A steroid derivative with paclitaxel-like effects on tubulin polymerization

Article abstract of DOI:10.1124/mol.57.3.568

The endogenous estrogen metabolite 2-methoxyestradiol has modest antimitotic activity that may result from a weak interaction at the colchicine binding site of tubulin, but it nevertheless has in vivo antitumor activity. Synthetic efforts to improve activ

Full text of DOI:10.1124/mol.57.3.568

0026-895X/00/030568-08
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MOLECULAR PHARMACOLOGY, 57:568–575 (2000).
A Steroid Derivative with Paclitaxel-Like Effects on Tubulin
Polymerization
PASCAL VERDIER-PINARD, ZHIQIANG WANG, ARASAMBATTU K. MOHANAKRISHNAN, MARK CUSHMAN, and
ERNEST HAMEL
Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis,
National Cancer Institute, Frederick Cancer Research and Development Center, Frederick, Maryland (P.V.-P., E.H.); and Department of
Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, Indiana
(Z.W., A.K.M., M.C.)
Received September 2, 1999; accepted December 10, 1999
This paper is available online at http://www.molpharm.org
ABSTRACT
The endogenous estrogen metabolite 2-methoxyestradiol has apparently distinct modes of action. Simple expansion of the B
modest antimitotic activity that may result from a weak inter- ring to seven members resulted in a compound comparable to
action at the colchicine binding site of tubulin, but it neverthe- 2EE in its ability to inhibit tubulin polymerization and colchicine
less has in vivo antitumor activity. Synthetic efforts to improve binding to tubulin. Acetylation of the hydroxyl groups in this
activity led to compounds that increased inhibitory effects on analog and in 2EE essentially abolished these inhibitory prop-
cell growth, tubulin polymerization, and binding of colchicine to erties. The introduction of a ketone functionality at C6, together
tubulin. This earlier work was directed at modifications in the with acetylation of the hydroxyls at positions 3 and 17, pro-
steroid A ring, which is probably analogous to the colchicine duced a compound with activity similar to that of paclitaxel, in
tropolonic C ring. One of the most active analogs prepared was that the agent enhanced tubulin polymerization into polymers
2-ethoxyestradiol (2EE). We report here that different modifica- that were partially stable at 0°C. The acetyl group at C17, but
tions in the steroid B ring of 2EE yield compounds with two not that at C3, was essential for this paclitaxel-like activity.
Tubulin, the building block of microtubules, is the target of tropolonic A ring analogs of 2ME to enhance the analogy to
many antimitotic drugs (for a review, see Hamel, 1996). the C ring of colchicine and found several of these to be highly
Three major pharmacological sites are present on tubulin: active inhibitors of tubulin assembly. Because allocolchici-
the colchicine site, the vinca alkaloid domain, and the taxoid noids, with a seven-member B ring but six-member C ring,
site. The latter is fully expressed only in tubulin polymers such as compound 1 (Iorio, 1984), are often more active than
with a substructure of well defined protofilaments (Parness the corresponding colchicinoids (Kang et al., 1990), we de-
and Horwitz, 1981; Takoudju et al., 1988). Among antimitotic cided to synthesize steroid derivatives with an expanded B
agents that appear to bind at the colchicine site are synthetic ring, with the two isomers of compound 2 being our ultimate
analogs of estradiol, such as diethylstilbestrol and the major target. We were encouraged to find the intermediate com-
endogenous metabolite of estradiol, 2-methoxyestradiol pound 5 (see reaction scheme in Fig. 2) active as an inhibitor
(2ME; structure in Fig. 1, with comparison with the structure of assembly, but ketone 8, representing the next step in the
of colchicine; D’Amato et al., 1994). 2ME also has significant synthesis, after deprotection of 7, was inactive.
antiangiogenic properties and in vivo antitumor activity (Fot-
In our previous work, we often analyzed protected estradiol
sis et al., 1994; Klauber et al., 1997). Based on the ability of analog intermediates bearing acetyl groups at positions C2
2ME to inhibit the binding of colchicine to tubulin, we pro- and/or C17. Invariably, these compounds had minimal or no
posed that its A ring might bear structural analogy to the C effect on tubulin polymerization (Cushman et al., 1995,
ring of colchicine.
