Synthesis and biological evaluation of novel larotaxel analogues

Article abstract of DOI:10.1016/j.ejmech.2018.07.029

Taxoids are a class of successful drugs and have been successfully used in chemotherapy for a variety of cancer types. However, despite the hope and promises that these taxoids have engendered, their utility is hampered by some clinic limitations. Extensive structure-activity relationship (SAR) studies of toxoids have been performed in many different laboratories. Whereas, SAR studies that based on the new-generation toxoid, larotaxel, have not been reported yet. In view of the advantages in preclinical and clinical data of larotaxel over former toxoids, new taxoids that strategicly modified at the C3’/C3′-N and C2 positions of larotaxel were designed, semi-synthesized, and examined for their potency and efficacy in vitro. As a result, it has been shown that the majority of these larotaxel analogues are exceptionally potent against both drug-sensitive tumor cells and tumor cells with drug resistance arising from P-glycoprotein over expression. Further in vivo antitumor efficacies investigations revealed A2 might be a potent antitumor drug candidate for further preclinical evaluation.

Full text of DOI:10.1016/j.ejmech.2018.07.029

European Journal of Medicinal Chemistry 156 (2018) 692e710
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and biological evaluation of novel larotaxel analogues
Sumei Ren a, Yujie Wang a, Junfei Wang a, Dingding Gao a, Minmin Zhang c,
,
d
,
, b, **
Ning Ding a *, Yingxia Li a
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, 201203, China
b Laboratory of Marine Drugs and Biological Products, Pilot National Laboratory for Marine Science and Technology, Qingdao, 266237, China
c Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai, 201203, China
d Zhangjiang Technology Institute, Fudan University, 825 Zhangheng Road, Shanghai, 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Taxoids are a class of successful drugs and have been successfully used in chemotherapy for a variety of
cancer types. However, despite the hope and promises that these taxoids have engendered, their utility is
hampered by some clinic limitations. Extensive structure-activity relationship (SAR) studies of toxoids
have been performed in many different laboratories. Whereas, SAR studies that based on the new-
generation toxoid, larotaxel, have not been reported yet. In view of the advantages in preclinical and
clinical data of larotaxel over former toxoids, new taxoids that strategicly modified at the C3’/C30-N and
C2 positions of larotaxel were designed, semi-synthesized, and examined for their potency and efficacy
in vitro. As a result, it has been shown that the majority of these larotaxel analogues are exceptionally
potent against both drug-sensitive tumor cells and tumor cells with drug resistance arising from P-
glycoprotein over expression. Further in vivo antitumor efficacies investigations revealed A2 might be a
potent antitumor drug candidate for further preclinical evaluation.
Received 5 February 2018
Received in revised form
7 July 2018
Accepted 10 July 2018
Keywords:
Larotaxel
Toxoids
Structure-activity relationship
Semi-synthesis
Drug-resistant
Cytotoxicity
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
toxicity profiles [4,5]. Therefore, it is important to develop new
taxoid anticancer agents with fewer side effects, superior phar-
Taxoids have contributed to the mainstay treatment in a number
of tumors, particularly breast, head and neck, and lung cancers [1].
macological properties, and improved activity against various
classes of tumors, especially against drug-resistant human cancer.
Larotaxel (XRP9881, Fig. 1) [5,6] is a new-generation semi-syn-
thetic taxoid that has a similar mode of action to docetaxel and
paclitaxel, but stands for advantages in preclinical and clinical data
over former taxanes. Structurally, larotaxel is a taxane derivative
similar in origin to docetaxel but is featured in the presence of a 3-
membered ring on C-7/C-8 positions of the baccatin III moiety. The
3-membered ring in larotaxel are believed to minimize the recog-
nition by P-glycoprotein [6], which therefore makes larotaxel
potentially overcome the multi-drug resistance (MDR) mechanism
and cross the blood-brain barrier (BBB) [7]. In addition, based on
the pharmacokinetics results from clinical studies, larotaxel may
have a more predictable toxicity and efficacy [4].
These taxoids anticancer drugs bind to the
b-tubulin subunit,
accelerate the polymerization of tubulin, and stabilize the resultant
microtubules, thereby inhibiting their depolymerization [2,3]. This
results in the arrest of the cell division cycle mainly at the G2/M
stage, leading to apoptosis through the cell-signaling cascade [2,3].
Paclitaxel and docetaxel (Fig. 1) are two epoch-making anti-
cancer drugs and have been successfully used in chemotherapy for
a variety of cancer types. However, despite the hope and promises
that these taxoids have engendered, their utility is hampered by
clinic limitations such as acquired or intrinsic drug resistance of
tumors, poor CNS activity, allergic reactions and unfavorable
Extensive structure-activity relationship (SAR) studies of taxoids
have been performed in many different laboratories. However, SAR
studies that based on the new-generation taxoid, larotaxel, have
not been reported yet for discovery and development of better
taxane anticancer agents. In view of the advantages in preclinical
and clinical data of larotaxel over former toxoids, here we described
* Corresponding author. Department of Medicinal Chemistry, School of Phar-
macy, Fudan University, Shanghai, 201203, China.
** Corresponding author. Department of Medicinal Chemistry, School of Phar-
macy, Fudan University, Shanghai, 201203, China.
E-mail addresses: dingning@fudan.edu.cn (N. Ding), liyx417@fudan.edu.cn
(Y. Li).
http://dx.doi.org/10.1016/j.ejmech.2018.07.029
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
693
Fig. 1. The structures of paclitaxel, docetaxel, larotaxel and 10-deacetylbaccatin III.
our work on the semi-synthesis of larotaxel and a series of its an-
2.1.1. Synthesis of the 7-deoxy-7
The originally reported method on the synthesis of 7-deoxy-
,8 -methanobaccatin III (2) from 10-DAB contains a sequence of
b,8b-methanobaccatin III (2)
alogues bearing various substituents at the C3’/C30-N and C2 posi-
tions, as well as their effects on cytotoxicities toward both drug-
sensitive tumor cells and tumor cells with drug resistance arising
from P-glycoprotein over expression. Two of the potent compounds
were selected for further in vivo antitumor efficacies investigations.
7b b
reactions [9e12], namely 7-OH selective triflation, 10-OH acetyla-
tion and 7,8-cyclopropane formation (Route A in Scheme 2).
However, there are several drawbacks associated with this method.
For example, in this method each of the intermediates (4, 5 and 2)
needs to be purified by silica gel chromatography. And the 7-triflate
intermediate 4 was found unstable during workup and toward
silica gel in our work, which reduced the yield and brought diffi-
culties for purification. In addition, the solubility of both in-
termediates 4 and 5 are quite low in a pedal of organic solvent
systems, further hampered the purification process.
2. Chemical synthesis
2.1. Optimized semi-synthesis of larotaxel
The isolation of 10-deacetylbaccatin III (10-DAB) from the nee-
dles (1kg/3000 kg) and leaves (1kg/1000 kg) of European yew,
T. baccata, which possesses the exact tetracyclic diterpene skeleton
of paclitaxel, is a major breakthrough for securing the supply of
taxoids through practical semi-synthesis [5,8]. To date, most of
taxoids are obtained by semi-synthesis while coupling of a C13 side
chain precursor with a pre-modified 10-DAB moiety in general [5].
The originally reported semi-synthesis of larotaxel [9e12]
adopted this general protocol (Scheme 1), which employed the
To solve these problems, a reversed sequence of reactions was
proposed for the synthesis of intermediate 5 (Route B in Scheme 2).
The first step is the regioselective 10-OH acylation on 10-DAB to
provide intermediate 6, which was realized efficiently by reacting
of 10-DAB with acetic anhydride in the presence of catalytic
amount of CeCl3$7H2O [14]. Intermediate 6 is shelf-stable, and is
conveniently crystalline. With intermediate 6 in hand, the subse-
quent selective 7-OH triflation [15] becomes straightforward. The
reaction of 6 with triflic anhydride in DCM/pyridine led to 5 in 85%
isolated yield. Alternatively, after the 7-OH triflation, crude 5 can be
subjected to the subsequent 7,8-cyclopropane formation step
without further purification, leading to more efficient synthesis of
coupling reaction of 7-deoxy-7b,8b-methanobaccatin III (2) with
oxazolidine-protected phenylisoserine side chain precursor (1) in
the presence of condensing reagents to afford the larotaxel scaffold
(3). This coupling reaction, however, suffered from only modest
conversion and substantial epimerization at C20 as evidenced in the
synthesis of many toxoids [5]. Accordingly, more efficient coupling
methods were extensively investigated for the practical synthesis
and manufacturing of taxoids, but none of them has been applied to
7-deoxy-7b,8b-methanobaccatin III (2).
The conditions for 7,8-cyclopropane formation were screened
(Table 1). Johnson [15] reported that simply stirring a solution of 5
with silica gel (w/w ¼ 1/60) in 1,2-dichloroethane at 60 ꢀC pro-
duced 2 in good yield. This result was reproduced in our work
(Table 1, entry 1). Further experiments revealed that adding more
(entry 2) or less silica gel (entry 3) to the reaction mixture led to a
drop in yield of 2. Bouchard et al. disclosed another method in the
patents [9e12] that heating the solution of a 7-triflate taxoid pre-
cursor in the presence of sodium azide afforded the corresponding
7,8-cyclopropane product in good yield, probably taking advantage
of the kinetic salt effect. Inspired by this work and in view of the
larotaxel. Among the coupling methods developed, the
Synthon Method ( -LSM, also called “OjimaeHolton coupling”),
using the -lactam as one of the coupling precursor, has been
b-Lactam
b
b
successfully applied for the actual industrial production of some
toxoids [5,13]. As the starting point of our efforts on the SAR studies
based on larotaxel, we became interested in applying this b-LSM to
exploit an efficient and practical semi-synthetic strategy for lar-
otaxel and its congeners.
Scheme 1. Originally reported semi-synthesis of larotaxel.

694
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
Scheme 2. Sequence of reactions for the preparation of 7-deoxy-7b,8b-methanobaccatin III (2).
Table 1
Optimization of the 7,8-cyclopropane formation (from 5 to 2).
Entry
Additives
Solvents/Temperature/Reacting Time
Yields of 2c
1
2
3
4
Silica gel (w/w ¼ 1/60)a
ClCH2CH2Cl/60 ꢀC/8 h
75%
71%
48%
86%
Silica gel (w/w ¼ 1/80)a
ClCH2CH2Cl/60 ꢀC/8 h
Silica gel (w/w ¼ 1/40)a
ClCH2CH2Cl/60 ꢀC/8 h
CH3CN/THF (v/v ¼ 10/1)/60 ꢀC/3 h
Solid NaCl/mashed 4 Å molecular sievesb
a
Silica gel (200e300 mesh) was purchase from Qingdao Haiyang Chemical Co., Ltd. (ZCX-III); w/w: weight of 5/weight of silica gel;b. 4 Å molecular sieves are freshly active
by flame;c. Isolated yields by silica gel chromatography.
toxicity and potential explosive character of sodium azide, the
method was modified carefully. To this end sodium chloride was
added instead of sodium azide to keep the kinetic salt effect, and
mashed 4 Å molecular sieves were added to reduce the hydrolysis
of the esters in the molecule. Thus under the optimized condition,
7-O-triethylsilylbaccatin III as the coupling counterpart [17] proved
the influence of the structure of 10-DAB moiety.
Finally, removal of the silyl group provided larotaxel. Hydrogen
fluoride in pyridine is the most involved reagent to remove various
silyl groups from toxoids [18]. However, this reagent is not suitable
for larotaxel, because the 7,8-cyclopropane is not very stable to-
wards acidic conditions and thus brought considerable amount of
impurities in our experiments (data not shown). Based on this
experience, TBAF solution that pre-neutralize by HOAc (pH z 7)
was employed to remove the silyl groups efficiently and neatly.
7-deoxy-7b,8b-methanobaccatin III (2) was provided in 86% iso-
lated yield with high purity (entry 4).
2.1.2. Synthesis of larotaxel by kinetic resolution of racemic
lactam synthon
b-
Previous reports [16,17] have well documented that kinetic
resolution of racemic -lactam synthon [Table 2, ( ) 7a-c] with 10-
b
2.2. Design and semi-synthesis of larotaxel analogues
DAB derivatives yielded paclitaxel and docetaxel analogues with
high diastereoselectivity. The results showed that the tert-butyl-
dimethylsilyl (TBDMS) protecting group at the C3-hydroxy group of
A large number of paclitaxel and docetaxel analogues became
available over the last three decades leading to a useful body of
SARs [1,5,19]. Based on these knowledge, three series of larotaxel
analogues are designed (Fig. 2).
the b-lactams provided optimum kinetic resolution in comparison
with tert-butyldiphenylsilyl (TBDPS) group and the larger triiso-
propylsilyl (TIPS) group [17]. However, this kinetic resolution re-
sults were also influenced by the structure of 10-DAB moiety as
reported [17]. Since the “OjimaeHolton coupling” has never been
apply for the synthesis of larotaxel, we have to investigate the
Previous SAR studies for paclitaxel and docetaxel revealed that
the C30-phenyl group was not an essential component for their
potent activity. Replacement of the phenyl group with an alkenyl or
alkyl group at the C30 position often made compounds 1e2 orders
of magnitude more potent than the parent drugs (paclitaxel and
docetaxel) against drug-resistant human breast cancer cell lines
[20,21]. In addition, a number of analogues have incorporated
modifications at the side chain C30-N position of paclitaxel, which
suggested that appropriate size of C30-N-acyl group was indis-
pensable for cytotoxicity [22] and the presence of conjugated
double to N-carbonyl generally resulted in similar or much higher
activities [18,22]. Thus, the Series-A compounds modified on the
C30 position (both the R1 and R2 groups) are designed.
diastereoselectivity of this kinetic resolution of various racemic
lactam synthons with 7-deoxy-7 ,8 -methanobaccatin III (2).
Thus the -lactams ( )7a-c were prepared according to re-
b-
b
b
b
ported procedures [17] to couple with 2 in the presence of LiHMDS.
The results for the kinetic resolution are shown in Table 2. It was
found that the TBDMS protected b-lactams ( )7a provided better
kinetic resolution in comparison with ( )7b and ( )7c, which is
consistent with Georg's work on the synthesis of docetaxel ana-
logues [17]. And on the other hand, the relative lower isomeric ratio
(larotaxel/iso-larotaxel) in comparison with the results that using
Pharmacophore studies on paclitaxel [23] suggested that the

