Analogues of Y27632 increase gap junction communication and suppress the formation of transformed NIH3T3 colonies.

BACKGROUND: Constitutive activation of RhoA-dependent RhoA kinase (ROCK) signalling is known to promote cellular transformation and the ROCK inhibitor Y-27632 has the ability to suppress focus formation of RhoA transformed NIH3T3 cells. METHODS: Sixty-four novel structural analogues of Y27632 were synthesised and tested for their ability to persistently inhibit the transformation of NIH3T3 cells by Rho guanidine exchange factor 16 (ARHGEF16) or Ras. In vitro kinase inhibitor profiling, co-culture of transformed cells with non-transformed cells and a novel Lucifer yellow/PKH67 dye transfer method were used to investigate their mode of action. RESULTS: Four Y27632 analogues inhibited transformed focus formation that persisted when the compound was withdrawn. No toxicity was observed against either transformed or non-transformed cells and the effect was dependent on co-culture of these two cell types. In vitro kinase inhibitor profiling indicated that these compounds had reduced activity against ROCK compared with Y27632, targeting instead Aurora A (AURKA), p38 (MAPK14) and Hgk (MAP4K4). Dye transfer analysis showed they increased gap junction intercellular communication (GJIC) between transformed and non-transformed cells. CONCLUSIONS: These data are the first to suggest that transient blockade of specific kinases can induce a persistent inhibition of non-contact inhibited transformed colony formation and can also remove pre-formed colonies. These effects could potentially be mediated by the observed increase in GJIC between transformed and non-transformed cells. Selection of kinase inhibitors with this property may thus provide a novel strategy for cancer chemoprevention.

Comparison of different busulfan analogues for depletion of hematopoietic stem cells and promotion of donor-type chimerism in murine bone marrow transplant recipients.

Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy.

The effect of ifosfamide and its metabolites on intracellular glutathione levels in vitro and in vivo.

The effect of ifosfamide and its metabolites on intracellular levels of glutathione in P388 cells in vitro has been studied. It is demonstrated that glutathione depletion occurs only in the presence of 4-hydroperoxyifosfamide and chloroacetaldehyde. In contrast isophosphoramide mustard had no effect on glutathione levels in intact cells. The concentration of 4-hydroperoxyifosfamide required to reduce glutathione levels by 50% was approximately 1 mM and this represents a concentration far in excess of that achievable in patients receiving the drug. However the concentration of chloroacetaldehyde (approximately 100 microM) required to reduce intracellular levels of glutathione to a similar extent is attained in patients receiving ifosfamide. The glutathione levels in lymphocytes isolated from a patient undergoing an eight hour infusion of ifosfamide showed a marked decrease to about 30% of their original value. We conclude that ifosfamide causes glutathione depletion in vivo and the majority of this can be accounted for by the production of chloroacetaldehyde.

Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates.

Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.

The suitability of carboplatin solutions for 14-day continuous infusion by ambulatory pump: an HPLC-dynamic FAB study.

The stability of aqueous carboplatin solutions over 14 days has been studied at 37 and 60 degrees C. High-performance liquid chromatography and dynamic FAB mass spectrometry studies have shown that carboplatin solutions were stable at 37 degrees C but degraded at 60 degrees C. Fluid loss through evaporation was significant at the higher temperature.

Dimethanesulphonate esters in receptor mapping studies. 2. Antitumour activities of alkyl and alkoxy dimethanesulphonates substituted on a benzene nucleus.

The antitumour activities of 15 novel aromatic dimethanesulphonate esters were studied. Several alkyl and alkoxy compounds have shown good antitumour activity whilst similar isomers have proved ineffective as antitumour agents. These differences in activity have been correlated with the length of the sidechain substituents and their relative flexibilities.

Antimitotic activity of diaryl compounds with structural features resembling combretastatin A-4.

Series of diaryl ethers, amines and amides have been synthesized and tested for antitumor activity. These diaryl compounds possess some of the structural features of combretastatin A-4 (a potent antimitotic agent). They were designed to discover whether transferring these structural motifs from stilbenes to heterosubstituted diaryl compounds would enhance their biochemical activities. Molecular modeling studies suggested that these diaryl compounds could adopt conformations similar to combretastatin A-4. However, although some agents (5-7) were cytotoxic and others (10 and 12) could interact with tubulin, none were as potent as combretastatin A-4.

Isolation of aurantiamide acetate from Arisaema erubescens.

Aurantiamide acetate (N-benzoyl-1-phenylalanyl-1-pheylalaninol acetate) has been isolated by chromatographic separation of a methanol extract of Arisaema erubescens and its structure confirmed by synthesis.

