Advancements in Phosphodiesterase 5 Inhibitors: Unveiling Present and Future Perspectives.

Phosphodiesterase 5 (PDE5) inhibitors presented themselves as important players in the nitric oxide/cGMP pathway, thus exerting a profound impact on various physiological and pathological processes. Beyond their well-known efficacy in treating male erectile dysfunction (ED) and pulmonary arterial hypertension (PAH), a plethora of studies have unveiled their significance in the treatment of a myriad of other diseases, including cognitive functions, heart failure, multiple drug resistance in cancer therapy, immune diseases, systemic sclerosis and others. This comprehensive review aims to provide an updated assessment of the crucial role played by PDE5 inhibitors (PDE5-Is) as disease-modifying agents taking their limiting side effects into consideration. From a medicinal chemistry and drug discovery perspective, the published PDE5-Is over the last 10 years and their binding characteristics are systemically discussed, and advancement in properties is exposed. A persistent challenge encountered with these agents lies in their limited isozyme selectivity; considering this obstacle, this review also highlights the breakthrough development of the recently reported PDE5 allosteric inhibitors, which exhibit an unparalleled level of selectivity that was rarely achievable by competitive inhibitors. The implications and potential impact of these novel allosteric inhibitors are meticulously explored. Additionally, the concept of multi-targeted ligands is critically evaluated in relation to PDE5-Is by inspecting the broader spectrum of their molecular interactions and effects. The objective of this review is to provide insight into the design of potent, selective PDE5-Is and an overview of their biological function, limitations, challenges, therapeutic potentials, undergoing clinical trials, future prospects and emerging uses, thus guiding upcoming endeavors in both academia and industry within this domain.

Discovery of novel 5-methoxybenzothiophene hydrazides as metabolically stable Clk1 inhibitors with high potency and unprecedented Clk1 isoenzyme selectivity.

Clk1 kinase is a key modulator of the pre-mRNA alternative splicing machinery which has been proposed as a promising target for treatment of various tumour types, Duchenne's muscular dystrophy and viral infections such as HIV-1 and influenza. Most reported Clk1 inhibitors showed significant co-inhibition of Clk2 and Clk4 in particular, which limits their usefulness for deciphering the individual roles of the Clk1 isoform in physiology and disease. Herein, we present a new 5-methoxybenzothiophene scaffold, enabling for the first time selective inhibition of Clk1 even among the isoenzymes. The 3,5-difluorophenyl and 3,5-dichlorophenyl derivatives 26a and 27a (Clk1 IC50 = 1.4 and 1.7 nM, respectively) showed unprecedented selectivity factors of 15 and 8 over Clk4, and selectivity factors of 535 and 84 over Clk2. Furthermore, 26a and 27a exhibited good growth inhibitory activity in T24 cancer cells and long metabolic half-lives of almost 1 and 6.4 h, respectively. The overall favorable profile of our new Clk1 inhibitors suggests that they may be used in in vivo disease models or as probes to unravel the physiological or pathogenic roles of the Clk1 isoenzyme.

An overview of cdc2-like kinase 1 (Clk1) inhibitors and their therapeutic indications.

Over the past decade, Clk1 has been identified as a promising target for the treatment of various diseases, in which deregulated alternative splicing plays a role. First small molecules targeting Clk1 are in clinical trials for the treatment of solid cancer, where variants of oncogenic proteins derived from alternative splicing promote tumor progression. Since many infectious pathogens hi-jack the host cell's splicing machinery to ensure efficient replication, further indications in this area are under investigation, such as Influenza A, HIV-1 virus, and Trypanosoma infections, and more will likely be discovered in the future. In addition, Clk1 was found to contribute to the progression of Alzheimer's disease through causing an imbalance of tau splicing products. Interestingly, homozygous Clk1 knockout mice showed a rather mild phenotype, opposed to what might be expected in view of the profound role of Clk1 in alternative splicing. A major drawback of most Clk1 inhibitors is their insufficient selectivity; in particular, Dyrk kinases and haspin were frequently identified as off-targets, besides the other Clk isoforms. Only few inhibitors were shown to be selective over Dyrk1A and haspin, whereas no Clk1 inhibitor so far achieved selectivity over the Clk4 isoform. In this review, we carefully compiled all Clk1 inhibitors from the scientific literature and summarized their structure-activity relationships (SAR). In addition, we critically discuss the available selectivity data and describe the inhibitor's efficacy in cellular models, if reported. Thus, we provide a comprehensive overview on the current state of Clk1 drug discovery and highlight the most promising chemotypes.