1997). When a similar screening assay was performed with
These findings have led to synthetic efforts to prepare compound 7 (Fig. 2), the diacetate of 8, we observed a stim-
steroid derivatives with better activity than 2ME. Among the ulation of assembly qualitatively similar to that which occurs
compounds we prepared, 2-ethoxyestradiol (2EE) had greater with the potent anticancer drug paclitaxel (Schiff et al., 1979;
inhibitory effects on tubulin polymerization and was 10-fold Hamel et al., 1981; Schiff and Horwitz, 1981). This study
more cytotoxic than 2ME (Cushman et al., 1995). Taking describes our initial analysis of this observation, as well as
another tack, Miller et al. (1997) prepared seven-member the expected inhibitory effects of compound 5. Finally, we
ABBREVIATIONS: 2ME, 2-methoxyestradiol; 2EE, 2-ethoxyestradiol; MAP, microtubule-associated protein; MES, 2-(N-morpholino)ethanesul-
fonate.
568

A Paclitaxel-Like Steroid Derivative
569
prepared and evaluated the two monoacetate analogs 9 and were all examined for inhibitory effects on tubulin polymer-
10 (Fig. 2) and demonstrated that 9 was inactive with tubulin ization and on the binding of [3H]colchicine to tubulin. Only
but that 10 was almost as active as 7.
compound 5 had significant activity, and in Table 1 we sum-
marize our findings, together with simultaneously obtained
data with 2ME and 2EE. [The compound resulting from
deacetylation of compound 3, as previously reported (Cush-
man et al., 1997), does inhibit tubulin assembly and colchi-
cine binding, with activities differing little from those of
2ME.] Estramustine phosphate was also examined and was
found to be much less potent as an inhibitor of tubulin poly-
merization, in agreement with the results of Da¨hlof et al.
(1993). Estramustine phosphate also interacts with MAPs
[see Tew et al. (1992) for a review]. Expanding the steroid B
ring to seven members failed to improve on the activity
Experimental Procedures
Materials. Electrophoretically homogeneous bovine brain tubulin
and heat-treated microtubule-associated proteins (MAPs; Hamel and
Lin, 1984), 2EE (Cushman et al., 1995), and compound 3 (Cushman
et al., 1997) were prepared as described previously. The synthesis of
compounds 5 to 10 was performed as outlined in Fig. 2, with details
of their preparation to be presented elsewhere. GTP, repurified by
anion exchange chromatography, was obtained from Sigma Chemi-
cal Co. (St. Louis, MO). 2ME was obtained from Aldrich Chemical
(Milwaukee, WI). [3H]Colchicine was purchased from DuPont-New
England Nuclear (Boston, MA). Paclitaxel and [3H]paclitaxel were observed with 2EE in either assay. As an inhibitor of assem-
obtained from the Drug Synthesis and Chemistry Branch, National
Cancer Institute. Estramustine phosphate was a generous gift from
Kabi Pharmaceuticals (Helsingborg, Sweden).
Methods. Tubulin polymerization was followed turbidimetrically
at 350 nm in Gilford model 250 spectrophotometers equipped with
electronic temperature controllers. Temperature in the cuvettes
rises at ϳ0.5°C/s and falls at ϳ0.1°C/s in the 0–37°C range. Specific
reaction conditions are described for the individual experiments.
bly, compound 5 was closer in activity to 2EE, but as an
inhibitor of colchicine binding, its activity was closer to that
of 2ME. We emphasize that compound 6, the diacetate of 5,
neither inhibited colchicine binding to tubulin nor signifi-
cantly affected tubulin assembly. Estramustine phosphate
had no effect on the binding of [3H]colchicine to tubulin,
which is consistent with reports of others (Laing et al., 1997;
Panda et al., 1997) that this agent may have a distinct
binding site on tubulin.
