Antitumor agents. 141. Synthesis and biological evaluation of novel thiocolchicine analogs: N-acyl-, N-aroyl-, and N-(substituted benzyl)deacetylthiocolchicines as potent cytotoxic and antimitotic compounds.

Three series of novel thiocolchicine analogs, N-acyl-, N-aroyl-, and N-(substituted benzyl)-deacetylthiocolchicinoids, have been synthesized and evaluated for their cytotoxicity against various tumor cell lines, especially solid tumor cell lines, and for their inhibitory effects on tubulin polymerization in vitro. Most of these compounds showed strong inhibitory effects on tubulin polymerization comparable to that obtained with thiocolchicine and greater than that obtained with colchicine. Only compounds with a long side chain at the C(7) position, such as 22-24, did not inhibit tubulin polymerization. Several of the active N-aroyldeacetylthiocolchicine analogs had positive optical rotations, in contrast to the negative optical rotation observed with most colchicinoids. This property might be attributed to a reversal of biaryl configuration from the normal aS to aR. Therefore, the N-aroyl analogs were further evaluated by circular dichroism, which readily distinguishes between the aS and aR biaryl configurations. This latter technique demonstrated that the active N-aroyl analogs do have an aS configuration despite their positive optical rotations. However, comparison of 1H NMR and UV spectral data of N-(substituted benzyl)-deacetylthiocolchicines with those of corresponding N-aroyldeacetylthiocolchicines suggested a different biaryl dihedral angle [even though these compounds have the same aS biaryl configuration]. The similar tubulin binding properties of these compounds suggest that a biaryl dihedral angle of 53 degrees is not essential for colchicinoid-tubulin interaction. The increased cytotoxicity of N-(substituted benzyl)deacetylthiocolchicines compared to the N-aroyldeacetylthiocolchicines may be attributed to different lipophilicity, drug uptake, or drug metabolism in the tumor cells. The side chain at the C(7) position affects inhibition of tubulin polymerization and the cytotoxic activity of colchicinoids as a function of its size and its contribution to lipophilicity.

Synthesis and tubulin binding of novel C-10 analogues of colchicine.

A series of novel C-10 derivatives of colchicine have been prepared and evaluated for inhibition of in vitro microtubule assembly and of [3H]colchicine binding to tubulin. The C-10 substituent of colchicine was replaced by halogens, alkyl and alkoxy groups, and hydrogen. Many of these compounds are available by nucleophilic substitution of 10-fluoro-10-demethoxycolchicine (9) without concomitant formation of ring contraction products. Compound 9 is prepared by reaction of (diethylamino)sulfur trifluoride with colchiceine. Unlike most reactions of colchiceine, the colchicine rather than the isocolchicine regiosiomer is the predominant product of this reaction. It was found that modification of the C-10 substituent of colchicine had a relatively minor effect on the potency of the colchicinoids. The electronic nature of the substituent had no significant effect on the efficacy of the compound, indicating that hydrogen bonding or polar interactions between the C-10 substituent of colchicinoids and an amino acid in the colchicine binding site on tubulin are not present in the colchicine-tubulin complex. A decrease in activity was observed with increasing length of the alkyl chain bonded to the C-10 position, but potency was less affected when the alkyl groups were positioned in close proximity to the C-10 carbon of the tropone ring. It is concluded that the steric rather than the electronic properties of the C-10 substituent are the predominant determinants of activity in this series.

Effect of the B ring and the C-7 substituent on the kinetics of colchicinoid-tubulin associations.

