(Thio, oxo and seleno) semicarbazone complexes with zinc and their use for treating cancer

Abstract

The invention provides organic complexes of Zn.sup.2+ that are useful for treating cancer, as well as compositions and kits comprising such complexes, and intermediate monomer compounds that are useful for the preparation of such complexes.

Claims

1. A complex comprising Zn.sup.2+ and a compound of formula (I): ##STR00025## or an ion or poly-ion thereof; or a solvate thereof, wherein: n is 0, 1, 2, 3, or 4; Y is O, S, or Se; each R.sup.1 is independently selected from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, hydroxyl, and halo; R.sup.2 is selected from the group consisting of H and (C.sub.1-C.sub.6)alkyl that is optionally substituted with aryl; and R.sup.a and each R.sup.b are each independently selected from H, (C.sub.1-C.sub.6)alkyl, aryl, and aryl(C.sub.1-C.sub.6)alkyl; wherein the ratio of the number of compounds of formula (I) or ions or poly-ions thereof or solvates thereof to zinc Zn.sup.2+ ions is about 2:1; provided the compound is not: ##STR00026##

2. The complex of claim 1 wherein: n is 0; R.sup.2 is (C.sub.1-C.sub.4)alkyl; Y is S or Se; and R.sup.a and each R.sup.b are each independently selected from H and (C.sub.1-C.sub.6)alkyl; or a solvate thereof.

3. The complex of claim 1 that comprises a compound selected from the group consisting of: ##STR00027## and ions and poly-ions thereof; and solvates thereof.

4. The complex or of claim 1, wherein R.sup.a is selected from (C.sub.1-C.sub.6)alkyl, aryl, and aryl(C.sub.1-C.sub.6)alkyl; or a solvate thereof.

5. The complex of claim 1, wherein R.sup.a and each R.sup.b are each independently selected from (C.sub.1-C.sub.6)alkyl, aryl, and aryl(C.sub.1-C.sub.6)alkyl; or a solvate thereof.

6. A pharmaceutical composition, comprising a complex of claim 1, or a solvate thereof, and a pharmaceutically acceptable carrier.

7. An injectable pharmaceutical formulation comprising, a complex of claim 1, or a solvate thereof, and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the X-ray structure of compound 12 (ZMC1 Zn); ORTEP drawing from X-ray crystallographic data.

(2) FIG. 2 illustrates the three day cell growth inhibition assays comparing three human tumor cell lines, TOV112D (p53-R175H), H460 (p53-WT), and H1299 (p53-null) for compound 6.

(3) FIG. 3 shows the superiority of the Zinc (II) complex 12 over the monomeric form 6 (ZMC1) in vitro: Zinc (II) complex 12 exhibits greater potency over monomeric form 6 (ZMC1). A cell growth inhibition assay was performed with 12 using monomer 6 as a control. Note the increased potency of the complex with tumor cell death approaching 100 percent.

(4) FIG. 4 shows PAB1620 antibody recognition of wildtype conformation after treatment of mutant p53 with compound 6.

(5) FIG. 5 illustrates data from Example 13 demonstrating that the Zinc (II) complex ZN1 exhibits greater potency over the monomeric form ZMC1 in vivo.

DESCRIPTION OF THE INVENTION

(6) The following definitions are used, unless otherwise described: halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to. Aryl denotes a phenyl radical or an ortho-fused bicyclic carbocyclic radical having about nine to ten ring atoms in which at least one ring is aromatic. Heteroaryl encompasses a radical of a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X) wherein X is absent or is H, O, (C.sub.1-C.sub.4)alkyl, phenyl or benzyl, as well as a radical of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms comprising one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(X).

(7) The term allyl as used herein refers to a substituent, molecular fragment, or radical having the chemical formula CH.sub.2CHCH.sub.2.

(8) The term benzyl as used herein refers to a substituent, molecular fragment, or radical having the chemical formula CH.sub.2C.sub.6H.sub.5.

(9) The term butyl as used herein refers to a four-carbon alkyl radical, substituent, or molecular fragment having the chemical formula C.sub.4H.sub.9.

(10) The term cyclopropyl as used herein refers to a radical, substituent, or molecular fragment having a chemical structure derived from cyclopropane and having the chemical formula C.sub.3H.sub.5.

