Synthesis of B-ring homologated estradiol analogues that modulate tubulin polymerization and microtubule stability

Article abstract of DOI:10.1021/jm0001119

2-Methoxyestradiol is a cytotoxic human metabolite of estradiol with the ability to bind to the colchicine site of tubulin and inhibit its polymerization, and its 2-ethoxy analogue is even more potent. On the basis of a hypothetical relationship between the structures of colchicine and 2- methoxyestradiol, a B-ring-expanded 2-ethoxyestradiol analogue was synthesized in which the B-ring of the steroid is replaced by the B-ring of colchicine. The synthesis relied on the B-ring expansion of available 6-keto estradiol derivatives as opposed to a total synthesis of the homologated steroid framework. The relative configurations of the acetamido substituents in both epimers of the final product were determined by NOESY NMR and confirmed by X-ray crystallography. The epimer having the 6α-acetamido substituent was more active as an inhibitor of tubulin polymerization, and it was also more cytotoxic than the 6β-epimer. These results are consistent with the proposed structural resemblance of 2-methoxyestradiol and colchicine. Several of the synthetic intermediates proved to be potent inhibitors of tubulin polymerization. On the other hand, a 3,17β- diacetylated, B-ring-expanded analogue of 2-ethoxyestradiol having a ketone at C-6 resembled paclitaxel (Taxol) in its ability to enhance tubulin polymerization and stabilize microtubules. The corresponding 3-acetate and the 17β-acetate were both synthesized, and it was determined that the 17β- acetate, but not the 3-acetate, conferred on the steroid derivative its paclitaxel-like activity.

Full text of DOI:10.1021/jm0001119

J . Med. Chem. 2000, 43, 2419-2429
2419
Syn th esis of B-Rin g Hom ologa ted Estr a d iol An a logu es th a t Mod u la te Tu bu lin
P olym er iza tion a n d Micr otu bu le Sta bility
Zhiqiang Wang,† Donglai Yang,† Arasambattu K. Mohanakrishnan,† Phillip E. Fanwick,‡ Priya Nampoothiri,§
Ernest Hamel,§ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, and
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, and Screening Technologies Branch,
Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute,
Frederick Cancer Research and Development Center, Frederick, Maryland 21702
Received March 7, 2000
2-Methoxyestradiol is a cytotoxic human metabolite of estradiol with the ability to bind to the
colchicine site of tubulin and inhibit its polymerization, and its 2-ethoxy analogue is even more
potent. On the basis of a hypothetical relationship between the structures of colchicine and
2-methoxyestradiol, a B-ring-expanded 2-ethoxyestradiol analogue was synthesized in which
the B-ring of the steroid is replaced by the B-ring of colchicine. The synthesis relied on the
B-ring expansion of available 6-keto estradiol derivatives as opposed to a total synthesis of the
homologated steroid framework. The relative configurations of the acetamido substituents in
both epimers of the final product were determined by NOESY NMR and confirmed by X-ray
crystallography. The epimer having the 6R-acetamido substituent was more active as an
inhibitor of tubulin polymerization, and it was also more cytotoxic than the 6â-epimer. These
results are consistent with the proposed structural resemblance of 2-methoxyestradiol and
colchicine. Several of the synthetic intermediates proved to be potent inhibitors of tubulin
polymerization. On the other hand, a 3,17â-diacetylated, B-ring-expanded analogue of
2-ethoxyestradiol having a ketone at C-6 resembled paclitaxel (Taxol) in its ability to enhance
tubulin polymerization and stabilize microtubules. The corresponding 3-acetate and the 17â-
acetate were both synthesized, and it was determined that the 17â-acetate, but not the 3-acetate,
conferred on the steroid derivative its paclitaxel-like activity.
In tr od u ction
how these two structures are related. On the basis of
the observation that the dimethoxylated compounds 3
and 4 were inactive, as were the corresponding conge-
ners containing single methoxyl groups at positions 3
or 4, D’Amato et al. proposed that the A-ring of 2-meth-
oxyestradiol (1) is functionally equivalent to the C-ring
of colchicine (2) and that the CD rings of the steroid
correspond to the trimethoxybenzene A-ring of colchi-
cine.12 On the basis of this hypothesis, Macdonald et
al. synthesized a series of 2-methoxyestradiol (1) ana-
logues in which the A-ring of the steroid was replaced
by variously substituted tropone rings.22,23 Most of the
resulting compounds, including the estratropone 5, were
more potent as inhibitors of tubulin polymerization than
2-methoxyestradiol (1) itself.
The human metabolite 2-methoxyestradiol (1) inhibits
angiogenesis1-8 and the growth of solid tumors in vivo,1,9
both of which have been demonstrated in several animal
models. In cancer cell cultures, 2-methoxyestradiol (1)
produces cytotoxic effects that are associated with
inhibition of DNA synthesis and mitosis, uneven chro-
mosome distribution, faulty spindle formation, and an
increase in the number of abnormal metaphases.10,11
The available evidence indicates that these effects result
from inhibition of tubulin polymerization by 2-methoxy-
estradiol, which binds to the colchicine binding site.12
Other possible mechanisms have also been suggested,
including those involving p53,13-18 nitric oxide syn-
thase,6 stress-activated protein kinase,5 p34cdc2,19 and
proliferating cell nuclear antigen (PCNA).19 There is
current interest in the development of 2-methoxyestra-
diol (1) as an anticancer agent, and several studies have
appeared describing the synthesis of 2-methoxyestradiol
(1) analogues, including several compounds that are
more potent as inhibitors of tubulin polymerization and/
or as cytotoxic agents than the parent compound.20-23
Since both 2-methoxyestradiol (1) and colchicine (2)
bind to the same site on â-tubulin, it is logical to ask
Encouraged by these results and also by the fact that
the allocolchicinoids, such as compound 6,24 which have
a 7-membered B-ring and a 6-membered C-ring, are
often more active than the corresponding colchicinoids,25
we decided to synthesize a 2-methoxyestradiol congener
7 in which the B-ring is replaced by the corresponding
B-ring of colchicine. An ethoxyl group was used in 7
instead of a methoxyl group because of our previous
finding that 2-ethoxyestradiol (8) is significantly more
cytotoxic in cancer cell cultures than 2-methoxyestradiol
(1) itself.20 This strategy has resulted in a series of
B-ring-expanded steroid analogues that modulate tu-
bulin polymerization and microtubule stability. As
described herein, several of these compounds resemble
* Corresponding author. Tel: 765-494-1465. Fax: 765-494-6790.
E-mail: cushman@pharmacy.purdue.edu.
† Department of Medicinal Chemistry and Molecular Pharmacology,
Purdue University.
‡ Department of Chemistry, Purdue University.
§ Frederick Cancer Research and Development Center.
10.1021/jm0001119 CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/17/2000

2420 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Wang et al.
Sch em e 1a
paclitaxel (Taxol) in their effects on tubulin and micro-
tubules, while others are similar to colchicine.26
Resu lts a n d Discu ssion
Syn th esis. A review of the literature revealed two
references for the preparation of the B-homoestrane
system, both of which involved total syntheses of the
B-homosteroid ring system.27,28 Limiting our endeavors
to routes starting from readily available steroids, we
decided to investigate an alternative strategy based on
the B-ring expansion of a suitably substituted estradiol
derivative.
2-Ethoxy-6-ketoestradiol (9) was synthesized from
estradiol as previously described21 and then protected
as the bis(tert-butyldimethylsilyl) ether 10 (Scheme 1).
