Synthesis of paclitaxel-BGL conjugates

Article abstract of DOI:10.1016/j.bmc.2012.07.031

Four kinds of symmetrically branched oligoglyceryl trimeric (BGL003)-paclitaxel conjugates and a corresponding heptameric (BGL007) conjugate were synthesized. Molecular weights of all the compounds were less than two times that of paclitaxel. The anti-tumor activity of the most water-soluble BGL003 conjugate was examined and found to be preserved in spite of the chemical modification that is displacement of the N3′-debenzoyl residue with the BGL003 succinyl residue.

Full text of DOI:10.1016/j.bmc.2012.07.031

Bioorganic & Medicinal Chemistry 20 (2012) 5559–5567
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of Paclitaxel–BGL Conjugates
Hisao Nemoto a, , Ayato Katagiri a, Masaki Kamiya a, Tomoyuki Kawamura a, Tsuyoshi Matsushita a,
⇑
Kosuke Matsumura a, Tomohiro Itou a, Hatsuhiko Hattori a, Miho Tamaki b, Keisuke Ishizawa b,
Licht Miyamoto b, Shinji Abe c, Koichiro Tsuchiya b
a Department of Pharmaceutical Chemistry, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
b Department of Medical Pharmacology, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
c Practice Room for Clinical Pharmacy, Institute of Health Biosciences, Graduate School of The University of Tokushima, 1-78, Sho-machi, Tokushima 770-8505, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four kinds of symmetrically branched oligoglyceryl trimeric (BGL003)–paclitaxel conjugates and a corre-
sponding heptameric (BGL007) conjugate were synthesized. Molecular weights of all the compounds
were less than two times that of paclitaxel. The anti-tumor activity of the most water-soluble BGL003
conjugate was examined and found to be preserved in spite of the chemical modification that is displace-
ment of the N30-debenzoyl residue with the BGL003 succinyl residue.
Received 2 July 2012
Revised 14 July 2012
Accepted 14 July 2012
Available online 25 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Water-solubility
Paclitaxel
Glycerol
Anti-tumor agent
Symmetrically branched oligoglycerols
1. Introduction
Our developed symmetrically branched oligoglycerols (BGL)9
have no asymmetric centers when the target medicinal molecule
Paclitaxel (1)1 is one of the most well known and commonly
used anti-tumor agents via injection (Fig. 1).2 Because 1 is very
poorly water-soluble (0.25
is clinically ineffective, 1 must be administered via injection as
an aqueous solution containing a surfactant and ethanol, which of-
ten cause undesired clinical side effects.4
Thus, several syntheses of chemically modified paclitaxel deriv-
atives have been reported.5 For example, Battaglia et al. reported a
succinic derivative, although water-solubility was not specifically
mentioned.5a Greenwald et al. reported a derivative possessing a
polyethylene glycol region, with a distributed molecular weight
of more than 5–50 times that of 1. Therefore, appropriate doses
of the entire drug package would be nonsensically increased if this
derivative were to be clinically used.5b Mandai et al. reported
derivatives of a diastereomeric mixture of sugars with a glucolate
linker,5c which may be prohibited due to guidelines adopted by
governments of advanced countries. Although inclusion complexes
of 1 by a protein,6 a micelle,7 or a liposome8 have also been devel-
oped, use of a chemically single molecule is preferable because
quality control of a single molecule is generally simple and highly
reproducible.
is connected at the apex of BGL. Using BGL003 (2) (the trimer of
BGL),10 some medicinal molecules were converted to the corre-
l
g/mL),3 and 1 via oral administration
sponding water-soluble derivatives, in which water-solubility
9a,12,13
was increased 1000–5000 times,
was increased only 1.5–2 times.
and molecular weight
The water-solubilizing
9b,12,13
effect is independent of pH because a number of neutral polar
functionalities that is primary hydroxyl groups are available for
water-solubilization. These characteristic features are attributed
to the molecular design and synthetic method of BGL. Accordingly,
we started to examine the covalent bond formation of BGL (BGLa-
tion) for paclitaxel with a linker possessing two carboxylates such
as succinate or glutarate to synthesize advanced and potentially
water-soluble derivatives, which would be diastereomerically
and enantiomerically pure.
2. Results and discussion
In this paper, we report the synthesis of paclitaxel–BGL conju-
gates. The three linking points are illustrated using white arrows
in Fig. 1. Chemical derivatization of free hydroxyl groups on 1
has been well studied.14,15 For example, the hydroxyl group at
C20 is most reactive for acylation or silylation, that at C7 is the sec-
ond most reactive, and that at C1 is hardly reactive. Panchagnula
reported that activity as an anti-tumor agent was similar even if
the C30-benzoyl moiety was substituted with other large acyl
⇑
Corresponding author. Tel./fax: +81 886337284.
E-mail address: nem@ph.tokushima-u.ac.jp (H. Nemoto).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.031

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H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
Scheme 1. Preparation of a BGLating agent for 3 and 4.
Figure 1. Structure of BGL003 and Paclitaxel (1) with examined BGLating points.
Scheme 2. Synthesis of 3.
Figure 2. Synthesized paclitaxel derivatives via BGLation at C20, C7 and C30.
groups,16 and a method for the preparation of C30-debenzoylated
paclitaxel is known.17 Based on this information, 3, 4 and 5 were
chosen as reasonable targets (Fig. 2). It is noted that the molecular
weights of 3–5 are only 1.26–1.38 times greater than that of 1,
which would minimize the dosage increase of the entire drug pack-
age given equivalent clinical activities of 3–5 to that of 1.
For BGLation to synthesize 3 and 4, the succinylated BGL003
possessing benzylidene (Bnd) groups 7 was prepared from 69c,18
using succinic anhydride with triethylamine in the presence of a
catalytic amount of 4-(N,N-dimethylamino)pyridine (DMAP) in
dichloromethane (CH2Cl2) at room temperature for 16 h in 84%
yield (Scheme 1).
Scheme 3. Synthesis of 4.
avoid competition between the condensation of the hydroxyl
group on the target molecule with those on the BGL terminus if
the Bnd groups were absent. In contrast, in the synthesis of 5,
The first target 3 was synthesized in 79% overall yield from 1 via
the condensation reaction of 1 and 7 using DMAP and 4-(N,N-
dimethylamino)propyl carbodiimide (EDC) hydrochloride (92%
yield), followed by hydrogenolysis of the two Bnd groups of the
resulting 8 using palladium hydroxide in methanol under hydro-
gen atmosphere (86% yield).
The second target 4 was synthesized from the known silylated
derivative 914 in 77% overall yield. The condensation reaction of
9 and 7 using diisopropylcarbodiimide and 4-dimethylaminopyrid-
inium p-toluenesulfonate afforded 10 in 93% yield. Treatment of
HFꢀpyridine in THF gave 11 in 95% yield. Finally, 4 was successfully
synthesized in 88% yield from 11 via similar hydrogenolysis as for
3.
In the syntheses illustrated in Schemes 2 and 3, the Bnd groups
on BGL were removed after BGLation of the target molecules, to
Scheme 4. Preparation of a BGLating agent for 5.

H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
5561
Scheme 5. Synthesis of 5.
the protecting groups on BGL were removed prior to BGLation, be-
cause amino groups are generally acylated much faster than hydro-
xyl groups, and several kinds of activated esters, which are reactive
only toward amino groups but not hydroxyl groups, are available.
Therefore, N-succinimidyl ester 13 was prepared from 7 via 12 as
follows (Scheme 4). Condensation of 7 and N-hydroxysuccinimide
(HOSu) using EDC gave 12 in 72% yield. Hydrogenolysis of 12 to re-
move the two Bnd groups afforded 13 in quantitative yield. The
activated ester 13 was used for BGLation immediately after isola-
tion via filtration to remove the palladium catalyst and subsequent
concentration to remove methanol. The N–O bond in the N-succin-
imidyl ester moiety of 13 was preserved during the hydrogenoly-
sis.19 Purification of 13 by silica gel column chromatography was
unsuccessful although NMR data of the crude product could be ob-
tained. When 13 was left for more than half a day at room temper-
ature, a non-negligible amount of unidentified byproducts and
HOSu were observed, probably due to intra- or intermolecular
alcoholysis of the N-succinimidyl ester moiety.
Figure 3. Anti-tumor activity of 5.
preserved in spite of the chemical modification that is debenzoyla-
tion and BGLation.
