Design and synthesis of an aminobenzo-15-crown-5-labeled estradiol tethered with disulfide linkage

Article abstract of DOI:10.1016/j.tet.2004.09.109

A novel measuring method (electroimmunoassay) of 17β-estradiol (E ₂) in urine or blood was proposed on the basis of a competition between E₂ and a labeled E₂ against an immobilizing antibody. To evaluate the principle, 3-{4-[17β-hydroxy-1,3,5(10)- estratrien-3-yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-octahydro-5,8,11,14, 17-pentaoxabenzocyclopentadecen-2-yl) succinamic acid (1) was designed and synthesized as a novel aminobenzo-15-crown-5-containing E₂ tethered with disulfide linkage. Two thiol-intermediates 5b and 19c were efficiently synthesized from mercaptosuccinic acid 7 and 4,4′-dithiodibutyric acid 12, respectively. Formations of disulfide linkages from less reactive thiols were examined and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) could be employed for the formation of an unsymmetrical disulfide 20c from 5b and 19c. Then, removal of TMS- and allyl-protecting groups in 20c successfully afforded the crown ether-containing E₂ 1. Graphical Abstract

Full text of DOI:10.1016/j.tet.2004.09.109

Tetrahedron 60 (2004) 11911–11922
Design and synthesis of an aminobenzo-15-crown-5-labeled
estradiol tethered with disulfide linkage
*
Shinya Harusawa, Kazufumi Yoshida, Chihiro Kojima, Lisa Araki and Takushi Kurihara
Osaka University of Pharmaceutical Sciences, 4-20-1, Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 27 August 2004; accepted 21 September 2004
Available online 19 October 2004
Abstract—A novel measuring method (electroimmunoassay) of 17b-estradiol (E2) in urine or blood was proposed on the basis of a
competition between E2 and a labeled E2 against an immobilizing antibody. To evaluate the principle, 3-{4-[17b-hydroxy-1,3,5(10)-
estratrien-3-yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-octahydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-2-yl) succinamic acid
(1) was designed and synthesized as a novel aminobenzo-15-crown-5-containing E2 tethered with disulfide linkage. Two thiol-intermediates
5b and 19c were efficiently synthesized from mercaptosuccinic acid 7 and 4,40-dithiodibutyric acid 12, respectively. Formations of disulfide
linkages from less reactive thiols were examined and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) could be employed for the formation of an
unsymmetrical disulfide 20c from 5b and 19c. Then, removal of TMS- and allyl-protecting groups in 20c successfully afforded the crown
ether-containing E2 1.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
accurately monitored in the blood and urine, can be
developed, it would be helpful for many women to maintain
the health or to detect of medical problems in the early
stage.
Estrogens are the hormones mainly responsible for the
growth of girls into sexually-mature women, in which 17b-
estradiol (E2) is the most abundant and potent.1,2 In
menopausal women, a decline in estrogen production results
in discomforts (e.g., hot flashes), changes in the metabolic
pathways of several organs, and the loss of essential bone
minerals leading to osteoporosis.3 Estrogen also plays an
important role in the maintenance of healthy blood-vessels
and blood-lipid profiles.4
We are now engaged in developing a novel measuring
method of E2 in urine or blood (termed as electro-
immunoassay) in collaboration with a certain electric
company. The principle of the assay and an equipment of
the E2 sensor are shown in Figure 1. The assay is a solid
phase-electroimmunoassay, based on competition between
E2 and a labeled E2 against an immobilizing antibody. The
principle of the assay is assumed as follows [the following
headings (1)–(6) correspond to those of Figure 1]: (1) anti-
E2 antibody is coated to a solid phase (agarose bead, etc.) to
form an immobilizing antibody (a), (2) next, labeled E2
molecule (b) is bound to a, giving a complex X, (3) then, an
electrode (c) is inserted into the complex X filled in a pipe of
the equipment [Y], and a working electrode (d) covered
with hydrophobic molecular film (insulating film) is placed
close to the counter-electrode (c), (4) when the tip of the
pipe is dipped in urine, a cross reaction occurs between
urinary E2 and the labeled E2 (b), (5) the liberated b is
absorbed to the hydrophobic film on the surface of the
working electrode d, and electric conduction is simul-
taneously caused between the two electrodes, (6) the
concentration of E2 in the urine is measured by an
alternating current (AC) resistance.
The E2 level varies throughout the menstrual cycle and on a
day-to-day basis.5 After about 2 weeks from the first day of
menses, the E2 level eventually reaches a peak and a sudden
surge in the production of luteinizing hormone (LH surge)
occurs, both of which trigger ovulation.6
Nowadays, women with problems related to pregnancy and
abortion constitute a major number of gynecological
patients.7 Therefore, it is very important that the woman
herself knows her menstrual cycles, menstruation days and
ovulation days. Estrogen tests may be also done to aid in the
diagnosis of many metabolic conditions. If a compact and
personal E2 sensor, by which the E2 level can be easily and
Keywords: 17b-Estradiol; Labeled estradiol; 40-Aminobenzo-15-crown-5;
Unsymmetrical disulfides; 4-Phenyl-1,2,4-triazoline-3,5-dione.
* Corresponding author. Tel.: C81 72 690 1086; fax: C81 72 690 1086;
e-mail: kurihara@gly.oups.ac.jp
40-Aminobenzo-15-crown-5 (2)8 may be first adopted as a
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.109

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S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
Figure 1. Principle of the electroimmunoassay.
marker in Figure 1. As the relationship between the
concentration of 2 and electrode-response could be
formulated, the concentration of 2 could be quantitatively
calculated from the numerical value arising from the
electrode-response.9 Anti-E2-glucuronide antibody (FKA-
238e)10 can be employed in this assay. Data of the cross
reactivity of FKA-238 with many steroids apparently show
that the sugar moiety of the E2-glucuronide is not required
as the antigen-recognition sites of FKA-238, as shown in
Figure 2, while the phenolic moiety and steroid D ring
containing secondary-OH are necessary.11 Therefore,
affinity of free E2 having phenolic-OH against the antibody
may be approximately equivalent to that of the E2-
glucuronide.
carboxylate into the spacer might make the molecule water-
soluble. Further, the solubility in water would be increased
by trapping of the sodium cation into the benzo-15-crown-5
(BC) residue. Herein, we describe the synthesis of a crown
ether-containing E2 1 tethered with a disulfide linkage,
which may be formed by oxidative coupling of two thiol
units. In this synthetic study, we found that 4-phenyl-1,2,4-
triazoline-3,5-dione (PTAD) can employed for formation of
unsymmetrical disulfides from two less reactive thiols.
2. Results and discussion
2.1. Synthesis of unsymmetrical disulfides
We first aimed at a synthesis of carboxylic acid 4, in which
the phenolic-OH was linked to an ester bond in the bridged
structure (Fig. 4). A crucial disulfide bond formation was
investigated in this synthetic study. Thiols 5 containing a
crown ether were synthesized from mercaptosuccinic acid
78, as shown in Scheme 1. Oxidation of 7 with alkaline
hydrogen peroxide followed by treatment of Ac2O12
afforded 2,20-dithiodisuccinic anhydride0 9 as a viscous
brown oil. Reaction of the crude 9 with 4 -amino BC ether
28 resulted in a disuccinamic acid 10 with a disulfide
linkage.13 Treatment of 10 with trimethylsilyldiazo-
methane14 gave a dimethyl disuccinamate 11a in 81%
overall yield from the starting 7. The following reduction of
11a did not proceed under mild reducing conditions such as
H2/Pd-black/MeOH,15 Ph3P/H2O–dioxane,16 Mg/THF–
MeOH,17 or Zn/AcOH,18 while the use of activated zinc
in a mixture of THF and saturated aqueous ammonium
chloride19 under reflux successfully afforded a thiol-
intermediate 5a in 76% yield. Further, diallyl disuccinamate
Figure 2. Antigen-recognition site of anti-E2-glucuronide (FKA-238).
We thus needed a labeled antigen b to evaluate the principle
in Figure 1. On the basis of the structural requirements
described above, we designed a candidate in which the
aromatic-NH2 of 2 was attached through a spacer group to
the phenolic-OH of E2 (Fig. 3). Introduction of sodium

S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11913
Figure 3. Design of a crown ether-labeled estradiol soluble in water.
Figure 4. Retrosynthesis of carboxylic acid 4 tethered with an ester bond.

