Cell Uptake of Steroid-BODIPY Conjugates and Their Internalization Mechanisms: Cancer Theranostic Dyes.

Estradiol-BODIPY linked via an 8-carbon spacer chain and 19-nortestosterone- and testosterone-BODIPY linked via an ethynyl spacer group were evaluated for cell uptake in the breast cancer cell lines MCF-7 and MDA-MB-231 and prostate cancer cell lines PC-3 and LNCaP, as well as in normal dermal fibroblasts, using fluorescence microscopy. The highest level of internalization was observed with 11ß-OMe-estradiol-BODIPY 2 and 7α-Me-19-nortestosterone-BODIPY 4 towards cells expressing their specific receptors. Blocking experiments showed changes in non-specific cell uptake in the cancer and normal cells, which likely reflect differences in the lipophilicity of the conjugates. The internalization of the conjugates was shown to be an energy-dependent process that is likely mediated by clathrin- and caveolae-endocytosis. Studies using 2D co-cultures of cancer cells and normal fibroblasts showed that the conjugates are more selective towards cancer cells. Cell viability assays showed that the conjugates are non-toxic for cancer and/or normal cells. Visible light irradiation of cells incubated with estradiol-BODIPYs 1 and 2 and 7α-Me-19-nortestosterone-BODIPY 4 induced cell death, suggesting their potential for use as PDT agents.

Synthesis and spectral properties of estrogen- and androgen-BODIPY conjugates.

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.

BODIPY-17α-ethynylestradiol conjugates: Synthesis, fluorescence properties and receptor binding affinities.

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.

PET imaging using 64Cu-labeled sulfophthalocyanines: synthesis and biodistribution.

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.

Phthalocyanine-peptide conjugates via palladium-catalyzed cross-coupling 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.

Structure-activity relationships of mono-substituted trisulfonated porphyrazines for the photodynamic therapy (PDT) of cancer.

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 to >2 weeks, and gave 41% tumour cure >3 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 and >90% 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.

Pd-catalyzed Heck reaction for the synthesis of isomeric metallo tetravinylsulfo phthalocyanines and their photosensitizing properties.

Tetrasulfonated zinc phthalocyanine (ZnPcS(4)) is a potent sensitizer for photodynamic therapy of various medical conditions. Depending on its mode of preparation the material consists of mixtures of ortho and meta sulfonated derivatives as well as regioisomers with different photodynamic potency. To study the effect of the site of substitution on biological parameters that contribute to overall photodynamic efficacy, a series of pure ortho- and meta-tetravinylsulfonated metallo phthalocyanines MPc-o/m-(VS)(4) were prepared from the corresponding tetraiodo phthalocyanines using the palladium-catalyzed cross-coupling reaction (Heck reaction). Compared to the tetrasulfonated phthalocyanines, the tetravinylsulfonated derivatives are more hydrophobic and their Q-band absorption maxima are red-shifted. While their in vitro phototoxicity is in the same range as the mixed isomeric sulfonated derivatives, ortho-substituted MPc-o-(VS)(4) are more photocytotoxic as compared to their corresponding meta analogs. Although the central metal ion and site of substitution affect aggregation, the tendency to aggregate did not correlate with photodynamic potency nor did overall cell uptake. Instead, intracellular localization at both mitochondrial and lysosomal membranes appears to be the major factor explaining the augmented activity of the ortho substituted derivatives.

Bromines on N-allyl position of cationic porphyrins affect both radio- and photosensitizing properties.

With the aim to develop improved dual-action sensitizers suitable for both photodynamic therapy (PDT) and radiotherapy, we prepared a series of metal and metal-free cationic porphyrins, brominated either on beta- or N-allyl positions. Photo- and radiosensitizing efficacy was evaluated in MDA-MB-231 breast cancer cells incubated with 1 muM porphyrin and treated with graded doses of visible light or 0-6 Gy of 60Co gamma irradiation. Metabolic activity after PDT or cell survival after gamma irradiation were estimated by a colorimetric (MTT) or clonogenicity assay, respectively. The highest photo- and radiosensitizing activities were observed with the porphyrins substituted with bromines on N-allyl positions. The non-metalated N-allyl bromoporphyrin exhibited the highest photocytotoxicity (LD50=4.1+/-0.6 J cm(-2), compared to 15.3+/-2.2 J cm(-2) for the non-brominated analog). The radiosensitizing capacity of the cationic porphyrins was also affected by these substitutions with the non-metalated N-allyl bromo analog showing the best improvement (LD50=1.2+/-0.4 Gy vs. 3.6+/-0.9 Gy for the non-brominated analog). The increased photodynamic and radiosensitizing potencies due to bromine addition hold potential for the development of new, improved drugs for cancer treatment in combination with photodynamic and radiation therapy.