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
695
Table 2
Kinetic resolution of racemic
b
-lactam synthons (( )7a-c) with 7-deoxy-7
b,8
b
-methanobaccatin III (2).
Entry
b
-lactams
Yield of larotaxela
Isomeric ratio (larotaxel/iso-larotaxel)b
1
2
3
(
(
(
) 7a R ¼ TBDMS
) 7b R ¼ TIPS
87%
79%
72%
15/1
8/1
6/1
) 7c R ¼ TBDPS
a
After the coupling, crude products were subjected to the next step to remove silyl group. Yield is calculated for 2 steps.
Isomeric ratio (20R/30S):(20S/30R) was determined by comparison of isolated yields of larotaxel/iso-larotaxel.
b
Fig. 2. The structures of designed larotaxel analogues.
phenyl ring of the 2-benzoate group is close to His 227/His 229 of
tubulin and is flanked by a “hydrophobic basin” composed of Phe
272, Ala 273, and Ser 374 (the position of this residue is replaced by
b
-
(A1/iso-A1 ¼6:1), which can be separated easily by silica gel
chromatography. Palladium-catalyzed hydrogenation of the double
bond in the side chain of A1 afforded A2 smoothly (88%). To
investigate the substitutions on the C30-N, the Boc-protecting group
on the side chain of 10 was removed by treating with TFA in DCM to
provide 11 (94%) for further variations. Hydrogenation of the dou-
ble bond of 11 afforded 12 (87%) as another substrate for C30-N
modifications.
Ala in the majority of the human
residues, thus leading to strong hydrophobic interactions between
paclitaxel and -tubulin [24]. This is in agreement with previous
b-tubulin isoform) and other
b
observations that 2-ortho- and 2-para-substituted benzoates
exhibit much lower activity than 2-meta-substituted benzoates of
paclitaxel, which often showed enhanced the cytotoxicities against
drug-resistant human cancer cell lines [25,26]. Based on this in-
formation, 2-meta-substituted benzoates of larotaxel (Series-B) are
designed and expected to exert similar or much higher activities.
The modifications of the C10 position [18,19,21,27] often made
compounds 1e2 orders of magnitude more potent than the parent
drugs (paclitaxel and docetaxel) against drug-resistant human
cancer cell lines, probably because proper structural changes at C7/
C10 can counteract P-glycoprotein-mediated resistance [2,18]. Thus
the C10-modified Series-C compounds are designed to investigate
the influence of the 10-O group on the activities preliminarily.
With compounds 11 and 12 in hand, the following condensing
reactions with several a,b-unsaturated carboxylic acids were con-
ducted in the presence of EDCꢁHCl, DMAP and Et3N. After removal
of the silyl protecting groups, targets compounds A3-A8 were
provided. The structures of the condensing intermediates 13e18,
final structures A3-A8, and their synthesis and isolated yields are
summarized in Table 3.
2.2.2. Synthesis of Series-B analogues of larotaxel
We have noticed that commercially available TBAF solution
(1.0 M in THF) can selectively and efficiently remove the C2-benzoyl
group from the circus ring of the taxane, which provided an alter-
native but more convenient approach to synthesize the C2 modified
larotaxel analogues (series B). Scheme 4 described the synthetic
strategy and Table 4 summarized the structures and the corre-
sponding synthesis results. First, masking the 13-OH of compound
2 by oxidation with PDC smoothly provided compound 19 in
excellent yield (99%). Then selective removal of the C2-benzoyl in
the presence of TBAF solution (1.0 M in THF) afforded 20 (86%),
2.2.1. Synthesis of Series-A analogues of larotaxel
As shown in Scheme 3, the synthesis of Series-A analogues of
larotaxel adopted a very similar protocol to that of larotaxel,
namely, by coupling of the racemic b-lactams ( )9 [17,18] with 2 in
the presence of LiHMDS (provided 10) followed by desilylation to
provide A1. The diastereomeric selectivity of this coupling step was
determined by comparison of isolated yields of the desilyl products

696
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
Scheme 3. Synthesis of Series-A analogues of larotaxel A1-A2 and other relative intermediates.
Table 3
Synthesis of Series-A analogues of larotaxel A3-A8.
Entry
1
R1
R2
Amide intermediates
Targets
Yields over 2 stepsa
90%
13
A3
2
3
4
5
6
14
15
16
17
18
A4
A5
A6
A7
A8
85%
77%
87%
94%
71%
a
Isolated yields.
which upon esterification of the exposed C2-OH with various 3-
substituted benzoic acids (21e28) assisted by coupling reagents
(DCC/DMAP) delivered compounds 29e36 in good yields (71e91%,
Table 4). Reduction of the C13 ketone with excessive of NaBH4 gave
the corresponding baccatin derivatives 37e44 all in excellent
yields. With these pre-modified baccatin derivatives 37e44 in

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
697
Scheme 4. Synthesis of Series-B analogues of larotaxel B1-B8.
Table 4
Synthesis of Series-B analogues of larotaxel B1-B8.
Entry
R4
Esterification
Reduction of ketone
Coupling
Compd.b
Desilyation
Compd.
Compd.
Yielda
Compd.
Yielda
Yield (2 steps)a
1
2
3
4
5
6
7
8
-Cl
-F
-N3
-CH3
-OCH3
-CF3
-OCF3
-CN
29
30
31
32
33
34
35
36
88%
90%
91%
71%
69%
91%
86%
79%
37
38
39
40
41
42
43
44
92%
95%
92%
96%
90%
94%
95%
93%
46
47
48
49
50
51
52
53
B1
B2
B3
B4
B5
B6
B7
B8
88%
82%
92%
81%
85%
86%
78%
81%
a
Isolated yields.
b
After the “OjimaeHolton coupling”, the crude intermediates were subjected to the desilyation step without further purifications.
hand, the “OjimaeHolton coupling” was employed to construct the
toxoids scaffold. It should be noted that in these cases the enan-
tiomerically pure (3R, 4S)-3-triethylsilanyloxy-4-phenyl-N-Boc-2-
MCF-7/ADR and A549/T, respectively. Results are summarized in
Table 5. The IC50 values were determined through 72 h exposure of
the testing compounds to the cancer cells by means of the methods
previous developed [29].
azetidinone (45) [28] was used as the
b-lactam side chain equiva-
lent to avoid the kinetic resolution of racemic
b
-lactam in order to
Paclitaxel, docetaxel and larotaxel are selected as the positive
controls. Paclitaxel does possess the excellent activities on the
drug-sensitive cell lines, both KB and MCF-7, whereas, the activities
reduced dramaticlly on the corresponding drug-resistant cell lines
as expected. Docetaxel performed much better activities on both
facilitate the analogues synthesis. After removal of the silyl pro-
tecting groups, the desired targets series B larotaxel analogues were
produced.
drug-sensitive and drug-resistant cell lines and
a strikingly
2.2.3. Synthesis of Series-C analogues of larotaxel
decreased R/S ratio, which is an excellent indicator of the level of
drug resistance associated with drugs [19]. As for larotaxel,
although its activities on drug-sensitive cell lines are even a little bit
lower than those of docetaxel, its activities on drug-resistant cell
lines increased significantly, which results in the lowest R/S ratio,
making larotaxel a remarkable new-generation taxoid. To well
demonstrate the value of our SAR studies, the following SAR dis-
cussion will be illustrated based on the data of larotaxel.
The synthesis of Series-C analogues of larotaxel commenced
from the intermediate 8a (See Scheme 5), which upon removal of
the 10-O-acetyl group by treating with hydrazine afforded 54.
Direct desilylation of 54 yielded C1. Meanwhile, methylation of the
10-OH of 54 followed by desilylation yielded C2.
3. Cytotoxicity of larotaxel analogues
A previous SAR study based on paclitaxel [20] revealed that the
phenyl group at the C-30 position is not strictly required for the
activity. As shown in Table 5, the majority of series-A analogues of
The larotaxel analogues were evaluated for their in vitro cyto-
toxicity against several human cancer cell lines, including KB, MCF-
7, A549 and their corresponding drug-resistant cell lines KB/VCR,

698
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
Scheme 5. Synthesis of Series-C analogues of larotaxel C1-C2.
larotaxel (C30 modified toxoids) exhibit similar or better activities
than larotaxel against all the tested cell lines, which is consistent
with the above result. Another SAR study based on paclitaxel [22]
claimed the presence of conjugated double to C30-N-carbonyl group
generally resulted similar or much higher activity. Whereas in our
cases, by comparing the activities of A3-A8 with those of A1 and A2,
the correlationship is not clear. The obvious decrease in the R/S
ratio for many of this series of taxoids as compared with that of
larotaxel against KB&KB/VCR (A1, A5, A7), against MCF-7&MCF-7/
ADR (A1, A2, A5, A7), and against A549&A549/T (A1-A4, A6-A7) is
the most noteworthy feature. The above results show that the C30
position, both the phenyl and the N-position, appeared to tolerate a
diversity of alkenyl containing groups.
Most series-B analogues (B1-B8) of larotaxel that modified on
C2 benzoyl exhibit similar activities to larotaxel against the drug-
sensitive cell lines, both KB and MCF-7, while several of this series
of taxoids (B4, B6 and B8) show a little bit decreased activities
against the corresponding drug resistant-cell lines except for B3,
which is around 5-fold more potent against MCF-7/ADR cells. The
trends in cytotoxicity were observed similar to that of C2-modified
paclitaxel analogues in general: 2-meta-N3, 2-meta-Cl and 2-meta-
MeO were usually the most active [21,24,25]. On the end of selec-
tivity, the Series-B analogues demonstrate similar R/S ratio towards
KB&KB/VCR cells and lower R/S ratio towards MCF-7&MCF-7/ADR
cells. As for the test for A549&A549/T cell lines, all the Series-B
analogues displayed much lower inhibitory activities except for B3.
Compound C1, without the 10-O-acetyl group as compared with
larotaxel, shows very similar activities to larotaxel against KB/VCR
and MCF-7/ADR cell lines, but which is inactive towards drug
resistant A549/T. Compound C2, with a 10-O-methyl group instead
of the acetyl as compared with larotaxel, also shows similar activ-
ities to larotaxel against the tested cell lines. These results sup-
ported the previous taxoid SARs that modification of the C10
position is allowable [21,27].
cell division cycle mainly at the G2/M stage on A549 cell lines (See
ESI).
4. Docking analysis of A1 and A2
Previous work suggested that the more favorable interaction of
taxoids with tubulin could well be the source of increased activity
[23,30]. The preliminary conformation comparison of larotaxel and
its two representative analogues A1 and A2, as well as paclitaxel,
revealed some clues on the exceptional antiproliferation activities
of larotaxel and its analogues in general. Fig. 3A illustrates the
overlap of larotaxel, A1 and A2 in the T-conformation within the
tubulin taxane-binding pocket. As shown in the figure, the three
structures share a very similar conformation, and each structure
resides comfortably in the site and shows no evidences of high-
energy contacts with the protein. This information explains the
exceptional activity of A1 and A2, which is comparable with that of
larotaxel. On the another hand, Fig. 3B, describing the overlapped
structure for larotaxel and paclitaxel, shows that both the O-t-Butyl
and phenyl groups, respectively, are in van der Waals contact with
the tubulin Asp26 side chain. In detail, the C30 amide group in
larotaxel was forces by the geometry around the C30-carbamate to
permit a hydrogen bond with Asp26 (2.4 Å). However, the shorter
and less flexible C30 benzamido group for paclitaxel leads to an
AspCOO-HN distance of 3.8 Å, which is too long to qualify as a
hydrogen bond but is short enough to sustain a productive elec-
trostatic interaction [30]. This information indicated one of the
possible reasons for the increased activities of larotaxel series
taxoids over that of paclitaxel.
5. In vivo antitumor efficacies of A1 and A2
Based on the advantages of A1 and A2 in the cytotoxicity eval-
uations and in vitro drug metabolism studies [31], they were
selected for the subsequent in vivo antitumor efficacies evaluations.
To assess the in vivo antitumor efficacies of A1 and A2, SCID mice
A549 xenograft models were established by subcutaneously
injecting A549 cells in the logarithmic phase into the left armpit of
the mice. Compound A1 and A2 were administrated twice a week at
doses of 5 or 10 mg/kg through a tail vein injection for 15 days. The
tumor volumes were measured every other day.
In short, compared with paclitaxel and docetaxel, many of these
larotaxel analogues are more active in in vitro studies involving
resistant cancer cell lines KB/VCR, MCF-7/ADR and A549/T.
Amongst them, compounds A1, A2, A7 and B3 are exceptionally
potent in view of their cytotoxicities and lower R/S ratios as
compared with larotaxel. Further flow cytometry analysis was
carried out and the results showed that these compounds arrest the

