We report a solid-phase synthesis of 3-acyltetramic acids that are components of naturally occurring antibiotics. The products were obtained in satisfactory purity by a novel cyclization-cleavage strategy via a Dieckmann condensation.Combinatorial chemistry started with the sequential synthesis of peptide libraries on solid support and is now being applied towards the synthesis of catalysts, polymers, drug-like molecules and very recently towards more complex natural products like epothilones. 1 The elaboration of combinatorial synthesis methods for various classes of interesting organic backbone structures as e.g. natural products represents a challenge for modern organic chemistry in the pharmaceutical industry. Whereas natural product chemistry has typically concentrated on the most efficient synthesis of one particular compound in the past, combinatorial chemistry aims rather at the simultaneous synthesis of structurally related compounds -compound libraries. Ideally, such new combinatorial synthetic procedures should be amenable towards parallel or split-mix automated robotics synthesis. We present here a facile, solid-phase synthesis that illustrates the above mentioned approach for the synthesis of 3-acyltetramic acids.3-Acyltetramic acids are substructures observed in several natural products that exhibit antibiotic activities. 2 A range of solution phase synthetic strategies have been reported. 2,3 A novel synthesis and purification method for tetramic acid derivatives has been published recently that takes advantage of the acidic nature of tetramic acids by using cationic ion exchanger resins to adsorb and release the final products selectively. 4 Our synthetic strategy is an extension of the published results and allows to generate combinatorial libraries of 3-acyltetramic acid derivatives using a solid phase support. The compounds are obtained in high purity and tedious purification of reaction intermediates or end products that may occur in several tautomeric forms and tend to form complexes or salts with metals and bases, respectively, is not necessary.The synthesis starts with Fmoc-protected amino acids that are linked via an ester bond to a high-loading hydroxymethyl polystyrene resin. After standard deprotection, the free amine 1 is reacted with aldehydes to give the corresponding azomethines 2. The progress of the reaction is easily monitored on beads by ATR-IR following the disappearance of free NH 2 groups. To achieve complete conversion for a broad range of aliphatic and aromatic aldehydes this condensation step had to be repeated. Subsequent acid catalyzed reduction with sodium cyanoborohydride in DMF gave the reductive alkylation products 3. This reduction step had to be carried out twice in order to achieve almost quantitative conversion as revealed by attenuated total reflection infrared spectroscopy of single resin beads (ATR-IR) by the disappearance of the C=N absorption at 1640 cm -1 . Attempts to perform the reductive N-alkylation in a single step using e.g. NaCNBH 3 , aldehyde and Mg...

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Background: BAL27862 is a novel small molecule, inducing apoptosis in cancer cells through microtubule destabilization. A series of amino acid-derived BAL27862 prodrugs was evaluated for solubility and in vivo conversion into drug. The Lys-prodrug BAL101553 was further compared with BAL27862 in animal models of human cancer. Methods: Kinetic solubility of compounds was determined by diluting DMSO stock solutions with aqueous buffer. Pharmacokinetics and prodrug conversion were evaluated in mice. In vivo efficacy was analyzed in colon carcinoma SW480 and patient-derived mammary MaCa4151 xenografts. MTD dosing was used in all cases, adapted for each mouse strain. Results: All amino acid-derived prodrugs showed significantly increased aqueous solubility compared to BAL27862, most pronounced at pH 3. At pH 5 and 6.5, the dibasic Lys-prodrug BAL101553 proved to be the most soluble compound (>200 μM). In vivo conversion rates differed significantly between the prodrugs. Highest exposure to the parent drug was obtained with the Lys, Ala and Gly derivatives, whereas the exposure achieved with other amino acid prodrugs (e.g. Phe, Asn, Ser, Trp) was more than twofold lower. Combining high solubility, good conversion and oral bioavailability, BAL101553 was selected for further evaluation. In tumor models, BAL27862 was rapidly distributed into SW480 tumors after i.v. administration of drug or