An Automated, Open-Source Workflow for the Generation of (3D) Fragment Libraries.

The recent success of fragment-based drug discovery (FBDD) is inextricably linked to adequate library design. To guide the design of our fragment libraries, we have constructed an automated workflow in the open-source KNIME software. The workflow considers chemical diversity and novelty of the fragments, and can also take into account the three-dimensional (3D) character. This design tool can be used to create large and diverse libraries but also to select a small number of representative compounds as a focused set of unique screening compounds to enrich existing fragment libraries. To illustrate the procedures, the design and synthesis of a 10-membered focused library is reported based on the cyclopropane scaffold, which is underrepresented in our existing fragment screening library. Analysis of the focused compound set indicates significant shape diversity and a favorable overall physicochemical profile. By virtue of its modular setup, the workflow can be readily adjusted to design libraries that focus on properties other than 3D shape.

Ultrasound-directed enzyme-prodrug therapy (UDEPT) using self-immolative doxorubicin derivatives.

Background: Enzyme-activatable prodrugs are extensively employed in oncology and beyond. Because enzyme concentrations and their (sub)cellular compartmentalization are highly heterogeneous in different tumor types and patients, we propose ultrasound-directed enzyme-prodrug therapy (UDEPT) as a means to increase enzyme access and availability for prodrug activation locally. Methods: We synthesized ß-glucuronidase-sensitive self-immolative doxorubicin prodrugs with different spacer lengths between the active drug moiety and the capping group. We evaluated drug conversion, uptake and cytotoxicity in the presence and absence of the activating enzyme ß-glucuronidase. To trigger the cell release of ß-glucuronidase, we used high-intensity focused ultrasound to aid in the conversion of the prodrugs into their active counterparts. Results: More efficient enzymatic activation was observed for self-immolative prodrugs with more than one aromatic unit in the spacer. In the absence of ß-glucuronidase, the prodrugs showed significantly reduced cellular uptake and cytotoxicity compared to the parent drug. High-intensity focused ultrasound-induced mechanical destruction of cancer cells resulted in release of intact ß-glucuronidase, which activated the prodrugs, restored their cytotoxicity and induced immunogenic cell death. Conclusion: These findings shed new light on prodrug design and activation, and they contribute to novel UDEPT-based mechanochemical combination therapies for the treatment of cancer.

Metallodrugs in cancer nanomedicine.

Metal complexes are extensively used for cancer therapy. The multiple variables available for tuning (metal, ligand, and metal-ligand interaction) offer unique opportunities for drug design, and have led to a vast portfolio of metallodrugs that can display a higher diversity of functions and mechanisms of action with respect to pure organic structures. Clinically approved metallodrugs, such as cisplatin, carboplatin and oxaliplatin, are used to treat many types of cancer and play prominent roles in combination regimens, including with immunotherapy. However, metallodrugs generally suffer from poor pharmacokinetics, low levels of target site accumulation, metal-mediated off-target reactivity and development of drug resistance, which can all limit their efficacy and clinical translation. Nanomedicine has arisen as a powerful tool to help overcome these shortcomings. Several nanoformulations have already significantly improved the efficacy and reduced the toxicity of (chemo-)therapeutic drugs, including some promising metallodrug-containing nanomedicines currently in clinical trials. In this critical review, we analyse the opportunities and clinical challenges of metallodrugs, and we assess the advantages and limitations of metallodrug delivery, both from a nanocarrier and from a metal-nano interaction perspective. We describe the latest and most relevant nanomedicine formulations developed for metal complexes, and we discuss how the rational combination of coordination chemistry with nanomedicine technology can assist in promoting the clinical translation of metallodrugs.

A Doxorubicin-Glucuronide Prodrug Released from Nanogels Activated by High-Intensity Focused Ultrasound Liberated ß-Glucuronidase.

