Azetidine derivatives are a popular class of heterocycles in pharmacologically active molecules due to their rigidity, smaller size, and lower lipophilicity compared to pyrrolidines and piperidines, while maintaining similar basicity. However, azetidines exhibit unique reactivity dependent on stereoelectronic factors and reaction conditions, leading to potential instabilities and rearrangements during synthesis. In our attempt to prepare Abrocitinib analogues by replacing the cis-cyclobutane-1,3-diamine scaffold with azetidin-3-amine, we discovered an unforeseen rearrangement of the N-(azetidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine substructure to the tricyclic (2,3-dihydro-1H-imidazo[1,2-c]pyrrolo[3,2-e]pyrimidin-2-yl)methanamine. These findings highlight the need for careful characterization of azetidine-containing compounds when based on N-(azetidin-3-yl)pyrimidin-4-amine substructures and suggest potential methods for preparation of new tricyclic scaffolds.

The design of inhaled selective phosphatidylinositol 3-kinase delta (PI3K delta) inhibitors for the treatment of inflammatory lung diseases was pursued. Knowledge-based design of a novel isocoumarin scaffold that was able to adopt a propeller-shape topology ensured the desired PI3K delta selectivity. Achievement of low nanomolar cellular potencies through hinge binder group optimization, reduction of intrinsic permeability through head group optimization to extend lung retention, and screening of crystalline forms suitable for administration as dry powders culminated in the identification of compound 18. This novel inhaled selective PI3K delta inhibitor displayed durable anti-inflammatory activity in a disease-relevant rat model of Th-2-driven acute lung inflammation and safe in vitro and in vivo preclinical profiles. Therefore, compound 18 showed the appropriate discovery profile and was progressed to clinical trials in healthy volunteers and chronic obstructive pulmonary disease (COPD) patients as CHF-6523.

We report the characterization of potent and selective ROCK inhibitors identified through a core-hopping strategy. A virtual screening workflow, combining ligand- and structure-based methods, was applied on a known series of ROCK inhibitors bearing an acetamido-thiazole scaffold. The most promising replacement of the central core was represented by a benzoazepinone ring, which was selected as a starting point for a subsequent chemical exploration. The overall design approach exploited previous SARs available for congeneric series and crystallographic information to optimize the hinge-binding group as well as the terminal aromatic moiety interacting with the glycine-rich loop. The introduction of elongated and flexible charged groups led to compound 15, which exhibited sub-nanomolar potencies in biochemical and cellular assays, as well as a remarkable selectivity over PKA. HDX studies not only supported the postulated binding mode of compound 15 but also confirmed the crucial role of specific ROCK peptide segments in driving ligand selectivity.

The development of molecules embedding two distinct pharmacophores acting as muscarinic antagonists and β2 agonists (MABAs) promises to be an excellent opportunity to reduce formulation issues and boost efficacy through cross-talk and allosteric interactions. Herein, we report the results of our drug discovery campaign aimed at improving the therapeutic index of a previous MABA series by exploiting the super soft-drug concept. The incorporation of a metabolic liability, stable at the site of administration but undergoing rapid systemic metabolism, to generate poorly active and quickly eliminated fragments was pursued. Our SAR studies yielded MABA 29, which demonstrated a balanced in vivo profile up to 24 h, high instability in plasma and the liver, as well as sustained exposure in the lung. In vitro safety and non-GLP toxicity studies supported the nomination of 29 (CHF-6366) as a clinical candidate, attesting to the successful development of a novel super-soft MABA compound.

Transforming growth factor (TGF)-β-induced myofibroblast transformation and alterations in mesenchymal-epithelial interactions contribute to chronic lung diseases such as chronic obstructive pulmonary disease (COPD), asthma and pulmonary fibrosis. Rho-associated coiled-coil-forming protein kinase (ROCK) consists as two isoforms, ROCK1 and ROCK2, and both are playing critical roles in many cellular responses to injury. In this study, we aimed to elucidate the differential role of ROCK isoforms on TGF-β signaling in lung fibrosis and repair. For this purpose, we tested the effect of a non-selective ROCK 1 and 2 inhibitor (compound 31) and a selective ROCK2 inhibitor (compound A11) in inhibiting TGF-β-induced remodeling in lung fibroblasts and slices; and dysfunctional epithelial-progenitor interactions in lung organoids. Here, we demonstrated that the inhibition of ROCK1/2 with compound 31 represses TGF-β-driven actin remodeling as well as extracellular matrix deposition in lung fibroblasts and PCLS, whereas selective ROCK2 inhibition with compound A11 did not. Furthermore, the TGF-β induced inhibition of organoid formation was functionally restored in a concentration-dependent manner by both dual ROCK 1 and 2 inhibition and selective ROCK2 inhibition. We conclude that dual pharmacological inhibition of ROCK 1 and 2 counteracts TGF-β induced effects on remodeling and alveolar epithelial progenitor function, suggesting this to be a promising therapeutic approach for respiratory diseases associated with fibrosis and defective lung repair.

