A Chemical Strategy for the Preparation of Multimodified Peptide Imaging Probes.

Multimodality probes appear of great interest for innovative imaging applications in disease diagnosis. Herein, we present a chemical strategy enabling site-specific double-modification and cyclization of a peptide probe exploiting native chemical ligation (NCL) and thiol-maleimide addition. The synthetic strategy is straightforward and of general applicability for the development of double-labeled peptide multimodality probes.

Structure-Activity Relationships of Triple-Action Platinum(IV) Prodrugs with Albumin-Binding Properties and Immunomodulating Ligands.

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure-activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment.

Fabrication of self-healing pectin/chitosan hybrid hydrogel via Diels-Alder reactions for drug delivery with high swelling property, pH-responsiveness, and cytocompatibility.

Self-healing hydrogels with pH-responsiveness could protect loaded drugs from being destroyed till it arrives to the target. The pectin-based hydrogel is a candidate due to the health benefit, anti-inflammation, antineoplastic activity, nontoxicity, and biospecific degradation, et al. However, the abundant existence of water-soluble branched heteropolysaccharide chains influenced its performance resulting in limitation of the potential. In the present study, we prepared a series of self-healing pectin/chitosan hydrogels via the Diels-Alder reaction. Moreover, pectin/chitosan composite hydrogel was prepared as a contrast. By comparison, it can be seen that the Diels-Alder reaction greatly improved the cross-linking density of hydrogels. The self-healing experiments showed excellent self-healing performance. In different swelling mediums, significant transformation in the swelling ratio was shown, indicating well-swelling property, pH- and thermo-responsiveness. The drug loading and release studies presented high loading efficiency and sustained release performance. The cytotoxicity assay that showed a high cell proliferation ratio manifested great cytocompatibility.

BF3-OEt2 Catalyzed C3-Alkylation of Indole: Synthesis of Indolylsuccinimidesand Their Cytotoxicity Studies.

A simple and efficient BF3-OEt2 promoted C3-alkylation of indole has been developed to obtain3-indolylsuccinimidesfrom commercially available indoles and maleimides, with excellent yields under mild reaction conditions. Furthermore, anti-proliferative activity of these conjugates was evaluated against HT-29 (Colorectal), Hepg2 (Liver) and A549 (Lung) human cancer cell lines. One of the compounds, 3w, having N,N-Dimethylatedindolylsuccinimide is a potent congener amongst the series with IC50 value 0.02 µM and 0.8 µM against HT-29 and Hepg2 cell lines, respectively, and compound 3i was most active amongst the series with IC50 value 1.5 µM against A549 cells. Molecular docking study and mechanism of reaction have briefly beendiscussed. This method is better than previous reports in view of yield and substrate scope including electron deficient indoles.

Synthesis of Michael Adducts as Key Building Blocks for Potential Analgesic Drugs: In vitro, in vivo and in silico Explorations.

BACKGROUND: Organocatalytic asymmetric Michael addition is a strong approach for C-C bond formation. The objective of the study is to design molecules by exploiting the efficiency of Michael Adducts. We proceeded with the synthesis of Michael adducts by tailoring the substitution pattern on maleimide and trans-ß-nitro styrene as Michael acceptors. The synthesized compounds were evaluated for dual cyclooxygenases (COX) and lipoxygenase (LOX) inhibition. METHODS: The compounds (4, 9-11) were synthesized through Michael additions. The cyclooxygenases (COX-1 and 2) and lipoxygenase (5-LOX) assays were used for in vitro evaluations of compounds. After the acute toxicity studies, the in vivo analgesic potential was determined with acetic acid induced writhing, tail immersion, and formalin tests. Furthermore, the possible roles of adrenergic and dopaminergic receptors were also studied. Extensive computational studies were performed to get a better understanding regarding the binding of this compound with protein target. RESULTS: Four Michael adducts (4, 9-11) were synthesized. Compound 4 was obtained in enantio- and diastereopure form. The stereopure compound 4 showed encouraging COX-1 and-2 inhibitions with IC50 values of 128 and 65 µM with SI of 1.94. Benzyl derivative 11 showed excellent COX-2 inhibition with the IC50 value of 5.79 µM and SI value 7.96. Compounds 4 and 11 showed good results in in vivo models of analgesia like acetic acid test, tail immersion, and formalin tests. Our compounds were not active in dopaminergic and adrenergic pathways and so were acting centrally. Through extensive computational studies, we computed binding energies, and pharmacokinetic predictions. CONCLUSION: Our findings conclude that our synthesized Michael products (pyrrolidinedione 4 and nitroalkane 11) can be potent centrally acting analgesics. Our in silico predictions suggested that the compounds have excellent pharmacokinetic properties. It is concluded here that dual inhibition of COX/LOX pathways provides a convincing step towards the discovery of safe lead analgesic molecules.

