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Photoimmunotherapy (PIT) combines the specificity of antibodies with the cytotoxicity of light activatable photosensitizers (PS) and is a promising new cancer therapy. We designed and synthesized, in a highly convergent manner, the silicon phthalocyanine dye WB692-CB2, which is novel for being the first light-activatable PS that can be directly conjugated via a maleimide linker to cysteines. In the present study we conjugated WB692-CB2 to a humanized antibody with engineered cysteines in the heavy chains that specifically targets the prostate-specific membrane antigen (PSMA). The resulting antibody dye conjugate revealed high affinity and specificity towards PSMA-expressing prostate cancer cells and induced cell death after irradiation with red light. Treated cells exhibited morphological characteristics associated with pyroptosis. Mechanistic studies revealed the generation of reactive oxygen species, triggering a cascade of intracellular events involving lipid peroxidation, caspase-1 activation, gasdermin D cleavage and membrane rupture followed by release of pro-inflammatory cellular contents. In first in vivo experiments, PIT with our antibody dye conjugate led to a significant reduction of tumor growth and enhanced overall survival in mice bearing subcutaneous prostate tumor xenografts. Our study highlights the future potential of the new phthalocyanine dye WB692-CB2 as PS for the fluorescence-based detection and PIT of cancer, including local prostate tumor lesions, and systemic activation of anti-tumor immune responses by the induction of pyroptosis.

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BACKGROUND/AIM: Breast cancer (BC) is the most common malignant disease worldwide. Localized stages of BC can be successfully treated by surgery. However, local recurrence occurs in about 4-10% of patients, requiring systemic treatments that impair the patients' quality of life and shortens life expectancy. Therefore, new therapeutic options are needed, which can be used intraoperatively and contribute to the complete removal of residual tumor cells in the surgical area. In the present study, we describe a cysteine-modified variant of the anti-HER2 antibody trastuzumab, that was coupled to the silicon phthalocyanine photosensitizer dye WB692-CB1 for the photoimmunotherapy (PIT) of BC. MATERIALS AND METHODS: The cysteine modified trastuzumab variant was cloned and expressed in Expi293F cells. After purification via immobilized affinity chromatography, the antibody was coupled to the dye. Cell binding of the antibody and the antibody dye conjugate was measured by flow cytometry. After incubation of BC cells with the conjugate and activation of the dye by irradiation with red light, cell viability was determined. RESULTS: The antibody and the conjugate showed specific binding to HER2-expressing BC cells. Treatment of the HER2high BC cell line SK-BR-3 with the conjugate followed by irradiation with a red light dose of 32 J/cm2 led to complete cell killing within 24 h. CONCLUSION: Our novel antibody dye conjugate represents a promising candidate for intraoperative treatment of localized BC, aiming to eliminate residual tumor cells in the surgical area and potentially reduce local recurrence, thereby improving recovery prospects for BC patients.

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BACKGROUND/AIM: The standard treatment for localized prostate cancer involves surgical removal of the prostate with curative intent. However, when tumor cells persist in the operation site, there is high risk of local recurrence and tumor spread, leading to stressful follow-up treatments, impaired quality of life, and reduced overall survival. This study examined photoimmunotherapy (PIT) as a new treatment option for prostate cancer cells. MATERIALS AND METHODS: We generated conjugates consisting of either a humanized antibody or Fab fragments thereof targeting the prostate specific membrane antigen (PSMA), along with our silicon phthalocyanine photosensitizer dye WB692-CB1. PSMA-expressing prostate cancer cells were incubated with the antibody dye or Fab dye conjugates and cell binding was measured using flow cytometry. Cells were irradiated with varying doses of red light for dye activation, and cytotoxicity was determined by erythrosin B staining and subsequent analysis using a Neubauer counting chamber. RESULTS: Specific cytotoxicity was induced with the antibody dye conjugate in the prostate cancer cells in a light dose-dependent manner. Treatment of the cells with the Fab dye conjugate resulted in lower cytotoxicity, which could be attributed to a reduced binding affinity and a reduced dye uptake of the Fab fragment. CONCLUSION: Our new antibody dye and Fab dye conjugates offer potential for future intraoperative PIT in patients with localized prostate cancer, with the aim to ensure complete removal of tumor cells from the surgical area, to avoid local recurrence, and to improve clinical outcome.

