Auckland Cancer Society Research Centre

Hypoxia-selective prodrugs of minor groove alkylators

Group leader



To exploit the extreme potency and selective binding properties to N3 of adenine in the minor groove of natural antibiotics of the cyclopropylindoline class and to utilize their potential as anti-cancer drugs by conversion to hypoxia-selective prodrugs.

Research overview

CC-1065 and the duocarmycins are natural (bacterial) products of the cyclopropylindoline class that bind to the minor groove of dsDNA and selectively alkylate N3 of adenine (NB this is one property that differentiates cyclopropylindolines from other alkylating drugs that are used in cancer treatment, which generally alkylate N7 of guanine). These compounds and their analogues are extremely potent cytotoxins and demonstrated broad spectrum anti-cancer activity in pre-clinical models. Notably, four synthetic analogues (adozelesin, carzelesin, and bizelesin by Upjohn, and KW-2189 by Kyowa Hakko Kogyo) were tested in clinical trials in the 90s but none continued beyond Phase II due to myelotoxicity preventing therapeutic concentrations to be achieved. In order to improve tumour-selectivity chemists under the leadership of Dr. Moana Tercel substituted the phenol in the 5-position with an amino group, which lends itself to (synthetic) conversion to a nitro group. This results in deactivation of the chloromethyl leaving group that results in a marked attenuation of cytotoxicity.


A pathophysiological characteristic of tumours is the presence of regions with severe hypoxia, which quite often borders necrotic tissue (another feature of tumours). Since the first step of reduction of a nitro group to the corresponding amine is an obligate one-electron reduction that leads to the nitro radical anion, which can be back-oxidised by oxygen (futile or redox cycling), tumour hypoxia can, in principle, be used to improve tumour-selectivity, which is arguably a major reason that limits the therapeutic index or window of many types of chemotherapy and, in particular, of alkylating agents. For example, the prototype nitrochloromethylbenzindoline (nitroCBI) prodrug SN 26438 is reduced by hypoxic cells in vitro as well as in vivo to the corresponding aminoCBI SN 26440 with an increase in cytotoxic potency of up to 50-fold depending on the cell line and experimental conditions.

With ongoing funding from the New Economy Research Fund we have undertaken an extensive lead discovery and optimisation programme to explore the structure-activity relationships of nitro- and aminoCBIs with cytotoxic potency and bioreductive (enzymatic) activation of the prodrugs. In addition, we are measuring drug diffusion through 3D cell cultures (the multicellular layer model (MCL) that we developed as a model for the extravascular tumour compartment to study drug transport as well as oxygen gradients within tumour tissue). In combination with plasma pharmacokinetics and anti-tumour activity against hypoxic cells in human tumour xenograft models we have recently commenced spatially-resolved PK/PD modelling with our lead compound SN 30726 to facilitate further development.

As part of our continuing studies with SN 30726 we are measuring DNA adducts formed by SN 30726 in conjunction with the hypoxia marker EF5, which is used to identify (label) hypoxic cells in MCLs or tumours. The latter is done through staining with specific antibodies against EF5 conjugates and visualising these with fluorescence microscopy or fluorescence-activated cell sorting (FACS) undertaken at the Biomedical Imaging Research Unit (BIRU) and the Flow Cytometry facility, respectively. DNA adducts of SN 30726 are measured after thermal depurination resulting in free aminoCBI-adenine adduct. Dr. Moana Tercel and her team have synthesised authentic aminoCBI-adenine adduct (SN 32057) and its poly-deuterated form as internal standard to facilitate quantitative measurement by triple-quadrupole mass spectrometry interfaced with liquid chromatography (LC-MS/MS). Using this ultra-sensitive analytical technique we can routinely detect as little as ca. 1 pg (ca. 1 fmol) of SN 32057 on column.

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Group members


Tercel, M., Atwell, G.J., Yang, S., Stevenson, R.J., Botting, K.J., Boyd, M., Smith, E., Anderson, R.F., Denny, W.A., Wilson, W.R., Pruijn, F.B. Hypoxia-activated prodrugs: substituent effects on the properties of nitro secoo-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (nitroCBI) prodrugs of DNA minor groove alkylating agents. Journal of Medicinal Chemistry, 2009, 52(22), 7258-7272. Read more

Milbank, J.B., Stevenson, R.J., Ware, D.C., Chang, J.Y., Tercel, M., Ahn, G.O., Wilson, W.R., Denny, W.A. Synthesis and evaluation of stable bidentate transition metal complexes of 1-(chloromethyl)-5-hydroxy-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1HH-pyrrolo[3,2-f]quinoline (seco-6-azaCBI-TMI) as hypoxia selective cytotoxins. Journal of Medicinal Chemistry, 2009, 52(21), 6822-6834. Read more

Wilson, W.R., Stribbling, S.M., Pruijn, F.B., Syddall, S.P., Patterson, A.V., Liyanage, H.D.S., Smith, E., Botting, K.J., Tercel, M. Nitro-chloromethylbenzindolines: hypoxia-activated prodrugs of potent adenine NN3 DNA minor groove alkylators. Molecular Cancer Therapeutics, 2009, 8(10), 2903-2913. Read more

Ahn, G., Botting, K.J., Patterson, A.V., Ware, D.C., Tercel, M. and Wilson W.R. Radiolytic and cellular reduction of a novel hypoxia-activated cobalt(III) prodrug of a chloromethylbenzindoline DNA minor groove alkylator. Biochemical Pharmacology, 2006, 71, 1683-1694. Read more

Tercel, M., Stribbling, S.M., Sheppard, H., Siim, B. G., Wu, K., Pullen, S.M., Botting, J., Wilson, W.R., Denny, W.A. Unsymmetrical DNA Cross-Linking Agents: Combination of the CBI and PBD Pharmacophores. Journal of Medicinal Chemistry, 2003, 46, 2132-2151. Read more

Tercel, M., Gieseg, M.A., Milbank, J.B., Boyd, M., Fan, J-Y., Tan, K.L., Wilson, W.R., Denny, W.A. Cytotoxicity and DNA Interaction of the Enantiomers of 6-Amino-3-(chloromethyl)-1-[(5,6,7-trimethoxyindol-2-yl)carbonyl]indoline (Amino-secoo-CI-TMI). Chemical Research in Toxicology, 1999, 12, 700-706. Read more

Milbank, J.B., Tercel, M., Atwell, G.J., Wilson, W.R., Hogg, A., Denny, W.A. Synthesis of 1-substituted 3-(chloromethyl)-6-aminoindoline (6-amino-secoo-CI) DNA minor groove alkylating agents and structure-activity relationships for their cytotoxicity. Journal of Medicinal Chemistry, 1999, 42(4), 649-658. Read more

Tercel, M., Gieseg, M.A., Denny, W.A., Wilson, W.R. Synthesis and cytotoxicity of amino-secoo DSA: an amino analogue of the DNA alkylating agent duocarmycin SA. Journal of Organic Chemistry, 1999, 64, 5946-5953. Read more

Atwell, G.J., Tercel, M., Boyd, M., Wilson, W.R., Denny, W.A. Synthesis and cytotoxicity of 5-amino-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbonyl]-1,2-dihydro-3HH-benz[e]indole (amino-seco-CBI-TMI) and related 5-alkylamino analogues: new DNA minor groove alkylating agents. Journal of Organic Chemistry, 1998, 63, 9414-9420. Read more