Base on these promising results, synthesis of other BGL–paclit-
axel conjugates based on the structure of 5 was carried out. The
N-succinimidyl ester possessing amide bond 20 was prepared as
illustrated in Scheme 6. The alcohol 69c was converted to azide
16 in 88% yield via sulfonate 15. Reduction of the azide functional-
ity of 16, acylation of the resulting amino group of 17 with glutaric
anhydride,20 followed by condensation with HOSu gave 19 in 70%
yield. Finally, hydrogenolysis of 19 afforded 20, which was imme-
diately used for the reaction with 14.17
The N-succinimidyl ester possessing BGL007 22 was prepared
from 21,9c via hydrogenolysis to remove the four Bnd groups
(Scheme 7), and immediately used for the condensation with 14.17
For the synthesis of two additional BGL conjugates 2321 and
24,22 the reaction was examined without using additives such as
HOBt and DMA to simplify purification of 23 and 24 (Scheme 8).
It was contrastive that the use of as small an amount of BGLating
agent as possible was prioritized in the case of 5. With 4.0 equiv
of 20 for 48 h, 14 disappeared by thin layer chromatography to af-
ford 23 in 68% yield. With 20 equiv of 22 for 40 h, 24 was afforded
in 80% yield. Even when BGLation was carried out using glyceryl
heptamers (BGL007), the molecular weight of 24 is less than two
times that of 1.
The simplest conditions [C30-debenzoylated paclitaxel (14)17
with 1.1 equivalents of 13 at room temperature for more than
20 h without any additive in various solvents such as THF, DMF
and DMSO] gave 5 in unsatisfactory (10–30%) yield (Scheme 5).
After several attempts, reaction conditions for 5 were found, in
which the minimum amount of BGLating agent 13 among all our
attempts (1.52 equiv vs 14) was used, with N-hydroxybenzotria-
zole (HOBt) and N,N-dimethylaniline (DMA) as additives.
To determine the water-solubility, partition coefficients of 1, 3,
4 and 5 were measured using a water/1-octanol system (Table 1).
Although their o/w ratios indicated that 4 and 5 can be signifi-
cantly more water-soluble than 1 as expected from our previous
reports, 11–13 3 was surprisingly as water-insoluble as 1. It is inter-
esting that the location of BGLation can dramatically influence the
water-solubility, probably due to the overall dipole moment of the
entrie molecule.
Since the water-solubility of 5 was higher than those of 3 and 4,
the anti-tumor activity of 5 (3.51 lmol/kg) was examined using
3. Conclusion
male nude mice (6-8 weeks old, BALB/c), inoculated subcutane-
ously with A549 human lung cancer cell, by intraperitoneal injec-
tion at the 1st, 4th and 7th days (indicated by black triangles at the
bottom of Fig. 3). A solution of each compound in DMSO/saline
Five kinds of paclitaxel–BGL conjugates, each of which is a
chemically single compound of less than two times the molecular
weight of paclitaxel (1), were synthesized starting from 1. Two pro-
cedures, deprotection after BGLation and deprotection prior to
BGLation, were demonstrated. Significantly, the BGLation location
unexpectedly influenced the water-solubilization. The anti-tumor
activity of 30-debenzoylated paclitaxel possessing BGL003 was
examined and found to be preserved in spite of chemical modifica-
tion. Further synthetic and evaluation studies are underway in our
laboratories.
(1:1, 100 lL) was intraabdominally administrated via injection.
The area (mm2) of tumor tissue was calculated by longest diame-
ter ꢁ shortest diameter. It was found that the size of the tumor tis-
sue by both 1 and 5 was significantly smaller than in the control
(vehicle), suggesting that anti-tumor activity was certainly
Table 1
Partition Coefficient of 1, 3, 4 and 5
Compound
Water layer (
lM)
1-Octanol layer (
lM)
O/W
log P o/w
4. Experimental section
4.1. General
1
3
4
5
0.55
0.51
1.00
3.44
97.3
92.4
98.2
99.1
177
181
98
2.25
2.26
1.99
1.46
29
IR spctra were measured by Nihon Bunko FT-IR 6200 spectrom-
eter. 1H and 13C NMR spectra were measured by JEOL JMN-AL400
(p < 0.001).

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H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
Scheme 6. Preparation of 20.
Scheme 7. Preparation of 22.
at 400 MHz, and JEOL JMN-AL300 at 75 MHz, respectively in deu-
triolized chloroform or methanol (CDCl3 or CD3OD). Chemical
shifts were indicated by d value with tetramethylsilane as an inter-
nal standard. Multiplicity is abbreviated as s: singlet, d: doublet, t:
triplet, q: quartet, quint: quintet, m: multiplet. High resolution
Scheme 8. Syntheses of 23 and 24.

H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
5563
mass spectra (HRMS) were measured by Waters LCT PRIMIER using
Electronically Sprayed Injection–Time-of-Fight (ESI-TOF). All the
reactions were carried out under argon atmosphere unless other-
wise noted. Reactions were monitored by thin layer chromatogra-
phy of Merck Silicagel 60 F254 (0.25 mm) when it was applicable.
Purifications were performed with Silica gel 60 N purchased from
KANTO unless otherwise noted. Dichloromethane (CH2Cl2) was
distilled over phosphorus pentoxide. Pyridine was distilled from
potassium hydroxide. Triethylamine (TEA) and N,N-dimethylfor-
maide (DMF) was distilled over calcium hydride. Anhydrous
tetrahydrofuran (THF) was purchased from KANTO.
100.9 (CH ꢁ 2), 84.3 (CH), 80.8 (C), 78.9 (C), 76.2 (CH2), 75.4
(CH), 74.9 (CH), 74.2 (CH), 71.9 (CH ꢁ 2), 71.6 (CH), 71.0 (CH),
70.8 (CH), 68.9 (CH2), 68.8 (CH2), 68.4 (CH2), 68.3 (CH2),
66.3(CH2), 66.1 (CH2), 58.3 (C), 52.7 (CH), 45.4 (CH), 43.0 (C),
35.4 (CH2 ꢁ 2), 29.1 (CH2), 28.9 (CH2), 26.6 (CH3), 22.5 (CH3),
22.0 (CH3), 20.7 (CH3), 14.6 (CH3), 9.4 (CH3); HRMS (ESI-TOF) m/z
calcd for C74H81NO23Na [M+Na]+ 1374.5097, found 1374.5150.
4.1.3. 20-[4-(1,3-Bis(1,3-dihydroxypropan-2-yloxy)propan-2-
yloxy)-4-oxobutanoyl]-paclitaxel (3)
To a solution of 8 (102 mg, 0.0752 mmol) in methanol (1.0 mL)
was added palladium hydroxide (0.020 g, 0.014 mmol) and the
suspension was stirred for 15 h under hydrogen atmosphere. After
inflow of argon gas to turn hydrogen gas out, the resulting suspen-
sion was filtered. The filtrate was concentrated in vacuo. The resi-
due was purified by silica gel column chromatography eluted with
chloroform/methanol (9/1) to afford 3 as a colorless gummy solid
(75.7 mg, 0.0643 mmol, 86% yield). FT-IR (neat): 3447, 2937,
4.1.1. 4-(1,3-Bis(2-phenyl-1,3-dioxan-5-yloxy)propan-2-yloxy)-
4-oxobutanoic acid (7)
To a solution of 69c (1.23 g, 2.95 mmol) in CH2Cl2 (10 mL) were
added 4-(N,N-dimethylamino)pyridine (DMAP) (0.040 g, 0.29
mmol), TEA (0.84 mL, 6.0 mmol) and succinic anhydride (0.33 g,
3.29 mmol), and the mixture was stirred for 16 h at room temper-
ature. The resulting solution was poured into a saturated aqueous
solution of copper(II) sulfate (CuSO4aq) (200 mL) and extracted
with ethyl acetate (300 mL ꢁ 1, 50 mL ꢁ 2). The combined organic
layers were washed with brine (200 mL), dried over anhydrous so-
dium sulfate (Na2SO4), and concentrated in vacuo. The residue was
purified by silica gel column chromatography eluted with ethyl
acetate to afford 7 as a colorless oil (1.27 g, 2.46 mmol, 84% yield).