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S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
Scheme 1. Reagents and conditions: (a) 8 N NaOH, 30%H2O2, rt, 2 h; (b) Ac2O, 60 8C, 3 h; (c) 2, MeCN, 50 8C, 2 h; (d) for 11a: TMSCHN2, THF, MeOH, rt,
10 min; (e) for 11b: allyl alcohol, WSC, DMAP, THF, rt, 3 h; (f) satd aqueous NH4Cl, Zn, THF, reflux, 16 h (for 5a), 24 h (for 5b).
11b, mentioned in the next section was synthesized by
esterification of 10 with allyl alcohol in the presence of 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
(WSC)/cat. DMAP in 46% overall yield from 7.20 Disulfide
11b was similarly converted into a thiol 5b (71%).
gave 14 in 85% yield. The reduction of 14 was carried out
by the previous zinc-aqueous ammmonium chloride, but
afforded thiol 6 in only 46% yield with recovery of
estadiolacetate 13. On the other hand, use of dried
ammonium chloride increased the yield of 6 to 70%.
Another steroidal thiol 6 was synthesized from 4,40-
dithiodibutyric acid 128 (Scheme 2). Treatment of a mixture
of 12 and b-estradiol-17 acetate 138 with DCC afforded only
a small amount of 4-dithiodibutanoate 14. Since Tanabe et
al. recently reported use of dimethylsulfamoyl chloride and
N,N-dimethylamine for esterification and amidation,21 we
applied it to the synthesis of 14. The new reagent system
Unsymmetrical disulfides can be prepared by the reaction of
sulfenyl derivatives with thiols.22a,b In the case of the
Mukaiyama method,22c reaction of a thiol RSH with diethyl
azodicarboxylate (DEAD) gives an N-alkanesulfenyl-
hydrazodicarboxylate, to which another thiol R’SH is then
added, producing the disulfide RSSR’. Oxidative coupling
of the two different thiols 5a and 6 using DEAD afforded an
Scheme 2.

S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11915
Table 1. Synthesis of unsymmetrical disulfide 3
Entry
Reaction conditions (h)
(ii)
Product (%)
(i)
DEAD, MeCN, rt, 1
DIAD, MeCN, rt, 1
3
15
1
2
3
4
Reflux, 5
Reflux, 17
—
15
10
—
40
—
—
—
10
TMAD, CH2Cl2, reflux, 1a
PTAD, MeCN, rt, 0.3
rt, 4
a TLC indicated no reaction
.
expected mixed-disulfide 3 in only 15% yield (Table 1,
entry 1). Other similar oxidizing agents, diisopropyl
azodicarboxylate (DIAD) or tetramethylazodicarboxamide
(TMAD)23 did not improve the reaction (entries 2 and 3).
Evidently, the low yield is due to the lower reactivity of 5a
and 6, because of small occupying of SH groups in the large
molecules. These results suggested to us that 4-phenyl-
1,2,4-triazoline-3,5-dione (PTAD),8,24 which is a powerful
dienophile of Diels–Alder reaction,25 might be able to effect
the dehydrogenation of two structurally different thiols to
disulfides. When thiol 6 was added to a characteristic red
solution of PTAD (1.0 equiv) in dichloromethane at rt, the
color faded to pale yellow (in ca 20 min), indicating the
formation of an N-alkanesulfenyl 4-phenylurazole inter-
mediate. Then, the secondary thiol 5a (1.0 equiv) was added
to the resulting solution and the whole was stirred at rt for
4 h. The expected disulfide 3 was obtained in an acceptable
yield (40%), along with a succinimide 15 (10%) cyclized by
internal elimination of methanol (entry 4). When the more
nucleophilic primary thiol 6 was reversely added to an
adduct formed from 5a and PTAD, the yield of 3 was only
10%. While Pilipenko et al. only used PTAD as an oxidant
for conversions of 2-mercaptopyridines into 2,20-dipyridyl
disulfides,26 PTAD has not been done for the synthesis of
unsymmetrical disulfides. This preliminary use of PTAD
was further extended in the preparation of unsymmerical
disulfides 20, as described in the next section.
led to the original E2-17-acetate 13. Therefore, we modified
the target compound to carboxylic acid 1 connected to
phenolic-OH of E2 with a stable ether-bonding (Fig. 3).
2.2. Synthesis of an amino BC containing-E2
The right-halves 19a–c of 1 were synthesized, as shown in
Scheme 3. Diborane-reduction27 of 128 (93%) followed by
bromination28 (97%) afforded a dibromide 17. Williamson
reaction of 17 with E2 in the presence of K2CO3 resulted in a
symmetrical disulfide 18a (75%),29 which was subsequently
reduced to a mercaptoestratrien-17-ol 19a (81%) by Zn-
NH4Cl. Alternatively, the C-17 hydroxy groups of 18a were
subjected to acetylation or trimethylsilylation to give
acetate 18b (80%) or TMS-ether 18c (97%). Their zinc
reductions also provided protected estradienylacetates 19b
(92%) and 19c (97%), respectively. Although diacetate 18b
could be also obtained by Mitsunobu reaction30 of diol 16
with E2-acetate 13 in the presence of DEAD and Ph3P in
58% yield (Scheme 3, Eq. 1), separation of 18b from 13
remaining in the reaction mixture was tedious.
Oxidative couplings between the right-half thiols 19 with
thiols 5 having a BC ether were performed using PTAD
(Table 2). The coupling of 19a (R1ZH) having a free
hydroxy group with left-half thiol 5a (R2ZMe) did not
proceed, while the reaction of protected thiols 19b (R1ZAc)
or 19c (R1ZTMS) with 5a supplied disulfides 20a or 20b in
50 or 62% yield, respectively (Table 2, entries 2 and 3). The
formation of 20a from 19b was accompanied with a small
amount of succinimide 21 as a byproduct (Scheme 4). The
reaction of 19c with allyl ester 5b afforded disulfide 20c in
41% yield.
It was, however, found that the CO–O bond of the phenolic
ester in 3 thus obtained was feasibly cleaved at the further
removal steps of two protecting groups: an acetyl group at
the steroid C-17 and a methyl group of the ester moiety
(Fig. 4). Treatment of disulfide 3 with standard hydrolysis-
conditions (aqueous methanolic 1 N NaOH or LiOH) easily