(11) The term ethyl as used herein refers to an alkyl substituent, radical, or molecular fragment having the chemical formula C.sub.2H.sub.5.

(12) The term heterocycle as used herein includes 4-, 5-, and 6-membered rings containing one or more heteroatoms selected from N, O, and S. In one embodiment, heterocycle includes azetidino, pyrrolidino, and piperidino.

(13) The term isopropyl as used herein refers to a propyl with a group attached to the secondary carbon.

(14) The term methyl as used herein refers to an alkyl derived from methane and containing one carbon atom bonded to three hydrogen atoms and having the chemical formula CH.sub.3.

(15) The term propyl as used herein refers to a linear three-carbon alkyl substituent, molecular fragment, or radical having the chemical formula C.sub.3H.sub.7.

(16) The term phenyl refers to a cyclic group of atoms, radical, substituent, or molecular fragment having the chemical formula C.sub.6H.sub.5.

(17) The term ion refers to a compound bearing a charge. The term poly-ion refers to a compound bearing more than one charge (e.g. two charges). For example, an ion of a compound of formula (I) can be prepared by deprotonating a compound of formula (I) to provide the corresponding ion of the compound of formula (I) that bears a 1 charge. Similarly, an poly-ion (e.g. a dianion) of a compound of formula (I) can be prepared by deprotonating a compound of formula (I) to provide the corresponding poly-ion of the compound of formula (I) that bears, for example, a 2 charge. Ions of the compounds of formula (I) may be incorporated into the complexes of the invention.

(18) It will be appreciated by those skilled in the art that compounds of the invention having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound of the invention, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

(19) When a bond in a compound formula herein is drawn in a non-stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities. When a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted. In one embodiment, the compound may be at least 51% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted.

(20) Specific values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents.

(21) Specifically, (C.sub.1-C.sub.6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C.sub.1-C.sub.6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; aryl can be phenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide) or quinolyl (or its N-oxide).

(22) In one embodiment, R.sup.a and R.sup.b taken together with the nitrogen to which they are attached form a 4-, 5-, or 6-membered heterocycle that is optionally substituted with one to three substituents independently selected from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, hydroxyl, halo, aryl, and heteroaryl. In another embodiment, R.sup.a and R.sup.b taken together with the nitrogen to which they are attached form an azetidino, pyrrolidino, or piperidino ring that is optionally substituted with one to three substituents independently selected from (C.sub.1-C.sub.4)alkyl, (C.sub.1-C.sub.4)alkoxy, hydroxyl, halo, aryl, and heteroaryl.

(23) In one embodiment, the complexes of the invention have a K.sub.d Zn.sup.2+ in the range from about 1 nM-500 nM.

(24) In one embodiment, the complexes of the invention have a K.sub.d Zn.sup.2+ in the range from about 10-200 nM.

(25) The ability of ZMC1, NTA (Zn.sup.2+-binding homolog), and A6 (Example 10) to increase intracellular [Zn.sup.2+].sub.free was evaluated by treating cells with the fluorescent Zn.sup.2+ indicator FluoZin-3-AM (FZ3-AM) in complete media and imaging them using confocal microscopy. In both HEK293 (non-cancer, p53-WT) and TOV112D (ovarian cancer, p53-R175H) cells, ZMC1 increased intracellular [Zn.sup.2+].sub.free as indicated by increased fluorescence, but NTA and A6 did not. This result is consistent with the metallochaperone (MC) model for ZMC1 function and explains the inability of NTA and A6 to reactivate p53-R175H at micromolar concentrations.

(26) ##STR00003##

(27) To determine whether ZMC1 is an ionophore the fluorescent Zn.sup.2+ indicator RhodZin-3 (RZ-3) was encapsulated inside 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes; the ability to transport Zn.sup.2+ in and out was monitored by fluorescence. Zn.sup.2+ alone was unable to permeate the liposomal membrane as indicated by the lack of RZ-3 fluorescence increase. Addition of ZMC1 caused a dose-dependent increase in the rate of RZ-3 fluorescence increase, indicating that ZMC1 can facilitate the transport of Zn.sup.2+ into the liposomes.

