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The chemistry of gem-dibromovinyl derivatives has undergone a renaissance through the application of palladium catalysis and has been applied to pyrrole substituted gem-dibromovinyl BODIPY. gem-Dibromovinyl BODIPYs (substituted at either the [Formula: see text]-position of 8-phenyl or the [Formula: see text]-position of the pyrrole rings) were studied for cross-coupling reactions using Sonogashira, Suzuki, Heck and Stille conditions, and with phosphonates and thiols. The assigned structures were supported by MS and 1H NMR, [Formula: see text]C NMR, X-ray diffraction analysis as well as optical spectroscopy. The conjugates were investigated for their absorption, fluorescence and solvatochromic properties in different solvents. Substitution at the [Formula: see text]-position of 8-phenyl derivatives of gem-diethynyl BODIPYs did not induce any shift in the absorption maximum, while the [Formula: see text]-position pyrrole substituted derivatives showed a red shift. Aromatic compounds gave larger red shifts as compared to the aliphatic substituted analogs.

Fluorescence and SPECT/PET imaging are powerful tools currently in use by the scientific community and receiving a great attention for the development of dual-modality imaging agents. BODIPYs are among the most promising candidates to be used for such functions due their excellent absorbance and fluorescence properties as well as their ease of radiolabeling without compromising their biological properties. In this manuscript we present an overview of BODIPY radiolabeling methods and their relevance to the development of multimodality agents.

Cytochrome P450 46A1 (CYP46A1) or cholesterol-24-hydroxylase is responsible for cholesterol metabolism and homeostasis in the human brain. More recently its activity has been linked to brain function and disease. The anti-HIV drug efavirenz activates CYP46A1 at low drug levels while inhibiting the enzyme activity at the high dose used in clinical practice. Synthetic analogs and hydroxylated metabolites of efavirenz enhance CYP46A1 activity, with reduced unwanted enzyme inhibition at higher concentrations. These observations provide a platform for structural modifications of efavirenz to modulate CYP46A1 activity as a therapeutic target of brain disorders such as Alzheimer's disease, for which currently no treatment is available.

This review focuses on progress in the development of different approaches to the design of steroid ([Formula: see text] estrogens, androgens, cholesterol) conjugates with coordination assemblies of metalloporphyrins, phthalocyanines and related complexes. Porphyrins and phthalocyanines have received considerable attention due to their novel composition, intriguing spectroscopic, photophysical, and redox properties, and potential application in light-harvesting and optoelectronic devices. With the development of more efficient imaging and therapeutic applications, these bio-conjugates are evaluated as multimodality agents (PET, fluorescence imaging) to monitor the mechanism of action of biologically active components in living systems and as agents for molecular recognition, oxygen atom transfer and catalysis. The tetrapyrrole components, which can be coupled via covalent and various non-covalent linkages, may exhibit strong interactions through efficient photo-induced electron and/or energy transfer processes.

To develop receptor based fluorescence ligands for imaging breast and prostate cancer, a series of estrogen-, testosterone- and 19-nortestosterone conjugates linked to BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or aza-BODIPY, were prepared. Their synthesis involves attachment of iodo derivatives of differently substituted BODIPY and aza-BODIPY analogs to the C17α-position of the steroid moieties using either the Sonogashira coupling or Click reaction. The UV-Vis absorption spectra of the conjugates range from 500 to 710nm with fluorescence emission properties ranging from 520 to 700nm, facilitating observations in living cells and tissues. Selection of the site of substitution, as well as the type of substituents on the steroidal moiety and the use of different linkers, provides a library of fluorescing conjugates to explore the effect of structural modifications on biological properties.

In vivo imaging of estrogen receptor (ER) densities in human breast cancer is a potential tool to stage disease, guide treatment protocols and follow-up on treatment outcome. Both positron emission tomography (PET) and fluorescence imaging have received ample attention to detect ligand-ER interaction. In this study we prepared BODIPY-estradiol conjugates using 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) as fluorescent probe and estradiol derivatives as ligand and established their relative binding affinity (RBA) for the ERα. The synthesis of the conjugates involves attachment of a BODIPY moiety to the C17α-position of estradiol using Sonogashira or click reactions of iodo-BODIPY or aza-BODIPY with various 17α-ethynylestradiol (EE2) derivatives. The highest RBA for the ERα was observed with the EE2-BODIPY conjugate (7) featuring a linear eight carbon spacer chain. Cell uptake studies and in vivo imaging experiments in an ER-positive mouse tumor model are in progress.

A family of new asymmetric and symmetric 1,3,7,9-tetramethyl-4,4-bora difluoro-diaza-s-indacene (BODIPY) derivatives, bearing gem-dibromovinyl substituents, was synthesized by the Corey-Fuchs olefination method. One or two gem-dibromovinyl moieties were attached at either the p-position of 5-phenyl, or the β-position of the pyrrole ring, directly or, through phenyl spacers. The assigned structures were supported by MS, NMR (1H, 13C, 19F), X-ray diffraction analysis and for some compounds 2D HSQC and 11B NMR as well as optical spectroscopy. Their absorption and fluorescence properties and solvatochromism in different solvents were investigated. The highest absorption and emission maxima were obtained for compounds having two gem-dibromovinyl groups attached directly or through the phenyl spacer. The best correlation (R-coefficient) between the solvent and spectral properties of the BODIPYs were obtained using the refractive index of the solvent. Although these compounds are structurally quite similar, their solid states show remarkable differences in the crystal system, clearly revealing two distinct patterns of gem-dibromovinyl orientation and torsion angles of the 5-phenyl ring and the indacene plane. Hirshfeld surface analysis data were used to visualize various intermolecular interactions.