11916
S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
Scheme 3. (a) BH3$SMe2, THF, 0 8C, 0.5 h, then rt, 2.5 h; (b) CBr4, Ph3P, CH2Cl2, rt, 1 h; (c) E2, K2CO3, acetone, reflux, 24 h; (d) Ac2O, py, 100 8C, 1 h;
(e) TMSCl, Et3N, THF, rt, 16 h; (f) dry NH4Cl, Zn, THF, reflux, 3.5 h (for 18a and 18c), 6 h (for 18b); (g) 13, DEAD, Ph3P, CH2Cl2, 40 8C,16 h.
During this synthetic study, Arisawa and Yamaguchi31a
reported an interesting formation of unsymmetrical
disulfides using a novel exchange reaction of symmetrical
disulfides catalyzed by a rhodium complex.31a,b However,
the desired disulfides 20c could not be obtained by the
disulfide exchange reaction from the corresponding
disulfides 11b and 18c.31c
Next, removal of the protecting groups of disulfides 20a
(R1ZAc and R2ZMe) was investigated. Hydrolysis of 20a
with aqueous 1 N LiOH–MeOH or aqueous 1 N KHCO3–
MeOH led to a symmetrical disulfide 18b (Scheme 4), the
formation of which indicated an easy cleavage of the S–S
bond under alkaline conditions. Treatment of 20a in the
presence of cat. TsOH using a water separator afforded
Table 2. Synthesis of unsymmetrical disulfides 20
Entry
19
R1
5
R2
Time (h)a
20
Yield (%)
b
1
2
3
4
19a
19b
19c
19c
H
Ac
TMS
TMS
5a
5a
5a
5b
Me
Me
Me
allyl
4
4
20
4
—
—
20a
20b
20c
50c
62
41
a Time of step (ii).
b The used thiols were recovered.
c Succinimide 21 was accompanied as byproduct.

S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11917
Scheme 4. Reagent and conditions: (a) Table 2, entry 2; (b) 1 N KHCO3, MeOH, rt; (c) 1 N LiOH, MeOH, rt; (d) cat. p-TsOH, benzene, reflux, 6 h, Dean–Stark
water separator; (e) 1 N HCl, THF, reflux, 24 h; (f) 1 N NaOH, THF, rt.
succinimide 21 (91%) which was identical with the
compound contaminated during the formation of 20a from
19b (Table 2, entry 2). The 1H NMR spectra of 21 exhibited
characteristic C4-methylene peaks (Hb, Hc) at 3.04 (dd) and
3.30 (dd) ppm, respectively, showing their coupling
constants Jb–c (18.8 Hz), Jb–a (9.4 Hz), and Jc–a
(4.0 Hz), as shown in Scheme 4. Alkaline hydrolysis of 21
also brought about the formation of 18b, while reflux of
succinimide 21 in aqueous 1 N HCl produced only 22 with a
free hydroxy group at steroid C-17, the succinimide ring and
disulfide bond remaining intact.
by silica gel chromatography with a solvent system
(H2O:MeOH:CHCl3Z0.5:25:65) as a mixture of dia-
stereomers (1:3, 13C NMR analysis34) in 37% yield. When
succinamic acid 1 thus obtained was added in saturated
aqueous sodium bicarbonate, it dissolved with visual
bubbling.35 The structure of 1 was further confirmed by
2
3
3
From these results, we selected 20c (Table 2, run 4) with
TMS and allyl groups as suitable protecting groups which
might enable removal of the protecting groups under neutral
or acidic conditions (Scheme 5).32 It is known that the
cleavage of the allyl group is accomplished by palladium-
catalyzed reaction with an allyl scavenger that attacks the
allyl group, forming a p allyl palladium complex. Further,
Nagakura et al.33 recently reported that sulfinic acid or its
salt in the presence of a catalytic amount of Pd(PPh3)4 was
highly effective for facilitating the carbon–oxygen bond
cleavage of allyl esters. After TMS removal of 20c in
methanolic citric acid (96%),32 the final deallylation of the
resulting allylic ester 23 provided a crude carboxylic acid 1
by treatment of 23 with sodium p-toluenesulfinate in the
presence of catalytic amounts of Pd(PPh3)4 and PPh3.33
Purification of the crude product was not easy, but further
efforts clarified that the succinamic acid 1 could be isolated
Scheme 5. Reagents and conditinos: (a) citric acid, CH2Cl2, MeOH, rt, 1 h;
(b) (i) (Ph3P)4Pd, Ph3P, TolSO2Na, THF, MeOH, rt, 16 h; (ii) SiO2
chromatography, H2O:MeOH:CHCl3 (0.5:25:65).

11918
S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
conversion into a methyl ester 24 (67%), which was
correlated to the previous disulfide 20b, since desilylation
of 20b afforded 24 (Scheme 6).
4.1.1. 2,20-Dithiodisuccinic acid (8). To 8 N aqueous
NaOH (18 ml) was added gradually mercaptosuccinic acid
78 (10 g, 67 mmol) at 0 8C to avoid an exothermic reaction
and then 30% H2O2 (5.2 ml) was added slowly to the
solution at rt. The reaction mixture was stirred at rt for 2 h
and subsequently acidified to pH 1 by addition of 20%
