Synthesis and Biological Evaluation of Novel 3'-Difluorovinyl Taxoids.

A series of 3'-difluorovinyl taxoids with C10 modifications, as well as those with C2 and C10 modifications, were strategically designed to block the metabolism by cytochrome P-450 3A4 enzyme and synthesized. These novel difluorovinyl taxoids were evaluated for their cytotoxicity against drug-sensitive human breast (MCF7), multidrug-resistant (MDR) human ovarian (NCI/ADR), human colon (HT-29) and human pancreatic (PANC-1) cancer cell lines. 3'-Difluorovinyl taxoids exhibit several to 16 times better activity against MCF7, HT-29 and PANC-1 cell lines and up to three orders of magnitude higher potency against NCI/ADR cell line as compared to paclitaxel. Structure-activity relationship study shows the critical importance of the C2 modifications on the activity against MDR cancer cell line, while the C10 modifications have a rather minor effect on the potency with some exceptions. The effect of the C2 modifications on potency against MCF7 cell line increases in the following order: H < F < Cl

Evaluation of combined bevacizumab and intraperitoneal carboplatin or paclitaxel therapy in a mouse model of ovarian cancer.

PURPOSE: To evaluate the pharmacokinetics of bevacizumab following IP and IV administration, and to investigate combined bevacizumab therapy (IP or IV) with IP paclitaxel or carboplatin in a mouse model of ovarian cancer. METHODS: Bevacizumab pharmacokinetics were investigated following IV or IP dosing, and mice bearing peritoneal A2780 xenografts were treated with vehicle, IV or IP bevacizumab, IP paclitaxel, IP paclitaxel with co-administration of IV or IP bevacizumab, IP carboplatin, and IP carboplatin with co-administration of IV or IP bevacizumab. Survival time was defined as the time to death or the time to reach 120% of baseline body weight. RESULTS: Following IP administration, bevacizumab was rapidly absorbed and bioavailability was 92.8%. Median survival time, which was 33 days for control mice, was increased by 24% with IP paclitaxel. IP carboplatin failed to increase survival time when administered alone. IV and IP bevacizumab increased survival time by 42 and 33%. Combined bevacizumab and IP paclitaxel was superior to paclitaxel alone (P = 0.01 for IV and P = 0.04 for IP bevacizumab), and combined bevacizumab and IP carboplatin was superior to carboplatin alone (P = 0.002 for IV and P = 0.02 for IP bevacizumab). There were no significant differences in survival between groups receiving bevacizumab IV or IP, either alone (P = 0.586), in combination with paclitaxel (P = 0.467), or in combination with carboplatin (P = 0.149). CONCLUSIONS: Following IP administration to mice, bevacizumab demonstrates rapid and near complete absorption. Bevacizumab therapy, initiated prior to IP carboplatin or paclitaxel administration, increased survival time significantly in mice, and results were not dependent on the route of bevacizumab administration (IV vs. IP).

Design and synthesis of de novo cytotoxic alkaloids by mimicking the bioactive conformation of paclitaxel.

Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics.

Use of an anti-vascular endothelial growth factor antibody in a pharmacokinetic strategy to increase the efficacy of intraperitoneal chemotherapy.

