16.49, 109.91 , 105.65, 96.57, 64.05, 52.18, 40.8, 33.57, 16.78, 14.37, 8.94, 8.83; HRMS m/z [M.f calcd for C_2?H_J9AuaF ₆OPS: 777 J 982, found:

777.1957; anal, calcd for C₂₇¾AaCIFN₇0₄PS: C 38.6 , H 4.68, N 1 .67, found: C 38.13, H 4.25, 10.79.

Cell pro lifer a tien ¾ssa y

The human non-small cell lung cancer cell lines, NC1-H4 0 (large cell) and NCI- H i 975 (adenocarcinoma), were obtained from the American Type Culture Collection (Rockville, MD, USA). Both cefl lines were cultured in RPMI-1640 media (HyCloiie) supplemented with 10 % fetal bovine serum (FBS), 10 % penstrep (P&S). 10 % L-gintamrae, and 1 .5g/L aHCCh, Cells were incubated at a constant temperatore at 37 °C in a humidified atmosphere containing 5 % C€½ and were subculfured every 2 to 3 days in order to maintain ceils in logarithmic growth. The cytotoxicity studies were carried out according to a standard protocol using the Celt titer 96 aqueous nonradioactive eel! proliferation assay kit (Prauvega, Madison, W^'i), Stock solutions (10 mM) of all drugs were made i DMF and serially diluted with media prior to incubation with cancer ceils. 1C« values were calculated from dose- response curves using sigmoids! curve fits in GraphPad Prism (GraphPad Software, La Jolla, CM

Thus, in an embodiment, the present invention relates to compounds, compositions and methods comprising the compounds of Formula 1 or Formula la:

FORMULA I

FORMULA la

wherein X is halo, ^", H₂0, ^«OC(0)R_¾ -Pi( ¾)qC¾)3, nitrate, sulfate or a earbene of struc ture

;

wherein q is 0, 1, 2, 3, 4, 5, or 6;

Xt and Xj are independently halo,

nitro, amino, - NHC(O)(R_J0)> -NHC(O)0(R.jo>, -C(0)NHRj,,, or -OC(Q}NHE_{f 0};

M is l inear gold(l), square planar p!atinum(IlX gold(Hl), or an octahedral metal including but not limited to plat um(IV), rulhenmm(II), foiheniumCIll), rhod um HI), sodiuraflii); Rs and s are ammo, ammonia or pyridine groups or together with the platinum atom to which they axe attached, Rt and R? form the ring ~NHHC¾)_V~NH;;~ wherein v is 1. 2, 3, or 4 or R; and R.₂ together can be any of the following groups a-h;

wherein A is H, -C¾, OOfe, Cf¼ or NCb;

s is -N(R.2fi)- or S; wherein ¾ is hydrogen or Ci-C¼alkyl;

RA is hydrogen, Ci.f, alkyl, or CH Rjs;

E is -iCi h_. r.

R is a direct bond, -NH- or Cj-Csaikylene;

or R5 and X together with the atoms io which they are attached form a ~ or ?-merabered ring, wherein said 6- or 7~.membered ring contains a linking group ~C(0)0- or ~OC(0}-; R? is hydrogen, methyl, ^«CH(R|7_.)(R½)_> -C JO-R^, or - OC(0)-RJS; wherein

5 is hydrogen or Cj-s alkyl;

3 _S is hydrogen, alkyl, -0!(Κ)ί)Χ¾ο phenyl, rtaphthyl, C3.6 cycloalkyi norbo yl, adamantyl, a natural or unnatural amino acid or a peptide;

Rj.> is hydrogen or C alkyl;

R₂₀ is hydrogen. CM alkyl;

R_s is -NR₅,r, -C(0) NR_{i >}- NR_{3 C(0)-, -0-, -S-, -OC{0)-, and -CfO) O- R;B is -H or -Chalky!;

R<> is hydrogen, C <_> alkyl, phenyl, napiuhyi,

cycloalkyl, norbomyl, adamantyl, a natural or unnatural amino acid or a peptide;

R₁₂ and Ri₂ are independently hydrogen, hydroxy!, Cj.?; alkyl, -OCH₃,-CF_s, ⁽¾;

R 4 and R25 are independently hydrogen or Cj-jaJk l; and

Z Is independently one or more halo or nitro. or one or more connterions suffic ent to balance die charge of the compound.

When M is an octahedral metal, it should be recognized that all possible geometric cis ar«J trans isomers of X, R 1 , and R2 are possible. It should be noted that in one embodiment, other octahedral metals besides those listed above can potentially be used as a possible variables for M.

in an embodiment, when M is Au(i), the compounds, compositions and methods of the present invention that incorporate the formulas (compounds arid compositions) of the presen invention can be derived from compounds where X. is trans to ¾.

la an embodiment, X_? is F or CI and X₂ is F or CI. In an embodiment X;> is F and X; is F or CI. In an embodiment, X₂ is F or CI and X_} is CI, in a variation, X₂ is F and X₅ is CI. is an embodiment, X is -PC-CHi-CH;;);:. In an embodiment. R₃ is -NH or S. in art embodiment, ₅ and R? together are ethyl or -NHCH¾. In a embodiment, R₄ is H or C¾ in an embodiment, RJJ is H or C¾. In an embodiment, E is methylene. In an embodiment, R;> is methyl or ethyl , hi a variation, R₉ is ethyl.

