Ho H. Lee

Hypoxia-selective antitumor agents. 16. Nitroarylmethyl quaternary salts as bioreductive prodrugs of the alkylating agent mechlorethamine

Nitrobenzyl quaternary salts of nitrogen mustards have been previously reported as hypoxia-selective cytotoxins. In this paper we describe the synthesis and evaluation of a series of heterocyclic analogues, including pyrrole, imidazole, thiophene, and pyrazole examples, chosen to cover a range of one-electron reduction potentials (from -277 to -511 mV) and substitution patterns. All quaternary salt compounds were less toxic in vitro than mechlorethamine, and all were more toxic under hypoxic than aerobic conditions, although the differentials were highly variable within the series. The most promising analogue, imidazole 2, demonstrated DNA cross-linking selectively in hypoxic RIF-1 cells, and was active in vivo in combination with radiation or cisplatin. However, 2 also produced unpredictable toxicity in vivo, suggestive of nonspecific nitrogen mustard release, and this has restricted further development of these compounds as hypoxia-selective cytotoxins.

Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.

Synthesis and structure-activity relationships of soluble 8-substituted 4-(2-chlorophenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of the Wee1 and Chk1 checkpoint kinases.

Pyrrolo[3,4-c]carbazoles bearing solubilising basic side chains at the 8-position retain potent Wee1 and Chk1 inhibitory properties in isolated enzyme assays, and evidence of G2/M checkpoint abrogation in several cellular assays. Co-crystal structure studies confirm that the primary binding to the Wee1 enzyme is as described previously, with the C-8 side chains residing in an area of bulk tolerance.

Preparation and Antitumour Properties of the Enantiomers of a Hypoxia‐Selective Nitro Analogue of the Duocarmycins

Racemic 2‐{[1‐(chloromethyl)‐5‐nitro‐3‐{5‐[2‐(dimethylamino)ethoxy]indol‐2‐carbonyl}‐1,2‐dihydro‐3H‐benzo[e]indol‐7‐yl]sulfonyl}aminoethyl dihydrogen phosphate, a synthetic nitro derivative of the duocarmycins, is a hypoxia‐selective prodrug active against radiation‐resistant tumour cells at nontoxic doses in mice. An intermediate in the synthesis of this prodrug was resolved by chiral HPLC and the absolute configuration assigned by X‐ray crystallography. The intermediate was used to prepare the prodrug′s enantiomers, and also the enantiomers of the active nitro and amino metabolites. In vitro analysis in the human cervical carcinoma cell line SiHa showed that both nitro enantiomers are hypoxia‐selective cytotoxins, but the “natural” S enantiomer is at least 20‐fold more potent. Examination of extracellular amino metabolite concentrations demonstrated no enantioselectivity in the hypoxia‐selective reduction of nitro to amino. Low levels of amino derivative were also found in aerobic cell suspensions, sufficient to account for the observed oxic toxicity of the nitro form. At an equimolar dose in SiHa‐tumour bearing animals, the (−)‐R enantiomer of the prodrug was inactive, while the (+)‐S enantiomer caused significantly more hypoxic tumour cell kill than the racemate. At this dose, the combination of (+)‐S‐prodrug and radiation eliminated detectable colony‐forming cells in four out of five treated tumour‐bearing animals.

Abstract A247: Mechanism of action of the hypoxia-activated irreversible pan-HER inhibitor SN29966.

Hypoxia occurs in most human tumors and is associated with disease progression, treatment resistance and poor patient outcome. We have developed the hypoxia-activated prodrug SN29966, designed to release the irreversible pan-HER inhibitor SN29926, following one-electron reduction by hypoxic cells (Smaill et al, Mol Cancer Ther., 2009; 8(12 Suppl), C46). Pharmacokinetic (PK) studies in nude mice bearing A431 tumor xenografts indicated SN29966 has a long tumor half-life (>3 days) and releases SN29926 in tumors. SN29966 demonstrated single agent activity in nude mice bearing A431 and SKOV3 xenografts, inducing striking tumor regressions in both models (Patterson et al, Mol Cancer Ther., 2009; 8(12 Suppl), B76). PR509 and PR610, clinical candidates developed from SN29966, are currently undergoing comparative evaluation with Phase I trials anticipated in early 2012. The single-agent antitumor activity of SN29966 is arguably counter-intuitive given that it is designed to target hypoxic cells within tumors. This activity may arise from a number of contributing mechanisms including; (i) bioactivity of the unreduced prodrug; (ii) local redistribution of released inhibitor in the tumor; (iii) liver metabolism and circulating inhibitor and (iv) a long tumor half-life allowing for targeting of both chronic and cycling hypoxia. To critically assess the relative contribution of each to the mechanism of action of SN29966 we performed a number of studies. We prepared SN31950, a prodrug of SN29926 designed to be incapable of one-electron fragmentation. In target modulation and anti-proliferative assays SN31950 showed no hypoxia-dependent activity. The murine A431 tumor PK of SN29966 and SN31950 demonstrated that at an equimolar dose (20 μmol/kg, ip), both prodrugs gave comparable tumor exposures (AUC0–72h: SN31950, 50 μmol*h/kg; SN29966, 57 μmol*h/kg). In contrast, the tumor exposure of SN29926 released from each prodrug differed by 40-fold (AUC0–72h: SN29926 from SN31950, 0.3 μmol*h/kg; SN29926 from SN29966, 12 μmol*h/kg). Plasma exposure of each prodrug was comparable, as were levels of SN29926 in plasma (presumed mainly due to hepatic prodrug metabolism). Consistent with the observed lack of inhibitor release in A431 tumors, SN31950 was inactive against A431 tumors in growth delay assays. To confirm the hypoxia-dependent nature of SN29966 inhibitor release in A431 tumors we re-oxygenated tumors in mice breathing 100% oxygen at 2.5 atm in a hyperbaric chamber. Accordingly, mice showed a marked reduction (56%, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A247.