Steroids linked to porphyrin fluorophores are being developed to improve the selectivity of photosensitizers for photodyanamic therapy and fluorescence imaging of steroid-hormone positive cancers. A number of porphyrin derivatives substituted with one or two steroid moieties coupled via a 17[Formula: see text]-ethynyl group of estradiol, testosterone and 19-nortestosterone were synthesized using Pd(II) as a catalyst.

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 ((64)Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of (64)Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

BODIPY and aza-BODIPY fluorophores linked to steroids are being developed as multimodal-imaging agents to monitor the mechanism of action of biologically active components in living systems.

A series of BODIPYs (boron-dipyrromethene dyes; 4,4-difluoro-5-aryl-4-bora-3a,4a-diaza-s-indacene) derivatives, bearing an aldehyde group at different pyrrole positions, were transformed into gem-dibromovinyl analogs using the Corey-Fuchs or Lautens olefination method. The gem-dibromovinyl moieties (one or two) are symmetric/asymmetric and attached at α- or β-positions of the pyrrole ring, directly or through the extension of a β-position substituted phenyl spacer ring. The molecular structures of the resultant dyes were assigned using MS, 1H, 13C, 19F NMR, X-ray diffraction and for some compounds 2D HSQC and 11B NMR. The absorption, fluorescence and solvatochromism properties were investigated in different solvents. The highest absorption and emission maxima were obtained for compounds having two gem-dibromovinyl groups attached directly at the α-positions. The best correlation (R-coefficient) of absorption between the solvents and spectral properties of the BODIPYs was obtained using the refractive index of the solvent. The R-coefficient of emission was obtained only for compounds having gem-dibromo vinyl moieties at α-positions. Their solid states clearly reveal distinct patterns of gem-dibromovinyl orientation, torsion angles of the 5-phenyl ring and the indacene plane. Hirshfeld surface analyses were used to visualize various intermolecular interactions.

The interaction of primary autoxidation products of cholesterol, 25- and 20ξ-hydroperoxides, with the four principal cholesterol-metabolizing cytochrome P450 enzymes is reported. Addition of cholesterol 25-hydroperoxide to CYP27A1 and CYP11A1 induced well-defined spectral changes while generating 25-hydroxycholesterol as major product along with small amounts of triols. The 20ξ-hydroperoxides induced spectral shifts in CYP27A1 and CYP11A1, yet glycol metabolites were detected only with CYP11A1. CYP7A1 and CYP46A1 failed to give metabolites with any of the hydroperoxides. A P450 hydroperoxide-shunt reaction is proposed, where the hydroperoxides serve both as donor for reduced oxygen and as substrate. For the first time, CYP27A1 is shown to mediate the reduction of cholesterol 25-hydroperoxide to 25-hydroxycholesterol, a role of potential significance for cholesterol-rich tissues with high oxidative stress. CYP27A1 may participate in these tissues in removal of harmful autoxidation products, while providing a complementary source for 25-hydroxycholesterol, a modulator of immune cell function and mediator of viral cell entry.

Cyclotron production of 99mTc is a promising route to supply 99mTc radiopharmaceuticals. Higher 99mTc yields can be obtained with medium-energy cyclotrons in comparison to those dedicated to PET isotope production. To take advantage of this capability, evaluation of the radioisotopic purity of 99mTc produced at medium energy (20–24 MeV) and its impact on image quality and dosimetry was required. Methods: Thick 100Mo (99.03% and 99.815%) targets were irradiated with incident energies of 20, 22, and 24 MeV for 2 or 6 h. The targets were processed to recover an effective thickness corresponding to approximately 5-MeV energy loss, and the resulting sodium pertechnetate 99mTc was assayed for chemical, radiochemical, and radionuclidic purity. Radioisotopic content in final formulation was quantified using γ-ray spectrometry. The internal radiation dose for 99mTc-pertechnetate was calculated on the basis of experimentally measured values and biokinetic data in humans. Planar and SPECT imaging were performed using thin capillary and water-filled Jaszczak phantoms. Results: Extracted sodium pertechnetate 99mTc met all provisional quality standards. The formulated solution for injection had a pH of 5.0−5.5, contained greater than 98% of radioactivity in the form of pertechnetate ion, and was stable for at least 24 h after formulation. Radioisotopic purity of 99mTc produced with 99.03% enriched 100Mo was greater than 99.0% decay corrected to the end of bombardment (EOB). The radioisotopic purity of 99mTc produced with 99.815% enriched 100Mo was 99.98% or greater (decay corrected to the EOB). The estimated dose increase relative to 99mTc without any radionuclidic impurities was below 10% for sodium pertechnetate 99mTc produced from 99.03% 100Mo if injected up to 6 h after the EOB. For 99.815% 100Mo, the increase in effective dose was less than 2% at 6 h after the EOB and less than 4% at 15 h after the EOB when the target was irradiated at an incident energy of 24 MeV. Image spatial resolution and contrast with cyclotron-produced 99mTc were equivalent to those obtained with 99mTc eluted from a conventional generator. Conclusion: Clinical-grade sodium pertechnetate 99mTc was produced with a cyclotron at medium energies. Quality control procedures and release specifications were drafted as part of a clinical trial application that received approval from Health Canada. The results of this work are intended to contribute to establishing a regulatory framework for using cyclotron-produced 99mTc in routine clinical practice.