FT-IR (neat): 2863, 1735, 1453, 1392, 1344, 1238, 1215, 1155,
1734, 1647, 1540, 1490, 1373, 1243, 1153, 1070, 909, 731 cmꢂ1
;
1H NMR (CDCl3, 400 MHz): d 8.16 (d, J = 8.6 Hz, 2H), 7.80 (d,
J = 7.4 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.54–7.49 (m, J = 9.6,
5.9 Hz, 3H), 7.45–7.38 (m, J = 3.0 Hz, 6H), 7.37–7.32 (m, 1H), 6.32
(s, 1H), 6.21 (t, J = 8.9 Hz, 1H), 5.96 (dd, J = 9.2, 3.4 Hz, 1H), 5.69
(d, J = 6.6 Hz, 1H), 5.49 (d, J = 3.6 Hz, 1H), 5.04 (quint, J = 5.2 Hz,
1H), 4.97 (dd, J = 9.6, 1.5 Hz, 1H), 4.43–4.41 (m, 1H), 4.31 (d,
J = 8.4 Hz, 1H), 4.21 (d, J = 8.6 Hz, 1H), 3.81–3.56 (m, 14H), 3.49–
3.41 (m, 2H), 2.86–2.61 (m, 6H), 2.57–2.50 (m, 1H), 2.45 (s, 3H),
2.35 (dd, J = 15.5, 9.4 Hz, 1H), 2.23 (s, 3H), 2.17–2.11 (m, 1H),
1.93 (s, 3H), 1.91–1.89 (m, 1H), 1.69 (s, 3H), 1.26 (s, 1H), 1.23 (s,
4H), 1.15 (s, 3H). 13C NMR (CDCl3, 75 MHz): d 203.0 (C), 171.6
(C), 171.4 (C), 171.0(C), 169.9 (C), 168.1 (C), 167.3 (C), 166.7 (C),
142.0 (C), 136.7 (C), 133.5 (C), 132.8 (C), 131.8 (CH), 130.1
(CH ꢁ 2), 129.1 (C), 128.9 (CH ꢁ 2), 128.6 (CH ꢁ 2), 128.5
(CH ꢁ 3), 128.4 (CH), 127.2 (CH ꢁ 2), 126.6 (CH ꢁ 2), 84.3 (CH),
81.0 (CH ꢁ 2, C), 78.8 (C), 76.3 (CH2), 75.5 (CH), 74.8 (CH), 74.4
(CH), 72.1 (CH), 71.9 (CH), 71.6 (CH), 67.8 (CH2), 67.7 (CH2), 61.9
(CH2), 61.8 (CH2 ꢁ 2), 61.7 (CH2), 58.2 (C), 52.8 (CH), 45.7 (CH),
43.1 (C), 35.7 (CH2), 35.3 (CH2), 29.1 (CH2), 28.8 (CH2), 26.5
(CH3), 22.5 (CH3), 21.9 (CH3), 20.8 (CH3), 14.6 (CH3), 9.6 (CH3);
HRMS (ESI-TOF) m/z calcd for C60H73NO23Na [M+Na]+ 1198.4471,
found 1198.4474.
1091, 1009, 982, 915, 759, 700 cmꢂ1 1H NMR (CDCl3, 400 MHz):
;
d 7.52–7.47 (m, 4H), 7.39–7.31 (m, 6H), 5.53 (s, 2H), 5.23 (quint,
J = 5.0 Hz, 1H), 4.39–4.32 (m, 4H), 4.04–3.98 (m, 4H), 3.86–3.74
(m, 4H), 3.33 (quint, J = 1.5 Hz, 2H), 2.68–2.63 (m, 2H), 2.59–2.54
(m, 2H); 13C NMR (CDCl3, 75 MHz): d 176.7 (C), 171.7 (C), 138.0
(C ꢁ 2), 128.8 (CH ꢁ 2), 128.1 (CH ꢁ 4), 126.0 (CH ꢁ 4), 101.0
(CH ꢁ 2), 71.8 (CH), 70.9 (CH ꢁ 2), 68.8 (CH2 ꢁ 2), 68.4 (CH2 ꢁ 2),
66.4 (CH2 ꢁ 2), 29.0 (CH2), 28.6 (CH2); HRMS (ESI-TOF) m/z calcd
for C27H32O10Na [M+Na]+ 539.1893, found 539.1901.
4.1.2. 20-[4-(1,3-Bis(cis-2-phenyl-1,3-dioxan-5-yloxy)propan-2-
yloxy)-4-oxobutanoyl]-paclitaxel (8)
To a solution of 1 (70.0 mg, 0.0820 mmol) in CH2Cl2 (1.0 mL)
were added
7
(50.8 mg, 0.0983 mmol), DMAP (12.0 mg,
0.0983 mmol) and 3-(N,N-dimethylamino)propyl carbodiimide
hydrochloride (EDC) (18.9 mg, 0.0983 mmol), and the mixture
was stirred for 5 h at room temperature. The resulting solution
was poured into CuSO4aq (30 mL) and extracted with ethyl acetate
(50 mL ꢁ 1, 25 mL ꢁ 2). The combined organic layers were washed
with brine (30 mL), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluted with hexane/ethyl acetate (1/2) to afford 8 as a colorless
gummy solid (102 mg, 0.0752 mmol, 92% yield). FT-IR (neat):
3502, 3065, 2975, 2250, 1732, 1660, 1603, 1580, 1522, 1488,
1453, 1372, 1240, 1153, 1093, 1016, 948, 912, 846, 799, 731,
4.1.4. 7-[4-(1,3-Bis(cis-2-phenyl-1,3-dioxan-5-yloxy)propan-2-
yloxy)-4-oxobutanoyl]-2’-TBS-paclitaxel (10)
To a solution of 914 (53.6 mg, 0.0554 mmol) in CH2Cl2 (2.0 mL)
were added 7 (41.6 mg, 0.0804 mmol), 4-dimethylaminopyridini-
um p-toluenesulfonate (31.6 mg, 0.107 mmol) and diisoproyl car-
bodiimide (0.033 mL, 0.220 mmol), and the mixture was stirred
for 12 h at room temperature. The resulting solution was poured
into CuSO4aq (30 mL) and extracted with ethyl acetate
(50 mL ꢁ 1, 25 mL ꢁ 2). The combined organic layers were washed
with brine (30 mL), dried over Na2SO4, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
eluted with hexane/ethyl acetate (1/1) to afford 10 as a colorless
gummy solid (75.5 mg, 0.0515 mmol, 93% yield). FT-IR (neat):
3440, 2930,1735, 1662, 1484, 1452, 1372, 1240, 1154, 1094,
648 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 8.15 (d, J = 7.4 Hz, 2H),
;
7.81 (d, J = 7.4 Hz, 2H), 7.63 (t, J = 7.2 Hz, 1H), 7.56–7.31 (m,
20H), 7.17 (q, J = 9.1 Hz, 1H), 6.31 (s, 1H), 6.23 (t, J = 8.8 Hz, 1H),
5.98 (dd, J = 9.0, 3.2 Hz, 1H), 5.69 (d, J = 7.0 Hz, 1H), 5.51 (s, 1H),
5.48 (s, 2H), 5.15 (quint, J = 4.9 Hz, 1H), 4.97 (d, J = 8.7 Hz, 1H),
4.45 (m, 1H), 4.35–4.19 (m, 6H), 4.00–3.86 (m, 5H), 3.85–3.68
(m, 6H), 3.32 (s, 1H), 3.27 (s, 1H), 2.73 (m, 2H), 2.65 (m, 2H),
2.56 (m, 1H), 2.45 (s, 3H), 2.34 (dd, J = 15.3, 9.3 Hz, 1H), 2.22 (s,
3H), 2.19–2.11 (m, 1H), 1.94 (s, 3H), 1.91 (m, 1H), 1.69 (s, 3H),
1.24 (s, 3H), 1.14 (s, 3H); 13C NMR (CDCl3, 75 MHz): d 203.6 (C),
171.6 (C), 171.0(C), 170.9(C), 169.7 (C), 167.8 (C), 167.0 (C), 166.8
(C), 142.6 (C), 138.0 (C), 137.9 (C), 136.8 (C), 133.5 (CH), 133.4
(C), 132.6 (C), 131.8 (CH), 130.1 (CH ꢁ 2), 129.0 (C), 128.9
(CH ꢁ 2), 128.7 (CH ꢁ 2), 128.6 (CH), 128.5 (CH ꢁ 2), 128.3 (CH),
128.0 (CH ꢁ5), 127.1 (CH ꢁ 2), 126.5 (CH ꢁ 2), 125.9 (CH ꢁ 4),
1018, 982, 838, 757, 699 cm–1 1H NMR (CDCl3, 400 MHz): d 8.14
;
(d, J = 7.2 Hz, 2H), 7.75 (d, J = 7.4 Hz, 2H), 7.62 (t, J = 7.4 Hz, 1H),
7.55–7.30 (m, 20H), 7.09 (d, J = 8.9 Hz, 1H), 6.29–6.21 (m, 2H),