Development of novel conformationally restricted selective Clk1/4 inhibitors through creating an intramolecular hydrogen bond involving an imide linker.

As prime regulators of pre-mRNA alternative splicing, different Clk isoforms were found to be overexpressed in various tumour types and have received much attention recently as potential targets for cancer therapy. Several studies have reported potent small-molecule Clk1/4 inhibitors with promising cellular anti-cancer activities; however, their clinical use was generally hampered by their compromised selectivity against off-targets, mainly Clk2 and Dyrk1A. In this study, we present a novel series of N-aroylated 5-methoxybenzothiophene-2-carboxamides (imides) as potent and selective Clk1/4 inhibitors. Potency of this series was found to be mainly dependent on the presence of an intramolecular H-bond between an ortho-methoxy group and the imide NH, that stabilizes a nearly coplanar conformation of high affinity to the ATP binding pocket(s) of Clk1/4. The two most potent hits in this series, compounds 20 (4-fluoro-2-methoxy) and 31 (5-chloro-2-methoxy) had cell free Clk1 IC50s of 4 and 9.7 nM, respectively, besides an unprecedented selectivity over Clk2 with 62- and 50-times higher affinities towards Clk1, respectively. 20 and 31 also exhibited remarkable selectivity over most common off-targets including Dyrk1A. Moreover, compounds 26 (2-ethoxy) and 31 showed growth inhibitory activities in T24 cancer cells with GI50s of <0.1 and 1.1 µM, respectively.

Discovery of novel 6-hydroxybenzothiazole urea derivatives as dual Dyrk1A/α-synuclein aggregation inhibitors with neuroprotective effects.

A role of Dyrk1A in the progression of Down syndrome-related Alzheimer's disease (AD) is well supported. However, the involvement of Dyrk1A in the pathogenesis of Parkinson's disease (PD) was much less studied, and it is not clear whether it would be promising to test Dyrk1A inhibitors in relevant PD models. Herein, we modified our previously published 1-(6-hydroxybenzo[d]thiazol-2-yl)-3-phenylurea scaffold of Dyrk1A inhibitors to obtain a new series of analogues with higher selectivity for Dyrk1A on the one hand, but also with a novel, additional activity as inhibitors of α-synuclein (α-syn) aggregation, a major pathogenic hallmark of PD. The phenyl acetamide derivative b27 displayed the highest potency against Dyrk1A with an IC50 of 20 nM and high selectivity over closely related kinases. Furthermore, b27 was shown to successfully target intracellular Dyrk1A and to inhibit SF3B1 phosphorylation in HeLa cells with an IC50 of 690 nM. In addition, two compounds among the Dyrk1A inhibitors, b1 and b20, also suppressed the aggregation of α-synuclein (α-syn) oligomers (with IC50 values of 10.5 µM and 7.8 µM, respectively). Both compounds but not the Dyrk1A reference inhibitor harmine protected SH-SY5Y neuroblastoma cells against α-syn-induced cytotoxicity, with b20 exhibiting a higher neuroprotective effect. Compound b1 and harmine were more efficient in protecting SH-SY5Y cells against 6-hydroxydopamine-induced cell death, an effect that was previously correlated to Dyrk1A inactivation in cells but not yet verified using chemical inhibitors. The presented dual inhibitors exhibited a novel activity profile encouraging for further testing in neurodegenerative disease models.

Discovery of Hydroxybenzothiazole Urea Compounds as Multitargeted Agents Suppressing Major Cytotoxic Mechanisms in Neurodegenerative Diseases.

Multiple factors are causally responsible and/or contribute to the progression of Alzheimer's and Parkinson's diseases. The protein kinase Dyrk1A was identified as a promising target as it phosphorylates tau protein, α-synuclein, and parkin. The first goal of our study was to optimize our previously identified Dyrk1A inhibitors of the 6-hydroxy benzothiazole urea chemotype in terms of potency and selectivity. Our efforts led to the development of the 3-fluorobenzyl amide derivative 16b, which displayed the highest potency against Dyrk1A (IC50 = 9.4 nM). In general, the diversification of the benzylamide moiety led to an enhanced selectivity over the most homologous isoform, Dyrk1B, which was a meaningful indicator, as the high selectivity could be confirmed in an extended selectivity profiling of 3b and 16b. Eventually, we identified the novel phenethyl amide derivative 24b as a triple inhibitor of Dyrk1A kinase activity (IC50 = 119 nM) and the aggregation of tau and α-syn oligomers. We provide evidence that the novel combination of selective Dyrk1A inhibition and suppression of tau and α-syn aggregations of our new lead compound confers efficacy in several established cellular models of neurotoxic mechanisms relevant to neurodegenerative diseases, including α-syn- and 6-hydroxydopamine-induced cytotoxicities.