Various zinc phthalocyanines were substituted at ortho or meta positions with 1,3,5,7-tetramethyl-8-phenyl-4,4-difluoroboradiazaindacene (BODIPY) moieties via Pd catalyzed Sonogashira-coupling reactions, at either meso or β positions of the BODIPY. UV–Vis and fluorescence emission spectra of the conjugates exhibit panchromatic behavior due to their absorption over a broad spectral region from ultraviolet to visible and energy transfer from the excited BODIPY substituent to the phthalocyanine core. Some conjugates show red-shifted Q-bands that correlate to the planarity and dihedral angle between the subunits, as well as the position and number of attached BODIPY and phthalocyanine moieties.

Methods for the preparation of a variety of covalently linked Pc–Pc homo-dimers (ortho and meta), heterodimers and Pc–Pc triads linked through NH and bridged viaN-aryl (phenyl) groups were developed. The palladium catalyzed intermolecular carbon–nitrogen coupling reaction (Buchwald–Hartwig) between Pc-iodo and Pc-amino phthalocyanine derivatives was used for these studies. Photophysical data reveal energy transfer between the Pc moieties resulting in the appearance of new red-shift Q-bands that correlate to the planarity and dihedral angle between the subunits. The shift and the nature of Q-band depend on the number of phenyl groups, the number of Pc and the position of attachment on the phenyl ring.

Several phthalocyanine dimers, trimers, and oligomers bridged via aryl (phenyl) groups were prepared using the Suzuki–Miyaura cross coupling reaction of phthalocyanine-boronate ester and various halide derivatives under palladium catalyst reaction conditions. Photophysical data reveal energy transfer between the Pc moieties resulting in the appearance of new red-shifted Q-bands. The shift and the nature of Q-band depend on the number of phenyl groups, the number of Pc, and the position of attachment on the phenyl ring.

The synthesis of a series of new fluorinated phthalocyanines and porphyrins as potential bimodal probes is evaluated. In these complexes, zinc phthalocyanine functions as a fluorescence imaging moiety while attachment of an aliphatic chain of different lengths bearing a tosylate group permits introduction of fluorine via nucleophilic substitution of the tosylate group. Using short-lived [18F]fluoride gives the analogous 18 F -radiolabelled tracer rendering the bimodal probe suitable for both fluorescence and PET imaging.

Rhodium-catalyzed carbon–carbon bond forming reactions using phthalocyanine-boronate ester were investigated. The reaction of Pc-boronate ester with substituted aldehydes gave 1,2-addition products (secondary alcohols), while α,β-unsaturated carbonyl compounds (aldehydes, ketones, esters, and amides) furnished 1,4-addition products. The reaction of Pc-substituted alkynes with o -formyl and acetyl phenylboronic acid gave 1 H - and 1-methyl inden-1-ol annulation derivatives. All reactions were performed in mixtures of organic–aqueous solvents.

The synthesis of a series of new zinc phthalocyanine-peptide conjugates targeting the gastrin-releasing peptide (GRP) and integrin receptors is reported. Two alternative synthetic methods based on Sonogashira cross-coupling of an iodinated zinc phthalocyanine with acetylenic bombesin or arginine-glycine-aspartic acid (RGD) derivatives, either in solution or on solid phase, are presented. The water-soluble conjugates were screened for their photodynamic efficacy against several cancer cell lines expressing different levels of GRP and integrin receptors, and their intracellular localization was evaluated via confocal fluorescence microscopy. Variations in photocytotoxicity between the conjugates correlate to differences in hydrophobicity as well as receptor-mediated cell uptake. In the case of the phthalocyanine-bombesin conjugate, competition experiments confirm the involvement of the GRP receptor in both the phototherapeutic activity as well as intracellular localization. These findings warrant further in vivo studies to evaluate the potential of this conjugate as photosensitizer for photodynamic therapy (PDT) of cancers overexpressing the GRP receptor.

An easy synthetic method for the preparation of pyrazine-2,3-dicarbonitrile 5,6-bis-substituted derivatives using Pd-catalyzed cross coupling reaction (Sonogashira, Suzuki, Heck) is described. The reaction conditions allow preparing symmetrical and unsymmetrical bis-substituted products in moderate to high yields, as precursors for the preparation of highly substituted symmetrical and non-symmetrical aza-phthalocyanines with well-defined spectral properties.

Sulfonated metallo phthalocyanines (MPcS(n)) are second generation photosensitizers advanced for photodynamic therapy of various medical applications. A series of ZnPcS(n) was demetallated and subsequently converted to the corresponding [(64)Cu]CuPcS(n) in 40-50% isolated yields and >98% radiochemical purities. Tumor-bearing mice were injected with the (64)Cu-labeled products and subjected to 3-h dynamic PET imaging studies. Biodistribution patterns showed characteristic differences between the various derivatives. Tumor uptake was detected only for the amphiphilic derivatives [(64)Cu]CuPcS(2) and [(64)Cu]CuPcS(3)C(6) (1-1.5%ID/g). The biological data suggest that PET imaging with [(64)Cu]CuPc can be used to establish structure-PDT efficacy relationships for Pc-based photosensitizers.

The reaction of Ni (II) complexes of etioporphyrins I, octaethylporphyrins, coproporphyrinsI tetramethylester, methyl pyrroporphyrin XXI ethylester and mesoporphyrin IX dimethyl ester derivatives with N-bromosuccinimide and triethylamine trihydrofluoride gave mono- through tetra-meso-brominated derivatives as the principal products. β-brominated products were also detected with the simultaneous loss of the β-alkyl substituents. Reaction of octaethylporphyrin Cu (II) was slow yielding meso-mono and dibromo derivatives as major products. Prolongation of the reaction time resulted in the formation of tri- and tetrabromo products substituted at both meso- and β-positions. Our results show that the central metal ion plays a major role in these bromination reactions.