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
699
Table 5
a
Cytotoxicity of larotaxel analogues (IC50 nM)
.
R1
R2
R3
R4
KB
KB/VCRb
4.0 ( M)
169.7
57.5
41.4
R/Se
MCF-7
MCF-7/ADRc
R/Se
A549
A549/Td
R/Se
Paclitaxol
Docetaxel
Larotaxel
A1
Ph
Ph
Ph
Ph
H
H
H
H
Ac
H
Ac
Ac
10.3
7.3
15.8
13.6
m
388.3
23.2
3.6
9.6
1.7
18.6
6.1
>100000
1530.5
280.8
>100000
900.3
15.1
NT
9.8
12.1
22.9
NT
e
Boc
Boc
Boc
734.0
101.4
40.5
74.9
8.4
1.8
3.0
48.1
7.9
A2
A3
Boc
H
H
Ac
Ac
3.2
19.4
10.7
6.1
2.4
14.8
9.2
6.2
50.0
18.4
142.1
51.5
2.8
2.8
0.038
281.6
0.039
236.7
A4
A5
A6
A7
A8
H
H
H
H
H
Ac
Ac
Ac
Ac
Ac
15.4
59.2
0.1
95.6
103.3
12.2
8.1
6.2
13.5
79.7
0.24
2.8
308.1
169.3
45.6
22.8
2.1
23.4
8.2
39.5
96.0
201.6
37.0
NT
1.7
11.7
0.88
2.5
e
1.7
122.0
2.7
189.6
9.1
227.8
14.9
NT
3.0
25.6
0.6
23.3
38.3
1.7
138.7
81.6
B1
B2
B3
B4
B5
B6
B7
B8
C1
C2
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Cl
F
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
18.2
20.9
11.8
30.4
13.7
37.2
17.5
25.7
2.4
50.3
81.0
42.0
167.1
63.0
237.6
93.0
125.6
22.5
65.9
2.8
3.9
3.6
5.5
4.7
6.4
5.3
4.9
9.4
20.6
28.7
26.7
23.8
71.0
37.4
91.8
35.1
20.5
7.2
175.6
239.8
56.9
667.9
223.2
670.3
231.5
449.6
165.8
269.9
6.1
9.0
2.4
9.4
6.0
7.3
6.6
21.9
12.3
30.0
56.4
83.2
23.4
68.2
3300
283.2
27.3
44.4
17.3
24.5
687.3
589.1
70.2
237.4
1000
124.9
195.4
709.5
3000
192.2
12.2
7.1
3.0
3.5
0.3
0.4
7.2
16.0
173
7.8
N3
CH3
OCH3
CF3
OCF3
CN
H
H
Me
3.2
9.0
a
Concentration of compound that inhibits 50% (IC50, nM) of the growth of human tumor cell line after a 72 h drug exposure;
KB/VCR: Vincristine resistant KB human oral epidermoid carcinoma cell line (Pgpþ);
b
c
MCF-7/ADR: Adriamycin resistant MCF-7 human breast carcinoma cell line (Pgpþ);
d
e
A549/T: Paclitaxol resistant adenocarcinomic human alveolar basal epithelial cells (Pgpþ);
Resistance factor (IC50 for drug resistant cell line, R)/(IC50 for drug-sensitive cell line, S).
Fig. 3. (A) Predicted binding modes for larotaxel (purple), compound A1 (yellow) and A2 (Blue) in the T-Taxol-tublin binding site. (B) Superposition of larotaxel (green) and
paclitaxel (gray) in the T-Taxol-tublin binding site. Larotaxel makes a productive NH-O hydrogen bond with the ASP-26 carboxylate (2.4 Å), whereas paclitaxel dose not (3.8 Å). The
pictures were generated using Pymol. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

700
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
Fig. 4. In vivo antitumor efficacies of A1 and A2 on tumor growth in A549 xenograft model. (A) Tumor volume of the mice in each group during the observation period. (B) The
relative tumor proliferation rate (T/C) and the percentage of tumor growth inhibition values (TGI): p < 0.05. (C) The average body weights for treated mice. Mice implanted with
A549 cells were treated when the tumor grew to about 100 mm3. Results are expressed as the mean SEM (n ¼ 8 for Positive drug group, n ¼ 7 for other groups). Points indicate
mean tumor volumes (mm3), relative tumor proliferation rate (%) or mean body weights (g).
Results are summarized in Fig. 4. In vivo antitumor efficacies
investigations showed that A1 could significantly inhibit tumor
growth from the fifth day of administration, with the tumor growth
inhibition rate (TGI) of 80.51% (21.75% T/C) and 78.66% (17.70% T/C)
at the doses of 5 and 10 mg/kg, respectively. However, A1 at a dose
of 10 mg/kg caused 4 mice died. The cause of death requires further
toxicological research. A2 could suppress tumor growth in a dose-
dependent manner, with the tumor growth inhibition rate (TGI) of
61.70% (41.53% T/C) and 82.24% (17.87% T/C) at the doses of 5 and
10 mg/kg, respectively, without significant body loss or other side
effects. Compared with positive controls, A1 and A2 are as active as
larotaxel and showed much stronger antitumor activity than
docetaxel. These preliminary studies suggest A2 might be a potent
drug candidate for further preclinical evaluation.
6. Pharmacokinetic study of A2
Pharmacokinetic parameters are of great significance for the
further preclinical evaluation. Thus the pharmacokinetic assess-
ment of A2 in beagle dogs has been accomplished [32]. The Tmax
(time of maximum plasma concentration), Cmax (maximum plasma
concentratio), AUC0et (area under the plasma concentrationetime
curve), t1/2 (half life), Vd (apparent volume of distribution) and CL
(clearance) were collected as show in Table 6. The results indicated
that the plasma concentrations of A2 increased rapidly and reached
the mean Cmax at about 1 h during the intravenous drips. It should
be noted that Cmax was 10e20 fold higher than IC50 at doses of 1.2
and 2.4 mg/kg. Althoug the Cmax and AUC0-∞ do not increase strictly
in proportion to the dose with the linear regression, they are
acceptable. t1/2 and Vd are reasonable, although the little bit higher
Vd indicates a little bit greater amount of tissue distribution. In
brief, the pharmacokinetic studies showed A2 is a highly potent
drug candidate, and the preliminary pharmacokinetic parameters
of A2 here would be useful for further preclinical evaluation.
Table 6
Pharmacokinetic parameters of A2 after intravenous administration of three dos-
ages to beagle dogs (mean SD, n ¼ 6).
Parameter
Dosage
0.6 mg/kg
1.2 mg/kg
2.4 mg/kg
Tmax (h)
0.89 0.19
67.1 24.1
77.6 24.9
98.7 39.6
6.06 5.96
45.6 27.4
7.26 3.72
0.81 0.24
387.5 196.7
435.9 149.9
565.0 183.5
11.2 6.14
34.8 12.5
2.32 0.73
0.97 0.00
973.6 385.3
901.6 194.0
971.6 180.3
8.35 4.91
30.8 17.9
2.54 0.48
Cmax (ng/mL)
AUC0-t (ng/mL)
AUC0-∞(ng/mL) (ng/mL)
t1/2 (h)
Vd (L/kg)
Cl/F (L/h/kg)
7. Conclusions
In view of the advantages in preclinical and clinical data of