The kinetics of four B-ring derivatives of colchicine binding to tubulin has been examined quantitatively. The bindings of deacetamidocolchicine, deacetylcolchicine, demecolcine, and N-methyl-demecolcine to tubulin were biphasic processes. The association rate constants were determined as a function of temperature, and the thermodynamic parameters for the transition states of the fast phase were calculated. The kinetic parameters for the formation of the deacetylcolchicine-tubulin, demecolcine-tubulin, and N-methyldemecolcine-tubulin complexes were very similar to each other, but different from the parameters for the colchicine-tubulin association. In particular, the global activation enthalpies for the formation of the three aminocolchicinoid-tubulin complexes were 3-5 kcal/mol greater than the global activation enthalpy of colchicine binding to tubulin. These results indicate that electronic rather than steric properties of the B-ring substituent are of greater importance in the activation enthalpy of colchicinoids binding to tubulin. In contrast, the global activation enthalpy for deacetamidocolchicine, which lacks a substituent on the C-7 carbon, binding to tubulin was virtually identical to the global activation enthalpy previously found for the colchicine analog that lacks the B ring, 2-methoxy-5-(2,3,4-trimethoxyphenyl)tropone, binding to tubulin (Bane, S., Puett, D., Macdonald, T. L., & Williams, R. C., Jr. (1984) J. Biol. Chem. 259, 7391-7398). This result demonstrates that the carbons of the B ring are not involved in the transition state for the formation of colchicinoid-tubulin complexes. The first-order dissociation rate constants of the colchicinoid-tubulin complexes were determined at 37 degrees C. The dissociation profiles of the colchicinoid-tubulin complexes also consisted of two phases.(ABSTRACT TRUNCATED AT 250 WORDS)

Antitumor agents. 139. Synthesis and biological evaluation of thiocolchicine analogs 5,6-dihydro-6(S)-(acyloxy)- and 5,6-dihydro-6(S)-[(aroyloxy)methyl]-1,2,3-trimethoxy-9-(methylthio)-8H- cyclohepta[a]naphthalen-8-ones as novel cytotoxic and antimitotic agents.

A series of novel thiocolchicine analogs, 5,6-dihydro-6(S)-(acyloxy)-and 5,6-dihydro-6(S)-[(aroyloxy)-methyl]-1,2,3-trimethoxy-9-(methylthi o)-8H- cyclohepta[a]naphthalen-8-ones, possessing a six-membered ring B, have been synthesized and evaluated for their cytotoxicity against various tumor cell lines, including solid tumor cell lines, and for their interaction with tubulin. The configuration of the parent alcohol (compound 5) was established unequivocally as (aR,6S) by X-ray crystallographic analysis. The side chain at the C(6) position is in a pseudoaxial orientation. The optical properties and 1H NMR data indicated that these compounds have the same conformations in solution as in the solid state. Biological results showed that compounds (5, 6, 14, 15, 17, and 18) bearing a small side chain at C(6) demonstrate high potency in inhibiting tubulin polymerization and binding of radiolabeled colchicine to tubulin. The most cytotoxic compounds were 14, 15, 17, and 18, with good activity against several solid tumor cell lines. To explain the strong antitubulin activity of compound 5 (with an aR configured biaryl system in contrast to the aS configuration previously described for colchicinoids, allocolchicinoids, and steganacin) we speculate that a rapid atropisomerism equilibrium must exist for 5 and its active derivatives. This equilibrium would yield adequate amounts of aS-configured conformers that interact, strongly with tubulin. Since the optically inactive 18 is also a potent inhibitor of tubulin, the configuration of the side chain of these six-membered ring B analogs cannot be essential for their binding to tubulin. Instead we propose that the size of ring B and of its side chain play important roles in tubulin binding activity by affecting the rotation of the rings A and C along their linking C-C bond axis.

Role of the B-ring substituent in the fluorescence of colchicinoid-tubulin and allocolchicinoid-tubulin complexes.