It should be understood that the methods, compounds and compositions of the present invention are generic. For example, the unique modular approach can be used to generate other ΪΤΚ.Ϊ pharmacophores targeting various cancer-related kinases. For example, the structure of WZ4002, an irreversible EGFR inhibitor and AVL-2 2, a Button's tyrosine kinase (BTK) inhibitor, a clinically approved drug, can be easily modified to allow attachment of a monofunctional platinum and/or gold m iety (see scheme 7 - shown with platinum).

it should be understood that modifications can be made to the present invention without departing from the spirit and scope of the present invention. For example and in one embodiment, rather than. Slaving a uinazoh^'tie group as a central heterocycle, it is contemplated and therefore withi the scope of the inventio that a pyrimidine moiety, or a benzene moiety might occupy the position occupied by the qiiinazolme. For example, modifications might be made to known cancer drugs to add the platinum moiety using the methodology as disclosed in the present invention to produce modified derivatives of ponatiiiib, XZ4002 or to AVL-292 (or other known cancer drugs) as shown in scheme 7. Scheme

R1 = sfkyl, ester

:R2 = a!kyl, ar l

:N^AN = en, pn, feytecta, (NH₃)₂, tmpda, (RR,RS.S5)-2,3-tiabn o - 0, 1 , 2, 3

X = NH, NHC(O), CH_S, O

Accordingly, in an aiiemate embodiment, the compounds, compositions and methods of present invention include compounds of Formula II:

+

FORMULA H

wherein X is halo, H₂0, -OC(0}R₉, -Ρ((-€Ηΐ)¾ΟΗ. 3_? nitrate or sulfate;

wherein q is 0, 1, 2, 3, 4, 5, or 6;

Xi is independently halo,

nitro, ammo, - HC(OKRio), - H:C(0)0(Riy), -C(0) HRjo_; or -OC{ )NHR₁₀;

M is An or Pi and if M is An, Rj and ₂ are not. present;

Rj and Rj are amino, ammonia or pyridine groups or together with the platinum atom to which they are attached, R_¾ and R? form the ring -ΝΗ_:?-(€Ι-Ϊ;_?Χ'- ¾~ wherein, is ί , 2, 3, or 4 or R._s md ^'Ri together can be any of the following groups a-h;

wherein A is II -C¾, -OCHj, CF₃ or N<¾;

R.5 is -N(R¾)- or S; wherein 26 is hydrogen or CrOsalkyi

R is hydrogen, Ct-₆ alkyl, or CH₂-R_{2;

E is -·«¾),,-;

Rs is a direct bond, -HE- or CVCsalkvlene; or Rs and X together with the atoms to which they are attached form 6- or 7-membered ring, wherein said 6- or 7~raeffibered ring contains a Sinking group -0(0)0- or -0C(0)-; R? is hydrogen, methyl -CHi^'R.nXRis), ~C(O)0~R₅*, or - OC(0)-R₁₈; wherein

Rj? is hydrogen or C_t^ alkyl;

is is hydrogen, C _<> alkyi, -CHiRt&XRa)), phenyl, naphthyl,€¾„<5 cycioa!kyl, norhornyl, adamantyl, a natural or unnatural amino acid or a peptide;

R ; is hydrogen or Cj.?, alky!;

ji> is hydrogen, C alkyl;

R* is -NR»-, -C(0) NR NR ΟΌΚ -0-, ~S~, -0C(0}-, and ~C(0) O- Ro is -H or -Chalky!;

Rj. and Rn are independent! y hydrogen, hydroxyl, CM alkyl, -OCH ,-CF₃, NO;?:

Ri is CVaalkoxy;

Ri5 is N-methyl-piperazine or -^H^'CHa^OCH;?;

R and 5 are independentl hydrogen or C_halk !;

and

Z is independently one or more halo or itro, or one or more counterions sufficient to balance the charge of the compound.

hi an embodiment, the present invention relates to compounds of Formulas 1 and/or 11, to compositions (e.g., pharmaceutical compositions) containing those compounds aid to methods of using those compounds.

Those of skill in the art will recognize that in formulas ί and II, if M - Ait and X - negatively charged residue, e.g., halide, there will be no counter ion, Z, and no net charge on the molecule.

i an embodiment, the pharmaceutical composition, may contain pharmaceutically acceptable salts, solvates, and prodrugs thereof, and may contain diluents, excipients, carriers, or other substances necessary to increase the bioavailability or extend the lifetime of the compounds of the present invention.

Subjects that may be treated by the compounds and compositions of the present invention include, but are not limited to, horses, cows, sheep, pigs, mice, dogs, cats, primates such as ehimpat ees, gorillas, rhesus monkeys, and, humans. In a embodiment, a subject is a uman in need of cancer treatment.

The pharmaceutical compositions containing a compound of the invention may be in a form suitable for injection either by itself or alternatively, using liposomes, micelles, and/or nanospheres. The pharmaceutical composition suitable for injection can be made as disclosed i Lammcrs, T. et ai, J. Controlled Release, 1.61 , 175-18? (2012), or in Barenholz, Y., J.

Controlled Release, 160, 1 17-134, (2012), both of which, are incorpora ted by reference m their entireties. Alternatively, compositions intended for injectio may be prepared according to any known method, and such compositions may contain one or more agents selected from the group consisting of solvents, co-solvents, solubiiizing agents, wetting agents, suspending agents, emulsifying agents, thickening agents, chelating agents, antioxidants, reducing agents, antimicrobial preservatives, buffers, pH adjusting agents, bulking agents, protectants, tonicity adjusters, and special additives. Moreover, other non-toxic pharmaceutically-acceptable excipients which are suitable for the .manufacture of injectab!es may be used.

