Design, synthesis and biological evaluation of nuclear receptor-degradation inducers

Article abstract of DOI:10.1016/j.bmc.2011.09.041

Compounds that regulate the function(s) of nuclear receptors (NRs) are useful for biological studies and as candidate therapeutic agents. Most such compounds are agonists or antagonists. On the other hand, we have developed specific protein degradation in

Full text of DOI:10.1016/j.bmc.2011.09.041

Bioorganic & Medicinal Chemistry 19 (2011) 6768–6778
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of nuclear
receptor-degradation inducers
Yukihiro Itoh a, , Risa Kitaguchi a, , Minoru Ishikawa a, Mikihiko Naito b, Yuichi Hashimoto a,
⇑
a Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
b National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Compounds that regulate the function(s) of nuclear receptors (NRs) are useful for biological studies and
as candidate therapeutic agents. Most such compounds are agonists or antagonists. On the other hand,
we have developed specific protein degradation inducers, which we designated as SNIPERs (Specific
and Nongenetic IAPs-dependent Protein ERasers), for selective degradation of target proteins. SNIPERs
are hybrid molecules consisting of an appropriate ligand for the protein of interest, coupled to a ligand
for inhibitor of apoptosis proteins (IAPs), which target the bound protein for polyubiquitination and pro-
teasomal degradation. We considered that protein knockdown with SNIPERs would be a promising alter-
native approach for modulating NR function. In this study, we designed and synthesized degradation
inducers targeting retinoic acid receptor (RAR), estrogen receptor (ER), and androgen receptor (AR). These
newly synthesized RAR, ER, and AR SNIPERs, 9, 11, and 13, respectively, were confirmed to significantly
reduce the levels of the corresponding NRs in live cells.
Received 29 August 2011
Revised 21 September 2011
Accepted 22 September 2011
Available online 29 September 2011
Keywords:
Protein knockdown
Protein degradation inducer
RAR
ER
AR
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Many agonists or antagonists for NRs have been found and syn-
thesized, and have often been used as regulators of NRs or thera-
Nuclear receptors (NRs), which are activated by small-molecu-
lar ligands, are transcription factors that regulate gene expression
in the nucleus and play key roles in biological functions such as
embryonic development and homeostasis.1 In addition, NRs are
targets for the treatment of various diseases.2 Among NRs, retinoic
peutic agents.3 For example, all-trans retinoic acid (ATRA, 1),
Am80 (2), and Ch55 (3) (Fig. 1) are agonists for retinoic acid recep-
tors (RARs).4 In particular, ATRA (1) and Am80 (2) have been used
as therapeutic agents for patients with acute promyelocytic leuke-
mia (APL).5 In addition, bicalutamide6 (4) ( Fig. 1), which is an
androgen receptor (AR) antagonist, and tamoxifen7 (5) ( Fig. 1),
which is an estrogen receptor (ER) antagonist, have been used for
the treatment of prostate cancer and breast cancer, respectively.
However, although AR antagonists, which show anti-androgenic
activity toward wild-type AR, are used as therapeutic agents for
the treatment of prostate cancer, it has been reported that some
AR antagonists shows androgenic activity toward some mutant
ARs.8 In addition, conventional AR antagonists are not effective to
treat polyglutamine expansion mutation in AR such as spinal and
bulbar muscular atrophy (SBMA).9 In regard to breast cancer, ac-
quired resistance to tamoxifen (5) is a substantial problem.10 As
a new approach, which might avoid some of the difficulties associ-
ated with the use of agonists or antagonists, we have focused on
protein knockdown of NRs.
acid receptors (RARa, RARb, and RARc) are receptors of all-trans
retinoic acid (ATRA). Retinoids modulate the growth and differen-
tiation of a wide variety of normal and transformed cells, and ATRA
is involved in the control of embryonic development and cell dif-
ferentiation. Estrogen receptors (ER
a and ERb) are activated by
the endogenous hormone 17b-estradiol (E2). Estrogens play an
important role in the initiation and progression of breast cancer,
and overexpression of ER
risk of breast cancer.2c Androgen receptor (AR) is a receptor of
androgens, typically testosterone and/or its active form, 5 -dihy-
a appears to be associated with increased
a
drotestosterone (DHT), which are endogenous ligands essential
for the development and maintenance of the male reproductive
system and secondary male sex characteristics. Among the patho-
physiological effects elicited by androgens, a role as endogenous
tumor promoters, especially for prostate tumor, is well known.
This action is considered to be mediated by androgen-binding to
AR.
Several groups have reported techniques for inducing target
protein-selective degradation in cells. Proteolysis-targeting chime-
ric molecules (PROTACs)11 represent a pioneering approach in the
field of down-regulating proteins by the use of peptides. Proteins
targeted by these PROTACs include methionine aminopeptidase 2
(MetAP-2), ER, AR, aryl hydrocarbon receptor (AhR), and FK506
binding protein (FKBP). On the other hand, we have developed
⇑
Corresponding author. Tel.: +81 3 5841 7847; fax: +81 3 5841 8495.
E-mail address: hashimot@iam.u-tokyo.ac.jp (Y. Hashimoto).
These authors equally contributed to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.041

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
6769
Figure 1. Structures of compounds 1–5.
protein knockdown with small-molecular protein-degradation
inducers (SNIPERs; Specific and Nongenetic IAPs-dependent Pro-
tein ERasers).12 SNIPERs are bifunctional compounds which are de-
signed by conjugating an IAPs-recognition structure with a target
protein-recognition structure. This approach to targeting proteins
for degradation involves three steps. First, its two recognition
structures allow SNIPER to form an artificial (nonphysiological)
complex linking IAPs,13 which have ubiquitin ligase (E3) activity,
with the target protein. Next, the target protein is polyubiquitinat-
ed by IAPs. Finally the polyubiquitinated protein is degraded by
proteasome (Fig. 2a). Previously, we adopted methyl bestatin
(MeBS, 6a) or BE04 (6b) (Fig. 2b) as a ligand for cellular inhibitor
of apoptosis protein 1 (cIAP1), which is one of the IAPs, and ATRA
as a ligand for cellular retinoic acid binding protein II (CRABP-II),
and synthesized CRABP-II degradation inducers 7 and 8. ATRA rec-
ognizes not only CRABP-II, but also RAR. However, we utilized
structure-based drug design to obtain 7 and 8, which induce selec-
tive degradation of CRABP-II, but not RARs (introduction of a sub-
stituent at the C4 position of ATRA results in loss of ATRA’s binding
affinity for RARs). The protein knockdown strategy has several
advantages, especially, (1) small-molecular degradation inducers
possesses sufficient activity, permeability, and stability in cells to
be employed in cellular assays, (2) target protein-selective protein
knockdown is expected to be useful for biological studies and dis-
ease treatment, (3) suppression or degradation of cIAP1 itself,
which is overexpressed in several human cancers, should not viti-
ate the anticancer effect, and both cancer-related protein and
cIAP1 degradation would be useful for cancer treatment (actually
we found that degradation of both CRABP-II and cIAP1 (7) is more
effective than degradation of CRABP-II alone (8) to inhibit the pro-
liferation of neuroblastoma cells).12b
This protein knockdown strategy is expected be generally
adaptable to a wide range of proteins by replacing ATRA with a
specific ligand for the target protein. In addition, the functions of
many disease-related proteins could be inhibited by protein
knockdown if suitable ligands were available. To examine the
scope of the protein knockdown strategy using SNIPERs, we fo-
cused on NRs, because some NRs are known to be cancer-related,
and conventional antagonists have not proved entirely satisfac-
tory. Herein, we describe the design, synthesis and biological eval-
uation of SNIPERs for RAR, ER, and AR. The developed compounds
were expected to exhibit antagonist-like activity via down-regula-
tion (knockdown) of the NRs. They should be useful as tools for
biological studies and candidate therapeutic agents that would
overcome some of the problems encountered with conventional
antagonists.
a
Step 2; Ubiquitination of target proteins
Ub
Ub
Ub
Ub
Ub
IAPs recognition
structure
target
protein
IAPs
specific ligand for
target protein
Step 3; proteasomal degradation
SNIPER
Step 1; formation of ternary complex
b
Figure 2. (a) Protein degradation (protein knockdown) by SNIPERs. (b) Chemical structures of cIAP1 ligands 6a,b, and protein degradation inducers 7 and 8.