Results
Stimulation of Tubulin Assembly Induced by Gluta-
Inhibition of Tubulin Polymerization by Compound mate by Compounds 7 and 10. In Fig. 3A, the effect of
5. The newly synthesized steroid derivatives described here compound 7 on glutamate-induced assembly is presented.
Fig. 1. Structures of colchicine,
2ME, 2EE, and related com-
pounds. The chemical names of
compounds 1 and 2 are as follows:
compound 1, N-acetylcolchinol O-
methyl ether; compound 2, 6-acet-
amido-2-ethoxy-B-homo-estra-
1,3,5(10)-trien-3,17␤-diol.

570
Verdier-Pinard et al.
Increasing amounts of the agent, up to ϳ10 ␮M, which is near at higher concentrations of compound 7. Deacetylation of com-
stoichiometric with the 12 ␮M tubulin concentration, resulted pound 7 to yield compound 8 eliminated this stimulatory activ-
in progressively more rapid reactions that all reached the same ity, as shown by curve 6 in Fig. 3A. The reaction mixture
turbidity plateau. The polymer formed was highly stable at 0°C represented by curve 6 contained 40 ␮M compound 8.
Fig. 2. Synthetic pathway to diacetate 7 and monoacetates 9 and 10. The reagents and reaction conditions indicated by the letters above the arrows
are as follows: a, KOH, methanol, 4 h at room temperature; b, tert-butyldimethylsilyl chloride, imidazole, N,NЈ-dimethylformamide, 18 h at room
temperature; c, first CH2Br2, lithium diisopropylamide, tetrahydrofuran, Ϫ78°C, 3 h, and then butyllithium added and the temperature increased to
0°C, 2.5 h; d, tetrabutylammonium fluoride, tetrahydrofuran, 4 h at room temperature; e, tosylhydrazine, methanol, 24 h at room temperature; f, first
catecholborane, CHCl3, and then sodium acetate, water, refluxed for 3 h; g, acetic anhydride, pyridine, 24 h at room temperature; h, CrO3, acetic acid,
12–14°C for 40 min; i, KOH, methanol, 3.5 h at Ϫ5°C to room temperature; j, KHCO3, methanol, 65°C for 1 h; and k, 1-acetyl-1H-1,2,3-triazolo[4,5-
b]pyridine, aqueous NaOH, tetrahydrofuran, 1.5 h at room temperature. Ac, acetyl; Et, ethyl; TBDMS, tert-butyldimethylsilyl; Ts, tosyl. The chemical
names of compounds 7 to 10 are as follows: compound 7, 3,17␤-diacetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-triene; compound 8, 2-ethoxy-6-oxo-
B-homo-estra-1,3,5(10)-trien-3,17␤-diol; compound 9, 3-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-17␤-ol; and compound 10, 17␤-acetoxy-2-
ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol.

A Paclitaxel-Like Steroid Derivative
571
Compounds 9 and 10 were synthesized to determine observed with 10 ␮M compound 7 or 10. Compounds 7 and 10
whether both acetyl groups were required for this ability to had no inhibitory effect on the binding of [3H]colchicine to
enhance polymer formation. The addition of a single acetyl at tubulin.
