Colchicine Analogues Research Articles

Microtubules Regulate Local Ca2+ Spiking in Secretory Epithelial Cells

The role of the cytoskeleton in regulating Ca(2+) release has been explored in epithelial cells. Trains of local Ca(2+) spikes were elicited in pancreatic acinar cells by infusion of inositol trisphosphate through a whole cell patch pipette, and the Ca(2+)-dependent Cl(-) current spikes were recorded. The spikes were only transiently inhibited by cytochalasin B, an agent that acts on microfilaments. In contrast, nocodazole (5-100 micrometer), an agent that disrupts the microtubular network, dose-dependently reduced spike frequency and decreased spike amplitude leading to total blockade of the response. Consistent with an effect of microtubular disruption, colchicine also inhibited spiking but neither Me(2)SO nor beta-lumicolchicine, an inactive analogue of colchicine, had any effect. The microtubule-stabilizing agent, taxol, also inhibited spiking. The nocodazole effects were not due to complete loss of function of the Ca(2+) signaling apparatus, because supramaximal carbachol concentrations were still able to mobilize a Ca(2+) response. Finally, as visualized by 2-photon excitation microscopy of ER-Tracker, nocodazole promoted a loss of the endoplasmic reticulum in the secretory pole region. We conclude that microtubules specifically maintain localized Ca(2+) spikes at least in part because of the local positioning of the endoplasmic reticulum.

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.

Antitumor agents. 199. Three-dimensional quantitative structure-activity relationship study of the colchicine binding site ligands using comparative molecular field analysis.

Inhibitors of tubulin polymerization interacting at the colchicine binding site are potential anticancer agents. We have been involved in the synthesis of a number of colchicine site agents, such as thiocolchicinoids and allocolchicinoids, which are colchicine analogues, and 2-phenyl-quinolones and 2-aryl-naphthyridinones, which are the amino analogues of cytotoxic antimitotic flavonoids. The most cytotoxic of the latter compounds strongly inhibit binding of radiolabeled colchicine to tubulin, and these agents therefore probably bind in the colchicine site of tubulin. We have applied conventional CoMFA and q(2)-GRS CoMFA to identify the essential structural requirements for increasing the ability of these compounds to form tubulin complexes. The CoMFA model for the training set of 51 compounds yielded cross-validated R(2) (q(2)) values of 0.637 for conventional CoMFA and 0.692 for q(2)-GRS CoMFA. The predictive power of this model was confirmed by successful activity prediction for a test set of 53 compounds with known potencies as inhibitors of tubulin polymerization. The activities of 88% of the compounds were predicted with absolute value of residuals of less than 0.5. The predictive q(2) values were 0.546 for conventional CoMFA and 0.426 for q(2)-GRS CoMFA. The conventional CoMFA model with the highest predictive q(2) (0.546) was analyzed in detail in terms of underlying structure-activity relationships.

Autopalmitoylation of tubulin.

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.

Microtubule cytoskeleton involvement in muscarinic suppression of voltage-gated calcium channel current in guinea-pig ileal smooth muscle.

1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.

Differential regulation of mitogen-activated protein kinases by microtubule-binding agents in human breast cancer cells.

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and beta-lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNKI or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H2O2- or doxorubicin-induced JNKI activity, but had no effect on JNKI activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca2+ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of antimicrotubule drugs.

IKP104-induced decay of tubulin: role of the A-ring binding site of colchicine.