The poor pharmacokinetics and selectivity of low-molecular-weight anticancer drugs contribute to the relatively low effectiveness of chemotherapy treatments. To improve the pharmacokinetics and selectivity of these treatments, the combination of a doxorubicin-glucuronide prodrug (DOX-propGA3) nanogel formulation and the liberation of endogenous ß-glucuronidase from cells exposed to high-intensity focused ultrasound (HIFU) were investigated in vitro. First, a DOX-propGA3-polymer was synthesized. Subsequently, DOX-propGA3-nanogels were formed from this polymer dissolved in water using inverse mini-emulsion photopolymerization. In the presence of bovine ß-glucuronidase, the DOX-propGA3 in the nanogels was quantitatively converted into the chemotherapeutic drug doxorubicin. Exposure of cells to HIFU efficiently induced liberation of endogenous ß-glucuronidase, which in turn converted the prodrug released from the DOX-propGA3-nanogels into doxorubicin. ß-glucuronidase liberated from cells exposed to HIFU increased the cytotoxicity of DOX-propGA3-nanogels to a similar extend as bovine ß-glucuronidase, whereas in the absence of either bovine ß-glucuronidase or ß-glucuronidase liberated from cells exposed to HIFU, the DOX-propGA3-nanogels hardly showed cytotoxicity. Overall, DOX-propGA3-nanogels systems might help to further improve the outcome of HIFU-related anticancer therapy.

Potent and Prolonged Innate Immune Activation by Enzyme-Responsive Imidazoquinoline TLR7/8 Agonist Prodrug Vesicles.

Synthetic immune-stimulatory drugs such as agonists of the Toll-like receptors (TLR) 7/8 are potent activators of antigen-presenting cells (APCs), however, they also induce severe side effects due to leakage from the site of injection into systemic circulation. Here, we report on the design and synthesis of an amphiphilic polymer-prodrug conjugate of an imidazoquinoline TLR7/8 agonist that in aqueous medium forms vesicular structures of 200 nm. The conjugate contains an endosomal enzyme-responsive linker enabling degradation of the vesicles and release of the TLR7/8 agonist in native form after endocytosis, which results in high in vitro TLR agonist activity. In a mouse model, locally administered vesicles provoke significantly more potent and long-lasting immune stimulation in terms of interferon expression at the injection site and in draining lymphoid tissue compared to a nonamphiphilic control and the native TLR agonist. Moreover, the vesicles induce robust activation of dendritic cells in the draining lymph node in vivo.

PEG-pHPMAm-based polymeric micelles loaded with doxorubicin-prodrugs in combination antitumor therapy with oncolytic vaccinia viruses.

An enzymatically activatable prodrug of doxorubicin was covalently coupled, using click-chemistry, to the hydrophobic core of poly(ethylene glycol)-b-poly[N-(2-hydroxypropyl)-methacrylamide-lactate] micelles. The release and cytotoxic activity of the prodrug was evaluated in vitro in A549 non-small-cell lung cancer cells after adding ß-glucuronidase, an enzyme which is present intracellularly in lysosomes and extracellularly in necrotic areas of tumor lesions. The prodrug-containing micelles alone and in combination with standard and ß-glucuronidase-producing oncolytic vaccinia viruses were also evaluated in vivo, in mice bearing A549 xenograft tumors. When combined with the oncolytic viruses, the micelles completely blocked tumor growth. Moreover, a significantly better antitumor efficacy as compared to virus treatment alone was observed when ß-glucuronidase virus treated tumor-bearing mice received the prodrug-containing micelles. These findings show that combining tumor-targeted drug delivery systems with oncolytic vaccinia viruses holds potential for improving anticancer therapy.

Synthesis of dihydrouracils spiro-fused to pyrrolidines: druglike molecules based on the 2-arylethyl amine scaffold.

The synthesis of a small library of dihydrouracils spiro-fused to pyrrolidines is described. These compounds are synthesized from beta-aryl pyrrolidines, providing products with the 2-arylethyl amine moiety, a structural feature often encountered in compounds active in the central nervous system. The b-aryl pyrrolidines are synthesized through a three-step methodology that includes a Knoevenagel condensation reaction, a 1,3-dipolar cycloaddition reaction, and a nitrile reduction.

Solution-phase parallel annulation of (thio)hydantoins to tetrahydroisoquinolines and tetrahydro-beta-carbolines containing the 2-arylethyl amine scaffold.

The one-step solution-phase parallel synthesis of two structurally diverse libraries of pharmacologically important compounds is described. The presented compounds combine three privileged structures: the 2-arylethyl amine moiety, a tetrahydro(hetero)areno[c]pyridine, and a (thio)hydantoin. These compounds are synthesized by annulation of a hydantoin or a 2-thiohydantoin ring to tri- or tetracyclic scaffolds, containing the 2-arylethyl amine moiety and a tetrahydroisoquinoline, a tetrahydro-beta-carboline, or a tetrahydrofuro[3,2-c]pyridine. The annulation leads to pharmacologically relevant structural motifs such as imidazopyrroloisoquinolines, dioxoloimidazopyrroloisoquinolines, furoimidazopyrrolopyridines, and imidazopyrrolopyridoindoles. Both libraries were obtained with quantitative yields. The 36-membered hydantoin library was obtained with purities from 57 to 100% (90% average) and the 32-membered thiohydantoin library with purities from 73 to 100% (94% average).