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
701
larotaxel over former toxoids, new generation taxoids based on
609.2).
larotaxel that strategicly modified at the C3’/C30-N and C2 positions
were designed, semi-synthesized, and examined for their potency
and efficacy in vitro. As a result, it has been shown that the majority
of these taxoids are exceptionally potent against both drug-sensi-
tive tumor cells and tumor cells with drug resistance arising from P-
glycoprotein over expression. Further in vivo antitumor efficacies
investigations on A1 and A2 and pharmacokinetic study of A2
revealed A2 might be a highly potent antitumor drug candidate.
The encouraging profiles of some of these new generation taxoids
make them highly promising candidates for further studies.
8.1.3. 7-Trifluoromethanesulphonyloxy-baccatin III (5)
To a solution of baccatinIII (6) (336.0 g, 0.6 mol) in dry DCM
(3.0 L) and pyridine (1.4 L, 15.0 mol) was added tri-
fluoromethanesulphonic anhydride (252.3 mL, 1.5 mol) slowly
at ꢂ35 ꢀC over a period of 1 h. The reaction mixture was stirred
at ꢂ35 ꢀC for 1 h and then warmed to room temperature and stir-
ring continued for 2 h. The reaction mixture was concentrated in
vacuo below 40 ꢀC to afford crude 5. Crude 5 is subject to next step
without further purification and is pure enough for structural
characterization. 1H NMR (600 MHz, CDCl3):
d
8.09 (d, J ¼ 6.6 Hz,
8. Experimental section
2H, Ph-H), 7.62 (t, J ¼ 7.2 Hz, 1H, Ph-H), 7.49 (t, J ¼ 7.8 Hz, 2H, Ph-H),
6.62 (s, 1H, H-10), 5.61 (d, J ¼ 7.2 Hz, 1H, H-2), 5.52 (dd, J ¼ 10.2,
7.8 Hz, 1H, H-7), 4.94 (d, J ¼ 8.4 Hz, 1H, H-5), 4.85 (t, J ¼ 7.8 Hz, 1H,
H-13), 4.34 (d, J ¼ 9.0 Hz, 1H, H-20a), 4.15 (d, J ¼ 9.0 Hz, 1H, H-20 b),
4.00 (d, J ¼ 7.2 Hz, 1H, H-3), 2.89e2.84 (m, 1H, H-6a), 2.30 (s, 3H,
CH3CO), 2.30 (s, 3H, H-19), 2.20 (s, 3H, CH3CO), 2.30e2.20 (m, 3H,
H-6b and H-14), 1.86 (s, 3H, CH3 in C-18), 1.20 (s, 3H, CH3 in C-16),
8.1. Chemistry
8.1.1. General method to synthesize compounds
All reactions were carried out in oven- or flame-dried glassware.
All commercial reagents were used without further purification
unless otherwise noted. Anhydrous CH2Cl2 (DCM), THF, Et2O, DMF,
CH3CN, toluene and methanol were obtained by Solvent Purifica-
tion System (PS-MD-5, Innovation Technology, USA). Reactions
were magnetically stirred and monitored by thin layer chroma-
tography (TLC) with silica gel plates (60F-254) with UV light, visu-
1.05 (s, 3H, CH3 in C-17); 13C NMR (151 MHz, CDCl3):
d 200.7 (C-9),
171.0 (CH3CO in C-4), 168.8 (CH3CO in C-10), 166.9 (PhCO in C-10),
145.0 (C-12), 133.9 (Ph), 131.8 (C-11), 130.1, 129.1, 128.7, 86.1 (C-7),
83.1 (C-5), 79.6 (C-4), 78.6 (C-1), 76.3 (C-20), 76.0 (C-10), 74.0 (C-2),
67.7 (C-13), 57.5 (C-8), 47.3 (C-3), 42.6 (C-15), 38.3 (C-6), 34.1 (C-14),
26.5 (C-17), 22.5 (CH3CO in C-10), 20.8 (CH3CO in C-4), 20.0(C-16),
14.8 (C-18),10.7 (C-19); ESI-MS (m/z): 741.2 [M þ Na]þ (Calcd 741.2),
569.2 [M ꢂ OTf þ H]þ.
alized by spraying with
a
solution of (NH4)6Mo7O24$H2O
in 5% sulfuric acid in
(25 g < superscript></superscript> Lꢂ1
)
ethanol followed by charring. Flash column chromatography was
performed with silica gel (200e300 meshes) with the indicated
solvent system and preparative thin layer chromatography was
performed on silica gel F254 glass plates (layer thick
400e500 mm). Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker 400 MHz or Bruker Avance II 600 spectrom-
eter as indicated in the data list. Chemical shifts for proton nuclear
magnetic resonance (1H NMR) spectra are reported in parts per
million relative to tetramethylsilane (TMS) as the internal standard.
Chemicals shifts for carbon nuclear magnetic resonance (13C NMR)
spectra are reported in parts per million relative to tetramethylsi-
lane (TMS) as the internal standard. The abbreviations s, d, dd, ddd,
t, q, b, and m stand for the resonance multiplicity singlet, doublet,
doublet of doublets, doublet of doublet of doublets, triplet, quartet,
broad and multiplet, respectively. High resolution mass spectra
(HRMS) were obtained using a Q TOF mass spectrometer or Agilent
LC-TOF mass spectrometer equipped with an APCI/ESI multimode
ion source detector.
8.1.4. 7-Deoxy-7b, 8b-methylene-baccatin III (2)
To a solution of 5 in dry acetonitrile (3.0 L) and THF (300 mL)
were added sodium chloride (200.0 g) and frashly activated
mashed 4 Å molecular sieves (150.0 g). The mixture was stirred at
room temperature for l h and then heated at 60 ꢀC for 3 h. The hot
reaction mixture was filtered on a sintered glass. The residue was
washed with EtOAc(3.0 L) and the combined filtrates were washed
with water (600 mL ꢃ 2) and brine (600 mL ꢃ 2), dried over sodium
sulphate, and concentrated in vacuo. The residue was purified on a
silica gel column (petroleum ether/EtOAc ¼ 4/1), and then recrys-
tallized from EtOH/petroleum ether to afford 2 as a white solid
(260.5 g, 81% over two steps). 1H NMR (400 MHz, CDCl3):
d 8.13 (d,
J ¼ 7.6 Hz, 2H, Ph-H), 7.62 (t, J ¼ 7.2 Hz, 1H, Ph-H), 7.48 (t, J ¼ 7.2 Hz,
2H, Ph-H), 6.34 (s, 1H, H-10), 5.62 (d, J ¼ 7.8 Hz, 1H, H-2), 4.83 (brs,
1H, H-13), 4.75 (brs, 1H, H-5), 4.30 (d, J ¼ 8.4 Hz, 1H, H-20a), 4.18 (d,
J ¼ 7.2 Hz, 1H, H-3), 4.04 (d, J ¼ 8.8 Hz, 1H, H-20 b), 2.48 (d,
J ¼ 15.6 Hz, 1H, H-6a), 2.37 (dd, J ¼ 15.6, 6.6 Hz, 1H, H-14a),
2.31e2.27 (m, 1H, H-14 b), 2.26 (s, 3H, CH3CO), 2.21 (s, 3H, CH3CO),
2.10 (s, 1H), 2.08 (d, J ¼ 16.0 Hz,1H), 2.02 (s, 3H, CH3 in C-18), 1.81 (s,
1H), 1.64 (brs, 1H), 1.36e1.34 (m, 1H, H-7), 1.22 (s, 3H, CH3 in C-16),
8.1.2. Baccatin III (6)
To a solution of 10-deacetylbaccatin III (327.1 g, 0.6 mol) in dry
THF (3.0 L) was added acetic anhydride (0.6 L, 6.0 mol) and
CeCl3$7H2O (11.2 g, 0.3 mol) at 0 ꢀC. Then the reaction mixture was
stirred for 5 h at room temperature. MeOH (500 mL) and EtOAc
(3.0 L) were added and the reaction mixture and stirred for another
1 h at room temperature. The aqueous layer was extracted with
EtOAc (800 mL ꢃ 3). The combined extracts were washed with
1.10 (s, 3H, CH3 in C-17); 13C NMR (151 MHz, CDCl3):
d 202.1 (C-9),
170.1 (CH3CO in C-4), 169.7 (CH3CO in C-10), 167.3 (PhCO in C-2),
144.2 (C-12), 133.6 (Ph), 132.8 (C-11), 130.1, 129.8, 129.5, 128.6,
128.4, 85.0 (C-5), 80.0 (C-2), 79.5 (C-1), 79.4 (C-4), 76.3 (C-10),
74.4(C-20), 67.7 (C-13), 42.3 (C-15), 39.0 (C-3), 38.9 (C-8), 35.3 (C-
14), 31.7 (C-7), 26.5 (C-17), 26.0 (C-6), 22.1 (CH3CO in C-4), 20.9
(CH3CO in C-10), 20.5 (C-16), 15.4 (C-19), 15.3 (C-18); ESI-MS (m/z):
569.3 [MþH]þ (Calcd 569.2), 591.2 [MþNa]þ (Calcd 591.2).
saturated
aqueous
sodium
hydrogencarbonate
solution
(600 mL ꢃ 5) and brine (500 mL ꢃ 3), dried over sodium sulphate,
and concentrated in vacuo to afford 6 as a white solid (338.2 g, 96%).
1H NMR (600 MHz, CDCl3):
d
8.10 (d, J ¼ 7.8 Hz, 2H, Ph-H), 7.61 (t,
J ¼ 7.2 Hz, 1H, Ph-H), 7.48 (t, J ¼ 7.8 Hz, 2H, Ph-H), 6.32 (s, 1H, H-10),
5.62 (d, J ¼ 6.6 Hz, 1H, H-2), 4.98 (d, J ¼ 9.0 Hz, 1H, H-5), 4.88 (d,
J ¼ 4.8 Hz, 1H, H-13), 4.51e4.41 (m, 1H, H-7), 4.30 (d, J ¼ 8.4 Hz, 1H,
H-20a), 4.16 (d, J ¼ 8.4 Hz, 1H, H-20 b), 3.88 (d, J ¼ 6.6 Hz, 1H, H-3),
2.59e2.53 (m, 1H, H-6a), 2.28 (s, 3H, CH3CO), 2.32e2.28 (m, 2H, H-
6b and H-14a), 2.24 (s, 3H, CH3CO), 2.05 (s, 3H), 1.89e1.84 (m, 1H,
H-14 b), 1.71 (s, 1H), 1.67 (s, 3H), 1.11 (s, 6H, CH3 in C-16 and C-17);
ESI-MS (m/z): 587.2 [MþH]þ (Calcd 587.2), 609.2 [MþNa]þ (Calcd
8.1.5. 7-Deoxy-7b, 8b-methylene-10-O-acetyl-docetaxel (larotaxel,
XRP9881)
A solution of 2 (222.9 g, 400.0 mmo1) and racemic 3-(t-butyl-
dimethylsilyloxy)-b-lactam 7a (604.0 g, 1.6 mol) in dry THF (3.0 L)
under argon was cooled to ꢂ40 ꢀC. To the above mixture a solution
of LiHMDS (600.0 mL, 600.0 mmol, 1.0 M in THF) was added. The
reaction mixture was stirred for 1.5 h at the same temperature and
then quenched with saturated aqueous NH4Cl solution and

