The Mediator complex-associated cyclin-dependent kinase CDK8 has been implicated in human disease, particularly in colorectal cancer where it has been reported as a putative oncogene. Here we report the discovery of 109 (CCT251921), a potent, selective, and orally bioavailable inhibitor of CDK8 with equipotent affinity for CDK19. We describe a structure-based design approach leading to the discovery of a 3,4,5-trisubstituted-2-aminopyridine series and present the application of physicochemical property analyses to successfully reduce in vivo metabolic clearance, minimize transporter-mediated biliary elimination while maintaining acceptable aqueous solubility. Compound 109 affords the optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to animal models of cancer.

We demonstrate a designed scaffold-hop approach to the discovery of 2,8-disubstituted-1,6-naphthyridine- and 4,6-disubstituted-isoquinoline-based dual CDK8/19 ligands. Optimized compounds in both series exhibited rapid aldehyde oxidase-mediated metabolism, which could be abrogated by introduction of an amino substituent at C5 of the 1,6-naphthyridine scaffold or at C1 of the isoquinoline scaffold. Compounds 51 and 59 were progressed to in vivo pharmacokinetic studies, and 51 also demonstrated sustained inhibition of STAT1(SER727) phosphorylation, a biomarker of CDK8 inhibition, in an SW620 colorectal carcinoma human tumor xenograft model following oral dosing.

The synthesis of the 16-membered core structure of leiodermatolide 40 has been achieved in 26 linear steps starting from (R)-Roche ester. The key steps in the synthesis of 40 are a Stille cross-coupling between two main fragments 11 and 33 having roughly equal size. For the trisubstituted C4/C5 double bond a carbometalation reaction followed by a Suzuki coupling was used. A Yamaguchi macrolactonization furnished macrolactone 39.

By the replacement of an acetate with propionate by means of organic synthesis, a range of zearalenone analogues were prepared that feature an allylic methyl group. For the synthesis of the aliphatic region of the analogues, we used an asymmetric alkylation to yield pentenol derivatives 16 and ent-16. By means of hydroboration the corresponding aldehydes were secured. These were coupled with 2-pentynol derivate 23 by means of a Carreira acetylide addition. Further routine steps led to the sulfones 29 and 45, respectively. After merging them with 2-bromobenzaldehyde 9 in a Julia-Kocienski reaction, metalation, carboxylation, and protecting-group manipulations gave the seco acids 35 and 49. By means of lactonization under Mitsunobu (alcohol activation) or Trost-Kita conditions (carboxyl activation), all four possible macrocyclic ketone stereoisomers were accessible. In all, considering various protecting-group decorations, 16 analogues were obtained and tested for cytotoxicity (L929 mouse fibroblast cell line). Whereas most of the analogues were less active than zearalenone (IC(50)=9.4 μM), the resorcinol derivatives were comparable, with one stereoisomer (40 b) being slightly more active (IC(50)=6.6 μM). These results were also reflected in the binding assays to Hsp90 in which 40 b showed a dissociation constant (K(d)) value of 130 nM.

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The diagnostic value of serum concentrations of squamous-cell carcinoma (SCC) antigen was evaluated on 48 untreated patients with squamous-cell carcinoma of the lung or oro-maxillo-facial region. To test for specificity SCC antigen was also looked for in 55 patients with various other epithelial neoplasms, in 18 with inflammatory lung disease, and in 43 with liver and 34 with renal function abnormalities. The sensitivity rate of SCC antigen in squamous-cell carcinoma of the lung was 53%, in carcinoma of the oromaxillofacial region 34%. Both groups differed significantly from all other groups, except for patients with abnormal renal function more than half of whom had raised serum concentrations. The proportion of false-positive SCC values was 11% each for patient with inflammatory lung disease and other epithelial neoplasias, and 7% for patients with abnormal liver function. SCC is not a specific marker for squamous-cell carcinoma. Its clinical value probably lies mainly in monitoring the course of such tumours.

