David C. Ware

Pulse radiolysis investigation on the mechanism of the catalytic action of Mn(II)-pentaazamacrocycle compounds as superoxide dismutase mimetics.

The mechanism for the catalytic dismutation of superoxide by the Mn(II) pentaazamacrocyclic compound M40403 ([manganese(II) dichloro-(4 R,9 R,14 R,19 R)-3,10,13,20,26 pentaazatetracyclo [20.3.1.0 (4,9).0 (14,9)] hexacosa-1(26),-22(23),24-triene], SODm1) and two 2,21-dimethyl analogues has been investigated using pulse radiolysis. The initial rate of reaction between superoxide and the manganese compounds was found to be dependent on structure and pH, with the resulting transient adducts possessing spectral characteristics of the metal center being oxidized to Mn(III). Values for the p K a of the transient adducts (p K a = 5.65 +/- 0.05; 5.3 +/- 0.1 and <5 for SODm1, SODm2 and SODm3, respectively) were obtained from spectrophotometric and conductivity measurements. Reaction of these transient adducts with further superoxide was highly structure dependent with the 2 S,21 S-dimethyl derivative (SODm2) being highly catalytically active at pH 7.4 ( k cat = 2.35 x 10 (8) M (-1) s (-1)) compared to SODm1 ( k cat = 3.55 x 10 (6) M (-1) s (-1)). In contrast the 2 R,21 R-dimethyl derivative (SODm3) showed no dismutation catalysis at all. The reaction rates of the initial complexes with HO 2 (*) were significantly lower than with O 2 (*-), and it is proposed that O 2 (*-) is the main reactant in the catalytic cycle at pH 7.4. Variable temperature studies revealed major differences in the thermodynamics of the catalytic cycles involving SODm2 or SODm1. In the case of SODm2, the observed high entropic contribution to the activation energy is indicative of ligand conformational changes during the catalytic step. These results have provided the basis for a new mechanism for the catalytic dismutation of superoxide by Mn(II)-pentaazamacrocycle SOD mimetics.

EXPLOITING TUMOR HYPOXIA THROUGH BIOREDUCTIVE RELEASE OF DIFFUSIBLE CYTOTOXINS: THE COBALT(III)-NITROGEN MUSTARD COMPLEX SN 24771

PURPOSE To assess the oxygen dependence of a novel cobalt-nitrogen mustard complex, SN 24771, designed to release a diffusible cytotoxic metabolite in hypoxic tumor microenvironments. METHODS AND MATERIALS Oxygen dependence of cell killing was assessing in well-stirred single cell suspensions obtained by enzymatic dissociation of EMT6 spheroids, using a sensitive oxygen electrode to measure oxygen concentrations in solution. Cell killing in intact EMT6 spheroids was also compared with that in single cell suspensions. RESULTS Cytotoxicity of SN 24771 in single cell suspensions was inhibited by very low concentrations of oxygen. The C50 value (O2 required for 50% inhibition of log cell kill) was ca. 0.02% O2 at 1 h, and the K value (O2 required to give a cytotoxic potency equal to the average of that at zero and infinite O2) was of a similar order. However, intact spheroids were much more sensitive to SN 24771 than could be accounted for by the K curve for single cell suspensions, this estimate being based on published data for the oxygen concentration profile in these spheroids. CONCLUSION The cytotoxicity of SN 24771 is inhibited appreciably at oxygen concentrations which are too low to provide radiosensitization. In this respect, SN 24771 resembles organic bioreductive drugs such as quinones and nitroaromatic compounds. However, the extensive killing observed in multicellular spheroids is consistent with release of a diffusible nitrogen mustard on reduction. Bioreductive drugs with a low K value for activation, but which release a diffusible cytotoxin, may have desirable properties as tumor radiosensitizers.

Design and synthesis of cobalt(III) nitrogen mustard complexes as hypoxia selective cytotoxins. The X-ray crystal structure of bis(3-chloropentane-2,4-dionato)(RS-N,N′-bis(2-chloroethyl)ethylenediamine)cobalt(III) perchlorate, [Co(Clacac)2(bce)]ClO4

The design, synthesis and biological evaluation of [Co(Meacac)2(dce)]+, a cobalt(III) nitrogen mustard complex that is effective as a hypoxia selective cytotoxin for potential anti-cancer use, is reported.