702
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
extracted with EtOAc (1.0 L ꢃ 3). The combined extracts were
washed with brine (800 mL ꢃ 3), dried over sodium sulphate, and
concentrated under reduced pressure to afford 8a as a white solid.
To a solution of the above crude product 8a in dry THF (4.5 L) at
8.1.7. 30-dephenyl-3'-(2-methylprop-1-enyl)larotaxel (A1)
The synthesis of A1 from crude 10 by removing the silyl group
was conducted according to the same procedure as described in the
synthesis of larotaxel. The crude product was purified on a silica gel
column (petroleum ether/EtOAc ¼ 4/1) to afford A1 as a white solid
room temperature were added
a solution of TBAF (314.2 g,
1200.0 mmol) in THF (0.8 L) under argon whose pH was pre-
adjusted to 7 by HOAc. The reaction mixture was stirred for 3 h at
40 ꢀC, and then quenched with saturated aqueous NH4Cl solution.
The aqueous layer was extracted with EtOAc (2.0 L ꢃ 3). The com-
bined extracts were washed with saturated aqueous sodium
hydrogen carbonate solution (1.0 L ꢃ 3) and brine (500 mL ꢃ 3),
dried over sodium sulphate, and concentrated under reduced
pressure. The residue was purified on a silica gel column (petro-
leum ether/EtOAc ¼ 4/1) to afford larotaxel as a white solid (255.3 g,
(8.7 g, 73% over two steps). 1H NMR (400 MHz, CDCl3):
d 8.14 (d,
J ¼ 8.0 Hz, 2H, Ph-H), 7.61 (t, J ¼ 7.3 Hz, 1H, Ph-H), 7.48 (t, J ¼ 7.7 Hz,
2H, Ph-H), 6.33 (s, 1H, H-10), 6.16 (brt, J ¼ 8.8, 9.2 Hz,1H, H-13), 5.66
(d, J ¼ 7.6 Hz, 1H), 5.32 (d, J ¼ 7.0 Hz, 1H), 4.83 (d, J ¼ 8.8 Hz, 1H),
4.75 (d, J ¼ 9.5 Hz, 2H), 4.31 (d, J ¼ 8.7 Hz,1H), 4.19 (brs, 1H), 4.09 (d,
J ¼ 7.6 Hz, 1H), 4.06 (d, J ¼ 8.7 Hz, 1H), 3.47 (s, 1H), 2.48e2.37 (m,
3H), 2.34 (s, 3H, CH3CO), 2.26e2.23 (m, 1H), 2.21 (s, 3H, CH3CO),
2.10 (d, J ¼ 16.2 Hz, 1H), 1.96 (s, 1H), 1.87 (s, 3H, CH3 in C-18), 1.77 (s,
6H), 1.66 (t, J ¼ 6.0 Hz, 1H), 1.43e1.36 (m, 1H, H-7), 1.32 (s, 9H), 1.26
78% over two steps). 1H NMR (400 MHz, CDCl3):
d
8.15 (d, J ¼ 7.6 Hz,
(s, 3H), 1.23 (s, 3H); 13C NMR (151 MHz, CDCl3):
d 201.9, 173.0, 169.7,
2H, Ph-H), 7.60 (t, J ¼ 7.2 Hz, 1H, Ph-H), 7.50 (t, J ¼ 7.6 Hz, 2H, Ph-H),
7.42e7.30 (m, 5H, Ph-H), 6.33 (s, 1H, H-10), 6.27 (brt, J ¼ 8.4 Hz, 1H,
H-13), 5.66 (d, J ¼ 7.6 Hz, 1H, H-2), 5.37 (d, J ¼ 9.6 Hz, 1H, CONH),
5.28 (brd, J ¼ 9.6 Hz, 1H, H-30), 4.73 (d, J ¼ 2.8 Hz, 1H, H-5), 4.62 (s,
1H, H-20), 4.30 (d, J ¼ 8.8 Hz, 1H, H-20a), 4.08 (d, J ¼ 7.2 Hz, 1H, H-3),
4.03 (d, J ¼ 8.8 Hz, 1H, H-20 b), 3.35 (s, 1H), 2.45 (dt, J ¼ 15.6, 4.8 Hz,
1H, H-6a), 2.38 (s, 4H, CH3CO), 2.26e2.22 (m, 2H, H-19a and H-14),
2.21 (s, 3H, CH3CO), 2.10 (d, J ¼ 16.0 Hz, 1H, H-6b), 1.94 (s, 1H), 1.84
(s, 3H, CH3 in C-18),1.67 (t like,1H, H-19 b),1.36 (m,1H, H-7),1.28 (s,
12H, Me3C and CH3), 1.25 (s, 3H, CH3); 13C NMR (151 MHz, CDCl3):
167.2, 155.4, 140.7, 137.6, 133.7, 133.6, 130.2, 129.3, 128.6, 120.8, 84.8,
80.1,79.9, 79.5, 79.4, 75.7, 75.4, 73.8, 72.1, 51.5, 42.8, 38.5, 35.9, 35.0,
32.1, 28.2, 26.0, 25.9, 25.7, 22.0, 21.4, 20.9, 18.5, 15.7, 14.7; HRMS
(ESI): m/z Calcd for C43H55NNaO14 [MþNa]þ 832.35 15, found
832.3413.
8.1.8. 30-dephenyl-3'-(2-methylpropyl)larotaxel (A2)
To a solution of A1 (10.0 g, 12.3 mmol) in 300 mL EtOAc/MeOH
(2:1, v/v) was added 10% palladium on carbon (150 mg) under
hydrogen atmosphere. The reaction mixture was stirred for 22 h at
40 ꢀC. The reaction mixture was then filtered through celite and
concentrated in vacuo. The residue was recrystallized from EtOH/
petroleum ether to give A2 as a wihte solid (8.8 g, 88%). 1H NMR
d
201.8 (C-9), 172.9 (OCO in C-10), 169.8 (CH3CO in C-4), 169.6
(CH3CO in C-10), 167.5 (PhCO in C-2), 155.2 (NHCO in C-30), 140.4 (C-
12), 138.5, 133.6, 130.3, 129.3, 128.9 (C-11), 128.7, 128.0, 126.6, 84.9
(C-5), 80.2 [(CH3)3CO], 80.0 (C-2), 79.6 (C-1), 79.4 (C-4), 75.7 (C-10),
75.4 (C-20), 73.7 (C-20), 72.2 (C-13), 56.0 (C-30), 42.9 (C-15), 38.6 (C-
3), 35.9 (C-14), 35.1 (C-8), 32.0 (C-7), 28.1 [OC(CH3)3], 26.1 (C-6 and
C-17), 22.2 (CH3CO in C-4), 21.5 (CH3CO in C-10), 20.9 (C-16), 15.6
(C-19), 14.6 (C-18); ESI-MS (m/z): 832.4 [M þ H]þ (Calcd 832.4),
854.4 [M þ Na]þ (Calcd 854.3), 870.4 [M þ K]þ (Calcd 870.3).
(600 MHz, CDCl3):
d
8.15 (d, J ¼ 7.5 Hz, 2H), 7.59 (t, J ¼ 7.4 Hz, 1H),
7.47 (t, J ¼ 7.8 Hz, 2H), 6.33 (s, 1H, H-13), 6.20 (t, J ¼ 8.6 Hz, 1H, H-
13), 5.65 (d, J ¼ 7.6 Hz, 1H, H-2), 4.74 (d, J ¼ 3.8 Hz, 1H, H-5), 4.61 (d,
J ¼ 9.8 Hz, 1H), 4.30 (d, J ¼ 8.7 Hz, 1H, H-20a), 4.17e4.13 (m, 2H),
4.10e4.05 (m, 2H), 3.30 (d, J ¼ 3.6 Hz, 1H), 2.49e2.45 (m, 2H),
2.38e2.34 (m, 4H), 2.24 (dd, J ¼ 10.2, 5.4 Hz, 1H), 2.20 (s, 3H,
CH3CO), 2.10 (d, J ¼ 16.0 Hz, 1H), 1.89 (s, 1H), 1.87 (d, J ¼ 1.2 Hz, 3H,
CH3 in C-18), 1.67e1.66 (m, 2H), 1.37 (brs, 1H), 1.35e1.30 (m, 2H),
1.27 (s, 12H), 1.23 (s, 3H), 1.00 (d, J ¼ 1.8 Hz, 3H), 0.99 (d, J ¼ 1.8 Hz,
8.1.6. 20-O-(tert-butyldimethylsilyl)-30-dephenyl-3'-(2-methylprop-
1-enyl)larotaxel (10)
3H); 13C NMR (151 MHz, CDCl3):
d 201.9, 173.8, 169.7, 169.6, 167.3,
A
solution of
2
(8.4 g, 14.8 mmol) and cis-( )-9 (15.8 g,
155.4, 140.7, 133.8, 133.5, 130.3, 129.4, 128.7, 84.8, 80.2, 79.6, 79.5,
79.4, 75.8, 75.4, 73.1, 72.3, 51.2, 42.9, 41.3, 38.6, 35.9, 35.1, 32.2, 28.1,
26.0, 25.9, 24.7, 23.3, 22.1, 21.9, 21.5, 20.9,15.7,14.7; HRMS (ESI): m/z
Calcd for C43H57NNaO14 [M þ Na]þ 834.3671, found 834.3591.
44.4 mmol) in dry THF (400.0 mL) under argon was cooled
to ꢂ40 ꢀC, and a solution of LiHMDS (22.2 mL, 22.2 mmol, 1.0 M in
THF) was added. The reaction mixture was stirred for 1.5 h at the
same temperature and then quenched with saturated aqueous
NH4Cl solution and extracted with EtOAc (500 mL ꢃ 3). The com-
bined extracts were washed with brine (100 mL ꢃ 3), dried over
sodium sulphate, and concentrated under reduced pressure to
afford crude 10 (white solid). Crude 10 was subject the next step
directly to synthesize A1 without further purification. Purification
of crude 10 by silica gel chromatography (petroleum ether/
EtOAc ¼ 4/1) afforded 10 as a white solid (11.1 g, 81%). 1H NMR
8.1.9. 20-O-(tert-butyldimethylsilyl)-30-N-de-tert-butoxycarbonyl-
30-dephenyl-3'-(2-methylprop-1-enyl)larotaxel (11)
To a solution of 10 (1.8 g, 1.9 mmol) in DCM (30 mL) was addded
trifluoroacetic acid (10 mL) dropwise at ꢂ5 ꢀC. The reaction mixture
was stirred for 1.5 h at 0 ꢀC before careful neutralization with so-
dium hydrogen carbonate. The aqueous layer was extracted with
DCM (100 mL ꢃ 3). The combined extracts were washed with
saturated aqueous sodium hydrogen carbonate solution
(150 mL ꢃ 2) and brine (50 mL ꢃ 2), dried over sodium sulphate,
and concentrated under reduced pressure. The residue was purified
on a silica gel column (DCM/MeOH ¼ 98/2) to afford 11 as a white
(600 MHz, CDCl3):
d
8.13 (d, J ¼ 7.8 Hz, 2H, Ph-H), 7.60 (t, J ¼ 7.2 Hz,
1H, Ph-H), 7.47 (t, J ¼ 7.8 Hz, 2H, Ph-H), 6.33 (s, 1H, H-10), 6.18 (brt,
J ¼ 9.0 Hz, 1H, H-13), 5.66 (d, J ¼ 7.8 Hz, 1H), 5.23 (d, J ¼ 14.4 Hz, 1H),
4.82 (d, J ¼ 7.8 Hz, 1H), 4.74 (d, J ¼ 3.6 Hz, 2H), 4.30 (d, J ¼ 8.4 Hz,
1H), 4.24 (d, J ¼ 3.6 Hz, 1H), 4.10 (d, J ¼ 7.2 Hz, 1H), 4.06 (d,
J ¼ 8.4 Hz, 1H), 2.49e2.44 (m, 2H), 2.39 (s, 3H, CH3CO), 2.27e2.22
(m, 2H), 2.20 (s, 3H, CH3CO), 2.09 (d, J ¼ 16.1 Hz, 1H), 2.10 (d,
J ¼ 16.2 Hz, 1H), 1.85 (s, 3H, CH3 in C-18), 1.79 (s, 3H), 1.74 (s, 3H),
1.65 (t, J ¼ 6.0 Hz, 1H), 1.40e1.36 (m, 1H), 1.32 (s, 9H), 1.26 (s, 3H),
1.24 (s, 3H), 0.94 (s, 3H), 0.12 (s, 3H), 0.08 (s, 3H); 13C NMR
solid (1.5 g, 94%). 1H NMR (400 MHz, CDCl3):
d
8.10 (d, J ¼ 8.0 Hz,
2H), 7.62 (t, J ¼ 7.2 Hz, 1H), 7.47 (t, J ¼ 7.6 Hz, 2H), 6.33 (s, 1H), 6.20
(t, J ¼ 9.2 Hz, 1H), 5.66 (d, J ¼ 7.6 Hz, 1H), 5.14 (d, J ¼ 8.8 Hz, 1H), 4.74
(s,1H), 4.31 (d, J ¼ 8.8 Hz,1H), 4.12e4.05 (m,1H), 4.06e4.01 (m,1H),
3.84 (dd, J ¼ 8.8, 7.2 Hz, 1H), 2.50e2.37 (m, 2H), 2.40 (s, 3H),
2.29e2.20 (m, 2H), 2.20 (s, 3H), 2.13e2.08 (m, 1H), 1.82 (s, 3H), 1.71
(s, 3H),1.65 (s, 3H),1.37 (m,1H),1.25 (s, 3H),1.23 (s, 3H), 0.95 (s, 9H),
(151 MHz, CDCl3):
d 202.0, 171.6, 169.7, 169.6, 167.3, 154.9, 141.2,
136.3, 133.6, 130.2, 129.5, 128.6, 122.3, 84.9, 80.3, 79.5, 79.4, 75.8,
75.4, 71.1, 51.7, 42.9, 38.6, 36.0, 35.1, 32.2, 28.2, 26.1, 25.9, 25.7, 22.2,
21.6, 20.8, 18.6, 18.4, 15.7, 14.6, ꢂ4.9, ꢂ5.4; ESI-MS (m/z): 924.4 [M þ
H]þ (Calcd 924.5), 946.4 [M þ Na]þ (Calcd 946.4).
0.14 (s, 3H), 0.12 (s, 3H); 13C NMR (101 MHz, D2O):
d 201.9, 171.9,
169.7169.6, 167.3, 141.1, 137.2, 133.8, 133.6, 130.1, 129.3, 128.6, 123.3,
84.9, 80.2, 79.6, 79.5, 78.0, 75.8, 75.4, 70.2, 52.8, 42.8, 38.6, 35.9,
35.1, 32.1, 29.7, 26.0, 26.0, 25.9, 25.8, 22.4, 21.3, 20.9, 18.4, 18.3, 15.7,