Tubulin as a target for anticancer drugs: agents which interact with the mitotic spindle.

Tubulin is the biochemical target for several clinically used anticancer drugs, including paclitaxel and the vinca alkaloids vincristine and vinblastine. This review describes both the natural and synthetic agents which are known to interact with tubulin. Syntheses of the more complex agents are referenced and the potential clinical use of the compounds is discussed. This review describes the biochemistry of tubulin, microtubules, and the mitotic spindle. The agents are discussed in relation to the type of binding site on the protein with which they interact. These are the colchicine, vinca alkaloid, rhizoxin/maytansine, and tubulin sulfhydryl binding sites. Also included are the agents which either bind at other sites or unknown sites on tubulin. The literature is reviewed up to October 1997.

Dimethanesulphonate esters in receptor mapping studies. 1. Benzene 1,2-, 1,3- and 1,4-diol, dimethanol and diethanol dimethanesulphonates and anti-tumour activity.

The anti-tumour activities of nine aromatic dimethanesulphonate esters were examined. Their biological effects have been related to both their chemical reactivity and to the computer-modelled vectorial positions of the centres of alkylation of the compounds. One compound (19) has shown activity between sensitive and resistant Yoshida tumours in vivo and also shows the highest activity between these two cell lines in vitro. In its minimal energy form, this compound would require to interact with converging nucleophilic centres about 6 A apart, and it is tentatively suggested that this may be an appropriate dimension for a receptor which is required to be alkylated in order to show anti-tumour activity.

Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro.

There have been many reports that high soya-based diets reduce the risk of certain types of cancer. This effect may be due to the presence of high levels of isoflavones derived from the soya bean, particularly genistein which has been shown to be a protein tyrosine kinase (PTK) inhibitor and have both oestrogenic and anti-oestrogenic properties. We have examined the effect of genistein and a number of novel synthetic analogues on both normal (IEC6, IEC18) and transformed (SW620, HT29) intestinal epithelial cell lines. Responses were compared to those elicited by oestradiol, the anti-oestrogen tamoxifen, and the tyrosine kinase inhibitor tyrphostin. Genistein and tamoxifen were potent inhibitors of cell proliferation. Of seven novel isoflavones tested, none were more potent inhibitors than genistein, and all displayed similar relative activities across the different cell lines. In addition to inhibiting cell proliferation, cell death via apoptosis was observed when the cells were exposed to the isoflavones and all but one exhibited PTK inhibitory activity. These data suggest that by reducing proliferation and inducing apoptosis, possibly due in part to PTK inhibition, isoflavones may have a role in protecting normal intestinal epithelium from tumour development (reducing the risk) and may reduce colonic tumour growth.

Molecular modeling of the interaction of anthracenyl-amino acid topoisomerase inhibitors with the DNA sequence d(CGTACG).

Anthracenyl-amino acid and dipeptide conjugates represent new classes of topoisomerase (topo) inhibitors. To investigate the structural basis for their different selectivity against topo I and II and varying potency, the binding of six compounds to d(CGTACG) was studied by molecular modeling. Modeling data were in good agreement with physical data showing that five compounds intercalated DNA with the anthraquinone chromophore orientated in parallel to the long dimension of the d(CpG) base pairs and the amino acid placed in the minor groove. Differences in binding modes emerged which correlated to different biological properties. The amino acid chain of the topo I inhibitor (NU/ICRF 600, gly-phe) extended significantly out from the helical axis horizontal. The amino acid side chains of two topo II inhibitors (NU/ICRF 510, arginine and NU/ ICRF 512, methionine) were inserted into the minor groove, whereas the C-terminal groups (hydrazide) of two potent topo II inhibitors (NU/ICRF 500 and 506, serine) were placed into the minor groove while the amino acid side chains pointed away from the minor groove. These data provide structural information which may prove valuable in rational design of second generation analogs.

Antimitotic and cell growth inhibitory properties of combretastatin A-4-like ethers.

A series of diarylamines, diaryl and arylbenzyl ethers based on combretastatin A-4 was prepared and evaluated for anticancer activity. 2-Methoxy-5-(3',4',5'-trimethoxyphenoxymethyl)phenol was the most active (IC50, K562 20 nM) and caused significant G2/M cell cycle arrest.

Cytotoxic Michael-type amine adducts of alpha-methylene lactones alantolactone and isoalantolactone.