Studies on the role of Rho-associated protein kinase (ROCK) in experimental pulmonary artery hypertension (PAH) relies mainly on the use of pharmacological inhibitors. However, interpreting these data is hampered by the lack of specificity of commonly utilized inhibitors. To fill this gap, we have selected and characterized a novel ROCK inhibitor, Compound 3, previously described in a patent. Inhibitory potency of Compound 3 against enzymatic activity of ROCK-1 and 2 (IC50 = 10 ± 3.1 and 7.8 ± 0.5 nM, respectively) was accompanied by a strong vasodilating effect in phenylephrine pre-contracted isolated rat pulmonary artery rings (IC50 = 51.7 ± 9.1 nM) as well as in aortic rings (IC50 = 45.5 ± 1.1 nM). Compound 3 showed a remarkable selectivity towards ROCK 1 and 2 when tested against a large panel (>400) of human kinases. A partial explanation for its selectivity is provided from docking simulations within ROCK-1. Pharmacokinetic studies showed that Compound 3 is suitable for a twice daily administration without significant accumulation upon repeated dosing. In rats with monocrotaline (MCT)-induced pulmonary hypertension, therapy with Compound 3, (1 and 3 mg/kg, s.c., b.i.d.), started 14 days after induction of the disease, attenuated right ventricle systolic pressure (RVSP) increase. Morphometric histological analysis showed that Compound 3, at both doses, counteracted MCT-induced medial thickening of lung distal arterioles with an effect comparable to macitentan (10 mg/kg, p.o., q.d.). Compound 3 is a potent and highly selective ROCK inhibitor that ameliorates hemodynamic parameters and counteracts pulmonary vascular remodeling in experimental PAH.

Studies aiming at elucidating the pathophysiological roles of Rho kinase (ROCK) in experimental pulmonary artery hypertension (PAH) mainly relies on a limited number of not very selective pharmacological inhibitors. From a patent we have identified trans-6-((4-aminocyclohexyl)amino)-5-fluoro-2-methoxynicotinamide (Compound 3), a potent ROCK1 (IC 50 =12.5±1.0 nM) and 2 (IC 50 =7.9±0.9 nM) inhibitor with an excellent selectivity profile when counter screened against a large (u003e400) panel of human kinases. Monocrotaline (MCT, 60 mg/Kg s.c.) was utilized in male Wistar rats to induce, in 4 weeks, the development of severe PAH. Interventional treatment with Compound 3 (1 and 3 mg/kg s.c., b.i.d.), were started at 14 days after MCT, when the disease is already rapidly progressing. As comparator was utilized macitentan (10 mg/kg, p.o., q.d.), an endothelin receptor antagonist currently in clinical use. Right ventricle pressure (RVP) in MCT rats (57.3±4.9 mm Hg) was attenuated by compound 3 at 1 mg/kg (37.2±2 mm Hg) and 3 mg/kg (35.2±3.5 mm Hg) as well as by macitentan (31.5±2.5 mm Hg). By morphometric analysis of lung tissue stained for α-smooth muscle actin (α-SMA) we found that Compound 3, at both doses, as well as macitentan, significantly counteracted the medial thickening and muscularization of both small and medium distal pulmonary arterioles. Compound 3 is a novel highly selective ROCK inhibitor, that, in experimental PAH, not only ameliorates hemodynamic parameters but also counteracts pulmonary vascular remodeling. This study highlights a therapeutic potential for pharmacological inhibition of ROCK in PAH.

Parallel PNA:PNA duplexes were synthesized and conjugated with meso-tris(pyridyl)phenylporphyrin carboxylic acid at the N-terminus. The introduction of one porphyrin unit was shown to affect slightly the stability of the PNA:PNA parallel duplex, whereas the presence of two porphyrin units at the same end resulted in a dramatic increase of the melting temperature, accompanied by hysteresis between melting and cooling curves. The circular dichroism (CD) profile of the Soret band and fluorescence quenching strongly support the occurrence of a face-to-face interaction between the two porphyrin units. Introduction of a L-lysine residue at the C-terminal of one strand of the parallel duplex induced a left-handed helical structure in the PNA:PNA duplex if the latter contains only one or no porphyrin moiety. The left-handed helicity was revealed by nucleobase CD profile at 240-280 nm and by the induced-CD observed in the presence of the DiSC(2)(5) cyanine dye at 500-550 nm. Surprisingly, the presence of two porphyrin units led to the disappearance of the nucleobase CD signal and the absence of CD exciton coupling within the Soret band region. In addition, a dramatic decrease of induced CD of DiSC(2)(5) was observed. These results are in agreement with a model where the porphyrin-porphyrin interactions cause partial loss of chirality of the PNA:PNA parallel duplex, forcing it to adopt a ladder-like conformation. Chirality 27:864-874, 2015. (c) 2015 Wiley Periodicals, Inc.