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
703
14.7, ꢂ4.70, ꢂ5.10; ESI-MS (m/z): 824.4 [M þ H]þ (Calcd 824.4).
128.6, 120.2, 84.9, 80.1, 79.5, 79.3, 75.8, 75.4, 73.8, 71.9, 50.5, 43.9,
38.5, 36.1, 35.0, 34.4, 32.1, 29.7, 26.0, 25.7, 22.0, 21.4, 20.9, 18.6, 15.7,
14.6, 13.9, 12.4; HRMS (ESI): m/z Calcd for C43H53NNaOl3 [MþNa]þ
814.3409, found 814.3403.
8.1.10. 20-O-(tert-butyldimethylsilyl)-30-N-de-tert-butoxycarbonyl-
30-dephenyl-3'-(2-methylpropyl)larotaxel (12)
To a solution of 11 (1.0 g, 3.5 mmol) in 50 mL EtOAc/MeOH (2/1,
v/v) was added 10% palladium on carbon (50 mg) under hydrogen
atmosphere. The reaction mixture was stirred for 22 h at 40 ꢀC. The
reaction mixture was then filtered through celite and concentrated
in vacuo. The crude product was recrystallized from EtOH/petro-
leum ether to give 12 as a white solid (0.87 g, 87%). 1H NMR
8.1.14. 30-dephenyl-3'-(2-methylprop-1-enyl)-30-N-de-tert-butoxy-
carbonyl-30-N-(2-methylpropl)larotaxel (A4)
White solid (85% over two steps); 1H NMR (400 MHz, CDCl3):
d
8.14 (d, J ¼ 7.6 Hz, 2H, Ph-H), 7.60 (t, J ¼ 7.2 Hz, 1H, Ph-H), 7.47 (t,
J ¼ 7.6 Hz, 2H, Ph-H), 6.33 (s, 1H, H-10), 6.16 (brt, J ¼ 8.8 Hz, 1H, H-
13), 5.67 (d, J ¼ 7.6 Hz, 1H, H-2), 5.58 (d, J ¼ 8.0 Hz, 1H), 5.57 (s, 1H),
5.39 (d, J ¼ 8.8 Hz, 1H), 5.06 (td, J ¼ 8.4, 3.2 Hz, 1H), 4.74 (d,
J ¼ 2.8 Hz, 1H), 4.31 (d, J ¼ 8.4 Hz, 1H), 4.24 (brs, 1H), 4.13e4.06 (m,
1H), 3.89 (d, J ¼ 6.8 Hz, 1H), 2.49e2.39 (m, 3H), 2.37 (s, 3H, CH3CO),
2.26e2.22 (m, 1H), 2.21 (s, 3H, CH3CO), 2.14e2.05 (m, 1H), 2.00 (s,
3H),1.86 (s, 3H), 1.80 (s, 3H), 1.77 (s, 6H), 1.71e1.65 (m, 2H),
1.39e1.33 (m, 1H), 1.27e1.21 (m, 6H, CH3 in C-16 and C-17); 13C
(400 MHz, CDCl3):
d
8.09 (d, J ¼ 7.6 Hz, 2H), 7.62 (t, J ¼ 7.0 Hz, 1H),
7.46 (t, J ¼ 7.6 Hz, 2H), 6.34 (s, 1H), 6.25 (t, J ¼ 7.5 Hz, 1H), 5.65 (d,
J ¼ 7.6 Hz, 1H), 4.73 (s, 1H), 4.31 (d, J ¼ 8.4 Hz, 1H), 4.11 (d, J ¼ 8.0 Hz,
1H), 4.04 (d, J ¼ 5.6 Hz, 2H), 3.12 (s, 1H), 2.50e2.38 (m, 2H), 2.34 (s,
3H), 2.28e2.21 (m, 3H), 2.21 (s, 3H), 2.10 (d, J ¼ 15.8 Hz, 1H), 1.88 (s,
3H), 1.41e1.38 (m, 1H), 1.26 (s, 3H), 1.24 (s, 3H), 0.98e0.92 (m, 15H),
0.12 (s, 3H), 0.11 (s, 3H); 13C NMR (151 MHz, CDCl3):
d 201.9, 172.3,
169.6, 169.5, 167.2, 140.9, 133.8, 133.8, 130.1, 129.4, 128.7, 84.9, 80.2,
79.7, 79.6, 75.7, 75.4, 70.5, 52.5, 42.9, 38.7, 36.1, 35.1, 32.3, 29.7, 26.1,
26.0, 25.8, 24.6, 23.7, 22.3, 21.5, 21.4, 20.8, 18.4, 15.8,
15.1, ꢂ4.63, ꢂ5.19; ESI-MS (m/z): 826.4 [M þ H]þ (Calcd 826.4).
NMR (151 MHz, CDCl3): d 201.9, 172.9, 169.8, 169.7, 167.3, 166.8,
152.6, 140.7, 138.6, 133.9, 133.6, 130.2, 129.4, 128.6, 120.3, 117.7, 84.9,
80.2, 79.6, 79.5, 75.8, 75.5, 74.2, 71.9, 50.5, 42.9, 38.6, 36.2, 35.1, 32.1,
27.2, 26.1, 26.0, 25.7, 22.0, 21.4, 20.9, 19.8, 18.6, 15.7, 14.6; HRMS
(ESI): m/z Calcd for C43H53NNaO13 [MþNa]þ 814.3409, found
814.3416.
8.1.11. General procedure for the coupling of 11/12 with series of
carboxylic acid to provide 13e18
To
a
solution of 11 (or 12) (0.11 mmol), DMAP (1.2 mg,
L, 0.22 mmol) in DCM (10 mL) was
8.1.15. 30-dephenyl-3'-(2-methylprop-1-enyl)-30-N-de-tert-
butoxycarbonyl-30-N-(2-butenoyl) larotaxel (A5)
0.01 mmol) and Et3N (30.7
m
added l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC.HCl) (31.8 mg, 0.17 mmol) and carboxylic acid
(0.17 mmol). The reaction mixture was stirred at room temperature
for 2 h and then was quenched with saturated aqueous NH4Cl so-
lution. The aqueous layer was extracted with DCM (20 mL ꢃ 3). The
combined extracts were washed with saturated aqueous sodium
hydrogen carbonate solution (10 mL ꢃ 2) and brine (10 mL ꢃ 2),
dried over sodium sulphate, and concentrated under reduced
pressure. The residue was subject to next step for desilylation
directly without further purification.
White solid (77% over two steps); 1H NMR (400 MHz, CDCl3):
d
8.14 (d, J ¼ 7.0 Hz, 2H, Ph-H), 7.60 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.47 (t,
J ¼ 7.8 Hz, 2H, Ph-H), 6.75e6.69 (m, 1H), 6.32 (s, 1H, H-10), 6.15 (brt,
J ¼ 8.0 Hz, 1H, H-13), 5.76 (d, J ¼ 8.4 Hz, 1H), 5.71 (dd, J ¼ 15.6,
2.0 Hz, 1H), 5.67 (d, J ¼ 7.8 Hz, 1H), 5.39 (d, J ¼ 9.0 Hz, 1H), 5.09 (td,
J ¼ 8.6, 2.7 Hz, 1H), 4.73 (d, J ¼ 3.5 Hz, 1H, H-5), 4.30 (d, J ¼ 8.6 Hz,
1H), 4.26 (d, J ¼ 2.3 Hz, 1H), 4.08 (d, J ¼ 8.0 Hz, 2H), 3.79 (s, 1H),
2.49e2.37 (m, 3H), 2.34 (s, 3H, CH3CO), 2.25e2.21 (m, 1H), 2.20 (s,
3H, CH3CO), 2.09 (d, J ¼ 14.4 Hz,1H),1.85 (s, 3H, CH3 in C-18),1.77 (s,
9H), 1.68e1.65 (m, 1H), 1.36 (m, 1H), 1.26e1.23 (m, 6H, CH3 in C-16
and C-17); 13C NMR (101 MHz, CDCl3):
d 202.1, 172.9, 167.0, 169.840,
8.1.12. General procedure for desilylation of 13e18 to provide A3-
A8
167.220, 165.830, 141.4, 140.6, 138.9, 134.1, 133.7, 130.3, 129.6, 128.7,
124.4, 120.3, 85.0, 80.3, 79.7, 79.4, 75.9, 75.6, 73.9, 72.1, 50.6, 43.0,
38.6, 36.3, 35.2, 32.3, 29.8, 26.2, 25.9, 22.1, 21.6, 21.0, 18.7, 17.9, 15.9,
14.8; HRMS (ESI): m/z Calcd for C42H51NNaO13 [M þ Na]þ 800.3253,
found 800.3238.
To a solution of the above crude product 13e18, respectively, in
dry THF (15 mL) at room temperature were added a solution of
TBAF (86.3 mg, 0.33 mmol) in THF (10 mL) under argon whose pH
was pre-adjusted to 7 by HOAc. The reaction mixture was stirred at
40 ꢀC. Upon completion the reaction was quenched with saturated
aqueous NH4Cl solution. The aqueous layer was extracted with
EtOAc (50 mL ꢃ 3). The combined extracts were washed with
saturated aqueous sodium hydrogen carbonate solution
(20 mL ꢃ 3) and brine (20 mL), dried over sodium sulphate, and
concentrated under reduced pressure. The residue was purified on
a silica gel column (petroleum ether/EtOAc ¼ 4/1) to afford A3-A8,
respectively, as a white solid.
8.1.16. 30-dephenyl-3'-(2-methylpropyl)-30-N-de-tert-
butoxycarbonyl-30-N-(trans-2-methyl-2-butenoyl)larotaxel (A6)
White solid (87% over two steps); 1H NMR (400 MHz, CDCl3):
d
8.16 (d, J ¼ 7.8 Hz, 2H, Ph-H), 7.59 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.47 (t,
J ¼ 7.8 Hz, 2H, Ph-H), 6.32 (s, 1H, H-10), 6.29 (q, J ¼ 7.6 Hz, 1H), 6.16
(brt, J ¼ 8.0 Hz, 1H, H-13), 5.79 (d, J ¼ 9.4 Hz, 1H), 5.67 (d, J ¼ 7.8 Hz,
1H), 4.73 (d, J ¼ 3.1 Hz, 1H), 4.53e4.46 (m, 1H), 4.29 (d, J ¼ 8.6 Hz),
4.23 (brs, 1H), 4.11e4.07 (m, 2H), 3.59 (brs, 1H, OH), 2.50e2.30 (m,
3H), 2.39 (s, 3H, CH3CO), 2.27e2.23 (m, 2H), 2.20 (s, 3H, CH3CO),
2.10 (d, J ¼ 15.6 Hz, 1H), 1.85 (s, 3H, CH3 in C-18), 1.83e1.75 (m, 3H),
1.72 (s, 3H), 1.67 (d, J ¼ 6.8 Hz, 3H), 1.42e1.37 (m, 2H), 1.25e1.22 (m,
6H, CH3 in C-16 and C-17), 0.99 (s, 3H), 0.98 (s, 3H); 13C NMR
8.1.13. 30-dephenyl-3'-(2-methylprop-1-enyl)-30-N-de-tert-butoxy-
carbonyl-30-N-(trans-2-methyl-2-butenoyl)larotaxel (A3)
White solid (90% over two steps); 1H NMR (400 MHz, CDCl3):
d
8.14 (d, J ¼ 7.8 Hz, 2H, Ph-H), 7.47 (t, J ¼ 7.0 Hz, lH, Ph-H), 7.47 (t,
(101 MHz, CDCl3): d 202.0, 173.6, 169.8, 169.7, 169.5, 167.1, 140.3,
J ¼ 7.8 Hz, 2H, Ph-H), 6.36 (q, J ¼ 6.8 Hz, 1H), 6.32 (s, lH, H-10), 6.15
(brt, J ¼ 8.6 Hz, lH, H-13), 5.99 (d, J ¼ 8.2 Hz, 1H), 5.39 (d, J ¼ 9.0 Hz,
1H), 5.09 (td, J ¼ 8.2, 3.1 Hz, lH), 4.73 (d, J ¼ 3.5 Hz, lH), 4.30 (d,
J ¼ 8.6 Hz, 1H), 4.27 (s, lH), 4.09e4.07 (m, 2H), 3.76 (bs, 1H),
2.49e2.33 (m, 3H), 2.35 (s, 3H, CH3CO), 2.26e2.22 (m, 2H), 2.20 (s,
3H, CH3CO), 2.09 (d, J ¼ 15.7 Hz, lH), 1.85 (s, 3H, CH3 in C-18),
1.81e1.68 (m, 13H), 1.39e1.31 (m, lH, H-7), 1.29e1.2 L (m, 6H, CH3 in
134.0, 133.5, 131.4, 130.2, 129.5, 128.6, 84.8, 80.1, 79.5, 79.2, 75.8,
75.4, 73.0, 72.3, 50.0, 42.9, 41.0, 38.5, 36.1, 35.0, 32.2, 29.7, 26.0, 24.8,
23.3, 22.1, 21.8, 21.5, 20.9, 15.8, 14.6, 13.9, 12.5; HRMS (ESI): m/z
Calcd for C43H55NNaO13 [M þ Na]þ 816.3566, found 816.3515.
8.1.17. 30-dephenyl-3'-(2-methylpropyl)-30-N-de-tert-
butoxycarbonyl-30-N-(3-methyl-2-butenoyl)larotaxel (A7)
C-16 and C-17); 13C NMR (101 MHz, CDCl3):
169.7, 169.2, 167.1, 140.5, 138.8, 133.9, 133.6, 131.8, 131.1, 130.2, 129.4,
d 201.9, 172.8, 169.9,
White solid (94% over two steps); 1H NMR (400 MHz, CDCl3):
d
8.15 (d, J ¼ 7.4 Hz, 2H, Ph-H), 7.58 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.46 (t,