After many unfruitful attempts involving the well-
studied Tiffeneau-Demjanov rearrangement29 and the
diazomethane ring expansion reaction,30 the key trans-
formation of the six-membered B-ring ketone 10 to the
corresponding seven-membered ring ketone 11 was
successfully carried out by employing Taguchi’s method.31
This method relies on the nucleophilic addition of
dibromomethyllithium to a carbonyl group and smooth
decomposition of a â-oxido carbenoid, generated by the
a
Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 23
°C (18 h); (b) (1) CH2Br2, LDA, THF, -78 °C (3 h), (2) BuLi, -78
to 0 °C (2.5 h); (c) Bu4NF, THF, 23 °C (6 h); (d) TsNHNH2, MeOH,
23 °C (24 h); (e) (1) catecholborane, CHCl3, (2) NaOAc, H2O, reflux
(6 h); (f) Ac2O, pyridine, 23 °C (24 h); (g) CrO3, AcOH, 12-14 °C
(40 min); (h) KHCO3, CH3OH, 65 °C (1 h); (i) KOH, MeOH, -5 to
23 °C (3.5 h); (j) 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine, 1 N
NaOH, THF, 23 °C (1.5 h).
action of n-butyllithium on the dibromomethyl inter-
mediate, to afford the lithium enolate of the ring-

B-Ring Homologated Estradiol Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2421
Sch em e 2a
expanded carbonyl compound. The present case em-
ployed a one-pot modification of the Taguchi method in
which lithium diisopropylamide was used to generate
dibromomethyllithium from methylene bromide. Treat-
ment of the initial adduct with n-butyllithium and
workup of the reaction mixture afforded the desired
product 11 in 62% yield based on consumption of
starting material. The starting material was recovered
in 25-30% yields even when an excess of the reagents
was employed, so proton abstraction from the carbonyl
compound may compete effectively with anion addition
to the carbonyl. It is noteworthy that the rearrangement
of the â-oxido carbenoid intermediate proceeded in a
highly regioselective fashion, affording the 7-oxo product
11 instead of the corresponding 6-oxo ring-expanded
product. The observed regioselectivity of the reaction
might be expected, because in â-oxido carbenoids de-
rived from 2-cyclohexenone, the main product results
from migration of the sp2 hybridized alkene carbon as
opposed to the sp3 hybridized alkane carbon.32
The following reactions were performed in order to
relocate the carbonyl from the 7-position in 11 to the
6-position in 16 (Scheme 1). Deprotection of 11 with
TBAF afforded the diol 12. Reaction of the carbonyl
group in 12 with tosylhydrazine afforded the corre-
sponding tosylhydrazone 13. Reduction of 13 with
catecholborane and thermal decomposition of the inter-
mediate diazine provided the desired product 14.33
Acetylation of the diol 14 yielded the diacetate 15, which
was oxidized at the benzylic position with chromium
trioxide in acetic acid to afford intermediate 16. The
phenolic acetate of 16 could be selectively deacetylated
with potassium bicarbonate in methanol to provide the
phenol 17, while treatment of 16 with potassium
hydroxide in methanol resulted in deacetylation of both
acetates to produce the diol 18. The phenolic hydroxyl
group of 18 was selectively acetylated with 1-acetyl-1H-
1,2,3-triazolo[4,5-b]pyridine with sodium hydroxide as
the base in THF, resulting in the formation of the
monoacetate 19.34
As displayed in Scheme 2, an alternative ring en-
largement strategy was investigated, resulting in a one-
pot conversion of the diacetate 2021 to the two possible
ketones 12 and 18.35 Treatment of 20 with trimethyl-
silyldiazomethane-boron trifluoride etherate in meth-
ylene chloride afforded a mixture of R-trimethylsilyl
ketones 23 and 24, which was converted to the corre-
sponding desilylated mixture of ketones 25 and 16 by
hydrochloric acid in the presence of silica gel in ether.
The 1H NMR spectrum of the reaction product indicated
a 1:1 mixture of both diacetates 25 and 16, which could
not be separated chromatographically. However, after
basic hydrolysis of the mixture of diacetates 25 and 16,
the resulting mixture of diols 12 and 18 could be
separated chromatographically to afford both products
in 31% and 24% yields, respectively. The mechanism of
the critical ring-expansion reaction most likely involves
axial attack of trimethylsilyldiazomethane on the 6-keto
group in 20 to afford a mixture of adducts 21 and 22.
Migration of the bond antiperiplanar to the diazonium
group in 21 and 22 then results in both silylated ketones
23 and 24, respectively. In comparison to the B-ring
expansions performed with these reagents on A-ring
saturated steroids, which were highly regioselective,35
a
Reagents and conditions: (a) BF3‚OEt2, TMSCHN2, CH2Cl2,
-20 °C (3 h); (b) HCl, SiO2, Et2O, 23 °C (4 h); (c) (i) KOH, MeOH,
23 °C (4 h), (ii) HCl.
the present reaction on steroid 20, having an aromatic
ring, proceeded with poor regioselectivity. The poor
regioselectivity in the present case might possibly result
from the less sterically demanding aromatic A-ring in
comparison with the saturated steroid A-ring in the
example reported in the original study of the reaction,
so that intermediate 21 forms in addition to 22.35
Alternatively, assuming that the initial addition of
trimethylsilyldiazomethane is reversible, the migratory
aptitude of the aromatic A-ring in the present case
might be greater than the saturated steroidal A-ring
reported in the original example.
The conversion of intermediate 16 to the desired
product 7, in which the B-ring of 2-ethoxyestradiol 8 is
replaced by the colchicine (6) B-ring, is outlined in
Scheme 3. The reductive amination of intermediate 16,
followed by acetylation, yielded a mixture of the aceta-
mides 26 and 27 in a ratio of 1:2, respectively.36 The
6R- and 6â-epimers, 26 and 27, were separated by
careful chromatography on silica gel. The two acetates

2422 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Wang et al.
Ta ble 1. Cytotoxicities of B-Ring Homologated Estradiol
Sch em e 3a
Analogues
compd
MGMa (µM)
compd
MGMa (µM)
1
7
8
1.3
7.0
0.076
0.13
11.5
15.5
18
28
29
31
32
33
34
35
14.4
90.4
24.0
51.3
1.32
42.7
0.079
0.27
9
12
13
14
15
16
2.48
14.5
>100
a
Mean graph midpoint for growth inhibition of all human
cancer cell lines tested.
of 29 with acetic anhydride in pyridine then afforded
the diacetate 30. The oximes 31 and 33 were obtained
in the usual way by reaction of 18 and 12 with
hydroxylamine hydrochloride in pyridine. The alkene
32 was synthesized by treatment of the tosylhydrazone
13 with an excess of methyllithium in THF.
a
Reagents and conditions: (a) (1) NH4OAc, NaCNBH3, MeOH,
65-70 °C (48 h), (2) Ac2O, pyridine, 23 °C (14 h); (b) NaOH, MeOH,
23 °C (5 h).
F igu r e 1. ORTEP diagram resulting from X-ray crystal-
lography of 7. Most of the hydrogen atoms and a water
molecule were omitted for clarity.
of both 26 and 27 were saponified with sodium hydrox-
ide in methanol to afford the desired product 7 and its
C-6 epimer 28. A detailed NMR analysis was performed
in order to elucidate the configuration at C-6 in each
epimer, as well as to determine the preferred conforma-
tion of the 7-membered ring. Although the proton
coupling constants were not very informative as far as
the assignment of the relative configuration of the
substituent at C-6 was concerned, the NOESY spectrum
revealed a very strong, diagnostic NOE between the
methine protons at H-6 and H-9 in diastereomer 28 that
was absent in its epimer 7. This structure was confirmed
by X-ray analysis (Figure 1).