Phthalocyanines (Pc) were conjugated with peptide moieties to improve their target selectivity for potential use as fluorescence and/or positron emission tomography (PET) probes in medical imaging. Three synthetic methods based on palladium-catalyzed cross-coupling reactions (Sonogashira, Buchwald-Hartwig, and Suzuki-Miyaura) were investigated. Using these methods, a series of peptides monofunctionalized with Pc at the N/C-terminal position or on a phenylalanine side chain was obtained in good yields and characterized.

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.

The impact of lipophilicity on biological parameters critical to photodynamic efficacy was analyzed for a new generation of trisulfobenzo(mononaphtho)porphyrazines. The porphyrazines were substituted on the naphtho ring with linear alkynyl side chains of various lengths. When compared to the analogous phthalocyanine structures, the added benzo ring in the porphyrazine structures increased the lipophilicity for analogs with short alkynyl chains, while this effect disappeared for analogs with longer side chains. In aqueous media, the analogous phthalocyanine series showed aggregation tendencies. In contrast, no correlation between aggregate formation and the length of the alkynyl side chain was evident in the porphyrazine series. At low concentrations, the length of the side chain did not affect cell uptake, while phototoxicity towards EMT-6 mouse tumour cells showed a parabolic relationship, where the hexynyl derivative showed the highest activity. The trisulfonated porphyrazines localized at intracellular organelles, plasma and perinuclear membranes, but could not be found in the nucleus. Total cell uptake of dye did not correlate with phototoxicity, suggesting that localization in certain intracellular organelles, and distribution into critical intracellular sites are important determinants of their photodynamic activity. The hexynyl trisulfonated zinc porphyrazine derivative (ZnNPcS(3)C(6)) showed the strongest in vitro photodynamic activity and therefore was further studied in an EMT-6 mouse tumour model. An i.v. dose of 1 micromol of ZnNPS(3)C(6) per kg, followed 24 h later by activation with light, induced 100% tumour necrosis within 24 h post-PDT. This treatment delayed tumour volume doubling time from 5 days to2 weeks, and gave 41% tumour cure3 weeks post-PDT. Applying the same light dose fractionated (5 min on, 2 min off), further improved tumour response, leading to a doubling time of 26 days and a 73% tumour cure. At the administered 1 micromol kg(-1) dye dose, no skin phototoxicity was observed and90% blood clearance was observed within 5 h post-injection. Compared to the analogous trisulfo monohexynyl zinc phthalocyanine, the new trisulfobenzo(mononaphthohexynyl)porphyrazine provided a broader range of excitation wavelengths, and improved photodynamic potency, while apparently being free of unwanted systemic side effects.

An efficient one-step method for the synthesis of covalently linked phthalocyanine–porphyrin (Pc–Por) hetero-dyads and Pc–Por–Pc hetero-triads, connected directly through C–C bonds, is presented. The procedure utilizes a Pd-mediated Suzuki cross-coupling reaction of a Pc–boronate synthon and a halogenated porphyrin. The two chromophores are directly linked, without spacer, through the β-pyrrolic-, meso-, and para-position of the meso-phenyl group of the porphyrins. The electronic absorption spectrum of the Pc–Por–Pc triad shows red-shifted, split Q-bands reflecting energy transfer.

The purpose of the research is to study the effect of acute inhibition of intravascular lipolysis on myocardial substrate selection during hypertriglyceridemia using in vivo radiotracer analysis and positron emission tomography.We induced acute hypertriglyceridemia in vivo using an intravenous infusion of Intralipid 20% (IL) without and with acute inhibition of fatty acid delivery from circulating triglycerides with injection of Triton WR-1339 (TRI) during a euglycemic-hyperinsulinemic clamp in Wistar rats. We determined the effect of TRI on myocardial uptake of circulating triglycerides and free fatty acids using intravenous injection of [(3)H]-triolein and [(14)C]-bromopalmitate, respectively. Myocardial blood flow, oxidative metabolism, and metabolic rate of glucose (MMRG) were determined using micro-positron emission tomography (microPET) with [(13)N]-ammonia, [(11)C]-acetate, and 2-deoxy-2-[F-18]fluoro-D: -glucose (FDG).TRI reduced myocardial incorporation of [(3)H]-triolein but not [(14)C]-bromopalmitate showing that it selectively reduces myocardial fatty acid delivery from circulating triglycerides but not from free fatty acids. IL reduced myocardial blood flow and MMRG by 37% and 56%, respectively, but did not affect myocardial oxidative metabolism. TRI did not abolish the effect of IL on myocardial blood flow and MMRG.Hypertriglyceridemia acutely reduces myocardial blood flow and MMRG in rats, but this effect is not explained by increased myocardial fatty acid delivery through intravascular triglyceride lipolysis.