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BACKGROUND/AIM: Although there are curative treatment options for non-muscle-invasive bladder cancer, the recurrence of this tumor is high. Therefore, novel targeted therapies are needed for the complete removal of bladder cancer cells in stages of localized disease, in order to avoid local recurrence, to spare bladder cancer patients from stressful and expensive treatment procedures and to increase their quality of life and life expectancy. This study tested a new approach for the photoimmunotherapy (PIT) of bladder cancer. MATERIALS AND METHODS: We generated a cysteine modified recombinant version of the antibody cetuximab targeting the epidermal growth factor receptor (EGFR) on the surface of bladder cancer cells. Then, we coupled the novel photoactivatable phthalocyanine dye WB692-CB1 via a maleimide linker to the free cysteines of the antibody. PIT was performed by incubating bladder cancer cells with the antibody dye conjugate followed by irradiation with visible red light. RESULTS: The conjugate was able to induce specific cytotoxicity in EGFR-positive bladder cancer cells in a light dose-dependent manner. Enhanced cytotoxicity in RT112 bladder cancer cells was evoked by addition of a second antibody dye conjugate targeting HER2 or by repeated cycles of PIT. CONCLUSION: Our new antibody dye conjugate targeting EGFR-expressing bladder cancer cells is a promising candidate for the future PIT of bladder cancer patients.

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Starting from ortho-bromostyrenes, 12 6,7-benzotropolones were synthesized in 3 or 4 steps. Br/Li exchanges provided the respective ortho-lithiostyrenes. The latter were acylated by a Weinreb amide, if unhindered, or hydroxyalkylated by an aldehyde, if hindered (an oxidation ensuing thereafter). Acetonide-containing benzannulated nonatrienones resulted. Ring-closing olefin metatheses converted them into acetonide-containing benzocycloheptadienones. Treatment with aq. HCl caused hydrolyses followed by ß-elimination reactions giving 6,7-benzotropolones.

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The 5S, 8'R, and 10'R configurations of militarinone B (3), which is a natural product from Paecilomyces militaris, should equal those in its biosynthetic precursor, militarinone C. The configuration at C-1' emerged from syntheses of the militarinone B candidates 1''S- and 1''R-(5S,8'R,10'R)-3 from the building blocks 9, 11, 14, and 15a while introducing TMB as a more acid-labile N-protecting group for ß-ketoamides than DMB. Comparisons of 1''S- and 1''R-(5S,8'R,10'R)-3 with natural militarinone B (3; reisolated from Nature) revealed identity versus distinctness.

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The first stereoselective total synthesis of the dimeric naphthoquinonopyrano-γ-lactone (-)-crisamicin A was realized (13 steps, 5% overall yield). 1,4,5-Trimethoxynaphthalene, reached in five known steps, was brominated at C-3 to install a but-3-enoic ester by an ensuing Heck coupling. An asymmetric Sharpless dihydroxylation followed and gave a ß-hydroxy-γ-lactone with >99.9% ee. Its OH substituent and acetaldehyde established the dihydropyran ring in a completely diastereoselective oxa-Pictet-Spengler cyclization. The 2,3-fused anisole moiety allowed the C5-H bond under Hartwig's conditions to be borylated. This set the stage for engaging the resulting C5-B bond in an oxidative dimerization, which led to a binaphthohydroquinon-5-yl. The latter was advanced to synthetic crisamicin A by a double CAN oxidation (→ a binaphthoquinon-5-yl) and a double demethylation.

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The (polyenoyl)tetramic acid militarinone C (1) heads a family of seven members. Before our work, the configuration of C-5 was unknown whereas the configurations of C-8' and C-10' were either (R,R) or (S,S). We synthesized the four stereoisomers of constitution 1, which conform with these insights. This included cross-coupling both enantiomers of the western building block (8) with both enantiomers of the eastern building block (9). The specific rotations of the resulting 1 isomers suggested that natural 1 is configured like the coupling partners (S)-8 and (R,R)-9. This conclusion was corroborated by degrading natural 1 to alcohol 35 and by proving its configurational identity with synthetic (R,R)-35.

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An asymmetric methanolysis of glutaric anhydride and 6 ensuing steps gave veratrol-annulated dimethylcyclo-heptenone diastereomers with 99% ee; ring closures occurred by Friedel-Crafts acylations of carboxylic acids obtained by stereospecific hydrogenolyses of a pair of diastereomeric δ-lactones. The mentioned cycloheptenones and Ph-NH-NH2 underwent Fischer indole syntheses providing the tetracyclic indoles cis- and trans-14a, respectively. Double lithiations with BuLi and quenchings with ClPPh2 furnished the diphosphanes cis- and trans-15 with perfect (P)- and (M)-atropselectivity, respectively.

