Synthesis of Six-Membered Rings and Inhibitors of Protein Kinases

The six-membered rings have a priviledged presence in both natural products and synthetic compounds such as drug molecules. Multiple methods to prepare them in the laboratory have been developed. The Diels-Alder reaction provides several pathways toward the construction of substituted six-membered rings with a high degree of regio-, diastereo- and enantioselectivity. It can be considered to be the most important and powerful carbon-carbon bond-forming reaction of all, in synthetic organic chemistry. A practical synthetic method for the preparation of hexahydrocinnolines was developed here, as part of continuing research on polymer-supported pericyclic reactions in preparation of biologically interesting compounds. Some cinnoline derivatives from the literature were reported to show interesting biological properties, such as antimicrobial activity and inhibition of cancer cell lines. Hexahydro-1,2,4-triazolocinnoline-1,3-diones and related compounds were synthesized via aza Diels-Alder reaction on solid-phase. Protein kinases are key regulators of cell function that constitute one of the largest and most functionally diverse gene families. By adding phosphate groups to substrate proteins, kinase driven phosphorylation plays a significant role in a wide range of cellular processes. More than 500 protein kinase genes are present in the the human genome, constituting about 2% of all human genes. They regulate many cellular processes such as growth, differentiation, and proliferation. Protein kinases are seen as potential therapeutic targets since their mutation and dysregulation is causal in multiple human diseases, including metabolic, immunological disorders, and cancer. The consistent structure of the catalytic site among protein kinases sets limits for the development of protein kinase inhibitors. Some protein kinases, however, have regulatory domains as part of their structure, such as protein kinase C (PKC), whose regulatory (C1) domain is unique and is found only in a small number of kinases. This offers a selectivity advantage, thus making the C1-domain an attractive drug target. In fact, the utilization of the the X-ray crystal structure of the PKCδ C1b domain, with molecular modeling, led to the discovery, in this work, of novel C1 domain ligands, the tricyclic γ-amino alcohols. Synthesis of these compounds was achieved by the utilization of the Diels-Alder reactions. In the process of modifying a naturally occurring deep-blue colored hydrocarbon guaiazulene, a novel aminoguaiazulene derivative was synthesized. This novel derivative undergoes ring annulation reactions with 1,2-dicarbonyl reagents to yield tricyclic δ-lactams, types of benzo[cd]azulenes. Benzo[cd]azulenes derived from guaiazulene, are colorful synthetic carbocyclics with interesting chemical and biological properties. Some of the benzo[cd]azulenes synthesized in this study were recently characterized as selective Pim kinase inhibitors. Pim kinases have become intriguing targets for cancer therapy that possess unique structural features, among protein kinases, that offer a great potential in the design of selective Pim-inhibitors. Based on the promising Pim-kinase inhibition results from multiple cell-based assays, a further modification of the benzo[cd]azulenes was conducted, where some interesting findings in their chemical behavior were observed; new phenolic benzo[cd]azulene compounds were formed, with potent Pim-inhibitory activities. The benzo[cd]azulenes developed in this study were found to be useful research compounds, potential Pim-selective kinase inhibitors, and putative anti-cancer drug candidates. The new synthetic methods detailed in this study will be valuable tools in the further development of additional benzo[cd]azulenes and related systems in the future.Luonnossa esiintyvistä orgaanisista molekyyleistä valtaosalla on rengasrakenne. Näistä kuusirenkaisilla yhdisteillä on merkittävä asema. Luonnonaineiden lisäksi kuusirengasrakenne on yleinen monissa synteettisissä molekyyleissä kuten