Tubulin, the major subunit protein of microtubules, has a tendency to lose its ability to assemble or to interact with ligands in a time-dependent process known as decay. Decay involves the increase in exposure of sulfhydryl groups and hydrophobic areas. The antimitotic drug IKP104 [2-(4-fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone] accelerates the decay of tubulin [Ludueña et al. (1995) Biochemistry 34, 15751-15759]. In the presence of colchicine, however, IKP104 stabilizes tubulin against decay. We have shown that the stability and the acceleration of the decay of tubulin are mediated respectively by the high- and low-affinity binding site(s) of IKP104 [Chaudhuri et al. (1998) J. Protein Chem. 17, 303-309]. To better understand the mechanism by which colchicine protects tubulin from IKP104-induced decay, we examined the effect of colchicine and its analogues on this process. We found that IKP104 unfolds tubulin in a process involving a specific domain where colchicine interacts, although the binding sites of these two drugs are distinctly different. 2-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropolone (MTPT), the bicyclic analogue of colchicine that lacks the B-ring, can also protect tubulin from IKP104-induced decay. An A-ring analogue of colchicine, 3,4,5-trimethoxybenzaldehyde (TMB), can also stop IKP104-induced unfolding of tubulin significantly. Interestingly, the C-ring analogue of colchicine, tropolone methyl ether (TME), does not prevent this process. Our results thus suggest that neither the B-ring nor the C-ring binding regions of colchicine are involved in the IKP104-induced decay and that the A-ring binding site of colchicine on tubulin plays a crucial role in IKP104-induced decay.

General features of the recognition by tubulin of colchicine and related compounds.

The kinetic mechanisms of the binding to tubulin of colchicine and eight different analogues have been studied to elucidate details of the recognition mechanism. All of the analogues follow a two step binding mechanism i.e. binding occurs via an initial step with low affinity, followed by an isomerisation of the initial complex leading to the final high affinity state. For several analogues the kinetic and thermodynamic data of both processes are compared here. For all the analogues the delta G1 degree of initial binding at 25 degrees C varies between -13.3 and -28.8 kJ. mol-1. For the second step delta G2 degrees varies between -2.4 and -27 kJ. mol-1. These limited ranges of free energy change are, however, obtained by a great variety of enthalpy changes and compensatory entropy changes. Comparison of the data for the first and second steps indicates that structural alterations of the drugs always change the thermodynamic parameters of the two steps, and the changes in the first and the second steps are in opposite directions. The fact that this range of experimental behaviour can be incorporated into a general mechanism encourages the extension of these investigations to other colchicine analogues and related compounds with potential pharmaceutical applications.

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition.

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C'). Specifically, excision of the 2- or 3-methoxy groups weakened binding by ca. 0.4 kcal mol-1, while that of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol-1. The effect on the inhibition of microtubule assembly, expressed as the equilibrium constant for the binding of the tubulin-drug complex to the end of a microtubule, was more complex and strongly dependent on the nature of ring C (or C'). This was attributed to the abilities of various groups on ring C' to overcome the wobbling in the tubulin-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the mechanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in the interactions that key oxygen atoms of ring C of COL or C' of structural analogues establish with the protein. It is proposed that the 4-methoxy group of ring A serves as a key attachment point for immobilization of the drugs on the protein.

ChemInform Abstract: Stereochemical Variations on the Colchicine Motif. Part 3.The First Colchicine Analogues with an Eight‐Membered B‐Ring. Structure, Optical Resolution and Inhibition of Microtubule Assembly.

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Stereochemical variations on the colchicine motif. Part 4. A remote metalation approach toward a colchicine analog with a five-membered B-ring.

Attempts to prepare a colchicine analog with a 5-membered B-ring by remote metalation of N,N-diethyl-3,4,5-trimethoxy-2-(5'-methoxy-4'-oxo-2', 5',7'-cycloheptatrienyl)-benzamide (2) led to ring contraction of the methoxytropone ring to the p-methoxycarbonylphenyl derivative (3). Dynamic 1H NMR investigations showed that the biaryl amide 2 exists as a mixture of diastereomers due to hindered rotation around both aryl-aryl and aryl-amide bonds, with rotational barriers of ca. 63 kJ mol-1. The colchicine and allocolchicine analogs 2 and 3 do not notably affect tubulin polymerization, despite the structural similarities with active analogs. The reduced tubulin binding activity of 2 and 3 may be a result of increasing steric bulk.

