Philip L. Fuchs

Folate-conjugated metal chelates have been proposed as potential imaging agents for cancers that overexpress folate receptors.  In a previous study, folic acid was linked through its g-carboxyl group to deferoxamine (DF), and the ⁶⁷Ga-labeled complex ([⁶⁷Ga]DF-folate) was examined for in vivo tumor targeting efficiency in athymic mice with a human tumor cell implant.  Although superb tumor-to-background contrast was obtained, slow heptabiliary clearance would compromise imaging of abdominal tumors such as ovarian cancer.  In the present study, folic acid was conjugated to an alternative chelator, diethylenetriaminepentaacetic acid (DTPA), via an ethylenediamine spacer.  The desired DTPA-folate(g) regioisomer was synthesized by two different approaches, purified by reversed phase column chromatography, and characterized mainly by analytical HPLC, mass spectroscopy, and NMR.   In cultured tumor cells, uptake of [¹¹¹In]DTPA-folate(g) was found to be specific for folate receptor-bearing cells, and the kinetics of uptake were similar to those of free folate and other folate-conjugated molecules.  In the normal rat, intravenously administered  [¹¹¹In]DTPA-folate(g) was found to be rapidly excreted into the urine, giving intestinal levels of radiotracer 10-fold lower than those observed with [⁶⁷Ga]DF-folate(g) at 4 h.  In a preliminary mouse imaging study, a folate receptor-positive KB cell tumor was readily visualized by g scintigraphy 1 h following intravenous administration of [¹¹¹In]DTPA-folate(g).

Reaction of folic acid (1) with excess trifluoroacetic anhydride provides access to both the previously unknown N¹⁰-(trifluoroacetyl)pyrofolic acid (8) and pyrofolic acid (9).  Reaction of either of these materials with hydrazine selectively affords pteroyl hydrazide (13), which may be oxidized to pteroyl azide (27) on a large scale (62% overall from 1 without the need for chromatography).  Treatment of 27 with differentially protected glutamates provides a convenient and high-yielding synthesis of differentially protected, optically pure folates. 

Anhydride 3 is a useful reagent for the synthesis of triply linked drug conjugates. Examples using paclitaxel are provided. Conversion of the azido moiety to a primary amine in the presence of substrates bearing labile ester functionality requires the use of a tin/mercaptan reducing system which includes methanol exchange equilibrium to effect nitrogen-tin bond scission.

anti-Oximes of 2-pyridylacetic acid esters are rapidly transformed to pyridine-2-carbonitrile under a variety of conditions while syn-oximes bearing tert-butyl esters can be conveniently deprotected to the corresponding carboxylic acid with subsequent fragmentation to the nitrile.

A series of Taxol derivatives, tethered at C2' and C7 to glutamate and folate, have been synthesized for evaluation as prodrugs which release Taxol via the hydrolytic lability of their alpha-alkoxy and alpha-amino esters. The half-life for hydrolysis of these materials was determined in pH 7 and pH 5 buffer. The in vitro cytotoxicity has been assessed in cell culture against A-549 lung cancer, MCF-7 breast cancer, and HT-29 colon cancer. Selected agents were further screened for folate binding with free folic acid. One agent (54), further evaluated in animal studies, was found to increase the lifespan in mice, but was less effective than Taxol itself.