QUINONE ANITUTMOR DRUGS
Although Adriamycin (doxorubicin) and related anthracycline antitumor drugs are amongst the most important antitumor drugs in clinical use, their mode of biological activ
ity is not completely understood. A mechanism proposed more than two decades ago is the alkylation and cross-linking of DNA via a reductive activation process. Establishing this mechanism has been difficult because of the instability of DNA-drug adducts and the lack of a good mechanistic hypothesis. The research group is now developing a structure based mechanism for the alkylation which first involves sacrificial drug-produced formaldehyde or catalytic drug-produced formaldehyde. Subsequent linking of the amino group of the drug to the amino group of a guanine base of double stranded DNA produces a drug-DNA virtual crosslink. The structure of the virtual crosslink is established by crystallography in 5'-CGCGCG-3' double stranded oligonucleotide, amongst other techniques. This discovery has led to the synthesis of dimeric anthracycline-formaldehyde conjugates (Doxoform, Daunoform, and Epidoxoform) which are active against resistant cancer cells. Activity against resistant cancer results in part because a resistance mechanism inhibits drug catalyzed formaldehyde production. Another resistance mechanism keeps the drugs from their nuclear target. Doxoform, Daunoform and Epidoxoform also overcome this mechanism as shown in photomicrographs of drug-treated resistant breast cancer cells. A picture of resistant breast cancer cells treated with Epidoxoform was featured on the cover of the July 1999 issue of Chemical Research in Toxicology. Of the three anthracycline-formaldehyde conjugates, Doxoform has been targeted for further development because of its exceptional activity against a wide variety cancer cells. A comparison of the activity of Doxoform with doxorubicin (Dox) against a panel of 60 human cancer cells by the National Cancer Institute shows a broad spectrum of activity.
Recent experiments now show the dimeric doxorubicin-formaldehyde conjugate, Doxoform (DoxF), to be a short-lived prodrug to the monomeric formaldehyde conjugate, Doxazolidine (Doxaz). Doxazolidine then directly cross-links DNA to induce cancer cell death. Doxoform and Doxazolidine show similar activity against a .
This technology (U.S. patent 6,677,309, Jan. 12, 2004) and subsequent improvements are available for licensing through the University of Colorado Office of Technology Transfer.
Current research focuses on the design, synthesis, and evaluation of doxorubicin-formaldehyde conjugates targeted to receptors overexpressed by lung cancer cells and/or immature vascular endothelial cells associated with the angiogenesis of the respective metastatic lesions. An early design incorporated an acyclic doxorubicin-formaldehyde conjugate as an N-Mannich base of salicylamide tethered to a targeting group. The salicylamide served to release the doxorubicin formaldehyde conjugate with a time constant of about 1 hr.Targets of interest included amino peptidase N, alpha-v beta-3 integrin, epidermal growth factor tyrosine kinase domain, estrogen receptor, antiestrogen binding sites, androgen receptor, and matrix metalloproteinase II.An example targeted to the estrogen receptor and to antiestrogen binding sites used hydroxytamoxifen as the targeting group. A second example targeted to the integrin avb3 used a cyclic pentapeptide related to Celengitide. This example is featured on the cover of the December 2004 issue of Molecular Cancer Therapeutics.
A limitation of the early design was the lower level of activity of an acyclic doxorubicin-formaldehyde conjugate relative to that of the cyclic conjugate, Doxazolidine. A current design employs Doxazolidine protected as a carbamate tethered to a self-eliminating spacer with a carboxylesterase cleavable site. The construct is thus targeted to cancer cells that overexpress carboxylesterase enzymes. Analogies include the clinical prodrug of 5-fluorouracil, Capecitabine, and the clinical prodrug of a camptothecin derivative, Irinotecan, that are substrates for carboxylesterase enzymes.
NUCLEIC ACID PHOTOCHEMISTRY
5-Bromouracil, 5-iodouracil, and 5- iodocytosine are chromophores readily incorporated into RNA and DNA oligonucleotides enzymatically or by solid state synthesis. Oligonucleotides bearing these chromophores are reactive in photocrosslinking to associated proteins. Multiple photochemical mechanisms are accessible through careful control of excitation wavelength using laser techniques. Photocrosslinking of nucleoprotein complexes followed by sequencing establishes residues in proximity at the nucleoprotein interface. The research group is presently developing the photocrosslinking technology to select and identify photo reactive nucleic acids, from a large combinatorial library of nucleic acids, which will bind and photocrosslink to a target protein and to no other protein in high yield. The protocol is called PhotoSELEXand is based upon the SELEX protocol for the selection of high affinity nucleic acid aptamers from a combinatorial library of nucleic acid sequences. Selected photo reactive nucleic acids, photoaptamers, may be useful in medical diagnostics to identify a protein characteristic of a pathological or disease state.
AMINO-CARBOXY-STABILIZED CARBON RADICALS
The 2-oxomorpholin-3-yl radical is an excellent example of a captodative radical, one that is stabilized by the synergetic interaction with an electron withdrawing substituent and an electron donating substituent, the electron withdrawing substituent being the carboxyl substituent and the electron donating substituent being the amino substituent.The magnitude of the stabilization is of current interest. TM-3 radical is formed by dissolving its dimer, TM-3 dimer, at ambient temperature; the radical simply exists in equilibrium with the dimer. An important property of TM-3 and related radicals under investigation is their ability to donate a single electron to a reducible substrate. Some oxomorpholinyl radicals combine reversibly with molecular oxygen to form peroxides which have potential as photo initiators of free radical polymerization.
