Synthesis of new betulinic acid/betulin-derived dimers and hybrids with potent antimalarial and antiviral activities.

Severe malaria and viral infections cause life-threatening diseases in millions of people worldwide every year. In search for effective bioactive hybrid molecules, which may possess improved properties compared to their parent compounds, a series of betulinic acid/betulin based dimer and hybrid compounds carrying ferrocene and/or artesunic acid moieties, was designed and, synthesized de novo. Furthermore, they were analyzed in vitro against malaria parasites (growth inhibition of 3D7-strain P. falciparum-infected erythrocytes) and human cytomegalovirus (HCMV). From this series of hybrids/dimers, the betulinic acid/betulin and artesunic acid hybrids 11 and 12 showed the most potent activities against P. falciparum and HCMV. On the strength of results, additive and/or synergistic effects between the natural or semisynthetic products, such as betulinic acid-/betulin- and artesunic acid-derived compounds, are suggested on the basis of putatively complex modes of antimicrobial action. This advantage may be taken into account in future drug development.

Artemisinin-(Iso)quinoline Hybrids by C-H Activation and Click Chemistry: Combating Multidrug-Resistant Malaria.

A substantial challenge worldwide is emergent drug resistance in malaria parasites against approved drugs, such as chloroquine (CQ). To address these unsolved CQ resistance issues, only rare examples of artemisinin (ART)-based hybrids have been reported. Moreover, protein targets of such hybrids have not been identified yet, and the reason for the superior efficacy of these hybrids is still not known. Herein, we report the synthesis of novel ART-isoquinoline and ART-quinoline hybrids showing highly improved potencies against CQ-resistant and multidrug-resistant P. falciparum strains (EC50 (Dd2) down to 1.0 nm; EC50 (K1) down to 0.78 nm) compared to CQ (EC50 (Dd2)=165.3 nm; EC50 (K1)=302.8 nm) and strongly suppressing parasitemia in experimental malaria. These new compounds are easily accessible by step-economic C-H activation and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click reactions. Through chemical proteomics, putatively hybrid-binding protein targets of the ART-quinolines were successfully identified in addition to known targets of quinoline and artemisinin alone, suggesting that the hybrids act through multiple modes of action to overcome resistance.

Synthesis of Artemisinin-Derived Dimers, Trimers and Dendrimers: Investigation of Their Antimalarial and Antiviral Activities Including Putative Mechanisms of Action.

Generation of dimers, trimers and dendrimers of bioactive compounds is an approach that has recently been developed for the discovery of new potent drug candidates. Herein, we present the synthesis of new artemisinin-derived dimers and dendrimers and investigate their action against malaria parasite Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV). Dimer 7 was the most active compound (EC50 1.4 nm) in terms of antimalarial efficacy and was even more effective than the standard drugs dihydroartemisinin (EC50 2.4 nm), artesunic acid (EC50 8.9 nm) and chloroquine (EC50 9.8 nm). Trimer 4 stood out as the most active agent against HCMV in vitro replication and exerted an EC50 value of 0.026 µm, representing an even higher activity than the two reference drugs ganciclovir (EC50 2.60 µm) and artesunic acid (EC50 5.41 µm). In addition, artemisinin-derived dimer 13 and trimer 15 were for the first time both immobilized on TOYOPEARL AF-Amino-650M beads and used for mass spectrometry-based target identification experiments using total lysates of HCMV-infected primary human fibroblasts. Two major groups of novel target candidates, namely cytoskeletal and mitochondrial proteins were obtained. Two putatively compound-binding viral proteins, namely major capsid protein (MCP) and envelope glycoprotein pUL132, which are both essential for HCMV replication, were identified.

Access to new highly potent antileukemia, antiviral and antimalarial agents via hybridization of natural products (homo)egonol, thymoquinone and artemisinin.

Hybridization of natural products has high potential to further improve their activities and may produce synergistic effects between linked pharmacophores. Here we report synthesis of nine new hybrids of natural products egonol, homoegonol, thymoquinone and artemisinin and evaluation of their activities against P. falciparum 3D7 parasites, human cytomegalovirus, sensitive and multidrug-resistant human leukemia cells. Most of the new hybrids exceed their parent compounds in antimalarial, antiviral and antileukemia activities and in some cases show higher in vitro efficacy than clinically used reference drugs chloroquine, ganciclovir and doxorubicin. Combined, our findings stress the high potency of these hybrids and encourages further use of the hybridization concept in applied pharmacological research.

Vinculin phosphorylation at residues Y100 and Y1065 is required for cellular force transmission.