5.77–5.69 (m, 2H), 5.61 (dd, J = 10.6, 7.1 Hz, 1H), 5.49 (s, 2H),
5.21 (quint, J = 5.0 Hz, 1H), 4.97 (d, J = 9.0 Hz, 1H), 4.67 (d, J = 2.0,
1H), 4.35–4.28 (m, 5H), 4.21 (d, J = 8.5 Hz, 1H), 3.99–3.95 (m,
5H), 3.84–3.77 (m, 4H), 3.40–3.36 (m, 2H), 2.77–2.55 (m, 8H),
2.46–2.37 (m, 1H), 2.20–2.11 (m, 4H), 1.97 (s, 3H), 1.90–1.85 (m,
1H), 1.81 (s, 3H), 1.20 (s, 3H), 1.16 (s, 3H). 13C NMR (CDCl3,
75 MHz): d 201.9 (C), 172.1 (C), 171. 4(C ꢁ 2), 169.8 (C), 169.0

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H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
(C), 167.0 (C), 166.8 (C), 140.5 (C), 138.1 (C ꢁ 3), 134.0 (C), 133.6
(CH), 132.6 (CH), 131.7 (CH), 130.1 (CH ꢁ 2), 129.0 (C), 128.7
(CH ꢁ 2), 128.6 (CH ꢁ 4), 128.0 (CH ꢁ 5), 127.9 (CH), 126.9
(CH ꢁ 2), 126.3 (CH ꢁ 2), 126.0 (CH ꢁ 5), 100.9 (CH ꢁ 2) 83.8
(CH), 80.8 (C), 78.4 (C), 76.1 (CH2), 75.0 (CH), 74.9 (CH), 74.3
(CH), 71.7 (CH), 71.3 (CH), 71.1 (CH), 70.9 (CH), 70.8 (CH), 68.9
(CH2 ꢁ 2), 68.4 (CH2), 68.3 (CH2), 66.3 (CH2 ꢁ 2), 55.7 (C), 55.4
(CH), 46.6 (CH), 43.1 (C), 35.3 (CH2), 33.0 (CH2), 28.8 (CH2), 28.7
(CH2), 26.1 (CH3), 25.2 (CH3 ꢁ 3), 22.7 (CH3), 21.1 (CH3), 20.4
(CH3), 17.8 (C), 14.3 (CH3), 10.6 (CH3), ꢂ5.53 (CH3), ꢂ6.16 (CH3);
J = 8.5 Hz, 1H), 4.10 (d, J = 1.2 Hz, 1H), 3.88 (d, J = 6.8 Hz, 1H),
3.84–3.60 (m, 12H), 3.53–3.46 (m, 2H), 3.03–2.77 (m, 3H) 2.73–
2.52 (m, 5H), 2.37 (s, 3H), 2.31 (d, J = 9.0 Hz, 2H), 2.16 (s, 3H),
1.87–1.78 (m, 9H), 1.20 (s, 3H), 1.15 (s, 3H). 13C NMR (CDCl3,
75 MHz): d 201.7 (C), 172.5 (C ꢁ 2), 172.3(C), 171.7 (C), 170.5 (C),
169.1 (C), 167.4 (C), 166.5 (C), 140.5 (C), 138.0 (C), 133.6 (C, CH),
132.5(C), 131.7 (CH), 130.0 (CH ꢁ 2), 128.9 (C), 128.7 (CH ꢁ 3),
128.6 (CH), 128.5 (CH ꢁ 2), 127.9 (CH), 127.0 (CH ꢁ 2), 126.9
(CH ꢁ 2), 83.7 (CH), 81.0 (CH ꢁ 2), 80.8 (C), 78.2 (C), 76.2 (CH2),
75.2 (CH), 74.1 (CH), 72.9 (CH), 71.8 (CH), 71.6 (CH), 71.5 (CH),
67.9 (CH2 ꢁ 2), 61.7 (CH2), 61.6 (CH2 ꢁ 2), 61.5 (CH2), 55.8 (C),
55.0 (CH), 46.9 (CH), 43.0 (C), 35.3 (CH2), 33.0 (CH2), 28.9
(CH2 ꢁ 2), 26.3 (CH3), 22.4 (CH3), 20.7 (CH3), 20.6 (CH3), 14.3
(CH3), 10.7 (CH3); HRMS (ESI–TOF) m/z calcd for C60H73NO23Na
[M+Na]+ 1198.4471, found 1198.4467.
HRMS (ESI–TOF) m/z calcd for
C
80H95NO23SiNa [M+Na]+
1488.5962, found: 1488.5955.
4.1.5. 7-[4-(1,3-Bis(cis-2-phenyl-1,3-dioxan-5-yloxy)propan-2-
yloxy)-4-oxobutanoyl]-paclitaxel (11)
To a solution of 10 (75.5 mg, 0.0515 mmol) in THF (1.0 mL)
were added pyridinium fluoride (0.5 mL) and pyridine (1.5 mL),
and the mixture was stirred for 20 h at room temperature. The
resulting mixture was poured into CuSO4aq (30 mL) and extracted
with ethyl acetate (50 mL ꢁ 1, 25 mL ꢁ 2). The combined organic
layers were washed with brine (30 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography eluted with hexane/ethyl acetate (1/3) to af-
ford 11 as a colorless gummy solid (65.8 mg, 0.0487 mmol, 95%
yield). FT–IR (neat): 3438, 3014, 1733, 1652, 1602, 1581, 1486,
4.1.7. 1,3-Bis(2-phenyl-1,3-dioxan-5-yloxy)propan-2-yl 2,5-
dioxopyrrolidin-1-yl succinate (12)
To a solution of 7 (232 mg, 0.900 mmol) in CH2Cl2 (5.0 mL) were
added HOSu (104 mg, 0.900 mmol) and EDC (172 mg, 0.900 mmol),
and the mixture was stirred for 1 h at room temperature. The
resulting mixture was poured into CuSO4aq (50 mL) and extracted
with ethyl acetate (50 mL ꢁ 1, 25 mL ꢁ 2). The combined organic
layers were washed with brine (50 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography eluted with hexane/ethyl acetate (1/3) to af-
ford 12 as a colorless gummy solid (237 mg, 0.403 mmol, 90%
yield). FT–IR (neat): 2860, 1815, 1784, 1740, 1454, 1390, 1239,
1452, 1238, 845, 755, 667 cmꢂ1 1H NMR (CDCl3, 400 MHz): d
;
8.12 (d, J = 7.4 Hz, 2H), 7.76 (d, J = 7.4 Hz, 2H), 7.63 (t, J = 7.2 Hz,
1H), 7.53–7.31 (m, 20H), 7.07 (d, J = 8.9 Hz, 1H), 6.20–6.13 (m,
2H), 5.80 (dd, J = 9.0, 3.2 Hz, 1H), 5.67 (d, J = 6.9 Hz, 1H), 5.56 (dd,
J = 10.4, 7.3 Hz, 1H), 5.50 (s, 1H), 5.49 (s, 1H), 5.19 (quint,
J = 5.0 Hz, 1H), 4.92 (d, J = 9.0 Hz, 1H), 4.79 (dd, J = 4.7, 2.5 Hz,
1H), 4.35–4.26 (m, 6H), 4.18 (d, J = 8.5 Hz, 1H), 4.00–3.93 (m,
4H), 3.91 (d, J = 6.8 Hz, 1H), 3.85–3.76 (m, 4H), 3.62 (d, J = 4.8 Hz,
1H), 3.41–3.36 (m, 2H), 2.74–2.53 (m, 5H), 2.37 (s, 3H), 2.32 (dd,
J = 8.9, 3.3 Hz, 1H), 2.15 (s, 3H), 1.86–1.78 (m, 7H), 1.20 (s, 3H),
1.16 (s, 3H). 13C NMR (CDCl3, 75 MHz): d 201.8 (C), 172.4 (C),
172.2 (C), 171.4 (C), 170.3 (C), 169.0 (C), 167.1 (C), 166.8 (C),
140.3 (C), 138.1 (C ꢁ 2), 138.0 (C), 133.7 (CH), 133.6 (C), 132.8
(C), 131.8 (CH), 130.1 (CH ꢁ 2), 129.0 (C), 128.8 (CH ꢁ 2), 128.7
(CH ꢁ 2), 128.6 (CH ꢁ 3), 128.1 (CH ꢁ 5), 127.0 (CH ꢁ 4), 126.0
(CH ꢁ 5), 100.9 (CH ꢁ 2) 83.7 (CH), 80.8 (C), 78.2 (C), 76.2 (CH2),
75.1 (CH), 74.1 (CH), 73.0 (CH), 71.8 (CH), 71.7 (CH), 71.4 (CH),
70.9 (CH ꢁ 2), 68.9 (CH2 ꢁ 2), 68.4 (CH2 ꢁ 2), 66.3 (CH2 ꢁ 2), 55.9
(C), 54.7 (CH), 46.7 (CH), 43.0 (C), 35.3 (CH2), 33.0 (CH2), 28.8
(CH2 ꢁ 2) 26.2 (CH3), 22.3 (CH3), 20.6 (CH3), 20.5 (CH3), 14.3
(CH3), 10.5 (CH3); HRMS (ESI-TOF) m/z calcd for C74H81NO23Na
[M+Na]+ 1374.5097, found 1374.5088.