Control of the Structural Stability of the Tubulin Dimer by One High Affinity Bound Magnesium Ion at Nucleotide N-site

Tubulin liganded with GTP at the N-site in the alpha-subunit and with GDP at the E-site in the beta-subunit (GDP-tubulin) reversibly binds one high affinity Mg2+ cation (Kb = 1.1 x 10(7) M-1), whereas tubulin liganded with GTP at both subunits (GTP-tubulin) binds one more high affinity Mg2+. The two cation binding loci are identified as nucleotide sites N and E, respectively. Mg2+ at the N-site controls the stability and structure of the alphabeta-tubulin dimer. Mg2+ dissociation is followed by the slow release of bound nucleotide and functional inactivation. Mg2+ bound to the N-site significantly increases the thermal stability of the GDP-tubulin dimer (by 10 degrees C and approximately 50 kcal mol-1 of experimental enthalpy change). However, the thermal stability of Mg2+-liganded GDP- and GTP-tubulin is the same. Mg2+ binding to the N-site is linked to the alphabeta-dimer formation. The binding of Mg2+ to the alpha-subunit communicates a marked enhancement of fluorescence to a colchicine analogue bound to the beta-subunit. Colchicine, in turn, thermally stabilizes Mg2+-depleted tubulin. The tubulin properties described would be simply explained if the N-site and the colchicine site are at the alpha-beta dimerization interface. It follows that the E-site would be at the beta-end of the tubulin dimer, consistent with the known functional role of the E nucleotide gamma-phosphate and coordinated cation controlling microtubule stability.

The first colchicine analogue with an eight membered B-ring. Structure, optical resolution and inhibition of microtubule assembly

Synthesis, crystal structure, enantiomeric resolution, and CD spectroscopy of the first colchicine derivative with an eight-membered B-ring lactam obtained via a Beckmann reaction, is described and only one of the stable atropisomeric enantiomers arrests microtubule assembly. Graphic

Mechanism of tubulin-colchicine recognition: a kinetic study of the binding of the colchicine analogues colchicide and isocolchicine.

Colchicide (IDE) is a colchicine (COL) analogue in which the C-10 methoxy group is replaced by a hydrogen atom. Its binding to tubulin is accompanied by a quenching of the protein fluorescence. The fluorescence decrease shows a monoexponential time dependence. The observed rate constant increases in a non-linear way with the total concentration of IDE, allowing the determination of a binding constant for an initial binding site (K1=5300+/-300 M-1) and the rate constant for the subsequent isomerization (k2=0.071+/-0.002 s-1) at 25 degrees C. The rate constant, k-2, for the reversed isomerization can be determined by displacement experiments. Despite the minor alteration of the C-ring substituent, the kinetic and thermodynamic parameters of binding are substantially different from those of COL itself, for both steps. In isocolchicine (ISO) the carbonyl oxygen atom and the methoxy groups of the C-ring have been interchanged. Its binding to tubulin only results in small fluorescence and absorbance changes. Therefore competition experiments with MTC [2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4, 6-cycloheptatrien-1-one] were performed. ISO competes rapidly and with low affinity with MTC. Fluorimetric titrations of tubulin with MDL (MDL 27048 or trans-1-(2,5 dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2-methyl-2-propen-1 -one) in the presence and absence of ISO give evidence for the existence of a second, slow-reacting low-affinity site for ISO that is not accessible to MTC or MDL. The relevance of these results for the recognition of COL is analysed.

The mechanism of tubulin-colchicine recognition--a kinetic study of the binding of a bicyclic colchicine analogue with a minor modification of the A ring.