The biodistribution and tumour uptake of a series of 16alpha-[(18)F]fluoroestradiol ([18F]FES) derivatives was determined in oestrogen receptors-positive (ER+) tumour-bearing mice to assess the impact of substituents, formulation and specific activity on target tissue uptake.MC4-L2 and MC7-L1 murine ER+ cells were inoculated in Balb/c mice. The animals were injected with various [(18)F]FES derivatives substituted with 2- or 4-fluorine and/or an 11beta-methoxy group. The radiopharmaceuticals were formulated in 10% ethanol/saline or 10% ethanol/lipid emulsion. The organs were counted, and radioactivity concentrations were expressed as the percentage of the injected dose per gram tissue (%ID/g). To estimate the effect of specific activity on tumour uptake, the 4-fluoro-11beta-methoxy-16alpha-[(18)F]-fluoroestradiol (4F-M[(18)F]FES) was co-injected with different concentrations of non-radioactive estradiol to give an in vivo competitive inhibition curve.4F-M[(18)F]FES exhibited the highest average uterine uptake (%ID/g = 15.7 +/- 2.1). The highest uptake by the two mammary tumours was observed with [(18)F]FES (%ID/g = 3.1 and 3.4 +/- 0.3) and 11beta-methoxy-16alpha[(18)F]-fluoroestradiol (M-[(18)F]FES) (%ID/g = 3.2 and 3.3 +/- 0.6), followed by 4F-M[(18)F]FES (%ID/g = 2.5 and 2.3 +/- 0.3). The formulation had little influence on the biodistribution pattern. Co-injection with a total mass of estradiol10(-10) mol blocked 4F-M[(18)F]FES tumour uptake.All of the radiolabelled estradiol derivatives achieved significant target tissue uptake in vivo, both in ER+ tumours and the uterus. The formulation had little impact on the biodistribution of these compounds but some compounds (4F-M[(18)F]FES, M-[(18)F]FES and [(18)F]FES) had more favourable target tissue uptake and target-to-background ratios.

Brominated derivatives of tetrapyridinium copper porphyrin were prepared via bromination of the β-positions (pyrrole rings) and/or the peripheral alkyl side-chains attached to the pyridine moieties. The radiosensitizing properties of these new cationic, brominated porphyrins were tested on MDA-MB-231 breast cancer cells in vitro using a 60 Co source or an X-ray irradiator. The non-brominated porphyrin and the porphyrin containing bromines at β-positions only were devoid of any radiosensitizing activity. However, a pronounced radiosensitizing effect was observed with the porphyrin containing bromo atoms at both β-positions and the peripheral side-chains. A similar radiosensitizing effect was detected for different radiation energies, suggesting that high energy photons could be used to treat tumors in conjunction with this novel brominated, porphyrin-based radiosensitizer.

Sequential two-photon (2-gamma) activated copper tetrasulfophthalocyanine (CuPcS(4)) was shown capable of inactivating acetylcholinesterase (ACE). ACE activity was measured photometrically by the Ellman method. Simultaneous irradiation of ACE in the presence of CuPcS(4) with 514 nm (183 mW/cm(2)) and 670 nm (86 mW/cm(2)) continuous wave (CW) light induced a 20-50% increase in enzyme inhibition as compared to one-photon (1-gamma) irradiation, using either 514- or 670-nm (CW) light at the same fluences. The enzyme activity was not affected by CuPcS(4) or light alone, decreased linearly with the irradiation time, and was shown to be oxygen-dependent. We conclude that the photoinactivation of ACE with sequential 2-gamma irradiation involves reactive oxygen species produced by the interaction of the upper excited T(n) state of CuPcS(4) with molecular oxygen. As CuPcS(4) shows little activity as a conventional 1-gamma photosensitizer, unwanted side effects such as prolonged skin sensitivity are eliminated rendering 2-gamma photodynamic therapy advantageous for the treatment of selected medical applications.

The stereoselective synthesis of (Z)-β-iodovinyl ketones/esters of Morita-Baylis-Hillman (MBH) porphyrin adducts has been achieved with a tandem formation of C - C and C - I bonds in a three-component reaction. The reaction is promoted by MgI 2 as a Lewis acid, as well as an iodine source for a Michael-type addition. The MBH adducts are efficiently oxidized to the corresponding ketones with Dess-Martin periodinane (DMP). These scaffolds are further used in a palladium-catalyzed Sonogashira carbon-carbon coupling reaction to obtain highly extended porphyrin systems.

Iodine in isopropenyl acetate (IPA) is a highly efficient catalyst for the acetylation of a variety of alcohols, phenols and amines under solvent free conditions. Primary, secondary, tertiary alcohols, amines and mono to polyhydroxy phenols and anilines with electron donating or withdrawing substituents can be easily acetylated in good to excellent yield at 85–90 °C.

The aim of this study was to determine the effect of hyperinsulinemia on myocardial and hepatic distribution and metabolism of 14(R,S)-[18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA). Mitochondrial retention and intracellular lipid incorporation of [18F]FTHA were compared to that of [14C]-2-bromopalmitate or [14C]palmitate during hyperinsulinemic clamp vs. saline infusion in male Wistar rats. Mitochondrial 18F activity was increased in the heart (1.7 ± 0.4 vs. 0.5 ± 0.1% ID/g, P < 0.05), whereas it was reduced in the liver (1.1 ± 0.3 vs. 1.8 ± 0.4% ID/g, P < 0.05) during insulin vs. saline infusion, respectively. Mitochondrial [14C]-2-bromopalmitate activity was affected by insulin in a similar way in both tissues. The fractional esterification of [18F]FTHA into triglycerides was impaired compared to [14C]palmitate in both tissues, and [18F]FTHA was insensitive to the shift of esterification of fatty acids into complex lipids in response to insulin. [18F]FTHA is sensitive to insulin-induced modifications of free fatty acid oxidative metabolism in rats but is insensitive to changes in nonoxidative fatty acid metabolism.

Silica gel supported InBr 3 or InCl 3 (15–20 mol %) were explored as a new solid-support catalysts for the facile and efficient oxidation, under solvent free conditions, of 2′-hydroxychalcones and flavanones to yield the corresponding flavones in >80% yield. The catalysts are easily prepared, stable, and efficient under mild reaction conditions.