1206, 1153, 1091, 1011, 915, 845, 800, 732, 701, 648 cmꢂ1 1H
;
NMR (CDCl3, 400 MHz): d 7.52–7.46 (m, 4H), 7.39–7.31 (m, 6H),
5.48 (s, 2H), 5.25 (quint, J = 5.2 Hz, 1H), 4.35–4.26 (m, 4H), 4.00–
3.94 (m, 4H), 3.86–3.76 (m, 4H), 3.36 (quint, J = 1.5 Hz, 2H), 2.91
(t, J = 6.8 Hz, 2H), 2.80 (s, 2H), 2.76 (t, J = 6.6 Hz, 2H); 13C NMR
(CDCl3, 75 MHz): d 170.5 (C), 168.9 (C ꢁ 2), 167.7 (C), 138.1
(C ꢁ 2), 128.8 (CH ꢁ 2), 128.1 (CH ꢁ 4), 126.0 (CH ꢁ 4), 100.9
(CH ꢁ 2), 72.0 (CH), 70.8 (CH ꢁ 2), 68.8 (CH2 ꢁ 2), 68.3 (CH2 ꢁ 2),
66.3 (CH2 ꢁ 2), 28.5 (CH2), 25.9 (CH2), 25.2(CH2 ꢁ 2); HRMS (ESI-
TOF) m/z calcd for
C
31H35NO12Na [M+Na]+ 636.2057, found
636.2040.
4.1.8. 1,3-Bis(1,3-dihydroxypropan-2-yloxy)propan-2-yl 2,5-
dioxopyrrolidin-1-yl succinate (13)
To a solution of 12 (40.0 mg, 0.065 mmol) in ethanol (2.0 mL)
was added palladium hydroxide (10.0 mg, 0.0071 mmol) and the
suspension was stirred for 15 h under hydrogen atmosphere. After
inflow of argon gas to turn hydrogen gas out, the resulting suspen-
sion was filtered. The filtrate was concentrated in vacuo. The resi-
due, crude 13 (19.0 mg) was immediately used in the next reaction
without further purification.
4.1.6. 7-[4-(1,3-Bis(1,3-dihydroxypropan-2-yloxy)propan-2-
yloxy)-4-oxobutanoyl]-paclitaxel (4)
To a solution of 11 (206 mg, 0.152 mmol) in methanol (2.0 mL)
was added palladium hydroxide (0.011 g, 0.071 mmol) and the
suspension was stirred for 58 h under hydrogen atmosphere. After
inflow of argon gas to turn hydrogen gas out, the resulting suspen-
sion was filtered. The filtrate was concentrated in vacuo. The resi-
due was purified by silica gel column chromatography eluted with
chloroform/methanol (8/1) to afford 4 as a colorless gummy solid
(158 mg, 0.134 mmol, 88% yield). FT–IR (neat): 3420, 2942, 2250,
1734, 1647, 1603, 1579, 1522, 1487, 1452, 1372, 1240, 1158,
4.1.9. (1S,2S,4S,9S,10S,15S,3R,7R,12R)-4,12-Diacetyloxy-1,9-
dihydroxy-10,14,17,17-tetramethyl-11-oxo-2- phenylcar-
bonyloxy-6-oxatetracyclo [11.3.1.0<3,10>.0<4,7>]heptadec-13-
en-15-yl (3S,2R)-2-hydroxy-3-{3-[(2-[2-hydroxy-1-
(hydroxymethyl)ethoxy]-1-{[2-hydroxy-1-(hydroxymethyl)
ethoxy]methyl}ethyl)oxycarbonyl]propanoylamino}-3-
phenylpropanoate (5)
To a solution of 14 (13.8 mg, 18.4
added 13 (12.0 mg, 28 mol), 1-hydroxybenzotriazol monohydrate
(0.60 mg, 37 mol) and N,N-dimethylaniline (2.3 L, 18.0 mol),
lmol) in THF (0.3 mL) were
l
1108, 1067, 979, 913, 846, 729 cm–1 1H NMR (CDCl3, 400 MHz):
;
l
l
l
d 8.11 (d, J = 7.3 Hz, 2H), 7.79–7.76 (m, 2H), 7.62 (t, J = 7.4 Hz,
1H), 7.53–7.46 (m, 5H), 7.43–7.30 (m, 5H), 6.19–6.13 (m, 2H),
5.78 (dd, J = 8.7, 2.3 Hz, 1H), 5.66 (d, J = 6.9 Hz, 1H), 5.56 (dd,
J = 10.4, 7.2 Hz, 1H), 5.11 (quint, J = 4.8 Hz, 1H), 4.94 (d, J = 9.0 Hz,
1H), 4.80 (dd, J = 5.3, 2.7 Hz, 1H), 4.31 (d, J = 8.5 Hz, 1H), 4.18 (d,
and the mixture was stirred for 19 h at room temperature. The
resulting mixture was poured into CuSO4aq (10 mL) and extracted
with ethyl acetate (20 mL ꢁ 1, 10 mL ꢁ 2). The combined organic
layers were washed with brine (10 mL), dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel

H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
5565
column chromatography eluted with chloroform/methanol (8/1) to
afford 5 as a colorless gummy solid (14.0 mg, 13.0 mol, 73%
yield). FT–IR (neat): 3385, 2926, 2348, 2251, 1723, 1662, 1539,
(100 mL) and extracted with ethyl acetate (100 mL ꢁ 1, 50 mL ꢁ 2).