(28) Of the two control compounds, A6 (Example 10) shuttled Zn.sup.2+ into the liposomes, but NTA did not. NTA binds Zn.sup.2+ with an affinity similar to that of ZMC1, but it cannot cross either liposomal or cellular membranes, likely because it possesses negative charges. A6, on the other hand, lacks charges and is similar in structure to ZMC1, but binds Zn.sup.2+ weakly (K.sub.d=1.1 M). It can function as an ionophore in conditions of the liposome experiments where external [Zn.sup.2+].sub.free was 10 M. However, in complete media containing 10% fetal bovine serum (FBS), Zn.sup.2+-binding proteins from the serum (e.g. albumin) necessarily compete for Zn.sup.2+ with any putative MC, making the effective [Zn.sup.2+].sub.free much lower than [Zn.sup.2+].sub.total. A6 therefore likely does not increase intracellular [Zn.sup.2+].sub.free in culture because K.sub.d,A6 is greater than extracellular [Zn.sup.2+].sub.free. Thus, both an appropriate Zn.sup.2+ K.sub.d and ionophore activity influence ZMC1 activity.

(29) To determine whether ZMC1 can traverse lipid bilayers as a free compound, the [Zn.sup.2+].sub.free gradient was reversed by adding a large excess of metal ion chelator EDTA to the solution outside of the liposomes; fluorescence was monitored in the presence and absence of ZMC1. EDTA alone did not cause a significant decrease in RZ-3 fluorescence as the liposomal membranes are impermeable to EDTA. After subsequent addition of ZMC1, there was a time dependent decrease in RZ-3 fluorescence. This result indicates that free ZMC1 crossed the liposomal membranes, bound internal Zn.sup.2+, and transported it back outside the liposome where the metal was then bound by the much stronger chelator EDTA. Thus, ZMC1 can cross biological membranes both as free drug and drug-Zn.sup.2+ complex, and therefore can transport Zn.sup.2+ into cells without becoming trapped as either species.

(30) To ensure that the fluorescence results were due to Zn.sup.2+ transport and not to non-specific disruption of liposomal membranes, a liposomal leakage assay was performed using the self-quenching fluorophore calcein. When calcein is encapsulated at concentrations above 4 mM its fluorescence is decreased via self-quenching. Leakage is detected by a fluorescence increase as the dye dilutes and its fluorescence dequenches. At the highest concentrations of ZMC1 and ZnCl.sub.2 a significant fluorescence increase was not detected. Disruption of liposomes can also be detected by alteration of their size distribution. The size distribution of liposomes treated with the highest concentrations of ZnCl.sub.2 and ZMC1 was identical to that of untreated liposomes. Together, these data indicate the liposomal membranes remained intact upon ZMC1 treatment, and therefore the RZ-3 fluorescence changes are attributable only to specific Zn.sup.2+ transport.

(31) Characterization of ZMC1-Mediated Zn.sup.2+ Transport in Live Cells

(32) To extend the investigation of ZMC1 as an ionophore to living systems, ZMC1-mediated Zn.sup.2+ transport was quantified in cells. The kinetics of intracellular [Zn.sup.2+].sub.free increase was measured by loading HEK293 and TOV112D cells with FZ3-AM, treating the cells with ZMC1 and ZnCl.sub.2, and monitoring fluorescence by time-lapse microscopy. To minimize the potential for Zn.sup.2+ contamination and contributions from poorly defined elements in complete media (e.g. FBS), cells were treated and imaged in Ca.sup.2+ and Mg.sup.2+-free Earle's Balanced Salt Solution supplemented with 10 mM HEPES pH 7.4 (EBSS/H ()Ca/Mg). Excess ZnCl.sub.2 with the Zn.sup.2+ ionophore pyrithione (PYR) was used as a positive control. Excess membrane-permeable Zn.sup.2+ chelator N,N,N,N-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) was used as a negative control. When treated with ZnCl.sub.2 alone or ZMC1 alone, neither cell type showed an increase in intracellular [Zn.sup.2+].sub.free. When treated with both ZMC1 and ZnCl.sub.2, both cell lines showed a time dependent increase at two different ZnCl.sub.2 concentrations, demonstrating that both ZMC1 and extracellular Zn.sup.2+ are required. When the fluorescence increases were fit to first-order exponentials, both concentrations of ZnCl.sub.2 yielded identical half-lives in their respective cell types, which we combine to report t.sub.1/2 (HEK293)=12420 s and t.sub.1/2 (TOV112D)=15650 s (meanSD, n=4).