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On the 1 H NMR timescale, 2,2'-biindolyls with (R)-configured (1-alkoxyprop)-2-yl, (1-hydroxyprop)-2-yl, or (1-siloxyprop)-2-yl substituents at C-1 and C-1' are atropisomerically stable at <0 °C and interconvert at >30 °C. A 2,2'-biindolyl (R,R)-17 a of that kind and achiral (!) brominating reagents gave the atropisomerically stable 3,3'-dibromobiindolyls (M)- and/or (P)-18 a at best atropselectively-because of point-to-axial asymmetric inductions-and atropdivergently, exhibiting up to 95 % (M)- and as much (P)-atropselectivity. This route to atropisomerically pure biaryls is novel and should extend to other substrates and/or different functionalizations. The dibromobiindolyls (M)- and (P)-18 a furnished the biindolyldiphosphanes (M)- and (P)-14 without atropisomerization. These syntheses did not require the resolution of a racemic mixture, which distinguishes them from virtually all biaryldiphosphane syntheses known to date. (M)- and (P)-14 acted as ligands in catalytic asymmetric allylations and hydrogenations. Remarkably, the ß-ketoester rac-25 c was hydrogenated trans-selectively with 98 % ee; this included a dynamic kinetic resolution.

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tert-Butyl 2,5-diaryl-3-oxopent-4-ynoates, obtained from arylacetylenes and the acid chloride of tert-butyl 2-phenylmalonate, represent strongly enolized ß-ketoesters. Their C≡C bonds were activated by Ag(I) salts so that de- tert-butylating π-cyclizations occurred. The latter followed a 6- endo- dig mode giving 3,6-diaryl-4-hydroxy-2-pyrones, or a 5- exo- dig mode giving ( Z)-configured 2-aryl-4-(arylmethylidene)tetronic acids ("pulvinones"). Perfectly selective pyrone formations were induced by AgSbF6 in methanol and equally selective pulvinone formations by Ag2CO3 and DABCO in acetonitrile.

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Hexahydropyridazines with CH2 PAr2 groups at both N atoms are newly designed 1,4-diphosphanes and were synthesized for the first time. Their N atoms assume a single configuration under the influence of stereocenters at C-5 and C-6. In the solid state, these N-atoms bind the CH2 PAr2 substituents axially. Combined with Pd0 , N,N'-chiral diphosphanes of this kind catalyzed Tsuji-Trost type allylations of dialkyl malonates with racemic 1,3-diphenylallyl acetate efficiently and with up to 91 % ee.

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The Cs2CO3-mediated annulations ("Deslongchamps annulations") of three spirocyclic benzoquinone monoketals 5b-d with an ester or acyl substituent at C-2 to two tert-butyl esters of γ,δ-unsaturated ß-ketocarboxyl acids ("Nazarov reagents"; 2a,b) were monitored 1H NMR spectroscopically. This revealed that a primary product, by all likelihood the Michael adduct, forms fast and prior to the appearance of the Deslongchamps adduct. These primary products form reversibly. This was proved by two crossover and four scavenging experiments. Therein, components already incorporated in one of the mentioned primary products ended up in Deslongchamps adducts different from the one, which would have resulted if the respective primary product had reacted alone. However, these experiments leave open whether our primary products are intermediates en route to Deslongchamps products or whether they represent dead ends.

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1,1'-Biphenyl-2,2'-diphosphanes with an achiral bridge spanning C-5 and C-5' form atropisomers that are enantiomers. Accessing them in an atropisomerically pure form requires resolving a racemic mixture thereof or of a bis(phosphane oxide) precursor. 1,1'-Biphenyl-2,2'-diphosphanes with a homochiral bridge spanning C-5 and C-5' form atropisomers that are diastereomers. We synthesized the first compound of this kind 1) atropselectively and 2) under thermodynamic control-seemingly a first-time exploit in diphosphane synthesis. The selectivity-inducing step was a high-temperature reduction of two non-interconverting bis(phosphane oxide) atropisomers (60:40 mixture). It furnished the desired diphosphane atropisomerically pure (and atropconvergently because the yield was 67 %). This diphosphane proved worthwhile in Tsuji-Trost allylations, the Hayashi addition of phenylboronic acid to cyclohexenone, and the asymmetric hydrogenation of methyl acetoacetate (up to 95 % yield and 95 % ee).

