Thomas E. Rawson

A class of 2,4-bisanilinopyrimidine Aurora A inhibitors with unusually high selectivity against Aurora B.

The two major Aurora kinases carry out critical functions at distinct mitotic stages. Selective inhibitors of these kinases, as well as pan-Aurora inhibitors, show antitumor efficacy and are now under clinical investigation. However, the ATP-binding sites of Aurora A and Aurora B are virtually identical, and the structural basis for selective inhibition has therefore not been clear. We report here a class of bisanilinopyrimidine Aurora A inhibitors with excellent selectivity for Aurora A over Aurora B, both in enzymatic assays and in cellular phenotypic assays. Crystal structures of two of the inhibitors in complex with Aurora A implicate a single amino acid difference in Aurora B as responsible for poor inhibitory activity against this enzyme. Mutation of this residue in Aurora B (E161T) or Aurora A (T217E) is sufficient to swap the inhibition profile, suggesting that this difference might be exploited more generally to achieve high selectivity for Aurora A.

A pentacyclic aurora kinase inhibitor (AKI-001) with high in vivo potency and oral bioavailability.

Aurora kinase inhibitors have attracted a great deal of interest as a new class of antimitotic agents. We report a novel class of Aurora inhibitors based on a pentacyclic scaffold. A prototype pentacyclic inhibitor 32 (AKI-001) derived from two early lead structures improves upon the best properties of each parent and compares favorably to a previously reported Aurora inhibitor, 39 (VX-680). The inhibitor exhibits low nanomolar potency against both Aurora A and Aurora B enzymes, excellent cellular potency (IC50 < 100 nM), and good oral bioavailability. Phenotypic cellular assays show that both Aurora A and Aurora B are inhibited at inhibitor concentrations sufficient to block proliferation. Importantly, the cellular activity translates to potent inhibition of tumor growth in vivo. An oral dose of 5 mg/kg QD is well tolerated and results in near stasis (92% TGI) in an HCT116 mouse xenograft model.

Benzodiazepine peptidomimetic inhibitors of farnesyltransferase.

A structural survey of protein Zn2+ binding geometries was instigated based upon the functional requirement of Ras farnesyltransferase for Zn2+. The Cys-X-X-Cys motif found in Zn(2+)-binding proteins such as aspartate transcarbamylase was used as a template to devise a bidentate-coordination model for Cys-A1-A2-X peptide inhibitors. Accordingly, replacement of the central dipeptide with the hydrophobic scaffold 3-amino-1-carboxymethyl-2,3-dihydro-5- phenyl-1H-1,4-benzodiazepin-2-one (BZA) yielded a peptidomimetic inhibitor, Cys(BZA)Met, of moderate potency (IC50 = 400 nM). N-Methylation of the cysteine amide improved potency almost 100-fold (IC50 = 0.3-1 nM). The increased affinity presumably correlates with a preferred conformation of the inhibitor which maximizes a hydrophobic interaction between the scaffold and the enzyme, and the proper presentation of cysteine and methionine to allow bidentate coordination at Zn2+. These non-peptide inhibitors have been shown to block farnesylation of the Ras protein in intact cells and provide lead compounds for the development of new cancer therapeutic agents.

The synthesis of chiral 3-oxo-6-[(phenylmethoxy)-carbonyl]-2-piperazineacetic acid esters designed for the presentation of an aspartic acid side chain. A subsequent novel Friedel Crafts reaction

Abstract The syntheses of (2 S , 6 R )-, and (2 S , 6 S )-3-oxo-6-[(phenylmethoxy)carbonyl]-2,-piperazineacetic acid methyl esters from L - or D -serine and dimethyl- D -malate are described. Acylation of the (2 S , 6 S ) isomer with 3-methoxyphenylacetyl chloride, hydrogenolysis of the benzyl ester, followed by treatment with oxalyl chloride then aluminum chloride led to an unexpected tricyclic product into which a C 2 O 2 unit had been incorporated.

Preparation and biological activity of novel tricyclic GPIIb/IIIa antagonists

Antagonists of the glycoprotein GPIIb/IIIa are a promising class of antithrombotic agents offering potential advantages over present antiplatelet agents (i.e., aspirin and ticlopidine). Novel tricyclic nonpeptidal GPIIb/IIIa antagonists have been prepared and evaluated in vitro as antagonists of fibrinogen binding to the purified GPIIb/IIIa receptor and as inhibitors of platelet aggregation. The work presented demonstrates the robustness of the benzodiazepinedione (BZDD) scaffold, which can be functionalized at the N1-C2 amide as well as at C7, to provide structural diversity and allow optimization of the physiochemical and pharmacological properties of the BZDD based GPIIb/IIIa antagonists. In addition, the resulting new class of tricyclic GPIIb/IIIa antagonists could be used to probe for additional binding interactions on the GPIIb/IIIa receptor and perhaps lead to BZDD based GPIIb/IIIa antagonists with increased potency. The tricyclic molecules reported herein demonstrate that a heterocyclic ring can be fused to the benzodiazepinedione scaffold with retention of anti-aggregatory potency and in the case of tetrazole 30i, increased potency relative to the bicyclic analogue 1c.