The efficacy of intraperitoneal chemotherapy for ovarian cancers is limited by poor penetration of drug into peritoneal tumors. Based on pharmacokinetic theory that suggests that penetration depth is primarily determined by the rate of drug removal via tumor capillaries, we have hypothesized that co-administration of antiangiogenic therapy will allow for decreased drug removal, increased drug concentrations in tumor, and increased efficacy of intraperitoneal chemotherapy. Pharmacokinetic modeling was conducted to simulate the effect of tumor blood flow on tumor concentrations of topotecan. Simulations predicted that tumor blood flow reductions, as potentially achieved by antiangiogenic therapy, would lead to substantial increases in tumor concentrations after intraperitoneal chemotherapy but would lead to a slight decrease after systemic chemotherapy. Pharmacokinetic studies performed using the A2780 xenograft tumor model showed that animals receiving combined intraperitoneal topotecan and an anti-vascular endothelial growth factor (VEGF) monoclonal antibody had approximately 6.5-fold higher (p = 0.0015) tumor topotecan concentrations compared with animals receiving intraperitoneal topotecan alone, whereas there was no significant (p = 0.16) difference for systemic topotecan. Therapeutic studies conducted with two different drugs, topotecan and cisplatin, showed that animals receiving combined intraperitoneal chemotherapy and anti-VEGF therapy displayed superior survival relative to animals treated with chemotherapy alone (i.e., cisplatin or topotecan), anti-VEGF alone, or intravenous chemotherapy with concomitant anti-VEGF therapy. Combined intraperitoneal topotecan and anti-VEGF resulted in the complete cure of four of 11 mice. The proposed combination of antiangiogenic therapy and intraperitoneal chemotherapy, which was predicted to be beneficial by pharmacokinetic simulations, may provide substantial benefit to patients with peritoneal malignancies.

Design, synthesis, and biological evaluation of novel C14-C3'BzN-linked macrocyclic taxoids.

Novel macrocyclic paclitaxel congeners were designed to mimic the bioactive conformation of paclitaxel. Computational analysis of the "REDOR-Taxol" structure revealed that this structure could be rigidified by connecting the C14 position of the baccatin moiety and the ortho position of C3'N-benzoyl group (C3'BzN), which are ca. 7.5 A apart, with a short linker (4-6 atoms). 7-TES-14beta-allyloxybaccatin III and (3R,4S)-1-(2-alkenylbenzoyl)-beta-lactams were selected as key components, and the Ojima-Holton coupling afforded the corresponding paclitaxel-dienes. The Ru-catalyzed ring-closing metathesis (RCM) of paclitaxel-dienes gave the designed 15- and 16-membered macrocyclic taxoids. However, the RCM reaction to form the designed 14-membered macrocyclic taxoid did not proceed as planned. Instead, the attempted RCM reaction led to the occurrence of an unprecedented novel Ru-catalyzed diene-coupling process, giving the corresponding 15-membered macrocyclic taxoid (SB-T-2054). The biological activities of the novel macrocyclic taxoids were evaluated by tumor cell growth inhibition (i.e., cytotoxicity) and tubulin-polymerization assays. Those assays revealed high sensitivity of cytotoxicity to subtle conformational changes. Among the novel macrocyclic taxoids evaluated, SB-T-2054 is the most active compound, which possesses virtually the same potency as that of paclitaxel. The result may also indicate that SB-T-2054 structure is an excellent mimic of the bioactive conformation of paclitaxel. Computational analysis for the observed structure-activity relationships is also performed and discussed.

Design, synthesis, and biological evaluation of new-generation taxoids.

Novel second-generation taxoids with systematic modifications at the C2, C10, and C3'N positions were synthesized and their structure-activity relationships studied. A number of these taxoids exhibited exceptionally high potency against multidrug-resistant cell lines, and several taxoids exhibited virtually no difference in potency against the drug-sensitive and drug-resistant cell lines. These exceptionally potent taxoids were termed "third-generation taxoids". 19 (SB-T-1214), 14g (SB-T-121303), and 14i (SB-T-1213031) exhibited excellent activity against paclitaxel-resistant ovarian cancer cell lines with mutations in beta-tubulin as well, wherein the drug resistance is mediated by the beta-tubulin mutation. These taxoids were found to possess exceptional activity in promoting tubulin assembly, forming numerous very short microtubules similar to those formed by discodermolide. Taxoids 19 and 14g also showed excellent cytotoxicity against four pancreatic cancer cell lines, expressing three to four multidrug-resistant genes. Moreover, taxoid 19 exhibited excellent in vivo efficacy against highly drug-resistant CFPAC-1 pancreatic as well as DLD-1 human colon tumor xenografts in mice.

Syntheses and Structure-Activity Relationships of Novel 3'-Difluoromethyl and 3'-Trifluoromethyl-Taxoids.