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The kodaistatins A-D are strongly anti-diabetic natural products from Aspergillus terreus that hold some promise of a novel diabetes cure. However, considerations of that kind face two drawbacks: 1) The kodaistatins A-D contain a heavily substituted pulvinone/cyclopentenone combination; 2) they are 1,2-diols, the 3D structures of which have not been assigned yet. However, we can exclude two of the four possible stereostructures. We conclude that kodaistatin A is a trans-, not a cis-diol from NMR comparisons with a pair of cis, trans-isomeric kodaistatin models, which we synthesized in 11 and 12 steps, respectively. The stereocenters of the diol moiety arose from stereocomplementary, highly diastereoselective aldol additions of a lithium enolate or the corresponding silyl ketene acetal. The cyclopentenone moieties stemmed from intramolecular aldol additions and ensuing dehydrations. The requisite enolates were obtained by the reduction of α-bromoketones with samarium diiodide.

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We have accomplished the first total synthesis of an isomerically pure naphthoquinonopyrano-γ-lactone dimer, γ-actinorhodin, in eleven steps. Two steps exploit pairs of peri-MeO groups as unusual selectivity controls. The respective MeO groups convey the steric bulk of a bromo or iodo substituent located ortho to one MeO group as steric hindrance into the vicinity of the second MeO group. This relay effect was indispensable for exerting regiocontrol in an aromatic bromination and diastereocontrol in an oxa-Pictet-Spengler cyclization. The absolute configuration of our target compound was established in an asymmetric Sharpless dihydroxylation of a ß,γ-unsaturated ester, which was synthesized in a Heck coupling of a bromoiodonaphthalene with ethyl vinylacetate. The dihydroxylation provided the γ-hydroxylactone moiety of the bromonaphthalene that was used as the substrate in the oxa-Pictet-Spengler cyclization. Dimerization to the core of γ-actinorhodin occurred by two Suzuki couplings.

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Type-18 or -23 benzocycloheptadienones are readily prepared by ring-closing olefin metatheses. Adding Br2 to 23 and eliminating HBr gave the bromoolefin 28 using DBU or its isomer iso-28 using DABCO, both with near-perfect regiocontrol. Both 28 and iso-28 underwent Sonogashira, Suzuki, Negishi, and Heck couplings as well as Pd-catalyzed alkoxycarbonylations. Hydrolysis of the resulting α-ketoketals and enolization of the liberated α-diketones delivered a portfolio of hitherto unknown 3,4-benzotropolones. The 8-ethoxycarbonylated dimethyl-3,4-benzotropolone 50 obtained by this route was dimethylated to give goupiolone A (52). This synthesis encompasses 9 steps from 22, that is, half as many as the only previous synthesis (19 steps). A variant of our route afforded the 1,8-dibromide 54. Coupling with excess phenylboronic acid and ketal hydrolysis provided the diphenylated benzotropolone 56 and suggests a strategy, by which the natural bispulvinone aurantricholone (7) might be reached.

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At −78 °C, primary dialkylmagnesium compounds reacted with diarylsulfoxides when 1.5 equiv of the dilithium salt of (S)-BINOL was added as a promotor. Alkyl aryl sulfoxides resulted in up to quantitative yield and with up to 97% ee. This demonstrates the feasibility of asymmetric sulfinylations by achiral sulfinylating agents (from the perspective of Alkyl2Mg) as well as the feasibility of asymmetric sulfoxide­magnesium exchanges (from the perspective of Ar2SO).

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We synthesized the dextrorotatory enantiomers of gregatin B (1.3 g scale) and E, establishing their diene side chains in the last step by Heck coupling with variable iodoolefins. Their counterpart was synthesized in 30% overall yield and with high enantiopurity (97% ee) from benzyl tiglate, beginning with an asymmetric dihydroxylation. The stereostructure of gregatin E followed from HPLC comparisons between the four synthetic stereoisomers of this compound and natural gregatin E, which we isolated from Cadophora gregata.

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40 years ago spectroscopy, derivatization, and degradation revealed the structures of α-lipomycin and its aglycon ß-lipomycin except for the configurations of their side-chain stereocenters. We synthesized all relevant ß-lipomycin candidates: the (12R,13S) isomer has the same specific rotational value as the natural product. By the same criterion the (12R,13S)-configured D-digitoxide is identical to α-lipomycin. We double-checked our assignments by degrading α- and ß-lipomycin to the diesters 33 and 34 and proving their 3D structures synthetically.