Aqueous suspensions may contain the active compounds in an admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethy!ceilulose, methyicel!uJose, hydroxypropylraethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide such as lecithin, or condensation products of an alkylene oxide with fatty acids, for example

polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaeihyl-eneoxycethanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as

poiyoxyethyleue sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more coloring agents.

Oily suspensions may be formulated b suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil sesame oil or coconut oil, or in a mineral oil such as a liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an ami-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active compound in admixture with a dispersing or wetting agent, suspending agent and. one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients., for example, sweetening, flavoring, and coloring agents may also be present. The pharmaceutical compositions of the invention may also be in the form of oi -in- water emulsions. The oily phase may be a vegetable oil, for example, olive oil or arachis oil, or a mineral oil, for example a liquid paraffin, or a mixture thereof. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth.

naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbUan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.

The pharmaceutics! compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known methods using suitable dispersing or wetting agents and suspending agents described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptahle diluent or solvent, for example as a solution in 1 ,3-butanedioi. Among the acceptable vehicles and solvents that may be employed are water, sterile water for injection (SWF1), Ringer^'s solution, and isotonic sodium chloride solution, in addition, sterile, fixed oils are conveniently employed as solvent or suspending medium. For this purpose, any bland fixed oil may be employed using synthetic mono- or diglycerides. in addition, fatty acids such as oleic acid find use in the preparation of injectabl.es.

Thus, in another embodiment, the present invention provides a pharmaceutical formulation solution comprising a compound of Formulas 1 and/or ff or a salt thereof.

A solution of the invention may be provided, in a sealed container, especially one made of glass, either in a unit dosage form or in. a multiple dosage form.

Any pharmaceutically acceptable salt of a compound of Formulas 1 and/or II may be used .for preparing a solution of the invention. Examples of suitable salts may be, for instance, the salts with mineral inorganic acids such as hydrochloric, hydrobromic, sulfuric, phosphoric, nitric and the like, and the salts with certain organic acids such as acetic, succinic, tartaric, ascorbic, citric, glutamic, benzoic, methanesulfonic, ethanesulfomc and the like. In an embodiment, the compound of Formulas ί and/or 11 is a hydrochloric acid salt including a mono, di, or trihydrochloride.

Any solvent which is pharmaceutically acceptable and which is able to dissolve the compounds of Formulas I and/or 11 or a pharmaceutically acceptable salt thereof may be used. The solution of the invention may also contain one or more additional components such as a co-solubilizing agent (which may be the same as a solvent), a tonicity adjustment agent, a stabilizing agent, a preservative, or mixtures thereof. Examples of solvents, co-solubilk g agents, tonicity adjustment agents, stabilizing agents and preservatives which may suitable for a solution formulation are described below.

Suitable solvents and co-solubilizing agents may include, but are not. limited to, water; sterile water for injection (S^'WFi); physiological saline; alcohois, e.g. ethanol, benzyl alcohol and the like; glycols and polyalcohols, e.g. propyleneglycoi, glycerin and the like; esters of po!ya!cohols, e.g. diacetine, triacetine and the like; polyglycols and polyethers, e.g.

Dolyethyleneglycol 400, propyleneglycoi methyiethers and the like; dioxoianes, e.g.

isopropyliden lycerin and the like; dimethylisosorbide; pyrrolidone derivatives, e.g. 2- pyrroiidone, N-medryl-2-pyrroiidone, polyvinylpyrrolidone (eo-solubilizing agent only) and the like; polyoxyethyienated fatty alcohols; esters of polyoxyethyienated fatty acids;

po!ysorbates, e.g., Tween™, polyoxyethylene derivatives of polypropylenegiycols, e.g., Pluronics™.

Suitable tonicity adjustment agents may include, but are not limited to,

pharmaceutically acceptable inorganic chlorides, e.g. sodium chloride; dextrose; lactose; mamiitol; sorbitol and. the like.

Preservatives suitable for physiological administration may be, for instance, esters of ^•parahydroxybenzoic acid (e.g., methyl, ethyl, propyl and butyl esters, or mixtures of them), chlorocresol and the tike.

Suitable stabilizing agents include, but are not limited to, monosaccharides (e.g., galactose, fructose, and fucose), disaccharid.es (e.g., lactose), polysaccharides (e.g.. dexiran), cyclic oligosaccharides (e.g., alpha-, beta-, gamma-cyclodextrin), aliphatic polyols (e.g., mamiitol, sorbitol, and thioglycerol), cyclic polyols (e.g. inositol) and organic solvents (e.g., ethyl alcohol and glycerol).

The above mentioned sol vents and co-solubi!izing agents, tonicity adjustment agents, stabil izing agents and 'preservatives can be used alone or as a mixture of two or more of them in a solution formulation.

In an embodiment, a pharmaceutical solution formulation may comprise compound of Formulas Ϊ aod/or II or a pharmaceutically acceptable salt thereof, and an agent selected from the group consisting of sodium chloride solution (i.e., physiological saline), dextrose, mamiitol, or sorbi tol, wherein the agent is in an amount of less than or equal to 5%. The pB of such a formulation may also be adjusted to improve the storage stability using a

pharmaceutically acceptable acid or base.