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
tion. Compound 18 was obtained from compound 17 via protec-
2. Chemistry
tion of the hydroxyl group, bromination and nucleophilic
substitution reaction. Solvolysis of acetate 18 with a catalytic
amount of sodium methoxide and oxidation with MnO2 yielded
aldehyde 19. Horner–Wadsworth–Emmons reaction of 16 and
19 afforded olefin 20 (E/Z mixture). Treatment of BBr3 with 20
gave compound 21 by deprotection of the methoxy group and
Compounds 9–13 prepared for this study are shown in Figure
3. Scheme 1 shows the preparation of compound 24, which is an
intermediate for synthesis of compounds 9 and 10. Acid 14 was
treated with 2.2 equiv of MeLi to give ketone 15. Compound 16
was prepared by bromination of 15, followed by Arbuzov reac-
Figure 3. Structures of NR SNIPERs and their negative controls.
Scheme 1. Reagents and conditions: (a) MeLi, THF, Et2O, ꢀ78 °C to room temperature, 85%; (b) Br2, AcOH, 0 °C; (c) P(OEt)3, THF, 60 °C, 59% (2 steps); (d) NaH, MeI, DMF, 0 °C
to room temperature, 100%; (e) N-bromosuccinimide (NBS), azobisisobutyronitrile (AIBN), CCl4, 70 °C; (f) AcOK, DMF, room temperature, 57% (2 steps); (g) NaOMe, MeOH,
room temperature, 86%; (h) MnO2, CH2Cl2, room temperature, 94%; (i) NaOMe, MeOH, room temperature, 76%; (j) BBr3, CH2Cl2, 0 °C; (k) DBU, toluene, reflux, 61% (2 steps); (l)
tert-butyl 3-bromopropylcarbamate, K2CO3, DMF, 90 °C, 98%; (m) HCl, 1,4-dioxane, CH2Cl2, room temperature, quant.

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
6771
1,4-addition of bromine. HBr elimination from 21 using DBU
afforded E-olefin 22 as a single geometrical isomer. Treatment
of 22 with tert-butyl 3-bromopropylcarbamate,14 followed by
deprotection of the tert-butoxycarbonyl (Boc) group, gave amine
24. Compounds 9 and 10 were prepared by means of the proce-
dure outlined in Scheme 2. Reduction of azide 2515 in the pres-
ence of PPh3 and H2O yielded amine 26. Condensation of amine
26 with acids 2712a,b afforded amides 28. Hydrolysis of esters 28
under an acidic condition gave acids 29. Condensation of acids
29 with amine 24 afforded amides 30. Removal of the Fmoc
group and methyl group of 30 gave compounds 9 and 10.
Compound 11 and negative control 12 were synthesized by
means of the procedure shown in Scheme 3. Compound 33 was pre-
pared by oximation of estrone (31) and O-(carboxylmethyl)hydrox-
ylamine (32). Amidation of 33 and 34a,b12b gave amides 35 and 12.
Removal of the Fmoc group of 35 with DBU12a,b,16 gavecompound 11.
Compound 13 was synthesized from compound 25, dihydrotes-
tosterone (DHT; 37) and compound 4012b via the route shown in
Scheme 4. Removal of the tert-butyl group of ester 25 gave acid
36. Condensation of DHT (37) and 36 afforded ester 38, and reduc-
tion of the azide group of 38 resulted in the formation of amine 39.
Amine 39 was treated with acid 40 to provide amide 41. Finally,
Scheme 2. Reagents and conditions: (a) PPh3, H2O, THF, room temperature; (b) 27a or 27b, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole
hydrate (HOBtꢁH2O), THF, room temperature, 89–100% (from 27); (c) HCl, 1,4-dioxane, CH2Cl2, room temperature, quant; (d) 24, EDCI, HOBtꢁH2O, THF, room temperature, 51–
71% (from 29); (e) LiOH, MeOH, H2O, room temperature, 61–83%.
Scheme 3. Reagents and conditions: (a) pyridine, room temperature, 100%; (b) 34a or 34b, EDCI, HOBtꢁH2O, CH2Cl2, room temperature, 60–64%; (c) DBU, n-C12H25SH, CH2Cl2,
room temperature, 61%.

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
Scheme 4. Reagents and conditions: (a) HCl, 1,4-dioxane, CH2Cl2, room temperature; (b) EDCI, N,N-dimethylaminopyridine (DMAP), CH2Cl2, room temperature, 45% (2 steps;
from 25); (c) PPh3, H2O, THF, room temperature; (d) 40, EDCI, HOBtꢁH2O, CH2Cl2, room temperature, 97% (2 steps; from 38); (e) HCl, 1,4-dioxane, CH2Cl2, 0 °C, 90%.
a
b
c
d
e
Figure 4. (a) Western blot-detection of RAR
detection of RAR in HT1080 after 6-h treatment with MG132, which was added to the culture 30 min prior to the addition of 9. (c) The influence of BE04 (6b) on RAR
Western blot detection of RAR in HT1080 after 24-h treatment with each reagent (30 M). (d) Western blot detection of RAR in HT1080 cells expressing FLAG-tagged cIAP1
a
and CRABP-II levels in HT1080 cells expressing FLAG-tagged cIAP1 after 24-h treatment with each reagent. (b) Western blot-
a
a
level.
a
l
a
after 24-h treatment with each reagent (30
lM). (e) Western blot detection of RAR
a
in HT1080 cells after 6-h treatment with LE540, which was added to the culture 30 min
prior to the addition of 9.
a
b
Figure 5. (a) Western blot detection of ERa in MCF-7 cells after 24-h treatment with (a) BE04 (6b), estrone (31), compound 11 and (b) negative control 12.

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
6773
compound 13 was obtained by removal of the Boc group under
acidic conditions.