Because of the potent biological activity previously
observed for the 6-oximino derivative of 2-ethoxyestra-
diol (8),21 the two methoximes 29 and 30, as well as the
oxime 31, were prepared in the present series. The
methoxime 29 was synthesized by treatment of 18 with
methoxylamine hydrochloride in pyridine. Acetylation
Biologica l Resu lts. Most of the new compounds
were tested in the National Cancer Institute’s screen
of approximately 55 different human cancer cell cultures
of diverse tumor origins. The resulting mean graph
midpoints (MGMs) for 50% growth inhibition are listed
in Table 1. The MGM is based on a calculation of the
average GI50 for all of the cell lines tested in which GI50
values above and below the test range (10-4-10-8 M)
are taken as the maximum (10-4 M) and minimum (10-8
M) drug concentrations in the screening test.37 The table
includes the previously published values for 2-meth-
oxyestradiol (1), 2-ethoxyestradiol (8), 2-ethoxy-6-ke-
toestradiol (9), 2-ethoxy-6-oximinoestradiol (34), and
2-ethoxy-6-methoximinoestradiol (35).21
The most interesting compound in the series proved
to be 16, which unexpectedly accelerated tubulin po-
lymerization and stabilized the microtubules toward

B-Ring Homologated Estradiol Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2423
Ta ble 2. Interactions of Steroid Derivatives with Tubulin
Part I. Apparent Stimulators of Assemblya
compound
enhancement of assembly rate EC50 (µM) ( SD
paclitaxel
0.83 ( 0.1
>400
15
16
17
19
20
25
28
30
5.5 ( 1
8.2 ( 1
360 ( 7
>400
>400
>200
>200
Part II. Inhibitors of Tubulin Polymerizationb
inhib of colchicine bindingc
inhibition of
polymerization
IC50 (µM) ( SD
% inhib ( SD
compd
expt Id
expt IIe
1
7
8
9
12
13
14
18
29
31
32
33
1.4 ( 0.2
6.9 ( 0.1
0.66 ( 0.06
3.0 ( 0.05
8.1 ( 0.7
80 ( 20
0.97 ( 0.3
130 ( 40
30 ( 4
19 ( 3
60 ( 10
51 ( 6
19 ( 6
87 ( 4
56 ( 7
F igu r e 2. Effects of weakly active stimulatory steroid deriva-
tives on tubulin polymerization. Reaction mixtures were
prepared as described in the text and contained the described
components as well as the following: curve 1, no further
addition; curve 2, 7.5 µM 16; curve 3, 100 µM 16; curve 4, 400
µM 15; curve 5, 200 µM 28; curve 6, 400 µM 20; curve 7, 400
µM 25; and curve 8, 200 µM 30. The figure is a composite of
several experiments.
57 ( 10
0.85 ( 0.1
99 ( 30
a
Stimulators of tubulin polymerization were evaluated as
b
described in the Experimental Section. Inhibitors of tubulin
polymerization were evaluated as described in the Experimental
Section. c Reaction mixtures contained 1.0 µM tubulin, 5.0 µM
[3H]colchicine, and 50 µM inhibitor. Expt I data are from ref 21.
d
e Expt II data are from ref 27.
cold-induced depolymerization.26 These effects resemble
those produced by the potent anticancer agent paclitaxel
(Taxol). The monoacetate 17, in which the C-17 hydroxyl
group is acetylated, proved to be almost as potent as
16, but the alternative monoacetate 19, in which the
phenolic hydroxyl group was acetylated, displayed
minimal taxoid activity. Thus, the 17-acetate, but not
the 3-acetate, appears to be necessary for the taxoid
activity of 16. Both 16 and 17 were less potent than
paclitaxel in their effects on tubulin and as cytotoxic
agents in cancer cell cultures, but the possibility exists
that they could serve as lead compounds for the devel-
opment of steroid derivatives having more potent and
more useful taxoid activities.
yielded 50% of this rate were determined (EC50 values).
The data we obtained are tabulated in part I of Table 2.
Compound 16 itself yielded an EC50 value of 5.5 µM,
while that for 17 was 8.2 µM. For comparison, paclitaxel
had an EC50 value of 0.83 µM. The only other apparently
stimulatory compound that yielded an EC50 value within
the concentration ranges we could study was compound
19, with an EC50 value of 360 µM. The remaining five
compounds listed in part I of Table 2 all seemed to have
some ability to enhance tubulin assembly, and turbidity
curves obtained at the highest concentrations we could
examine (based on the concentrations of the stock
solutions) are shown in Figure 2. This figure also shows
typical curves obtained without drug (curve 1) and with
compound 16 near its EC50 value (curve 2) and at 100
µM (curve 3).
Besides enhancement of tubulin assembly, another
important property of paclitaxel, shared by compound
16, is stabilization of polymer to cold-induced disas-
sembly. Although in the current studies the stabilizing
effect of 16 was less pronounced than observed previ-
ously,26 a variety of stabilization patterns was observed
with the stimulatory steroid derivatives. Two param-
eters, relative to the control without drug, are apparent
from the reaction curves shown in Figure 2. First, upon
reaching a depolymerizing temperature, the control
disassembles rapidly. Second, the disassembly of the
control polymer is essentially complete (electron micro-
graphs rarely show surviving polymer, and the slight
change in the baseline is generally interpreted as
representing partial denaturation of tubulin during the
course of the reaction).
In evaluating the newly synthesized compounds de-
scribed here for their effects on tubulin polymerization,
we noted that a number of other agents also appeared
to have slight activity as stimulators of tubulin as-
sembly. Since their relative activities could provide
useful structure-activity information, we decided to
design a more quantitative assay to compare the activi-
ties of these compounds. We continued to use glutamate-
and GTP-induced assembly of purified tubulin as the
basic assay system, and we found that the difference
between the reaction without steroid and that with
compound 16 was maximal at a reaction temperature
of 24 °C (see Figure 2). The reaction parameter we
decided to use for compound comparisons was the
maximum rate of turbidity change (in terms of ∆A350
units) in the presence of 100 µM 16 (see below for
further details; in the absence of steroid this rate was
18 milliunits of absorbance/min, and with 100 µM 16,
160 milliunits/min). Compound concentrations that

2424 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Wang et al.
In reaction mixtures containing compound 16, the
rate of depolymerization was markedly reduced, and the
reversal of the assembly reaction was not complete. Only
compounds 17 (not shown, but see ref 26) and 25 (Figure
2, curve 7) yielded similar stability patterns. Compound
17 is the analogue of 16 lacking only the C-3 acetyl
group. Compound 25 had both acetyl groups, but the
ketone group was at C-7 (rather than at C-6 in 16).
MGM of 0.079 µM and had an IC50 of 1.1 µM for
inhibition of tubulin polymerization.21 Similarly, the
methoxime 29 displayed an MGM of 24 µM and an IC50
value for inhibition of tubulin polymerization of 30 µM,
even though the closely related methoxime 35, having
a six-membered B-ring, had an MGM of 0.27 µM and
an IC50 value for inhibition of tubulin polymerization
of 1.4 µM. These differences underscore a high degree
of structural specificity attached to the biological effects
of the oxime 34 and the methoxime 35.
It is also worth contrasting the deacetylated/diacetate
pairs 9/20 and 18/16. As noted previously,21 compound
9 has reasonable activity as an inhibitor of tubulin
polymerization (IC50 value, 3.0 µM in the current
evaluation) and is among the more cytotoxic steroid
derivatives we have prepared, whereas 20 has minimal
activity as a stimulator of tubulin polymerization (EC50
value > 400 µM). In contrast, 18 is a weak inhibitor of
assembly (IC50 value, 130 µM), while 16 is the most
potent of the stimulatory steroid derivatives. In addi-
tion, shifting the ketone moiety to the C-7 carbon versus
the C-6 carbon results in substantial enhancement of
activity as an inhibitor of assembly (cf. 12 vs 18) but
substantial loss of activity as a stimulator of assembly
(cf. 25 vs 16).
In conclusion, the synthesis of a 2-ethoxyestradiol
analogue has been executed in which the B-ring is
replaced by the corresponding 7-membered B-ring
present in colchicine. An investigation of the biological
activities of the synthetic intermediates led to the
unexpected observation that two compounds in the
series resemble paclitaxel in that they accelerate tubulin
polymerization and stabilize microtubules, whereas
other members of the series show the expected ability
to inhibit tubulin polymerization. One possibility is that
the taxoid compounds 16 and 17 may “cross over” from
the colchicine binding site on tubulin to the paclitaxel
binding site, or at least partially occupy the paclitaxel
binding site. The recent determination of the structure
of the tubulin dimer in docetaxel-stabilized, antiparallel
zinc protofilaments by electron crystallography has
revealed the structure of the protein complex containing
GTP, GDP, and docetaxel.38 In addition, the available
information on colchicine binding, in conjunction with
the structure of the protein, has allowed a hypothetical
model to be constructed of the binding of colchicine to
tubulin.39 Although it is clear how the structure of the
2-ethoxyestradiol-colchicine hybrid 7 would be over-
lapped with the proposed structure of colchicine in the
colchicine binding pocket, it is not obvious on first
inspection how the structure of the taxoid-like com-
pounds 16 and 17 resembles that of paclitaxel. In any
case, compounds 16 and 17 represent the first com-
pounds in the steroid series to have demonstrable
paclitaxel-mimetic properties. Further work is needed
to define the structure-activity relationships of the
compounds in this series.