lääkeaineissa. Näiden valmistamiseksi laboratoriossa on kehitetty suuri joukko erilaisia menetelmiä. Yksi merkittävimmistä on Diels-Alder-reaktio, joka kuuluu laajempaan perisyklisten reaktioiden ryhmään. Yhtenäistä perisyklisille reaktioille on konjugoitu syklinen siirtymätila, jonka seurauksena reaktiotuote syntyy ilman välituotetta. Diels-Alder-reaktiolla voidaan valmistaa tehokkaasti kuusirenkaisisia yhdisteitä ja reaktio on usein stereo- ja regioselektiivinen. Reaktio ei rajoitu pelkästään hiiltä sisältävien renkaiden syntetisointiin, vaan erilaisia heteroatomeja kuten typpeä ja happea sisältäviäkin kuusirenkaita voidaan valmistaa. Diels-Alder-reaktion katsotaan olevan ehkäpä jopa tärkein ja tehokkain menetelmä uusien hiili-hiilisidosten muodostamiseksi synteettisessä orgaanisessa kemiassa. Reaktio ei rajoitu pelkästään laboratorioon, vaan myös luonnosta on löydetty entsyymejä jotka katalysoivat Diels-Alder-reaktiota. Tutkimusryhmässämme on valmistettu biologisesti mielenkiintoisia heterosyklisiä yhdisteitä kiinteäfaasitekniikalla, jossa reaktiot tapahtuvat kiintokantajan (polymeerin) pinnalla. Kiinteä-faasitekniikan etuina on mm. polymeeriin sidotun tuotteen helppo eristys ja puhdistus. Lisäksi suuria reagenssiylimääriä on mahdollista käyttää, sillä ne voidaan suodattaa helposti erilleen tuotteesta. Kirjallisuudessa esiintyvillä kinnoliinijohdannaisilla on havaittu mm. antibakteerisia ja syöpäsolujen kasvua estäviä vaikutuksia. Tutkimuksessa kehitettiin kiinteäfaasimenetelmä heksahydrokinnoliinirakenteen syntetisoimiseksi hetero-Diels-Alder-reaktiolla, jossa reaktiivinen typpi-typpikaksoissidos osallistuu dienofiilinä sykloadditioon polymeeriin sidotun dieenin kanssa. Reaktiotuotteiden irrotus kiintokantajista tapahtui happokäsittelyllä ja tämän jälkeen ne puhdistettiin pylväskromatografisin menetelmin. Lopuksi selvitettiin kinnoliiniyhdisteiden kyky estää 77 proteiinikinaasin aktiivisuutta. Proteiinikinaasit ovat kinaasientsyymejä, joiden substraatti on toinen proteiini. Liittämällä fosfaattiryhmän kohdeproteiiniinsa proteiinikinaaseilla on merkittävä rooli solunsisäisessä viestinnässä. Ihmisen genomi sisältää yli 500 proteiinikinaasigeeniä, joka käsittää noin 2 % koko ihmisen perimästä. Proteiinikinaasit säätelevät monia keskeisiä solun toimintaan vaikuttavia prosesseja, kuten kasvua, erilaistumista ja jakautumista. Proteiinikinaasit ovat potentiaalinen lääkevaikutuskohde, sillä näiden entsyymien mutaatiot ja säätelyhäiriöt ovat osallisena useissa ihmisen vakavissa sairauksissa kuten syövissä. Joillakin proteiinikinaaseilla, kuten proteiinikinaasi C:llä on erillinen regulatorinen C1b-domeeni osana proteiinirakennetta. Väitöstyössä käytettiin hyväksi tunnettua proteiinikinaasi C:n C1b:n kiderakennetta ja C1b-domeeniin sitoutuvan forboliesterin rakennetta, joiden avulla mallinnettiin C1b-domeeniin uudentyyppinen inhibiittoriyhdiste. Tämän kolmirengasrakenteisen, kuusi stereokeskusta sisältävän, γ-aminoalkoholin syntetisoinnissa käytettiin hyväksi peräkkäisiä Diels-Alder-reaktioita, joiden avulla yhdisteen molekyylirunko valmistettiin. Tutkimusryhmässämme on kehitetty menetelmiä luonnossa esiintyvän guajatsuleeni-hiilivedyn muokkaamiseksi. Tämän seskviterpeeneihin kuuluvan atsuleenin kemiallisesti muokatuilla johdoksilla on havaittu Pim-1 ja Pim-3-kinaasien toimintaa estävä vaikutus. Tutkimuksessa kehitettiin menetelmä aminoryhmän liittämiseksi guajatsuleenin rakenteeseen. Uusi aminoguajatsuleeni reagoi 1,2-dikarbonyyliyhdisteiden kanssa muodostaen δ-laktaamirakenteisia, typpiatomin sisältäviä, bentso[cd]azuleeneja. Tutkimuksessa muokattiin myös kemiallisesti jo aikaisemmin ryhmässämme syntetisoituja bentso[cd]atsuleenirakenteita, tarkoituksena valmistaa potentiaalisia ja selektiivisiä Pim-kinaasi-inhibiittoreita. Samalla havaittiin, että osalla näistä yhdisteitä on biologisten ominaisuuksien lisäksi myös ainutlaatuisia kemiallisia ominaisuuksia. Tässä tutkimuksessa valmistetut ja karakterisoidut bentso[cd]atsuleeniyhdisteet ovat osoittautuneet hyödyllisiksi tutkimuskäytössä ja voivat olla myös mahdollisia syöpälääkekandidaatteja tulevaisuudessa

DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZAZULENE-BASED COMPOUNDS

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A covalent calmodulin inhibitor as a tool to study cellular mechanisms of K-Ras-driven stemness

This article is part of the Research Topic Ras and other GTPases in Cancer: From Basic to Applied Research https://doi.org/10.3389/978-2-88974-081-9Recently, the highly mutated oncoprotein K-Ras4B (hereafter K-Ras) was shown to drive cancer cell stemness in conjunction with calmodulin (CaM). We previously showed that the covalent CaM inhibitor ophiobolin A (OphA) can potently inhibit K-Ras stemness activity. However, OphA, a fungus-derived natural product, exhibits an unspecific, broad toxicity across all phyla. Here we identified a less toxic, functional analog of OphA that can efficiently inactivate CaM by covalent inhibition. We analyzed a small series of benzazulenones, which bear some structural similarity to OphA and can be synthesized in only six steps. We identified the formyl aminobenzazulenone 1, here named Calmirasone1, as a novel and potent covalent CaM inhibitor. Calmirasone1 has a 4-fold increased affinity for CaM as compared to OphA and was active against K-Ras in cells within minutes, as compared to hours required by OphA. Calmirasone1 displayed a 2.5-4.5-fold higher selectivity for KRAS over BRAF mutant 3D spheroid growth than OphA, suggesting improved relative on-target activity. Importantly, Calmirasone1 has a 40-260-fold lower unspecific toxic effect on HRAS mutant cells, while it reaches almost 50% of the activity of novel K-RasG12C specific inhibitors in 3D spheroid assays. Our results suggest that Calmirasone1 can serve as a new tool compound to further investigate the cancer cell biology of the K-Ras and CaM associated stemness activities.Peer reviewe

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 μM–44 μM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1α. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1α-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 μM–44 μM in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases

Carotid shunt provides cerebral protection during emergency coronary artery bypass grafting in a patient with bilateral high grade carotid stenosis: a case report

<p>Abstract</p> <p>Background</p> <p>Management of patients with co-existent coronary and carotid disease is a controversial and challenging issue. The risk for stroke after coronary artery bypass grafting (CABG) in patients with hemodynamically significant carotid stenosis is up to 30%. In these patients a common practice is to proceed first with the restoration of cerebral perfusion and then perform the coronary revascularization. The rationale is that this strategy will reduce perioperative neurological morbidity and mortality. However, what happens when the carotid procedure is acutely complicated by cardiac instability which necessitates the interruption of the carotid procedure?</p> <p>Case report</p> <p>We describe a case of a patient with unstable angina and high grade asymptomatic bilateral carotid stenosis who underwent emergency combined CABG and carotid endarterectomy (CEA). Due to hemodynamic instability, ST-T changes, hypotension and bradycardia, upon completion of endarterectomy we placed a carotid shunt and the patient was put on cardiopulmonary bypass through median sternotomy. After triple CABG (duration of 90 minutes) we concluded the interrupted CEA procedure with primary closure of the carotid arteriotomy with the shunt in place. The postoperative course was uneventful and the patient was discharged after a week. In extreme cases with bilateral severe carotid stenosis and coronary artery disease where the carotid procedure should be interrupted, we suggest the use of carotid shunt which can provide adequate cerebral perfusion giving time to cardiac surgeon to perform the life saving cardiac procedure first.</p

Preparation and characterization of dentin phosphophoryn-derived peptide-functionalized lignin nanoparticles for enhanced cellular uptake