Synthesis and Evaluation of Stable Bidentate Transition Metal Complexes of 1-(Chloromethyl)-5-hydroxy-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) as Hypoxia Selective Cytotoxins

A series of metal complexes were prepared as potential prodrugs of the extremely toxic DNA minor groove alkylator 1-(chloromethyl)-5-hydroxy-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-2,3-dihydro-1H-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) and close analogues. The pyrrolo[3,2-f]quinoline cytotoxins were prepared from 2-methoxy-4-nitroaniline in a nine-step synthesis involving a Skraup construction of a quinoline intermediate, its appropriate functionalization, and a final radical cyclization. The metal complexes were prepared from these and the labile metal complex synthons [Co(cyclen)(OTf)(2)](+), [Cr(acac)(2)(H(2)O)(2)](+), and [Co(2)(Me(2)dtc)(5)](+). The cobalt complexes were considerably more stable than the free effectors and showed significant attenuation of the cytotoxicity of the latter, with IC(50) ratios (complex/effector) of 50- to 150-fold, and substantial hypoxic cell selectivity, with IC(50) ratios (oxic/hypoxic cells) of 20- to 40-fold. The cobalt complexes were also efficiently activated by ionizing radiation, with G values for loss of the compound close to the theoretical value for one-electron reduction of 0.68 micromol/J. This work extends earlier observations that cobalt cyclen complexes are suitable for both the bioreductive and radiolytic release of potent pyrrolo[3,2-f]quinoline effectors.

Optimization of the Auxiliary Ligand Shell of Cobalt(III)(8-hydroxyquinoline) Complexes as Model Hypoxia-Selective Radiation-Activated Prodrugs

Abstract Ahn, G-O., Ware, D. C., Denny, W. A. and Wilson, W. R. Optimization of the Auxiliary Ligand Shell of Cobalt(III)(8-hydroxyquinoline) Complexes as Model Hypoxia-Selective Radiation-Activated Prodrugs. Radiat. Res. 162, 315–325 (2004). A potential approach for activating prodrugs in hypoxic regions of tumors is to use ionizing radiation, rather than bioreductive enzymes, to effect reduction. This study investigates radiolytic release of 8-hydroxyquinoline (8-HQ), as a model for hydroxyaza-chloromethylbenzindoline DNA minor groove alkylators, from Co(III) complexes under hypoxia. 8-HQ release, measured by HPLC, showed higher efficiency (one-electron stoichiometry) when the auxiliary ligand was a tetraazamacrocycle [e.g. 1,4,7,10-tetraazacyclododecane (cyclen)] rather than a triazamacrocycle [1,4,7-triazacyclononane (TACN)]. These complexes differ from the bioreductive cobalt complex SN 24771 in that their reduction provides stable cobalt-containing products rather than free (aquated) Co2+. Radiolytic release of 8-HQ from Co(cyclen)(8-HQ) and Co(TACN)(CN)(8-HQ) was also demonstrated in deoxygenated human plasma, selectively in the absence of oxygen, again with higher efficiency for the cyclen system. The cobalt complexes were >1000-fold less potent than free 8-HQ as inhibitors of cell proliferation and were metabolically stable in aerobic and hypoxic cell cultures. Investigation of cell uptake of total cobalt, by inductively coupled plasma mass spectrometry, showed that these complexes enter cells but do not accumulate to the high concentrations seen with SN 24771. The results demonstrate the feasibility of masking the cytotoxicity of hydroxyquinoline-based cytotoxins as Co(III) complexes and demonstrate the utility of cyclen-based auxiliary ligands for optimizing radiolytic activation of these novel prodrugs under hypoxia.

Synthesis and characterization of cationic bis-aziridine cobalt(III) complexes containing schiff base ligands

Abstract The syntheses of cationic bis-aziridine (Az) complexes, [Co(SB)(Az) 2 ]Cl, where SB is a Schiff base ligand (SB = acacen, salen, salpn, hapen, happn or saloph) by oxidation of cobalt(II) in basic solution containing the ligands are described. A corresponding series of methylamine complexes [Co(SB)(CH 3 NH 2 ) 2 ]Cl was also prepared (SB = acacen, salen, salpn, hapen or saloph). Both series of complexes were characterized by 1 H NMR spectroscopy, while the aziridine complexes and selected methylamine complexes were also investigated by 13 C NMR spectroscopy.

Porphyrin complexes containing coordinated BOB groups: synthesis, chemical reactivity and the structure of [BOB(tpClpp)]2+

The reactions of boron halides with free base porphyrins under conditions where partial hydrolysis of the boron halides can occur give diboron porphyrin complexes containing BOB moieties in which each boron is bonded to two porphyrin nitrogen atoms. BF(3).OEt(2) with H(2)(por) gives B(2)OF(2)(por) (por = tpp, ttp, tpClpp, oep) which has an asymmetric structure in which one boron lies in the porphyrin plane (B(ip)) while the other lies above it (B(oop)). BCl(3).MeCN with H(2)(por) gives B(2)O(2)(BCl(3))(2)(por) which contains a four-membered B(2)O(2) ring and is stable only in the presence of excess BCl(3). BBr(3) with Li(2)(tpClpp) gives the dicationic complex [B(2)O(tpClpp)](2+) as its [BBr(4)](-) salt, and is the first example of a boron porphyrin containing three-coordinate boron to be structurally characterised. B(2)O(2)(BCl(3))(2)(por) can be chromatographed on basic alumina to give the hydroxyboron complex B(2)O(OH)(2)(por), which is deduced from its NMR spectra and DFT calculations to have a structure analogous to B(2)OF(2)(por). The OH protons are shifted upfield to near delta -4 (B(oop)-OH) and -10 (B(ip)-OH) by the diamagnetic porphyrin ring current. The reaction of either B(2)O(2)(BCl(3))(2)(por) or B(2)O(OH)(2)(por) (por = ttp, tpClpp) with alcohols (ROH, R = Et, 4-C(6)H(4)CH(3)) gives B(2)O(OR)(2)(por), which can in turn be converted to B(2)O(OR)(OH)(por) by repeated chromatography. The reaction of PhBCl(2) with H(2)(por) (por = ttp, tpClpp) gives B(2)O(Ph)(OH)(por) which has been characterised by spectroscopy in concert with DFT calculations. It is a further example of the B(2)OF(2)(por) structural type, in which the phenyl group is coordinated to the out-of-plane boron and the OH group to the in-plane boron, as are its derivatives B(2)O(Ph)(X)(tpClpp) (X = F, OEt). Steric drivers for the facile hydrolysis of haloboron porphyrins relative to their dipyrromethene and expanded porphyrin counterparts are discussed.

