Fine-tuning of radiative properties by "mild" substituents: searching for a perfectly soft chromophore.

Controlling spectral properties to achieve desired characteristics is an attractive goal in application-oriented research, e.g., in the design of fluorescence sensors. "Soft" chromophores, molecules with strong spectral responses to internal or external perturbations are good candidates for such studies. In this work, absorption, fluorescence, and magnetic circular dichroism (MCD) spectra were obtained for a series of porphyrins, substituted at the meso-positions with n-hexyl groups. As the number of substituents increases from 1 to 4, significant changes are observed. The intensity of the S0-S1 transition (Qx) in the 0-0 region strongly decreases in mono-substituted porphyrin, but upon additional substitutions it increases to values larger than in the parent, unsubstituted molecule. Such behavior can be explained, using the perimeter model, by changes in the energy splittings between the two highest (HOMO) and two lowest (LUMO) frontier molecular orbitals. Single substitution makes porphyrin a nearly perfect soft chromophore, but upon introduction of a larger number of n-hexyl groups it is transformed into a hard one. DFT simulations incorrectly predict a continuous transition from a soft to hard chromophore, because the calculated ordering of two HOMO orbitals is opposite to that obtained by experiment. On the other hand, for those porphyrins that can be classified as hard chromophores, the calculations nicely reproduce contributions of Franck-Condon and Herzberg-Teller terms to absorption and fluorescence spectra.

A thermally responsive biopolymer conjugated to an acid-sensitive derivative of paclitaxel stabilizes microtubules, arrests cell cycle, and induces apoptosis.

Poor aqueous solubility limits the therapeutic index of paclitaxel as an anti-cancer drug. Synthesis of soluble prodrugs of paclitaxel, or conjugation of the drug to macromolecular carriers have been reported to increase its water-solubility. Macromolecular drug carriers have an added advantage of targeting the drug to the tumor site due to the abnormal tumor blood and lymphatic vasculature. This study describes a thermally responsive macromolecular carrier, elastin-like polypeptide (ELP) for the delivery of paclitaxel. Paclitaxel was bound to ELP by conjugation with the 6-maleimidocaproyl hydrazone derivative of paclitaxel, an acid-sensitive paclitaxel prodrug, for the potential treatment of breast cancer. Focused hyperthermia above a specific transition temperature at the site of a tumor causes ELP to aggregate and accumulate, thereby increasing the local concentration of the drug cargo. The paclitaxel prodrug described here bears an acid-sensitive linker that is cleavable at the lysosomal/endosomal pH, which allows a controlled intracellular release of the drug. The ELP-delivered paclitaxel in the presence of hyperthermia inhibits MCF-7 cell proliferation by stabilizing the microtubule structures, arresting the cells at the G2/M stage, and inducing apoptosis in a manner similar to conventional paclitaxel. It also inhibits proliferation of a paclitaxel resistant MCF-7 cell line. These data provide an in vitro proof of concept for the use of ELP as a delivery vehicle of paclitaxel.

Helimeric porphyrinoids: stereostructure and chiral resolution of meso-tetraarylmorpholinochlorins.

The synthesis and chiral resolution of free-base and Ni(II) complexes of a number of derivatives of meso-tetraphenylmorpholinochlorins, with and without direct ß-carbon-to-o-phenyl linkages to the flanking phenyl groups, is described. The morpholinochlorins, a class of stable chlorin analogues, were synthesized in two to three steps from meso-tetraphenylporphyrin. The conformations and the relative stereostructures of a variety of free-base and Ni(II) complexes of these morpholinochlorins were elucidated by X-ray diffractometry. Steric and stereoelectronic arguments explain the relative stereoarray of the morpholino-substituents, which differ in the free-base and Ni(II) complexes, and in the monoalkoxy, ß-carbon-to-o-phenyl linked morpholinochlorins, and the dialkoxy derivatives. The Ni(II) complexes were all found to be severely ruffled whereas the free-base chromophores are more planar. As a result of the helimeric distortion of their porphyrinoid chromophores, the ruffled macrocycles possess a stable inherent element of chirality. Most significantly, resolution of the racemic mixtures was achieved, both by classical methods via diastereomers and by HPLC on a chiral phase. Full CD spectra were recorded and modeled using quantum-chemical computational methods, permitting, for the first time, an assignment of the absolute configurations of the chromophores. The report expands the range of known pyrrole-modified porphyrins. Beyond this, it introduces large chiral porphyrinoid π-systems that exist in the form of two enantiomeric, stereochemically stable helimers that can be resolved. This forms the basis for possible future applications, for example, in molecular-recognition systems or in materials with chiroptic properties.