The focal adhesion protein vinculin connects the actin cytoskeleton, through talin and integrins, with the extracellular matrix. Vinculin consists of a globular head and tail domain, which undergo conformational changes from a closed auto-inhibited conformation in the cytoplasm to an open conformation in focal adhesions. Src-mediated phosphorylation has been suggested to regulate this conformational switch. To explore the role of phosphorylation in vinculin activation, we used knock-out mouse embryonic fibroblasts re-expressing different vinculin mutants in traction microscopy, magnetic tweezer microrheology, FRAP and actin-binding assays. Compared to cells expressing wild-type or constitutively active vinculin, we found reduced tractions, cytoskeletal stiffness, adhesion strength, and increased vinculin dynamics in cells expressing constitutively inactive vinculin or vinculin where Src-mediated phosphorylation was blocked by replacing tyrosine at position 100 and/or 1065 with a non-phosphorylatable phenylalanine residue. Replacing tyrosine residues with phospho-mimicking glutamic acid residues restored cellular tractions, stiffness and adhesion strength, as well as vinculin dynamics, and facilitated vinculin-actin binding. These data demonstrate that Src-mediated phosphorylation is necessary for vinculin activation, and that phosphorylation controls cytoskeletal mechanics by regulating force transmission between the actin cytoskeleton and focal adhesion proteins.

Access to Artemisinin-Triazole Antimalarials via Organo-Click Reaction: High In Vitro/In Vivo Activity against Multi-Drug-Resistant Malaria Parasites.

Malaria is one of the most widespread diseases worldwide. Besides a growing number of people potentially threatened by malaria, the consistent emergence of resistance against established antimalarial pharmaceuticals leads to an urge toward new antimalarial drugs. Hybridization of two chemically diverse compounds into a new bioactive product is a successful concept to improve the properties of a hybrid drug relative to the parent compounds and also to overcome multidrug resistance. 1,2,3-Triazoles are a significant pharmacophore system among nitrogen-containing heterocycles with various applications, such as antiviral, antimalarial, antibacterial, and anticancer agents. Several marketed drugs possess these versatile moieties, which are used in a wide range of medical indications. While the synthesis of hybrid compounds containing a 1,2,3-triazole unit was described using Cu- and Ru-catalyzed azide-alkyne cycloaddition, an alternative metal-free pathway has never been reported for the synthesis of antimalarial hybrids. However, a metal-free pathway is a green method that allows toxic and expensive metals to be replaced with an organocatalyst. Herein, we present the synthesis of new artemisinin-triazole antimalarial hybrids via a facile Ramachary-Bressy-Wang organocatalyzed azide-carbonyl [3 + 2] cycloaddition (organo-click) reaction. The prepared new hybrid compounds are highly potent in vitro against chloroquine (CQ)-resistant and multi-drug-resistant Plasmodium falciparum strains (IC50 (Dd2) down to 2.1 nM; IC50 (K1) down to 1.8 nM) compared to CQ (IC50 (Dd2) = 165.3 nM; IC50 (K1) = 302.8 nM). Moreover, the most potent hybrid drug was more efficacious in suppressing parasitemia and extending animal survival in Plasmodium berghei-infected mice (up to 100% animal survival and up to 40 days of survival time) than the reference drug artemisinin, illustrating the potential of the hybridization concept as an alternative and powerful drug-discovery approach.

Autofluorescent antimalarials by hybridization of artemisinin and coumarin: in vitro/in vivo studies and live-cell imaging.

Malaria is one of our planet's most widespread and deadliest diseases, and there is an ever-consistent need for new and improved pharmaceuticals. Natural products have been an essential source of hit and lead compounds for drug discovery. Antimalarial drug artemisinin (ART), a highly effective natural product, is an enantiopure sesquiterpene lactone and occurs in Artemisia annua L. The development of improved antimalarial drugs, which are highly potent and at the same time inherently fluorescent is particularly favorable and highly desirable since they can be used for live-cell imaging, avoiding the requirement of the drug's linkage to an external fluorescent label. Herein, we present the first antimalarial autofluorescent artemisinin-coumarin hybrids with high fluorescence quantum yields of up to 0.94 and exhibiting excellent activity in vitro against CQ-resistant and multidrug-resistant P. falciparum strains (IC50 (Dd2) down to 0.5 nM; IC50 (K1) down to 0.3 nM) compared to reference drugs CQ (IC50 (Dd2) 165.3 nM; IC50 (K1) 302.8 nM) and artemisinin (IC50 (Dd2) 11.3 nM; IC50 (K1) 5.4 nM). Furthermore, a clear correlation between in vitro potency and in vivo efficacy of antimalarial autofluorescent hybrids was demonstrated. Moreover, deliberately designed autofluorescent artemisinin-coumarin hybrids, were not only able to overcome drug resistance, they were also of high value in investigating their mode of action via time-dependent imaging resolution in living P. falciparum-infected red blood cells.