Two series of cytotoxic (IC50, K562 cell line, 1-24 microM) alpha-aminomethyl substituted lactones 3 and 4 were prepared by stereoselective Michael-type addition of amines to alantolactone (1) and isoalantolactone (2). The lactones 1 and 2 and their amine adducts induce apoptosis and act as alkylating agents.

The interaction of chalcones with tubulin.

The chalcone 3,4,3',4',5'-pentamethoxychalcone is a potent cytotoxic agent. A series of chalcones and (E)-4-(4'-hydroxyphenyl)but-3-en-2-one were prepared and assessed for their ability to inhibit cell growth in vitro. The cytotoxicity correlates with their ability to bind to tubulin as measured by immunofluorescence, cell cycle analysis and disruption of microtubule assembly. Some of the chalcones were shown to bind to the type II oestrogen receptor.

Novel syntheses of cis and trans isomers of combretastatin A-4.

A high-yielding, two-step stereoselective synthesis of the anticancer drug (Z)-combretastatin A-4 (1) has been devised. The method uses the Perkin condensation of 3,4,5-trimethoxyphenylacetic acid and 3-hydroxy-4-methoxybenzaldehyde followed by decarboxylation of the cinnamic acid intermediate using copper and quinoline. The iodine-catalyzed isomerization of the Z isomer 1 results in complete conversion to the E isomer. The Suzuki cross-coupling of an aryl boronic acid and vinyl bromide has also been successfully employed to produce both Z and E isomers of combretastatin A-4 stereoselectively. Both methods are far superior to the current five-step Wittig synthesis in which both isomers are produced nonstereoselectively.

Bioactivity and molecular modelling of diphenylsulfides and diphenylselenides.

Bis(2-bromo-4,5-dimethoxyphenyl)sulfide (5) and bis(2-bromo-4,5-dimethoxyphenyl) selenide (7) have been shown to block cells in the G2/M phase of the cell cycle, whereas the debromo (4,6) equivalents do not. The biobromoselenide (7) is cytotoxic to tumour cells in vitro and has been shown to increase the mitotic index of treated cells. These biological effects are consistent with disruption of the mitotic apparatus. This agent does not inhibit microtubule assembly in vitro, but does bind to tubulin. Molecular modelling of these structures indicates that their spatial and electronic structures may make an important contribution to the biological activity.

Synthesis and anticancer activities of 4-oxobenzopyrano[2,3-d]pyrimidines.

Several 2-aryl-4-oxoxbenzopyrano[2,3-d]pyrimidines have previously been shown to exhibit in vivo antitumor activity in mice with P388 lymphocytic leukemia. In the present study, a series of novel substituted benzopyrano[2,3-d]pyrimidines have been prepared and tested for cytotoxic activity against a panel of cancer cell lines including the P388 lymphocytic leukemia cell line. The unsubstituted parent compound, some methoxylated derivatives and a cyclohexyl derivative all exhibited potent cytotoxic activity (IC50 values 0.3-0.64 microM). A number of derivatives, including the unsubstituted parent pyrimidine, were shown to cause a significant perturbation in cell cycle kinetics with an observed 2- to 3-fold increase in cells in the G2/M phase of the cell cycle. Furthermore, a polymethoxylated derivative, 2-(3,4,5-trimethoxyphenyl)-9-methoxy-4-oxo-2,3-dihydrobenzopyrano[ 2,3-d]pyrimidine 13, was shown to be selectively active against a number of human ovarian cell lines.

Potent antimitotic and cell growth inhibitory properties of substituted chalcones.

A series of substituted chalcones was synthesised and screened for cytotoxic activity against the K562 human leukaemia cell line. (E)-3-(3"-Hydroxy-4"-methoxyphenyl)-2-methyl-1-(3',4',5'- trimethoxyphenyl)-prop-2-en-1-one [IC50 (K562) 0.21 nM] was found to be the most active. A relationship between the conformation and cytotoxicity of the chalcones is discussed.

Linked parallel synthesis and MTT bioassay screening of substituted chalcones.

A 644-membered library of chalcones was prepared by parallel synthesis using the Claisen-Schmidt base-catalyzed aldol condensation of substituted acetophenones and benzaldehydes. The cytotoxicity of these chalcones was conveniently determined upon the crude products directly in 96-well microtiter test plates by the conventional MTT assay. This method revealed seven chalcones of IC(50) less than 1 microM of which 4'-hydroxy-2,4,6,3'-tetramethoxychalcone (5a) was the most active [IC(50) (K562), 30 nM]; it causes cell cycle arrest at the G(2)/M point and binds to tubulin at the colchicine binding site.