2-Methoxy-5-(2',3',4'-trimethoxy)-2,4,6-cycloheptatrien-1-one (MTC) is a colchicine analogue that lacks the B ring. 2-Methoxy-5-(2',4'-dimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MD) is an A-ring analogue of MTC, in which one methoxy group is replaced by a hydrogen atom. This paper describes the kinetic features of MDC binding to tubulin, and compares its behaviour with MTC to analyse the effect of the A-ring modification on the recognition process by tubulin. Binding is accompanied by a strong enhancement of MDC fluorescence and quenching of protein fluorescence. The kinetic and thermodynamic parameters were obtained from fluorescence stopped-flow measurements. The kinetics are described by a single exponential, indicating that this drug does not discriminate between the different tubulin isotypes. The observed pseudo-first-order rate constant of the fluorescence increase upon binding increases in a non-linear way, indicating that this ligand binds with a similar overall mechanism as colchicine and MTC, consisting of a fast initial binding of low affinity followed by a slower isomerisation step leading to full affinity. The K1 and k2 values for MDC at 25 degrees C were 540 +/- 65 M(-1) and 70 +/- 6 s(-1) respectively. From the temperature dependence, a reaction enthalpy change (deltaH(o)1) of the initial binding of 49 +/- 11 kJ/mol(-1) and an activation energy for the second step of 28 +/- 9 kJ/mol(-1) were calculated. Displacement experiments of bound MDC by MTC allowed the determination of a rate constant of reverse isomerisation of 0.60 +/- 0.07 s(-1) at 25 degrees C and the activation energy of 81 +/- 6 kJ/mol(-1). The overall binding constant was (6.3 +/- 0.2) x 10(4) M(-1) at 25 degrees C. Combination of these results with the kinetic parameters for association gives a full characterisation of the enthalpy pathway for the binding of MDC. The pathway of MDC is shown to differ considerably from that of MTC binding. Since its structural difference is located in ring A, this result indicates the use of ring A in the first step. The kinetics of the binding of MDC in the presence of some A-ring colchicine analogues (podophyllotoxin, 3',4',5'-trimethoxyacetophenone and N-acetylmescaline) and a C-ring analogue (tropolone methyl ether) suggest that the A and C rings are involved in the binding of MDC.

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV).

Tubulin exists as various isoforms, which differ in their assembly, drug-binding properties, and the dynamic properties of the microtubules they compose. One of the most striking differences in drug binding among the isoforms is observed with colchicine, which binds much better to the alphabeta(II) and alphabeta(IV) isoforms than to the alphabeta(III) isoform. Here we have studied the interaction of these isoforms with 2-methoxy-5-(2',3',4'-trimethoxyphenyl) tropone (MTPT), an analog of colchicine that lacks the B-ring. The kinetics of association and dissociation were studied fluorometrically, and the kinetic parameters for the two-step binding were determined for different beta-tubulin isoforms. The apparent on-rate constants for alphabeta(II), alphabeta(III) and alphabeta(IV) were 13358, 4558 and 10828 M(-1) s(-1), the off-rate constants (k(-2)) were 0.04, 0.03 and 0.02 s(-1), and the affinity constants are 3.33 x 10(5), 1.56 x 10(5) and 5.44 x 10(5) M(-1), respectively. The differences in kinetic parameters among different beta-tubulin isoforms are greatly reduced when the B-ring is removed. Our results indicate that the B-ring plays a major role in determining the isoform differences, and the results might be of importance for designing tissue-specific analogs of colchicine for cancer chemotherapy.

Colchicine binding to tubulin monomers: a mechanistic study.

The kinetic and thermodynamic parameters for colchicine-tubulin and deacetamidocolchicine-tubulin interaction, under the condition where tubulin is predominantly in its dissociated state (approximately 80% monomer), have been determined. We observe that the kinetic parameters exihibit marked change when colchicine interacts with the monomeric form of tubulin rather than with the dimeric form of tubulin. The reaction of colchicine with tubulin monomers is characterized by an enhanced association rate which is a consequence of the lowering of activation energy. Colchicine-tubulin interaction, which is only poorly reversible, becomes partially reversible under this condition. Differences were also noticed in the thermodynamic parameters: the reaction of colchicine with tubulin monomers is enthalpy driven with small positive entropy, while with tubulin dimers a large positive entropy change was reported. However, no such changes in the binding parameters were observed for the reaction involving deacetamidocolchicine (a colchicine analog devoid of a side chain at the C-7 position of B-ring) with tubulin monomers. We therefore conclude that a single subunit of tubulin is capable of binding colchicine and that the unusual properties of colchicine-tubulin interactions such as the slow association rate, high activation energy, and the poor reversibility are due to the possible contact(s) of the C-7 substituent (in the B-ring) of colchicine with the other subunit of tubulin.

