Design, Synthesis and Characterization of ABCG2 Inhibitors with a Focus on Water Solubility and Stability in Plasma

ATP-binding cassette (ABC) transporters are gatekeepers at all major tissue barriers, e.g. the intestinal barrier or the blood-brain barrier (BBB). On one hand, these proteins extrude endogenous substrates and on the other hand, they protect cells against the entry of numerous xenobiotics, including a variety of drugs such as anticancer agents or neuropharmaceuticals. The three main drug transporters are ABCB1, ABCC1 and ABCG2, with ABCG2 being predominant at the human BBB. They constitute a severe impediment for the oral bioavailability and brain penetration of drugs, and confer multidrug resistance (MDR) to cancer cells. Thus, inhibitors of ABCG2 are required as molecular tools for examinations on the patho(physiological) role of this transporter and as potential drugs to overcome tissue barriers (especially the BBB) and MDR in cancer. So far, the discovery of ABCG2 inhibitors had largely been driven by potency, while drug-like properties – imperative for application in vivo – had been overlooked. Moreover, the mechanism of transport inhibition had been enigmatic. Therefore, the present thesis aimed at creating novel ABCG2 inhibitors with improved drug-like properties, especially stability in blood plasma and water solubility, and at elucidating the underlying transport inhibition mechanism. Tariquidar analogs had been described as potent and selective ABCG2 inhibitors. Yet, their susceptibility to hydrolysis limited their applicability in vivo. Chapter 2 of this thesis comprises the synthesis (performed by Prof. König’s group) and characterization (performed by our group, including the author of this thesis) of new tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in the previous tariquidar analog UR-ME22-1. CuAAC (“click” reaction) gave access to a triazole core as a substitute for the labile amide group and the unstable ester moiety was replaced by acyl groups. An HPLC assay proved enhanced stability in blood plasma. Compounds UR-MB108 and UR-MB136 inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM, thus belong to the most potent ABCG2 inhibitors described so far. Compound UR-MB108 was highly selective for ABCG2, while its PEGylated analog UR-MB136 showed some potency at ABCB1. Both UR-MB108 and UR-MB136 produced an ABCG2 ATPase-depressing effect, which is in agreement with a precedent cryo-EM study identifying UR MB136 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation of ABCG2. As reference substances, compounds UR-MB108 and UR-MB136 allow additional mechanistic studies on ABCG2 inhibition. Stable in blood plasma, they are also applicable in vivo. Chapter 3 of this thesis is focused on water solubility, which is an indispensable prerequisite for all bioactive compounds – seemingly trivial, however often underestimated. Many drug development failures have been attributed to poor aqueous solubility. ABCG2 inhibitors are especially prone to be insoluble since they have to address the extremely large and hydrophobic multidrug binding site in ABCG2. A case in point is UR-MB108, which showed high potency (79 nM), but very low aqueous solubility (78 nM). To discover novel potent ABCG2 inhibitors with improved solubility, a fragment-based approach was pursued. Substructures of UR-MB108 were optimized and the fragments ‘enlarged’ to obtain inhibitors, in part supported by molecular docking studies. Syntheses were achieved, i.a., via Sonogashira coupling, click chemistry and amide coupling. A kinetic solubility assay revealed that UR-MB108 and most of the new inhibitors did not precipitate during the short time period of the applied biological assays. The solubility of the compounds in aqueous media at equilibrium was investigated in a thermodynamic solubility assay, where UR-Ant116, UR-Ant121, UR-Ant131 and UR-Ant132 excelled with solubilities between 1 µM and 1.5 µM – an up to 19-fold improvement compared to UR-MB108. Moreover, these novel N-phenyl-chromone-2-carboxamides inhibited ABCG2 in a Hoechst 33342 transport assay with potencies in the low three-digit nanomolar range, reversed MDR in ABCG2-overexpressing cancer cells, were non-toxic and proved stable in blood plasma. Altogether, the aforementioned compounds are attractive candidates for in vivo studies and in vitro assays requiring long-term incubation, both needing sufficient solubility at equilibrium. UR-Ant121 and UR-Ant132 were highly ABCG2-selective, a precondition for developing PET tracers. The triple ABCB1/C1/G2 inhibitor UR-Ant116 qualifies for potential therapeutic applications, given the concerted role of the three transporter subtypes at many tissue barriers, e.g. the BBB. Taken together, this thesis provides ABCG2 inhibitors that are potent and effective as well as water-soluble, stable in blood plasma and nontoxic, which may be of interest as molecular biological tools and as drugs for patients with cancer or brain disorders