The surface modification of nanoparticles (NPs) using different ligands is a common strategy to increase NP−cell interactions. Here, dentin phosphophoryn‐derived peptide (DSS) lignin nanoparticles (LNPs) are prepared and characterized, the cellular internalization of the DSS‐functionalized LNPs (LNPs‐DSS) into three different cancer cell lines is evaluated, and their efficacy with the widely used iRGD peptide is compared. It is shown that controlled extent of carboxylation of lignin improves the stability at physiological conditions of LNPs formed upon solvent exchange. Functionalization with DSS and iRGD peptides maintains the spherical morphology and moderate polydispersity of LNPs. The LNPs exhibit good cytocompatibility when cultured with PC3‐MM2, MDA‐MB‐231, and A549 in the conventional 2D model and in the 3D cell spheroid morphology. Importantly, the 3D cell models reveal augmented internalization of peptide‐functionalized LNPs and improve antiproliferative effects when the LNPs are loaded with a cytotoxic compound. Overall, LNPs‐DSS show equal or even superior cellular internalization than the LNPs‐iRGD, suggesting that DSS can also be used to enhance the cellular uptake of NPs into different types of cells, and release different cargos intracellularly.Peer reviewe

Mechanism of the Oxidation of Heptafulvenes to Tropones Studied by Online Mass Spectrometry and Density Functional Theory Calculations

We have identified the most likely reaction mechanism for oxidizing heptafulvenes to the corresponding tropones by experimental and theoretical investigations. The experimental studies were done by coupling a three-dimensional printed miniaturized reactor with an integrated electrospray ionization needle to a mass spectrometer. Using the experimentally observed ions as a basis, nine alternative reaction pathways were investigated with density functional theory calculations. The lowest energy reaction pathway starts with the formation of an epoxide that is opened upon the addition of a second equivalent of the oxidizing species meta-chloroperoxybenzoic acid. The adduct formed then undergoes a Criegee-like rearrangement to yield a positively charged hemiketal, which on deprotonation dissociates into acetone resembles a Hock-like rearrangement. and tropone. Overall, the reaction mechanism resembles a Hock-like rearrangement.Peer reviewe

Novel Small Molecule Hsp90/Cdc37 Interface Inhibitors Indirectly Target K-Ras-Signaling

Simple SummaryThe correct folding of proteins is essential for their activity. Therefore, cells have evolved protein-folding chaperones, such as Hsp90. Interestingly, in several cancer cells, Hsp90 appears to have a role that is more important than normal. The current working model suggests that, with the help of its co-chaperone, Cdc37, it stabilizes mutant kinases. However, Hsp90, together with Cdc37, assists additional proteins that may be relevant in cancer. We demonstrate that the Hsp90-dependent stability of the transcription factor HIF-1 alpha and one of its downstream transcriptional targets, galectin-3, is important to maintain the elevated activity of the major oncogene KRAS. This is because galectin-3 stabilizes the MAPK-signaling complexes of K-Ras, which is called a nanocluster. In addition, we identified six drug-like small molecules that inhibit the Hsp90/Cdc37 protein interface at low micro molar concentrations. Given the co-occurrence of mutant KRAS with high HIF-1 alpha and high galectin-3 levels in pancreatic cancer, our results suggest an application of Hsp90 inhibitors in this cancer type.The ATP-competitive inhibitors of Hsp90 have been tested predominantly in kinase addicted cancers; however, they have had limited success. A mechanistic connection between Hsp90 and oncogenic K-Ras is not known. Here, we show that K-Ras selectivity is enabled by the loss of the K-Ras membrane nanocluster modulator galectin-3 downstream of the Hsp90 client HIF-1 alpha. This mechanism suggests a higher drug sensitivity in the context of KRAS mutant, HIF-1 alpha-high and/or Gal3-high cancer cells, such as those found, in particular, in pancreatic adenocarcinoma. The low toxicity of conglobatin further indicates a beneficial on-target toxicity profile for Hsp90/Cdc37 interface inhibitors. We therefore computationally screened >7 M compounds, and identified four novel small molecules with activities of 4 mu M-44 mu M in vitro. All of the compounds were K-Ras selective, and potently decreased the Hsp90 client protein levels without inducing the heat shock response. Moreover, they all inhibited the 2D proliferation of breast, pancreatic, and lung cancer cell lines. The most active compounds from each scaffold, furthermore, significantly blocked 3D spheroids and the growth of K-Ras-dependent microtumors. We foresee new opportunities for improved Hsp90/Cdc37 interface inhibitors in cancer and other aging-associated diseases

Tricyclic Benzo[cd]azulenes Selectively Inhibit Activities of Pim Kinases and Restrict Growth of Epstein-Barr Virus-Transformed Cells

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