5,10-A2B2-type meso-substituted porphyrins--a unique class of porphyrins with a realigned dipole moment.

Current applications in porphyrin chemistry require the use of unsymmetrically substituted porphyrins. Many current industrial interests in optics and biomedicine require systems with either push-pull (electron-donating and -withdrawing groups) or amphiphilic systems (hydrophobic and hydrophilic groups). In this context we present the class of 5,10-A(2)B(2)-type porphyrins for which two different substituents are positioned in diagonally opposite meso positions. Thus, the intramolecular dipole moment in these tetrapyrroles is positioned along a ß-ß vector passing through two pyrrole rings. This is opposite to the situation of the frequently used 5,15-A(2)BC porphyrins for which the dipole moment is oriented along a meso-meso axis. We have elaborated syntheses of the 5,10-A(2)B(2) porphyrins by using transition-metal-catalyzed transformations of 5,10-A(2) porphyrins or direct substitutions reactions thereof; this gives the target molecules in 22-77% overall yields. The compounds exhibit interesting structural, spectroscopic, and optical features and can serve as building blocks for new porphyrin arrays and applications.

Stepwise conversion of two pyrrole moieties of octaethylporphyrin to pyridin-3-ones: synthesis, mass spectral, and photophysical properties of mono and bis(oxypyri)porphyrins.

Free-base octaethylporphyrin (OEP) was converted in two steps (beta,beta'-dihydroxylation and oxidative diol cleavage with concomitant aldol condensation) to the corresponding oxypyriporphyrin. This conversion was previously described to be applicable only to the Ni(II) complex of OEP. Modified diol cleavage conditions made this reaction sequence now applicable to free-base OEP. The single-crystal structure of the resulting free-base oxypyriporphyrin was determined, proving its near-perfect planarity. The reaction sequence can also be applied to oxypyriporphyrin itself, generating the unprecedented bacteriochlorin-type bis(oxypyri)porphyrin as two separable isomers. The ground-state (UV/Vis and fluorescence spectroscopies) and excited-state (transient triplet-triplet absorption, triplet lifetimes, and triplet EPR spectroscopy) photophysical properties of all chromophores are compared with those of OEP, chlorins, and oxochlorins. The pyridone-modified porphyrins possess unique spectroscopic signatures that distinguish them from regular porphyrins or chlorins. The presence of the pyridone moiety alters the ESI(+) collision-induced fragmentation properties of these oxypyriporphyrins only to a minor degree when compared with those of OEP or chlorins, confirming their stability.

Oxazolochlorins. 2. Intramolecular cannizzaro reaction of meso-tetraphenylsecochlorin bisaldehyde.

Using mildly basic reaction conditions, the periodate-induced diol cleavage of meso-tetraphenyl-2,3-diolchlorin allows for the generation and isolation of the corresponding hitherto elusive free base secochlorin bisaldehyde. An intramolecular Cannizzaro reaction of this porphyrinoid generates three pyrrole-modified, oxazole-based porphyrins: the known porpholactol (2-oxa-3-hydroxychlorin) as the major product, known porpholactone (2-oxa-3-oxoporphyrin), and a novel porpholactol dimer that is linked through an acetal functionality. The structure of the dimer was confirmed by (1)H NMR spectroscopy, X-ray diffractometry, and ESI(+) collision-induced fragmentation mass spectrometry. The chromophores in the dimer are coupled electronically only to a minor extent. A mechanism to rationalize the formation of all products is advanced.