Synthesis of hydantoins and thiohydantoins spiro-fused to pyrrolidines: druglike molecules based on the 2-arylethyl amine scaffold.

The synthesis of a 144-compound library of hydantoins and thiohydantoins spiro-fused to pyrrolidines is described. These compounds are synthesized from beta-aryl pyrrolidines, providing products with the 2-arylethyl amine moiety, a structural feature often encountered in compounds active in the central nervous system. All possible stereoisomers of the two-stereocenter products are synthesized. The 80-membered hydantoin sublibrary was obtained with yields ranging from 58 to 100% (87% average) and purities from 51 to 100% (87% average) and the 64-membered thiohydantoin sublibrary was obtained with yields ranging from 65 to 100% (89% average) and purities from 67 to 100% (93% average).

Synthesis of tetrahydro-beta-carbolines and tetrahydroisoquinolines fused to pyrrolidines and solution-phase parallel acylation.

A structurally diverse library of potentially pharmacologically important compounds employing classical synthesis methods is described. These compounds are synthesized from beta-aryl pyrrolidines, providing products with the 2-arylethyl amine moiety, a structural feature often encountered in compounds active in the central nervous system. Tri- and tetracyclic scaffolds were obtained using the Pictet-Spengler reaction, resulting in hexahydropyrrolo[3,4-c]isoquinolines 1-3, an octahydropyrrolo[3',4':5,6]pyrido[3,4-b]indole 4, and a hexahydrofuro[2,3-d]pyrrolo[3,4-b]pyridine 5. These scaffolds were further derivatized in parallel fashion to make a 32-membered amide library with yields from 62 to 100% (90% average) and purities from 63 to 100% (93% average).

Synthesis of spirohydantoins and spiro-2,5-diketopiperazines via resin-bound cyclic alpha,alpha-disubstituted alpha-amino esters.

A seven-step solid-phase synthesis of spirohydantoins and an eight-step solid-phase synthesis of spiro-2,5-diketopiperazines is reported. Key intermediate in the synthesis of both compound libraries is the resin-bound cyclic alpha,alpha-disubstituted alpha-amino ester, which can be obtained after selective homogeneous reduction of the aliphatic nitro ester using tin(II) chloride dihydrate. Nitro ester, in turn, is synthesized by a high-pressure-assisted [4 + 2] cycloaddition of resin-bound nitro alkene and butadiene, whereas nitro alkene is obtained by a Knoevenagel condensation of resin-bound nitro acetate with an imine. Novel spirohydantoins are obtained by isocyanate coupling with the resin-bound amino ester 5, followed by cyclization cleavage using a base. Novel spiro-2,5-diketopiperazines are obtained by PyBOP coupling of a Fmoc-protected amino acid with resin-bound amino ester, followed by Fmoc deprotection and an acid-assisted cyclization cleavage. After preparation of seven different resin-bound alpha,alpha-disubstituted alpha-amino esters, a 7 x 8 compound library of spirohydantoins was synthesized using eight different isocyanates, and a 7 x 8 compound library of spiro-2,5-diketopiperazines was synthesized using eight different Fmoc amino acids.

A methylester of the glucuronide prodrug DOX-GA3 for improvement of tumor-selective chemotherapy.