3. Results and discussion
3.1. RAR SNIPER
First, we attempted to identify an RAR SNIPER in order to confirm
that the protein knockdown strategy is applicable to NRs. ATRA (1),
Am80 (2), and Ch55 (3) are RAR agonists, but ATRA (1) and Am80 (2)
also bind to CRABP-II.17 On the other hand, the binding affinity of
Ch55 (3) for CRABP-II is quite low (almost no binding).4b,c Therefore
we expected to obtain high selectivity for RAR degradation by the
use of Ch55 (3) for an RAR SNIPER. Additionally, previous studies
suggested that introduction of a substituent at the ortho-position
to the carboxyl group of Ch55 (3) would not affect the binding affin-
ity of the compound for RAR.14 Using BE04 (6b) (Fig. 2b) as the bind-
ing moiety for cIAP1 has been established to be effective.12b,18 Based
on these results, we designed and synthesized compound 9 as an
RAR SNIPER (Fig. 3). We also prepared a negative control 10
(Fig. 3), which would not bind to cIAP1.12b
Figure 6. Western blot detection of AR in MCF-7 cells after 24-h treatment with
BE04 (6b), DHT (37) and compound 13.
bind to cIAP1, did not decrease ER
indicate that compound 11 induced protein knockdown of ER
The result of protein knockdown for AR is shown in Figure 6.
Compound 13 (30 M; lane 5) decreased the AR level, compared
with the control (lane 1), and did so more potently than BE04
(6b, 30 M; lane 2), DHT (37, 30 M; lane 3), or the mixture of
BE04 (6b) and DHT (37) (30 M each; lane 4). Thus, conjugation
a (Fig. 5b). Collectively, these data
a.
l
l
l
l
of BE04 (6b) and DHT (37) within a single molecule is also impor-
tant for AR degradation-inducing activity. These results suggested
that compounds 11 and 13 effectively decrease ERa and AR levels,
We evaluated the RARa degradation induced by compound 9 by
means of Western blot analysis. Treatment of HT1080 cells
expressing FLAG-tagged cIAP1 with compound 9 decreased the
respectively, and may be useful both as tools for biological studies
and candidate anticancer agents, serving to inhibit NR functions
through knockdown of the receptors.
RAR
a level in a concentration-dependent manner (Fig. 4a). Com-
pound 9 had no influence on the amount of CRABP-II, while
compound 7 ( Fig. 2b), which induces degradation of CRABP-II,
did not induce RARa degradation (Fig. 4a). This indicated that com-
pound 9 shows selectivity for RAR, as we had expected.
4. Conclusion
We investigated the influence of proteasome inhibitors on the
RAR
was blocked by proteasome inhibitor MG132. Thus, the decrease
of RAR induced by 9 can be attributed to proteasomal degradation.
We confirmed that the negative control 10 and BE04 (6b) had no
effect on RAR level (Fig. 4c and d). In addition, RAR antagonist,
LE540, inhibited degradation of RAR induced by compound 9
a level in 9-treated cells (Fig. 4b). The reduction of RARa by 9
Based on the structures of the RAR agonist Ch55 (3), AR agonist
DHT (37), and ER agonist estrone (31), we designed and synthe-
sized RAR, AR, and ER degradation inducers, consisting of the
appropriate NR ligand coupled to a ligand of cellular inhibitor of
apoptosis protein 1 (cIAP1), which targets the bound NR protein
for polyubiquitination and proteasomal degradation. We con-
firmed that these newly synthesized RAR, AR, and ER SNIPERs, 9,
11, and 13, respectively, were effective for protein knockdown of
the corresponding NR, significantly reducing the levels of RAR,
AR, and ER, respectively, in live cells. These compounds are ex-
pected to be useful as new NR regulators for biological studies
and treatment of NR-related diseases.
a
a
a
(Fig. 4e). Thus, conjugation of Ch55 (3) and BE04 (6b) within a
single molecule, thah is, affording a compound which binds simulta-
neously to both RAR
inducing activity. Collectively, these data confirm that compound 9
degraded RAR by protein knockdown. It is noteworthy that CRABP-
II and RAR could be independently and selectively degraded by the
a and cIAP1, is essential for RARa degradation-
a
a
use of 7 and 9, respectively, despite the fact that both proteins recog-
nize ATRA.
5. Experimental section
5.1. Chemistry
3.2. ER and AR SNIPERs
Next, we attempted to create SNIPERs targeting other NRs, that
is, ER and AR. We used the structures of an ER agonist, estrone (31;
Scheme 3) and an AR agonist, DHT (37; Scheme 4) for these SNIP-
ERs, respectively. It has been reported that introduction of substit-
uents to C-17 position of steroid structure kept binding affinity to
the corresponding NRs.11c,19 Therefore, we designed compounds 11
and 13 using a similar strategy to that which we had employed for
the development of the RAR SNIPER (Fig. 3).
Proton nuclear magnetic resonance spectra (1H NMR) and car-
bon nuclear magnetic resonance spectra (13C NMR) were recorded
on a JEOL JNMGX500 (500 MHz) spectrometer in the indicated sol-
vent. Chemical shifts (d) are reported in parts per million relative to
the internal standard tetramethylsilane. High-resolution mass
spectra (HRMS) and fast atom bombardment (FAB) mass spectra
were recorded on a JEOL JMA-HX110 mass spectrometer. Other
chemical reagents and solvents were purchased from Aldrich,
Merck, Tokyo Kasei Kogyo, Wako Pure Chemical Industries, and
Kanto Kagaku and used without purification. Flash column chro-
matography was performed using silica gel 60 (particle size
0.060–0.210 mm) supplied by Kanto Kagaku.
The biological activities of synthetic compounds 11 and 13 were
evaluated by Western blotting using human mammary tumor MCF-
7 cells, which express ER
regulated ER levels (Fig. 5a). We confirmed that the decrease in ER
was not caused by a partial structure of 11 or by a mere mixture of
BE04 (6b) and estrone (31). BE04 (6b, 30 M; lane 2), estrone (31,
30 M; lane 3), and the mixture of BE04 (6b) and estrone (31)
aand AR. Compound 11 significantly down-
a
a
l
5.1.1. 1-(3,5-Di-tert-butylphenyl)ethanone (15)
l
1.6 N MeLi in Et2O (300 lL, 0.480 mmol) was added to a solu-
(30
(30
l
l
M each; lane 4) did not decrease ER
M; lane 5). Thus, conjugation of BE04 (6b) and estrone (31)
degradation-inducing
activity. In addition, the negativecontrol 12(Fig. 3), which wouldnot
a as much as did 11
tion of 14 (50.7 mg, 0.0216 mmol) in THF (1.0 mL) at ꢀ78 °C. The
mixture was stirred at room temperature for 5 h, then poured into
water and extracted with AcOEt. The organic layer was washed
within a single molecule is important for ER
a

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Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
with brine and dried over MgSO4. Filtration, evaporation of the sol-
vent in vacuo and purification by flash column chromatography
(AcOEt/n-hexane = 1/25) gave 42.5 mg (85%) of 15 as a yellow
oil; 1H NMR (CDCl3, 500 MHz, d; ppm) 7.81 (2H, d, J = 1.8 Hz),
7.65 (1H, t, J = 1.8 Hz), 2.63 (3H, s), 1.36 (18H, s); MS (FAB) m/z:
233 (MH+).
4.75 (2H, d, J = 6.1 Hz), 3.93 (3H, s), 3.88 (3H, s), 1.79 (1H, t,
J = 6.1 Hz); MS (FAB) m/z: 197 (MH+).
A mixture of methyl 4-(hydroxymethyl)-2-methoxybenzoate
(738 mg 3.76 mmol) and MnO2 (3270 mg, 37.6 mmol) in CH2Cl2
(20 mL) was stirred at room temperature for 28 h. The MnO2 was
removed by filtration and the filtrate was concentrated in vacuo.