N-alkylated cyclen cobalt(III) complexes of 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol DNA alkylating agent as hypoxia-activated prodrugs

A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).

Organoimido complexes of tungsten(IV) containing π-acceptor ligands

Abstract Reaction of PhCCPh with [WCl2(NR)(PMe3)3] gives the cis-dichloro-trans- phosphine complexes [WCl2(NR)(PhCCPh)(PMe3)2] [R = Ph (1), R = CHMe2 (3)] in which the π-acceptor acetylene ligand lies cis to the imido function. For 1 v(CC) is at 1760 cm−1 and for 3, 1740 cm−1. Me3SiCCSiMe3 and PhCCPh do not replace phosphine ligands from [WCl2(NPh)(PMe3)3] or [WCl2(NPh)(PMePh2)3]. Reaction of PhCCPh with [WCl2(NPh)(PMe2Ph)3] gives [WCl2(NPh)(PhCCPh)(PMe2Ph)2] (2). Alkyl substituted acetylenes do not react cleanly with [WCl2(NPh)(PMe3)3] but HCCH gives [WCl2(NPh)(HCCH)(PMe3)2] (4). Reaction of PhCCH with [WCl2(NR)(PMe3)3] gives [WCl2(NR)(PhCCH)(PMe3)2] [R = Ph (5), R = CHMe2 (6)]. The phenylacetylene ligand gives rise to asymmetry in 5 and 6 leading to AB system 31P{1H} NMR spectra. Different values of 1J(PW) for the two phosphine ligands in both complexes indicate small differences in WP bonding. The acetylenic protons and carbons in the 1H and 13C{1H} NMR spectra of the phenylacetylene complexes couple differently to the two phosphines. For 5 3J(HP) cis and trans = 5.90 and 17.26 Hz, 2J(C[H]P) cis and trans = 5.29 and 21.99 Hz and 2J(C[Ph]P) cis and trans = 4.88 and 15.63 Hz. The 13C{1H} NMR acetylenic carbon resonance positions in complexes 1, 3, 5 and 6 suggest that an isopropylimido ligand is a better π-donor than a phenylimido ligand, allowing more metal-acetylene π-back donation. Reduction of [WCl3(NPh)(PMe3)2] with Na/Hg amalgam under CO gives [WCl2(NPh)(CO)(PMe3)2] (7) for which v(CO) at 1925 cm−1 indicates considerable π- backbonding.

Synthesis, structures and hypoxia-selective cytotoxicity of cobalt(III) complexes containing tridentate amine and nitrogen mustard ligands

Cobalt(III) complexes containing the tridentate nitrogen mustard ligand N,N-bis(2-chloroethyl)diethylenetriamine (DCD) and the non-alkylating analogs N,N-diethyldiethylenetriamine (DED) and diethylenetriamine (dien) have been synthesised and the mustard complexes evaluated as potential hypoxia-selective anticancer drugs. The complexes were prepared from the precursor trans-K2[Co(acac)(CO3)(NO2)2]·H2O in a charcoal-catalyzed substitution reaction. Reaction of this precursor with dien·3HCl in water resulted in the isolation of two isomeric products, mer- and s-fac-[Co(dien)(acac)(NO2)]+. Reaction in methanol with DED·3HCl gave one product, mer-[Co(DED)(acac)(NO2)]ClO4, while from the reaction with DCD·3HCl both the tridentate mustard complex mer-[Co(DCD)(acac)(NO2)]ClO4 and, in low yield, a complex containing DCD coordinated in a bidentate fashion, trans-Co(η2-DCD)(acac)(NO2)2, were isolated. The two DCD complexes were characterized by X-ray structure determinations, which confirm the tridentate coordination with a long cobalt–tertiary nitrogen bond distance in the former, and bidentate coordination with the mustard nitrogen as a pendant arm in the latter. The bidentate complex, which contains a free nitrogen mustard, had cytotoxicity similar to that of the free ligand, but cytotoxicity was successfully masked in the tridentate complex, which showed modest hypoxic selectivity (five-fold) in a clonogenic assay.