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7α-cyano derivatives of the (17α,20E/Z)-[ 125 I ]iodovinyl-(CIVE) and 16α-[ 125 I ]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17α,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17α-ethynyl group of the 7α-cyano-17α-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [ 125 I ]-CIVE isomers were prepared via the same stereospecific reaction. [ 125 I ]-CIE was prepared from 7α-cyano-16β-bromoestradiol via halogen exchange with Na 125 I . Addition of the 7α-cyano group to 16α-iodoestradiol did not affect estrogen receptor binding affinity (RBA of CIE is 115). However the estrogenic potential of CIE, as measured by the capacity to stimulate the expression of the pS2 gene, was reduced to 1% as compared to that of estradiol. Addition of a 7α-cyano group to the (17α,20E/Z)-IVE isomers reduced the RBA to 21 and 36, respectively, while the estrogenic potential was reduced to 2–3% of that of estradiol. Uterus uptake in immature rats of the 125 I -labeled CIVE 20E-isomer and the 16α-iodo CIE peaked at 0.5 h post injection while the (17α,20Z)-CIVE isomer showed a maximum only past 5 h post injection. Uptake of all three 125 I -labeled 7α-cyanoestrogens was suppressed by the co-injection of non-radioactive estradiol confirming the role of estrogen receptors in the localization process. Uterus retention pattern differ substantially from those of the analogues 7α-methylestrogens, which were previously shown to give high maximum 125 I -uptake values at 2 h post injection. Overall our data indicate that addition of a 7α-cyano group to 123 I -labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.

Photo-excited Zn-phthalocyanine (ZnPc) interactions with four (hydro)peroxides (ROOR′) were studied by T–T absorption decay and fluorescence quenching. Triplet state interactions are weak (logkq∼4) and independent from the quencher used, while singlet quenching is variable (logkq 4.5–9.4). EPR and spin-trapping show that at low ZnPc concentrations the primary decomposition products are alkoxyl radicals. At high ZnPc concentrations, photosensitization of ROOH gives rise to peroxyl radicals. The (hydro)peroxide reducing potentials were estimated following AM1 MO-computations. Our experimental data and theoretical predictions (Marcus–Saveant theory) are consistent with a charge transfer mechanism to account for the alkoxyl radical formation.

We prepared the 2,16α- and 4,16α-difluoroestradiols and their 11β-methoxy derivatives via two different pathways. The first route permits large scale synthesis and characterization of the final products while the second route was selected to allow for fluorination as a final step to facilitate labeling with the short-lived [18F]fluorine. The former route involves successive electrophilic fluorinations of intermediate bistrimethylsilyl enol ethers and 16α-fluoroestrones followed by reduction of the 17-ketone and chromatographic separation of the isomeric products. The second route proceeds via electrophilic substitution of estrone or 11β-methoxyestrone with N-fluoropyridinium salt to give the 2- and 4-fluoro derivatives followed by conversion to the reactive 16β,17β-cyclic sulfates. Stereoselective opening of the cyclic sulfates via nucleophilic fluorination with Me4NF and subsequent removal of the protecting ether and sulfate groups via rapid hydrolysis in acidic ethanol, gave the desired 16α-fluoro derivatives. The latter procedure is readily adapted for radiolabeling with 18F by substituting Me4NF for 18F− in acetonitrile. Preliminary biological data suggest that the addition of both a 4-fluoro and 11β-methoxy group onto 16α-[18F]fluoroestradiol (FES) may provide an improved radiopharmaceutical for positron emission tomography (PET) imaging of estrogen receptor densities in breast cancer patients.

Different pH-sensitive, randomly- and terminally-alkylated N-isopropylacrylamide (NIPAM) copolymers were synthesized and used to prepare pH-responsive polymeric micelles (PM). These copolymers were modified from previously-studied copolymers by incorporating an additional hydrophilic monomer, N-vinyl-2-pyrrolidone (VP) to decrease uptake by the mononuclear phagocyte system (MPS) and improve localization in tumors. VP lowered the phase transition pH of the copolymers but did not affect the onset of micellization. The in vitro cytotoxicity of the copolymers was evaluated on EMT-6 mouse mammary tumor cells in comparison to Cremophor EL (CRM). The anticancer photosensitizer aluminum chloride phthalocyanine (AlClPc) was loaded into the PM with a standard dialysis procedure. Biodistribution and in vivo photodynamic activity were then evaluated in Balb/c mice bearing intradermal EMT-6 tumors. All NIPAM copolymers demonstrated substantially lower cell cytotoxicity than the control surfactant CRM. In vivo, similar AlClPc tumor uptake was observed for the PM and CRM formulations. However, the PM appeared to exhibit greater activity in vivo than CRM formulation at an AlClPc subtherapeutic dose. Therefore, NIPAM-based copolymers containing VP units represent promising alternatives for the formulation of poorly water-soluble phthalocyanines.

Palladium-catalyzed copper-free Sonogashira cross-coupling reactions were performed on a protected and unprotected monoiodotriglycerol substituted Zn(II) phthalocyanine with various alkynes in organic and aqueous medium. From the silyl protected alkyne derivative, a protected homo coupled dimeric product was also prepared using Glaser coupling. All products were purified by reverse phase HPLC and the assigned structures were confirmed by mass and UV–vis spectroscopy.