(18)F-labelling of A-ring substituted 16alpha-fluoro-estradiols as potential radiopharmaceuticals for PET imaging.

The 2-methoxy derivative of estradiol is currently in Phase II clinical trial as an anticancer agent while the 4-methyl derivative has been shown to interact with cytoplasmic and nuclear estrogen receptors in rat pituitary gland and hypothalamus. We hypothesize that the 16alpha-(18)F-analogs of these estrogens could be suitable radiotracers to evaluate action mechanisms of the parent compounds. In this study we report the synthesis of the 16alpha-(18)F and 16alpha-(19)F-analogs of the A-ring substituted estradiols in high yield via stereoselective opening of the intermediate 16beta,17beta-O-cyclic sulfones with [(18)F]F(-) or F(-) followed by deprotection.

Synthesis and biological activities of nucleoside-estradiol conjugates.

Nucleosides were coupled to estradiol via a 17alpha-ethynyl spacer group using Pd(II) as a catalyst. The conjugates were evaluated in vitro for estrogen receptor (ER) binding affinity and cytotoxicity against cell lines with and without ER. The highest receptor binding affinities (RBA approximately 3) were observed with conjugates coupled via a relative long spacer group, while none of the conjugates exhibited cytotoxicity against either cell lines.

Structure-photodynamic activity relationships of substituted zinc trisulfophthalocyanines.

To identify optimal features of metalated sulfophthalocyanine dyes for their use as photosensitizers in the photodynamic therapy of cancer, we synthesized a series of alkynyl-substituted trisulfonated phthalocyanines and compared their amphiphilic properties to a number of parameters related to their photodynamic potency. Varying the length of the substituted alkynyl side-chain modulates the hydrophobic/hydrophilic properties of the dyes providing a linear relationship between their n-octanol/water partition coefficients and retention times on reversed-phase HPLC. Aggregate formation of the dyes in aqueous solution increased with increasing hydrophobicity while monomer formation was favored by the addition of serum proteins or organic solvent. Trisulfonated zinc phthalocyanines bearing hexynyl and nonynyl substituents exhibited high cellular uptake with strong localization at the mitochondrial membranes, which coincided with effective photocytotoxicity toward EMT-6 murine mammary tumor cells. Further increase in the length of the alkynyl chains (dodecynyl, hexadecynyl) did not improve their phototoxicity, likely resulting from extensive aggregation of the dyes in aqueous medium and reduced cell uptake. Aggregation was evident from shifts in the electronic spectra and reduced capacity to generate singlet oxygen. When monomerized through the addition of Cremophor EL all sulfonated zinc phthalocyanines gave similar singlet oxygen yields. Accordingly, differences in the tendency of the dyes to aggregate do not appear to be a determining factor in their photodynamic potency. Our results confirm that the latter in particular relates to their amphiphilic properties, which facilitate cell uptake and intracellular localization at photosensitive sites such as the mitochondria. Combined, these factors play a significant role in the overall photodynamic potency of the dyes.

Synthesis of the 7alpha-cyano-(17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones: potential radioligands for androgen and progesterone receptors.

We report the preparation of the 7alpha-cyano derivative of the isomeric (17alpha,20E/Z)-[125I]iodovinyl-19-nortestosterones (IVNT) together with their binding affinity for the androgen receptor (AR) and their biodistribution in two different animal models. The cyano group was introduced at the 7alpha-position by hydrocyanation of 4,6-estradien-17beta-ol-3-one with diethylaluminum cyanide. Selective protection of the A-ring enone system as the dienol ether followed by ethynylation and deprotection under base and acid hydrolysis condition gave 7alpha-cyano-17alpha-ethynyl-19-nortestosterone. The stannyl derivatives were prepared by addition of tri-n-butylstannyl hydride and converted stereospecifically to the corresponding [125I]iodovinyl analog using [125I]NaI and H2O2. The [125I]iodovinylsteroids were intravenously administered to male rats and estrogen-primed immature female rats and tissue uptake was measured up to 6h post-injection. Co-administration of NLP-004 or ORG-2058, highly selective ligands for the progesterone receptor, to the female rats did not affect uterus uptake of the 125I-ligands. However co-injection of testosterone to DES-primed male rats induced a marked increase in prostate uptake of the 20Z-isomer of 7alpha-cyano-[125I]-IVNT. The relative binding affinity (RBA) of either 7alpha-cyano-(17alpha,20E/Z)-IVNT isomer for the AR is low (RBA=4 and 3, respectively, versus 100 for 5alpha-dihydrotestosterone (DHT)), suggesting the absence of a possible role of the AR in the localization process. These findings contrast previously reported data for the analogous 7alpha-methyl-[125I]-IVNT where co-administration of testosterone was shown to result in a 50% drop in prostate uptake. These data indicate that the addition of an electron withdrawing 7alpha-cyano group to 123I-labeled nortestosterone derivatives does not improve their potential to serve as SPECT agents for the imaging of AR densities in the prostate.