A series of novel 3'-difluoromethyl-taxoids and 3'-trifluoromethyl-taxoids with modifications at the C2 and C10 positions were synthesized and evaluated for their in vitro cytotoxicities against human breast carcinoma (MCF7-S, MCF7-R, LCC6-WT, LCC6-MDR), non-small cell lung carcinoma (H460) and colon adenocarcinoma (HT-29) cell lines. These second-generation fluoro-taxoids exhibited several times to more than 20 times better potency than paclitaxel against drug-sensitive cancer cell lines, MCF7-S, LCC6-WT, H460, and HT-29. These fluoro-taxoids also possess two orders of magnitude higher potency than paclitaxel against drug-resistant cancer cell lines, MCF7-R and LCC6-MDR. Structure-activity relationship study shows the importance of the C10 modification for increasing the activity against multidrug-resistant cancer cell lines. Effects of the C2-benzoate modifications on the potency in the 3-difluoromethyl-taxoid series are very clear (i.e., F < MeO < Cl < N(3)), while those in the 3-trifluoromethyl-taxoid series are less obvious. Also, different trends in the sensitivity to the C2-substitution are observed between drug-sensitive cell lines and drug-resistant cancer cell lines that overexpress efflux pumps.

Modeling the combination of amphotericin B, micafungin, and nikkomycin Z against Aspergillus fumigatus in vitro using a novel response surface paradigm.

Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC(50)) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC(50) for a fixed-ratio concentration, but with concentrations normalized by the respective IC(50)s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology.

The 4'-O-benzylated doxorubicin analog WP744 overcomes resistance mediated by P-glycoprotein, multidrug resistance protein and breast cancer resistance protein in cell lines and acute myeloid leukemia cells.

BACKGROUND: The synthetic 4'-O-benzylated doxorubicin analog WP744 was designed to abrogate transport by the multidrug resistance (MDR)-associated ATP-binding cassette (ABC) proteins P-glycoprotein (Pgp) and multidrug resistance protein (MRP-1). We compared its uptake and cytotoxicity with those of doxorubicin and daunorubicin in cell lines overexpressing Pgp, MRP-1 or breast cancer resistance protein (BCRP) and in acute myeloid leukemia (AML) cells. METHODS: Cellular uptake was studied by flow cytometry and cytotoxicity in 96-h 96-well cultures in cell lines overexpressing Pgp, MRP-1 or wild type (BCRP(R482)) or mutant (BCRP(R482T), BCRP(R482G)) BCRP and in pre-treatment AML marrow cells. RESULTS: Uptake and cytotoxicity of WP744 were consistently greater than those of doxorubicin and daunorubicin at equimolar concentrations in all cell lines studied and in AML cells. CONCLUSION: WP744 overcomes transport by Pgp, MRP-1 and BCRP in cell lines and AML cells and is a promising agent for clinical development in AML and other malignancies with broad-spectrum multidrug resistance.

Effect of amphotericin B and micafungin combination on survival, histopathology, and fungal burden in experimental aspergillosis in the p47phox-/- mouse model of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47phox-/- knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 x 10(4) CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge (n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

Syntheses and evaluation of novel fatty acid-second-generation taxoid conjugates as promising anticancer agents.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA), linolenic acid, and linoleic acid were linked to the C-2' position of the second-generation taxoids that could overcome MDR caused by overexpressed ABC transporters. The new conjugates, tested in vivo, exhibited strong activity against drug-resistant colon cancer and drug-sensitive ovarian cancer xenografts in mice. Two of the new conjugates, DHA-SB-T-1214 and DHA-SB-T-1213, were found to achieve the total regression of drug-resistant and drug-sensitive tumors, respectively, in the animal models with substantially reduced systemic toxicity.

Use of the tubulin bound paclitaxel conformation for structure-based rational drug design.