position C3 yielded the inert compound 9, but the addition of
Stimulation by Compounds 7 and 10 of MAP-Induced
a single acetyl group at C17 yielded compound 10 with activ- Tubulin Assembly. As shown in Fig. 4, in experiments with
ity only slightly less than that of compound 7 (Fig. 3B). Much the optimum amount of our current MAP preparation (0.75
less dramatic paclitaxel-like effects were also observed with mg/ml MAP with 1.0 mg/ml tubulin), compounds 7 and 10
compound 3 and the ketone diacetate of compound 4, with enhanced tubulin assembly at 25°C, although their activities
these compounds at 40 ␮M having effects less than those appeared to be quantitatively lower than had been the case in
the glutamate reaction condition. A progressive increase in
TABLE 1
activity with up to 40 ␮M agent was seen with both com-
pounds (data presented in full only for compound 7), and at
all concentrations compound 10 was less stimulatory than 7
(Fig. 4 presents data with compound 10 only at 10 ␮M). In
addition, Fig. 4 demonstrates the effect of 10 ␮M paclitaxel
on the assembly reaction. With a temperature jump directly
from 0–25°C, the assembly reaction with paclitaxel was
much more extensive than that with compound 7 (not
shown). In part, this could be attributed to assembly stimu-
lated by paclitaxel that occurred before temperature equili-
bration (Grover et al., 1995), and this could be clearly dem-
onstrated by adding a 10°C step to the reaction sequence, as
was done in the experiment presented in Fig. 4.
Inhibition of tubulin polymerization and the binding of [3H]colchicine
to tubulin by compound 5, 2ME, and 2EE
Tubulin polymerization and colchicine-binding assays were performed as described
previously (D’Amato et al., 1994). In the polymerization assay, 12 ␮M tubulin was
preincubated with varying drug concentrations for 15 min at 26°C before the addi-
tion of GTP. The assembly reactions were followed turbidimetrically for 20 min at
26°C, and drug concentrations required for 50% reduction in the plateau turbidity
reading were determined. In the colchicine-binding assays, reaction mixtures con-
tained 1.0 ␮M tubulin, 5.0 ␮M [3H]colchicine, and inhibitors, if present, at 50 ␮M.
Incubation was for 10 min at 37°C. Previous studies have shown that at this time
point, the colchicine-binding reaction was about 50% complete in the control reaction
mixtures under the reaction conditions used. All experiments were performed twice,
and standard deviations are shown. In the colchicine-binding experiments, the
individual assays were also performed with duplicate samples.
Inhibition of
Polymerization
IC50
Inhibition of
Colchicine
Binding
Compound
It was possible that compounds 7 and 10 were not actually
acting like paclitaxel in enhancing microtubule assembly and
in stabilizing the microtubules formed in the course of the
turbidity studies shown in Fig. 4 but rather that these agents
were causing the formation of polymers of aberrant morphol-
ogy and temperature stability different from microtubules,
such as occurs with vinca alkaloids (Himes, 1991) or curacin
A (Hamel et al., 1995). We therefore examined polymer mor-
phology in the electron microscope. With compound 7, abun-
dant microtubules were formed that were indistinguishable
from those formed in the absence of drug. Even more impor-
tant, when the reaction mixtures were returned to 0°C for 15
min, no microtubules were visualized on grids prepared from
the reaction mixture without drug. In the presence of 40 ␮M
␮M Ϯ S.D.
1.3a
2.2 Ϯ 0.3
1.0 Ϯ 0.07
120 Ϯ 20
% inhibition Ϯ S.D.
5
2ME
2EE
56 Ϯ 7
60 Ϯ 10
87 Ϯ 4
0a
Estramustine phosphate
a Same value obtained in both experiments.
Fig. 4. Enhancement of MAP-induced tubulin assembly by compounds 7
and 10. Each reaction mixture contained 0.1 M 2-(N-morpholino)ethane-
sulfonate (MES) (pH 6.9 with NaOH in 0.5 M stock solution), 0.1 mM
GTP, 10 ␮M (1.0 mg/ml) tubulin, 0.75 mg/ml heat-treated MAPs, 4%
DMSO, and drug as indicated. Baselines were established at 0°C, with
drug added last, and the cuvette contents were observed for 10 min. There
was no change in turbidity in any reaction mixture (not shown). At this
point, the temperature controller was set at 10°C, and subsequent tem-
perature changes, as indicated, were made at the times indicated by the
vertical dashed lines. Curve 1, no drug; curve 2, 10 ␮M paclitaxel; curve
3, 10 ␮M compound 7; curve 4, 20 ␮M compound 7; curve 5, 40 ␮M
compound 7; and curve 6, 10 ␮M compound 10.