In the sol u tions of the inven tion the concentration of the compound of Formulas Ϊ and/or II or a pharmaceutically acceptable salt thereof may be less than 100 mg/mL, or less than 50 mg mL, or less than 10 mg/mL, or less than 1.0 rag mL aad greater than 0.01 mg/raL, or between 0.5 mg/mL and 5 mg/raL, or between 1 mg/mL and 3 mg/mL. In an embodiment, die concentration that is used is the idea! concentration to be sufficiently cytotoxic to the cancer ceils yet limit the toxicity on other cells.

Suitable packaging for the pharmaceutical solution formulations may be all approved containers intended for parenteral use, such as plastic and glass containers, ready-to-use syringes and the like, in an embodiment, the container is a sealed glass container, e.g. a vial or an ampoule. A hermetically sealed glass vial is particularly preferred.

According to an embodiment of the present invention, there is provided,, in a sealed glass container, a sterile, injectable solution comprising a compound of Formulas 1 and or II or a pharmaceutically acceptable salt thereof in a physiologically acceptable sol vent, and which has a pH of from 2.5 to 3.5. For solution formulations, various compounds of the present invention may be more soluble or stable for longer periods in solutions at a H lower than 6. Further, acid salts of the compounds of the present invention may be more soluble in aqueous solutions than their free base counter parts, but when the aeid salts are added to aqueous solutions the pH of ihe solution may be too low to be suitable for administration. Thus, solution formulations having a pH above pH 4.5 may be combined prior to

administration with a diluent solution of pH greater than ? such that the pH of the

combination formulation administered is pH 4.5 or higher. In one embodiment, the diluent solution comprises a pharmaceutically acceptable base such as sodium hydroxide. In another embodiment, the diluent solution is at pH of between 1 and 12. In another embodiment, the pB of the combined formulation administered is greater than. 5.0. In another embodiment, the pH of the combined formulation administered is between pH 5.0 and 7.0.

The invention, also provides a process for producing a sterile solution, with a pH of from 2.5 to 3.5 which process comprises dissolving a compound of Formulas I and/or II or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable solvent. Where a pharmaceutically acceptable acid salt of a compound of Formulas Ϊ and/or IT is used the pH of the solution may be adjusted using a pharmaceutically acceptable base or basic solution addin a physiologically acceptable acid or buffer to adjust the pH within a desired range. The method may further comprise passing the resulting solution through a ste.rilizt.ug filter- One or more additional components such as co-soiubiimng agents, tonicity

adjustment agents, stabilising agents and preservatives, for instance of the kind previously specified, may be added to the solution prior to passing the solution through the sterilizing filter. in a limber variation, the present invention contemplates combination therapies in which the compounds of the present invention cao. be used in conjunction with other cisplatin compounds. The efficacy of this combination therapy is likely to be enhanced because of the differeiii mechanisms and modes of aciion ihai first generation cisplatin compounds exbibii relative to the compounds of the present invention. It is also contemplated and therefore within the scope of the invention that other anti-neoplastic agents compounds can be used in conjunction with the compounds of the present invention. The anti-neoplastic

agents/compounds that can be used with the compounds of the present invention include cytotoxic compounds as well as non-cytotoxic compounds.

Examples include anti-tumor agents such as HERGEPTIN ^m {trastuzumab),

R1TUXAN™ (riii!ximab), ZEVALIN™ (ibriliimomab tiuxetanX LYMPHOCIDE™

(epratuxumab), GLEEVAC^m and BEXXAR^1M (iodine 131 tosiiiimoraab).

Other anti-neoplastic agents/compounds that can be used in conjunction with the compounds of the presen t in vention include aati-an.giogen.ic compounds such as ERB1TUX* O C-C225), DR (kinase domain receptor) inhibitory agents (e.g., antibodies and antigen binding regions that specifically bind to the kinase domain receptor), antt-VEGF agents (e.g., antibodies or antigen binding regions that specifically bind VEGF, or soluble VEGF receptors or a ligand binding region thereof) such as AVASTTN^m or VEGF-TRAP^iM, and anii-VEGF receptor agents (e.g., antibodies or antigen binding regions that specifically bind thereto), EGFR inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto) such as ABX-EGF (panitumnmab), IRESSA ^ίΜ (gefitinsb), TARCEVAⁱ (etSoiioib), anti-Angl and atiti-Ang2 agents (e.g., antibodies or antigen binding regions specifically binding thereto or to their receptors, e.g., Tie2 Tek), and anii~Tie2 kinase inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto).

Other anti-angiogenic compounds/agents that can be used in conjunction with the compounds of the present invention include Campath, ΪΪ..-8, B-FGF, Tek antagonists, anti- TWEAK agents (e.g., specifically binding antibodies or antigen binding regions, or soluble TWEAK receptor antagonists, ADAM distintegrin domain to antagonize the binding of integral to its Hgands, specifically binding anti-eph receptor and/or anti-ephrin antibodies or antigen binding regions, and anii-FDGF-BB antagonists (e.g., specifi.cally binding. antibodies or antigen binding regions) as well as antibodies or antigen binding regions specifically binding to FDGF-BB ligands, and PDGFR kinase inhibitory agents (e.g., antibodies or antigen binding regions that specifically bind thereto). Other aati-angiogenie/anti-tumor agents that can be used in conjunction with {^'he compounds of the present invention include: SD-7784 (Piker, USA); cilengitide. (.Merck KGaA, Germany, EPO 770622); pegaptanib octasodium, (Gilead Sciences, USA);