The glucuronide prodrug of doxorubicin, DOX-GA3, can be selectively activated in tumors by extracellular human beta-glucuronidase, resulting in a better therapeutic index than doxorubicin. DOX-GA3, however, is rapidly excreted by the kidney. We hypothesized that slow release of DOX-GA3 from its methylester, DOX-mGA3, by esterase activity in blood would result in improved circulation half-life (t(1/2)) of DOX-GA3. DOX-mGA3 was synthesized more efficiently with an overall yield of 60% as compared to 37% in the case of DOX-GA3. We showed that DOX-mGA3 was enzymatically converted to DOX-GA3 with a t(1/2) of approximately 0.5 min in mouse plasma to 2.5 h in human plasma, which was in agreement with differences in esterase activity between species. DOX-mGA3, similar to DOX-GA3, was at least 37-fold less potent than the parent drug doxorubicin in growth inhibition of four different human malignant cell lines in vitro. Incubation of OVCAR-3 cells with DOX-mGA3 in combination with an excess of human beta-glucuronidase (0.05 U mL(-1)) resulted in a similar growth inhibition to that of doxorubicin. Intravenous administration of DOX-mGA3 in FMa-bearing mice resulted in an area under the concentration versus time curve (AUC) of DOX-GA3 in tumor and most normal tissues that was 2.5- to 3-fold higher than after the same dose of DOX-GA3 itself. In tumor tissue, this was accompanied by a 2.7-fold increase in the AUC of doxorubicin from DOX-mGA3 than from DOX-GA3. In conclusion, an advantage of DOX-mGA3 over DOX-GA3 is that this prodrug can be produced with a higher yield. Another important advantage is the improved pharmacokinetics of the lipophilic DOX-mGA3 as compared to that of the hydrophilic DOX-GA3. This effect may even be more pronounced in man, because of the lower plasma esterase activity than measured in mice.

Plasmin-activated doxorubicin prodrugs containing a spacer reduce tumor growth and angiogenesis without systemic toxicity.

To generate doxorubicin (Dox) specifically at the tumor site, the chemotherapeutic agent was incorporated into a prodrug by linkage to a peptide specifically recognized by plasmin, which is overproduced in many cancers. ST-9905, which contains an elongated self-elimination spacer, is activated more rapidly in vitro by plasmin than is ST-9802. Prodrug activation in vitro depended on the level of urokinase produced by tumor cells and was inhibited by aprotinin, a plasmin inhibitor. Comparison of equimolar concentrations of ST-9905, ST-9802, and Dox in EF43.fgf-4 and MCF7 models revealed that both prodrugs, in sharp contrast to Dox, displayed antiproliferative and antiangiogenic activities without discernible toxicity. Although MCF7 cells are poor urokinase producers in vitro, prodrug efficacy in this model may be explained by production of plasmin by tumor-infiltrating host cells. Mice treated with equitoxic concentrations (maximum tolerated doses) of prodrugs showed 100% survival and negligible body weight loss, in contrast to results after Dox treatment. ST-9905 was substantially more effective than ST-9802 and induced similar tumor growth inhibition as Dox but without apparent toxicity. This finding may be explained by the elongated spacer, which facilitates enzymatic prodrug activation. These data validate both the use of elongated spacers in vivo and the concept of targeting anticancer prodrugs to tumor-associated plasmin.

High pressure: a promising tool for multicomponent reactions.

In this paper the application of high pressure in multicomponent reactions is discussed. Using high pressure the scope of certain multicomponent reactions can be increased. Reactions are described that can only be performed in a multicomponent fashion when high pressure catalysis is applied. An overview of high pressure catalysed multicomponent reactions is presented with special attention to the domino [4 + 2]/[3 + 2] cycloaddition reaction.

Design, synthesis, and biological evaluation of a dual tumor-specific motive containing integrin-targeted plasmin-cleavable doxorubicin prodrug.

The design, synthesis, and initial biological evaluation of a doxorubicin prodrug that contains a dual tumor specific moiety, which allows enhanced tumor recognition potential, is reported. Both a tumor-specific recognition site and a tumor selective enzymatic activation sequence are incorporated in the prodrug. The first tumor-specific sequence is the bicyclic CDCRGDCFC (RGD-4C) peptide that selectively binds alpha v beta 3 and alpha v beta 5 integrins. These integrins are highly overexpressed on invading tumor endothelial cells. The second tumor-specific sequence is a D-Ala-Phe-Lys tripeptide that is selectively recognized by the tumor-associated protease plasmin, which is involved in tumor invasion and metastasis. An aminocaproyl residue was incorporated as a spacer between the two peptide sequences, whereas a self-eliminating 4-aminobenzyl alcohol spacer was inserted between the plasmin substrate and doxorubicin. Although the prodrug showed a decreased binding affinity as compared with the unconjugated reference peptide, it was still a potent ligand for alpha v beta 3 and alpha v beta 5 integrin receptors. The synthesized construct also possessed plasmin substrate properties as demonstrated by doxorubicin release from 1 upon incubation with plasmin. The release of doxorubicin from 1 was not complete, possibly related to low prodrug solubility. In vitro prodrug 1 showed plasmin-dependent cytotoxicity for endothelial cells and HT1080 fibrosarcoma cells. On the basis of these in vitro results, derivatives of 1 with improved water solubility are considered good candidates for additional development and in vivo evaluation of this dual targeting concept.