aqueous H2SO4 (19 ml). The whole was extracted with Et2O
(!3) by salting-out techniques. The extract was dried and
evaporated to give 8 (10 g, quant) as pale brown powders;
1H NMR (DMSO-d6) d 2.69 (dd, JZ11.6, 3.2 Hz, 2H), 2.83
(ddd, JZ11.6, 6.4, 1.1 Hz, 2H), 3.75 (dt, JZ6.4, 3.2 Hz,
2H), 12.4–13.0 (br, 1H); HRMS (SIMS) calcd for
C8H11O8S2 [(MC1)C] 298.9894, found 298.9891.
4.1.2. 2,20-Dithiodisuccinic anhydride (9). Acetic
anhydride (5 ml) was added to 8 (900 mg, 3.0 mmol), and
the mixture was stirred at 60 8C for 3 h. Then, the acetic
anhydride was evaporated using an aspirator in a hood, and
the resulting residue was dissolved with EtOAc. The EtOAc
solution was washed with H2O, brine, dried, and evaporated
to give a black oil 9 (791 mg, quant); IR (nujol) 1860, 1780,
Scheme 6. Reagents and conditions: (a) TMSCHN2, THF, MeOH, rt, 0.5 h;
(b) citric acid, CH2Cl2, MeOH, rt, 0.5 h.
1
1040 cmK1; H NMR (CDCl3) d 3.16 (ddd, JZ19.8, 6.4,
3. Conclusion
2.1 Hz, 2H), 3.48 (dq, JZ19.8, 4.9 Hz, 2H), 4.28–4.40 (m,
2H); HRMS (SIMS) calcd for C8H7O6S2 [(MC1)C]
262.9683, found 262.9687.
In conclusion, we have described the design and synthesis of
a novel BC-containing antigen 1 for the newly proposed
measuring system of E2 in urine and blood, as shown in
Figure 1. The synthetic approach was briefly outlined in
Figure 5, in which PTAD was effective for the formation of
the unsymmetrical disulfide 20c from less reactive thiols 5b
and 19c, and TMS and allyl groups were selected as the
protecting groups of 20c. Further work on use of 1 in the
electroimmunoassay is under way and will be published in
due course.
4.1.3. 3,30-Dithiobis[N-(6,7,9,10,12,13,15,16-octahydro-
5,8,11,14,17-pentaoxa-benzocyclopentadecen-2-yl)]
disuccinamic acid (10). To a solution of 9 (791 mg,
3.0 mmol) in acetonitrile (20 ml) was added slowly 28
(1700 mg, 6.0 mmol) at 50 8C. The mixture was stirred at
the same temperature for 2 h, and the solvent was
evaporated to give a residue. The residue was washed
twice with hot benzene (20 ml) by decantation. The
collected benzene solutions were evaporated to give 10
1
(2490 mg, quant) as pale brown amorphous product; H
NMR (DMSO-d6) d 2.69–3.08 (m, 2H), 3.49–3.68 (m, 16H),
3.68–3.88 (m, 8H), 3.88–4.16 (m, 10H), 6.80–7.31 (m, 6H),
12.1–12.8 (br, 2H); HRMS (SIMS) calcd for
C36H49N2O16S2 [(MC1)C] 829.2520, found 829.2529.
4.1.4. Dimethyl 3,30-dithiobis[N-(6,7,9,10,12,13,15,16-
octahydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-
2-yl)] disuccinamate (11a). To a solution of the disuc-
cinamic acid (10, 300 mg, 0.36 mmol) in MeOH–THF (1:1,
4 ml) was added slowly 2.0 N hexane solution of
TMSCHN2 (0.36 ml, 0.72 mmol). After 10 min at rt, the
solvent was evaporated to give a residue which was
subsequently purified by column chromatography
(MeOH:CHCl3Z1:20) to give 11a (252 mg, 81%) as pale
Figure 5. The outline of the present synthetic study.
4. Experimental
4.1. General methods
The melting points were determined on a hot-stage
apparatus and were uncorrected. IR spectra were recorded
on a Shimadzu IR-435 spectrometer. 1H and 13C NMR
spectra were taken with tetramethylsilane as an internal
standard on a Varian Gemini-200, Varian Mercury-300 and
Varian UNITY INOVA-500 spectrometers. Reactions with
air- and moisture-sensitive compounds were carried out
under an argon atmosphere. Unless otherwise noted, all
extracts were dried over Na2SO4, and the solvent was
removed in a rotary evaporator under reduced pressure.
Unless otherwise stated, Fuji Silysia FL-60D silica gel, Fuji
Silysia BW-127ZH silica gel, and Merck 60F254 were used
for flash column chromatography, column chromatography
and thin-layer chromatography (TLC), respectively.
1
brown amorphous product; H NMR (CDCl3) d 2.72–2.98
(m, 2H), 2.98–3.29 (m, 2H), 3.64–3.81 (m, 22H), 3.81–3.98
(m, 8H), 3.98–4.16 (m, 10H), 6.66–7.02 (m, 6H); HRMS
(SIMS) calcd for C38H53N2O16S2 [(MC1)C] 857.2833,
found 857.2836.
4.1.5. Diallyl 3,30-dithiobis[N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-2-
yl)] disuccinamate (11b). To a solution of 10 (300 mg,
0.36 mmol), DMAP (4 mg, 0.04 mmol) and allyl alcohol
(0.49 ml, 7.25 mmol) in THF (6 ml) was added WSC
(174 mg, 0.91 mmol). The mixture was stirred for 3 h at rt
and evaporated to give a residue which was subsequently