Alphastatin, (BioAeta, UK); M-PGA, {Celgeiie, USA); ilomastat, (Arriva, USA,); emaxanib, (Pfizer, USA,); vafcalanib, (Novartis, Switzerland); 2-methoxy¾stradiol, (EntreMed, USA); TLC ELL- .12, (Elan, Ireland); auecortave acetate, (Afcon, USA); alpha-D148 Mat, (Amgen, USA); CEP-7055, (Cephalon, USA); anti-Vn Mab, (Cmce!l Netherlands)

DAOantiangiogenic, (ConjuChem, Canada); Angiocidin, (InKine Pharmaceutical, USA); KM-2550, (Kyowa Hakko, Japan); SU-0879, (Pfizer, USA); CGP-79787, (Novartis, Switzerland); the ARGENT technology of Ariad, USA; YIGSR-Stealth, (Johnson & Johnson, USA); fibrinogen-E fragment, (BioActa, UK); the angiogenesis inhibitors of Trigen, UK; TBC-I635, (Encysive Pharmaceuticals, USA): SC-236, (Pfizer, USA); ABT-567, (Abbott, USA); M.eiastatin, (EntreMed, USA); angiogenesis inhibitor, (Tripep, Sweden); maspin, (Sosei, Japan); 2-met.hoxyestradioL (Oncology Sciences Corporation, USA); ER.-6S203~00, CWVAX, USA); Benefm, (Lane Labs, USA); Tz-93, (Tsuraura, Japan); TAN-l 120, (Takeda, Japan); F -1 1 1 142, (Fujisawa, Japan); platelet factor 4, (RepliGen, USA); vascular endothelial growth factor antagonist, (Borean, Denmark); bevacizumab (pINN), (Geneirtecli, USA); XL 784, (Exeiixis, USA); XL 647, (Exeiixis, USA); MAb, alpha5beta3 integrin, second generation, (Applied Molecular Evolution, USA and Medlmmune, USA); gene therapy, retinopathy, (Oxford BioMedica, UK); enzastaurin. hydrochloride (USAN), (Lilly, USA); CEP 7055, (Cephalon, USA. and Sanofi-Synthelabo, France); BC 1 , (Genoa Institute of Cancer Research, Italy); angiogenesis inhibitor, (Alehemia, Australia); VEGF antagonist, (Regeueron, USA); rBPI 21 and BPI-derived antiangiogenic, (XOMA, USA); PI 88, (Progen, Australia); cilengitide (piNN), (Merck KGaA, German; Munich ^'Technical University, Germany, Seripps Clinic and Research Foundation, USA); eetuxirnab (INN), (Aventis, France); AVE 8062, (Ajinomoto, Japan); AS .1404, (Cancer Research Laboratory, New Zealand); SG 292, (Tehos, USA); Endostalm, (Boston. Children's Hospital USA); ATN 161, (Attention, USA); ANGIOSTATIN, (Bostoo. Children's Hospital USA); 2-methoxyesiradiol, (Boston Children's Hospital, USA); ZD 6474, (AstraZeneca, UK); ZD 6126, (Angiogene Pharmaceuticals, UK); PPI 2458, (Praecis, USA); AZD 9935, (AstraZeneca, UK); AZD 2171 , (AstraZeneca, UK); vatalanib (pINN), (Novartis, Switzerland and Schering AG, Germany); tissue factor pathway inhibitors, (EntreMed, USA); pegaptanib (Ptnn), (Gilead Sciences, USA); xan horrhizol, (Yorisei University, South Korea); vaccine, gene-based, VEGP-2, (Seripps Clinic and Research Foundation, USA); SPV5.2, (Sopratek, Canada); SDX 103, (University ofCaHfcraia at San Diego, USA); FX 478, (ProiX, USA); METASTATIN, (EntreMed, USA); troponin L (Harvard University, USA); SU 6668, (SUGEN, USA); OXI 4503, (OXiGENE, USA); o~guanidines, (Dimensional Pharmaceuticals, USA);

motuporamine C, (British Columbia University, Canada); CDP 7 1 , (Ceiltech Group, UK); aiipri od (plNN), (GlaxoSmithKlme, UK); E 7820, (Eisai, Japan); CYC 381 , (Harvard

University, USA); AE 941, (Aeterna, Canada); vaccine, angiogenesis, (EntreMed, USA); urokinase plasminogen aciivaior inhibitor, (Dendreon, USA); oglufanide (plNN), (Melmotte, USA); HIF-lalfa inhibitors, (Xenova, UK); CEP 5214, (Cephalon, USA); BAY RES 2622, (Bayer, Germany); Angioeklin, (InKine, USA); A6, ( Angstrom, USA); KR 31372, (Korea Research Institute of Chemical Technology, Sooth Korea); GW 2286, (GlaxoSmith line,

UK); EHT 0101 , (ExonHit, France); CP 868596, (Pfizer, USA); CP 564959, (QSI, USA); CP 547632, (Pfizer, USA); 786034, (GlaxoSmithKline, UK); R 633, (Krri Brewery, Japan); drug delivery system, intraocular, 2-me(hoxyeslradioI, (EntreMed, USA); anginex,