The most common pattern observed with weakly
stimulatory analogues was a rapid disassembly reaction,
differing little from that without drug, but with incom-
plete reversal. The extent of reversal was minimal with
compound 15 (Figure 2, curve 4) and more extensive
with compounds 20 (curve 6) and 30 (curve 8). With 20
and 30, the reversibility was similar to what was
observed with 16. These three compounds were all
acetylated at both positions C-3 and C-17. In addition,
compound 15 had an unsubstituted 7-membered B-ring,
20 had a 6-membered B-ring but a ketone substituent
at C-6, and 30 was a close analogue of 16, but with a
methoxime replacing the ketone at C-6. These results
would appear to emphasize the importance of the
7-membered B-ring and the ketone substituent at C-6
for taxoid-like activity in a steroid derivative.
Two compounds showed no apparent stabilization of
polymer, in that reaction mixtures containing these
compounds displayed disassembly patterns essentially
superimposable on the control reaction. These were
compounds 19 (data not shown) and 28 (Figure 2, curve
5). Compound 19 is structurally identical to compound
16, except that it lacks the C-17 acetyl group. Compound
28, however, was a diastereomer of the target compound
7, which inhibits the polymerization reaction (see
below). It thus retains the 7-membered B-ring but lacks
the acetate and ketone groups observed in most of the
other stimulatory compounds.
The remaining compounds in the series displayed the
expected ability to inhibit tubulin polymerization, al-
though a number of them were only marginally active
(13, 18, 29, 31, and 33). The target compound 7, in
which the acetamide at C-6 has the same absolute
configuration at the corresponding group in colchicine
(assuming the correctness of the proposed structural
analogy between these two compounds), proved to be
significantly more active than its C-6 epimer 28 as a
cytotoxic agent. Compound 7 also inhibited tubulin
polymerization, as opposed to the stimulation observed
with 28. However, in contrast to the enhanced activity
observed with expanded A-ring analogues,22,23 com-
pound 7 was about 10-fold less potent than 2-ethoxy-
estradiol (8) as an inhibitor of both tubulin polymeri-
zation and cell growth. The complete removal of the
acetamide from 7/28 resulted in the more potent ana-
logue 14. This compound was almost as potent as
2-ethoxyestradiol (8) as an inhibitor of tubulin polym-
erization, but 14 was less cytotoxic than 8. The biological
profile produced by the alkene 32 was very similar to
that observed with its saturated analogue 14.
Exp er im en ta l Section
Several of the compounds in the series were striking
in their biological inactivities. For example, both the
6-oximino and 7-oximino compounds 31 and 33 were
minimally active as inhibitors of tubulin polymerization
and as cytotoxic agents, even though the corresponding
oxime 34, having a six-membered B-ring, displayed an
Gen er a l. Melting points are uncorrected. Nuclear magnetic
resonance spectra for proton (1H NMR) were recorded on a
300 MHz spectrometer. The chemical shift values are ex-
pressed in ppm (parts per million) relative to tetramethylsilane
as internal standard. For data reporting, s ) singlet, d )
doublet, m ) multiplet, br ) broad, bs ) broad singlet.

B-Ring Homologated Estradiol Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2425
Microanalyses were performed at the Purdue Microanalysis
Laboratory, and all values were within (0.4% of the calculated
compositions. Column chromatography was carried out using
Merck silica gel (230-400 mesh). Analytical thin-layer chro-
matography (TLC) was performed on silica gel GF (Analtech)
glass-coated plates (2.5 × 10 cm with 250 µM layer and
prescored), and spots were visualized with UV light at 254 nm
or with 5% H2SO4 in ethanol. Most chemicals and solvents
were analytical grade and used without further purification.
Commercial reagents were purchased from Aldrich Chemical
Company (Milwaukee, WI).
h. The resultant mixture was filtered and the solid washed
with cold methanol to afford tosylhydrazone 13 as white
crystals (2.48 g, 96.5%). The analytical sample was recrystal-
lized from methanol: mp 179-181 °C; IR (KBr, cm-1) 3428
(br, OH, NH), 2922, 1624, 1594, 1511; 1H NMR (CDCH3) δ 7.88
(d, J ) 8.0 Hz, 2 H), 7.34 (d, J ) 8.0 Hz, 2 H), 7.33 (s, 1 H,
NH), 6.71 (s, 1 H), 6.68 (s, 1 H), 5.55 (s, 1 H), 4.11 (q, J ) 7.0
Hz, 2 H), 3.58 (d, J ) 19.1 Hz, 2 H), 3.39 (s, 1 H), 3.45 (t, J )
8.5, 1 H), 3.15 (d, J ) 19.1 Hz, 1 H), 2.46 (s, 3 H), 2.10-0.80
(m, 16 H), 0.78 (s, 3 H); CIMS (isobutane) m/ z (rel intensity)
360 (MH+, 100), 342 (51). Anal. (C28H36O5N2) C, H.
3,17â-Di-O-(ter t-bu tyldim eth ylsilyl)-2-eth oxy-6-oxoestr a-
d iol (10). Under argon, a solution of compound 9 (4.92 g, 14.9
mmol), imidazole (12.3 g, 178.4 mmol), and tert-butyldimeth-
ylchlorosilane (13.5 g, 89.6 mmol) in DMF (100 mL) was stirred
for 18 h at room temperature. The reaction mixture was poured
into an ice-cold sodium bicarbonate solution (250 mL), and the
product was extracted with ethyl acetate (200 mL, 150 mL,
100 mL). The organic layers were washed with water (200 mL)
and brine (2 × 100 mL), dried over sodium sulfate, and
evaporated to dryness. Crystallization of the residue from
methanol gave compound 10 (7.6 g, 91%), which was obtained
B-Hom o-2-eth oxy-3,17â-estr a d iol (14). Under argon, a
solution of 13 (2.27 g, 4.43 mmol) in anhydrous chloroform (150
mL) was cooled to 0 °C, and then catecholborane (44.3 mL,
1.0 M solution in THF, 44.3 mmol) was added dropwise. The
resultant mixture was stirred for 10 h, and sodium acetate
trihydrate (12.06 g, 88.62 mmol) was added in portions. The
mixture was allowed to warm to room temperature over 30
min and then heated under reflux for 6 h. The reaction mixture
was cooled to room temperature and filtered. The solid
material was washed with chloroform (100 mL), and the
combined filtrates were evaporated under reduced pressure
to dryness. The remaining oil was purified by chromatography
on a silica gel column with ethyl acetate-hexane 1:3 by
volume. The product was crystallized from ethyl acetate/
hexane to give compound 14 as white crystals (1.17 g, 80.1%):
mp 86-88 °C; IR (KBr, cm-1) 3490, 3259, 2925, 2861, 1607,
1
as white crystals: mp 150-151 °C; H NMR (CDCl3) δ 7.51
(s, 1 H), 6.78 (s, 1 H), 4.19 (q, J ) 6.9 Hz, 2 H), 3.65 (t, J ) 8.5
Hz, 1 H), 2.85-2.48 (m, 2 H), 2.45-1.1 (m, 14 H), 0.98 (s, 9
H), 0.86 (s, 9 H), 0.72 (s, 3 H), 0.15 (s, 6 H), 0.01 (s, 6 H); CIMS
(isobutane) m/ z (rel intensity) 559 (MH+, 100). Anal. (C32H54O4-
Si2) C, H.
1
1511; H NMR (CDCl3 + DMSO-d6) δ 6.81 (s, 1 H), 6.67 (s, 1
H), 5.46 (s, 1 H), 4.11 (dq, J ) 3.3 Hz, J ) 7.0 Hz, 2 H), 3.74
(t, J ) 8.2 Hz, 1 H), 2.86 (dt, J ) 14.6 Hz, J ) 7.3, 1 H), 2.64-
2.47 (m, 2 H), 2.14-1.22 (m, 18 H), 0.83 (s, 3 H); CIMS
(isobutane) m/ z (rel intensity) 330 (M+, 100). Anal. Calcd for
(C21H30O3) C, H.