(33) The steady-state intracellular [Zn.sup.2+].sub.free of both cell types was then quantified after treatment with the 2:1 ratio of ZMC1:ZnCl.sub.2. Cells were again loaded with FZ3-AM, treated with 1 M ZMC1 and 0.5 M ZnCl.sub.2 in EBSS/H ()Ca/Mg, and imaged as above. To normalize for differential dye loading, cells were then sequentially treated with excess PYR/ZnCl.sub.2, imaged, treated with TPEN, and imaged again. PYR/ZnCl.sub.2 and TPEN served to saturate and apoize the intracellular FZ3, respectively. In the absence of drug an intracellular [Zn.sup.2+].sub.free of 0.690.25 nM was measured for HEK293 cells and 0.710.19 nM was measured for TOV112D cells. These values reflect the lower limit of detection by FZ3-AM and are likely overestimates. Upon treatment with ZMC1 and ZnCl.sub.2 intracellular [Zn.sup.2+].sub.free rose to 18.14.7 nM for HEK293 cells and 15.82.5 nM for TOV112D cells. These concentrations are theoretically sufficient to reactivate 90% of p53-R175H based on the K.sub.d1 value of 2.1 nM measured for DBD-R175H.

Materials and Methods

(34) Reagents

(35) FZ3-AM, RZ-3 (K.sup.+ salt), and cell culture media were purchased from Life Technologies. DOPC was purchased from Avanti Polar Lipids. ZMC1 and A6 were similarly obtained. Zn.sup.2+(ZMC1).sub.2 was synthesized and crystallized. HEK293 and TOV112D cells were purchased from ATCC and maintained in DMEM+GlutaMAX with 10% FBS and 1 mg/mL penicillin-streptomycin under a 5% CO.sub.2 atmosphere at 37 C. All non-cell based experiments were conducted in 50 mM Tris pH 7.2, 0.1 M NaCl at 25 C.

(36) Liposome Import Assay

(37) DOPC-liposomes were prepared by film rehydration and extrusion followed by gel filtration and diluted to an OD.sub.600=0.06 in buffer. The size distribution of the liposomes was determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS. Fluorescence measurements were taken on a Horiba Fluoromax-4 spectrofluorimeter in a 55 mm quartz cuvette with .sub.ex/.sub.em=550/572 nm for RZ-3 and 490/515 nm for calcein. Initial Zn.sup.2+ import/export was quantified by fitting the first 10-30 s of data after each treatment to a line and converted to units of flux using the following Eqn 1:

(38) $\begin{array}{cc}{J}_i=\frac{F}{t}.Math.\left(\frac{F_{\mathrm{ma}x}-F_{min}}{\left[\mathrm{RZ}3\right]}\right).Math.\left(\frac{\mathrm{SA}}{\mathrm{Vol}}\right)& \mathrm{Eqn}1\end{array}$
where J.sub.i is the initial flux, F/t is the slope of the fit line, F.sub.max is RZ-3 fluorescence in the presence of saturating Zn.sup.2+ and 1% TritonX-100, F.sub.min is RZ-3 fluorescence in the presence of excess EDTA and 1% TritonX-100, [RZ3] is the concentration of encapsulated RZ-3, and SA/Vol is the surface area to volume ratio calculated assuming hollow spheres of the mean diameter determined by DLS.
Intracellular [Zn.sup.2+].sub.free Imaging