Derivatives of nitrogen mustard anticancer agents with improved cytotoxicity

In previous studies, we demonstrated that esters of bendamustine containing a basic moiety are far more cytotoxic anticancer agents than their parent compound and that the substitution of the labile ester moiety by a branched ester or an amide markedly increases stability in the blood plasma. In the current study, we showed that this substitution was bioisosteric. Aiming at increased cytotoxicity, we introduced the same modification to related nitrogen mustards: 6‐isobendamustine, chlorambucil, and melphalan. The synthesis was accomplished using the coupling reagents N,N′‐dicyclohexylcarbodiimide or 2‐(1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminium tetrafluoroborate. Cytotoxicity against a panel of diverse cancer cells (carcinoma, sarcoma, and malignant melanoma) was assessed in a kinetic chemosensitivity assay. The target compounds showed cytotoxic or cytocidal effects at concentrations above 1 µM: a striking enhancement over bendamustine and 6‐isobendamustine, both ineffective against the selected cancer cells at concentrations up to 50 µM, and a considerable improvement over chlorambucil, showing some potency only against the sarcoma cells. Melphalan was almost as effective as the target compounds—derivatization only provided a small improvement. The novel cytostatics are of interest as model compounds for analyzing a correlation between cytotoxicity and membrane transport and for the treatment of malignancies

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Stabilities of neutral and basic esters of bendamustine in plasma compared to the parent compound: Kinetic investigations by HPLC

Esters of the cytostatic bendamustine (1), previously demonstrated to be much more potent than the parent compound as antiproliferative agents in vitro, were investigated by HPLC for stability in buffer and plasma, as well as against porcine liver esterase in the presence of different amounts of albumin. The hydrolysis of the nitrogen mustard moiety was retarded (for 1: approximately 130 vs. 11 min) in the presence of plasma proteins. For the derivatives, both cleavage of ester and nitrogen mustard moieties were analyzed. Enzymatic hydrolysis was very fast in case of 2-pyrrolidino-, 2-piperidino- and 2-(4-methylpiperazino)-ethyl esters, whereas methyl, ethyl, morpholinoethyl and branched 2-pyrrolidinoethyl esters were considerably more stable (half-lives between 41 and 116 min, compared to <5 min). Inhibition by physostigmine indicated unspecific cholinesterases to be involved in the rapid ester cleavage. Due to lower protein content and higher enzymatic activity in murine compared to human plasma, reduced stability of all investigated esters in mouse plasma (t½ <2 min) has to be taken into account with respect to the design of animal studies

Tariquidar-related triazoles as potent, selective and stable inhibitors of ABCG2 (BCRP)

Tariquidar derivatives have been described as potent and selective ABCG2 inhibitors. However, their susceptibility to hydrolysis limits their applicability. The current study comprises the synthesis and characterization of novel tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in our previous tariquidar analog UR-ME22-1 (9). CuAAC ("click" reaction) gave convenient access to a triazole core as a substitute for the labile amide group and the labile ester moiety was replaced by different acyl groups in a Sugasawa reaction. A stability assay proved the enhancement of the stability in blood plasma. Compounds UR-MB108 (57) and UR-MB136 (59) inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM and belong to the most potent ABCG2 inhibitors described so far. Compound 57 was highly selective, whereas its PEGylated analog 59 showed some potency at ABCB1. Both 57 and 59 produced an ABCG2 ATPase-depressing effect which is in agreement with our precedent cryo-EM study identifying 59 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation. Thermostabilization of ABCG2 by 57 and 59 can be taken as a hint to comparable binding to ABCG2. As reference substances, compounds 57 and 59 allow additional mechanistic studies on ABCG2 inhibition. Due to their stability in blood plasma, they are also applicable in vivo. The highly specific inhibitor 57 is suited for PET labeling, helping to further elucidate the (patho) physiological role of ABCG2, e.g. at the BBB. (c) 2020 Elsevier Masson SAS. All rights reserved

Tariquidar-related triazoles as potent, selective and stable inhibitors of ABCG2 (BCRP)

Tariquidar derivatives have been described as potent and selective ABCG2 inhibitors. However, their susceptibility to hydrolysis limits their applicability. The current study comprises the synthesis and characterization of novel tariquidar-related inhibitors, obtained by bioisosteric replacement of the labile moieties in our previous tariquidar analog UR-ME22-1 (9). CuAAC (“click” reaction) gave convenient access to a triazole core as a substitute for the labile amide group and the labile ester moiety was replaced by different acyl groups in a Sugasawa reaction. A stability assay proved the enhancement of the stability in blood plasma. Compounds UR-MB108 (57) and UR-MB136 (59) inhibited ABCG2 in a Hoechst 33342 transport assay with an IC50 value of about 80 nM and belong to the most potent ABCG2 inhibitors described so far. Compound 57 was highly selective, whereas its PEGylated analog 59 showed some potency at ABCB1. Both 57 and 59 produced an ABCG2 ATPase-depressing effect which is in agreement with our precedent cryo-EM study identifying 59 as an ATPase inhibitor that exerts its effect via locking the inward-facing conformation. Thermostabilization of ABCG2 by 57 and 59 can be taken as a hint to comparable binding to ABCG2. As reference substances, compounds 57 and 59 allow additional mechanistic studies on ABCG2 inhibition. Due to their stability in blood plasma, they are also applicable in vivo. The highly specific inhibitor 57 is suited for PET labeling, helping to further elucidate the (patho)physiological role of ABCG2, e.g. at the BBB.ISSN:0223-5234ISSN:1768-325