Reversible Diels-Alder Reactions with a Fluorescent Dye on the Surface of Magnetite Nanoparticles.

Diels-Alder reactions on the surface of nanoparticles allow a thermoreversible functionalization of the nanosized building blocks. We report the synthesis of well-defined magnetite nanoparticles by thermal decomposition reaction and their functionalization with maleimide groups. Attachment of these dienophiles was realized by the synthesis of organophosphonate coupling agents and a partial ligand exchange of the original carboxylic acid groups. The functionalized iron oxide particles allow a covalent surface attachment of a furfuryl-functionalized rhodamine B dye by a Diels-Alder reaction at 60 °C. The resulting particles showed the typical fluorescence of rhodamine B. The dye can be cleaved off the particle surface by a retro-Diels-Alder reaction. The study showed that organic functions can be thermoreversibly attached onto inorganic nanoparticles.

Pyrrole-2,5-dione analogs as a promising antioxidant agents: microwave-assisted synthesis, bio-evaluation, SAR analysis and DFT studies/interpretation.

A new series of pyrrole analogs were developed via the microwave irradiation synthesis. Consequently, got a high yield of the products. As pyrroles are familiar for showing various biological properties, all obtained compounds were screened for their antioxidant properties, most of the compounds showing significant activity. In fact, the motifs 5e, 5g, 5h and 5m showed outstanding antioxidant properties. Further, to enlighten the biologically energetic behavior underlying the antioxidant activity, compounds DFT studies were performed. Noteworthy results have been attained and the structure activity relationship (SAR) was discussed with the support of this results. It was found that highly biological active compounds exhibited a low HOMO-LUMO energy gap (Eg) and the high Eg value compounds show very low/negligible or inactive antioxidant activities. In other cases, compounds containing high HOMO energy levels also provide high antioxidant activity. The thought-provoking point of our results is that theoretical descriptors of the HOMO-LUMO energy gap and the highest occupied molecular orbital energy are important descriptors in the bioorganic research to support the biological experiments.

Novel Schiff bases derived from N-aryl maleimide derivatives as an effective antimicrobial agent: Theoretical and experimental approach.

A set of new Schiff bases of N-aryl 3- and 4-substituted maleimides has been prepared via condensation of N-aryl 3- and 4- substituted maleimides with p-toluene sulfonyl hydrazide in acidic medium at room temperature. The structures of synthesized compounds were characterized by IR, 1H NMR, 13C NMR, MS spectral data, and further confirmed by single-crystal x-ray crystallography for 5c. The computational study was carried out using Gaussian 09 software by using the B3LYP/6-311+G(d,p) basis set. Single-crystal study results showed much closeness with computational study results. These novel compounds were screened for their antimicrobial activity against two pathogenic bacteria such as Escherichia coli (ATCC 8739) and Staphylococcus aureus (ATCC25923) and two pathogenic fungi such as Aspergillus niger (MCIM10231) and Candida albicans (MTCC6275). The investigation of antimicrobial screening data showed that the most of tested compounds are moderate to good microbial inhibitors.