S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11919
dissolved with CHCl3. The organic layer was washed with
2 N HCl, brine, dried (MgSO4), and evaporated. The residue
was purified by column chromatography (MeOH:CHCl3Z
3:97) to give 11b (150 mg, 46%) as yellow amorphous
product; 1H NMR (CDCl3) d 2.76–2.99 (m, 2H), 2.99–3.34
(m, 2H), 3.68–3.79 (m, 16H), 3.79–3.94 (m, 8H), 4.01–4.18
(m, 10H), 4.52–4.77 (m, 4H), 5.15–5.44 (m, 4H), 5.76–6.02
(m, 2H), 6.66–7.09 (m, 6H); HRMS (SIMS) calcd for
C42H57N2O16S2 [(MC1)C] 909.3146, found 909.3151.
4.1.9. 17-Acetoxy-1,3,5(10)-estratrien-3-yl 4-mercapto-
butanoate (6). By the same as for the preparation of 5a, a
mixture of 14 (415 mg, 0.5 mmol), activated zinc (98 mg,
1.5 mmol), and dried ammonium chloride (80 mg,
1.5 mmol) in THF (8 ml) was refluxed for 3 h followed by
column chromatography (Mallincrodt 6447) to yield 6
(292 mg, 70%) as a white powder (which was susceptible to
oxidation into disulfide 14 in storage); mp 72–73 8C
(colorless needles from 5% benzene in acetonitrile; 1H
NMR (CDCl3) d 0.83 (s, 3H), 1.24–1.59 (m, 8H), 1.70–1.78
(m, 1H), 1.85–1.92 (m, 2H), 2.06 (s, 3H), 2.05 (quint, JZ
7.0 Hz, 2H), 2.17–2.32 (m, 3H), 2.66 (dt, JZ8.5, 7.0 Hz,
2H), 2.69 (t, JZ7.0 Hz, 2H), 2.84–2.89 (m, 2H), 4.69 (dd,
JZ9.0, 8.0 Hz, 1H), 6.78 (d, JZ2.5 Hz, 1H), 6.83 (dd, JZ
8.6, 2.5 Hz, 1H), 7.27 (d, JZ8.6 Hz, 1H); 13C NMR
(CDCl3) d 12.0, 21.1, 23.2, 23.9, 26.0, 27.0, 27.5, 28.9, 29.5,
32.7, 36.8, 38.2, 42.8, 43.9, 49.8, 82.6, 118.5, 121.4, 126.4,
137.9, 138.1, 148.3, 171.2, 171.8.
4.1.6. Methyl 3-mercapto-N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-2-yl)
succinamate (5a). To a solution of 11a (300 mg,
0.35 mmol) in THF (6 ml) was added activated zinc
(68 mg, 1.00 mmol) and saturated aqueous ammonium
chloride solution (0.5 ml). The mixture was refluxed for
16 h to produce green precipitates. The supernatant liquid of
the mixture was separated and the remaining precipitate was
stirred in CHCl3–MeOH (1:1) to form a suspension. The
suspension was combined with the previous precipitate, and
the whole was filtered through a Celite pad. The filtrate was
washed with H2O, dried (MgSO4), and evaporated to give a
residue. It was purified by SiO2 column chromatography
(Mallincrodt 6447) using MeOH–CHCl3 (1:9) to give 5a
4.1.10. Methyl 3-{3-[17-acetoxy-1,3,5(10)-estratrien-3-
yloxycarbonyl]propyl-disulfanyl}-N-(6,7,9,10,12,13,15,
16-octahydro-5,8,11,14,17-pentaoxabenzocyclo-penta-
decen-2-yl) succinamate (3). To a solution of 6 (75 mg,
0.18 mmol) in acetonitrile (2 ml) was added PTAD (32 mg,
0.18 mmol). The resulting red solution changed to a pale
yellow solution by a stirring at rt for 20 min. Then, a
solution of 5a (84 mg, 0.20 mmol) in acetonitrile (1 ml) was
added to the mixture which was further stirred at rt for 4 h.
The solvent was evaporated to give a residue, which was
purified by flash chromatography with CHCl3 to give 3
(62 mg, 40%) and 15 (13 mg, 10%). 3: a pale yellow
amorphous product; 1H NMR (CDCl3) d 0.83 (s, 3H), 1.22–
1.59 (m, 7H), 1.70–1.78 (m, 1H), 1.84–1.92 (m, 2H), 2.06
(s, 3H), 2.08–2.16 (m, 2H), 2.17–2.32 (m, 3H), 2.60–2.72
(m, 2H), 2.79–2.89 (m, 4H), 3.10–3.39 (m, 2H), 3.71 (s,
3H), 3.73–3.77 (m, 8H), 3.84–3.98 (m, 5H), 4.07–4.12 (m,
4H), 4.69 (t, JZ8.4 Hz, 1H), 6.75–6.86 (m, 5H), 7.24–7.28
(m, 1H); HRMS (SIMS) calcd for C43H58NO8S2 [(MC1)C]
844.3397, found 844.3393. 15: oil; 1H NMR (CDCl3) d 0.83
(s, 3H), 1.23–1.78 (m, 8H), 1.85–1.91 (m, 2H), 2.06 (s, 3H),
2.10–2.32 (m, 5H), 2.66 (t, JZ9.1 Hz, 2H), 2.80–2.92 (m,
4H), 3.03 (dd, JZ18.7, 4.2 Hz, 1H), 3.32 (dd, JZ18.7,
9.3 Hz, 1H), 3.76 (m, 8H), 3.88 (m, 4H), 3.96 (dd, JZ9.3,
4.2 Hz, 1H), 4.11 (m, 4H), 4.69 (t, JZ5.4 Hz, 1H), 6.77–
6.86 (m, 4H), 7.24–7.28 (m, 2H).
1
(228 mg, 76%) as a pale yellow amorphous product; H
NMR (CDCl3) d 2.58–3.42 (m, 2H), 3.60–3.81 (m, 11H),
3.81–4.00 (m, 4H), 4.00–4.22 (m, 5H), 6.70–6.95 (m, 3H);
HRMS (SIMS) calcd for C19H28NO8S [(MC1)C]
430.1534, found 430.1540.
4.1.7. Allyl 3-mercapto-N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclopentadecen-2-yl)
succinamate (5b). By the same as for the preparation of 5a,
a mixture of 11b (546 mg, 0.60 mmol), activated zinc
(195 mg, 3.0 mmol), and saturated aqueous ammonium
chloride solution (1.0 ml) was reluxed for 24 h followed by
column chromatography (Mallincrodt 6447) to yield 5b
1
(387 mg, 71%) as a pale yellow amorphous product; H
NMR (CDCl3) d 2.60–3.32 (m, 2H), 3.60–3.80 (m, 8H),
3.80–4.00 (m, 4H), 4.00–4.22 (m, 5H), 4.52–4.76 (m, 2H),
5.16–5.43 (m, 2H), 5.76–6.00 (m, 1H), 6.68–7.01 (m, 3H);
HRMS (EIMS) calcd for C21H29NO8S [(MC1)C]
455.1612, found 455.1613.
4.1.8. Bis[17-acetoxy-1,3,5(10)-estratrien-3-yl] 4,40-
dithiodibutanoate (14). To a suspension of 4,40-dithio-
dibutyric acid 128 (723 mg, 3.0 mmol) in acetonitrile
(21 ml) were added b-estradiol-17 acetate 138 (1900 mg,
6.1 mmol), BuNMe2 (2.2 ml, 18.2 mmol), DMAP (75 mg,
0.61 mmol), Me2NSO2Cl (1.3 ml, 12.2 mmol) in that order.
The mixture was stirred at 45 8C for 1 h to produce white
precipitate. The precipitate was filtered and washed with
cold acetonitrile to give 14 (2150 mg, 85%) as a white
powder; mp 167–170 8C (colorless small needles from 5%
4.1.11. 4,40-Dithiodibutan-1-ol (16). To a suspension of
128 (2.00 g, 8.4 mmol) in THF (30 ml) was added slowly
2 M THF solution of BH3$SMe2 (12.0 ml, 24.0 mmol) over
30 min at 0 oC to give a pale brown precipitate. After the
resulting mixture was stirred at the same temperature for
30 min and further at rt for 2.5 h, methanol was added to
dissolve the precipitate. The solvent was evaporated to give
a residue, which was subsequently diluted with EtOAc. The
solution was washed with saturated aqueous sodium
bicarbonate, brine, and dried, and evaporated to give a
crude oil, which was subjected to a flash chromatography.
Elution with MeOH–CHCl3 (3:17) afforded 16 (1.63 g,
1
benzene in acetonitrile); H NMR (CDCl3) d 0.82 (s, 6H),
1.20–1.62 (m, 14H), 1.68–1.80 (m, 2H), 1.82–1.94 (m, 4H),
2.06 (s, 6H), 2.15 (quint, JZ6.9 Hz, 4H), 2.09–2.34 (m,
6H), 2.69 (t, JZ6.9 Hz, 4H), 2.81 (t, JZ6.9 Hz, 4H), 2.75–
2.90 (m, 4H), 4.68 (t, JZ8.3 Hz, 2H), 6.77 (d, JZ2.4 Hz,
2H), 6.83 (dd, JZ8.3, 2.4 Hz, 2H), 7.26 (d, JZ8.3 Hz, 2H);
HRMS (SIMS) calcd for C48H63O8S2 [(MC1)C] 831.3961,
found 831.3963; Anal. Calcd for C48H62O8S2: C, 69.37; H,
7.52; S, 7.72. Found: C, 69.15; H, 7.39; S, 7.84.
1
93%) as a pale yellow oil; H NMR (CDCl3) d 1.62–1.86
(m, 8H), 2.73 (t, JZ6.3 Hz, 4H), 3.67 (t, JZ6.3 Hz, 4H);
HRMS (EIMS) calcd for C8H18O2S2 (MC) 210.0747, found
210.0741.