5-Methoxybenzothiophene-2-Carboxamides as Inhibitors of Clk1/4: Optimization of Selectivity and Cellular Potency.

Clks have been shown by recent studies to be promising targets for cancer therapy, as they are considered key regulators in the process of pre-mRNA splicing, which in turn affects every aspect of tumor biology. In particular, Clk1 and -4 are overexpressed in several human tumors. Most of the potent Clk1 inhibitors reported in the literature are non-selective, mainly showing off-target activity towards Clk2, Dyrk1A and Dyrk1B. Herein, we present new 5-methoxybenzothiophene-2-carboxamide derivatives with unprecedented selectivity. In particular, the introduction of a 3,5-difluoro benzyl extension to the methylated amide led to the discovery of compound 10b (cell-free IC50 = 12.7 nM), which was four times more selective for Clk1 over Clk2 than the previously published flagship compound 1b. Moreover, 10b showed an improved growth inhibitory activity with T24 cells (GI50 = 0.43 µM). Furthermore, a new binding model in the ATP pocket of Clk1 was developed based on the structure-activity relationships derived from new rigidified analogues.

Extending the use of tadalafil scaffold: Development of novel selective phosphodiesterase 5 inhibitors and histone deacetylase inhibitors.

Herein we present the synthesis and characterization of a novel chemical series of tadalafil analogues that display different pharmacological profiles. Compounds that have the 6R, 12aR configuration and terminal carboxylic acid group at the side chain arising from the piperazinedione nitrogen were potent PDE5 inhibitors, with compound 11 having almost equal potency to tadalafil and superior selectivity over PDE11, the most common off-target for tadalafil. Modifying the stereochemistry into 6S, 12aS configuration and adopting the hydroxamic acid moiety as a terminal group gave rise to compounds that only inhibited HDAC. Dual PDE5/HDAC inhibition could be achieved with compounds having 6R, 12aR configuration and hydroxamic acid moiety as a terminal group. The anticancer activity of the synthesized compounds was evaluated against a diverse number of cell lines of different origin. The compounds elicited anticancer activity against cell lines belonging to lymphoproliferative cancer as well as solid tumors. Despite the previous reports suggesting anticancer activity of PDE5 inhibitors, the growth inhibitory activity of the compounds seemed to be solely dependent on HDAC inhibition. Compound 26 (pan HDAC IC50 = 14 nM, PDE5 IC50 = 46 nM) displayed the most potent anticancer activity in the present series and was shown to induce apoptosis in Molt-4 cells. HDAC isoform selectivity testing for compound 26 showed that it is more selective for HDAC6 and 8 over HDAC1 by more than 20-fold.

Development of novel amide-derivatized 2,4-bispyridyl thiophenes as highly potent and selective Dyrk1A inhibitors. Part II: Identification of the cyclopropylamide moiety as a key modification.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) is a potential target in Alzheimer's disease (AD) because of the established correlation between its over-expression and generation of neurofibrillary tangles (NFT) as well as the accumulation of amyloid plaques. However, the use of Dyrk1A inhibitors requires a high degree of selectivity over closely related kinases. In addition, the physicochemical properties of the Dyrk1A inhibitors need to be controlled to enable CNS permeability. In the present study, we optimized our previously published 2,4-bispyridyl thiophene class of Dyrk1A inhibitors by the synthesis of a small library of amide derivatives, carrying alkyl, cycloalkyl, as well as acidic and basic residues. Among this library, the cyclopropylamido modification (compound 4b) was identified as being highly beneficial for several crucial properties. 4b displayed high potency and selectivity against Dyrk1A over closely related kinases in cell-free assays (IC50: Dyrk1A = 3.2 nM; Dyrk1B = 72.9 nM and Clk1 = 270 nM) and inhibited the Dyrk1A activity in HeLa cells with high efficacy (IC50: 43 nM), while no significant cytotoxicity was observed. In addition, the cyclopropylamido group conferred high metabolic stability and maintained the calculated physicochemical properties in a range compatible with a potential CNS activity. Thus, based on its favourable properties, 4b can be considered as a candidate for further in vivo testing in animal models of AD.