Synthesis, structure, and optical properties of the platinum(II) complexes of indaphyrin and thiaindaphyrin.

The novel free base meso-di(5'-methylthien-2'-yl)thiaindaphyrin, 10, was prepared from the corresponding meso-tetra(thien-2-yl)porphyrin using a methodology analogous to that for the preparation of known meso-diphenylindaphyrin, 5: beta,beta'-Dihydroxylation of the porphyrin is followed by oxidative diol cleavage. The resulting aldehyde moieties undergo an acid-catalyzed intramolecular Friedel-Crafts alkylation of the adjacent meso-thienyl groups with concomitant oxidation. Insertion of Pt(II) into either of the chromophores is facile, producing 5Pt and 10Pt. The crystal structure of 5Pt, the first for any indaphyrin, shows that the conformation of the indaphyrinato ligand is strongly ruffled, while the N(4) donor set that coordinates the central Pt(II) maintains a near-perfect square-planar coordination geometry around the central metal ion (crystal data for C(44)H(24)N(4)O(2)Pt: triclinic space group P1 with a = 8.8735(4) A, b = 12.9285(6) A, c = 14.3297(6) A, alpha = 88.785(1) degrees, beta = 82.248(1) degrees, gamma = 72.422(1) degrees; Z = 2). The UV-vis and emission spectra, triplet yields, and lifetimes of the Pt(II) complexes 5Pt and 10Pt were determined. Both complexes luminesce (in EtOH at 77 K) in the NIR (5Pt: lambda(max-emission) = 864, 974 nm, lifetime 2 micros; 10Pt: lambda(max-emission) = 990, 1112, 1276 nm) with modest to low quantum yields (Phi(p) approximately 1% and approximately 6 x 10(-3) %, respectively).

In vitro and in vivo evaluation of doxorubicin conjugates with the divalent peptide E-[c(RGDfK)2] that targets integrin alphavbeta3.

Integrins, especially integrin alpha vbeta3, are attractive receptors for vascular targeting strategies. Recently, a divalent RGD peptidomimetic, E-[c(RGDfK)2], has been described that demonstrates increased uptake in human ovarian carcinoma OVCAR-3 xenograft tumors. Inspired by these results, we set out to develop doxorubicin conjugates with E-[c(RGDfK)2] by binding two different maleimide derivatives of doxorubicin to E-[c(RGDfK)2] that was thiolated with iminothiolane. In this way, two water-soluble derivatives were obtained, E-[c(RGDfK)2]-DOXO-1 and E-[c(RGDfK)2]-DOXO-2. In E-[c(RGDfK)2]-DOXO-1, doxorubicin was bound to the peptide through a stable amide bond, and in E-[c(RGDfK)2]-DOXO-2, a MMP-2/MMP-9 cleavable octapeptide was introduced between doxorubicin and the peptide. The rationale for a MMP-2/MMP-9-cleavable linker was that MMP-2 and MMP-9 bind to integrin alpha vbeta3 and both are overexpressed in tumor vasculature. In addition, analogous control doxorubicin-containing peptides bearing c(RADfK) that does not bind to integrin alpha vbeta3 were synthesized, i.e., c(RADfK)-DOXO-1 and c(RADfK)-DOXO-2. Whereas E-[c(RGDfK) 2]-DOXO-2 was cleaved effectively by MMP-2 and in OVCAR-3 tumor homogenates releasing a doxorubicin-tetrapeptide or doxorubicin as the final cleavage product, no release of doxorubicin was observed for E-[c(RGDfK)2]-DOXO-1. Proliferation of HUVEC in the presence of MMP-2-cleavable doxorubicin-containing peptides exhibited 6- to 10-fold increased inhibition compared to the amide-linked doxorubicin-containing peptides. In addition, inhibition of HUVEC sprouting during a 24 h exposure was approximately 3-fold stronger for E-[c(RGDfK) 2]-DOXO-2 and 20-fold stronger for the reference peptide conjugate c(RADfK)-DOXO-2 than for doxorubicin alone. In vivo studies in an OVCAR-3 xenograft model demonstrated no or only moderate antitumor efficacy for either E-[c(RGDfK)2], E-[c(RGDfK)2]-DOXO-1, E-[c(RGDfK)2]-DOXO-2, or c(RADfK)-DOXO-2, even at doses of 3 x 24 mg/kg doxorubicin equivalents, compared to an improved antitumor effect for doxorubicin at 2 x 8 mg/kg.