11920
S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
4.1.12. 4,40-Dithiodibutyl bromide (17). To a solution of
16 (250 mg, 1.19 mmol) in CH2Cl2 (8 ml) was added CBr4
(1250 mg, 3.81 mmol) and PPh3 (749 mg, 2.86 mmol) at
0 8C. The mixture was stirred at rt for 1 h and a small
amount of silica gel was added to the solution. The solvent
was evaporated to give a coated silica gel, which was
subsequently placed in a column (Mallincrodt 6447).
Chromatography using EtOAc–hexane (1:9) gave 17
(m, 2H), 2.70–2.90 (m, 8H), 3.63 (t, JZ8.4 Hz, 2H), 3.90–
4.00 (m, 4H), 6.60 (d, JZ2.6 Hz, 2H), 6.67 (dd, JZ8.6,
2.6 Hz, 2H), 7.18 (d, JZ8.6 Hz, 2H); HRMS (EIMS) calcd
for C50H78O4S2Si2 (MC) 862.4876, found 862.4881.
4.1.16. 3-(4-Mercaptobutoxy)-1,3,5(10)-estratrien-17-ol
(19a). By the same procedure as for the preparation of 5a,
a mixture of 18a (93 mg, 0.13 mmol), activated zinc
(43 mg, 0.65 mmol), and dried ammonium chloride
(35 mg, 0.65 mmol) in THF (5 ml) was refluxed for 3.5 h.
Filtration of the resulting mixture through a Celite pad,
evaporation, and column chromatography gave 19a (76 mg,
1
(387 mg, 97%) as a pale yellow oil; H NMR (CDCl3) d
1.78–2.06 (m, 8H), 2.72 (t, JZ7.2 Hz, 4H), 3.43 (t, JZ
7.2 Hz, 4H); HRMS (EIMS) calcd for C8H16O2Br2 [(MC
1)C] 333.9060, found 333.9054.
1
81%) as a white powder; H NMR (CDCl3) d 0.78 (s, 3H),
4.1.13. Bis{4-[17-hydroxy-1,3,5(10)-estratrien-3-yloxy]-
butyl} disulfide (18a). A suspension of 17 (85 mg,
0.25 mmol), E2 (345 mg, 1.27 mmol), and potassium
carbonate (263 mg, 1.91 mmol) in acetone (10 ml) was
refluxed for 24 h and then diluted with EtOAc. The resulting
mixture was washed with H2O, brine, dried, and evaporated
to give a residue. The residue was dissolved with EtOAc and
a small amount of silica gel was added to the solution. The
solvent was evaporated to give a coated silica gel, which
was subsequently placed in a column. Flash chromato-
graphy using EtOAc–hexane (3:7) gave 18a (137 mg, 75%)
1.12–1.60 (m, 9H), 1.64–2.00 (m, 7H), 2.04–2.24 (m, 2H),
2.26–2.36 (m, 1H), 2.60 (q, JZ7.0 Hz, 2H), 2.80–2.88 (m,
2H), 3.72 (t, JZ8.1 Hz, 1H), 3.94 (t, JZ6.2 Hz, 2H), 6.61
(d, JZ2.7 Hz, 1H), 6.68 (dd, JZ8.4, 2.7 Hz, 1H), 7.18 (d,
JZ8.4 Hz, 1H); HRMS (EIMS) calcd for C22H32O2S (MC)
360.2121, found 360.2128.
4.1.17. 3-(4-Mercaptobutoxy)-1,3,5(10)-estratrien-17-yl
acetate (19b). A mixture of 18b (240 mg, 0.3 mmol),
activated zinc (104 mg, 1.5 mmol), and dried ammonium
chloride (80 mg, 1.5 mmol) in THF (6 ml) was refluxed for
6 h to give 19b (222 mg, 92%) as a white powder; 1H NMR
(CDCl3) d 0.83 (s, 3H), 1.20–1.62 (m, 8H), 1.68–1.96 (m,
7H), 2.06 (s, 3H), 2.14–2.34 (m, 3H), 2.60 (q, JZ7.7 Hz,
2H), 2.80–2.90 (m, 2H), 3.94 (t, JZ6.9 Hz, 2H), 4.68 (t, JZ
8.3 Hz, 1H), 6.61 (d, JZ2.7 Hz, 1H), 6.68 (dd, JZ8.6,
2.7 Hz, 1H), 7.17 (d, JZ8.6 Hz, 1H); 13C NMR (CDCl3) d
12.5, 21.6, 23.6, 24.8, 26.6, 27.9, 28.4, 30.1, 37.1, 38.8,
43.1, 44.0, 50.0, 67.2, 82.6, 111.7, 114.1, 125.9, 132.1,
137.4, 156.2, 170.5; HRMS (EIMS) calcd for C24H34O3S
(MC) 402.2227, found 402.2225.
1
as white amorphous product; H NMR (CDCl3) d 0.78 (s,
6H), 1.12–1.62 (m, 16H), 1.64–1.76 (m, 2H), 1.80–1.98 (m,
12H), 2.02–2.24 (m, 4H), 2.24–2.36 (m, 2H), 2.70–2.80 (m,
4H), 2.80–2.88 (m, 4H), 3.68–3.78 (m, 2H), 3.90–3.98 (m,
4H), 6.61 (d, JZ2.4 Hz, 2H), 6.68 (dd, JZ8.7, 2.4 Hz, 2H),
7.18 (d, JZ8.7 Hz, 2H); HRMS (EIMS) calcd for
C44H62O4S2 (MC) 718.4086, found 718.4089.
4.1.14. Bis{4-[17-acetoxy-1,3,5(10)-estratrien-3-yloxy]-
butyl} disulfide (18b). A solution of 18a (200 mg,
0.28 mmol) in acetic anhydride (5 ml) and pyridine
(0.5 ml) was stirred at 100 8C for 1 h. Ice was added to
the mixture, and the resulting mixture was diluted with
EtOAc. The organic layer was washed with 2 N HCl,
saturated aqueous sodium bicarbonate, H2O, brine, dried
and evaporated. The residue was dissolved in EtOAc and a
small amount of silica gel was added to the solution. The
solvent was evaporated to give a coated silica gel, which
was subsequently placed in a column. Flash chromato-
graphy using EtOAc–hexane (1:9) gave 18b (179 mg, 80%)
as a white powder; 1H NMR (CDCl3) d 0.82 (s, 6H), 1.16–
2.00 (m, 20H), 1.86 (m, 8H), 2.06 (s, 6H), 2.10–2.36 (m,
6H), 2.68–2.70 (m, 8H), 3.94 (m, 6H), 4.68 (t, JZ8.5 Hz,
2H), 6.61 (d, JZ3.0 Hz, 2H), 6.68 (dd, JZ8.7, 3.0 Hz, 2H),
7.17 (d, JZ8.7 Hz, 2H); HRMS (EIMS) calcd for
C48H66O6S2 (MC) 802.4297, found 802.4299.
4.1.18. 4-[17-Trimethylsilyloxy-1,3,5(10)-estratrien-3-
yloxy]butanethiol (19c). A mixture of 18c (424 mg,
0.49 mmol), activated zinc (96 mg, 1.50 mmol), and dried
ammonium chloride (78 mg, 1.47 mmol) in THF (10 ml)
was refluxed for 3.5 h. The mixture was evaporated to give a
residue, which was dissolved with hexane. Filtration of the
solution through a Celite pad and evaporation gave 19c
(413 mg, 97%) as a white amorphous product; RfZ0.54 on
1
silica gel TLC (EtOAc–hexaneZ1:9); H NMR (CDCl3) d
0.10 (s, 9H), 0.74 (s, 3H), 1.08–1.72 (m, 9H), 2.08–2.36 (m,
2H), 2.60 (q, JZ7.4 Hz, 2H), 2.79–2.87 (m, 2H), 3.63 (t,
JZ8.3 Hz, 1H), 3.94 (t, JZ6.1 Hz, 2H), 6.60 (d, JZ2.6 Hz,
1H), 6.68 (dd, JZ8.4, 2.6 Hz, 1H), 7.18 (d, JZ8.4 Hz, 1H);
13C NMR (CDCl3)d 0.8, 11.8, 23.6, 24.8, 26.8, 27.7, 28.4,
30.2, 31.0, 31.2, 37.3, 39.1, 43.6, 44.3, 49.9, 67.2, 81.7,
111.7, 114.1, 126.0, 132.4, 137.6, 156.1, HRMS (EIMS)
calcd for C25H40O2SSi (MC) 432.2516, found 432.2519.
4.1.15. Bis{4-[17-trimethylsilyloxy-1,3,5(10)-estratrien-
3-yloxy]butyl} disulfide (18c). To a solution of 18a
(1.05 g, 1.47 mmol) in THF (40 ml) were added chloro-
trimethylsilane (1.87 ml, 1.47 mmol) and triethylamine
(2.00 ml, 14.7 mmol). The resulting mixture was stirred at
rt for 16 h and evaporated to give a residue, which was
subsequently dissolved in hexane. The solution was washed
with saturated aqueous sodium bicarbonate, dried, and
evaporated to give 18c (1.22 g, 97%) as amorphous product;
4.1.19. Methyl 3-{4-[17-acetoxy-1,3,5(10)-estratrien-3-
yloxy]butyl-disulfanyl}-N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclo-pentadecen-2-
yl) succinamate (20a). To a solution of 19b (60 mg,
0.15 mmol) in CH2Cl2 (1 ml) was added PTAD (26 mg,
0.15 mmol). The solution was stirred at rt for 1 h. Then, a
solution of 5a (65 mg, 0.15 mmol) in CH2Cl2 (1 ml) was
added to the mixture which was stirred at rt for 4 h to give
20a (62 mg, 50%) as a pale yellow amorphous product; 1H
NMR (CDCl3) d 0.82 (s, 3H), 1.18–1.97 (m, 15H), 2.06 (s,
1
RfZ0.48 on silica gel TLC (EtOAc–hexaneZ1:9, v/v); H
NMR (CDCl3) d 0.10 (s, 18H), 0.73 (s, 6H), 1.08–1.72 (m,
16H), 1.78–2.00 (m, 14H), 2.10–2.22 (m, 2H), 2.22–2.34