The residue was purified by flash column chromatography
(AcOEt/n-hexane = 1/2 to AcOEt only) to give 687 mg (94%) of 19
as a colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 10.03 (1H,
s), 7.90 (1H, d, J = 7.9 Hz), 7.48 (2H, m), 3.98 (3H, s), 3.93 (3H, s),
1.79 (1H, t, J = 6.1 Hz); MS (FAB) m/z: 195 (MH+).
5.1.2. Diethyl 3,5-di-tert-butylbenzoylmethylphosphonate (16)
Br2 (620 mg, 3.38 mmol) was added to solution of 15 (694 mg,
2.99 mmol) in AcOH/THF (5.0 mL/5.0 mL) at 0 °C. The reaction mix-
ture was stirred for 4 h, then concentrated and purified by flash
column chromatography (AcOEt/n-hexane = 1/10) to afford a crude
bromine compound that was used in the next reaction without fur-
ther purification. A mixture of the crude bromine compound and
5.1.5. (E)-Methyl 4-[3-(3,5-di-tert-butylphenyl)-3-oxoprop-1-
enyl]-2-hydroxybenzoate (22)
P(OEt)3 (623
l
L, 3.60 mmol) in MeCN (5.0 mL) was heated at reflux
A solution of 16 (132 mg, 0.680 mmol) in MeOH (3.0 mL) was
added to a solution of NaOMe (109 mg, 2.02 mmol) in MeOH
(3.0 mL) with cooling in an ice-bath and the mixture was stirred
for 30 min. A solution of 19 (240 mg, 0.651 mmol) in MeOH
(4.0 mL) was added, and the resulting mixture was stirred at room
temperature for 36 h, then acidified with 2 N HCl and extracted
with AcOEt. The organic layer was washed with brine, and dried
over MgSO4. Filtration and evaporation of the solvent in vacuo gave
202 mg of 20 (E/Z mixture) as a yellow oil. 1 N BBr3 in CH2Cl2
(2.0 mL, 2.0 mmol) was added to a solution of 20 (201 mg,
0.492 mmol) in 3.0 mL of CH2Cl2 at 0 °C. The mixture was stirred
for 1 h, poured into water and extracted with AcOEt. The organic
layer washed with brine, and dried over MgSO4. Filtration, evapo-
ration of the solvent in vacuo and purification of the residue by
flash column chromatography (AcOEt/n-hexane = 1/10) gave
155 mg of a yellow solid. A mixture of the yellow solid (134 mg)
temperature for 8 h. The reaction mixture was concentrated and
purified by flash column chromatography (AcOEt/n-hexane = 1/
11) to give 647 mg (59%; 2 steps) of 16 as a colorless solid; 1H
NMR (CDCl3, 500 MHz, d; ppm) 7.86 (2H, d, J = 1.8 Hz), 7.66 (1H,
t, J = 1.8 Hz), 4.15 (4H, m), 3.67 (1H, s), 3.63 (1H, s), 1.36 (18H, s),
1.29 (6H, t, J = 6.7 Hz); MS (FAB) m/z: 369 (MH+).
5.1.3. Methyl 4-(acetoxymethyl)-2-methoxybenzoate (18)
A solution of 17 (3000 mg, 18.1 mmol) in DMF (5.0 mL) was
added to a suspension of 60% NaH in oil (1019 mg, 25.5 mmol) in
DMF (10 mL) with cooling in an ice-bath and the mixture was
stirred for 30 min, then MeI (3.5 mL, 56.2 mmol) was added to it.
Stirring was continued at room temperature for 18 h, then the mix-
ture was acidified with 2 N HCl and extracted with AcOEt. The or-
ganic layer was washed with brine, and dried over MgSO4.
Filtration, evaporation of the solvent in vacuo and purification of
the residue by flash column chromatography (AcOEt/n-hex-
ane = 1/4) gave 3252 mg (100%) of methyl 2-methoxy-4-meth-
ylbenzoate as a colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm):
7.72 (1H, d, J = 8.5 Hz), 6.79 (1H, d, J = 6.7 Hz), 6.78 (1H, s), 3.90
(3H, s), 3.87 (3H, s), 2.37 (3H, s); MS (FAB) m/z: 181 (MH+).
AIBN (578 mg, 3.52 mmol) was added to a solution of methyl 2-
methoxy-4-methylbenzoate (3172 mg, 17.6 mmol) and NBS
(3446 mg, 19.4 mmol) in CCl4 (40 mL) and the resulting mixture
was heated at 70 °C for 8 h. After cooling, the reaction mixture
was concentrated and purified by flash column chromatography
(AcOEt/n-hexane = 1/6 to 1/3) to give a crude bromine compound
that was used in the next reaction without further purification.
AcOK (9062 mg, 92.3 mmol) was added to a solution of the crude
bromine compound in DMF (10 mL) and the resulting mixture
was stirred at room temperature for 15 h, then poured into water
and extracted with AcOEt. The organic layer was washed with
brine, and dried over MgSO4. Filtration, evaporation of the solvent
in vacuo and purification of the residue by flash column chroma-
tography (AcOEt/n-hexane = 1/4 to 1/1) gave 2395 mg (57%; 2
steps) of 18 as a colorless solid; 1H NMR (CDCl3, 500 MHz, d;
ppm): 7.80 (1H, d, J = 7.9 Hz), 6. 69 (1H, d, J = 7.9 Hz), 6.95 (1H,
s), 5.12 (2H, s), 3.92 (3H, s), 3.89 (3H, s), 2.13 (3H, s); MS (FAB)
m/z: 239 (MH+).
and DBU (43 lL, 0.288 mmol) in toluene (1.0 mL) was heated at re-
flux temperature for 30 min, then cooled, concentrated and puri-
fied by flash column chromatography (AcOEt/n-hexane = 1/6 to 1/
3) to give 103 mg (46%; 3 steps) of 22 as a yellow solid; 1H NMR
(CDCl3, 500 MHz, d; ppm): 10.83 (1H, s), 7.88 (1H, d, J = 7.9 Hz),
7.83 (1H, d, J = 1.8 Hz), 7.70 (1H, d, J = 18.0 Hz), 7.65 (1H, t,
J = 1.2 Hz), 7.55 (1H, d, J = 16.9 Hz), 7.25 (1H, s), 7.14 (1H, dd,
J = 7.9, 1.8 Hz), 3.98 (3H, s), 1.39 (18H, s); MS (FAB) m/z: 395 (MH+).
5.1.6. (E)-Methyl 2-[3-(tert-butoxycarbonyl amino)propoxy]-4-[3-
(3,5-di-tert-butylphenyl amino)-3-oxoprop-1-enyl]benzoate (23)
A mixture of 22 (33.1 mg 0.0839 mmol), K2CO3 (47.2 mg,
0.342 mmol), KI (20.8 mg, 0.125 mmol) and tert-butyl 3-bromo-
propylcarbamate (79.6 mg, 0.334 mmol) in DMF (2.0 mL) was
heated at 90 °C for 5 h. After cooling, the reaction mixture was
poured into water and extracted with AcOEt. The organic layer
washed with brine, and dried over MgSO4. Filtration, evaporation
of the solvent in vacuo and purification of the residue by flash col-
umn chromatography (AcOEt/n-hexane = 1/3) gave 45.2 mg (98%)
of 23 as a yellow oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.94
(1H, d, J = 8.5 Hz), 7.82 (1H, d, J = 1.8 Hz), 7.72 (1H, d, J = 18.0 Hz),
7.69 (1H, t, J = 1.2 Hz), 7.51 (1H, d, J = 16.9 Hz), 7.30 (1H, d,
J = 7.9, Hz), 7.14 (1H, s), 5.92 (1H, br), 4.19 (2H, t, J = 5.4 Hz), 3.92
(3H, s), 3.43 (2H, m), 2.08 (2H, m), 1.46 (9H, s), 1.39 (18H, s); MS
(FAB) m/z: 552 (MH+).