Synthesis and biological properties of 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl- and 16alpha-[125I]iodo-estradiols.

The synthesis, receptor binding affinity, estrogenic potency and tissue distribution of the 7alpha-cyano derivatives of the (17alpha,20E/Z)-[125I]iodovinyl-(CIVE) and 16alpha-[125I]iodo-estradiols (CIE) are reported. The iodovinyl derivatives were prepared via the (17alpha,20E/Z)-tri-n-butylstannyl intermediates, derived from the addition of tri-n-butyl tin hydride to the 17alpha-ethynyl group of the 7alpha-cyano-17alpha-ethynylestradiol, using triethylborane as a catalyst. The no-carrier-added [125I]-CIVE isomers were prepared via the same stereospecific reaction. [125I]-CIE was prepared from 7alpha-cyano-16beta-bromoestradiol via halogen exchange with Na125I. Addition of the 7alpha-cyano group to 16alpha-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 7alpha-cyano group to the (17alpha,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 125I-labeled CIVE 20E-isomer and the 16alpha-iodo CIE peaked at 0.5h post injection while the (17alpha,20Z)-CIVE isomer showed a maximum only past 5h post injection. Uptake of all three 125I-labeled 7alpha-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 7alpha-methylestrogens, which were previously shown to give high maximum 125I-uptake values at 2h post injection. Overall our data indicate that addition of a 7alpha-cyano group to 123I-labeled estrogens does not improve their potential to serve as SPECT agents for the imaging of estrogen receptor densities in breast cancer.

Synthesis and biological activities of phthalocyanine-estradiol conjugates.

Phthalocyanine-based photosensitizers, coupled via a 17alpha-ethynyl group to estradiol using Pd(II) as a catalyst, were synthesized and evaluated for their estrogen receptor binding affinity and in vitro photocytotoxicity. The highest receptor binding affinities (RBA=8-13) were observed with lipophilic conjugates coupled via a relative long spacer group while the sulfonated analogues showed little binding affinities (RBA <2). The highest photocytotoxicity was observed with the sulfonated conjugates, the nature of the spacer group did not have a pronounced effect.

Synthesis of nitrile derivatives of estrogens.

The first time synthesis of 7alpha- and 11beta-nitrile estradiol is described. Reaction of 7alpha-cyano-19-nortestosterone with copper(II)bromide in acetonitrile at room temperature results in aromatization of the A-ring. Treatment of 11beta-cyano-19-nortestosterone-17-one under similar condition does not induce A-ring aromatization but rather results in bromination at the 2beta-position. However A-ring aromatized products are obtained when the latter compound is treated with Ac2O-Py-AcOCl, NBS and HCl.

18F-labeled difluoroestradiols: preparation and preclinical evaluation as estrogen receptor-binding radiopharmaceuticals.

A-ring fluorination of estradiol (ES) at position 2 or 4 decreases the rate of metabolism by blocking the formation of catechol estrogens, one of the major metabolic pathways of ES. We postulate that adding a 2- or 4-fluoro substituent to 16alpha-[18F]fluoroestradiol (FES), a positron emission tomography (PET) radiopharmaceutical used for estrogen receptor (ER) imaging, should prolong its blood circulation time, and thus, improve its localization in ER-rich target tissues. On such account, we prepared a series of FES derivatives substituted with a fluorine atom at C2 or C4, with or without an 11beta-OMe group, and we tested their binding affinities for the ER and different serum proteins including rat alphafetoprotein (AFP) and human sex hormone-binding globulin (SHBG). Labeling at the 16alpha-position was accomplished via nucleophilic substitution with [18F]F(-) on the reactive 16beta,17beta-cyclic sulfate intermediates. Decay corrected yields varied between 30 and 50% for a total synthesis time of 120 min, providing final products with specific activities >3000 Ci/mmol. The 18F-labeled analogs were evaluated for their biodistribution in immature female rats. Substitutions with the 4-F have little effect on binding affinities. Addition of the 2-F diminishes ER and AFP-binding affinities while augmenting the affinity for the SHBG. Addition of the 11beta-OMe decreases all binding affinities, particularly to AFP and SHBG. In contrast, biodistribution of the corresponding [16alpha-18F]fluoro analogs in immature female rats revealed that the presence of the 11beta-OMe group improves ER-mediated uterus uptake, with the 4,16alpha-[16alpha-18F]difluoro-11beta-methoxyestradiol showing the highest uptake values (15% ID at 1-h post-injection). These data suggest that the addition of both a 4-F and 11beta-OMe group onto FES may provide an improved radiopharmaceutical for PET imaging of ER densities in breast cancer patients.