BAL101553. Strikingly, BAL27862 was retained in tumor ∼1.5 times longer after BAL101553 (T1/2: 8.3h) vs. BAL27862 (T1/2: 5.4h) administration. In all models, a higher MTD was reached with the prodrug, related to ∼60% prodrug conversion in vivo. In MaCa4151 xenografts, twice-weekly i.v. administration of BAL101553 (17 mg/kg BAL27862 equivalents [BE]) elicited superior antitumor effects (final T/C=30%; p<0.05 vs. controls) as compared to BAL27862 (10 mg/kg; T/C=66%). Once-weekly dosing of BAL101553 (14 mg/kg BE) and BAL27862 (8 mg/kg) in the SW480 model resulted in final T/C's of 34% and 49% (both p<0.001), resp. Strikingly, fractionation of the same total weekly dose did not significantly affect outcome for either BAL101553 (3x per day once-weekly: T/C=40%; 3x per week: T/C=26%; both p<0.001) or BAL27862 (3x per day once-weekly: T/C=54%; 3x per week: T/C=54%; p=0.001 and p=0.002, resp.), suggesting antitumor response is related to AUC. Again, indications of superior antitumor responses were observed with all prodrug schedules in this model. Conclusions: BAL101553 has been identified as a highly soluble prodrug of BAL27862, which can be administered p.o. or i.v. in the absence of solubilizing excipients known to be associated with adverse side-effects. Its administration facilitates higher tumor exposure to the active agent, with more profound responses in some tumor models. These data, together with a flexible dosing potential, support profiling of BAL101553 in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1347. doi:10.1158/1538-7445.AM2011-1347

Background: BAL27862 is a synthetic small molecule, inducing apoptosis in cancer cells through a unique, destabilizing effect on microtubules (MT). BAL27862 can be administered intravenously (i.v.) and orally, and exhibits broad antitumor activity against a range of human tumor histotypes, including models refractory to conventional agents such as taxanes and vinca alkaloids. To improve water solubility, allowing a well tolerated formulation, a novel prodrug (BAL101553) has been developed. Materials and Methods: Solubility was determined by HPLC-quantitation of compound concentration in saturated solutions after filtration and appropriate dilution. Antiproliferative activity in vitro was analyzed by Crystal Violet assay. Effects on MTs were assessed by immunoblotting (IB) or immunofluorescence (IF) for α-tubulin. For pharmacokinetic (PK) studies, colon carcinoma SW480 xenografted mice were treated i.v. with BAL27862 or BAL101553 weekly for 4 weeks. Compound levels were determined in plasma, brain and tumors. Efficacy was assessed in a NSCLC A549 mouse xenograft model using weekly MTD doses. Results: BAL27862 is insoluble in water (<0.001 mg/mL), whereas the hydrochloride salt of BAL101553 has a solubility >100 mg/mL. Using tumor lines in vitro, BAL101553 elicited antiproliferative IC50s approx. double those of the parent drug (e.g. A549: 34.5 nM and 18.5 nM, respectively), consistent with its conversion to active drug in cell culture. Importantly, BAL101553's antiproliferative activity was associated with efficient MT depolymerization, characterized by the formation of multiple, tiny MT asters in dividing cells. The latter is a phenomenon unique to BAL27862 and is not observed with conventional MT-targeted agents like taxanes and vinca alkaloids. In vivo, BAL101553 was converted to active BAL27862. In mice, after i.v. administration of BAL27862 or BAL101553, brain and tumor concentrations equal to plasma levels were detected 5 min and 30 min post-administration, respectively. There was no brain or tumor accumulation over time, as drug concentrations paralleled those in plasma. Notably, a higher maximum tolerated dose was reached with BAL101553 as compared to BAL27862. In terms of exposure, this translated to a 20% lower Cmax and a 10% higher AUC of active drug with BAL101553. When administered i.v. to A549 xenograft-bearing mice, the prodrug was well tolerated, eliciting significant antitumor effects (p≤0.05), equivalent to the parent drug (final %T/C: 44% BAL27862 [10 mg/kg], 40% BAL101553 [28 mg/kg]). Conclusions: BAL101553 is a prodrug of BAL27862 with high water solubility, allowing i.v. administration in the absence of solubilizing excipients known to be associated with adverse side-effects. The in vitro profile, favorable PK characteristics and efficacy in animal tumor models support further evaluation of BAL101553 for the treatment of human cancers. 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 4419.