5.1.4. Methyl 4-formyl-2-methoxybenzoate (19)
A solution of 18 (227 mg, 0.953 mmol) in MeOH (2.0 mL) was
added to a solution of NaOMe (10.0 mg, 0.185 mmol) in MeOH
(1.0 mL) with cooling in an ice-bath and the mixture was stirred
for 22 h. The reaction mixture was acidified with 2 N HCl and ex-
tracted with AcOEt. The organic layer was washed with brine,
and dried over MgSO4. Filtration and evaporation of the solvent
in vacuo gave 160 mg (86%) of methyl 4-(hydroxymethyl)-2-
methoxybenzoate as a colorless oil; 1H NMR (CDCl3, 500 MHz, d;
ppm): 7.80 (1H, d, J = 7.9 Hz), 7.03 (1H, s), 6.94 (1H, d, J = 8.5 Hz),
5.1.7. (E)-Methyl 2-(3-aminopropoxy)-4-[3-(3,5-di-tert-
butylphenyl)-3-oxoprop-1-enyl]benzoate hydrochloride
(24ꢁHCl)
4 N HCl in 1,4-dioxane (500 lL, 2.00 mmol) was added to a
solution of 23 (28.1 mg, 0.0509 mmol) in CH2Cl2 (0.50 mL) with
cooling in an ice-bath and the resulting mixture was stirred at
room temperature for 4 h, then concentrated in vacuo to give crude
24ꢁHCl, which was used in the next step without further purifica-
tion; MS (FAB) m/z: 452 (MH+).

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
6775
5.1.8. tert-Butyl 2-{2-[2-(2-aminoethoxy)ethoxy]ethoxy}acetate
(26)
Triphenyl phosphine (151 mg, 0.576 mmol) was added to a
solution of 25 (151 mg, 0.522 mmol) and H2O (150 lL, 8.31 mmol)
in THF (3.0 mL) and the resulting mixture was stirred at room tem-
perature for 24 h. The reaction mixture was concentrated in vacuo
to give crude 26, which was used in the next step without further
purification.
(2H, m), 7.82 (2H, d, J = 1.8 Hz), 7.76 (1H, m), 7.69 (1H, d,
J = 15.8 Hz), 7.67 (1H, t, J = 1.8 Hz), 7.48 (1H, d, J = 15.8 Hz), 7.32–
7.18 (5H, m), 7.14 (1H, s), 4.30 (2H, m), 4.14 (3H, m), 4.00–3.40
(21H, m), 3.26 (1H, m), 3.13 (1H, m), 2.04 (2H, m), 1.80 (1H, m),
1.61 (2H, m), 1.38 (18H, s), 0.90 (3H, d, J = 6.7 Hz) 0.89 (3H, d,
J = 6.7 Hz); 13C NMR (CDCl3, 125 MHz, d; ppm): 191.49, 172.61,
171.91, 169.91, 169.74, 151.33, 143.48, 137.76, 129.36, 128.99,
127.74, 127.29, 127.23, 122.75, 120.51, 119.27, 113.92, 70.96,
70.10, 70.02, 69.69, 69.03, 69.39, 68.04, 67.02, 55.01, 52.79, 40.08,
39.12, 36.39, 36.22, 35.03, 31.41, 29.69, 24.81, 23.04, 21.57; MS
5.1.9. tert-Butyl 2-[2-(2-{2-[(S)-2-((2S,3R)-3-{[(9H-fluoren-9-
yl)methoxy]carbonyl amino}-2-hydroxy-4-phenylbutanamido)-
4-methylpentanamido]ethoxy}ethoxy)ethoxy]acetate (28a)
EDCI (46.2 mg, 0.241 mmol) was added to a solution of crude
26, 27a (45.2 mg, 0.0852 mmol) and HOBtꢁH2O (52.1 mg,
0.340 mmol) in CH2Cl2 (3.0 mL) with cooling in an ice-bath. The
resulting mixture was stirred at room temperature for 17 h. The
reaction was quenched with water, and the mixture was extracted
with AcOEt. The organic layer was washed with brine, and dried
over MgSO4. Filtration, evaporation of the solvent in vacuo and
purification of the residue by flash column chromatography
(MeOH/CHCl3 = 1/30) gave 59.1 mg (89%; from 27a) of 28a as a col-
orless oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.75 (2H, d,
J = 7.3 Hz), 7.51 (1H, d, J = 7.3 Hz), 7.39 (2H, t, J = 7.3 Hz), 7.35–
7.16 (6H, m), 6.81 (1H, br), 5.49 (1H, d, J = 8.5 Hz), 5.14 (1H, m),
4.49 (1H, m), 4.28 (4H, m), 4.13 (1H, m), 4.03 (3H, m), 3.80–1.38
(12H, m), 3.04 (1H, m), 2.96 (1H, m), 1.73 (1H, m), 1.58 (2H, m),
1.48 (9H, s), 0.86 (3H, d, J = 6.1 Hz), 0.86 (3H, d, J = 6.1 Hz); MS
(FAB) m/z: (MH+); 798 (MNa+), 776 (MH+).
(FAB) m/z: 917 (MH+); HRMS (FAB) calcd for C51H73N4Oþ
,
11
917.5276; found 917.5297.
5.1.13. (S)-tert-Butyl 2-[2-(2-{2-[4-methyl-2-(4-
phenylbutanamido)pentanamido]ethoxy}ethoxy)
ethoxy]acetate (28b)
Compound 28b (yield; 60.0 mg, 100% from 27b) was prepared
from 27b (36.1 mg, 0.114 mmol) and crude 26 using the same pro-
cedure as described for 28a; Colorless oil; 1H NMR (CDCl3,
500 MHz, d; ppm): 7.27 (2H, t, J = 7.3 Hz), 7.19–7.14 (3H, m), 6.82
(1H, m), 6.22 (1H, m), 4.49 (1H, dt, J = 3.6, 4.8 Hz), 4.01 (2H, s),
3.70 (4H, m), 3.62 (4H, m), 3.55 (2H, t, J = 4.9 Hz), 3.44 (2H, t,
J = 5.4 Hz), 2.64 (2H, t, J = 7.6 Hz), 2.21 (2H, t, J = 7.6 Hz), 1.95
(2H, quin, J = 7.3 Hz), 1.64 (2H, m), 1.53 (1H, m), 1.52 (9H, s),
0.93 (3H, d, J = 6.1 Hz), 0.92 (3H, d, J = 6.1 Hz); MS (FAB) m/z: 523
(MH+).