(Maastricht University, Netherlands, and Minnesota University, USA); A.BT 5 0, (Abbott, USA); AAL 993, (Novartis, Switzerland); VEGl, (ProteoraTech, USA); tumor necrosis factor-alpha inhibitors, (National Institute on Aging, USA); SU 1 1248, (Pfizer, USA and SUGEN USA); ABT 51 , (Abbott, USA); YH16, (Yantai Rongchang, China); S-3APG, (Boston Children's Hospital, USA and EntreMed, USA); MAb, KDR, (ImClone Systems, USA); MAb, aiphaS beta!, {Protein Design, USA); KDR kinase inhibitor, (Ceiltech Group, UK, and Johnson & Johnson, USA); GFB 1 16. (South Florida University, USA and Yale University, USA); CS 706, (Sankyo, Japan); conibretastatin A4 prodrugs, (Arizona State University, USA); ehondroitinase AC, (IBEX, Canada); BAY RES 2690, (Bayer, Germany); AGM 1470, (Harvard University, USA. Takeda, Japan, and TAP, USA); AG 13925, (Agouron, USA); Tetrathionro!ybdate, (University of Michigan, USA); GCS 100, (Wayne State University, USA) CV 247, (Ivy Medical, UK); CKD 732, (Chong Kun Dang, South Korea); MAb, vascular endothelium growth factor, (Xenova, UK); irsogladine (INN),

(Nippon Shinyaku, Japan); RG 13577, (Aventis, France); WX 360, (Wilex, Germany);

squaJamine (plN), (Geoaera, USA); RPi 4610, (Sima, USA); heparanase inhibitors, (InSight, Israel); L 3106, (Kolon, South Korea); Honokio!, (Emory University, USA); Z CDK, (Schering AG, Germany); ZK Anglo, (Schering AG, Germany); ZK 229561, (Novartis,

Switzerland, and Schering AG, Germany); XMP 300, (XOMA, USA); VGA 1 102, (Taisho, Japan); VEGF receptor modulators, (Pharmacopeia, USA); VE-cadherin-2 antagonists,

(ImClone Systems, USA); Vasostatin, (National Institutes of Health, US );vaccine, FSfc-i , (ImClone Systems, US ); TZ 93, (Tsumura, Japan); TomStatin, (Beth Israel Hospital, USA); truncated soluble FLT I (vascular endothelial growth factor receptor I X (Merck & Co, USA); Tie- 2 hgands, (Regenerou, USA); and, thmmbospondm 1 inhibitor, (Allegheny Health, Education and Research Foundation, USA).

it is contemplated and therefore within the scope of the in vention that the compounds of the present invention can be modified to target specific receptors or cancer cells or can be modified so that they can survive various in vivo environments. As examples, the compounds of the present invention can be modi fied so that th ey are com bined with dendrfmers or other cyclic sugars to form carboxylate dendrimers or other sugars. They may be combined with steroids such as estrogen to form carboxylate steroids like carboxylate estrogen, if the compounds of the present invention contain carboxylate functionalities, the carboxylate functionalities on these compounds may be modified so that they contain folic acid. Those of skill in the art will recognize that there are other modifications that can be made to the compounds of the present invention so that they can target specific receptors, ceils or provide stability to the compounds, it is contemplated that the compounds of the present invention can have modifications made that are covalent modifications, ionic modifications, modified so that they chela te to other compounds, or other undergo some other type of interaction that allows the compounds of the present invention to suit their use (such as hydrophobic or Van der Waa^'ls type interactions).

In a further variation, the compounds of the present invention can be used against solid tumors, cell lines, and ceil line tissue that demonstrate npregulated nucleotide excision repair and other upregulated resistance mechanisms. Thus, in an embodiment, the present invention discloses methods of treating cancer in an individual in need thereof by the use of a compound of Formulas I and/or II .

The Platinum-based and Gold-based compounds of the present invention may also be used against genitou inal (bladder, ovaries, testes) cancers and carcinomas of the head and neck, as well as colon cancers. Although the compounds of the present invention may also show promise against these various cancers and carcinomas, the compounds of the present invention show an altered spectrum of activity compared to previous drugs used against these various cancers and carcinomas, the compounds of the present invention also show excellent activity in cancers insensitive to the clinical platinums of the prior art.

In a variation, the compounds of the present invention can be used for treatin diseases of abnormal cel.! growth mi/or dysregulated apoptosis, s uch as cancer,

mesothioloma, bladder cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, ovarian cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of me endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the valva, bone cancer, ovarian cancer, cervical cancer, colon, cancer, rectal cancer, cancer of the anal region, stomach cancer, gastrointestinal (gastric, colorectal and duodenal), chronic lymphocytic leukemia , esophageal cancer, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer o f the adrenal gland, sarcoma of soft tis sue, canc er of the urethra, cancer of the penis, testicular cancer, hepatocellular cancer {hepatic and billiary duct), primary or secondary central nervous system tumors, primary or secondary brain tumors, Hodgkirfs disease, chronic or acute leukemias, chronic myeloid leukemia, lymphocytic lymphomas, lymphoblastic leukemia, follicular lymphoma, lymphoid malignancies ofT-ce!l or B-ceil. origin, melanoma, multiple myeloma, oral cancer, ovarian cancer, non-small cell lun cancer, prostate cancer, small ceil lung cancer, cancer of the kidney and ureter, renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of the central nervous system, primary central nervous system lymphoma, non-Hodgkin's lymphoma, spina! axis tumors, brains stem glioma, pituitary adenoma, adrenocort cal cancer, gall bladder cancer, cancer of the spleen, cholangiocarcmoma, fibrosarcoma, neuroblastoma, retinoblastoma, or a combination thereof, hi a variation, the compounds of the present invention can be used in the treatment of non-small cell lung cancer, pancreatic cancer, breast cancer, and ovarian cancer.