Label-free, high-throughput detection of P. falciparum infection in sphered erythrocytes with digital holographic microscopy.

Effective malaria treatment requires rapid and accurate diagnosis of infecting species and actual parasitemia. Despite the recent success of rapid tests, the analysis of thick and thin blood smears remains the gold standard for routine malaria diagnosis in endemic areas. For non-endemic regions, sample preparation and analysis of blood smears are an issue due to low microscopy expertise and few cases of imported malaria. Automation of microscopy results could be beneficial to quickly confirm suspected infections in such conditions. Here, we present a label-free, high-throughput method for early malaria detection with the potential to reduce inter-observer variation by reducing sample preparation and analysis effort. We used differential digital holographic microscopy in combination with two-dimensional hydrodynamic focusing for the label-free detection of P. falciparum infection in sphered erythrocytes, with a parasitemia detection limit of 0.01%. Moreover, the achieved differentiation of P. falciparum ring-, trophozoite- and schizont life cycle stages in synchronized cultures demonstrates the potential for future discrimination of even malaria species.

Synthesis of Artemisinin-Estrogen Hybrids Highly Active against HCMV, P. falciparum, and Cervical and Breast Cancer.

Artemisinin-estrogen hybrids were for the first time both synthesized and investigated for their in vitro biological activity against malaria parasites (Plasmodium falciparum 3D7), human cytomegalovirus (HCMV), and a panel of human malignant cells of gynecological origin containing breast (MCF7, MDA-MB-231, MDA-MB-361, T47D) and cervical tumor cell lines (HeLa, SiHa, C33A). In terms of antimalarial efficacy, hybrid 8 (EC50 = 3.8 nM) was about two times more active than its parent compound artesunic acid (7) (EC50 = 8.9 nM) as well as the standard drug chloroquine (EC50 = 9.8 nM) and was, therefore, comparable to the clinically used dihydroartemisinin (6) (EC50 = 2.4 nM). Furthermore, hybrids 9-12 showed a strong antiviral effect with EC50 values in the submicromolar range (0.22-0.38 µM) and thus possess profoundly stronger anti-HCMV activity (approximately factor 25) than the parent compound artesunic acid (7) (EC50 = 5.41 µM). These compounds also exerted a higher in vitro anti-HCMV efficacy than ganciclovir used as the standard of current antiviral treatment. In addition, hybrids 8-12 elicited substantially more pronounced growth inhibiting action on all cancer cell lines than their parent compounds and the reference drug cisplatin. The most potent agent, hybrid 12, exhibited submicromolar EC50 values (0.15-0.93 µM) against breast cancer and C33A cell lines.

Synthesis of Thymoquinone-Artemisinin Hybrids: New Potent Antileukemia, Antiviral, and Antimalarial Agents.

A series of hybrid compounds based on the natural products artemisinin and thymoquinone was synthesized and investigated for their biological activity against the malaria parasite Plasmodium falciparum 3D7 strain, human cytomegalovirus (HCMV), and two leukemia cell lines (drug-sensitive CCRF-CEM and multidrug-resistant subline CEM/ADR5000). An unprecedented one-pot method of selective formation of C-10α-acetate 14 starting from a 1:1 mixture of C-10α- to C-10ß-dihydroartemisinin was developed. The key step of this facile method is a mild decarboxylative activation of malonic acid mediated by DCC/DMAP. Ether-linked thymoquinone-artemisinin hybrids 6a/b stood out as the most active compounds in all categories, while showing no toxic side effects toward healthy human foreskin fibroblasts and thus being selective. They exhibited EC50 values of 0.2 µM against the doxorubicin-sensitive as well as the multidrug-resistant leukemia cells and therefore can be regarded as superior to doxorubicin. Moreover, they showed to be five times more active than the standard drug ganciclovir and nearly eight times more active than artesunic acid against HCMV. In addition, hybrids 6a/b possessed excellent antimalarial activity (EC50 = 5.9/3.7 nM), which was better than that of artesunic acid (EC50 = 8.2 nM) and chloroquine (EC50 = 9.8 nM). Overall, most of the presented thymoquinone-artemisinin-based hybrids exhibit an excellent and broad variety of biological activities (anticancer, antimalarial, and antiviral) combined with a low toxicity/high selectivity profile.

SYBR® Green I-Based Fluorescence Assay to Assess Cell Viability of Malaria Parasites for Routine Use in Compound Screening.