Integrin-targeted nano-sized polymeric systems for paclitaxel conjugation: a comparative study.

The generation of rationally designed polymer therapeutics via the conjugation of low molecular weight anti-cancer drugs to water-soluble polymeric nanocarriers aims to improve the therapeutic index. Here, we focus on applying polymer therapeutics to target two cell compartments simultaneously - tumour cells and angiogenic endothelial cells. Comparing different polymeric backbones carrying the same therapeutic agent and targeting moiety may shed light on any correlation between the choice of polymer and the anti-cancer activity of the conjugate. Here, we compared three paclitaxel (PTX)-bound conjugates with poly-l-glutamic acid (PGA, 4.9 mol%), 2-hydroxypropylmethacrylamide (HPMA, 1.2 mol%) copolymer, or polyethyleneglycol (PEG, 1:1 conjugate). PGA and HPMA copolymers are multivalent polymers that allow the conjugation of multiple compounds within the same polymer backbone, while PEG is a bivalent commercially available Food and Drug Administration (FDA)-approved polymer. We further conjugated PGA-PTX and PEG-PTX with the integrin αvß3-targeting moiety RGD (5.5 mol% and 1:1 conjugate, respectively). We based our selection on the overexpression of integrin αvß3 on angiogenic endothelial cells and several types of cancer cells. Our findings suggest that the polymer structure has major effect on the conjugate's activity on different tumour compartments. A multivalent PGA-PTX-E-[c(RGDfK)2] conjugate displayed a stronger inhibitory effect on the endothelial compartment, showing a 50% inhibition of the migration of human umbilical vein endothelial cell cells, while a PTX-PEG-E-[c(RGDfK)2] conjugate possessed enhanced anti-cancer activity on MDA-MB-231 tumour cells (IC50 = 20 nM versus IC50 300 nM for the PGA conjugate).

A practical synthesis of meso-monosubstituted, beta-unsubstituted porphyrins.

[formula: see text] A simple straightforward synthesis for meso-monosubstituted, beta-unsubstituted porphyrins is reported. Porphyrins of this type are easily prepared by condensation of dipyrromethane, pyrrole-2-carbaldehyde, and the desired aromatic or aliphatic aldehyde. The method can be used for a variety of functional groups with yields between 2 and 12%. In most cases, the 5, 15-disubstituted porphyrin is obtained as a second product but can be removed easily.

In vitro and in vivo evaluation of a paclitaxel conjugate with the divalent peptide E-[c(RGDfK)2] that targets integrin alpha v beta 3.