Alternatively, the compounds of the present invention, can be used in the treatment of non- small cell lung cancer, pancreatic, and ovarian cancer.

in an embodiment, the compounds of the present invention also have potential applications as antiviral and anti- Alzheimer's drugs.

Thus, it is contemplated thai the compounds, compositions and methods of the present invention, .may be useful for first- and/or second-line treatment option for cancers that are inherently resistant, or, Stave become resistant to clinical therapies, especially classical platinum drugs. The compounds of the present invention may provide an avenue to solve the urgent need .for mechanistically novel drugs for the life-prolonging/curative treatment of SCLC.

ft is contemplated and therefore within the scope of the present invention that any feature that is disclosed in the present invention can be combined with any other feature in the present invention. For example, if a variable is described with reference to formula I, it should be recognized that it is contemplated and therefore within the scope of the present invention that the defined variable can also be used with Formula If it is also contemplated that minor .modifications can be made to the present invention.. The following references are incorporated by reference in their entireties.

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Claims

We claim

1. a I or ia:

FORMULA I

FORMULA Ia

wherein. X is halo, OH^", H₂0, -OC(Q)Ri>, -P((~CH₂)qCt¾k nitrate, sulfate or a carbene of structure

;

wherein q is 0, i, 2, or 3;

5 and ^'2 are independently halo,

nitro, amino, - HC(0XR_K.), - HC(0)0(Rjo), -€(0) ¾«) or ^«OC(0)NHR_to; M is Au or Pi and if M is Au, Rs. and R_. are not present;

R.i and R_¾ are amino, ammonia or pyridine groups or together with the platinum atom to which they are attached, R_t and R₂ form the ring

whereiu v is I , 2, 3, or 4 or R₃ and Rj_. together can be any of the following groups a-h;

wherein A is H, -CH -OCHj, CF₃ or N<¾;

R* is - (R¾)- or S; wherein ¾; is hydrogen or Ci -C_halky! ;

R4 IS hydrogen, C alkyl, or CHj-R-._;j;

is a direct bond, -NH- or C{-C§alkylene;

or Rj and X together with the atoms to which they are attached form a 6- or 7- membered ring, wherein said 6- or 7-membered ring contains a linking group - C(0)0- or ~OCiO)-;

R₇ is hydrogen, methyl, ~CH(R, ₇)(R_i;<),, ~C(0)0~R_t¾ or - OC(0)-R _ls; wherein

Rt? is hydrogen or CM alkyl;

Ris is hydrogen, C alkyl, -CH{Ri; R2o), phenyl, naphthyi, C3-6 cycioalkyl, norbomyl, adaro.antyf a natural or unnatural amino acid or a peptide;

Rjy is hydrogen or CM alkyl;

jo is hydrogen, C M alkyl;

Rs is -NRs.r- -C(0 NR_u-₅- NR»._? C(O)-, -0-, -S-, -OC(0)-, and -C(0) O- Ri is --H or -Ci-ealkyi;

R>> is hydrogen. C alkyl, phenyl, naphthyi, C3-6 cycioalkyi, norbomyl, adamantyl, natural or unnatural amino acid or a peptide; Ru and Rt* are independently hydrogen, hydroxy!,

aikyi, -OC¾,-CF¾ N<¾

R;? and R;?s are independently hydrogen or Chalky!.;

and

Z is independently one or more halo or nitre, or one or more coun tenons sufficient to balance the charge of the compound,

2. The compound, pharmaceutically acceptable salt, or solvate of claim I wherein j is F or CI and X is F or CI,

3. The compound, pharmaceutically acceptable salt, or solvate of claim 1, wherein ? is F and X) is CI.

4. The compound, pharmaceutically acceptable salt, or solvate of claim i wherein M is Ai! and X is -P(-CH CH_s)₃.

5. The compound, pharmaceutically acceptable salt, or solvate of claim I wherein R₃ is -NH or S.

6. The compound, pharmaceutically acceptable salt, or solvate o f claim I wherein R> and R-7 together are ethyl or -NHQ¾.

7. The compound, pharmac uticaH acceptable salt, or solvate of claim 1 wherein ₄ is H or C¾.

8. The compound, pharmaceutically acceptable salt, or sol vate of claim 1 wherein R_}3 is H or CHs.

9. The compound, pharmaceutical y acceptable salt, or solvate of claim 1 wherein R* is ethyl.

10. The compound, pharmaceutically acceptable salt, or solvate of claim 1 wherein j is CI and Xj is F, wherein Rj is -NH or S, wherein R_$ and R- together are ethyl or - NBC¾, wherein E is .methylene, wherein R₄ is H. or CHj, wherein R»j> is H or CHj, and wherein R=> is ethyl .