Novel 20-carbonate linked prodrugs of camptothecin and 9-aminocamptothecin designed for activation by tumour-associated plasmin.

The first prodrugs of camptothecin and 9-aminocamptothecin that are activated by the tumour-associated protease plasmin are reported. The tripartate prodrugs consist of a tripeptide sequence recognised by plasmin, which is linked to the 20-hydroxyl group of the camptothecins via a 1,6-elimination spacer. After selective N-protection of 9-aminocamptothecin with an Aloc group, the promoiety (tripeptide-spacer conjugate) was linked to camptothecin or 9-Aloc-9-aminocamptothecin via a 20-carbonate linkage by reacting parent drugs with the p-nitrophenyl carbonate activated promoiety in the presence of DMAP. Both prodrugs showed to be stable in buffer solution and both parent drugs were released upon incubation in the presence of plasmin. Furthermore, the prodrugs showed an average 10-fold decreased cytotoxicity with respect to their parent drugs upon incubation in seven human tumour cell lines.

Beta-glucuronidase-mediated drug release.

The selective activation of a relatively non-toxic prodrug by an enzyme present only in the tumour should enhance the drug concentration at the tumour site and result in a better anti-tumour effect and a reduction in systemic toxicity as compared to conventional chemotherapy. beta-Glucuronidase is such an enzyme. It is normally expressed in the lysosomes of cells. In larger tumours, however, high levels of the enzyme are present in necrotic areas. Several glucuronide prodrugs have been synthesised that can be activated by beta-glucuronidase. They are relatively non-toxic due to their hydrophilic nature, which prevents them from entering cells and thus from contact with lysosomal beta-glucuronidase. The main problem of glucuronide prodrugs for clinical use is their fast renal clearance. Special attention should be paid to the development of new less hydrophilic prodrugs with slower clearance, as this would result in a prolonged exposure to beta-glucuronidase at the site of the tumour and a reduction of the amount of prodrug needed. A number of interesting anthracyclin-based glucuronide prodrugs have been synthesised and have shown favourable therapeutic effects compared to treatment with the parent drug. The tumoural levels of beta-glucuronidase can even be enhanced by two-step approaches, in which exogenous enzyme is targeted to the tumour by an antibody (ADEPT) or by the gene encoding the enzyme in transduced tumour cells (GDEPT). The ADEPT and GDEPT approaches in combination with glucuronide prodrugs have shown enhanced efficacy in experimental tumour models. Further improvement of ADEPT and GDEPT is warranted to optimise the tumour uptake and retention of antibody-enzyme fusion proteins and the efficiency and safety of current gene delivery methods. In conclusion, it is clear that glucuronide prodrugs hold promise for future use in the treatment of cancer in patients as monotherapy. Enhancement of the therapeutic effects of glucuronide prodrugs, also in patients with small tumour lesions, may possibly be achieved by techniques that target beta-glucuronidase specifically to the site of the tumour.

Synthesis of novel paclitaxel prodrugs designed for bioreductive activation in hypoxic tumour tissue.

The syntheses and preliminary evaluation of the first potential bioreductive paclitaxel prodrugs are described. These prodrugs were designed as potential candidates in more selective chemotherapy by targeting hypoxic tumour tissue. Aromatic nitro and azide groups were used as the bioreductive trigger. Generation of paclitaxel occurs after reduction and subsequent 1,6-elimination or 1,8-elimination. All prodrugs are stable in buffer and indeed give paclitaxel after chemical reduction of the aromatic nitro or azide functionality. In aerobic cytotoxicity assays several prodrugs exhibit diminished cytotoxicity. These compounds are interesting candidates for further biological evaluation.

Semisynthesis of Some 7-Deoxypaclitaxel Analogs from Taxine B.

Taxine B (3), isolated from the dried needles of Taxus baccata, was converted into six novel 7-deoxypaclitaxel analogs, 20, 21a,b, and 23-25, that have structural changes at C1, C2, and C4. A method for the introduction of the benzoyl function at C2, via a benzylidene acetal at C1-C2, will be revealed. All compounds showed very little or no measurable cytotoxic activity against some well-characterized human tumor cell lines, probably due to the nonacylated hydroxyl group at C4.