Response of microtubules to the addition of colchicine and tubulin–colchicine: evaluation of models for the interaction of drugs with microtubules

The effects of free drug and tubulin-drug complexes on steady-state GTP/GDP-associated microtubules and on equilibrium guanosine 5'[beta,gamma-imido]triphosphate-associated microtubules are compared. The addition of colchicine or the tubulin-colchicine complex (TuCol) to steady-state microtubules induces microtubule disassembly. Only limited disassembly of equilibrium microtubules is observed under similar conditions. Addition of colchicine or the bifunctional colchicine analogue 2-methoxy-5-(2'3',4'-trimethoxyphenyl)tropone to preassembled steady-state or equilibrium microtubules does induce disassembly, but establishment of the new steady state or equilibrium is very slow. These observations are related to the fact that TuCol readily adds to the microtubule end, but is only incorporated into the lattice with difficulty. As a result, microtubule growth is effectively inhibited and the critical concentration is significantly increased. Nevertheless, drug-induced disassembly can be extremely slow, because the frequency of addition reactions increases as the concentration of soluble dimers increases. The efficiency of incorporation of TuCol decreases as it concentration increases. The work further confirms the existence of colchicine-binding sites with low affinity (association constant KMT approximately 3 x 10(2) M-1) along the microtubule lattice. This value suggests that part of the colchicine-binding site on tubulin remains available in the polymer. The interaction of colchicine with these sites has no appreciable effect on microtubule dynamics. These observations are reproduced and rationalized by the model described elsewhere [Vandecandelaere, Martin, Bayley and Schilstra (1994) Biochemistry 33, 2792-2801], and the possibility that there are co-operative effects in the inhibition is considered.

A/C-Ring Colchicine Analogues: a Comparison of Molecular Conformations of the Minimized and Crystal Structures

Molecular mechanics and molecular orbital calculations have been used to determine the low-energy conformations of six biaryl analogues of colchicine lacking the seven-membered B-ring. A comparison of the conformations resulting from the different minimizations has been made, and these conformations were also compared with those found in the solid state for the respective biaryl analogues and the A/C-ring systems of colchicine and isocolchicine. The barriers to rotation about the A/C-linkage of the analogues were estimated from rotational plots. The MM+ calculations were not satisfactory for estimating the barriers, whilst the MMX, MAXIMIN2 and AM1 values, although agreeing on average only to within 16 kJ mol-1 , exhibited the expected trend in magnitude. This trend, however, did not correlate with the inhibition of tubulin polymerization to microtubules.

Zyn-Linked colchicines: Controlled-release lipophilic prodrugs with enhanced antitumor efficacy

Conjugation of antimitotic colchicine derivatives with proprietary lipophilic molecules (Zyn-Linkers™) via acid cleavable linkages (hydrazone or imine) produced prodrugs with enhanced antitumor activity. The pharmacodynamic properties (half-lives) of the pH-sensitive linkages were determined around values which might be expected in intracellular, especially lysosomal, environments. In buffered solutions at pH 4.1, 5.6, and 7.2, conjugates released 50% of the drug at rates from 0.1 h at pH 4.1 to over 3 months at neutral pH. In a tumor cell cytotoxicity assay, the slowest releasing molecules were up to 100-fold less active in vitro than the unlinked drug, which demonstrated that the linkages were stable and that a true prodrug had been generated. Binding of conjugate to cells, release of active drug and action on an intracellular target (tubulin) were demonstrated by the ability of the conjugates to block cells in the G2/M phase of the cell cycle 20 h after a 10 min exposure and was consistent with the rate of drug released. In vivo, a single injection of the Zyn-Linked conjugated colchicines enhanced the survival time of the mice to a greater extent than single doses of the unlinked colchicine analogues in a murine tumor model. Activity of the slowest releasing conjugate (ZYN 162) was shown to be comparable to a single LD 10 dose of doxorubicin. These data suggest that the Zyn-Linker conjugates described containing acid cleavable bonds form a body depot of prodrug which release an anti-mitotic agent and can significantly inhibit tumor growth.