Analogues of DNA minor groove cross-linking agents incorporating aminoCBI, an amino derivative of the duocarmycins: Synthesis, cytotoxicity, and potential as payloads for antibody–drug conjugates

A Pd-catalysed amination method is used to convert seco-CBI, a synthetic analogue of the alkylating subunit of the duocarmycin natural products, from the phenol to amino form. This allows efficient enantioselective access to the more potent S enantiomer of aminoCBI and its incorporation into analogues of DNA minor groove cross-linking agents. Evaluation in a panel of nine human tumour cell lines shows that the bifunctional agents containing aminoCBI are generally less cytotoxic than their phenolCBI analogues and more susceptible to P-glycoprotein-mediated resistance. However, all bifunctional agents are potent cytotoxins, some in the sub-pM IC50 range, with in vitro properties that compare favourably with established microtubule-targeted ADC payloads.

Influence of a Basic Side Chain on the Properties of Hypoxia-Selective Nitro Analogues of the Duocarmycins: Demonstration of Substantial Anticancer Activity in Combination with Irradiation or Chemotherapy

A new series of nitro analogues of the duocarmycins was prepared and evaluated for hypoxia-selective anticancer activity. The compounds incorporate 13 different amine-containing side chains designed to bind in the minor groove of DNA while spanning a wide range of base strength from pKa 9.64 to 5.24. The most favorable in vitro properties were associated with strongly basic side chains, but the greatest in vivo antitumor activity was found for compounds containing a weakly basic morpholine. This applies to single-agent activity and for activity in combination with irradiation or chemotherapy (gemcitabine or docetaxel). In combination with a single dose of γ irradiation 50 at 42 μmol/kg eliminated detectable clonogens in some SiHa cervical carcinoma xenografts, and in combination with gemcitabine using a well-tolerated multidose schedule, the same compound caused regression of all treated A2780 ovarian tumor xenografts. In the latter experiment, three of seven animals receiving the combination treatment were completely tumor free at day 100.

An improved synthesis of substituted dibenzo[l,4]dioxines

An improved general synthesis of substituted dibenzo[1,4]dioxines by reaction of catechol and substituted 1,2-dichloro- or 2-chloronitro-benzenes with metallic potassium in hexamethylphosphoramide is reported. The yields are generally superior to those in published methods, and in particular the reaction appears the one of choice for the synthesis of both the parent dibenzodioxine and the 1-carboxy derivative.

Abstract LB-297: Characterization of novel hypoxia-activated prodrugs of irreversible pan-HER inhibitors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Reversible HER1 (EGFR) inhibitors erlotinib and gefitinib are approved for use in non-small cell lung cancer (NSCLC), particularly demonstrating activity against tumors expressing mutant forms of HER1. Unfortunately, relapse invariably occurs in this patient population, with approximately half of patients having acquired an additional T790M mutation in HER1. Irreversible pan-HER inhibitors (BIBW2992, PF00299804) are under clinical evaluation in the context of NSCLC that has relapsed post-erlotinib/gefitinib treatment. However it is currently unclear whether these agents possess the therapeutic index in man necessary to gain approval in this setting (Regales et al., J. Clin. Invest. 2009, 119:3000-10), prompting the development of HER1T790M mutant-selective irreversible inhibitors (Zhou et al., Nature. 2009, 462: 1070-74). Hypoxia occurs in most human tumors and is associated with disease progression, resistance to conventional therapies and poor patient outcome. It can however be considered as an exploitable physiological target, as it supports tumor-selective bioreduction of prodrugs. We have developed hypoxia-activated prodrugs of irreversible pan-HER inhibitors as a strategy to broaden their therapeutic index. SN29966 is a prototype nitromethylaryl quaternary (NMQ) ammonium salt prodrug of an irreversible pan-HER inhibitor (SN29926), with masked cellular activity. Radiolytic reduction of SN29966 demonstrated fragmentation of the one-electron adduct to release SN29926 [kfrag 130 ± 10 s-1], and hypoxia-selective metabolism to SN29926 has been confirmed in all cell lines tested. SN32807 is the lead prodrug of this series, releasing the irreversible pan-HER inhibitor SN32793 in a hypoxia-dependent manner. In a panel of HER1/HER1T790M/HER2-expressing cell lines SN32807 showed hypoxia-dependent inhibition of proliferation (hypoxic/oxic IC50 ratios of 60, 11 and 20 in A431, H1975 and SKOV3 cells, respectively), a property SN32793 lacked (IC50 ratios 0.8-1.6). Tumor growth delay experiments (NIH-III nude mice) in large (500 mm3) HER1T790M-expressing H1975 tumor xenografts showed BIBW2992 (20 mg/kg, p.o. daily) failed to control tumor growth (i.e. progressive disease), with only a modest Tumor Growth Delay value of 35% [TGD % = [(T-C)/C]x100 where T and C are time to 4-fold increase in tumor volume from treatment day-1 for treated and control tumors, respectively]. In contrast, single-agent SN32807 (88 mg/kg; ip, q3dx8) demonstrated complete tumor responses in all animals during the treatment period (TGD value 246%; P <0.0005 vs controls; P <0.0005 vs BIBW-2992; log-rank test). Collectively the data indicates that SN32807 possesses a much improved therapeutic index relative to existing HER-family inhibitors, demonstrating marked activity against a HER1T790M-expressing erlotinib, gefitinib and BIBW2992 resistant tumor xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-297.