5.1.14. (S,E)-Methyl 4-[3-(3,5-di-tert-butylphenyl)-3-oxoprop-1-
enyl]-2-(3-{2-[2-(2-{2-[4-methyl-2-(4-phenylbutanamido)
pentanamido]ethoxy}ethoxy)ethoxy]acetamido}propoxy)
benzoate (30b)
5.1.10. 2-[2-(2-{2-[(S)-2-((2S,3R)-3-{[(9H-Fluoren-9-yl)methoxy]
carbonyl amino})-2-hydroxy-4-phenylbutanamido)-4-methyl-
pentanamido]ethoxy}ethoxy)ethoxy]acetic acid (29a)
Compound 30b (yield; 30.8 mg, 72%; 2 steps from 28b) was pre-
pared from crude 24 and 28b (24.5 mg, 0.0469 mmol) using the
same procedure as described for 28a and 30a; Colorless oil; 1H
NMR (CDCl3, 500 MHz, d; ppm): 7.87 (1H, d, J = 7.9 Hz), 7.82 (2H,
d, J = 1.8 Hz), 7.72 (1H, d, J = 15.9 Hz), 7.68 (1H, t, J = 1.8 Hz), 7.53
(1H, t, J = 15.8 Hz), 7.42 (1H, br), 7.16 (4H, m), 6.92 (1H, m), 5.52
(1H, m), 4.49 (1H, m), 4.17 (2H, m), 4.09 (2H, s), 3.91 (3H, s),
3.80–3.37 (14H, m), 2.62 (2H, t, J = 7.6 Hz), 2.21 (2H, t, J = 7.6 Hz),
2.19 (2H, t, J = 7.6 Hz), 1.96 (2H, m), 1.68 (2H, m), 1.51 (1H, m),
1.39 (18H, s), 0.92 (6H, d, J = 4.8 Hz); MS (FAB) m/z: 922 (MNa+),
900 (MH+).
4 N HCl in 1,4-dioxane (1000 lL, 4.00 mmol) was added to a
solution of 28a (31.8 mg, 0.0513 mmol) in CH2Cl2 (0.50 mL) with
cooling in an ice-bath and the resulting mixture was stirred at
room temperature for 5 h. The reaction mixture was concentrated
in vacuo to give crude 29a, which was used in next step without
further purification; MS (FAB) m/z: 720 (MH+).
5.1.11. Methyl 2-(3-{2-[2-(2-{2-[(S)-2-((2S,3R)-3-{[(9H-fluoren-
9-yl)methoxy]carbonyl amino}-2-hydroxy-4-phenylbutanamido)-
4- methylpentanamido]ethoxy}ethoxy)ethoxy]acetamido}
propoxy)-4-[(E)-3-(3,5-di-tert-butylphenyl)-3-oxoprop-1-enyl]
benzoate (30a)
Compound 30a (yield; 30.0 mg, 51%; 2 steps from 28a) was pre-
pared from crude 24 and 29a using the same procedure as de-
scribed for 28a; Colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm):
7.89 (1H, d, J = 7.9 Hz), 7.82 (2H, s), 7.75–7.68 (4H, m), 7.53–7.46
(4H, m), 7.38 (5H, m), 7.30–7.13 (7H, m), 5.60 (1H, d, J = 8.5 Hz),
5.47 (1H, m), 4.51 (1H, m), 4.32–4.09 (7H, m), 3.89 (3H, s), 3.72–
3.40 (14H, m), 3.04 (1H, m), 2.93 (1H, m), 2.07 (2H, m), 1.73 (1H,
m), 1.58 (2H, m), 1.38 (18H, s), 0.85 (6H, d, J = 5.5 Hz); MS (FAB)
m/z: 1175 (MNa+), 1153 (MH+).
5.1.15. (S,E)-4-[3-(3,5-Di-tert-butylphenyl)-3-oxoprop-1-enyl]-
2-(3-{2-[2-(2-{2-[4-methyl-2-(4-phenylbutanamido)
pentanamido]ethoxy}ethoxy)ethoxy]acetamido}propoxy)
benzoic acid (10)
Compound 10 (yield; 18.6 mg, 61%) was prepared from 30b
(30.8 mg, 0.0342 mmol) using the same procedure as described
for 9; Colorless oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.98 (1H,
d, J = 7.9 Hz), 7.83 (2H, d, J = 1.8 Hz), 7.72 (1H, d, J = 15.2 Hz),
7.69 (1H, t, J = 1.8 Hz), 7.52 (1H, d, J = 15.8 Hz), 7.33 (1H, d,
J = 7.3 Hz), 7.26 (1H, d, J = 7.3 Hz), 7.16 (3H, m), 6.39 (1H, m),
4.55 (1H, q, 6.1 Hz), 4.21 (2H, t, J = 5.4 Hz), 4.04 (2H, m), 3.72–
3.32 (14H, m), 2.62 (2H, t, J = 7.3 Hz), 2.23 (2H, t, J = 7.6 Hz),
2.10 (2H, t, J = 6.1 Hz), 1.96 (2H, quin, J = 7.3 Hz), 1.62 (2H, m),
1.53 (1H, m), 1.38 (18H, s), 0.93 (3H, d, J = 6.7 Hz), 0.92 (3H, d,
J = 6.7 Hz); 13C NMR (CDCl3, 125 MHz, d; ppm): 191.32, 173.26,
172.63, 170.30, 158.23, 151.37, 142.99, 141.32, 137.61, 132.87,
128.43, 128.39, 128.37, 127.35, 125.35, 124.94, 122.75, 122.35,
120.16, 113.28, 70.68, 70.54, 70.47, 70.13, 69.89, 69.58, 67.70,
57.24, 51.80, 41.37, 39.01, 36.57, 35.68, 35.41, 35.03, 31.39,
28.98, 27.08, 24.80, 22.79, 22.21; MS (FAB) m/z: 908 (MNa+),
886 (MH+); HRMS (FAB) calcd for C51H72N3O1þ0, 886.5218; found
886.5235.