Fig. 3. Enhancement of glutamate-induced tubulin assembly by com-
pounds 7 (A) and 10 (B). Each reaction mixture contained 0.8 M monoso-
dium glutamate (pH 6.6 with HCl in 2 M stock solution), 0.4 mM GTP, 12
␮M (1.2 mg/ml) tubulin, 4% (v/v) DMSO, and drug as indicated. Baselines
were established at 0°C, and at time zero the temperature controller was
set at 26°C. At the time indicated by the dashed line, the temperature
controller was set at 0°C. A, curve 1, no drug; curve 2, 2 ␮M compound 7;
curve 3, 5 ␮M compound 7; curve 4, 10 ␮M compound 7; curve 5, 40 ␮M
compound 7; and curve 6, 40 ␮M compound 8. B, curve 1, no drug; curve
2, 2 ␮M compound 10; curve 3, 5 ␮M compound 10; curve 4, 10 ␮M
compound 10; and curve 5, 40 ␮M compound 10.

572
Verdier-Pinard et al.
7 (Fig. 5) or 10 ␮M paclitaxel (not shown), abundant cold concentrations, obtained at 30°C, of 1.4, 0.06, and 0.50 mg/ml
stable microtubules were observed. for MAP- and GTP-dependent assembly without drug and in
When either MAPs or GTP was omitted from the reaction the presence of 10 ␮M paclitaxel or 10 ␮M 7, respectively,
mixture, with 10 ␮M tubulin no reaction occurred with either and 0.20 and 1.4 mg/ml for GTP-dependent, MAP-indepen-
no drug or 40 ␮M compound 7. In contrast, with 10 ␮M dent assembly with 10 ␮M paclitaxel or 10 ␮M 7 (no reaction
paclitaxel, a reaction was observed in both cases (cf. Hamel et occurred without drug at tubulin concentrations up to 4 mg/
al., 1981; Grover et al., 1995).
Quantification of Relative Effects of Paclitaxel and
ml).
At first glance, these data suggest that the affinity of
Compound 7 on Tubulin Polymerization. It is clear from paclitaxel for tubulin polymers is only 7- to 8-fold greater
the data of Fig. 4 that the activities of 7 and 10 are signifi- than that of compound 7, but this method may significantly
cantly less than that of paclitaxel. We wanted to put some underestimate the quantitative difference between the two
quantitative measurement on the difference. We found, how- compounds. We previously observed with paclitaxel analogs
ever, that we could demonstrate only minimal inhibition by that differences between compounds are magnified in restric-
compound 7 of the binding of [3H]paclitaxel to tubulin poly- tive reaction conditions and minimized in favorable reaction
mer, nor was compound 7 active in room temperature gluta- conditions (Grover et al., 1995; Kingston et al., 1998). Anal-
mate-dependent assay systems (Hamel et al., 1999) where no ogously, in recently reported studies, we found sarcodictyin A
polymerization reaction occurs in the absence of drug. We to be significantly less potent than paclitaxel in its interac-
therefore returned to the MAP/GTP system, and we exam- tions with tubulin under restrictive reaction conditions, and
ined the effect of 10 ␮M compound 7 on 40 ␮M tubulin. We it only weakly inhibited the binding of radiolabeled paclitaxel
did observe assembly without MAPs but not without GTP. to tubulin polymer (Hamel et al., 1999). However, under a
We therefore decided that measuring the critical concentra- favorable reaction condition (at room temperature), the
tion with and without MAPs with paclitaxel and compound 7 quantitative difference between paclitaxel and sarcodictyin A
should provide some idea of the comparative activity of these was only 2.5-fold. Compound 7 was inactive in this room
agents. With MAPs, a constant weight ratio to tubulin of 1:3 temperature assay. We therefore also determined the critical
was used, because the suboptimal concentration of MAPs concentration with 10 ␮M sarcodictyin A under the reaction
caused greater differences in the critical concentrations ob- conditions shown in Fig. 6B, obtaining a value of 1.0 mg/ml in
tained. The data are presented in Fig. 6, which yield critical the GTP only system (data not presented). Relative order of
Fig. 5. Cold-stable microtubules formed in the presence of compound 7. The reaction mixture was identical with that indicated by curve 5 in Fig. 4.