B-Hom o-3,17â-d i-O-(ter t-bu tyld im eth ylsilyl)-2-eth oxy-
7-oxoestr a d iol (11). Under argon, a well-stirred solution of
10 (6.38 g, 11.41 mmol) and dibromomethane (11.9 g, 68.5
mmol) in dry THF (190 mL) was cooled to -78 °C, and then
the solution was treated with lithium diisopropylamide mono-
(tetrahydrofuran) (30.4 mL, 1.5 M solution in cyclohexane, 45.6
mmol) dropwise over a period of 1.5 h. The mixture was stirred
for 3 h, and n-butyllithium (60 mL, 1.6 M solution in hexane,
96 mmol) was added to the mixture over a period of 1.5 h. The
resulting red solution was stirred for another 1 h at -78 °C
and 10 min at 0 °C, quenched by pouring into ice (150 g), and
extracted with ethyl acetate (3 × 100 mL). The organic layers
were washed with brine (3 × 100 mL), combined, dried over
sodium sulfate, and evaporated to dryness. Chromatography
of the residue (silica gel 230-400 mesh, methylene chloride:
hexane 7:3 by volume) yielded the starting material 10 (1.98
g) and pure compound 11 as an oil (2.78 g, 61.6% based on
the consumption of 10): IR (KBr, cm-1) 2955, 2930, 1709 (7-
CdO), 1511; 1H NMR (CDCl3) δ 6.78 (s, 1 H), 6.60 (s, 1 H),
4.04 (qd, J ) 7.0 Hz, J ) 1.5 Hz, 2 H), 3.66 (t, J ) 8.5 Hz, 1
H), 3.60 (d, J ) 20.1 Hz, 1 H), 3.28 (d, J ) 20.3, 1 H), 2.68 (dd,
J ) 11.3 Hz, J ) 8.5 Hz, 1 H), 2.34 (m, 1 H), 2.2-1.20 (m, 14
H), 0.98 (s, 9 H), 0.88 (s, 9 H), 0.78 (s, 3 H), 0.13 (s, 6 H), 0.05
(s, 6 H); CIMS (isobutane) m/ z (rel intensity) 573 (MH+, 100).
Anal. Calcd for (C33H56O4Si2) C, H.
B-Hom o-3,17â-d i-O-a cetyl-2-eth oxyestr a d iol (15). Acetic
anhydride (6 mL, 63 mmol) was added under argon at room
temperature to a solution of compound 14 (1.14 g, 3.45 mmol)
in anhydrous pyridine (12 mL). The resulting mixture was
stirred for 24 h and poured into an ice-water mixture (100
g). The compound was extracted with ethyl acetate (3 × 70
mL). The organic layers were washed with water (100 mL), a
solution of sodium bicarbonate (2 × 100 mL), and brine (2 ×
100 mL), dried over sodium sulfate, and evaporated to dryness.
Chromatography of the residue on a silica gel (230-400 mesh)
column using ethyl acetate-hexane 1:1 by volume gave
compound 15 (1.43 g, 100%), which was crystallized from ethyl
acetate/hexane to afford white crystals: mp 98-100 °C; IR
(KBr, cm-1) 2928, 2870, 1768, 1735, 1510; 1H NMR (CDCl3) δ
6.89 (s, 1 H), 6.75 (s, 1 H), 4.70 (t, J ) 8.7 Hz, 1 H), 4.06 (dq,
J ) 3.3 Hz, J ) 7.0 Hz, 2 H), 2.89 (dt, J ) 14.6 Hz, J ) 7.3,
1 H), 2.64-2.52 (m, 2 H), 2.29 (s, 3 H), 2.07 (s, 3 H), 2.23-
1.26 (m, 17 H), 0.88 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 415 (MH+, 48), 355 (100). Anal. Calcd for (C25H34O5)
C, H.
B-Hom o-2-eth oxy-7-oxo-3,17â-estr a d iol (12). Under ni-
trogen, a mixture of 11 (3.42 g, 5.97 mmol) and a 1.0 M solution
of tetrabutylammonium fluoride in THF (50 mL, 50 mmol) was
stirred at room temperature for 6 h. The reaction mixture was
poured into an ice-cold sodium bicarbonate solution (200 mL)
and extracted with ethyl acetate (3 × 100 mL). The organic
phase was washed with brine (2 × 100 mL), dried over sodium
sulfate, and evaporated to dryness. Chromatography of the
residue on silica gel, eluting with ethyl acetate:hexane 1:1 by
volume, gave compound 12 as white crystals (1.8 g, 87%): mp
210-212 °C; IR (KBr, cm-1) 3403 (br), 2970, 2855, 1688, 1582,
B-Hom o-3,17â-d i-O-a cetyl-2-eth oxy-6-oxoestr a d iol (16).
A solution of chromium trioxide (1.34 g, 13.4 mmol) in 90%
glacial acetic acid (13 mL) was added dropwise over a period
of 20 min at 13-15 °C to a well-stirred solution of compound
15 (1.29 g, 3.11 mmol) in glacial acetic acid (35 mL), and the
resulting mixture was stirred at room temperature for 15 min.
The mixture was poured into an ice/water mixture (300 g) and
extracted with ethyl acetate (3 × 200 mL). The combined
organic layers were washed with brine (2 × 100 mL), a solu-
tion of sodium bicarbonate (2 × 100 mL), and brine (2 ×
100 mL), dried over sodium sulfate, and evaporated to dry-
ness. Chromatography of the residue on a silica gel column
using 20% ethyl acetate in hexane gave pure compound 16
(925 mg, 69.3%), which was crystallized from ethyl acetate/
hexane to afford white crystals: mp 178-180 °C; IR (KBr,
cm-1) 2935, 2865, 1774, 1728, 1669, 1500; 1H NMR (CDCl3)
δ 7.36 (s, 1 H), 6.84 (s, 1 H), 4.72 (t, J ) 8.3 Hz, 1 H), 4.13
(q, J ) 7.0 Hz, 2 H), 2.64-2.52 (m, 3 H), 2.29 (s, 3 H), 2.07
(s, 3 H), 2.23-1.26 (m, 15 H), 0.94 (s, 3 H); CIMS (isobutane)
m/ z (rel intensity) 429 (MH+, 49), 341 (100). Anal. (C25H32O6)
C, H.
1
1510; H NMR (CDCl3 + DMSO-d6) δ 6.82 (s, 1 H), 6.69 (s, 1
H), 4.14 (q, J ) 7.0 Hz, 2 H), 3.69 (t, J ) 8.5 Hz, 1 H), 3.62 (d,
J ) 20.1 Hz, 1 H), 3.32 (d, J ) 19.8, 1 H), 2.68 (dd, J ) 11.3
Hz, J ) 8.5 Hz, 1 H), 2.34 (m, 1 H), 2.10-1.70 (m, 7 H), 1.50-
1.20 (m, 7 H), 0.88 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 345 (MH+, 100). Anal. (C21H28O4) C,H.
B-Hom o-2-eth oxy-3,17â-estr adiol-7-tosylh ydr azon e (13).
Under nitrogen and at room temperature, a mixture of
compound 12 (1.73 g, 5.02 mmol) and p-toluenesulfonhydrazide
(4.67 g, 25.1 mmol) in methanol (100 mL) was stirred for 24

2426 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Wang et al.
B-H om o-17â-O-a cet yl-2-et h oxy-3-h yd r oxyest r a -6-k e-
toestr a d iol (17). Under nitrogen, a solution of compound 16
(65 mg, 0.15 mmol) in methanol (10 mL) was deoxygenated
by bubbling through it a slow stream of nitrogen for 30 min.
