Pre-clinical studies of EC2629, a highly potent folate- receptor-targeted DNA crosslinking agent.

Folate receptor (FR)-targeted small molecule drug conjugates (SMDCs) have shown promising results in early stage clinical trials with microtubule destabilizing agents, such as vintafolide and EC1456. In our effort to develop FR-targeted SMDCs with varying mechanisms of action, we synthesized EC2629, a folate conjugate of a DNA crosslinking agent based on a novel DNA-alkylating moiety. This agent was found to be extremely potent with an in vitro IC50 ~ 100× lower than folate SMDCs constructed with various microtubule inhibitors.

Latent Warheads for Targeted Cancer Therapy: Design and Synthesis of pro-Pyrrolobenzodiazepines and Conjugates.

Pyrrolobenzodiazepines (PBDs) and their dimers (bis-PBDs) have emerged as some of the most potent chemotherapeutic compounds, and are currently under development as novel payloads in antibody-drug conjugates (ADCs). However, when used as stand-alone therapeutics or as warheads for small molecule drug conjugates (SMDCs), dose-limiting toxicities are often observed. As an elegant solution to this inherent problem, we designed diazepine-ring-opened conjugated prodrugs lacking the imine moiety.

Antiinflammatory Activity of a Novel Folic Acid Targeted Conjugate of the mTOR Inhibitor Everolimus.

Folate receptor (FR)-β has been identified as a promising target for antimacrophage and antiinflammatory therapies. In the present study, we investigated EC0565, a folic acid-derivative of everolimus, as a FR-specific inhibitor of the mammalian target of rapamycin (mTOR). Because of its amphiphilic nature, EC0565 was first evaluated for water solubility, critical micelle formation, stability in culture and FR-binding specificity.

Folate-vinca alkaloid conjugates for cancer therapy: a structure-activity relationship.

Vintafolide is a potent folate-targeted vinca alkaloid small molecule drug conjugate (SMDC) that has shown promising results in multiple clinical oncology studies. Structurally, vintafolide consists of 4 essential modules: (1) folic acid, (2) a hydrophilic peptide spacer, (3) a disulfide-containing, self-immolative linker, and (4) the cytotoxic drug, desacetylvinblastine hydrazide (DAVLBH). Here, we report a structure-activity study evaluating the biological impact of (i) substituting DAVLBH within the vintafolide molecule with other vinca alkaloid analogues such as vincristine, vindesine, vinflunine, or vinorelbine; (ii) substituting the naturally (S)-configured Asp-Arg-Asp-Asp-Cys peptide with alternative hydrophilic spacers of varied composition; and (iii) varying the composition of the linker module.

Tumor targeting using 67Ga-DOTA-Bz-folate--investigations of methods to improve the tissue distribution of radiofolates.

Introduction: Use of folic acid radioconjugates for folate receptor (FR) targeting is a promising strategy for imaging purposes as well as for potential therapy of cancer and inflammatory diseases due to the frequent FR overexpression found on cancer cells and activated macrophages. Herein, we report on preclinical results using a novel DOTA-Bz-EDA-folate conjugate radiolabeled with [(67)Ga]-gallium.

Methods: DOTA-Bz-EDA-folate was prepared by conjugation of ethylenediamine-(γ)-folate with 2-(p-isothiocyanobenzyl)-DOTA.

Design and regioselective synthesis of a new generation of targeted therapeutics. Part 3: Folate conjugates of aminopterin hydrazide for the treatment of inflammation.

Efficient syntheses of folate receptor (FR) targeting conjugates of the anti-inflammatory, aminopterin hydrazide, are described. 2-{4-Benzoylamino}-5-oxo-5-{N'-[2-(pyridin-2-yldisulfanyl)-ethoxycarbonyl]-hydrazino}-pentanoic acid is synthesized from commercially available 4-[(2-amino-4-imino-3,4-dihydro-pteridin-6-yl-methyl)-amino]-benzoic acid. Conjugation of this novel, activated aminopterin hydrazide to folic acid through cysteine-terminating (C-terminus), peptide/carbohydrate spacers results in highly water soluble conjugates which allow for the release of free aminopterin hydrazide within the endosomes of targeted cells.

Regioselective synthesis of folate receptor-targeted agents derived from epothilone analogs and folic acid.

Efficient regioselective syntheses of conjugates of folic acid and cytotoxic agents derived from natural epothilones are described. These folate receptor (FR) targeting compounds are water soluble and incorporate a hydrophilic peptide-based spacer unit and a reducible self-immolative disulfide-based linker system between the FR-targeting ligand and the parent drug.

Carbohydrate-based synthetic approach to control toxicity profiles of folate-drug conjugates.

To better regulate the biodistribution of the vinblastine-folate conjugate, EC145, a new folate-spacer that incorporates 1-amino-1-deoxy-D-glucitol-gamma-glutamate subunits into a peptidic backbone, was synthesized. Synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-D-glucitol-gamma-glutamate 20, suitable for Fmoc-strategy solid-phase peptide synthesis (SPPS), was achieved in four steps from delta-gluconolactone. Addition of alternating glutamic acid and 20 moieties onto a cysteine-loaded resin, followed by the addition of folate, deprotection, and cleavage, resulted in the isolation of the new folate-spacer: Pte-gammaGlu-(Glu(1-amino-1-deoxy-D-glucitol)-Glu)(2)-Glu(1-amino-1-deoxy-D-glucitol)-Cys-OH (21).