5.1.12. 2-[3-(2-{2-[2-(2-{(S)-2-[(2S,3R)-3-Amino-2-hydroxy-4-
phenylbutanamido]-4-methylpentanamido}ethoxy)ethoxy]
ethoxy}acetamido)propoxy]-4-[(E)-3-(3,5-di-tert-butylphenyl)-
3-oxoprop-1-enyl]benzoic acid (9)
LiOHꢁH2O (36.0 mg, 0.858 mmol) was added to a solution of 30a
(16.0 mg, 0.0139 mmol) in MeOH/H2O (1.0 mL/0.25 mL) and the
resulting mixture was stirred at room temperature for 2 h. The reac-
tion mixture was neutralized and concentrated in vacuo. The residue
was extracted with AcOEt. The organic solution was washed with
brine, concentrated in vacuo and purified by PTLC (CHCl3/MeOH/
25% NH3 aqueous solution = 6/1/1) to give 683 mg (64%) of 9 as a col-
orless solid; 1H NMR (CDCl3, 500 MHz, d; ppm): 8.19 (1H, br), 7.90

6776
Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
5.1.16. 2-({(E)-[(8R,9S,13S,14S)-3-Hydroxy-13-methyl-7,8,
9,11,12,13,15,16-octahydro-6H-cyclopenta[a]phenanthren-
17(14H)-ylidene]amino}oxy)acetic acid (33)
5.1.19. N-[2-(2-{2-[2-({(E)-[(8R,9S,13S,14S)-3-Hydroxy-13-
methyl-7,8,9,11,12,13,15,16-octahydro-6H-cyclopenta[a]
phenanthren-17(14H)-ylidene]amino}oxy)acetamido]
A mixture of estrone (31) (50.0 mg, 0.185 mmol) and O-(carbo-
xylmethyl)hydroxylamine (32) (61.0 mg, 0.555 mmol) in pyridine
(2.0 mL) was stirred at room temperature for 17 h. The reaction
mixture was poured into 10% aqueous citric acid and extracted
with AcOEt. The organic layer was washed with brine and dried
over MgSO4. Filtration, evaporation of the solvent in vacuo and
purification of the residue by flash column chromatography
(CHCl3/MeOH = 6/1) gave 64.0 mg (100%) of 33 as a colorless solid;
1H NMR (CD3CD, 500 MHz, d; ppm): 7.09 (1H, d, J = 8.6 Hz), 6.56
(1H, dd, J = 8.6, 2.5 Hz), 6.50 (1H, d, J = 2.4 Hz), 4.53 (2H, s), 4.84–
4.77 (2H, m), 2.66–2.55 (2H, m), 2.40–2.35 (1H, m), 2.26–2.21
(1H, m), 2.03 (1H, td, J = 12.8, 3.1 Hz), 1.98–1.91 (2H, m), 1.62
(1H, dt, J = 13.4, 4.3 Hz), 1.54–1.40 (5H, m), 0.97 (3H, s); MS
(FAB) m/z: 344 (MH+).
ethoxy}ethoxy)ethyl]-4-methyl-(S)-2-(4-
phenylbutanamido)pentanamide (12).
Compound 12 (yield; 43.0 mg, 60%) was prepared from 34b
(35.0 mg, 0.102 mmol) and 33 (51.0 mg, 0.0772 mmol) using the
same procedure as described for 28a; colorless solid; 1H NMR
(CDCl3, 500 MHz, d; ppm); 7.27 (1H, t, J = 7.3 Hz), 7.20–7.12 (3H,
m), 6.69 (1H, t, J = 5.5 Hz), 6.64 (1H, dd, J = 8.3, 2.8 Hz), 6.59 (2H,
d, J = 2.8 Hz), 6.14 (1H, d, J = 8.3 Hz), 4.50 (s, 2H), 4.44 (1H, dt,
J = 5.5, 8.6 Hz), 3.64–3.56 (6H, m), 3.52–3.50 (4H, m), 3.47–3.38
(2H, m), 2.86–2.82 (2H, m), 2.63 (2H, t, J = 7.3 Hz), 2.59–2.49 (2H,
m), 2.35–2.32 (1H, m), 2.21 (2H, t, J = 7.3 Hz), 2.19–1.89 (5H, m),
1.63–1.35 (10H, m), 0.93 (6H, d, J = 1.8 Hz), 0.92 (3H, s); 13C NMR
(CDCl3, 125 MHz, d; ppm): 172.83, 172.65, 172.26, 170.61,
153.88, 141.34, 137.89, 131.79, 128.46, 128.41, 126.37, 125.99,
115.40, 112.93, 72.58, 70.58, 70.38, 70.16, 69.93, 69.68, 52.96,
51.57, 44.64, 43.98, 41.55, 39.26, 38.71, 38.17, 35.76, 35.16,
34.08, 29.48, 27.23, 27.09, 26.13, 26.07, 24.80, 22.97, 22.89,
22.14, 17.28; MS (FAB) m/z: 733 (MH+); HRMS (FAB) calcd for
5.1.17. (9H-Fluoren-9-yl)methyl [(14S,17S,18R)-17-hydroxy-1-
({(E)-[(8R,9S,13S,14S)-3-hydroxy-13-methyl-7,8,9,11,12,13,15,
16-octahydro-6H-cyclopenta[a]phenanthren-17(14H)-
ylidene]amino}oxy)-14-isobutyl-2,13,16-trioxo-19-phenyl-6,9-
dioxa-3,12,15-triazanonadecan-18-yl]carbamate (35)
C
42H61N4Oþ, 733.4540; found 733.4554.
7
5.1.20. (5S,8R,9S,10S,13S,17S)-10,13-Dimethyl-3-oxohexadeca-
hydro-1H-cyclopenta[a]phenanthren-17-yl 2-{2-[2-(2-amino-
ethoxy)ethoxy]ethoxy}acetate (38)
Compound 35 (yield; 43.0 mg, 64%) was prepared from 34a
(35.0 mg, 0.102 mmol) and 33 (51.0 mg, 0.0772 mmol) using the
same procedure as described for 28a; Colorless solid; 1H NMR
(CDCl3, 500 MHz, d; ppm): 7.75 (2H, d, J = 7.3 Hz), 7.50 (2H, d,
J = 7.3 Hz), 7.39 (2H, t, J = 7.3 Hz), 7.31–7.18 (5H, m), 7.11 (1H, d,
J = 8.6 Hz), 6.76 (1H, br s), 6.70 (1H, t, J = 4.3 Hz), 6.62 (1H,
dd, J = 10.0, 2.5 Hz), 6.57 (1H, d, J = 2.5 Hz), 4.48 (2H, s), 4.46 (1H,
m), 4.30–4.18 (4H, m), 4.12 (1H, br), 3.50 (12H, m), 3.30
(1H, m), 3.04–2.94 (2H, m), 2.84–2.81 (2H, m), 2.60–2.46 (2H,
m), 2.34–2.30 (1H, m), 2.22 (1H, m), 2.20 (1H, m), 2.03–2.00
(1H, m), 1.92–1.87 (2H, m), 1.73–1.34 (8H, m), 0.92 (3H, s), 0.86
(3H, d, J = 6.1 Hz), 0.84 (3H, d, J = 6.1 Hz); MS (FAB) m/z: 987
(MH+).
4 N HCl in 1,4-dioxane (2.00 mL, 8.00 mmol) was added to a solu-
tion of 25 (220 mg, 0.760 mmol) in CH2Cl2 (2.0 mL) with cooling in
an ice-bath and the resulting mixture was stirred at room tempera-
ture for 24 h. The reaction mixture was concentrated in vacuo to give
crude 36. EDCI (250 mg, 1.30 mmol) was added to a solution of crude
36, DHT (37) (221 mg, 0.760 mmol) and DMAP (140 mg, 1.14 mmol)
in CH2Cl2 (7.6 mL) with cooling in an ice-bath. The resulting mixture
was stirred at room temperature for 18 h. The reaction was
quenched with water, and the mixture was extracted with AcOEt.
The organic layer was washed with brine, and dried over MgSO4. Fil-
tration, evaporation of the solvent in vacuo and purification of the
residue by flash column chromatography (AcOEt/n-hexane = 1/1)
gave 174 mg (45%; 2 steps) of 38 as a colorless solid; 1H NMR (CDCl3,
500 MHz, d; ppm): 4.68 (1H, t, J = 7.9 Hz), 4.14 (2H, s), 3.75–3.67
(10H, m), 3.39 (2H, t, J = 4.9 Hz), 2.43–1.99 (6H, m), 1.77–0.73
(22H, m); MS (FAB) m/z: (MH+); 506 (MH+).