A similar deoxygenated solution of KHCO3 (152 mg, 1.52
mmol) in water (1 mL) was added, and the reaction mixture
was stirred and heated at 65 °C (outer bath) for 1.5 h. The
reaction mixture was cooled to room temperature and neutral-
ized to pH ) 6 with 6 N HCl. The solvents were removed under
reduced pressure, and the residue was dissolved in a mixture
of ethyl acetate (50 mL) and water (50 mL). The ethyl acetate
layer was separated, dried over sodium sulfate, and evaporated
to dryness. Chromatography of the residue on silica gel, eluting
with ethyl acetate-hexane 1:2 by volume, yielded compound
17 (56 mg, 95%), which was easily crystallized from ethyl
acetate-hexane to afford white crystals: mp 240-242 °C; IR
(KBr, cm-1) 3385 (OH), 2941, 1721, 1663, 1614, 1511; 1H NMR
(CDCl3) δ 7.22 (s, 1 H), 6.75 (s, 1 H), 5.54 (s, 1 H), 4.72 (t, J )
8.5 Hz, 1 H), 4.19 (q, J ) 7 Hz, 2 H), 2.58 (m, 3 H), 2.55 (m, 1
H), 2.08 (s, 3 H), 1.94-1.27 (m, 14 H), 0.88 (s, 3 H); CIMS
(isobutane) m/ z (rel intensity) 387 (MH+, 100). Anal. Calcd.
for (C23H30O5) C, H.
mixture was neutralized with 6 M HCl, and the solvent was
removed. Extraction of the crude product with ethyl acetate,
followed by column chromatographic purification on silica gel,
eluting with 1:2 ethyl acetate-hexane, afforded compound 12
(0.26 g, 31%) and compound 18 (0.20 g, 24%). The IR and NMR
spectra of these compounds were identical with those of the
samples described above.
B-Hom o-6r-acetam ido-3,17â-di-O-acetyl-2-eth oxyestr a-
d iol (26) a n d B-Hom o-6â-a ceta m id o-3,17â-d i-O-a cetyl-2-
eth oxyestr a d iol (27). Sodium cyanoborohydride (493 mg, 7.8
mmol) was added to a solution of compound 16 (270 mg, 0.78
mmol) and ammonium acetate (4.0 g, 51.9 mmol) in anhydrous
methanol (20 mL) at room temperature under argon. The
reaction mixture was stirred at room temperature for 30 min
and then heated at reflux for 48 h. The mixture was cooled to
about 40 °C, and the solvent was removed under reduced
pressure. The residue was treated with saturated aqueous
NaHCO3 (50 mL) and ethyl acetate (3 × 50 mL). The ethyl
acetate layers were washed with brine (2 × 30 mL), combined,
dried over sodium sulfate, and evaporated to dryness. The
residue was dissolved in anhydrous pyridine (10 mL), and
acetic anhydride (4 mL) was added. The resulting mixture was
stirred at room temperature under argon for 14 h. The pyridine
and excess acetic anhydride were removed under reduced
pressure at 40-45 °C, and the residue was dissolved in ethyl
acetate (100 mL). The ethyl acetate solution was washed with
saturated NaHCO3 (50 mL) and brine (50 mL), dried over
sodium sulfate, and evaporated to dryness. Chromatography
of the residue on a silica gel column, eluting with ethyl
acetate-hexane, 7:2 by volume, yielded compound 26 (99 mg,
27%), which was dried under high vacuum at 40-50 °C to
provide a stable white foam: IR (KBr, cm-1) 3313, 2935, 1765,
1733, 1655, 1510; 1H NMR (CDCl3) δ 6.95 (s, 1 H, 4-ArH), 6.85
(s, 1 H, 1-ArH), 5.80 (d, J NH-6 ) 7.2 Hz, 1 H, NH), 5.13 (ddd,
J NH-6 ) 7.2 Hz, J 6-7 ) 11.7 and 6.4 Hz, 1 H, 6â-H), 4.73 (t, J
) 8.5 Hz, 1 H, 17R-H), 4.08 (qd, J ) 7.1 Hz, J ) 1.5 Hz, 2 H,
OCH2), 2.56 (m, 1 H), 2.28 (s, 3 H, 3-CH3CO), 2.07 (s, 3 H,
17-CH3CO), 1.95 (s, 3 H; 6-NCOCH3), 2.36-1.26 (m, 17 H),
0.90 (s, 3 H, 18-CH3); CIMS (isobutane) m/ z (rel intensity)
472 (MH+, 100). Anal. (C27H37NO6) C, H, N. The above
chromatography also gave compound 27 as a white foam (180
mg, 48%): IR (KBr, cm-1) 3313, 2935, 1765, 1734, 1655, 1510;
1H NMR (CDCl3) δ 6.88 (s, 1 H, 4-ArH), 6.78 (s, 1 H, 1-ArH),
5.68 (d, J NH-6 ) 8.5 Hz, 1 H, NH), 5.40 (ddd, J NH-6 ) 8.5 Hz,
J 6-7 ) 11.7 and 6.4 Hz, 1 H, 6R-H), 4.69 (t, J ) 8.5 Hz, 1 H,
17R-H), 4.08 (qd, J ) 7 Hz, J ) 1.5 Hz, 2 H, OCH2), 2.52 (m,
1 H), 2.30 (s, 3 H, 3-CH3CO), 2.10 (s, 3 H, 17-CH3CO), 2.07 (s,
6-NCOCH3, 3 H), 2.36-1.26 (m, 17 H), 0.88 (s, 3 H, 18-CH3);
CIMS (isobutane) m/ z (rel intensity) 472 (MH+, 100). Anal.
(C27H37NO6) C, H, N.
B-Hom o-2-eth oxy-6-oxo-3,17â-estr a d iol (18). Under ni-
trogen, a suspension of compound 16 (0.95 g, 2.2 mmol) in
anhydrous methanol (10 mL) was cooled to -5-0 °C, and a
20% solution of KOH in methanol (10 mL, KOH 1 g, 17.8
mmol) was added dropwise. The resulting mixture was allowed
to warm to room temperature and stirred for 3.5 h. The
mixture was cooled to 0 °C and neutralized with 3 N HCl to
pH ) 5, and the solid was allowed to precipitate in
a
refrigerator overnight. The resulting mixture was filtered to
afford the white solid 18 (725 mg, 95%), which was recrystal-
lized from methanol to afford analytical sample: mp 198-200
°C; IR (KBr, cm-1) 3497 (OH), 3281 (OH), 2935, 2882, 1650,
1608, 1508; 1H NMR (CDCl3) δ 7.22 (s, 1 H), 6.76 (s, 1 H),
4.19 (q, J ) 7 Hz, 2 H), 3.78 (t, J ) 8.5 Hz, 1 H), 2.54 (m, 3 H),
2.15-1.21 (m, 15 H), 0.90 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 345 (MH+, 100). Anal. (C21H28O4) C, H.
B-Hom o-3-O-a cetyl-2-eth oxy-6-oxo-3,17â-estr a d iol (19).
Under argon at room temperature, compound 18 (60 mg, 0.17
mmol) was dissolved in THF (4 mL), and 1 N NaOH (0.19 mL,
0.19 mmol) was added to the solution. After stirring for 20
min, a solution of 1-acetyl-1H-1,2,3-triazolo[4,5-b]pyridine (31
mg, 0.19 mmol) in THF (2 mL) was added dropwise to the
reaction mixture, which was stirred at room temperature for
1.5 h. The reaction mixture was poured into ice (20 g) and
neutralized to pH ) 6 with 2 N HCl, and it was extracted with
ethyl acetate (2 × 30 mL). The ethyl acetate layers were
combined, dried over sodium sulfate, and evaporated to
dryness. Chromatography of the residue on silica gel, eluting
with ethyl acetate-hexane, 1:3 by volume, gave compound 19
(61 mg, 90%), which precipitated from hexane to afford stable
white foam that was dried under vacuum at 40-50 °C: IR
(KBr, cm-1) 3449 (br), 2933, 1766, 1669, 1605, 1502; 1H NMR
(CDCl3) δ 7.36 (s, 1 H), 6.86 (s, 1 H), 4.14 (q, J ) 7 Hz, 2 H),
3.78 (t, J ) 8.5 Hz, 1 H), 2.59 (m, 3 H), 2.30 (s, 3 H), 2.15-
1.27 (m, 15 H), 0.89 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 387 (MH+, 100). Anal. (C23H30O5) C, H.