11. A pharmaceutical composition comprising compound, pharmaceutically acceptable salt, or solvate of Formula I or Formula la:

FORMUL A I

FORMULA la

whereiii X is halo, O ^", H₂0, -OC(0)R¾₅ -P(( ¾)qC¾)3, nitrate, sulfate or a earbene of struc ture

;

wherein q is 0, 1, 2, or 3;

Xi and Xz are independently halo, O^alkoxy, Cs^alkyl, Ca^lkya l, iCro, amino,. - HC(OKRjo), ~NHC(0)0(Rjo), -C(0)NHR_u>, or OC(0)NHR_U»;

M is A.a or Pi ant! if M is An, Rj and Rj are not present; Rj and Rj are amino, ammonia or pyridine groups or together with the platinum atom to which they are attached, Rj and j form the ring ~ ii;?-{CH2)_v- I:¾- wherem v is 1 , 2, 3, or 4 or j and R₂ together can be any of the following groups a~h;

wherein A is H, -€¾ -OCM3, OF* or NO_;5;

R₃ is -N(R½)- or S; wherein ;¾ is hydtogeu or

R4 is hydrogen,

alkyl, or CH.2-R.s₂;

E is ~C H₂

Rs is a direct bond, -NH- or Cj-Q_>alkylene;

or R5 and X together with the atoms to which they are attached form a 6- or 7- membered ring, wherein said 6- or 7-membered ring contains a linking group - CfO)0- or -OC(Ok

R7 is hydrogen, methyl, -CH(Ri7)(R_{!f ?} ~€(0)0-R_i¾ or - OC(C))~R_!S: wherein.

R j7 is hydrogen or Ci„6 alkyl;

Ris is hydrogen, C ; alkyl, -€H(K !_¾){¾(}), phenyl, naphthyl, eycioalky!, norbomyl, adamantyi, a natvaal or unnatural amino acid csr a peptide;

R -.j is hydrogen or C ; alkyl;

R₂₀ is hydrogen, alkyl;

R₈ is -NR._l3-, -C(0) NR_{i r},- NR» C(Oh -0-, -S-, -OC(0)-, and -C(0) O- j 3 is -H or -Cs-csalkyl;

Ry is hydrogen, Ci.g alkyl phenyl, naphthyl,€>;.₍, eycloalkyi, norbomyl, adamantyl, natural or unnatural amino acid or a peptide;

ji and R; are independently hydrogen, hydroxy!, al l,

NO?; ; 4 and « are independently hydrogen

and

Z is independently one or more halo or iiitro, or one or more eoanieriom sufficient to balance the charge of the compound and one or more pharmaceuticall acceptable diluents, excipients, or carriers.

12, The composition of claim 11, wherein Xj is F or CI and X? is F or C!,

13, The composition of claim 11 , wherein X₂ is F and X) is CI.

14. The composition of claim 1 K wherein M is Au and X is -P(-CH CHj)5.

1 5. The composition of claim .1 K wherein R₃ is -NH or S.

16. The composition of claim. .1 .1 _s wherein. R₅ and - together are ethyl or -NHCHj.

17. The composition of claim i i, wherein R is H or C¾ and wherein R^ is H or CH-t.

18. The composition of claim 1. 1, wherein R-._; is ethyl

19. A. method of treating cancer comprising administering to an individual in need thereof a compound, a pharmaceutically acceptable salt, or a solvate of Formula 1 or Formula 3

FORMUL A I

FORMULA la

wherein X is halo, O ^". H₂0, ~OC(0}¾ .P((-CH₂)qCH₃)3, niijrale, sulfate or a carbene of structure

_;

wherein q is 0, K 2, or 3;

Xj and .X.2 are independently halo,

nitm, amino, -NHCCOXRioi, -NHC(0)0(Rio), <¾0)NHRjo, or -OC(0)NHR_JO;

M is Au or Pi and if M is Ao₅ R_s aod R₂ are not present;

Rj and Rj are amino, ammonia or pyridine groups or together with the platinum atom to which they are attached, R¾ and R> form the ring ~NHHC¾X~ H₂- wherein v is 1, 2, 3, or 4 or Ri and j together can be any of the following groups a-h;

wherein A is H, -C%, -OCH¾ CF._? or N0₂;

s is - (R.;¾)~ or S; wherein. R¾ is hydrogen or CrC^alkyl;

R₄ IS hydrogen,

alkyl, or CHj- ;?;

E is -HC¾)_£r;

R? is a direct bond, - H- or Ct-€_f>alkyiene;

or R. and X together with the atoms to which ihey are attached form a 6- or ^'7- membered ring, wherein said 6- or 7-membered ring contains a linking group - (0)0- or -OC(0}-;

R₇ is hydrogen, methyl, -CH{R_i7XRts.k -OOXVRts, or - OC(0)-Ri₈; wherein

R - ; is hydrogen or C ; alky!;

Rj8 is hydrogen, Ci-6 alkyl, -CH(R<<>}(R;¾), phenyl, naphthy!, cycloalkyl, norborayl, adaroantyl, a ttaturai or unnatural amino acid or a peptide:

R j9 is hydrogen or

alkyl;

R2₀ is hydrogen, Cj.<i alkyl;

s is -N ;r, -C(G) NR_u-r NR_t3 C(0>, -0-, -S-, -OC(0 , and -C(0) O- i3 is -H or -Chalk I;

9 is hydrogen, C₅„ alkyl, phenyl, naphthyl, C^, cycloalkyl, .norbomyl, adaroantyl, a natural or unnatural amino acid or a peptide;

Ru and Rn are independently hydrogen, hydroxy! ,

alkyl, -OCH3,-CF„ Nt¾; R2 and R;25 are independently hydrogen or Chalky!;

and

Z is independently one or more halo or nitro, or one or more coirnterions sufficient to balance the charge of the compound.

20. The method of claim 1, wherein the cancer is non-small cell lung can cer.