Stereoselective synthesis of 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-one derivatives as PIM kinase inhibitors inspired from marine alkaloids.

We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.

Identification of unprecedented ATP-competitive choline kinase inhibitors.

In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.

Novel pyrrole carboxamide inhibitors of JAK2 as potential treatment of myeloproliferative disorders.

Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors.

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

Pyrrole-3-carboxamides as potent and selective JAK2 inhibitors.

We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.

Structural insight into maternal embryonic leucine zipper kinase (MELK) conformation and inhibition toward structure-based drug design.

Maternal embryonic leucine zipper kinase (MELK) is upregulated in several types of tumor, including breast, prostate, and brain tumors. Its expression is generally associated with cell survival, cell proliferation, and resistance to apoptosis. Therefore, the potential of MELK inhibitors as therapeutic agents is recently attracting considerable interest. Here we report the first structures of MELK in complex with AMP-PNP and with nanomolar inhibitors. Our studies shed light on the role of the MELK UBA domain, provide a characterization of the kinase active site, and identify key residues for achieving high potency, laying the groundwork for structure-based drug design efforts.

Discovery and optimization of pyrrolo[1,2-a]pyrazinones leads to novel and selective inhibitors of PIM kinases.

A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure-activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.

Discovery of NMS-E973 as novel, selective and potent inhibitor of heat shock protein 90 (Hsp90).

Novel small molecule inhibitors of heat shock protein 90 (Hsp90) were discovered with the help of a fragment based drug discovery approach (FBDD) and subsequent optimization with a combination of structure guided design, parallel synthesis and application of medicinal chemistry principles. These efforts led to the identification of compound 18 (NMS-E973), which displayed significant efficacy in a human ovarian A2780 xenograft tumor model, with a mechanism of action confirmed in vivo by typical modulation of known Hsp90 client proteins, and with a favorable pharmacokinetic and safety profile.

The generation of purinome-targeted libraries as a means to diversify ATP-mimetic chemical classes for lead finding.

The generation of novel chemotypes in support of our oncology research projects expanded in recent years from a canonical design of kinase-targeted compound libraries to a broader interpretation of purinome-targeted libraries (PTL) addressing the specificity of cancer relevant targets such as kinases and ATPases. Successful screening of structurally diverse ATP-binding targets requires compound libraries covering multiple design elements, which may include phosphate surrogate moieties in ATPase inhibitors or far reaching lipophilic residues stabilizing inactive kinase conformations. Here, we exemplify the design and preparation of drug-like combinatorial libraries and report significantly enhanced screening performance on purinomic targets. We compared overall hit rates of PTL with a simultaneously tested unbiased collection of 200,000 compounds and found consistent superiority of the targeted libraries in all cases. We also analyzed the performance of the largest targeted libraries in comparison with each other and often found striking differences in how a specific target responds to various chemotypes and to whole collections.

Discovery of Stereospecific PARP-1 Inhibitor Isoindolinone NMS-P515.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.

Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118): A Potent, Orally Available, and Highly Selective PARP-1 Inhibitor for Cancer Therapy.

The nuclear protein poly(ADP-ribose) polymerase-1 (PARP-1) has a well-established role in the signaling and repair of DNA and is a prominent target in oncology, as testified by the number of candidates in clinical testing that unselectively target both PARP-1 and its closest isoform PARP-2. The goal of our program was to find a PARP-1 selective inhibitor that would potentially mitigate toxicities arising from cross-inhibition of PARP-2. Thus, an HTS campaign on the proprietary Nerviano Medical Sciences (NMS) chemical collection, followed by SAR optimization, allowed us to discover 2-[1-(4,4-difluorocyclohexyl)piperidin-4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMS-P118, 20by). NMS-P118 proved to be a potent, orally available, and highly selective PARP-1 inhibitor endowed with excellent ADME and pharmacokinetic profiles and high efficacy in vivo both as a single agent and in combination with Temozolomide in MDA-MB-436 and Capan-1 xenograft models, respectively. Cocrystal structures of 20by with both PARP-1 and PARP-2 catalytic domain proteins allowed rationalization of the observed selectivity.

Key Intermediates in Combinatorial Chemistry: Access to Various Heterocycles from alpha,beta-Unsaturated Ketones on the Solid Phase.

The value of alpha,beta-unsaturated ketones as key intermediates for the combinatorial assembly of four different templates on the solid phase, namely pyrimidines, dihydropyrimidinones, pyridines, and pyrazoles, was explored with individual syntheses of variably substituted model compounds. Starting from aldehydes grafted on polystyrene support, the Wittig and the Claisen-Schmidt reaction conditions were adapted to efficiently prepare alpha,beta-unsaturated ketones on the solid phase. Further derivatization of the alpha,beta-unsaturated ketones to form pyrimidines succeeded with a number of amidines. In a feasibility study, the potential to obtain, in a modular fashion, other small heterocycles from the same intermediates was assessed. In this solid-phase approach alpha,beta-unsaturated carbonyl intermediates can act as a three-carbon component and a primary enamine is utilized to complement the system for pyridine ring formation. Instead, with N-methylurea a dihydropyrimidinone is obtained. As an alternative, substituted hydrazines are incorporated in one orientation, providing pyrazoles with defined regioisomerism. The study indicates that alpha,beta-unsaturated ketones grafted on the solid phase can take a pivotal role as branching points in a number of synthetic diversity schemes and, therefore, represent versatile intermediates for the efficient preparation of combinatorial small molecule libraries.

Trifluoroacetic anhydride-mediated solid-phase version of the Robinson-Gabriel synthesis of oxazoles.

A traceless solid-phase synthesis of oxazoles 4 via Robinson-Gabriel reaction of solid-supported alpha-acylamino ketones 2 has been achieved. The reaction requires that the cyclization precursor be linked to a benzhydrylic-type linker (compounds 2) and that trifluoroacetic anhydride be used as the cyclodehydrating agent. The solvent has a dramatic effect on the latter reaction, which goes to completion and follows a cyclative-type mechanism only when an ethereal solvent is used. Different synthetic routes have been investigated toward assembling compounds 2. The most straightforward one, which we have validated more extensively, comprises the reaction of Merrifield alpha-methoxyphenyl (MAMP) resin with an alpha-amino ketone to form compounds 1, which are, in turn, acylated. Other methodologies and strategies allowing for the synthesis of compounds 1 that have been investigated include direct alkylation of Rink amide resin; reductive amination of the latter with alpha-keto aldehydes; reaction of MAMP resin with alpha-amino alcohols, followed by oxidation; and protection of Rink amide resin with either 2,4-dinitrosulfonyl or allyl group, followed by alkylation and removal of protecting group. In addition, we disclose a novel variant of the Ugi four-component reaction that allows for the preparation of compounds 2 in a single synthetic step.