Hom ologa tion by Tr im eth ylsilyld ia zom eth a n e: B-Ho-
m o-2-eth oxy-7-oxo-3,17â-estr a d iol (12) a n d B-Hom o-2-
eth oxy-6-oxo-3,17â-estr a d iol (18). A solution of trimethyl-
silyldiazomethane (4.83 mL, 2 M in hexane, 9.7 mmol) was
added dropwise to a solution of intermediate 2021 (1 g, 2.41
mmol) in dichloromethane (40 mL) containing BF3‚OEt2 (1.5
mL, 12.2 mmol) at -20 °C. After 3 h, the reaction mixture
was poured over ice water (50 mL). The organic layer was dried
(Na2SO4), and the solvent was removed. The residue was
dissolved in diethyl ether (40 mL). Silica gel (20 g) and 6 M
HCl (1 mL) were added, and the reaction mixture was stirred
at room temperature for 4 h. Usual workup of the reaction
mixture gave a sticky residue that was then dissolved in
anhydrous methanol (30 mL). A 20% solution of KOH in
methanol (15 mL) was slowly added, and the reaction mixture
was stirred at room temperature for an additional 4 h. The
B-Hom o-6r-a ceta m id o-2-eth oxy-3,17-â-estr a d iol (7). A
solution of compound 26 (75 mg, 0.16 mmol) in methanol (14
mL) was deoxygenated by bubbling through it a slow stream
of nitrogen for 30 min. A similarly deoxygenated 1 M solution
of NaOH in water (1.6 mL, 16 mmol) was added and the
mixture stirred at room temperature for 5 h. The reaction
mixture was neutralized with acetic acid to pH ) 6, and then
the solvents were removed under reduced pressure. The
residue was dissolved in a mixture of ethyl acetate (60 mL)
and saturated sodium bicarbonate (50 mL). The organic layer
was separated and washed with brine (2 × 30 mL), dried over
sodium sulfate, and evaporated to dryness. Chromatography
of the residue on a silica gel column using methylene chloride-
acetone, 2:1 by volume, as the eluant gave the desired
compound 7 (42 mg, 68%), which solidified from acetone/
hexane/methylene chloride to afford a white solid: mp 196-
198 °C; IR (KBr, cm-1) 3328, 3313, 2937, 1650, 1512; 1H NMR
(CDCl3) δ 6.84 (s, 1 H, 4-ArH), 6.77 (s, 1 H, 1-ArH), 5.73 (d,
J NH-6 ) 7.2 Hz, 1 H, NH), 5.57 (s, 1 H, OH), 5.08 (ddd, J NH-6
) 7.2 Hz, J 6-7 ) 11.7 and 6.4 Hz, 1 H, 6â-H), 4.08 (qd, J ) 7
Hz, J ) 1.5 Hz, 2 H, OCH2), 3.75 (t, J ) 8.5 Hz, 1 H, 17R-H),
2.50 (m, 1 H), 2.33 (m, 1 H), 1.95 (s, 3 H, 6-NCOCH3), 2.18-
1.26 (m, 17 H), 0.85 (s, 3 H, 18-CH3); CIMS (isobutane) m/ z

B-Ring Homologated Estradiol Analogues
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2427
(rel intensity) 388 (MH+, 100). Anal. (C23H33NO4‚2/3CH3-
mixture was allowed to warm to room temperature after 2 h
and stirred for 20 h. The reaction mixture was poured into ice
(50 g), acidified with 6 N HCl, and extracted with ethyl acetate
(3 × 30 mL). The ethyl acetate extracts were washed with
saturated sodium sulfate (30 mL) and brine (2 × 30 mL),
combined, dried over sodium sulfate, and evaporated to
dryness. Chromatography of the residue on a silica gel column,
using ethyl acetate-hexane, 2:5 by volume, as the eluant,
yielded compound 32 (26 mg, 54%), which was crystallized
from ethyl acetate/hexane to afford white crystals: mp 167-
168 °C; IR (KBr, cm-1) 3503, 3231, 2930, 1603, 1510; 1H NMR
(CDCl3) δ 6.82 (s, 1 H), 6.70 (s, 1 H), 6.54 (d, J ) 10.1 Hz, 1
H), 6.06 (m, 1 H), 5.51 (s, 1 H), 4.15 (q, J ) 7.0 Hz, 2 H), 3.74
(t, J ) 8.7 Hz, 1 H), 2.21-1.12 (m, 16 H), 0.84 (s, 3 H, CH3);
CIMS (isobutane) m/ z (rel intensity) 329 (MH+, 100), 311 (52).
Anal. (C21H28O3‚1/4H2O) C, H.
COCH3) C, H, N.
B-Hom o-6â-a ceta m id o-2-eth oxy-3,17-â-estr a d iol (28). A
solution of compound 27 (178 mg, 0.38 mmol) in deoxygenated
methanol (14 mL) was treated as described for 7 to afford the
desired compound 28 (98 mg, 67%), which was solidified from
acetone/hexane/methylene chloride to afford a white solid: mp
>162 °C (dec); IR (KBr, cm-1) 3314, 2927, 1658, 1506; 1H NMR
(CDCl3) δ 6.78 (s, 1 H, 4-ArH), 6.72 (s, 1 H, 1-ArH), 5.71 (d,
J NH-6 ) 8.8 Hz, 1 H, 6R-CNH), 5.61 (s, 1 H, OH), 5.38 (ddd,
J NH-6 ) 8.8 Hz, 1 H, J 6-7 ) 11.7 and 6.4 Hz, 1 H, 6RH), 4.11
(qd, J ) 7.1 Hz, J ) 1.5 Hz, 2 H, OCH2), 3.75 (t, J ) 8.5 Hz,
1 H, 17R-H), 2.48 (m, 1 H), 2.10 (s, 3 H, 6-NCOCH3), 2.18-
1.26 (m, 18 H), 0.83 (s, 3 H, 18-CH3); CIMS (isobutane) m/ z
(rel intensity) 388 (MH+, 100). Anal. (C23H33NO4) C, H, N.
B-Hom o-2-eth oxy-6-m eth oxim in o-3,17â-estr a d iol (29).
To a solution of compound 16 (76 mg, 0.21 mmol) in pyridine
(10 mL) was added methoxylamine hydrochloride (354 mg, 4.23
mmol) in one portion under nitrogen. The resulting mixture
was heated at 100 °C for 3 h and then cooled to about 50 °C.
The pyridine was removed under reduced pressure. The
residue was dissolved in a mixture of ethyl acetate (50 mL)
and water (50 mL). The ethyl acetate solution was separated
and then washed with brine (2 × 30 mL), dried over sodium
sulfate, and evaporated to dryness. Chromatography of the
residue on silica gel, eluting with ethyl acetate-hexane, 3:5
by volume, gave compound 29 (78 mg, 94%), which was
crystallized from ethyl acetate/hexane to afford white cystals:
mp 181-183 °C; IR (KBr, cm-1) 3511, 3318, 2972, 2897, 1618,
1576, 1508; 1H NMR (CDCl3) δ 7.03 (s, 1 H), 6.74 (s, 1 H),
5.53 (s, 1 H), 4.17 (q, J ) 7 Hz, 2 H), 3.97 (s, 3 H), 3.78 (t, J
) 8.5 Hz, 1 H), 2.58 (m, 2 H), 2.35 (m, 1 H), 1.94 (m, 3 H),
1.78-1.27 (m, 12 H), 0.88 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 374 (MH+, 100). Anal. (C22H31NO4) C, H, N.
B-Hom o-2-eth oxy-7-oxim in o-3,17â-estr a d iol (33). Under
nitrogen at room temperature, compound 12 (60 mg, 0.17
mmol) was dissolved in anhydrous pyridine (5 mL), and
hydroxylamine hydrochloride (240 mg, 3.48 mmol) was added.