The combined organic layers were washed with brine (100 mL),
dried over Na2SO4, and concentrated in vacuo to afford crude 18
(1.31 g), which was used in the next reaction without further puri-
fication. To a solution of 18 (1.31 g) in CH2Cl2 (15 mL) were added
H–OSu (536 mg, 4.66 mmol) and EDC (893 mg, 4.66 mmol), and
the mixture was stirred for 3 h at 40 °C. The resulting mixture
was quenched with water (50 mL), and extracted with ethyl
acetate (50 mL ꢁ 1, 25 mL ꢁ 2). The combined organic layers were
washed with brine (50 mL), dried over Na2SO4, and concentrated in
vacuo. The residiue was purified by silica gel column chromatogra-
phy eluted with hexane/ethyl acetate (1/3) to afford 19 as a color-
less gummy solid (1.02 g, 1.63 mmol, 70% yield). FT-IR (neat):
2866, 1783, 1738, 1661, 1530, 1454, 1388, 1209, 1154, 1091,
l
1452, 1373, 1243, 1177, 1110, 1071, 1026, 979, 909, 854, 776,
731, 647 cmꢂ1 1H NMR (CD3OD, 400 MHz): d 8.11 (d, J = 7.7 Hz,
;
2H), 7.67 (t, J = 7.4 Hz, 1H), 7.57 (t, J = 7.6 Hz, 2H), 7.46–7.37 (m,
4H), 7.28 (t, J = 6.8 Hz, 1H), 6.47 (s, 1H), 6.16 (t, J = 8.9 Hz, 1H),
5.66 (d, J = 7.1 Hz, 1H), 5.45 (d, J = 4.1 Hz, 1H), 5.06 (t, J = 4.9 Hz,
1H), 4.99 (d, J = 9.4 Hz, 1H), 4.59 (q, J = 4.4 Hz, 1H), 4.32 (dd,
J = 10.6, 6.8 Hz, 1H), 4.19 (s, 2H), 3.82 (d, J = 7.0 Hz, 1H), 3.78–
3.47 (m, 13H), 3.31 (s, 4H), 2.61 (t, J = 14.0 Hz 4H), 2.51–2.42 (m,
1H), 2.34 (s, 3H), 2.25 (dd, J = 15.4, 9.4 Hz, 1H), 2.18 (s, 3H), 2.04
(dd, J = 15.4, 9.4 Hz, 1H), 1.93 (s, 3H), 1.85–1.76 (m, 1H), 1.66 (s,
3H), 1.19 (s, 4H), 1.17 (s, 3H); 13C NMR (CD3OD, 75 MHz): d
205.4 (C), 174.6 (C), 174.3(C), 174.2 (C), 172.1 (C), 171.5 (C),
167.8 (C), 142.3 (C), 140.2 (C), 135.0 (C), 134.8 (CH), 131.5(C),
131.3 (CH ꢁ 2), 129.9 (CH ꢁ 2), 129.8 (CH ꢁ 2), 129.0 (CH), 128.6
(CH ꢁ 2), 85.9 (CH), 83.2 (CH ꢁ 2), 82.4 (C), 79.1 (C), 77.5 (CH2)
76.9 (CH), 76.3 (CH), 74.9 (CH), 73.9 (CH), 72.5 (CH), 72.4 (CH),
69.6 (CH2ꢁ 2), 62.5 (CH2 ꢁ 4), 59.3 (C), 57.0 (CH), 47.9 (CH), 44.6
(C), 37.5 (CH2), 36.6 (CH2), 31.3 (CH2), 30.5 (CH2), 27.0 (CH3),
23.2 (CH3), 22.3 (CH3), 20.8 (CH3), 14.7 (CH3), 10.4 (CH3); HRMS
(ESI–TOF) m/z calcd for C53H69NO22Na [M+Na]+ 1094.4209, found
1094.4219.
1010, 755, 701 cmꢂ1 1H NMR (CDCl3, 400 MHz): d 7.50–7.44 (m,
;
4H), 7.39–7.32 (m, 6H), 6.79 (d, J = 8.4, 2H), 5.50 (s, 2H), 4.37–
4.27 (m, 5H), 4.00–3.93 (m, 4H), 3.80–3.75 (m, 2H), 3.71–3.65
(m, 2H), 3.35 (s, 2H), 2.60 (t, J = 6.8, 2H), 2.53 (s, 4H), 2.28 (t,
J = 6.8, 2H), 2.06 (t, J = 6.8, 2H); 13C NMR (CDCl3, 75 MHz): d
171.4 (C), 169.4 (C ꢁ 2), 168.2 (C), 138.1 (C ꢁ 2), 128.7 (C ꢁ 2),
128.0 (C ꢁ 4), 125.8 (C ꢁ 2), 100.9 (CH ꢁ 2), 70.6 (CH ꢁ 2), 68.9
(CH2 ꢁ 2), 68.3 (CH2 ꢁ 2), 65.8 (CH2 ꢁ 2), 48.6 (CH), 34.1 (CH2),
29.6 (CH2), 25.2 (CH2 ꢁ 2), 20.9 (CH2); HRMS (ESI-TOF) m/z calcd
for C32H38N2O11Na [M+Na]+ 649.2373, found 649.2350.
4.1.10. 5,50-(2-Azidopropane-1,3-diyl)bis(oxy)bis(2-phenyl-1,3-
dioxane) (16)
4.1.12. 2,5-Dioxopyrrolidin-1-yl 5-((1,3-bis((1,3-dihydroxy-
propan-2-yl)oxy)propan-2-yl)amino)-5-oxopentanoate (20)
To a solution of 69c (2.35 g, 5.64 mmol) in pyridine (12 mL) were
added p-toluenesulfonyl chloride (1.62 g, 8.46 mmol) and DMAP
(690 mg, 0.56 mmol), and the mixture was stirred for 20 h. The
resulting mixture was poured into CuSO4aq (200 mL) and ex-
tracted with ethyl acetate (200 mL ꢁ 1, 100 mL ꢁ 2). The combined
organic layers were washed with NaHCO3aq (200 mL), brine
(200 mL), dried over Na2SO4, and concentrated in vacuo. The resi-
due, crude 15 (3.49 g) was used for the next reaction without fur-
ther purification. To a solution of 15 (3.49 g) in anhydrous DMF
(20 mL) was added sodium azide (1.11 g, 16.9 mmol), and the mix-
ture was stirred for 2 h at 100 °C. The resulting mixture was poured
into NaHCO3aq (200 mL) and extracted with ethyl acetate
(200 mL ꢁ 1, 100 mL ꢁ 2). The combined organic layers were
washed with brine (200 mL), dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chromatog-
raphy eluted with hexane/ethyl acetate (1/1) to afford 16 as a col-
orless gummy solid (2.21 g, 5.01 mmol, 89% yield). FT–IR (neat):
A solution of 19 (47.6 mg, 76
with palladium hydroxide (3.0 mg, 21
at room temperature under hydrogen atmosphere. After inflow of
argon gas to remove hydrogen gas, the resulting suspension was
filtered. The filtrate was concentrated in vacuo, and the residue,
l
mol) in THF (2 mL) suspended
l
mol) was stirred for 2 h
crude 20 (34 mg, 76 lmol), was immediately used in the next reac-
tion without further purification.
4.1.13. (1S,2S,4S,9S,10S,15S,3R,7R,12R)-4,12-Diacetyloxy-1,9-
dihydroxy-10,14,17,17-tetramethyl-11-oxo-2-phenylcarbonyl-
oxy-6-oxatetracyclo[11.3.1.0<3,10>.0<4,7>]heptadec-13-en-15-
yl (3S,2R)-2-hydroxy-3-{4-[N-(2-[2-hydroxy-1- (hydroxymethyl)
ethoxy]-1-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}
ethyl) carbamoyl]butanoylamino}-3-phenylpropanoate (23)
To a solution of 1417 (14.2 mg, 19.0
added crude 20 (34 mg, 76 mol), and the mixture was stirred for
lmol) in DMF (0.2 mL) was
l
2099, 1154, 1092, 1010, 699 cm–1 1H NMR (CDCl3, 400 MHz): d
;
48 h at room temperature. The resulting mixture was poured into
NaHCO3aq (20 mL) and extracted with ethyl acetate (30 mL ꢁ 1,
10 mL ꢁ 2). The combined organic layers were washed with brine
(20 mL), dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography eluted with
chloroform/methanol (7/1) to afford 23 as a colorless gummy solid
7.52–7.47 (m, 4H), 7.39-7.31 (m, 6H), 5.52 (s, 2H), 4.38–4.31 (m,
4H), 4.04–3.98 (m, 4H), 3.85–3.75 (m, 5H), 5.15 (quint, J = 1.6 Hz,
2H), 13C NMR (CDCl3, 75 MHz): d 138.0 (C ꢁ 2), 128.7 (CH ꢁ 2),
128.0 (CH ꢁ 4), 125.9 (CH ꢁ 4), 100.9 (CH ꢁ 2), 71.1 (CH ꢁ 2),
68.5 (CH2 ꢁ 2), 68.3 (CH2 ꢁ 2), 67.5 (CH2 ꢁ 2), 60.0 (CH); HRMS
(ESI-TOF) m/z calcd for C23H27N3O6Na [M+Na]+ 464.1798, found
464.1797.