S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11921
3H), 2.11–2.35 (m, 4H), 2.72–2.90 (m, 4H), 3.71 (s, 3H),
3.68–3.82 (m, 8H), 3.82–4.01 (m, 7H), 4.06–4.18 (m, 4H),
4.68 (t, JZ7.8 Hz, 1H), 6.60 (d, JZ2.8 Hz, 1H), 6.67 (dd,
JZ8.4, 2.8 Hz, 1H), 6.73–6.95 (m, 3H), 7.18 (d, JZ8.4 Hz,
1H); HRMS (SIMS) calcd for C43H59NO11S2 [(MC1)C]
829.3526, found 829.3530.
4.1.23. 1-{4-[17-Hydroxy-1,3,5(10)-estratrien-3-yloxy]-
butyl-2-{N-(6,7,9,10,12,13,15,16-octahydro-5,8,11,14,17-
pentaoxabenzocyclopentadecen-2-yl)-2,5-dioxopyrroli-
din-3-yl} disulfide (22). A solution of 21 (8 mg, 0.01 mmol)
in THF (2 ml) was refluxed with 1 N HCl for 24 h to give 22
(6 mg, 75%) as a pale yellow amorphous product; 1H NMR
(CDCl3) d 0.78 (s, 3H), 1.12–2.03 (m, 15H), 2.03–2.36 (m,
4H), 2.71–2.94 (m, 4H), 3.04 (dd, JZ18.8, 3.8 Hz, 1H),
3.30 (dd, JZ18.8, 9.0 Hz, 1H), 3.62–3.81 (m, 9H), 3.81–
4.02 (m, 7H), 4.02–4.20 (m, 4H), 6.60 (d, JZ2.3 Hz, 1H),
6.66 (dd, JZ8.9, 2.3 Hz, 1H), 6.78 (d, JZ1.5 Hz, 1H), 6.83
(dd, JZ8.9, 1.5 Hz, 1H), 6.90 (d, JZ8.9 Hz, 1H), 7.16 (d,
JZ8.9 Hz, 1H); MS of 22 could not be measured owing its
thermal instability.
4.1.20. Methyl 3-{4-[17-trimethylsilyloxy-1,3,5(10)-
estratrien-3-yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,
15,16-octahydro-5,8,11,14,17-pentaoxabenzocyclo-pen-
tadecen-2-yl) succinamate (20b). A pale yellow amor-
1
phous product; H NMR (CDCl3) d 0.10 (s, 9H), 0.74 (s,
3H), 1.06–1.58 (m, 7H), 1.58–2.02 (m, 8H), 2.08–2.36 (m,
2H), 2.72–2.92 (m, 4H), 3.16–3.40 (m, 2H), 3.63 (t, JZ
8.3 Hz, 1H), 3.71 (s, 3H), 3.60–3.82 (m, 8H), 3.82–4.04 (m,
7H), 4.02–4.22 (m, 4H), 6.60 (d, JZ2.5 Hz, 1H), 6.67 (dd,
JZ8.0, 2.5 Hz, 1H), 6.74–6.96 (m, 3H), 7.18 (d, JZ8.0 Hz,
1H); HRMS (SIMS) calcd for C44H66NO10S2Si [(MC1)C]
860.3893, found 860.3902.
4.1.24. Allyl 3-{4-[17-hydroxy-1,3,5(10)-estratrien-3-
yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclo-pentadecen-2-
yl) succinamate (23). To a solution of 20c (340 mg,
0.38 mmol) in CH2Cl2–MeOH (1:1, 10 ml) was added citric
acid (37 mg, 0.19 mmol). The mixture was stirred at rt for
1 h. The solvent was evaporated to give a residue, which
was diluted with CH2Cl2. The resulting insoluble material
was filtered off through a Celite pad to give a filtrate.
Evaporation of the filtrate gave 23 (296 mg, 96%) as a pale
4.1.21. Allyl 3-{4-[17-trimethylsilyloxy-1,3,5(10)-estra-
trien-3-yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-
octahydro-5,8,11,14,17-pentaoxabenzocyclo-penta-
decen-2-yl) succinamate (20c). To a solution of 19c
(139 mg, 0.32 mmol) in CH2Cl2 (8 ml) was added PTAD
(68 mg, 0.39 mmol). The resulting red solution was changed
to a pale yellow solution by a stirring at rt for 1 h. Then, a
solution of 5b (147 mg, 0.32 mmol) in CH2Cl2 (4 ml) was
added to the mixture which was stirred at rt for 4 h. The
solvent was evaporated to give a residue, which was purified
by column chromatography (Mallinckrodt 6447) with
MeOH–CHCl3 (1:99) to give 20c (116 mg, 41%) as a pale
1
yellow amorphous product; H NMR (CDCl3) d 0.77 (s,
3H), 1.12–2.02 (m, 15H), 2.02–2.36 (m, 2H), 2.70–2.94 (m,
4H), 2.98–3.36 (m, 2H), 3.68–3.83 (m, 9H), 3.83–4.02 (m,
7H), 4.05–4.18 (m, 4H), 4.57–4.68 (m, 2H), 5.20–5.40 (m,
2H), 5.81–5.99 (m, 1H), 6.61 (d, JZ2.8 Hz, 1H), 6.68 (dd,
JZ8.4, 2.8 Hz, 1H), 6.73–6.98 (m, 3H), 7.18 (d, JZ8.4 Hz,
1H); HRMS (SIMS) calcd for C43H60NO10S2 [(MC1)C]
814.3655, found 814.3651.
1
yellow amorphous product; H NMR (CDCl3) d 0.10 (s,
9H), 0.73 (s, 3H), 1.08–1.73 (m, 8H), 1.73–2.02 (m, 7H),
2.08–2.36 (m, 2H), 2.72–2.92 (m, 4H), 2.98–3.42 (m, 2H),
3.63 (t, JZ8.0 Hz, 2H), 3.67–3.82 (m, 8H), 3.82–4.00 (m,
7H), 4.02–4.20 (m, 4H), 4.53–4.72 (m, 2H), 5.13–5.40 (m,
2H), 5.79–5.98 (m, 1H), 6.60 (d, JZ2.8 Hz, 1H), 6.67 (dd,
JZ8.4, 2.8 Hz, 1H), 6.74–6.95 (m, 3H), 7.18 (d, JZ8.4 Hz,
1H); HRMS (SIMS) calcd for C46H68NO10S2Si [(MC1)C]
886.405, found 886.4038.
4.1.25. 3-{4-[17-Hydroxy-1,3,5(10)-estratrien-3-yloxy]-
butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-octahydro-5,
8,11,14,17-pentaoxabenzocyclo-pentadecen-2-yl) suc-
cinamic acid (1). To a THF–MeOH (4:3, 7 ml) solution
of 23 (208 mg, 0.26 mmol) in a flask, which was blinded by
aluminum foil, were added Pd(PPh3)4 (15 mg, 0.01 mmol),
PPh3 (7 mg, 0.03 mmol), and sodium p-toluenesulfinate
(46 mg, 0.26 mmol). The mixture was stirred at rt for 16 h
and acidified with 2 N hydrochloric acid (0.15 ml). The
solvent was evaporated to give a residue, which was
chromatographed [BW-127ZH, H2O–MeOH–CHCl3
(0.5:25:65)] to give 1 (73 mg, 37%) as a pale yellow
amorphous product. It was dissolved with visual bubbling in
saturated aqueous sodium bicarbonate35; RfZ0.4, 0.5 [a
mixture of diastereomers (1:3)34, H2O–MeOH–CHCl3
4.1.22. 1-{4-[17-Acetoxy-1,3,5(10)-estratrien-3-yloxy]}-
butyl-2-{N-(6,7,9,10,12,13,15,16-octahydro-5,8,11,14,17-
pentaoxabenzocyclopentadecen-2-yl)-2,5-dioxo-
pyrrolidin-3-yl} disulfide (21). A solution of 20a (11 mg,
0.01 mmol) in benzene (15 ml) was refluxed with p-TsOH
(1 mg) for 6 h using a Dean–Stark water separator. The
solvent was evaporated to give a residue, which was
dissolved with CH2Cl2. The solution was washed with H2O,
dried (MgSO4), and evaporated to give a crude oil that was
purified by column chromatography with MeOH–CHCl3
(3:97) to give 21 (10 mg, 91%) as an amorphous product; 1H
NMR (CDCl3) d 0.82 (s, 3H), 1.20–1.98 (m, 15H), 2.05 (s,
3H), 2.12–2.34 (m, 4H), 2.72–2.94 (m, 4H), 3.04 (dd, JZ
18.8, 4.0 Hz, 1H), 3.30 (dd, JZ18.8, 9.4 Hz, 1H), 3.69–3.83
(m, 8H), 3.83–4.02 (m, 7H), 4.05–4.22 (m, 4H), 4.68 (t, JZ
8.0 Hz, 1H), 6.59 (d, JZ2.4 Hz, 1H), 6.66 (dd, JZ8.7,
2.4 Hz, 1H), 6.78 (d, JZ1.7 Hz, 1H), 6.83 (dd, JZ8.7,
1.7 Hz, 1H), 6.90 (d, JZ8.7 Hz, 1H), 7.16 (d, JZ8.7 Hz,
1H); HRMS (SIMS) calcd for C42H55NO10S2 (MC)
797.3264, found 797.3257.
1
(4:25:65)]; H NMR (CDCl3) d 0.78 (s, 3H), 1.08–2.50
(m, 17H), 2.62–2.94 (m, 4H), 3.03–3.20 (m, 2H), 3.56–3.75
(m, 9H), 3.75–3.99 (m, 7H), 3.99–4.20 (m, 4H), 6.50–6.70
(m, 2H), 6.81–7.18 (m, 3H), 7.19–7.36 (m, 1H); HRMS
(SIMS) calcd for C40H56NO10S2 [(MC1)C] 774.3342,
found 774.3337.
4.1.26. Methyl 3-{4-[17-hydroxy-1,3,5(10)-estratrien-3-
yloxy]butyldisulfanyl}-N-(6,7,9,10,12,13,15,16-octa-
hydro-5,8,11,14,17-pentaoxabenzocyclo-pentadecen-2-
yl) succinamate (24). To a MeOH–THF (1:1, 1 ml) solution
of 1 (9 mg, 0.01 mmol) was added slowly 2.0 N hexane
solution of TMSCHN2 (0.007 ml, 0.01 mmol). After 30 min
at rt, the solvent was evaporated to give a residue which was