Strategy to prevent drug-related hypersensitivity in folate-targeted hapten immunotherapy of cancer.

Cancer vaccine/immunotherapy rarely involves systemic administration of an immunogenic compound to an actively immunized host. We have developed such a strategy that utilizes folate to deliver antigenic haptens [e.g.

Properties influencing the relative binding affinity of pteroate derivatives and drug conjugates thereof to the folate receptor.

Purpose: Using in vitro competition assays, determine salient chemical features of pteroates and pteroate-drug conjugates which afford high affinity to the folate receptor.

Materials And Methods: Both folate binding protein-coated polystyrene plates and adherent human cell-based assays were used to evaluate the effects of assay temperature and buffer composition on pteroate/pteroate-drug conjugate binding affinity. Following assay selection and optimization, the relative binding affinities of ten vitamers and derivatives as well as seven pteroate-drug conjugates were evaluated.

N-methylation of the c3' amide of taxanes: synthesis of N-methyltaxol C and N-methylpaclitaxel.

A method has been developed for the methylation of the C3' amide of taxol C and paclitaxel. Taxol C and paclitaxel were sequentially silylated at the 2', 7, and 1-hydroxyl groups with tert-butyldimethylsilyl chloride, triethylsilyl chloride, and dimethylsilyl chloride, respectively. Subsequent reaction with potassium tert-butoxide and methyl iodide provided the corresponding N-methylated taxane derivatives.

Preclinical antitumor activity of a novel folate-targeted dual drug conjugate.

We have designed a new type of tumor-targeted agent by tethering two different drug molecules, with distinct biological mechanisms of action, to the same ligand. This compound, named EC0225, represents the "first in class" multidrug, folate receptor (FR)-targeted agent to be disclosed. It was constructed with a single folate molecule, extended by a hydrophilic peptide-based spacer, which was in turn attached to mitomycin and Vinca alkaloid units via two separate disulfide-containing linkers.

Folate receptor-specific antitumor activity of EC131, a folate-maytansinoid conjugate.

EC131, a new folate receptor (FR)-targeted drug conjugate, was prepared by covalently attaching the vitamin folic acid (FA) to a potent microtubule-inhibiting agent, maytansinoid DM1, via an intramolecular disulfide bond. When tested on cells in culture, EC131 was found to retain high affinity for FR-positive cells and to provide FR-specific cytotoxicity with an IC(50) in the low nanomolar range. The activity of EC131 was completely blocked in the presence of an excess of free FA, and no activity was detected against FR-negative cells.

An assembly concept for the consecutive introduction of unsymmetrical disulfide bonds: synthesis of a releasable multidrug conjugate of folic acid.

We describe the development of methodology which allows for the introduction of a second disulfide bond into a molecular framework with a pre-existing disulfide linker system. Compounds which contain an S-9-fluorenylmethyl-protected thiol and an additional disulfide linkage are deprotected in situ and trapped with an activated thiophile. This methodology allowed for the synthesis of the first molecule possessing two different biologically active agents covalently attached to a folate receptor targeting ligand unit via two disulfide-based release systems.

Design, synthesis, and antiproliferative and CDK2-cyclin a inhibitory activity of novel flavopiridol analogues.

The design and synthesis of a small library of 8-amidoflavone, 8-sulfonamidoflavone, 8-amido-7-hydroxyflavone, and heterocyclic analogues of flavopiridol is reported. The potential activity of these compounds as kinase inhibitors was evaluated by cytotoxicity studies in MCF-7 and ID-8 cancer cell lines and inhibition of CDK2-Cyclin A enzyme activity in vitro. The antiproliferative and CDK2-Cyclin A inhibitory activity of these analogues was significantly lower than the activity of flavopiridol.

Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part 1: EC145, a folic acid conjugate of desacetylvinblastine monohydrazide.

An efficient synthesis of the folate receptor (FR) targeting conjugate EC145 is described. EC145 is a water soluble derivative of the vitamin folic acid and the potent cytotoxic agent, desacetylvinblastine monohydrazide. Both molecules are connected in regioselective manner via a hydrophilic peptide spacer and a reductively labile disulfide linker.

Folate receptor specific anti-tumor activity of folate-mitomycin conjugates.

Purpose: Folate receptor (FR) targeted drug conjugates were prepared by covalently attaching the vitamin folate, to the potent anticancer drug, mitomycin C (MMC). One such conjugate, called EC72, was synthesized with an intramolecular disulfide bond, and it was found to exhibit efficacious anti-tumor activity against FR-expressing M109 tumors in a manner that yielded no gross or microscopic toxicity, even to FR-positive kidneys.

Methods: EC72's specificity was demonstrated by two methods: (1) blocking EC72's activity with an excess of co-administered folic acid (FA) in M109 tumor bearing mice and (2) the absence of therapeutic activity in mice bearing FR-negative tumors.