Owing to its fast and reliable assessment of parasite growth, the SYBR® Green I-based fluorescence assay is widely used to monitor drug susceptibility of malaria parasites. Its particular advantages are that it is a simple, one-step procedure and very cost-effective making it especially suited for high through put screening of newly developed drugs and drug combinations. Here we describe a SYBR® Green I-based fluorescence assay protocol to be used for routine screening of compounds and extracts in a research laboratory environment. A variation of the standard protocol is also provided allowing to address stage-specific effects of fast-acting drugs.

Deeper Insight into the Six-Step Domino Reaction of Aldehydes with Malononitrile and Evaluation of Antiviral and Antimalarial Activities of the Obtained Bicyclic Products.

The straightforward and efficient synthesis of complex aza- and carbobicyclic compounds, which are of importance for medicinal chemistry, is a challenge for modern chemical methodology. An unprecedented metal-free six-step domino reaction of aldehydes with malononitrile was presented in our previous study to provide, in a single operation, these bicyclic nitrogen-containing molecules. Presented here is a deeper investigation of this atom-economical domino process by extending the scope of aldehydes, performing post-modifications of domino products, applying bifunctional organocatalysts and comprehensive NMR studies of selected domino products. The thermodynamic aspects of the overall reaction are also demonstrated using DFT methods in conjunction with a semi-empirical treatment of van der Waals interactions. Furthermore, biological studies of seven highly functionalized and artemisinin-containing domino products against human cytomegalovirus (HCMV) and Plasmodium falciparum 3D7 are presented. Remarkably, in vitro tests against HCMV revealed five domino products to be highly active compounds (EC50 0.071-1.8 µm), outperforming the clinical reference drug ganciclovir (EC50 2.6 µm). Against P. falciparum 3D7, three of the investigated artemisinin-derived domino products (EC50 0.72-1.8 nm) were more potent than the clinical drug chloroquine (EC50 9.1 nm).

Synthesis of Novel Hybrids of Quinazoline and Artemisinin with High Activities against Plasmodium falciparum, Human Cytomegalovirus, and Leukemia Cells.

Many quinazoline derivatives have been synthesized over the last few decades with great pharmacological potential, such as antimalarial, anti-inflammatory, antimicrobial, anticancer, and antiviral. But so far, no quinazoline-artemisinin hybrids have been reported in the literature. In the present study, five novel quinazoline-artemisinin hybrids were synthesized and evaluated for their in vitro biological activity against malarial parasites (Plasmodium falciparum 3D7), leukemia cells (CCRF-CEM and CEM/ADR5000), and human cytomegalovirus. Remarkably, hybrid 9 (EC50 = 1.4 nM), the most active antimalarial compound of this study, was not only more potent than artesunic acid (EC50 = 9.7 nM) but at the same time more active than the clinically used drugs dihydroartemisinin (EC50 = 2.4 nM) and chloroquine (EC50 = 9.8 nM). Furthermore, hybrids 9 and 10 were the most potent compounds with regard to anticytomegaloviral activity (EC50 = 0.15-0.21 µM). They were able to outperform ganciclovir (EC50 = 2.6 µM), which is the relevant standard drug of antiviral therapy, by a factor of 12-17. Moreover, we identified a new highly active quinazoline derivative, compound 14, that is most effective in suppressing cytomegalovirus replication with an EC50 value in the nanomolar range (EC50 = 50 nM). In addition, hybrid 9 exhibited an antileukemia effect similar to that of artesunic acid, with EC50 values in the low micromolar range, and was 45 times more active toward the multidrug-resistant CEM/ADR5000 cells (EC50 = 0.5 µM) than the standard drug doxorubicin.

New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids.

In our ongoing search for highly active hybrid molecules exceeding their parent compounds in anticancer, antimalaria as well as antiviral activity and being an alternative to the standard drugs, we present the synthesis and biological investigations of 2nd generation 1,2,4-trioxane-ferrocene hybrids. In vitro tests against the CCRF-CEM leukemia cell line revealed di-1,2,4-trioxane-ferrocene hybrid 7 as the most active compound (IC50 of 0.01 µM). Regarding the activity against the multidrug resistant subline CEM/ADR5000, 1,2,4-trioxane-ferrocene hybrid 5 showed a remarkable activity (IC50 of 0.53 µM). Contrary to the antimalaria activity of hybrids 4-8 against Plasmodium falciparum 3D7 strain with slightly higher IC50 values (between 7.2 and 30.2 nM) than that of their parent compound DHA, hybrids 5-7 possessed very promising activity (IC50 values lower than 0.5 µM) against human cytomegalovirus (HCMV). The application of 1,2,4-trioxane-ferrocene hybrids against HCMV is unprecedented and demonstrated here for the first time.