Fluorescence spectra of several B-ring derivatives of allocolchicine in a variety of solvents have been obtained. The quantum yields of the allocolchicine derivatives decreased with amine substitution at the C-7 position [R = H greater than NH2 much greater than NHCH3 greater than N(CH3)2]. The relative fluorescent intensities of the aminoallocolchicinoids [R = NH2, NHCH3, N(CH3)2] bound to tubulin were significantly less than the fluorescent intensities of tubulin-bound allocolchicine (R = NHCOCH3) and deacetamidoallocolchicine (R = H). The low fluorescent intensities of the aminoallocolchicinoids in solvents and bound to tubulin could be explained by exciplex formation between the lone pair of electrons on the nitrogen and the allocolchicinoid pi-system, which leads to quenching of the allocolchicinoid fluorescence. Direct evidence for exciplex formation between the C-7 amine nitrogen and the allocolchicinoid pi-system was found in the emission spectrum of N-methyldemeallocolchicine [R = N(CH3)2] in dioxane. The quantum yields of the aminoallocolchicinoids in glycerol were higher than would be predicted on the basis of solvent dielectric effects. Glycerol appears to increase the quantum yields of the aminoallocolchicinoids through both viscosity and hydrogen-bonding effects. The latter effect serves to sequester the lone pair of electrons on the nitrogen, decreasing its ability to interact with the pi-system of the allocolchicinoid. It is concluded that the fluorescent properties of colchicinoids and allocolchicinoids in glycerol are not reliable indicators of the fluorescent properties of the molecules bound to tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of B-ring substituents on absorption and circular dichroic spectra of colchicine analogues.

Near-ultraviolet absorption and circular dichroic spectra of several B-ring derivatives of colchicine have been obtained in a variety of solvents. The spectra of the molecules in solvent were analyzed and compared with spectra of the molecules bound to tubulin. Absorption spectra of deacetamidocolchicine, deacetylcolchicine, demecolcine, and N-methyldemecolcine [B-ring substituents = H, NH2, NHCH3, and N(CH3)2, respectively] were analyzed by multiple differentiation of the spectrum. It was found that an amine substituent at the C-7 position on the B-ring of the colchicinoid affected the higher energy transition of the near-ultraviolet spectra of the colchicinoid in the absence of tubulin in a manner consistent with a hyperconjugative alteration of this transition. The fourth derivatives of the absorption spectra of all four molecules bound to tubulin were similar to each other and to colchicine. As was true in the case of colchicine, the negative near-ultraviolet circular dichroic band of the aminoclochicinoids was relatively unaffected by solvent, but the molar ellipticity of the band was greatly reduced with tubulin binding. It is concluded that the binding site environments of the B-ring analogues of colchicine, as probed by absorption and circular dichroic spectroscopy, are equivalent.

Synthesis and evaluation of 2-diazo-3,3,3-trifluopropanoyl derivatives of colchicine and podophyllotoxin as photoaffinity labels: reactivity, photochemistry, and tubulin binding.

Derivatives of the tubulin polymerization inhibitors colchicine and podophyllotoxin bearing the photoreactive 2-diazo-3,3,3-trifluoropropanoyl (DTFP) group were synthesized for evaluation as potential photoaffinity labels of the tubulin binding site. All labels were assayed for their ability to inhibit tubulin polymerization, and N-DTFP-deacetylthiocolchicine was shown to competitively inhibit tubulin-colchicine binding with a Ki of 4-5 microM. The tubulin off-rate of this analog was similar to that of podophyllotoxin, rather than to the relatively irreversibly bound colchicine. Photochemical solvent insertion reactions of the labels were investigated. Radioactive samples of the two most active labels were prepared and used in initial protein-labeling experiments, during which the fractional occupancy of tubulin and extent of covalent incorporation were determined. A rearrangement of DTFP amides was encountered which is relevant to the utility of this moiety for use in synthesis of photoaffinity labels.

Interactions of colchicine with tubulin.

Colchicine exerts its biological effects through binding to the soluble tubulin heterodimer, the major component of the microtubule. The colchicine-binding abilities of tubulins from a variety of sources are summarized, and the mechanism of colchicine binding to brain tubulin is explored in depth. The relationship between colchicinoid structure and tubulin binding activity provides insight into the structural features of colchicine responsible for high affinity binding to tubulin and is reviewed for analogs in the colchicine series. The thermodynamic and kinetic aspects of the association are described and evaluated in terms of the binding mechanism. Colchicine binding to tubulin results in unusual alterations in the low energy electronic spectra of colchicine. The spectroscopic features of colchicine bound to tubulin are discussed in terms of the nature of the colchicine-tubulin complex. Attempts to locate the high affinity colchicine binding site on tubulin are presented.

Binding of colchiceine to tubulin. Mechanisms of ligand association with tubulin.