(39) TOV112D or HEK293 cells (40,000 cells/well) were plated on either 8-well BD Falcon chambered culture slides (Corning Life Sciences) or 8-chambered #1.5 Nunc Lab-Tek II chambered coverglasses (Thermo Scientific) treated with poly-L-lysine. After 48 h, cells were washed 25 m in serum-free media and incubated with 1 M FZ3-AM for 40 m at 37 C. Cells were then washed 25 m in either EBSS/H ()Ca/Mg or phenol-red free DMEM+10% FBS containing the indicated treatments for 20 m before imaging. For nuclear colocalization, 1 g/mL Hoechst 33342 was also included. Cells were imaged using a Zeiss LSM510 META NLO confocal microscope equipped with 37 C. environmental control chamber. FZ3 and Hoechst 33342 were excited at 488 nm (argon laser) and 790 nm (Chameleon Ti:sapphire laser), respectively. To determine the kinetics of fluorescence change, each background-subtracted image in the time-lapse series was integrated in ImageJ and normalized to the integrated fluorescence of the first frame after treatment. For quantification of intracellular [Zn.sup.2+].sub.free, each cell was analyzed in the treated, 50 M PYR/ZnCl.sub.2 (1:1), and 100 M TPEN images by taking the mean fluorescence of an ROI inside the cell subtracted by an ROI immediately outside the cell measured in ImageJ. The [Zn.sup.2+].sub.free for each cell was then calculated by Eqn 2:

(40) $\begin{array}{cc}{\left[{\mathrm{Zn}}^{2+}\right]}_{\mathrm{free}}=\frac{F-F_{min}}{F_{\mathrm{ma}x}-F}.Math.K_d& \mathrm{Eqn}.2\end{array}$
Where F, F.sub.max, and F.sub.min are fluorescence in the treatment, PYR/ZnCl.sub.2, and TPEN images, respectively, and K.sub.d is that of FZ3 for Zn.sup.2+ (15 nM). To minimize the effects of outliers the lowest and highest 5% of cells in each series were rejected, and the remaining values averaged to give the value from that experiment. The number of cells analyzed in each trial ranged from 54-163. For nuclear colocalization, treated, PYR/ZnCl.sub.2, and TPEN treated images costained with Hoechst 33342 were aligned and each pixel subjected to Eqn. 2 in MATLAB (MathWorks). The resultant images were Gaussian mean filtered and false-colored by calculated [Zn.sup.2+].sub.free.
p53-R175H Immunofluorescence

(41) DMEM+10% FBS was treated with 5 g Chelex 100 resin per 100 mL media for 1 hour with gentle shaking. The media was then decanted and filtered through 0.2 m sterile filter. TOV112D cells were then incubated with 1 M ZMC1 in untreated media, Chelex-treated media, or media+10 M TPEN at 37 C. for 2 h, fixed, and stained with PAB240 and PAB1640.

(42) Assays:

(43) Cell growth inhibition assay using human tumor cell lines with different p53 status (wildtype, null, p53-R175H) were employed to determine if wildtype structure is restored to mutant p53 after treatment with a zinc metallochaperone. Compound 6 shown in FIG. 2 selectively killed the p53-R175H tumor cell line (TOV112D) while leaving the p53 wildtype (H460) and p53 null (H1299) cell lines undisturbed.

(44) When assayed side-by-side, the Zn (II) complex 12 exhibited greater potency in the TOV112D (p53-R175H) cells than the monomers 6 alone, FIG. 3.

(45) An immunofluorescence assay using conformation specific antibodies was used to determine if a test compound could induce a wildtype conformation of mutant p53. The results illustrated in FIG. 4 show PAB1620 antibody recognition of wildtype conformation after treatment of mutant p53 with compound 6. Compound 6 causes mutant p53 to refold into the wildtype conformation forcing cells to pro-apoptotic endpoint.

(46) The invention will now be illustrated by the following non-limiting Examples.

EXAMPLES

(47) Chemistry: General Method A.

(48) A general synthetic approach to the preparation thiosemicarbazone monomers is shown in Example 2.

(49) Chemistry: General Method B for the Synthesis of the [Zn(Thiosemicarbazone).sub.2] Complexes.

(50) A general synthetic approach to the preparation small molecule complexes with Zn (II) is shown in Scheme 1. Treatment of 6 with 0.5 equiv. of ZnCl.sub.2 and excess triethylamine in ethanol heated to reflux for 2 hours afforded, after cooling to ambient temperature, the crystalline complex 12 (Kovala-Demertzi, D., Yadav, P. N., Wiecek, J., Skoulika, S., Varadinova, T., and Demertzis, M. A. (2006) Zn(II) complexes derived from pyridine-2-carbaldehyde thiosemicarbazone and (1E)-1-pyridin-2-ylethan-1-one thiosemicarbazone. Synthesis, crystal structures and antiproliferative activity of Zn(II) complexes. Journal of Inorganic Biochemistry 100, 1558-1567). Recrystallization from the appropriate solvent afforded crystals suitable for X-ray crystallographic analysis. In each case, the protocol gave the complex with 2:1 stoichiometry where two monomers were deprotonated to form a complex with Zn with an overall neutral charge. FIG. 1 shows the ORTEP drawing of the X-ray structure of compound 12.