Thiol-Reactive Clickable Cryogels: Importance of Macroporosity and Linkers on Biomolecular Immobilization.

Macroporous cryogels that are amenable to facile functionalization are attractive platforms for biomolecular immobilization, a vital step for fabrication of scaffolds necessary for areas like tissue engineering and diagnostic sensing. In this work, thiol-reactive porous cryogels are obtained via photopolymerization of a furan-protected maleimide-containing poly(ethylene glycol) (PEG)-based methacrylate (PEGFuMaMA) monomer. A series of cryogels are prepared using varying amounts of the masked hydrophilic PEGFuMaMA monomer, along with poly(ethylene glycol) methyl ether methacrylate and poly(ethylene glycol) dimethacrylate, a hydrophilic monomer and cross-linker, respectively, in the presence of a photoinitiator. Subsequent activation to the thiol-reactive form of the furan-protected maleimide groups is performed through the retro Diels-Alder reaction. As a demonstration of direct protein immobilization, bovine serum albumin is immobilized onto the cryogels. Furthermore, ligand-directed immobilization of proteins is achieved by first attaching mannose- or biotin-thiol onto the maleimide-containing platforms, followed by ligand-directed immobilization of concanavalin A or streptavidin, respectively. Additionally, we demonstrate that the extent of immobilized proteins can be controlled by varying the amount of thiol-reactive maleimide groups present in the cryogel matrix. Compared to traditional hydrogels, cryogels demonstrate enhanced protein immobilization/detection. Additionally, it is concluded that utilization of a longer linker, distancing the thiol-reactive maleimide group from the gel scaffold, considerably increases protein immobilization. It can be envisioned that the facile fabrication, conjugation, and control over the extent of functionalization of these cryogels will make these materials desirable scaffolds for numerous biomedical applications.

Rhodium(III)-Catalyzed C-H Alkenylation: Access to Maleimide-Decorated Tryptophan and Tryptophan-Containing Peptides.

Maleimide is widely applied in many fields, especially in antibody-drug conjugations and peptide drugs. Herein, we develop a strategy for the C-H alkenylation of tryptophan and tryptophan-containing peptides, providing a synthetic route of decorating maleimide on peptides. The method has a high tolerance of functional groups and protecting groups. Furthermore, this method was applied to prepare peptide conjugation with molecules such as drugs and fluorescence probes. Moreover, macrocyclic peptides were obtained via this reaction.

One-pot construction of functionalized aziridines and maleimides via a novel pseudo-Knoevenagel cascade reaction.

An Ugi, novel pseudo-Knoevenagel, ring expansion cascade reaction was discovered and utilized for the synthesis of aziridinyl succinimides in one-pot. Subsequently, densely functionalized aziridines and maleimides have been designed and synthesized through similar cascade reactions. The target compounds were prepared by means of a mild reaction and a simple operation procedure, which could be applicable to a broad scope of starting materials. This series of novel cascade reactions generates opportunities for the tailored synthesis of a wide range of biologically active scaffolds through tuneable Ugi inputs. Discovery of compound 8i with comparable potency to sorafenib in liver cancer cell lines could provide a new avenue for liver cancer drug discovery.

Highly selective isomer fluorescent probes for distinguishing homo-/cysteine from glutathione based on AIE.

Two highly selective OFF-ON isomer fluorescent probes (1 and 2) for homo-/cysteine were designed and synthesized. The pyrene modified tetraphenylethylene derivative with AIE was used as luminescent group while maleimide was used as recognition group. These two isomer probes were found to be nearly nonfluorescent when treated with GSH. However, upon interaction with Cys or Hcy, the fluorescence was enhanced by 2000 folds in a wide pH range from 3 to 10. Experimental results and DFT calculation have demonstrated that the fluorescence OFF-ON switch of such thiol probes is resulted from the termination of the PET (photo-induced electron transfer) effect through the Michael addition reaction of maleimide unit and thiols. In addition, probe 1 and 2 exhibit excellent selectivity and sensitivity towards Cys, Hcy over GSH and other amino acids, which was confirmed by mass MS. We suggested that Michael addition reaction of these probes with GSH was prevented because of the stereo-hindrance effect. Furthermore, these two isomer probes were successfully used for imaging biothiols in living H1299 lung cancer cells.