Development of Selective Clk1 and -4 Inhibitors for Cellular Depletion of Cancer-Relevant Proteins.

In cancer cells, kinases of the Clk family control the supply of full-length, functional mRNAs coding for a variety of proteins essential to cell growth and survival. Thus, inhibition of Clks might become a novel anticancer strategy, leading to a selective depletion of cancer-relevant proteins after turnover. On the basis of a Weinreb amide hit compound, we designed and synthesized a diverse set of methoxybenzothiophene-2-carboxamides, of which the N-benzylated derivative showed enhanced Clk1 inhibitory activity. Introduction of a m-fluorine in the benzyl moiety eventually led to the discovery of compound 21b, a potent inhibitor of Clk1 and -4 (IC50 = 7 and 2.3 nM, respectively), exhibiting an unprecedented selectivity over Dyrk1A. 21b triggered the depletion of EGFR, HDAC1, and p70S6 kinase from the cancer cells, with potencies in line with the measured GI50 values. In contrast, the cellular effects of congener 21a, which inhibited Clk1 only weakly, were substantially lower.

Pharmacological inhibition of protein kinase C (PKC)ζ downregulates the expression of cytokines involved in the pathogenesis of chronic obstructive pulmonary disease (COPD).

The protein kinase PKCζ is involved in the fine regulation of the NF-κB transcriptional activity and, therefore, represents a potential pharmacological target in inflammatory diseases. We previously developed a selective, allosteric inhibitor (MA130) of PKCζ. Now, we investigated which of the NF-κB-regulated gene expressions are suppressed by MA130 after TNFα-stimulation of the macrophage model cell line U937. The analysis of gene expressions using a qPCR array revealed that many cytokines contributing to the pathogenesis and systemic inflammation in chronic obstructive pulmonary disease (COPD), including CCL2, CCL20, CSF2, CXCL1, CXCL10, IL1B and TNFα, were down-regulated by MA130 but not by a PKCζ-inactive control compound. Thus, we provided the first evidence that PKCζ is a potential target for the treatment of COPD by selective small molecules. MA130 inhibited only a subset of NF-κB-dependent gene expressions, suggesting that targeting PKCζ will be more tolerable than total inhibition of NF-κB activation.

Design and synthesis of novel tamoxifen analogues that avoid CYP2D6 metabolism.

Tamoxifen (TAM) is a widely used drug in the prophylaxis and treatment of breast cancer. TAM is metabolized to the more active 4-hydroxytamoxifen (4-OH-TAM) and endoxifen by cytochrome P450 (CYP) mainly CYP2D6 and CYP3A4 enzymes. Due to the genetic polymorphisms in CYP2D6 genes, high variation in the clinical outcomes of TAM treatment is observed among women of different populations. To address this issue, novel TAM analogues with possible altered activation pathways were synthesized. These analogues were tested for their antiproliferative action on MCF-7 breast cancer cell lines as well as their binding affinity for estrogen receptor (ER) ER-α and ER-ß receptors. These entire novel compounds showed better antiproliferative activity than did TAM on the MCF-7 cells. Moreover, compound 10 exhibited a half maximal growth inhibition (GI50) that was 1000 times more potent than that of TAM (GI50 < 0.005 µM vs 1.58 µM, respectively). Along with a broad spectrum activity on various cancer cell lines, all the TAM analogues showed considerable activity on the ER-negative breast cancer cell line. For further study, compound 10 was incubated in human liver microsomes (HLM), human hepatocytes (hHEP) and CYP2D6 supersomes. The active hydroxyl metabolite was detected after incubation in HLM and hHEP, implicating the involvement of other enzymes in its metabolism. These results prove that this novel series of TAM analogues might provide improved clinical outcomes for poor 2D6 metabolizers.

Structure-Based Design of Novel Tetrahydro-Beta-Carboline Derivatives with a Hydrophilic Side Chain as Potential Phosphodiesterase Inhibitors.

Tadalafil is a clinically approved phosphodiesterase-5 inhibitor for the treatment of erectile dysfunction and pulmonary arterial hypertension. It contains two chiral carbons, and the marketed isomer is the 6R, 12aR isomer with a methyl substituent on the terminal nitrogen of the piperazinedione ring. In this report, tadalafil analogues with an extended hydrophilic side chain on the piperazine nitrogen were designed to interact with particular hydrophilic residues in the binding pocket. This leads to analogues with moderate inhibitory activity on phosphodiesterase-5, even for isomers in which chiral carbons are of the S configuration.