(51) ##STR00004##

Example 1

(52) ##STR00005##

(53) (E)-N-(1-(6-methylpyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide (1). General Method A: To a solution of azetidine-1-carbothiohydrazide (156 mg, 1.19 mmol, 1.0 eq) and 1-(pyridin-2-yl)ethan-1-one (152 mg, 1.25 mmol, 1.05 eq) in DCM (6 ml) was added AcOH (4 drops). After stirring overnight at room temperature, the reaction was concentrated under reduced pressure and recrystallized from MeOH to afford 1 as a crystalline white solid (132 mg, 0.56 mmol, 47%). .sup.1H-NMR (400 MHz, CDCl.sub.3) 2.35 (m, 5H), 2.56 and 2.67 (E/Z s, 3H), 4.34 (m, 2H), 4.70 (m, 2H), 7.11 and 7.18 (E/Z d, J=7.52 Hz, 1H), 7.30 and 7.65 (E/Z d, J=8.00 Hz, 1H), 7.56 and 7.73 (E/Z t, J=7.84 Hz, 1H), 8.71 (s, 1H, NH). MS: 248.9 [M+H].sup.+.

Example 2

(54) ##STR00006##

(55) (E)-N-(1-(3-methylpyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide (2)Following General Method A for the condensation of azetidine-1-carbothiohydrazide and 1-(3-methylpyridin-2-yl)ethan-1-one the title compound 2 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, CDCl.sub.3) 2.27 (m, 6H), 2.43 (m, 2H), 4.37 (m, 4H), 7.19 and 7.26 (E/Z dd, J=7.72 Hz, 4.72 Hz, 1H), 7.56 and 7.61 (E/Z d, J=7.68 Hz, 1H), 8.40 and 8.66 (E/Z br. s, 1H, NH), 8.46 and 8.55 (E/Z d, J=3.76 Hz, 1H). MS: 248.9 [M+H].sup.+.

Example 3

(56) ##STR00007##

(57) (E)-N-methyl-N-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide (3): Following General Method A for the condensation of N-methylazetidine-1-carbothiohydrazide and 1-(pyridin-2-yl)ethan-1-one the title compound 3 was isolated as a yellow solid after recrystallization from EtOAc/Hex. .sup.1H-NMR (400 MHz, CDCl.sub.3) 2.10 (t, J=7.76 Hz, 1H), 2.14 (t, J=7.80 Hz, 1H), 2.48 (s, 3H), 3.56 (s, 3H), 4.18 (t, J=7.72 Hz, 4H), 7.38 (ddd, J=5.92 Hz, 4.80 Hz, 1.08 Hz, 1H), 7.76 Hz (dt, J =7.76 Hz, 1.76 Hz, 1H), 8.10 (d, J=8.04 Hz, 1H), 8.66 (m, 1H). MS: 248.9 [M+H].sup.+.

Example 4

(58) ##STR00008##

(59) (E)-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-carbothioamide (4): Following General Method A for the condensation of hydrazinecarbothioamide and 1-(pyridin-2-yl)ethan-1-one the title compound 4 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 2.39 (s, 3H), 7.38 (dd, J=7.24 Hz, 5.76 Hz, 1H), 7.79 (dt, J=9.17 Hz, 1.68 Hz, 1H), 8.13 (br. s, 1H, NH), 8.39 (br. s, 1H, NH), 8.43 (d, J=8.08 Hz, 1H), 8.58 (d, J=4.08 Hz, 1H), 10.30 (s, 1H, NH).