Polymer was assembled during a 20-min incubation at 37°C, and the reaction mixture was then chilled at 0°C for 20 min. An aliquot was placed on
a carbon-coated, Formavar-treated, 200-mesh copper grid. The sample was washed off by a stream of droplets of 0.5% uranyl acetate, with excess stain
wicked from the grid with filter paper. The grid was examined in a Zeiss model 10CA electron microscope (Magnification: A, 9,900ϫ; B, 159,000ϫ).

A Paclitaxel-Like Steroid Derivative
573
compound activity thus persists, with sarcodictyin A being tubulin assembly, but with glutamate plus Mg2ϩ, assembly
ϳ5-fold less active than paclitaxel and 40% more active than rate was reduced with formation of a morphologically unal-
7, but this critical concentration assay probably overesti- tered polymer with enhanced temperature stability. The
mates the biochemical activity of less active taxoid-mimetic elimination of Mg2ϩ converted 2ME to a pure inhibitor of
compounds.
assembly (D’Amato et al., 1994). Studies with [3H]2ME
showed that binding of 2ME to unpolymerized tubulin was
strongly inhibited by colchicine site drugs. Binding of 2ME to
glutamate polymer occurred in a 1:1 stoichiometry with tu-
bulin in a reaction minimally inhibited by colchicine-site
drugs, suggesting a distinct binding site for 2ME on polymer
or that 2ME binds in a site inaccessible to colchicine in
polymer (Hamel et al., 1996). Binding of [3H]2ME to polymer
was minimally inhibited by paclitaxel (E.H., unpublished
data).
Discussion
We originally evaluated the interaction of 2ME with tubu-
lin because Seegers et al. (1989) found that the compound
caused accumulation of mitotic MCF-7 cells with malformed
spindles. We observed little effect of 2ME on MAP-dependent
In subsequent synthetic studies, enhanced cytotoxicity cor-
related well with enhanced inhibition of both glutamate-
induced assembly and colchicine binding, with one of the
more active analogs being 2EE (Cushman et al., 1995, 1997).
In these studies, acetylation of the C3 and/or C17 hydroxyl
groups led to a substantial loss of activity, and this finding
continues with the contrast between the inhibition obtained
with compound 5 and the inactivity of compound 6. Differing
from these observations with steroid derivatives that inhibit
tubulin polymerization are our findings with compounds 7 to
10. The diacetate compound 7 had taxoid-mimetic properties
in both glutamate- and MAP-induced assembly reactions,
whereas the dialcohol compound 8 was inactive. Removal of
the C17 acetyl group yielded the inactive compound 9, but
activity was largely retained after removal of the C3 acetyl
group (compound 10). Figure 7 summarizes our current
knowledge of structure-activity relationships of compounds
related to 2ME.
Fig. 6. Critical concentrations of tubulin with paclitaxel and compound 7
in reaction mixtures containing MAPs plus GTP (A) or GTP only (B).
Reaction mixtures (0.25 ml) contained 0.1 M MES (pH 6.9), 100 ␮M GTP,
4% DMSO, the indicated tubulin concentration (with heat-treated MAPs
in 1:3 weight ratio to the tubulin, A only), and either 10 ␮M paclitaxel (Œ),
10 ␮M compound 7 (E), or no drug (). Cuvette contents were equili-
brated at 0°C, drugs were added, temperature was jumped to 30°C (ϳ1
min), and the turbidity changes after a 20-min incubation were mea-
sured.