(12.9 mg, 12.9
1721, 1648, 1542, 1458, 1374, 1276, 1121, 1072, 977, 748,
708 cmꢂ1 1H NMR (CD3OD, 400 MHz): d 8.13 (d, J = 7.3 Hz, 2H),
lmol, 68% yield). FT-IR (neat): 3365, 2924, 2853,
;
7.68 (t, J = 7.4 Hz, 1H), 7.58 (t, J = 7.6 Hz, 2H), 7.48–7.39 (m, 4H),
7.30 (t, J = 7.0 Hz, 1H), 6.48 (s, 1H), 6.17 (t, J = 8.8 Hz, 1H), 5.68
(d, J = 7.1 Hz, 1H), 5.48 (d, J = 4.5 Hz, 1H), 5.01 (d, J = 9.4 Hz, 1H),
4.61 (d, J = 4.6 Hz, 1H), 4.35 (dd, J = 10.8, 6.65 Hz, 1H), 4.21 (s,
2H), 4.15 (dt, J = 10.3, 5.2 Hz, 1H), 3.85 (d, J = 7.1 Hz, 1H), 3.79–
3.71 (m, 2H), 3.69–3.52 (m, 10H), 3.46-3.38 (m, 2H), 3.34–3.31
(m, 1H), 2.53–2.42 (m, 1H), 2.37–2.21 (m, 8H), 2.20 (s, 3H), 2.08
(dd, J = 15.5, 9.0 Hz, 1H), 1.95 (s, 3H), 1.91–1.77 (m, 3H), 1.68 (s,
3H), 1.22-1.16 (m, 6H); 13C NMR (CD3OD, 75 MHz): d 205.1 (C),
175.4 (C), 175.2(C), 174.5 (C), 171.8 (C), 171.3 (C), 167.6 (C),
142.1 (C), 140.0 (C), 135.0 (C), 134.6 (CH), 131.3(C), 131.1
(CH ꢁ 2), 129.7 (CH ꢁ 4), 128.9 (CH), 128.5 (CH ꢁ 2), 85.8 (CH),
83.1 (CH), 83.0 (CH), 82.3 (C), 79.0 (C), 77.5 (CH2) 76.8 (CH), 76.2
4.1.11. 2,5-Dioxopyrrolidin-1-yl 5-(1,3-bis(2-phenyl-1,3-dioxan-
5-yloxy)propan-2-ylamino)-5-oxopentanoate (20)
To a solution of 16 (1.03 g, 2.33 mmol) in THF (20 mL) was
added lithium aluminum hydride (265 mg, 6.99 mmol) in some
portions at 0 °C, and the mixture was stirred for 30 min at 0 °C,
and quenched with ethyl acetate (0.6 mL) and water (0.2 mL) at
0 °C. The resulting suspension was filtered through celite, and the
filtrate was dried over Na2SO4, and concentrated in vacuo to afford
the crude amine 17 (1.14 g), which was used in the next reaction
without further purification. To a solution of 17 (1.14 g) in CH2Cl2
(15 mL) were added TEA (0.65 mL, 4.07 mmol) and glutaric anhy-
dride (319 mg, 2.79 mmol), and the mixture was stirred for 8 h at
room temperature. The resulting mixture was poured into CuSO4aq

5566
H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
(CH), 74.7 (CH), 72.4 (CH), 72.3 (CH), 69.8 (CH2 ꢁ 2), 62.6 (CH2),
62.5 (CH2), 62.4 (CH2 ꢁ 2), 59.3 (C), 56.8 (CH), 51.0 (CH), 47.9
(CH), 44.6 (C), 37.5 (CH2), 36.6 (CH2), 36.1 (CH2), 36.0 (CH2), 27.0
(CH3), 23.3 (CH2), 23.2 (CH3), 22.4 (CH3), 20.8 (CH3), 14.7 (CH3),
10.5 (CH3); HRMS (ESI-TOF) m/z calcd for C54H72N2O21Na
[M+Na]+ 1107.4525, found 1107.4520.
References and notes
1. Wani, M. C.; Taylor, H. L.; Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. Soc.
1971, 93, 2325.
2. Schiff, P. B.; Fant, J.; Horwitz, S. B. Nature 1979, 277, 665.
3. Vyas, D. M.; Wong, H.; Crosswell, A. R.; Casazza, A. M.; Knipe, J. O.; Mamber, S.
W.; Doyle, T. W. Bioorg. Med. Chem. Lett. 1993, 3, 1357.
4. http://packageinserts.bms.com/pi/pi_taxol.pdf (English). http://www.bms.co.
jp/medical/if/IF_TX0809.pdf (Japanese).
5. Typical examples: (a) Battaglia, A.; Bertucci, C.; Bombardelli, E.; Cimitan, S.;
Guerrini, A.; Morazzoni, P.; Riva, A. Eur. J. Med. Chem. 2003, 38, 383; (b)
Greenwald, R. B.; Gilbert, C. W.; Pendri, A.; Conover, C. D.; Xia, J.; Martinez, A. J.
Med. Chem. 1996, 39, 424; (c) Mandai, T.; Okumoto, H.; Oshitari, T.; Nakanishi,
K.; Mikuni, K.; Hara, K.; Hara, K.; Iwatani, W.; Amano, T.; Nakamura, K.;
Tsuchiya, Y. Heterocycles 2001, 54, 561.
6. http://dailymed.nlm.nih.gov/dailymed/getFile.cfm?id=62063&type=pdf&
name=24d10449-2936-4cd3-b7db-a7683db721e4 (English) http://di.taiho.co.
jp/taiho/hp/fileDown loadMedia Content.do?_contentGroupId=2349&_psp
Code=75&_pspSubCode=1 (Japanese).
4.1.14. 2,5-Dioxopyrrolidin-1-yl 5-(5,11-bis((1,3-dihydroxy-
propan-2-yloxy)methyl)-1,15-dihydroxy-2,14-bis (hydroxy-
methyl)-3,6,10,13-tetraoxapentadecan-8-ylamino)-5-
oxopentanoate (21)
A solution of 209c (28.0 mg, 0.025 mmol) in ethanol (2.5 mL)
suspended with palladium hydroxide (10 mg, 0.071 mmol) was
stirred for 2 h at room temperature under hydrogen atmosphere.
The resulting suspension was filtered to remove palladium cata-
lyst. The filtrate was concentrated in vacuo to afford 21 (17.0 mg,
0.022 mmol), which was used in the next reaction without further
purification.
7. Kato, K. Drug Deliv. Sys. 2009, 24, 28.
8. Zang, J. A.; Anyarambhatla, G.; Ma, L.; Ugwu, S.; Xuan, T.; Sardone, T.; Ahmad, I.
Eur. J. Pharm. Biopharm. 2005, 59, 177.
9. (a) Preparation of BGL family: (a) Nemoto, H.; Wilson, J. G.; Nakamura, H.;
Yamamoto, Y. J. Org. Chem. 1992, 57, 435, and J. Org. Chem., 1995, 60, 1478.
[Erratum].; (b) Nemoto, H.; Araki, T.; Kamiya, M.; Kawamura, T.; Hino, T. A. Eur.
J. Org. Chem. 2007, 3003; (c) Nemoto, H.; Kamiya, M.; Minami, Y.; Araki, T.;
Kawamura, T. Synlett 2007, 2091; (d) Nemoto, H.; Atsushi, I.; Araki, T.; Katagiri,
A.; Kamiya, M.; Matsushita, T.; Hattori, H.; Mimura, Y.; Tomoda, Y.; Yamasaki,
M. Bioorg. Med. Chem. Lett. 2011, 4724.
10. Preparation of 2 on laboratory scale: (a) Nemoto, H.; Kamiya, M.; Nakamoto, A.;
Katagiri, A.; Yoshitomi, K.; Kawamura, T.; Hattori, H. Chem. Lett. 2010, 39, 856;
(b) Preparation of 2 on industrial scale: Hattori, H.; Matsushita, T.; Yoshitomi,
K.; Katagiri, A.; Nemoto, H. Synthesis, 2012, 44, ASAP (http://dx.doi.org/
10.1055/s-0031-1289809).
11. (a) Nemoto, H.; Iwamoto, S.; Nakamura, H.; Yamamoto, Y. Chem. Lett. 1993,
465; (b) Nemoto, H.; Cai, J.; Iwamoto, S.; Yamamoto, Y. J. Med. Chem. 1995, 38,
1673; (c) Yamamoto, Y.; Cai, J.; Nakamura, H.; Sadayori, N.; Asao, N.; Nemoto,
H. J. Org. Chem. 1995, 60, 3352; (d) Takagaki, M.; Ono, K.; Oda, Y.; Kikuchi, H.;
Nemoto, H.; Iwamoto, S.; Cai, J.; Yamamoto, Y. Cancer Res. 1996, 56, 2017; (e)
Nemoto, H.; Kikuishi, J.; Yanagida, S.; Kawano, T.; Yamada, M.; Harashima, H.;
Kiwada, H.; Shibuya, M. Bioorg. Med. Chem. Lett. 1999, 9, 205; (f) Yamaguchi, H.;
Suzawa, T.; Araki, T.; Kamiya, M.; Nemoto, H.; Yamasaki, M. Biochim. Biophys
Acta. 2008, 1780, 680; (g) Ishihara, A.; Yamauchi, M.; Kusano, H.; Mimura, M.;
Nakakura, M.; Kamiya, M.; Katagiri, A.; Kawano, M.; Nemoto, H.; Suzawa, T.;
Yamasaki, M. Int. J. Pharm. 2010, 391, 237; (h) Ishihara, A.; Yamauchi, M.;
Tsuchiya, T.; Mimura, M.; Tomoda, Y.; Katagiri, A.; Kamiya, M.; Nemoto, H.;
Suzawa, T.; Yamasaki, M. J. Biom. Sci., Polymer Ed. in press.