Dynamic positron emission tomography (PET), combined with constant infusion of 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG), enables real-time monitoring of transient metabolic changes in vivo, which can serve to understand the underlying physiology. Here we investigated characteristic changes in the tumour FDG-uptake profiles in relation to acute localized inflammatory responses induced by photodynamic therapy (PDT). Dynamic PET imaging with constant FDG infusion was used with EMT-6 tumour bearing mice. FDG time-activity uptake curves were measured simultaneously, in treated and reference tumours, for 3 hours, before, during and after PDT light treatment. Inflammation was studied when evoked, either by PDT using a trisulfonated porphyrazine photosensitizer, or lipopolysaccharide (LPS), and inhibited using indomethacin. The distinct transient patterns, characterized by drops and subsequent recovery of tumour FDG uptake rates, were also analysed using immunohistochemical markers for apoptosis, necrosis, and inflammation. Typical profiles for tumour FDG-uptake, consisted of a drop during PDT, followed by a gradual recovery period. Tumours treated with LPS, but not with light, showed a continuous increase in FDG-uptake during the 3 h experimental period. Treatment with indomethacin, inhibited the rise in FDG-uptake observed with either LPS or PDT. Tumour FDG-uptake profiles correlated with necrosis markers during PDT, and inflammatory response markers post-PDT, but not with an apoptosis marker at any time during or after PDT. Dynamic FDG-PET imaging combined with indomethacin reveals that, the drop in the tumour FDG-uptake rate during the PDT illumination phase reflects vascular collapse and necrosis, while the increased tumour FDG-uptake rate immediately post-illumination involves an acute localized inflammatory response. Dynamic FDG infusion and PET imaging, combined with the use of selective inhibitors, provides unique insight for deciphering the complex underlying processes leading to tumour response in PDT, and allows for rapid as well as cost effective optimization of PDT protocols.

Immunophototherapy of cancer combines the specificity of a monoclonal antibody (MAb) to an overexpressed tumor marker with the phototoxic properties of the conjugated dye. To analyze the potential role of internalisation of the dye on photo-induced cytotoxicity, we compared two target antigens, carcinoembryonic antigen (CEA) that does not internalise and ErbB2 that does. Human ovarian carcinoma SKOv3 cells that express a high level of ErbB2 were transfected with the CEA cDNA. Using FACS analysis, the resulting cell line, SKOv3-CEA-1B9, demonstrated comparable levels of expression of the two target antigens. Aluminium tetrasulfophthalocyanine (AlPcS 4) was covalently coupled to anti-CEA MAb 35A7, anti-ErbB2 MAb FSP77 and a non-specific MAb PX, via a five-carbon sulfonamide spacer chain (A 1) at molar ratios ranging from 6 to 9 moles of AlPcS 4 per mole of MAb. The 35A7-(AlPcS 4 A 1)8 conjugate induced 68% growth inhibition of the SKOv3-CEA-1B9 cell line after a 20 h incubation at 2.50 μg/ml (based on AlPcS 4 A 1 content) following light exposure. However, the FSP77-(AlPcS 4 A 1)6 conjugate gave a 51% growth inhibition for an AlPcS 4 A 1 concentration as low as 0.04 μg/ml after the same incubation time and exposure to the same light dose. At a 1.25 μg/ml AlPcS 4 A 1 concentration, the FSP77-(AlPcS 4 A 1)6 conjugate gave a 67% growth inhibition after an incubation time as short as 1 h, reaching a 96% inhibition after an 8 h incubation time. Using an unique cell line that expresses two different target antigens, we demonstrated a clear advantage of an internalising over a non-internalising MAb-dye conjugate in terms of phototoxic efficacy. In vivo evaluation of the photodynamic properties of the conjugates is in progress. © 2001 Cancer Research Campaign http://www.bjcancer.com © 2001 Cancer Research Campaign

The aim of this study was to evaluate the role of low-density lipoproteins (LDLs) as a vehicle for aluminum sulfophthalocyanine ( AlPcS ) to target tumor cells for photodynamic therapy (PDT). LDLs are biological particles containing an apolipoprotein which recognizes with high affinity specific receptors on many cell types, including cancer cells. LDL was loaded with AlPcS in two different manners. In the first procedure, the tetrasulfonated AlPcS4was covalently bound to the protein part of the LDL via amide bonds to 6-carboxypentylaminosulfonyl spacer chains attached to two of the four sulfonate groups, i.e. AlPcS4A2. In the second procedure, the AlPcS4was substituted with a linear dodecylaminosulfonyl chain, i.e. AlPcS4( C12) and non-covalently inserted into the phospholipid monolayer of the LDL. Both preparations contained over 50 moles AlPcS /mole LDL. They were tested in vitro for their phototoxic properties against the EMT-6 mouse mammary tumor cell line and the A-549 human lung adenocarcinoma cell line. Cell survival was assessed using the MTT colorimetric assay. Association of the free AlPcS4( C12) with LDL increased the in vitro phototoxicity of the dye substantially against both cell lines while the covalently loaded AlPcS4A2-LDL preparation showed little cytotoxicity, even at a 10-fold-higher light or drug doses. It was postulated that the covalent labeling of the protein moiety with the Pc greatly reduced LDL receptor recognition, rendering this derivative photo-inactive. Photodynamic therapy of EMT-6 tumor-bearing mice showed that the free and LDL-associated AlPcS4( C12) exhibited similar activities, with 100% cure one week post-PDT at 0.2 μmol kg−1. We conclude that the attached aliphatic chain of the AlPcS4( C12) greatly enhances the phototoxicity of the parent AlPcS4but that pre-association of the AlPcS4( C12) with LDL does not further augment its in vivo photodynamic efficacy.