Example 5

(60) ##STR00009##

(61) (E)-4-(pyridin-2-yl)-N-(1-(pyridin-2-yl)ethylidene)piperazine-1-carbothiohydrazide (5): Following General Method A for the condensation of 4-(pyridin-2-yl)piperazine-1-carbothiohydrazide and 1-(pyridin-2-yl)ethan-1-one the title compound 5 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 2.41 and 2.68 (E/Z s, 3H), 3.57 (t, J=5.28 Hz, 2H), 3.66 (t, J=5.30 Hz, 2H), 4.03 (t, J=4.80, 2H), 4.12 (m, 2H), 6.68 (app t, J=4.88 Hz, 1H), 6.85 (app. t, J =8.72 Hz, 1H), 7.40 and 7.57 (E/Z m, 1H), 7.57 (m, 1H), 7.84 and 8.03 (E/Z t, J=7.72 Hz, 1.72 Hz, 1H), 7.92 and 8.04 (E/Z d, J=7.88 Hz, 1H), 8.14 (m, 1H), 8.61 and 8.76 (E/Z d, J=4.16 Hz, 1H), 10.00 and 14.81 (E/Z s, 1H, NH). MS: 341.1 [M+H].sup.+.

Example 6

(62) ##STR00010##

(63) (E)-N-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide (6) Following General Method A for the condensation of azetidine-1-carbothiohydrazide and 1-(pyridin-2-yl)ethan-1-one the title compound 6 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 2.26 (m, 2H), 2.36 (s, 3H), 4.12 (m, 2H), 4.59 (m, 2H), 7.39 (m, 1H), 7.83 (dt, J=7.96 Hz, 1.76 Hz, 1H), 7.94 (d, J=8.04 Hz, 1H), 8.60 (d, J=4.00 Hz, 1H), 10.24 (s, 1H, NH). MS: 235.1 [M+H].sup.+.

Example 7

(64) ##STR00011##

(65) (E)-N,N-dimethyl-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-carbothioamide (7): Following General Method A for the condensation of N,N-dimethylhydrazinecarbothioamide and 1-(pyridin-2-yl)ethan-1-one the title compound 7 was isolated as a white solid after recrystallization from MeOH. MS: 223.1 [M+H].sup.+.

Example 8

(66) ##STR00012##

(67) (E)-N-methyl-2-(1-(pyridin-2-yl)ethylidene)hydrazine-1-carbothioamide (8): Following General Method A for the condensation of N-methylhydrazinecarbothioamide and 1-(pyridin-2-yl)ethan-1-one the title compound 8 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 2.39 (s, 3H), 3.07 (d, J=4.40 Hz, 3H), 7.40 (t, J=6.20 Hz, 1H), 7.82 (t, J=8.00 Hz, 1H), 8.43 (d, J=8.04 Hz, 1H), 8.59 (d, J=4.80 Hz, 1H), 8.64 (m, 1H, NH), 10.35 (s, 1H, NH). MS: 209.1 [M+H].sup.+.

Example 9

(68) ##STR00013##

(69) (E)-3,3-difluoro-N-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbothiohydrazide (9): Following General Method A for the condensation of 3,3-difluoroazetidine-1-carbothiohydrazide and 1-(pyridin-2-yl)ethan-1-one the title compound 9 was isolated as a white solid after recrystallization from MeOH. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 1.19 (t, J=7.24 Hz, 2H), 2.47 and 2.54 (E/Z s, 3H), 4.52 (t, J=11.8 Hz, 2H), 7.47 (m, 1H), 7.89 (t, J=7.40 Hz, 1H), 8.09 (d, J=8.04 Hz, 1H), 8.63 (d, J=4.80 Hz, 1H), 9.71 (s, 1H, NH). MS: 271.1 [M+H].sup.+.