Stereochemistry of the benzodiazepine based Ras farnesyltransferase inhibitors

Abstract Chiral benzodiazepine I is the preferred dipeptide turn mimic enantiomer employed in a series of Ras farnesyltransferase inhibitors. It was resolved as the camphorsulfonic acid salt of its methyl ester via a directed crystallization process. Crystallographic analysis of a derivative established R stereochemistry at C-3. The stereochemistry of the additional two chiral centers in derived inhibitor II is addressed.

Abstract A20: Development and validation of a novel acute myeloid leukemia xenograft model that is dependent on the JAK2V617F mutation for growth in vivo

Background: The Janus‐associated kinase (JAK) proteins are a family of non‐receptor tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) that play an important role in cellular survival, proliferation, and differentiation. A gain of function mutation (V617F) in the pseudo‐kinase domain of JAK2 is detected at high frequency in Philadelphia‐negative myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. To further elucidate the role of JAK2V617F on cell growth in vivo we established and validated a novel xenograft model using the acute myeloid leukemia cell line, SET2, which is heterozygous for the mutation. Materials and Methods: SET2 cells containing stable doxycycline‐inducible JAK2V617F shRNA clones were generated by lentiviral transduction. Cell proliferation and viability were assessed by incorporation of 3H‐Thymidine or by ATP quantitation. Cell‐cycle and apoptosis markers were evaluated by Western blotting using antibodies against cyclin D1, PIM1, cleaved caspase 3 and 7 and PARP. Phospho‐STAT5 and total STAT5 were measured using both western blotting and ELISA. A SET2 shRNA clone was selected for in vivo growth in SCID beige female mice and treated with doxycycline or a JAK2 inhibitor (JAK2i). Plasma and tumor drug levels were measured by LC/MS. Results: Genetic knockdown using a doxycycline‐inducible JAK2V617F shRNA confirmed SET2 cells are dependent on this gain of function mutation for growth based on inhibition of cell proliferation in the presence of doxycycline. The latter was corroborated with a selective JAK2i (Ki= 4nM) that potently inhibited SET2 cell viability (EC50 = 355nM). The JAK2i rapidly suppressed phosphorylation of STAT5 (EC50 = 313nM) and induced cycle arrest at the G0–G1 phase based on downregulation of cyclin D1 expression. The JAK2i also effectively inhibited expression of PIM‐1 kinase, a STAT5 target gene, and activated cleavage of procaspase 3 and 7 and DNA‐repair enzyme PARP to induce apoptosis in a time‐ and dose‐dependent manner. A SET2 xenograft model was established by in vivo selection of a variant of the doxycycline‐ JAK2V617F shRNA parental cell line. Its dependence on JAK2V617F was confirmed by treatment with doxcycline and oral administration of a JAK2i, which resulted in significant tumor growth inhibition in vivo. Investigation of the PK/PD relationship underlying the activity of JAK2i provided direct evidence of phospho‐STAT5 suppression and induction of apoptosis for its antitumor effect in vivo. Plasma drug levels correlated with the duration and magnitude of suppression. Conclusion: A JAK2V617F dependent SET2 xenograft model has been established and validated by shRNA gene knockdown and pharmacologically with a JAK2i in vivo. JAK2V617F inhibition results in suppression of STAT5 phosphorylation, cell‐cycle arrest and induction of apoptosis in vitro and in vivo. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A20.

Synthesis of 3-Amino-l-CarboxymethyI-Benzodiazepine (BZA) Peptidomimetics

Replacement of key structural or binding elements of a peptide lead with nonpeptide components can improve affinity and metabolic stability (1-5). Such a strategy was successfully applied to the generation of potent, cell-permeable inhibitors of Ras famesyltransferase (FTase) (6,7). The central pair of amino acids in the CAAX tetrapeptide was replaced with the nonpeptide scaffold 3-methylamino-1-carboxymethyl-2,3-dihydro-5-phenyl-1H-1,4-benzodiazepin-2-one, (N-Me)BZA, shown below.

The synthesis of a phorbol 12,13-bis-lactone

Abstract The synthesis of a phorbol derivative ( 1 ) containing a 24-membered ring 12,13-bis-lactone was achieved via bis-acylation of 20-methoxytritylphorbol with decynoic acid, followed by a copper mediated macrocyclic ring closure, deprotection and selective reduction.