The alpha(v)beta(3) integrin is overexpressed on proliferating endothelial cells such as those present in growing tumors as well as on tumor cells of various origins. Tumor-induced angiogenesis can be inhibited in vivo by antagonizing the alpha(v)beta(3) integrin with small peptides containing the arginyl-glycyl-aspartic acid (RGD) amino acid sequence. The divalent cyclic peptide E-[c(RGDfK)(2)] is a novel ligand-based vascular-targeting agent that binds integrin alpha(v)beta(3) and demonstrated high uptake in OVCAR-3 xenograft tumors. In this work, we coupled the 2'-OH-group of paclitaxel through an aliphatic ester to the amino group of E-[c(RGDfK)(2)] or the control peptide c(RADfK), thus obtaining the derivatives E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel. Subsequently, we investigated the activity of the paclitaxel derivatives using several well-established in vitro angiogenesis assays: using a standard 72 h endothelial cell proliferation assay, we showed that both E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel inhibit the proliferation of human umbilical vein endothelial cells (HUVEC) in a similar manner as free paclitaxel (IC(50) value approximately 0.4 nM), an observation that can be explained by the half-life of the paclitaxel ester bond in the conjugates of approximately 2h at pH 7. In contrast, a 30-min exposure of the cells to the three drugs showed a clear difference between free paclitaxel, E-[c(RGDfK)(2)]-paclitaxel and c(RADfK)-paclitaxel with IC(50) values of 10nM, 25 nM, and 60 nM, respectively. These differences are very likely due to the different routes of cellular entry of these three molecules. While the hydrophobic paclitaxel diffuses rapidly through the cell membrane, the charged peptide-containing derivative E-[c(RGDfK)(2)]-paclitaxel binds to the overexpressed alpha(v)beta(3) integrin in order to enter the cells via receptor-mediated endocytosis. The differences between the derivatives were further demonstrated using an endothelial cell adhesion assay. Inhibition of cell attachment was observed only with the E-[c(RGDfK)(2)]-paclitaxel derivative indicating its specificity to the growing endothelial cells. Furthermore, E-[c(RGDfK)(2)]-paclitaxel inhibited both endothelial cells migration and capillary-like tube formation. These results further demonstrate their antiangiogenic properties. In vivo studies in an OVCAR-3 xenograft model demonstrated no antitumor efficacy for either E-[c(RGDfK)(2)] or E-[c(RGDfK)(2)]-paclitaxel compared to moderate efficacy for paclitaxel.

Prodrug strategies in anticancer chemotherapy.

The majority of clinically approved anticancer drugs are characterized by a narrow therapeutic window that results mainly from a high systemic toxicity of the drugs in combination with an evident lack of tumor selectivity. Besides the development of suitable galenic formulations such as liposomes or micelles, several promising prodrug approaches have been followed in the last decades with the aim of improving chemotherapy. In this review we elucidate the two main concepts that underlie the design of most anticancer prodrugs: drug targeting and controlled release of the drug at the tumor site. Consequently, active and passive targeting using tumor-specific ligands or macromolecular carriers are discussed as well as release strategies that are based on tumor-specific characteristics such as low pH or the expression of tumor-associated enzymes. Furthermore, other strategies such as ADEPT (antibody-directed enzyme prodrug therapy) and the design of self-eliminating structures are introduced. Chemical realization of prodrug approaches is illustrated by drug candidates that have or may have clinical importance.

Synthesis of mono- and disubstituted porphyrins: A- and 5,10-A2-type systems.

General syntheses have been developed for meso-substituted porphyrins with one or two substituents in the 5,10-positions and no beta substituents. 5-Substituted porphyrins with only one meso substituent are easily prepared by an acid-catalyzed condensation of dipyrromethane, pyrrole-2-carbaldehyde, and an appropriate aldehyde using a "[2+1+1]" approach. Similarly, 5,10-disubstituted porphyrins are accessible by simple condensation of unsubstituted tripyrrane with pyrrole and various aldehydes using a "[3+1]" approach. The yields for these reactions are low to moderate and additional formation of either di- or monosubstituted porphyrins due to scrambling of the intermediates is observed. However, the reactions can be performed quite easily and the desired target compounds are easily removed due to large differences in solubility. A complementary and more selective synthesis involves the use of organolithium reagents for S(N)Ar reactions. Reaction of in situ generated porphyrin (porphine) with 1.1-8 equivalents of RLi gave the monosubstituted porphyrins, while reaction with 3-6 equivalents of RLi gave the 5,10-disubstituted porphyrins in yields ranging from 43 to 90 %. These hitherto almost inaccessible compounds complete the series of different homologues of A-, 5,15-A(2)-, 5,10-A(2)-, A(3)-, and A(4)-type porphyrins and allow an investigation of the gradual influence of type, number, and regiochemical arrangement of substituents on the properties of meso-substituted porphyrins. They also present important starting materials for the synthesis of ABCD porphyrins and are potential synthons for supramolecular materials requiring specific substituent orientations.