The reaction mixture was stirred at room temperature for 40
h. The pyridine was removed under reduced pressure at 30-
35 °C. The residue was dissolved in ethyl acetate (50 mL) and
water (30 mL), and the ethyl acetate was washed with brine
(2 × 20 mL), dried over sodium sulfate, and evaporated to
dryness. Chromatography of the residue on silica gel, eluting
with ethyl acetate-hexane, 1.25:1 by volume, yielded com-
pound 33 as off-white crystals (50.3 mg, 80.3%): mp 236-238
°C; IR (KBr, cm-1) 3292 (br), 2930, 1737, 1592, 1510; 1H NMR
(CDCl3 + DMSO-d6) δ 9.88 (s, 1 H), 7.03 (s, 1 H), 6.77 (s, 1 H),
6.71 (s, 1 H), 4.12 (q, J ) 7.0 Hz, 2 H), 3.86 (d, J ) 20.1 Hz, 1
H), 3.68 (t, J ) 8.5 Hz, 1 H), 3.43 (d, J ) 19.8, 1 H), 2.20-1.10
(m, 16 H), 0.84 (s, 3 H); CIMS (isobutane) m/ z (rel intensity)
360 (MH+, 100). Anal. (C21H29NO4‚1/6H2O) C, H, N.
B -H o m o -3,17â-d i-O -a c e t y l-2-e t h o x y -6-m e t h o x im i-
n oestr a d iol (30). Acetic anhydride (0.6 mL, 6.3 mmol) was
added under argon at room temperature to a solution of
compound 29 (46 mg, 0.12 mmol) in anhydrous pyridine (3
mL). The resulting mixture was stirred at room temperature
for 20 h and poured into ice water (50 g). The compound was
extracted with ethyl acetate (3 × 40 mL). The organic layers
were washed with sodium bicarbonate (50 mL) and brine (50
mL), dried over sodium sulfate, and evaporated to dryness.
Chromatography of the residue on a silica gel column, eluting
with ethyl acetate-hexane, 1:5 by volume, yielded compound
30 (51 mg, 90%), which was dried under high vacuum at 40-
50 °C as a stable white foam: IR (KBr, cm-1) 2934, 1770, 1734,
1614, 1500; 1H NMR (CDCl3) δ 7.12 (s, 1 H), 6.81 (s, 1 H),
4.72 (t, J ) 8.5 Hz, 1 H), 4.08 (q, J ) 7 Hz, 2 H), 3.96 (s, 3 H),
2.57 (m, 2 H), 2.38 (s, 3 H), 2.35 (m, 1 H), 2.06 (s, 3 H), 2.2-
1.2 (m, 15 H), 0.90 (s, 3 H); CIMS (isobutane) m/ z (rel
intensity) 458 (MH+, 100). Anal. (C26H35NO6) C, H, N.
B-Hom o-2-eth oxy-6-oxim in o-3,17â-estr a d iol (31). Under
nitrogen at room temperature, compound 18 (200 mg, 0.58
mmol) was dissolved in anhydrous pyridine (10 mL), and
hydroxylamine hydrochloride (807 mg, 11.6 mmol) was added.
The reaction mixture was stirred for 20 h. The pyridine was
removed under reduced pressure at 30-35 °C, and the residue
was dissolved in a mixture of ethyl acetate (50 mL) and water
(30 mL). The ethyl acetate layer was separated, washed with
brine (2 × 20 mL), dried over sodium sulfate, and evaporated
to dryness. Chromatography of the residue on silica gel, eluting
with ethyl acetate-hexane, 5:4 by volume, yielded compound
31 (198 mg, 95%), which was recrystallized from ethyl acetate
as white crystals: mp 226-228 °C; IR (KBr, cm-1) 3452, 3160,
3039, 2932, 2897, 1722, 1599, 1501; 1H NMR (CDCl3 + DMSO-
d6) δ 9.73 (s, 1 H), 6.98 (s, 1 H), 6.73 (s, 1 H), 4.15 (q, J ) 7.0
Hz, 2 H), 3.73 (t, J ) 8.5 Hz, 1 H), 2.63-1.17 (m, 18 H), 0.86
(s, 3 H); CIMS (isobutane) m/ z (rel intensity) 360 (MH+, 100).
Anal. Calcd for (C21H29NO4) C, H, N.
X-Ra y Cr ysta llogr a p h ic An a lysis of 7. A colorless plate
of 7 hydrate, C23H35NO5 (0.25 × 0.17 × 0.13 mm), was mounted
on glass fibers in a random orientation. Preliminary examina-
tion and data collection were performed with Mo KR radiation
(KR ) -/71073 Å) on a Nonius Kappa CCD diffractometer.
The cell constants and an orientation matrix for data collected
were obtained from least-squares refinement using the setting
angles of 4017 reflections in the ranges of 4 < θ < 27 °C. The
data were collected at 293 ( 1 K. A total of 4017 reflections
were collected, of which 2560 were unique. The structure was
solved by direct methods using SIR97. The remaining atoms
were located in succeeding difference Fourier syntheses.
Hydrogen atoms were included in refinement but restrained
to ride on the atom to which they were bound, where the
2
2
function minimized was ∑w(|Fo| - |Fc| )2. Refinement was
performed on an AlphaServer 2100 using SHELX-97. Crystal-
lographic drawings were done using programs ORTEP.
Tu bu lin Assa ys. Electrophoretically homogeneous tubulin
was purified from bovine brain as described previously.40
Determination of IC50 values for inhibition of polymerization
of purified tubulin was performed as described in detail
elsewhere,12 except that Beckman DU7400/7500 spectropho-
tometers equipped with “high performance” temperature
controllers were used. Unlike the manual control possible with
the previously used Gilford spectrophotometers, the polymer-
ization assays required use of programs provided by MDB
Analytical Associates, South Plainfield, NJ , since the Beckman
instruments are microprocessor-controlled. The Beckman in-
struments were unable to maintain 0 °C, and the lower
temperature in the assays fluctuated between 2 and 5 °C.
Temperature changes were, however, more rapid than in the
Gilford instruments with the jump from the lower temperature
to 26 °C taking about 20 s and the reverse jump about 95 s.
In most cases the Beckman instruments yield somewhat lower
IC50 values than were obtained previously with the Gilford
instruments (cf. values for 1 and 8 with those previously
reported). The reason for this is unknown. In brief, tubulin
was preincubated at 26 °C with varying compound concentra-
tions, reaction mixtures were chilled on ice, GTP (required for
the polymerization reaction) was added, and polymerization
B-Hom o-2-eth oxy-3,17â-estr a d iol-6-en e (32). A solution
of methyllithium in THF/cumene (1.0 M, 2 mL, 2.0 mmol) was
added dropwise to a stirred solution of compound 13 (75 mg,
0.15 mmol) in THF (10 mL) under argon at 0 °C. The reaction

2428 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Wang et al.
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was followed at 26 °C by turbidimetry at 350 nm. The extent
of polymerization after 20 min was determined. IC50 values
were determined graphically. All compounds were examined
in at least two independent assays.
For examination of compounds that enhanced tubulin
polymerization, the Gilford model 250 instruments, equipped
with electronic temperature controllers, were used because of
their superior temperature control. In addition, the Gilford
instruments have more stable readings and are thus more
reliable for rate comparisons. There was no drug-tubulin
preincubation in these studies. Reaction mixtures (0.25 mL)
contained 1.2 mg/mL (12 µM) tubulin, 4% (v/v) dimethyl
sulfoxide, 0.8 M monosodium glutamate (pH 6.6), 0.4 mM GTP,
and varying compound concentrations. Reaction mixtures were
transferred to cuvettes held at 0 °C, baselines were established,
and the temperature was jumped to 24 °C (about 50 s). The
reaction was followed for about 25 min, and the temperature
was reverse jumped to 0 °C (about 4 min). In these experiments
the A350 in each sample was followed continuously for 5 s, and
approximately three readings per minute were taken. Maximal
rates were directly determined from the tracings. The EC50
values were determined graphically, by direct comparison to
a simultaneous reaction with 100 µM compound 16.
Ack n ow led gm en t. This research was made possible
by Contract NO1-CM-67260 from the National Cancer
Institute, DHHS.
Note Ad d ed in P r oof: At the 91st Annual Meeting
of the American Association for Cancer Research, April
1-5, 2000, a steroidal natural product was reported to
have microtuble-stabilizing activity. Mooberry, S. L.;
Hernandez, A. H.; Tien, G.; Hemscheidt, T. K. Discovery
of a New Microtubule-Stabilizing Agent from a Tropical
Plant. Abstract #3523.
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