11922
S. Harusawa et al. / Tetrahedron 60 (2004) 11911–11922
11. Cross reactivities of FKA 238 with steroids is shown in the
data sheet of FKA 238.
subsequently purified by silica gel chromatography with
MeOH–CHCl3 (1:20) to give 24 (6 mg, 67%) as a brown
amorphous product; IR (CHCl3, cmK1) 1720, 1670; 1H
NMR (CDCl3) d 0.77 (s, 3H), 1.11–2.02 (m, 15H), 2.02–
2.36 (m, 2H), 2.68–2.90 (m, 4H), 3.14–3.42 (m, 2H), 3.70
(s, 3H), 3.63 (m, 9H), 3.81–4.01 (m, 7H), 4.01–4.20 (m,
4H), 6.60 (d, JZ2.8 Hz, 1H), 6.67 (dd, JZ8.6, 2.8 Hz, 1H),
6.73–6.95 (m, 3H), 7.17 (d, JZ8.6 Hz, 1H); 13C NMR
(CDCl3) d: 11.0, 23, 25.7, 26.1, 26.3, 27.3, 28.0, 29.8, 30.6,
35.7, 38.7, 38.9, 43.3, 44.0, 50.0, 52.2, 58.6, 67.1, 68.8,
69.4, 69.7, 70.6, 71.0, 76.5, 76.5, 76.8, 77.0, 77.3, 81.9,
106.7, 112.0, 114.5, 118.7, 126.3, 132.8, 138.0, 156.7,
171.4, 171.8; HRMS (SIMS) calcd for C41H58NO10S2
[(MC1)C] 788.3498, found 788.3502.
12. Cason, J. Org. Synth. Coll., 4, 630–633.
13. Disuccinamic acid 10 could not be reduced to a thiol 5 (RZH)
by zinc-saturated aqueous ammonium chloride.
14. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 1475–1478.
15. Zervas, B. L.; Theodoropoulos, D. M. J. Am. Chem. Soc. 1956,
78, 1359–1363.
16. Overman, L. E.; Smoot, J.; Overman, J. D. Synthesis 1974,
59–60.
17. Sridhar, M.; Vadivel, S. K.; Bhalerao, U. T. Synth. Commun.
1997, 27, 1347–1350.
18. Allen, C. F. H.; MacKay, D. D. Org. Synth. Coll., 2, 580–583.
19. Holton, R. A.; Crouse, D. J.; Williams, A. D.; Kennedy, R. M.
J. Org. Chem. 1987, 52, 2317–2318.
20. Yamazaki, S.; Sakamoto, M.; Suzuri, M.; Doi, M.; Nakazawa,
T.; Kobayashi, Y. J. Chem. Soc., Perkin Trans. 1 2001,
1870–1875.
Acknowledgements
We are grateful to Dr. M. Sakaguchi, Osaka University of
Pharmaceutical Science, for helpful discussions. This
research was partially supported by a Grant-in-Aid for
High Technology Research from the Ministry of Education,
Science, Sports and Culture, Japan.
21. Wakasugi, K.; Nakamura, A.; Tanabe, Y. Tetrahedron Lett.
2001, 42, 7427–7430.
22. (a) Smith, M. B.; March, J. March’s Advanced Organic
Chemistry. Reactions, Mechanisms, and Structure, 5th ed.;
Wiley Inter-Science: New York, 2001; pp 1543–1544. (b) Bao,
M.; Shimizu, M. Tetrahedron 2003, 59, 9655–9659 and
references cited therein. (c) Mukaiyama, T.; Takahashi, K.
Tetrahedron Lett., 5907–5908.
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34. The ratio of diastereomers was determined from 13C NMR
analysis of 24.
8. This was purchased from Aldrich Chemical Company.
9. Unpublished Data: in the low concentration, the relationship
between the concentration of 2 and electrode-response was
well approximated to a Langmuir’s adsorption isotherm:
DGZK1/(1CK2/C)CK3$C.
35. The sodium salt of 1 showed solubility in water, while its
triethylammonium carboxylate was an insoluble salt in water.
From these observations, the metal cation of the sodium salt is
assumed to be trapped by the BC ether in 1.
10. FKA 238 (COSMO FKA 238) was available from COSMO
BIO Co., Ltd.