Colchiceine, a closely related structural analog of colchicine possessing a C-ring tropolone, has been shown to be a potent inhibitor of microtubule assembly in vitro (I50 = 20 microM). The mechanism of inhibition is mediated through binding to tubulin (KA = 1.2 +/- 0.7 x 10(4) M-1), although potentially not through the colchicine receptor site. Supporting the hypothesis of an alternate receptor are the observation of colchiceine binding to the isolated colchicine-tubulin complex (KA = 2.2 +/- 1.0 x 10(4) M-1), the poor correlation between the competitive inhibition of colchicine binding (KI = 125 microM) and the inhibition of microtubule assembly, and different structure-activity relationships for colchiceine analogs as compared to the colchicine series.

Effect of tubulin binding and self-association on the near-ultraviolet circular dichroic spectra of colchicine and analogues.

Near-UV circular dichroic (CD) spectra of three colchicine analogues that differ at the C-10 position have been obtained in the presence and absence of tubulin. All three colchicine analogues show dramatic alterations in the low-energy near-UV CD band upon tubulin binding that cannot be mimicked by solvent, but in no event does the rotational strength of the CD band decrease to nearly zero as in the case of colchicine [Detrich, H. W., III, Williams, R. C., Jr., Macdonald, T. L., & Puett, D. (1981) Biochemistry 20, 5999-6005]. The effect of self-association of colchicine and one of the C-10 analogues, thiocolchicine, on the near-UV CD band was also investigated. A qualitative similarity was seen between the near-UV CD spectra of colchicine and thiocolchicine dimers and the spectra of these molecules bound to tubulin. These observations support the previous suggestion that ligands bound to the colchicine site on tubulin may be interacting with an aromatic amino acid in the colchicine binding site [Hastie, S. B., & Rava, R. P. (1989) J. Am. Chem. Soc. 110, 6993-7001].

Spectroscopic and kinetic features of allocolchicine binding to tubulin.

Allocolchicine is a structural isomer of colchicine in which colchicine's tropone C ring is replaced with an aromatic ester. In spite of the structural differences between the two ligands, the association parameters for both molecules binding to tubulin are quite similar. The association constant for allocolchicine binding to tubulin was determined by fluorescence titration to be 6.1 x 10(5) M-1 at 37 degrees C, which is about a factor of 5 less than that of the colchicine-tubulin association. In particular, analysis of the kinetics of the association of allocolchicine with tubulin yielded nearly equivalent activation parameters for the two ligands. The activation energy of the allocolchicine binding reaction was found to be 18.4 +/- 1.5 kcal/mol, which is only slightly less than the activation energy for colchicine binding to tubulin. This finding argues against conformational flexibility of the C ring as the structural feature of colchicine responsible for the slow kinetics of colchicinoid-tubulin binding reactions. Tubulin binding promote a dramatic enhancement of allocolchicine fluorescence. Unlike colchicine, the emission energy and intensity of the tubulin-bound allocolchicine fluorescence can be mimicked by solvent, and a general hydrophobic environment for the ligand binding site is indicated. The excitation spectrum of the protein-bound species, however, is shown to possess two bands which center at higher and lower energy than the energy maximum of the spectrum of the ligand in apolar solvents, indicating that properties of the colchicine binding site in addition to a low dielectric constant contribute to the fluorescence of the bound species.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of thiocolchicine with tubulin.

Thiocolchicine, a colchicine analog in which the C-10 methoxy is replaced with a thiomethyl moiety, was shown to bind with high affinity to the colchicine site on tubulin (Ka = 1.07 +/- 0.14 x 10(6) M-1 at 23 degrees C). Like colchicine, the association kinetics were biphasic, and the rate constants of both phases were temperature dependent. The rate constant of the fast phase of the association was 4 times greater than the rate constant for colchicine binding, and the activation energy was lower (19.1 +/- 1.8 kcal/mol). X-ray crystallographic analysis shows that thiocolchicine displays greater puckering of the tropone C ring than colchicine (Koerntgen, C. and Margulis, T. N. (1977) J. Pharm. Sci. 66, 1127-1131.). These results indicate that the conformation of the C ring may have little effect on the energetics of colchicinoids binding to tubulin.

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin.

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.