The synthesis of three key fragments of the novel 16-membered macrolide leiodermatolide is described. The stereotetrad-containing building block was prepared via a Marshall-Tamaru reaction on an aldehyde obtained by organocatalysis. For a second building block, a Marshall-Tamaru reaction was used as well. The side-chain fragment containing a hydroxy d-lactone could be obtained by intramolecular Reformatsky reaction.Leiodermatolide (1) is a potent antimitotic agent, recently isolated by the group of Amy Wright from the sponge Leiodermatium, which belongs to the order Lithistida (Scheme 1). 1 It displays cytotoxicity at nanomolar level against a variety of human tumor cell lines 2 while showing reduced toxicity to normal cell lines. Leiodermatolide does not show much similarity to other cytotoxic polyketides, however, it shares a carbamate function, for example with palmerolide 3 and discodermolide. 4 This novel polyketides features a 16-membered macrolide, with a six-membered lactone ring on the side chain and has nine stereocenters together with a Z,Z-and E,E-diene system. Although, the initial report of Wright et al. 1 just contained a flat structure of this macrolide, more recently additional data with stereochemical information as shown in Scheme 1 appeared on the web. 5Taking into account the remarkable potent antiproliferative activity and the unique structural features which are calling for proof, leiodermatolide (1) deserves attention for total synthesis. As outlined in the retrosynthetic plan in Scheme 1, we decided to remove part of the side chain by cutting the C18-C19 trans double bond (JuliaKocienski olefination). 6 For macrolactone formation a ring-closing metathesis approach was considered. 7,8 Alternatively, other C-C bond-forming reactions or lactonization reactions (Yamaguchi/Mitsunobu) might be options. The internal Z,Z-diene would come from an enyne precursor. This way, a Sonogashira cross-coupling followed by Z-selective reduction is obvious. This leads to two building blocks 4 and 5, both having roughly equal size. For these, we decided to apply a Marshall-Tamaru reaction 9,10 that would secure the anti stereochemistry at C6/C7 and C14/C15 carbons.The synthesis of alkyne 4 started from known aldehyde (+)-6, which was obtained via L-proline-catalyzed crossaldol reaction of a-silyloxyacetaldehyde using a known literature procedure (Scheme 2). 11,12 With this aldehyde in hand, which was used as a mixture of diastereomers, we tested the Marshall-Tamaru conditions hoping for separable diastereomeric diols. To our surprise, when (R)-mesylate 13 7 (2.0 equiv) was added into the reaction mixture containing Pd(OAc) 2 (0.05 equiv), Ph 3 P (0.05 equiv) and aldehyde 6 followed by slow addition of diethyl zinc (3.0 equiv) and stirred for 48 hours, diol 8 was isolated as a single isomer in 61% yield after chromatographic purification. This reaction outcome can be understood based on Felkin-Anh-like transition state A which is akin to attack of an E-enolate to an a-substituted aldehyde (Scheme 2). Due to an...

Background The Mediator complex-associated kinases CDK8 and CDK19 are cyclin C-dependent enzymes that, with MED12 and MED13, form the kinase module of the Mediator complex. CDK8 expression correlates with activation of β-catenin in colon and gastric cancers and has also been associated with increased mortality in colorectal, breast and ovarian cancers. CDK8 is located in a region of chromosome 13 known to undergo copy number gain in ∼60% of colorectal cancers and inducible shRNA-mediated knockdown of CDK8 protein reduces the growth of colorectal cancer human tumor xenograft animal models harboring CDK8 gene amplification. Results Here we report the discovery and evaluation of CCT251545, a potent, selective and orally bioavailable small molecule chemical probe for CDK8 and CDK19 that we identified from a cell-based WNT pathway screen [1]. We also report a structure-based design approach to the discovery of CCT251921, a potent, selective and orally bioavailable inhibitor of CDK8, with equipotent affinity for CDK19, that has optimised pharmacokinetic and pharmaceutical properties suitable for preclinical development. Furthermore, we describe the discovery of MSC2530818, a structurally differentiated back-up candidate with equivalent pharmacological profile to CCT251921, from a high throughput screen versus CDK8 and subsequent structure-based design. Taking advantage of these two structurally distinct and highly selective dual CDK8/19 modulators we were able to reliably define on-target effects of targeting both CDK8 and CDK19 in the cellular context and in in vivo animal models. We describe gene expression profiles resulting from dual inhibition of CDK8 and CDK19 to demonstrate robust modulation of WNT signalling and additional pathways, including stress and immune response, consistent with the multiple contexts in which Mediator complex is known to regulate gene transcription. We show that both CCT251921 and MSC2530818 exhibit potent cell-based and in vivo inhibition of STAT1SER727 phosphorylation, a target engagement biomarker of CDK8 inhibition, and further demonstrate in vivo antiproliferative activity in human tumour xenograft animal models of colorectal cancer and acute myeloid leukaemia at exposures where pharmacodynamics biomarker modulation is evident. Recent observations suggest CDK8 as a novel anticancer therapeutic target; here we will disclose, for the first time, comprehensive preclinical efficacy, toleration and safety findings for both CCT251921 and MSC2530818 which will inform on the potential for dual CDK8/19 inhibition in the clinical setting. References 1. Dale, T. et. al. Identification of a potent and selective chemical probe for exploring the role of Mediator complex-associated protein kinases CDK8 and CDK19 in human disease. 2015, Nat. Chem. Biol., 11, 973-980. Citation Format: Paul Clarke, Christina Esdar, Aurelie Mallinger, Kai Schiemann, Dennis Waalboer, Simon Crumpler, Christian Rink, Frank Stieber, Michel Calderini, Olajumoke Adeniji-Popoola, Maria-Jesus Ortiz-Ruiz, Rahul S. Samant, Paul Czodrowski, Djordje Musil, Daniel Schwarz, Klaus Schneider, Michael Busch, Mark Stubbs, Rosemary Burke, Robert TePoele, Sharon Gowan, Felix Rohdich, Florence Raynaud, Richard Schneider, Oliver Poeschke, Andree Blaukat, Klaus Urbahns, Paul Workman, Wolfgang Kaufmann, Stephanie Simon, Suzanne A. Eccles, Trevor Dale, Dirk Wienke, Julian Blagg. Discovery of preclinical development candidate inhibitors of the mediator complex-associated kinases CDK8 and CDK19 and evaluation of their therapeutic potential. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3025.