Photodynamic therapy is a binary treatment now accepted in clinic for various malignancies in several countries around the world. Phthalocyanine molecules are second-generation photosensitizers with enhanced photophysical and photochemical properties over those of porphyrins. They have been shown to be phototoxic against a number of cell types and tumor models. A great deal of research has been devoted to the elucidation of their mechanism of action and mode of cell death. The present paper reviews phthalocyanine pre-clinical anti-cancer research with emphasis on phthalocyanine induced apoptosis using a silicon phthalocyanine, Pc 4. A brief summary of the latest clinical results using phthalocyanines is presented.

A series of novel functionalized trisulfonated phthalocyanines were prepared in good to excellent yield using the monoiodo trisulfonated phthalocyanine as precursor for palladium-catalysed cross coupling reactions (Heck reaction and Buchwald amination).

Three new 125I-radioiodinated estrogens featuring a 13β-ethyl instead of the natural 13-methyl group, i.e. 18-homoestradiols, were synthesized and evaluated as potential estrogen receptor imaging agents. The 16α-iodo-18-methylestradiol and the 125I-labeled analog were synthesized from the corresponding 16β-bromo analog by the halogen-exchange method. The cis-bromohydrin precursor was obtained by bromination of an estrone enolacetate, followed by epimerization and reduction. The isomeric (17α,20E/Z)-iodovinyl-18-methylestradiols were prepared via the vinyltin intermediates. Treatment of 18-methyl-17α-ethynylestradiol with tri-n-butyltin hydride, in the presence of azobisisobutyronitrile as catalyst and heating at 90–100°C afforded the (17α,20E)-tri-n-butylstannyl isomer as the major product. Changing the catalyst for triethyl borane, at room temperature, mainly gave the 20Z-isomer. The nca 125I-labeled analogs were obtained from their corresponding tin intermediates upon treatment with [125I]NaI in the presence of H2O2. The 16α-[125I]iodo- and isomeric (17α,20E/Z)-[125I]iodovinyl-18-methylestradiols were evaluated for estrogen receptor-mediated uterine uptake in immature female rats. Homologation of the C13-methyl group did improve the uterine uptake of the iodovinyl derivatives, but also increased blood retention, resulting in lower target uptake ratios. In the case of the 16α-iodo analog uterine retention decreased upon C13-homologation.

Covalently linked phthalocyanine–phthalocyanine (Pc–Pc) heterodimers and Pc–Pc triad phthalocyanines connected through phenyl–phenyl (C–C) linkages are prepared using phthalocyanine-boronate (Suzuki Pc-synthon) and palladium catalyst.

Phthalocyanines (Pc), which are extensively studied as tumor localizing photosensitizers for photodynamic therapy, are transported by the blood circulatory system to target tissues. Binding interactions between human serum albumin and differently sulfonated aluminum phthalocyanines (AlPcSn; n = 1-4) were studied using optical and ESR spectroscopy. AlPcSn (n = 1-3) occupy one strong binding site and eight weaker sites. The high affinity binding site interactions differ with respect to the degree of sulfonation and isomeric composition of the Pc. Phthalocyanines without SO-3 groups on adjacent iso-indole rings exhibit a high affinity binding site constant of K approximately 3-4 x 10(7) M-1, while Pc with two or three adjacent SO-3 groups show binding for this high affinity site that is no longer independent, but cooperative (alpha = 2), with K approximately 2-6 x 10(6) M-1. Binding isotherms for AlPcS4 and its close analog, tempoyl spin-labeled SL-AlPcS3, do not approach saturation at high ligand concentrations. Competition analyses between AlPcSn and spin-labeled fatty acids (5- and 16-doxyl stearate isomers) reveal that all compounds participate in cooperative (allosteric) interactions with the high affinity binding site of 16-DS, while extruding 5-DS isomer from certain sites and increasing the binding affinity for the remaining. Protein conformational dynamics was studied by ESR spectroscopy using covalent (alkylation of Cys34 residue) and noncovalent spin labeling (employing SL-AlPcS3). Phthalocyanines perturb conformational dynamics parameters (tauc and S) depending on the degree of sulfonation and isomeric composition corresponding to the type of sites, i.e., independent or cooperative, occupied on the HSA molecule.

The goal of the present study was to determine whether ACTH and progesterone have any effect on LH secretion and pulse frequency in recently castrated rams. Six 2-year-old Corriedale rams were castrated in the winter. The day before castration, blood samples were taken in order to establish the precastration LH levels. The rams were divided into an untreated group (group U: n = 2) and a treated group (group T: n = 4). The first treatment consisted of the i.v. administration of 0.5 mg of ACTH on day 20 post-castration, immediately after the first sample had been taken. During the second treatment, subcutaneous progesterone implants were given to group T for 5 days. Control samplings were performed one week before each treatment. Prior to castration, the testosterone levels were low, while after castration they were below the detection limit of the assay. Cortisol and progesterone concentrations were basal before castration in all of the animals and after castration in group U and also in the control samplings for group T. ACTH treatment caused a significant increase in both cortisol and progesterone levels for 3 h (P0.001). Progesterone implants raised progesterone levels in group T, but cortisol levels remained basal. Before castration, all animals had low LH levels and hardly any pulse activity was seen. After castration, both the number of LH pulses and the mean LH production increased significantly in all of the animals (P0.01). During the ACTH trial, LH pulse frequency was significantly reduced for the first 4 h following ACTH administration (P = 0.013), however, no such differences occurred in the prior control period. No effect was seen on mean LH concentration during the ACTH treatment. Progesterone treatment did not have any effect on either the number of LH pulses nor on LH concentrations (P0.05).