The taxoid-mimetic properties of compounds 7 and 10 in-
cluded more rapid induction of polymer at reduced tempera-
tures, formation of microtubules stable to disassembly at 0°,
reduction in the tubulin critical concentration, and polymer
formation in 0.1 M MES without MAPs. In contrast to pac-
litaxel (Hamel et al., 1981; Schiff and Horwitz, 1981; Grover
Fig. 7. Current summary of structure-activity relationships among steroid derivatives related to 2ME.

574
Verdier-Pinard et al.
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␤-tubulin peptides 1-31 (Rao et al., 1994) and 217-231 (Rao et
al., 1995) and with ␤-Arg282 (Rao et al., 1999), observations
consistent with the location of docetaxel in the model of
tubulin derived by Nogales et al. (1998) from zinc-induced
tubulin polymer. Direct photoactivation of colchicine by illu-
mination of the tubulin-colchicine complex at 350 nm (absor-
bance maximum caused by the tropolone C ring) resulted in
cross-links between radiolabeled colchicine and ␤-tubulin
peptides 1-36 and 214-241 (Uppuluri et al., 1993). The anal-
ogy we proposed between the colchicine C ring and the 2ME
A ring (D’Amato et al., 1994) is supported by the structure-
activity work of Miller et al. (1997), who synthesized steroid
derivatives with tropolonic A rings with enhanced antitubu-
lin activity. These findings are thus highly suggestive that
there may be at least some overlap between the site at which
paclitaxel binds on tubulin polymer and the site the colchi-
cine C ring occupies in unpolymerized tubulin.
In contrast to the colchicine direct photoaffinity study, a
thiocolchicine derivative with a chemically reactive chloro-
acetyl group replacing the C3-methoxy reacted preferentially
with ␤-Cys354 (Bai et al., 1996), whereas replacing the C2-
methoxy group with the chloroacetyl group yielded an analog
that reacted with both ␤-Cys354 and ␤-Cys239 (R. Bai and E.
Hamel, in preparation). This seems to place the A ring of
colchicine between these residues. Downing and Nogales
(1998) were able to use these observations to place colchicine
within their model in a region of tubulin distinct from that
occupied by docetaxel, but it remains possible that the bind-
ing sites for the two drugs possess some common structural
elements. Furthermore, there must be some significant con-
formational change in tubulin on its polymerization, because
colchicine will not bind to microtubules (Wilson and Meza,
1973; Lee et al., 1974) and paclitaxel will not bind to unpo-
lymerized tubulin (Parness and Horwitz, 1981; Takoudju et
al., 1988).
We have not observed significant cytotoxicity of compound
7 or 10 in any cell line studied. This is probably not surpris-
ing, because the sarcodictyins also have feeble effects on cell
growth (Ciomei et al., 1997; Hamel et al., 1999). However, it
is likely that still more active steroid derivatives can be
synthesized or discovered in existing compound collections,
and we hope that our findings will encourage such efforts.
Finally, it has been long postulated that drug-binding sites
on tubulin represent sites for endogenous molecules that
regulate cellular microtubule structure and function. The
realization that low concentrations of antimitotic drugs can
have dramatic effects on microtubule dynamics (Wilson and
Jordan, 1995) strengthens this concept. It is thus of interest
that relatively minor structural changes in steroid deriva-
tives can have opposite effects on tubulin assembly and poly-
mer stability and, possibly, differing effects on microtubule
dynamic properties. Perhaps known or still undiscovered
steroids play important roles in regulating microtubule
structure and function in eucaryotic cells.
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A Paclitaxel-Like Steroid Derivative
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Send reprint requests to: Dr. E. Hamel, P.O. Box B, Building 469, Room
237, National Cancer Institute, Frederick Cancer Research and Development
Center, Frederick, MD 21702. E-mail: hamele@dc37a.nci.nih.gov