3-18F-fluoropropane-1-thiol and 18F-PEG4-1-thiol: Versatile prosthetic groups for radiolabeling maleimide functionalized peptides.

The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-18F-fluoropropane-1-thiol and 2-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (18F-fluoro-PEG4 thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix222/K2CO3 conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-18F-fluoropropyl) benzothioate and 18F-fluoro-PEG4 benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]2), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting 18F-fluoride ranged from 6 to 22% in a synthesis time of about 90 min. 18F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]2 maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. 18F-fluoro-PEG4-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to 18F-fluoropropyl-S-E[c(RGDfK)]2. Bone uptake was low for both indicating in vivo stability.

Enantioselective Intermolecular Excited-State Photoreactions Using a Chiral Ir Triplet Sensitizer: Separating Association from Energy Transfer in Asymmetric Photocatalysis.

Enantioselective catalysis of excited-state photoreactions remains a substantial challenge in synthetic chemistry, and intermolecular photoreactions have proven especially difficult to conduct in a stereocontrolled fashion. Herein, we report a highly enantioselective intermolecular [2 + 2] cycloaddition of 3-alkoxyquinolones catalyzed by a chiral hydrogen-bonding iridium photosensitizer. Enantioselectivities as high as 99% ee were measured in reactions with a range of maleimides and other electron-deficient alkene reaction partners. An array of kinetic, spectroscopic, and computational studies supports a mechanism in which the photocatalyst and quinolone form a hydrogen-bonded complex to control selectivity, yet upon photoexcitation of this complex, energy transfer sensitization of maleimide is preferred. The sensitized maleimide then reacts with the hydrogen-bonded quinolone-photocatalyst complex to afford a highly enantioenriched cycloadduct. This finding contradicts a long-standing tenet of enantioselective photochemistry that held that stereoselective photoreactions require strong preassociation to the sensitized substrate in order to overcome the short lifetimes of electronically excited organic molecules. This system therefore suggests that a broader range of alternate design strategies for asymmetric photocatalysis might be possible.

Preliminary evaluation of 18F­AlF­NOTA­MAL­Cys40­Exendin­4 in rodent heart after myocardial ischemia and reperfusion.

Glucagon­like peptide­1 (GLP­1) and its receptor (GLP­1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non­invasive method for monitoring GLP­1R expression. In the present study, a fluorine­18­labeled aluminum fluoride exendin­4 analog [18F­AlF conjugated with 1,4,7­triazacyclononanetriacetic acid (NOTA)­maleimide (MAL)­Cys40­exendin­4] was synthesized and evaluated in a rat MI/R model for GLP­1R imaging. NOTA­MAL­Cys40­exendin­4 was synthesized by coupling Cys40­exendin­4 with NOTA­MAL. NOTA­MAL­Cys40­exendin­4 was then conjugated with 18F­AlF to obtain 18F­AlF­NOTA­MAL­Cys40­exendin­4. The yield of 18F­AlF­NOTA­MAL­Cys40­exendin­4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP­1R and an appropriate signal­to­noise ratio. At 8 h post­MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post­MI/R compared with 8 h post­MI/R, but still remained higher compared with sham­operated groups (0.06±0.012 %ID/g; n=6). Pre­injected unlabeled exendin­4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F­AlF­NOTA­MAL­Cys40­exendin­4 demonstrated favorable characteristics for GLP­1R imaging following MI/R. PET imaging using 18F­AlF­NOTA­MAL­Cys40­exendin­4 in rodent hearts after MI/R revealed a dynamic pattern of GLP­1R upregulation.