A new computational docking protocol has been developed and used in combination with conformational information inferred from REDOR-NMR experiments on microtubule bound 2-(p-fluorobenzoyl)paclitaxel to delineate a unique tubulin binding structure of paclitaxel. A conformationally constrained macrocyclic taxoid bearing a linker between the C-14 and C-3'N positions has been designed and synthesized to enforce this "REDOR-taxol" conformation. The novel taxoid SB-T-2053 inhibits the growth of MCF-7 and LCC-6 human breast cancer cells (wild-type and drug resistant) on the same order of magnitude as paclitaxel. Moreover, SB-T-2053 induces in vitro tubulin polymerization at least as well as paclitaxel, which directly validates our drug design process. These results open a new avenue for drug design of next generation taxoids and other microtubule-stabilizing agents based on the refined structural information of drug-tubulin complexes, in accordance with typical enzyme-inhibitor medicinal chemistry precepts.

Design, synthesis and structure-activity relationships of novel taxane-based multidrug resistance reversal agents.

A series of novel taxane-based multidrug resistance (MDR) reversal agents (TRAs) has been designed and synthesized. Structure-activity relationship (SAR) study clearly indicates that modification of the C-7 position with hydrophobic arenecarbonylcinnamoyl groups brings about high potency against drug efflux mediated by P-glycoprotein (P-gp). Six TRAs exhibit ability to modulate a wide range of ATP-binding cassette (ABC) transporters, such as P-gp, multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein (BCRP), which may serve as novel broad-spectrum modulators of ABC transporters.

Design and synthesis of de novo cytotoxic alkaloids through mimicking taxoid skeleton.

Based on a common pharmacophore model and the hypothesis that the baccatin core of taxoids is a scaffold securing the proper orientation of the side chains, a bicyclic alkaloid scaffold was designed as a baccatin surrogate. Using this scaffold, two novel macrocyclic and open-chain 'taxoid-mimicking' compounds were synthesized. Two of these 'taxoid-mimics', 2 and 3, were found to possess cytotoxicity with micromolar level IC50 values against human breast cancer cell lines.

Structure-activity analysis of taxane-based broad-spectrum multidrug resistance modulators.

BACKGROUND: Clinical drug resistance is frequently associated with overexpression of the multidrug resistance (MDR) proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP). Taxanes are substrates for Pgp and MRP-1, but not BCRP. Taxane-based reversal agents (tRAs) are non-cytotoxic MDR modulators previously examined for broad-spectrum modulation of Pgp, MRP-1 and BCRP. MATERIALS AND METHODS: Modulation by tRAs was studied by flow cytometry and resistance to taxanes was studied in cytotoxicity assays in the parental HL60/wt, 8226/wt and MCF7/S, and the resistant HL60/ADR, 8226/Dox6, 8226/MR20 and MCF7 AdVp3000 cell lines. Amino acid sequence (BLAST) alignments were performed using ClustalW. RESULTS: Structure-activity analysis demonstrated greatest alignment of BCRP with the transmembrane 7-12 region of Pgp and identified tRA side groups that contributed or were detrimental to modulation. CONCLUSION: Identification of tRA side groups contributing to modulation of Pgp, MRP-1 and BCRP will allow the design of a next generation of tRAs and will optimize their potential clinical applicability.

Synthesis, molecular modeling, and evaluation of nonphenolic indole analogs of mycophenolic acid.

Based on the promising activity of an indole-3-carboxamide derivative, a nonphenolic analog of mycophenolic acid (MPA), we report herein the synthesis of a compound containing two important features for the activity of MPA, the ring methoxy and methyl. The synthesis was accomplished using two strategies; a method dependent on stepwise building of the hexenoate side chain followed by the indolecarboxamide ring system, and a convergent route that depended on 1,3-sigmatropic rearrangement as a key step. Docking experiments on both Chinese Hamster and Human Type-II inosine monophosphate dehydrogenase (IMPDH) showed that this compound has potential binding interactions with the NAD site. The analogs showed no activity against MCF7-S, MCF7-R, or IGR-OV1 cancer cells.