Enhanced Bioactivity of Internally Functionalized Cationic Dendrimers with PEG Cores

Hybrid dendritic-linear block copolymers based on a 4-arm polyethylene glycol (PEG) core were synthesized using an accelerated AB(2)/CD(2) dendritic growth approach through orthogonal amine/epoxy and thiol-yne chemistries. The biological activity of these 4-arm and the corresponding 2-arm hybrid dendrimers revealed an enhanced, dendritic effect with an exponential increase in cell internalization concomitant with increasing amine end-groups and low cytotoxicity. Furthermore, the ability of these hybrid dendrimers to induce endosomal escape combined with their facile and efficient synthesis makes them attractive platforms for gene transfection. The 4-arm-based dendrimer showed significantly improved DNA binding and gene transfection capabilities in comparison with the 2-arm derivative. These results combined with the MD simulation indicate a significant effect of both the topology of the PEG core and the multivalency of these hybrid macromolecules, on their DNA binding and delivery capablities

Long-term safety and efficacy of patisiran for hereditary transthyretin-mediated amyloidosis with polyneuropathy: 12-month results of an open-label extension study

© 2020 Elsevier Ltd. All rights reserved.Background: Hereditary transthyretin-mediated amyloidosis is a rare, inherited, progressive disease caused by mutations in the transthyretin (TTR) gene. We assessed the safety and efficacy of long-term treatment with patisiran, an RNA interference therapeutic that inhibits TTR production, in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy. Methods: This multicentre, open-label extension (OLE) trial enrolled patients at 43 hospitals or clinical centres in 19 countries as of Sept 24, 2018. Patients were eligible if they had completed the phase 3 APOLLO or phase 2 OLE parent studies and tolerated the study drug. Eligible patients from APOLLO (patisiran and placebo groups) and the phase 2 OLE (patisiran group) studies enrolled in this global OLE trial and received patisiran 0·3 mg/kg by intravenous infusion every 3 weeks with plans to continue to do so for up to 5 years. Efficacy assessments included measures of polyneuropathy (modified Neuropathy Impairment Score +7 [mNIS+7]), quality of life, autonomic symptoms, nutritional status, disability, ambulation status, motor function, and cardiac stress, with analysis by study groups (APOLLO-placebo, APOLLO-patisiran, phase 2 OLE patisiran) based on allocation in the parent trial. The global OLE is ongoing with no new enrolment, and current findings are based on the interim analysis of the patients who had completed 12-month efficacy assessments as of the data cutoff. Safety analyses included all patients who received one or more dose of patisiran up to the data cutoff. This study is registered with ClinicalTrials.gov, NCT02510261. Findings: Between July 13, 2015, and Aug 21, 2017, of 212 eligible patients, 211 were enrolled: 137 patients from the APOLLO-patisiran group, 49 from the APOLLO-placebo group, and 25 from the phase 2 OLE patisiran group. At the data cutoff on Sept 24, 2018, 126 (92%) of 137 patients from the APOLLO-patisiran group, 38 (78%) of 49 from the APOLLO-placebo group, and 25 (100%) of 25 from the phase 2 OLE patisiran group had completed 12-month assessments. At 12 months, improvements in mNIS+7 with patisiran were sustained from parent study baseline with treatment in the global OLE (APOLLO-patisiran mean change -4·0, 95 % CI -7·7 to -0·3; phase 2 OLE patisiran -4·7, -11·9 to 2·4). Mean mNIS+7 score improved from global OLE enrolment in the APOLLO-placebo group (mean change from global OLE enrolment -1·4, 95% CI -6·2 to 3·5). Overall, 204 (97%) of 211 patients reported adverse events, 82 (39%) reported serious adverse events, and there were 23 (11%) deaths. Serious adverse events were more frequent in the APOLLO-placebo group (28 [57%] of 49) than in the APOLLO-patisiran (48 [35%] of 137) or phase 2 OLE patisiran (six [24%] of 25) groups. The most common treatment-related adverse event was mild or moderate infusion-related reactions. The frequency of deaths in the global OLE was higher in the APOLLO-placebo group (13 [27%] of 49), who had a higher disease burden than the APOLLO-patisiran (ten [7%] of 137) and phase 2 OLE patisiran (0 of 25) groups. Interpretation: In this interim 12-month analysis of the ongoing global OLE study, patisiran appeared to maintain efficacy with an acceptable safety profile in patients with hereditary transthyretin-mediated amyloidosis with polyneuropathy. Continued long-term follow-up will be important for the overall assessment of safety and efficacy with patisiran.info:eu-repo/semantics/publishedVersio