Artificial Viral Capsid Dressed Up with Human Serum Albumin.

Capsid of tomato bushy stunt virus consists of an outer coat protein shell decorated on an internal skeleton comprising a ß-annulus motif. We mimicked this capsid structure with our artificial viral capsid dressed up with protein. We synthesized the ß-annulus peptide bearing a Cys at the C-terminal side and linked it with Cys34 of the human serum albumin (HSA) via a bismaleimide linker. The ß-annulus peptide-HSA conjugate self-assembled into spherical structures of a 50-70 nm size range in the Tris-HCl buffer, with the ζ-potential of assemblies of such conjugate revealing that HSA proteins were displayed on the outer surface of the artificial viral capsid. Interestingly, the critical aggregation concentration (CAC) of the conjugate in the Tris-HCl buffer at 25 °C was approximately 0.01 µM, or 1/2500 lower than that of the unmodified ß-annulus peptides, suggesting that the artificial viral capsids were stabilized via HSA modification. The present strategy of constructing protein nanocapsule by self-assembly of a ß-annulus peptide-protein conjugate is simpler than that of previously reported protein nanocapsules.

In vivo irreversible albumin-binding near-infrared dye conjugate as a naked-eye and fluorescence dual-mode imaging agent for lymph node tumor metastasis diagnosis.

Tumor metastases account for about 90% of cancer-related death, among which lymphatic metastases play a pivotal role. Therefore, high-efficiency sentinel lymph node (SLN) identification is significant for lymph node (LN) metastasis diagnosis in clinic. Herein, a novel in vivo covalent albumin-binding near-infrared (NIR) fluorescent IR820-maleimide conjugate (IR-Mal) is firstly designed as a SLN dual-mode imaging agent. The IR-Mal conjugate exhibits bright blue appearance and its large Stokes shift (over 100 nm) increases the fluorescent imaging resolution effectively. The fluorescence intensity of covalent albumin-binding IR-Mal (BSA-IR-Mal) complex is considerably stronger than that of IR-Mal. In vivo, IR-Mal could rapidly covalently bind the tissue interstitial albumin following subcutaneous administration and BSA-IR-Mal complexes could specifically accumulate on LN, and detect both normal and metastatic SLN through naked-eye and fluorescence imaging with high resolution. Moreover, the light stability and enhanced fluorescence intensity of BSA-IR-Mal complex facilitates its diagnosis accuracy. These findings suggest that such in vivo irreversible albumin-binding fluorescence conjugates could serve as a new agent for dual-mode imaging and have a great potential to be applied in the SLNs imaging and diagnosis.

In situ chemically crosslinked injectable hydrogels for the subcutaneous delivery of trastuzumab to treat breast cancer.