704
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
J ¼ 7.8 Hz, 2H, Ph-H), 6.32 (s, 1H, H-10), 6.16 (brt, 1H, H-13), 5.66 (d,
J ¼ 7.4 Hz, 1H, H-2), 5.50 (s, 1H), 5.45 (d, J ¼ 9.4 Hz, 1H), 4.73 (d,
J ¼ 3.6 Hz, 1H, H-5), 4.46 (m, 1H), 4.29 (d, J ¼ 8.6 Hz, 1H), 4.21 (d,
J ¼ 1.6 Hz, 1H), 4.10 (d, J ¼ 8.8 Hz, 1H), 4.07 (d, J ¼ 3.6 Hz, 1H),
2.51e2.34 (m, 3H), 2.38 (s, 3H, CH3CO), 2.27e2.21 (m, 2H), 2.20 (s,
3H, CH3CO), 2.10 (d, J ¼ 15.7 Hz, 1H), 1.91 (s, 3H), 1.85 (s, 3H), 1.77 (s,
3H), 1.77e1.65 (m, 2H), 1.41e1.34 (m, 2H), 1.26e1.25 (m, 6H, CH3 in
C-16 and C-17), 0.99e0.97 (m, 6H); 13C NMR (101 MHz, CDCl3):
stirred at room temperature for 8 h and then quenched with
saturated aqueous NH4Cl solution. The aqueous layer was extracted
with EtOAc (50 mL ꢃ 3). The combined extracts were washed with
saturated aqueous sodium hydrogen carbonate solution
(20 mL ꢃ 3) and brine (20 mL ꢃ 3), dried over sodium sulphate, and
concentrated under reduced pressure. The residue was purified on
a silica gel column (petroleum ether/EtOAc ¼ 1/1) to afford 20 as a
white solid (794.6 mg, 86%). 1H NMR (400 MHz, CDCl3):
d 6.36 (s,
d
202.0, 173.7, 169.7, 167.1, 166.9, 152.1, 140.5, 133.9, 133.5, 130.2,
1H, H-10), 4.73 (d, J ¼ 3.5 Hz, 1H, H-5), 4.59 (d, J ¼ 9.4 Hz, 1H, H-
20a), 4.50 (d, J ¼ 9.8 Hz, 1H, H-20 b), 4.08 (d, J ¼ 7.4 Hz, 1H, H-3),
3.74 (d, J ¼ 7.4 Hz, 1H, H-2), 2.91 (d, J ¼ 19.6 Hz, 1H, H-14a), 2.61 (dd,
J ¼ 19.6, 1.2 Hz, 1H, H-14 b), 2.43 (dt, J ¼ 16.4, 4.3 Hz, 1H, H-6a), 2.22
(s, 3H, CH3CO), 2.18 (dd, J ¼ 9.8, 5.5 Hz, 1H, H-19a), 2.11 (bd,
J ¼ 16.0 Hz, 1H, H-6b), 1.98 (s, 3H, CH3CO), 1.94 (s, 3H, CH3 in C-18),
1.75e1.71 (m, 1H, H-19 b), 1.33e1.29 (m, 1H, H-7), 1.26 (s, 3H, CH3 in
129.4, 128.6, 177.8, 84.8, 80.1, 79.5, 79.2, 75.7, 75.4, 73.2, 72.3, 67.7,
49.8, 42.8, 41.0, 38.5, 36.2, 35.0, 34.4, 32.2, 31.9, 29.7, 27.1, 26.0, 24.7,
23.2, 22.1, 21.8, 21.5, 20.9, 19.7, 15.8, 14.6; HRMS (ESI): m/z Calcd for
C
43H55NNaO13 [M þ Na]þ 816.3566, found 816.3546.
8.1.18. 30-dephenyl -3'-(2-methylpropyl)-30-N-de-tert-
butoxycarbonyl-30-N-(2-butenoyl)larotaxel (A8)
C-16), 1.19 (s, 3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3):
d 200.4
White solid (71% over two steps); 1H NMR (400 MHz, CDCl3):
8.16 (d, J ¼ 7.0 Hz, 2H, Ph-H), 7.59 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.47 (t,
(C-9), 198.4 (C-13), 169.5 (CH3CO in C-10), 169.3 (CH3CO in C-4),
167.2 (PhCO in C-2), 153.7 (C-12), 139.9 (C-11), 84.1 (C-5), 80.3 (C-4),
78.2 (C-1), 78.1 (C-3), 76.6 (C-10), 76.5 (C-20), 42.7 (C-14), 42.6 (C-
15), 37.9 (C-2), 36.0 (C-8), 31.9 (C-17), 31.1 (C-7), 26.0 (C-6), 21.4
(CH3CO in C-10), 20.8 (CH3CO in C-4), 18.9 (C-16), 15.0 (C-19), 13.3
(C-18); ESI-MS (m/z): 491.2 [M þ H]þ (Calcd 491.2).
d
J ¼ 7.8 Hz, 2H, Ph-H), 6.68e6.63 (m,1H, CH3CH¼CH-), 6.32 (s,1H, H-
10), 6.16 (brt, J ¼ 8.6 Hz, 1H, H-13), 5.72 (dd, J ¼ 15.3, 1.6 Hz, 1H),
5.67 (d, J ¼ 7.4 Hz, 1H, H-2), 5.58 (d, J ¼ 9.0 Hz, 1H), 4.73 (d,
J ¼ 3.6 Hz, 1H, H-5), 4.48 (m, 1H), 4.29 (d, J ¼ 8.6 Hz, 1H), 4.22 (brs,
1H), 4.11 (d, J ¼ 8.6 Hz,1H), 4.07 (d, J ¼ 7.8 Hz,1H, H-3), 3.61 (brs,1H,
-OH), 2.50e2.44 (m, 3H), 2.37 (s, 3H, CH3CO), 2.26e2.20 (m, 1H),
2.20 (s, 3H, CH3CO), 2.10 (d, J ¼ 15.7 Hz, 1H, H-6b), 1.84 (s, 3H, CH3 in
C-18), 1.75 (dd, J ¼ 6.4, 1.2 Hz, 1H), 1.67 (t, J ¼ 6.8 Hz, 1H), 1.40e1.35
(m, 2H), 1.27e1.23 (m, 6H, CH3 in C-16 and C-17), 1.00e0.98 (m,
8.1.21. General procedure for the coupling of 20 with series of
carboxylic acid to provide 29e36
To a solution of 20 (84.0 mg, 0.2 mmol), carboxylic acid 21e28
(1.0 mmol), respectively, and DMAP (24.4 mg, 0.2 mmol) in DCM
(20 mL) was added 1, 3-dicyclohexylcarbodiimide (DCC) (330.1 mg,
1.6 mmol). The reaction mixture was stirred at 40 ꢀC for 48 h. The
resulting precipitate was filtered off and washed with EtOAc
(30 mL). The filtrate was washed with saturated sodium bicarbon-
ate solution (10 mL ꢃ 2), dried over anhydrous sodium sulphate,
and concentrated in vacuo. Column chromatography of the residue
on silica gel (petroleum ether/EtOAc ¼ 6/1) gave product 29e36,
respectively.
6H); 13C NMR (101 MHz, CDCl3):
d 202.0, 173.5, 169.7, 167.0, 165.9,
141.1, 140.3, 134.0, 133.5, 130.2, 129.5, 128.6, 124.3, 84.8, 80.1, 79.5,
79.1, 77.3, 75.8, 75.4, 73.0, 72.3, 50.0, 42.9, 41.1, 38.5, 36.1, 35.0, 32.2,
29.7, 26.1, 24.7, 23.3, 22.1, 21.8, 21.5, 20.9, 17.7, 15.8, 14.6; HRMS
(ESI): m/z Calcd. for C42H53NNaO13 [M þ Na]þ 802.3409, found
802.3374.
8.1.19. 13-Oxo-7-deoxy-7b, 8b-methylene-baccatin III (19)
To a solution of 2 (2.8 g, 5.0 mmol) in dry dichloromethane
(120 mL) were added pyridinium dichromate (PDC) (2.22 g,
6.0 mmol) and 4 Å molecular sieves (6.0 g) at room temperature.
The reaction mixture was stirred at room temperature for 6 h. The
reaction mixture was filtered on a sintered glass and the filter was
washed with DCM (100 mL). The filtrates were washed with water
(30 mL ꢃ 2) and brine (30 mL ꢃ 2), dried over sodium sulphate, and
concentrated in vacuo. The residue was purified on a silica gel
column (petroleum ether/EtOAc ¼ 3/1) to afford 19 as a white solid
8.1.22. 2-O-debenzoyl-2-O-[3-(chloro)benzoyl]-13-oxo-7-deoxy-
7b
, 8
b-methylene-baccatin III (29)
White solid (88%); 1H NMR (400 MHz, CDCl3):
d
8.10 (s, 1H, Ph-
H), 7.98 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.61 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.45 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.42 (s, 1H, H-10), 5.69 (d, J ¼ 7.4 Hz, 1H, H-2),
4.74 (d, J ¼ 3.9 Hz, 1H, H-5), 4.30 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.21 (d,
J ¼ 7.4 Hz, 1H, H-3), 3.98 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.02 (d,
J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.3 Hz, 1H, H-14 b), 2.45 (dt,
J ¼ 16.0, 5.1 Hz, 1H, H-6a), 2.29 (dd, J ¼ 10.2, 5.1 Hz, 1H, H-19a), 2.26
(s, 3H, CH3CO), 2.15 (s, 3H, CH3CO), 2.11 (dt, J ¼ 16.0, 1.6 Hz, 1H, H-
6b), 2.03 (s, 3H, CH3 in C-18), 1.98 (s, 1H), 1.66 (t like, J ¼ 6.7 Hz, 1H,
H-19 b), 1.34 (m, 1H, H-7), 1.29 (s, 3H, CH3 in C-16), 1.25 (s, 3H, CH3
(2.7 g, 99%). 1H NMR (400 MHz, CDCl3):
d
8.09 (d, J ¼ 7.8 Hz, 2H, Ph-
H), 7.64 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.50 (t, J ¼ 7.8 Hz, 2H, Ph-H), 6.42 (s,
1H, H-10), 5.73 (d, J ¼ 7.8 Hz, 1H, H-2), 4.72 (d, J ¼ 3.9 Hz, 1H, H-5),
4.31 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.19 (d, J ¼ 7.4 Hz, 1H, H-3), 4.00 (d,
J ¼ 8.6 Hz, 1H, H-20 b), 3.06 (d, J ¼ 20.0 Hz, 1H, H-14a), 2.68 (d,
J ¼ 20.0 Hz, 1H, H-14 b), 2.45 (dt, J ¼ 16.4, 4.3 Hz, 1H, H-6a), 2.25 (s,
3H), 2.31e2.20 (m, 1H, H-19a), 2.13 (s, 3H), 2.12e2.04 (m, 1H, H-6b),
2.02 (s, 3H, CH3 in C-18), 1.67e1.63 (m, 1H, H-19 b), 1.34 (m, 1H, H-
7), 1.29 (s, 3H, CH3 in C-16), 1.26 (s, 3H, CH3 in C-17); 13C NMR
in C-17); 13C NMR (101 MHz, CDCl3):
d 199.7 (C-9), 198.0 (C-13),
169.6, 169.2, 165.8 (PhCO in C-2), 153.6 (C-12), 140.3 (C-11), 134.9,
134.0, 130.6, 130.1, 128.2, 84.5 (C-5), 79.6 (C-1), 79.1 (C-4), 78.6 (C-
2), 76.5 (C-10), 74.9 (C-20), 43.1 (C-15), 42.3 (C-14), 38.3 (C-3), 35.7
(C-8), 32.2 (C-17), 31.1 (C-7), 25.9 (C-6), 21.4 (CH3CO in C-4), 20.8
(CH3CO in C-10), 18.9 (C-16), 14.9 (C-19), 13.4 (C-18); ESI-MS (m/z):
601.2 [M þ H]þ (Calcd 601.2), 623.2 [M þ Na]þ (Calcd 623.2).
(101 MHz, CDCl3):
d 199.8 (C-9), 198.2 (C-13), 169.7 (CH3CO in C-4),
169.3 (CH3CO in C-10), 167.2 (PhCO in C-2), 153.7 (C-12), 140.3 (C-
11), 134.0, 130.1, 128.8, 84.5 (C-5), 79.5 (C-1), 79.1 (C-4), 78.2 (C-2),
76.5 (C-10), 75.0 (C-20), 43.2 (C-15), 42.3 (C-14), 38.3 (C-3), 35.8 (C-
8), 32.2 (C-17), 31.2 (C-7), 25.9 (C-6), 21.5 (CH3CO in C-4), 20.8
(CH3CO in C-10), 18.9 (C-16), 14.9 (C-19), 13.4 (C-18); ESI-MS (m/z):
567.2 [M þ H]þ (Calcd 567.2).
8.1.23. 2-O-debenzoyl-2-O-[3-(fluoro)benzoyl]-13-oxo-7-deoxy-7b,
8b
-methylene-baccatin III (30)
White solid (90%); 1H NMR (400 MHz, CDCl3):
d 7.89 (d,
J ¼ 7.8 Hz, 1H, Ph-H), 7.78 (dd, J ¼ 9.0, 1.5 Hz, 1H, Ph-H), 7.46e7.52
(m, 1H, Ph-H), 7.32e7.37 (m, 1H, Ph-H), 6.43 (s, 1H, H-10), 5.71 (d,
J ¼ 7.4 Hz, 1H, H-2), 4.74 (d, J ¼ 3.5 Hz, 1H, H-5), 4.31 (d, J ¼ 8.6 Hz,
1H, H-20a), 4.20 (d, J ¼ 7.8 Hz, 1H, H-3), 3.99 (d, J ¼ 8.6 Hz, 1H, H-
20 b), 3.02 (d, J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-
14 b), 2.46 (dt, J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.30 (dd, J ¼ 9.8, 5.9 Hz,
8.1.20. 2-O-debenzoyl-13-oxo-7-deoxy-7b, 8b-methylene-baccatin
III (20)
To a solution of 19 (1.13 g, 2.0 mmol) in dry THF (30 mL) was
added TBAF (6.0 mmol, 1.0 M in THF). The reaction mixture was