4.1.15. (1S,2S,4S,9S,10S,15S,3R,7R,12R)-4,12-Diacetyloxy-1,9-
dihydroxy-10,14,17,17-tetramethyl-11-oxo-2-phenylcarbonyl-
oxy-6-oxatetracyclo[11.3.1.0<3,10>. 0<4,7>]heptadec-13-en-15-
yl (3S,2R)-2-hydroxy-3-[4-(N-{2-(2-[2-hydroxy-1-
(hydroxymethyl)ethoxy]-1-{[2-hydroxy-1-(hydroxymethyl)
ethoxy]methyl}ethoxy)-1-[(2-[2-hydroxy-1-(hydroxymethyl)
ethoxy]-1-{[2-hydroxy-1-(hydroxymethyl) ethoxy]methyl}
ethoxy)methyl]ethyl}carbamoyl) butanoylamino]-3-
phenylpropanoate (24)
To a solution of 1417 (0.8 mg, 1.1
lmol) in DMF (0.5 mL) was
added crude 22 (17.0 mg, 0.022 mmol), and the mixture was stirred
for 40 h. The resulting mixture was concentrated in vacuo, and the
residue was purified by HPLC [ODS-80Ts column, 2.0 mm
id ꢁ 100 mm length, flow rate = 1.0 mL/min, linear gradient
(water/acetonitrile = 95/5–5/95 with 0.1% TFA for 1 h), retention
time of 24 = 30.5–38.5 min] to afford 24 as a colorless gummy solid
(1.2 mg, 0.87
lmol, 80% yield). FT–IR (neat): 3357, 2940, 1719,
12. Nemoto, H.; Kamiya, M.; Nakamoto, A.; Matsushita, T.; Matsumura, K.; Hattori,
H.; Kawamura, T.; Taoka, C.; Abe, S.; Ishizawa, K.; Miyamoto, L.; Tsuchiya, K.
Bioorg. Med. Chem. Lett. 2012. under revision.
1677, 1544, 1452, 1372, 1245, 1203, 1070, 906, 837, 801, 719 cmꢂ1
;
1H NMR (CD3OD, 400 MHz): d 8.13 (d, J = 7.3 Hz, 2H), 7.69 (t,
J = 7.4 Hz, 1H), 7.59 (t, J = 7.6 Hz, 2H), 7.48–7.39 (m, 4H), 7.30 (t,
J = 6.8 Hz, 1H), 6.48 (s, 1H), 6.16 (t, J = 9.0 Hz, 1H), 5.67 (d,
J = 7.2 Hz, 1H), 5.47 (dd, J = 8.6, 4.9 Hz, 1H), 5.00 (d, J = 9.4 Hz, 1H),
4.60 (d, J = 4.8 Hz, 1H), 4.34 (dd, J = 11.0, 6.7 Hz, 1H), 4.23–4.16
(m, 3H), 3.86-3.56 (m, 31H), 3.51–3.42 (m, 4H), 2.53-2.43 (m, 1H),
2.39–2.23 (m, 8H), 2.20 (s, 3H), 2.10–2.00 (m, 1H), 1.98–1.77 (m,
6H), 1.68 (s, 3H), 1.20 (s, 3H), 1.18 (s, 3H); 13C NMR (CD3OD,
75 MHz): d 205.1 (C), 175.8 (C), 175.2(C), 174.5 (C), 171.8 (C),
171.3 (C), 167.6 (C), 142.1 (C), 140.0 (C), 134.9 (C), 134.6 (CH),
131.3(C), 131.2 (CH ꢁ 2), 129.7 (CH ꢁ 4), 129.0 (CH), 128.5
(CH ꢁ 2), 85.8 (CH), 82.9 (CH ꢁ 4), 82.3 (C), 80.1 (CH ꢁ 2), 79.0 (C),
77.5 (CH2) 76.8 (CH), 76.2 (CH), 74.8 (CH), 72.4 (CH), 72.3 (CH),
70.9 (CH2 ꢁ 2), 70.7 (CH2 ꢁ 2), 70.6 (CH2 ꢁ 2), 62.4 (CH2 ꢁ 8), 59.3
(C), 57.0 (CH), 51.1 (CH), 47.9 (CH), 44.6 (C), 37.5 (CH2), 36.6
(CH2), 36.1 (CH2 ꢁ 2), 27.0 (CH3), 23.3 (CH2), 23.2 (CH3), 22.3
(CH3), 20.8 (CH3), 14.8 (CH3), 10.4 (CH3); HRMS (ESI–TOF) m/z calcd
for C66H96N2O29Na [M + Na]+ 1403.5996, found 1403.5996.
13. Nemoto, H.; Katagiri, A.; Kamiya, M.; Matsushita, T.; Hattori, H.; Matsumura,
K.; Itou, T.; Kawamura, T.; Kita, T.; Nishida, H.; Arakaki, N. Bioorg. Med. Chem.
Lett. 2012, 22, 5051. http://dx.doi.org/10.1016/j.bmcl.2012.06.019.
14. Forrest, M. L.; Yáñez, J. A.; Remsberg, C. M.; Ohgami, Y.; Kwon, G. S.; Davies, N.
M. Pharm. Res. 2008, 25, 194.
15. Sambandam, T. G.; Johnson, J. H.; Hand, B. J.; Howe, C. D.; Franke, R. R.; Bucher,
B. A.; Gallagher, R. T.; Plante, M. A.; Juchum, J. S.; Desimone, E. M. III; Yang, D. S.
PCT Int. Appl. 2003, US03/10556.
16. Panchagnula, R. Int. J. Pharm. 1998, 133, 191.
17. PCT Int. Appl. 1996, WO 96/23780.
18. It is well known that hydrogenation of the bridged tetra-substituted C@C bond
did not proceed at all under hydrogen atmosphere (1 atom), and skeleton of 1
was decomposed under strong acidic conditions.5 Therefore, Bnd group was
used for BGL protecting group. Additionally, because of the facility, the method
for the synthesis of BGL possessing Bnd9c is more recommendable than that of
BGL possessing Bn,9a as the inventor of BGL.10a,b
19. In our previous paper,11f hydrogenolysis of eight benzyl groups of BGL006
possessing N-hydroxysuccinimidyl ester was reported. In such a case, the N–O
bond was preserved to smoothly carry out the BGLation of the lysine side
chains of an artificial protein drug. The protection-free N-succinimidyl ester
decomposed after a couple of days at room temperature.
20. When succinic anhydride was used for the primary amine, more than 20% of
the cyclic imide illustrated below was obtained as an undesired byproduct.
Glutaric anhydride gave no such cyclic imide.9c
Acknowledgments
We thank the Japan Science and Technology Agency (JST) for
partial support for this project (Research for Adaptable and Seam-
less Technology Transfer Program through Target-driven R&D, JST
in 2009–2011) (Nemoto, Hattori, Miyamoto and Tsuchiya), and
the discretionary budget of the President of The University of
Tokushima (Abe, Tsuchiya and Nemoto: 2011–2012).
Undesired byproduct
21. Either 5 or 23 was not pro-drug of paclitaxel any more, because even the
selective cleavage of the amide bond at C3’ generate no paclitaxel.
Furthermore, ester bonds are generally cleaved faster than amide bonds in
biological circumstance. Therefore, we believe that 5 itself shows the anti-

H. Nemoto et al. / Bioorg. Med. Chem. 20 (2012) 5559–5567
5567
tumor activity but not some of the metabolites from 5 does not. Only one
possible metabolite is the compound generated by the hydrolysis of ester bond
between BGL and linker of 5. Therefore, the amide bond was introduced
instead of the ester bond in the case of 23.
22. If a paclitaxel–BGL conjugate is commercially available, to reduce the total
amount of water in the drug package will be desirable for patients because
price of drug package will be reduced and the required time for injection will
be shorten. From this point of view, 24 is more attractive than 5 or 23.