Example 10

(70) ##STR00014##

(71) (E)-N-(1-(pyridin-2-yl)ethylidene)azetidine-1-carbohydrazide (10): Azetidine (85 mg, 1.5 mmol) was dissolved in 5 mL methylene chloride and diisopropylethylamine (195 mg, 1.50 mmol, 1.0 equiv.) was added followed by triphosgene (267 mg, 0.9 mmol, 0.6 equiv.) and the reaction mixture stirred at ambient temperature for 4 h to generate the azetidine-1-carbonyl chloride in situ. Without workup, (E)-2-(1-hydrazonoethyl pyridine (203 mg, 1.5 mmol, 1.0 equiv.) was added directly to this mixture following by additional diisopropylethylamine 195 mg, 1.50 mmol, 1.0 equiv.) and the reaction continued to stir at ambient temperature for 72 h. The reaction was then evaporated to dryness and purified by silica gel chromatography (high purity grade silica gel, 32-64 microns, 230-400 mesh) eluted with a gradient of 5-10% methanol/methylene chloride with 1% triethylamine to give a white crystalline solid in selected middle fractions. These crystals were washed with 5:1 hexanes:methylene chloride, and dried under vacuum to give 24 mg of the title compound 10 as a free flowing white crystalline solid. .sup.1H-NMR (400 MHz, CDCl.sub.3) 2.28 (s, 3H), 2.32 (t, J=7.68 Hz, 1H), 2.36 (t, J=7.80 Hz, 1H), 4.35 (t, J=7.36 Hz, 4H), 7.24 (ddd, J=3.52 Hz, 2.48 Hz, 1.08 Hz, 1H), 7.66 (dt, J=7.84 Hz, 1.76 Hz, 1H), 7.81 (s, 1H, NH), 7.91 (d, J=8.08 Hz, 1H), 8.57 (d, J=4.20 Hz, 1H). MS: 219 [M+H].sup.+.

Example 11

(72) ##STR00015##

(73) (E)-N-(2,2-dimethyl-1-(pyridin-2-yl)propylidene)azetidine-1-carbothiohydrazide (11): Following General Method A for the condensation of azetidine-1-carbothiohydrazide and 2,2-dimethyl-1-(pyridin-2-yl)propan-1-one, the title compound 11 was isolated as a white solid after recrystallization from EtOAc/Hex. .sup.1H-NMR (400 MHz, CDCl.sub.3) 1.18 (s, 9H), 2.29 (t, J=7.76 Hz, 1H), 2.33 (t, J=7.84 Hz, 1H), 4.26 (t, J=7.36 Hz, 1H), 4.66 (t, J=7.72 Hz, 1H), 7.18 (d, J=7.76 Hz, 1H), 7.35 (ddd, J=6.00 Hz, 4.92 Hz, 1.12 Hz, 1H), 7.81 (dt, J=7.76 Hz, 1.76 Hz, 1H), 8.26 (br. s, 1H, NH), 8.76 (d, J=4.80 Hz, 1H). MS: 277.1 [M+H].sup.+.

Example 12

(74) ##STR00016##

(75) Synthesis of (12): To a suspension of 6 (190.8 mg, 0.814 mmol, 1 equiv.) in EtOH (20 ml) was added ZnCl.sub.2 (55.5 mg, 0.407 mmol, 0.5 equiv.). After 5 minutes, TEA (0.80 ml, excess) was added and the mixture was heated for 2 hours at reflux under nitrogen. Upon cooling to ambient temperature, a solid precipitated that was collected by filtration and washed with EtOH followed by Et.sub.2O. The solids were dried under high vacuum to give [Zn(ZMC1).sub.2] 12, (215 mg, 0.404 mmol, 99%) as a bright yellow solid. .sup.1H-NMR (400 MHz, DMSO-d.sub.6) 2.26 (overlapping tt, J=7.48 Hz, 7.40 Hz, 4H), 2.58 (s, 6H), 4.05 (m, 8H), 7.29 (dd, J=7.28 Hz, 5.60 Hz, 2H), 7.75 (m, 4H), 7.88 (dt, J=8.04 Hz, 1.52 Hz, 2H). Slow evaporation of [Zn(ZMC1).sub.2] from a 1:1 mixture of DCM/MeOH afforded yellow crystals that were suitable for X-ray crystallography. See X-ray ORTEP drawing (FIG. 1) and data tables.

(76) Cell-based TOV112D activity for representative compounds is shown in Table 1.

(77) TABLE-US-00001 TABLE 1 COMPOUND Activity Number Structure TOV112D 1 +++ 2 +++ 6 +++ 7 0 +++ 9 +++ 10 + 11 + 12 +++ +++, most active; ++, moderately active; +, less active

Example 13

(78) Mice were administered molar equivalents of monomer ZMC1 (5 mg/kg) versus complex ZN1 (5.6 mg/kg) IP daily and monitored for survival. Results are shown in FIG. 5.

(79) All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.