Recently, novel approaches for the delivery of therapeutic antibodies have attracted much attention, especially sustained release formulations. However, sustained release formulations capable of carrying a high antibody load remain a challenge for practical use. In this study, a novel injectable hydrogel composed of maleimide-modified γ-polyglutamic acid (γ-PGA-MA) and thiol end-functionalized 4-arm poly(ethylene glycol) (4-arm PEG-SH) was developed for the subcutaneous delivery of trastuzumab. γ-PGA-MA and 4-arm PEG-SH formed a hydrogel through thiol-maleimide reactions, which had shear-thinning properties and reversible rheological behaviors. Moreover, a high content of trastuzumab (>100 mg/mL) could be loaded into this hydrogel, and trastuzumab demonstrated a sustained release over several weeks through electrostatic attraction. In addition, trastuzumab released from the hydrogel had adequate stability in terms of its structural integrity, binding bioactivity, and antiproliferative effect on BT-474 cells. Pharmacokinetic studies demonstrated that trastuzumab-loaded hydrogel (Her-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, and 10 mg/mL trastuzumab) and trastuzumab/Zn-loaded hydrogel (Her/Zn-hydrogel-10, composed of 1.5% γ-PGA-MA, 1.5% 4-arm PEG-SH, 5 mM ZnCl2, and 10 mg/mL trastuzumab) could lower the maximum plasma concentration (Cmax) than the trastuzumab solution. Furthermore, Her/Zn-hydrogel-10 was better able to release trastuzumab in a controlled manner, which was ascribed to electrostatic attraction and formation of trastuzumab/Zn nanocomplexes. In a BT-474 xenograft tumor model, Her-hydrogel-10 had a similar tumor growth-inhibitory effect as that of the trastuzumab solution. By contrast, Her/Zn-hydrogel-10 exhibited a superior tumor growth-inhibitory capability due to the functionality of Zn. This study demonstrated that this hydrogel has potential as a carrier for the local and systemic delivery of proteins and antibodies. STATEMENT OF SIGNIFICANCE: Recently, novel sustained-release formulations of therapeutic antibodies have attracted much attention. However, these formulations should be able to carry a high antibody load owing to the required high dose, and these formulations remain a challenge for practical use. In this study, a novel injectable chemically cross-linked hydrogel was developed for the subcutaneous delivery of trastuzumab. This novel hydrogel possessed ideal characteristics of loading high content of trastuzumab (>100 mg/mL), sustained release of trastuzumab over several weeks, and maintaining adequate stability of trastuzumab. In vivo studies demonstrated that a trastuzumab-loaded hydrogel possessed the ability of controlled release of trastuzumab and maintained antitumor efficacy same as that of trastuzumab. These results implied that a γ-PGA-MA and 4-arm PEG-SH-based hydrogel has great potential in serving as a carrier for the local or systemic delivery of therapeutic proteins or antibodies.

Synthesis and biological evaluation of 3-aryl-4-indolyl-maleimides as potent mutant isocitrate dehydrogenase-1 inhibitors.

A series of 3-aryl-4-indolylmaleimide IDH1/R132H inhibitors with a novel structure was obtained by high-throughput screening and structure-based optimization. Most compounds such as 7a, 7d, 7h, 7i, 7k and 7o showed high inhibitory effects on IDH1/R132H and were highly selective against IDH1/WT, IDH2/WT, GDH, GK, and FBP. Evaluation of the biological activities and function at cellular level showed that compounds 7h, 7i and 7k could effectively suppress the production of 2-hydroxyglutaric acid in U87MG cells expressing IDH1/R132H. Additionally, 7h could reversed the differentiation block of the myeloid leukemic cell line, TF-1, caused by the overexpression of IDH1/R132H. We also explore the structure-activity relationship based on the experimental data, with an attempt to pave the way for future studies.

Synthesis and Characterization of the Selective, Reversible PKCß Inhibitor (9 S)-9-[(Dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, Ruboxistaurin (LY333531).

The demonstrated role of PKCß in  mediating amphetamine-stimulated dopamine efflux, which regulates amphetamine-induced dopamine transporter trafficking and activity, has promoted the research use of the selective, reversible PKCß inhibitor (9 S)-9-[(dimethylamino)methyl]-6,7,10,11-tetrahydro-9 H,18 H-5,21:12,17-dimethenodibenzo[ e,k]pyrrolo[3,4- h][1,4,13]oxadiazacyclohexadecine-18,20(19 H)-dione, ruboxistaurin. Despite the interest in development of ruboxistaurin as the mesylate monohydrate (Arxxant) for the treatment of diabetic retinopathy, macular edema, and nephoropathy, several crucial details in physicochemical characterization were erroneous or missing. This report describes the synthesis and full characterization of ruboxistaurin free base (as a monohydrate), including X-ray crystallography to confirm the absolute configuration, and of the mesylate salt, isolated as a hydrate containing 1.5 mol of water per mole.