Broad-spectrum modulation of ATP-binding cassette transport proteins by the taxane derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023.

PURPOSE: The taxanes paclitaxel and docetaxel are substrates for P-glycoprotein (Pgp), an ATP-binding cassette (ABC) transport protein associated with multidrug resistance (MDR). In contrast, the synthetic taxane ortataxel (BAY 59-8862, IDN-5109) is effective against Pgp-expressing cells by virtue of modulation of Pgp-mediated transport. The synthetic taxane tRA96023 also modulates Pgp and is noncytotoxic due to removal of the tubulin-binding side chain at the C-13 position of the taxane backbone. We studied the effects of ortataxel and tRA96023 on the other MDR-associated ABC transport proteins, multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP, MXR, ABCG2). METHODS: Modulation of mitoxantrone, daunorubicin and doxorubicin retention and cytotoxicity by ortataxel and tRA96023 was studied in established cell lines overexpressing Pgp, MRP-1 and wild type (BCRP(R482)) and mutant (BCRP(R482T)) BCRP, and was compared with modulation by the established Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C, respectively. RESULTS: Ortataxel effectively modulated drug retention and cytotoxicity in cell lines overexpressing MRP-1 and BCRP(R482), in addition to Pgp. tRA96023 modulated drug retention and cytotoxicity in cell lines overexpressing BCRP(R482) and Pgp, but not those overexpressing MRP-1. Neither ortataxel nor tRA96023 modulated BCRP(R482T). CONCLUSIONS: The synthetic taxane derivatives ortataxel and tRA96023 are broad-spectrum ABC protein modulators. Further studies will seek to identify a noncytotoxic synthetic taxane that modulates Pgp, MRP-1 and BCRP.

Structure-activity relationships of ring C-secotaxoids. 1. Acylative modifications.

The acylative modification of IDN 5390 (3a), a 7,8-secotaxoid under preclinical development, was investigated. A modest decrease of potency was observed upon acylation of the primary and the enolic hydroxyls, suggesting that, just like in paclitaxel, the hydroxyl groups in the upper right-hand sector are not critical for cytotoxicity. The activity of these analogues, and especially of the chemically robust carbonates 3c and 3d, makes it unlikely that the activity of IDN 5390 is due to in vivo oxidation to a fledgling 7-aldehyde and re-aldolization to the corresponding taxane derivative.

Induction of survivin expression by taxol (paclitaxel) is an early event, which is independent of taxol-mediated G2/M arrest.

Survivin is a novel anti-apoptotic protein that is highly expressed in cancer but is undetectable in most normal adult tissues. It was reported that taxol-mediated mitotic arrest of cancer cells is associated with survivin induction, which preserves a survival pathway and results in resistance to taxol. In this study, we provide new evidence that induction of survivin by taxol is an early event and is independent of taxol-mediated G(2)/M arrest. Taxol treatment of MCF-7 cells rapidly up-regulated survivin expression (3.5-15-fold) within 4 h without G(2)/M arrest. Lengthening the treatment of cells (48 h) with taxol resulted in decreased survivin expression in comparison with early times following taxol treatment, although G(2)/M cells were significantly increased at later times. Interestingly, 3 nm taxol induces survivin as effectively as 300 nm and more effectively than 3000 nm. As a result, 3 nm taxol is ineffective at inducing cell death. However, inhibition of taxol-mediated survivin induction by small interfering RNA significantly increased taxol-mediated cell death. Taxol rapidly activated the phosphatidylinositol 3-kinase/Akt and MAPK pathways. Inhibition of these pathways diminished survivin induction and sensitized cells to taxol-mediated cell death. A cis-acting DNA element upstream of -1430 in the survivin pLuc-2840 construct is at least partially responsible for taxol-mediated survivin induction. Together, these data show, for the first time, that taxol-mediated induction of survivin is an early event and independent of taxol-mediated G(2)/M arrest. This appears to be a new mechanism for cancer cells to evade taxol-induced apoptosis. Targeting this survival pathway may result in novel approaches for cancer therapeutics.