5.1.18. (S)-2-[(2S,3R)-3-Amino-2-hydroxy-4-
phenylbutanamido]-N-[2-(2-{2-[2-({(E)-[(8R,9S,13S,14S)-3-
hydroxy-13-methyl-7,8,9,11,12,13,15,16-octahydro-6H-
cyclopenta[a]phenanthren-17(14H)-ylidene] amino}oxy)
acetamido]ethoxy}ethoxy)ethyl]-4-methylpentanamide (11)
5.1.21. (14S,17S,18R)-[(5S,8R,9S,10S,13S,14S,17S)-10,13-Dimethyl-
3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] 18-
(tert-butylcarbonylamino)-17-hydroxy-14-isobutyl-13,16-dioxo-
19-phenyl-3,6,9-trioxa-12,15-diazanonadecan-1-oate (39)
Crude 39 was prepared from 38 (36.0 mg, 0.0712 mmol) using
the same procedure as described for 26. Compound 39 was used
in the next step without further purification.
DBU (12.0
l
L, 0.0759 mmol) was added to
a
solution of
L,
35 (25.0 mg, 25.3 mmol) and dodecyl mercaptan (8.00
l
0.0336 mmol) in CH2Cl2 (1.0 mL). The reaction mixture was stir-
red at room temperature for 2 h, then purified by PTLC (CHCl3/
MeOH/25% NH3 aqueous solution = 15/1/0.1) to give 11.9 mg
(61%) of 11 as a colorless oil; 1H NMR (CDCl3, 500 MHz, d;
ppm): 7.55 (1H, d, J = 8.5 Hz), 7.32–7.29 (2H, m), 7.24–7.21 (1H,
m), 7.12 (1H, d, J = 8.6 Hz), 6.90 (1H, t, J = 5.5 Hz), 6.70 (1H, t,
J = 5.5 Hz), 6.64 (1H, dd, J = 8.6, 2.5 Hz), 6.58 (1H, d, J = 4.5 Hz),
4.50 (2H, s), 4.45–4.42 (1H, m), 4.00 (1H, d, J = 2.4 Hz), 3.58–
3.34 (14H, m), 2.97 (1H, dd, J = 13.4, 4.9 Hz), 2.83 (2H, m),
2.61–2.49 (3H, m), 2.35 (1H, dd, J = 13.4, 3.4 Hz), 2.22 (1H, dt,
J = 13.4, 3.4 Hz), 2.05–2.01 (2H, m), 1.91 (2H, m), 1.73–1.37 (8H,
m), 0.93 (6H, s), 0.90 (3H, s); 13C NMR (CDCl3, 125 MHz, d;
ppm): 173.13, 172.50, 171.63, 170.49, 153.46, 137.95, 137.69,
131.68, 129.06, 128.47, 126.44, 126.16, 115.12, 112.64, 72.31,
70.29, 70.06, 69.93, 69.64, 69.50, 54.28, 52.71, 51.50, 44.42,
43.73, 40.44, 39.03, 38.94, 38.50, 37.85, 33.83, 29.43, 29.22,
26.97, 25.89, 25.85, 24.64, 22.75, 22.72, 21.48, 17.04; MS (FAB)
5.1.22. (14S,17S,18R)-[(5S,8R,9S,10S,13S,14S,17S)-10,13-Dimethyl-
3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-1-7-yl] 18-
(tert-butylcarbonylamino)-17-hydroxy-14-isobutyl-13,16-dioxo-
19-phenyl-3,6,9-trioxa-12,15-diazanonadecan-1-oate (41)
Compound 41 (yield; 20.0 mg, 96%; 2 steps from 38) was pre-
pared from crude 39 (36.0 mg, 0.0712 mmol) and 40 (9.70 mg,
0.0237 mmol) using the same procedure as described for 28a; yel-
low oil; 1H NMR (CDCl3, 500 MHz, d; ppm): 7.30–7.20 (5H, m), 6.87
(1H, s), 5.43 (1H, s), 5.04 (1H, s), 4.67 (1H, t, J = 9.2 Hz), 4.50–4.44
(1H, m), 4.16–4.08 (3H, m), 3.74–3.36 (7H, m), 3.06–2.98 (2H,
m), 2.40–2.07 (5H, m), 2.02–1.98 (1H, m), 1.75–1.25 (24H, m),
1.17 (1H, dt, J = 10.4, 4.3 Hz), 1.07–1.03 (1H, m) 1.01 (3H, s), 0.93
(3H, d, J = 6.1 Hz), 0.90 (3H, d, J = 6.2 Hz), 0.79 (3H, s), 0.75 (1H,
dt, J = 10.4, 4.3 Hz); MS (FAB) m/z: 770 (MH+).
m/z: 764 (MH+); HRMS (FAB) calcd for C42H62N5Oþ, 764.4598;
8
found 764.4590.

Y. Itoh et al. / Bioorg. Med. Chem. 19 (2011) 6768–6778
6777
5.1.23. (14S,17S,18R)-[(5S,8R,9S,10S,13S,14S,17S)-10,13-
Education, Culture, Sports, Science and Technology, Japan, and
the Japan Society for the Promotion of Science. This work was also
supported financially by the Takeda Science Foundation and the
Naito Foundation. We are grateful to Nippon Kayaku Co., especially
Dr. Keiko Sekine, for providing bestatin for chemical experiments,
and to Dr. Yukihide Tomari for help with Western blot detection.
Dimethyl-3-oxohexadecahydro-1H-cyclopenta[a]phenanthren-
17-yl] 18-amino-17-hydroxy-14-isobutyl-13,16-dioxo-19-
phenyl-3,6,9-trioxa-12,15-diazanonadecan-1-oate (13)
Compound 13 (yield; 2.40 mg, 90%) was prepared from 41
(3.00 mg, 0.0034 mmol) using the same procedure as described for
24; yellow amorphous solid; 1H NMR (CD3OD, 500 MHz, d; ppm):
7.40–7.31 (5H, m), 4.69 (1H, t, J = 8.6 Hz), 4.38 (1H, dt, J = 8.6,
6.7 Hz), 4.17 (2H, s), 4.14 (2H, d, J = 3.1 Hz), 3.72–3.59 (9H, m),
3.53 (2H, t, J = 6.1 Hz), 3.43–3.40 (2H, m), 3.12 (1H, dd, J = 14.0,
7.9 Hz), 2.94 (1H, dd, J = 7.3 Hz), 2.50 (1H, dt, J = 6.7, 15.3 Hz), 2.38
(1H, t, J = 14.0 Hz), 2.26–2.14 (2H, m) 2.07–2.01 (2H, m), 1.80–1.10
(18H, m), 1.08 (3H, s), 0.98 (3H, d, J = 6.1 Hz), 0.97 (3H, d,
J = 6.1 Hz), 0.85 (3H, s), 0.84–0.77 (1H, m); 13C NMR (CDCl3,
125 MHz, d; ppm): 211.73, 172.14, 171.08, 135.20, 129.67, 128.95,
127.49, 84.22, 53.71, 53.57, 50.35, 46.50, 44.60, 42.79, 38.43,
38.09, 36.81, 35.68, 35.13, 31.13, 28.70, 27.48, 24.87, 23.49, 23.20,
21.36, 20.87, 12.19, 11.44; MS (FAB) m/z: 770 (MH+); HRMS (FAB)
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9
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Grants-in-Aid for Scientific Research from The Ministry of

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