Artificial DNA cutters have been developed by us in our previous studies by combining two strands of pseudo-complementary peptide nucleic acid (pcPNA) with Ce(IV)-EDTA-promoted hydrolysis. The pcPNAs have two modified nucleobases (2,6-diaminopurine and 2-thiouracil) instead of conventional A and T, and can invade double-stranded DNA to activate the target site for the scission. This system has been applied to site-selective scissions of plasmid, λ-phage, E. coli genomic DNA, and human genomic DNA. Here, we have reported a still simpler and more convenient DNA cutter obtained by conjugating peptide nucleic acid (PNA) with a nuclear localization signal (NLS) peptide. This new DNA cutter requires only one PNA strand (instead of two) bearing conventional (non-pseudo-complementary) nucleobases. This PNA-NLS conjugate effectively activated the target site in double-stranded DNA and induced site-selective scission by Ce(IV)-EDTA. The complex formation between the conjugate and DNA was concretely evidenced by spectroscopic results based on time-resolved fluorescence. The target scission site of this new system was straightforwardly determined by the Watson-Crick base pairing rule, and mismatched sequences were clearly discriminated. Importantly, even highly GC-rich regions, which are difficult to be targeted by a previous strategy using pcPNA, were successfully targeted. All these features of the present DNA cutter make it promising for various future applications.

Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.

Fmoc- and Boc-protected modified monomers bearing 5-azidomethyluracil nucleobase were synthesized. Four different solid-phase synthetic strategies were tested in order to evaluate the application of this series of monomers for the solid-phase synthesis of modified PNA. The azide was used as masked amine for the introduction of amide-linked functional groups, allowing the production of a library of compounds starting from a single modified monomer. The azide function was also exploited as reactive group for the modification of PNA in solution via azide-alkyne click cycloaddition.

Enantiomeric analysis is one of the crucial points for the sensor technology, due to the increasing importance that enantiomerically pure compounds and drugs have in pharmaceutic and agrochemical applications. Enantiomeric luminescent sensors give different responses by interaction or reaction with chiral molecules, allowing one to assess their optical purity by spectroscopic measurements. Moreover, chemosensors have been developed to perform enantiomeric analysis of both luminescent and non-luminescent organic compounds. In the present chapter we focus on the recent advances in the sensing of chiral molecules by luminescent sensory systems, with the aim of outlining different mechanisms: fluorescence quenching by metal complexes, photoinduced electron transfer (PET) quenching, fluorescence enhancement by PET inhibition, analyte induced sensor conformational changes, modulation of excimer and exciplex formation, and aggregation induced emission enhancement (AIEE). Recent advances in the use of more elaborate techniques such as anisotropy measurements, gated detection, circularly polarized luminescence (CPL) and perspectives in the field are also discussed. Emphasis is given to the methods which have provided high enantioselectivity and which are amenable to fast screening procedures.

The K562 cell line has been proposed as a useful experimental system to identify anti-tumor compounds acting by inducing terminal erythroid differentiation. K562 cells exhibit a low proportion of hemoglobin-synthesizing cells under standard cell growth conditions, but are able to undergo terminal erythroid differentiation when treated with a variety of anti-tumor compounds. In this paper we report a screening study on a set of different modified C(5) uracil derivatives for the evaluation of their antiproliferative effect in connection with erythroid differentiation pathways, and for defining a new class of drug candidates for the treatment of chronic myelogenous leukemia. Activity of the derivatives tested can be classified in two effect: an antiproliferative effect linked to a high level of erythroid differentiation activity and an antiproliferative effect without activation of gamma globin genes The highest antiproliferative effect and erythroid induction was shown by compound 9, a thymine derivative bearing a n-octyl chain on nitrogen N(1), whereas thymine did not show any effect, suggesting the importance of the linear alkyl chain in position N(1). To our knowledge this compound should be considered among the most efficient inducers of erythroid differentiation of K562 cells. This work is the starting point for the quest of more effective and specific drugs for the induction of terminal erythroid differentiation, for leading new insights in the treatment of neoplastic diseases with molecules acting by inducing differentiation rather than by simply exerting cytotoxic effects.

The synthesis of C5 linked uracil dimers was carried out according to a model developed in order to bind adenine in DNA. N1-Alkylated uracil derivatives were synthesized from isoorotic acid (uracil-5-carboxylic acid) or thymine. The carboxylic acid derivatives were condensed with diamines in order to produce dimeric compounds or with monoamines in order to obtain reference monomeric compounds. Some of the derivatives, in particular the uracil dimers, showed antiproliferative and erythroid differentiation induction properties towards human chronic myelogenous leukemia K562 cells, thus indicating that these compounds could represent a new class of drugs useful for the development of antitumor therapy based on the ability to induce terminal differentiation.