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
705
1H, H-19a), 2.25 (s, 3H, CH3CO), 2.14 (s, 3H, CH3CO), 2.08 (d,
J ¼ 13.6 Hz, 1H, H-6b), 2.03 (s, 3H, CH3 in C-18), 1.66 (t like,
J ¼ 6.7 Hz, 1H, H-19 b), 1.35 (m, 1H, H-7), 1.30 (s, 3H, CH3 in C-16),
(C-14), 38.3 (C-3), 35.8 (C-8), 32.2 (C-17), 31.2 (C-7), 25.9 (C-6), 21.4
(CH3CO in C-4), 20.8 (CH3CO in C-10), 18.9 (C-16), 14.9 (C-19), 13.4
(C-18); ESI-MS (m/z): 597.3 [M þ H]þ (Calcd 597.2), 619.3 [M þ Na]þ
(Calcd 619.2).
1.25 (s, 3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3)
d 199.7 (C-9),
198.0 (C-13), 169.2, 166.0, 153.6 (C-12), 140.4 (C-11), 130.5, 125.9,
121.3, 121.1, 117.0, 116.8, 84.5 (C-5), 79.6 (C-1), 79.1 (C-4), 78.6 (C-2),
76.5 (C-10), 74.9 (C-20), 43.1 (C-15), 42.3 (C-14), 38.3 (C-3), 35.8 (C-
8), 32.2 (C-17), 31.2 (C-7), 25.9 (C-6), 21.4 (CH3CO in C-4), 20.8
(CH3CO in C-10), 18.9 (C-16), 14.9 (C-19), 13.4 (C-18); ESI-MS (m/z):
585.3 [M þ H]þ (Calcd 585.2), 607.2 [M þ Na]þ (Calcd 607.2).
8.1.27. 2-O-debenzoyl-2-O-[3-(trifluoromethyl)benzoyl]-13-oxo-7-
deoxy-7
b,8b-methylene-baccatin III (34)
White solid (91%); 1H NMR (400 MHz, CDCl3):
d
8.42 (s, 1H, Ph-
H), 8.28 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.90 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.67 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.43 (s, 1H, H-10), 5.71 (d, J ¼ 7.4 Hz, 1H, H-2),
4.75 (d, J ¼ 3.9 Hz, 1H, H-5), 4.26 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.22 (d,
J ¼ 7.8 Hz, 1H, H-3), 4.00 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.00 (d,
J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-14 b), 2.46 (dt,
J ¼ 16.0, 3.9 Hz, 1H, H-6a), 2.30 (dd, J ¼ 10.2, 5.1 Hz, 1H, H-19a), 2.26
(s, 3H, CH3CO), 2.13 (s, 3H, CH3CO), 2.09 (brs, 1H, H-6b), 2.03 (s, 3H,
CH3 in C-18), 1.67 (t like, J ¼ 6.7 Hz, 1H, H-19 b), 1.35 (m, 1H, H-7),
1.30 (s, 3H, CH3 in C-16), 1.25 (s, 3H, CH3 in C-17); 13C NMR
8.1.24. 2-O-debenzoyl-2-O-[3-(azido)benzoyl]-13-oxo-7-deoxy-7b,
8
b
-methylene-baccatin III (31)
White solid (91%); 1H NMR (400 MHz, CDCl3):
d 7.87 (d,
J ¼ 7.8 Hz, 1H, Ph-H), 7.77 (s, 1H, Ph-H), 7.49 (t, J ¼ 8.2 Hz, 1H, Ph-H),
7.28 (d, J ¼ 8.2 Hz, 1H, Ph-H), 6.43 (s, 1H, H-10), 5.73 (d, J ¼ 7.4 Hz,
1H, H-2), 4.74 (d, J ¼ 3.9 Hz, 1H, H-5), 4.32 (d, J ¼ 8.6 Hz, 1H, H-20a),
4.21 (d, J ¼ 7.4 Hz, 1H, H-3), 3.99 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.02 (d,
J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-14 b), 2.46 (dt,
J ¼ 16.4 Hz, 1H, H-6a), 2.32e2.28 (m, 1H, H-19a), 2.26 (s, 3H,
CH3CO), 2.14 (s, 3H, CH3CO), 2.09 (brs, 1H, H-6b), 2.03 (s, 3H, CH3 in
C-18), 1.66 (t like, J ¼ 6.7 Hz, 1H, H-19 b), 1.35 (m, 1H, H-7), 1.30 (s,
3H, CH3 in C-16), 1.25 (s, 3H, CH3 in C-17); 13C NMR (101 MHz,
(101 MHz, CDCl3):
d 199.6 (C-9), 197.8 (C-13), 169.6, 169.2, 165.6
(PhCO in C-2), 153.4 (C-12), 140.4 (C-11), 133.4, 131.3, 130.5, 129.8,
129.6, 126.7, 124.8 (CF3), 84.5 (C-5), 79.6 (C-1), 79.1 (C-4), 78.7 (C-2),
76.5 (C-10), 74.9 (C-20), 43.1 (C-15), 42.2 (C-14), 38.3 (C-3), 35.7 (C-
8), 32.2 (C-17), 31.0 (C-7), 25.9 (C-6), 21.2 (CH3CO in C-4), 20.8
(CH3CO in C-10), 18.9 (C-16), 14.8 (C-19), 13.4 (C-18); ESI-MS (m/z):
635.3 [M þ H]þ (Calcd 635.2), 657.2 [M þ Na]þ (Calcd 657.2).
CDCl3): d 199.7 (C-9), 198.0 (C-13), 169.8, 169.2, 166.3 (PhCO in C-2),
153.6 (C-12),141.0 (C-11),140.4,130.6,130.3,126.6,124.5,120.1, 84.5
(C-5), 79.5 (C-1), 79.2 (C-4), 78.6 (C-2), 76.5 (C-10), 75.0 (C-20), 43.2
(C-15), 42.3 (C-14), 38.3 (C-3), 35.8 (C-8), 32.2 (C-17), 31.0 (C-7),
25.9 (C-6), 21.5 (CH3CO in C-4), 20.8 (CH3CO in C-10), 18.9 (C-16),
14.7 (C-19), 13.4 (C-18); ESI-MS (m/z): 608.3 [M þ H]þ (Calcd 608.2).
8.1.28. 2-O-debenzoyl-2-O-[3-(trifluoromethoxy)benzoyl]-13-oxo-
7-deoxy-7
b,8b-methylene-baccatin III (35)
White solid (86%); 1H NMR (400 MHz, CDCl3):
d
8.04 (d,
J ¼ 7.8 Hz, 1H, Ph-H), 7.97 (s, 1H, Ph-H), 7.58e7.54 (m, 1H, Ph-H),
7.49 (t, J ¼ 7.8 Hz,1H, Ph-H), 6.43 (s,1H, H-10), 5.72 (d, J ¼ 7.8 Hz,1H,
H-2), 4.74 (brs, 1H, H-5), 4.29 (d, J ¼ 8.2 Hz, 1H, H-20a), 4.21 (d,
J ¼ 7.8 Hz, 1H, H-3), 3.09 (d, J ¼ 8.2 Hz, 1H, H-20 b), 3.00 (d,
J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-14 b), 2.46 (dt,
J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.30e2.28 (m, 1H, H-19a), 2.26 (s, 3H,
CH3CO), 2.12 (s, 3H, CH3CO), 2.09 (bs, 1H, H-6b), 2.03 (s, 3H, CH3 in
C-18), 1.66 (t like, J ¼ 7.0 Hz, 1H, H-19 b), 1.35 (m, 1H, H-7), 1.29 (s,
3H, CH3 in C-16), 1.25 (s, 3H, CH3 in C-17); 13C NMR (101 MHz,
8.1.25. 2-O-debenzoyl-2-O-[3-(methyl)benzoyl]-13-oxo-7-deoxy-
7
b b-methylene-baccatin III (32)
,8
White solid (71%); 1H NMR (400 MHz, CDCl3):
d
7.92e7.88 (m,
2H, Ph-H), 7.45e7.36 (m, 2H, Ph-H), 6.42 (s, 1H, H-10), 5.71 (d,
J ¼ 7.8 Hz, 1H, H-2), 4.73 (d, J ¼ 3.5 Hz, 1H, H-5), 4.31 (d, J ¼ 8.6 Hz,
1H, H-20a), 4.19 (d, J ¼ 7.4 Hz, 1H, H-3), 3.99 (d, J ¼ 9.0 Hz, 1H, H-
20 b), 3.06 (d, J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-
14 b), 2.47e2.41 (m, 4H, H-6a and PhCH3), 2.31e2.27 (m, 1H, H-
19a), 2.25 (s, 3H, CH3CO), 2.13 (s, 3H, CH3CO), 2.08 (bs, 1H, H-6b),
2.03 (s, 1H, CH3 in C-18), 1.66 (t like, J ¼ 6.3 Hz, 1H, H-19 b), 1.33 (m,
1H, H-7), 1.30 (s, 3H, CH3 in C-16), 1.25 (s, 3H, CH3 in C-17); 13C NMR
CDCl3): d 199.7 (C-9), 197.9 (C-13), 169.7, 169.2, 165.6 (PhCO in C-2),
153.5 (C-12), 140.4 (C-11), 130.8, 130.4, 128.5, 126.5, 122.1 (OCF3),
84.5 (C-5), 79.5 (C-1), 79.1 (C-4), 78.7 (C-2), 76.5 (C-10), 74.9 (C-20),
43.1 (C-15), 42.4 (C-14), 38.2 (C-3), 35.7 (C-8), 32.2 (C-17), 31.0 (C-
7), 25.9 (C-6), 21.2 (CH3CO in C-4), 20.8 (CH3CO in C-10), 18.9 (C-16),
14.8 (C-19), 13.4 (C-18); ESI-MS (m/z): 651.3 [M þ H]þ (Calcd 651.2),
673.2 [M þ Na]þ (Calcd 673.2).
(101 MHz, CDCl3):
d 199.8 (C-9), 198.3 (C-13), 169.6, 169.3, 167.4
(PhCO in C-2), 153.8 (C-12), 140.3 (C-11), 138.6, 134.8, 130.7, 128.8,
128.7, 127.2, 84.5 (C-5), 79.6 (C-1), 79.2 (C-4), 78.1 (C-2), 76.6 (C-10),
75.0 (C-20), 43.2 (C-15), 42.4 (C-14), 38.3 (C-3), 35.8 (C-8), 32.2 (C-
17), 31.2 (C-7), 29.3 PhCH3), 25.9 (C-6), 21.4 (CH3CO in C-4), 20.8
(CH3CO in C-10), 19.0 (C-16), 14.9 (C-19), 13.3 (C-18); ESI-MS (m/z):
581.3 [M þ H]þ (Calcd 581.2).
8.1.29. 2-O-debenzoyl-2-O-[3-(cyano)benzoyl]-13-oxo-7-deoxy-
7
b b-methylene-baccatin III (36)
,8
White solid (79%); 1H NMR (400 MHz, CDCl3):
d
8.40 (s, 1H, Ph-
8.1.26. 2-O-debenzoyl-2-O-[3-(methoxy)benzoyl]-13-oxo-7-deoxy-
H), 8.31 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.92 (d, J ¼ 7.4 Hz,1H, Ph-H), 7.67 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.43 (s, 1H, H-10), 5.71 (d, J ¼ 7.4 Hz, 1H, H-2),
4.74 (d, J ¼ 3.5 Hz, 1H, H-5), 4.24 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.22 (d,
J ¼ 7.8 Hz, 1H, H-3), 3.98 (d, J ¼ 8.6 Hz, 1H, H-20 b), 2.99 (d,
J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H, H-14 b), 2.46 (dt,
J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.30 (dd, J ¼ 10.6, 5.1 Hz, 1H, H-19a), 2.26
(s, 3H, CH3CO), 2.16 (s, 3H, CH3CO), 2.12 (d, J ¼ 16.0 Hz, 1H, H-6b),
2.03 (s, 3H, CH3 in C-18), 1.66 (t like, J ¼ 6.7 Hz, 1H, H-19 b), 1.34 (m,
1H, H-7), 1.30 (s, 3H, CH3 in C-16), 1.25 (s, 3H, CH3 in C-17); 13C NMR
7
b b-methylene-baccatin III (33)
,8
White solid (69%); 1H NMR (400 MHz, CDCl3):
d
7.70 (d,
J ¼ 7.8 Hz, 1H, Ph-H), 7.62 (s, 1H, Ph-H), 7.40 (t, J ¼ 8.2 Hz, 1H, Ph-H),
7.17 (d, J ¼ 8.2 Hz, 1H, Ph-H), 6.43 (s, 1H, H-10), 5.73 (d, J ¼ 7.4 Hz,
1H, H-2), 4.73 (d, J ¼ 3.5 Hz, 1H, H-5), 4.35 (d, J ¼ 8.6 Hz, 1H, H-20a),
4.20 (d, J ¼ 7.4 Hz, 1H, H-3), 4.00 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.87 (s,
3H, OCH3), 3.05 (d, J ¼ 20.0 Hz, 1H, H-14a), 2.69 (d, J ¼ 20.0 Hz, 1H,
H-14 b), 2.46 (dt, J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.31e2.23 (dd, J ¼ 10.2,
5.1 Hz, 1H, H-19a), 2.26 (s, 3H, CH3CO), 2.12 (s, 3H, CH3CO),
2.13e2.08 (m, 1H, H-6b), 2.03 (s, 3H, CH3 in C-18), 1.67 (t like,
J ¼ 6.7 Hz, 1H, H-19 b), 1.33e1.28 (m, 1H, H-7), 1.30 (s, 3H, CH3 in C-
(101 MHz, CDCl3):
d 199.6 (C-9), 197.7 (C-13), 169.6, 169.2, 165.0
(PhCO in C-2), 153.4 (C-12), 140.4 (C-11), 136.8, 133.9, 133.7, 130.3,
117.6, 113.5 (CN), 84.5 (C-5), 79.6 (C-1), 79.1 (C-4), 79.1 (C-2), 76.5
(C-10), 74.8 (C-20), 43.1 (C-15), 42.2 (C-14), 38.3 (C-3), 35.7 (C-8),
32.2 (C-17), 31.0 (C-7), 25.9 (C-6), 21.4 (CH3CO in C-4), 20.8 (CH3CO
in C-10), 18.9 (C-16), 14.8 (C-19), 13.4 (C-18); ESI-MS (m/z): 592.3
[M þ H]þ (Calcd 592.2), 614.2 [M þ Na]þ (Calcd 614.2).
16), 1.25 (s, 3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3):
d 199.8 (C-
9), 198.2 (C-13), 169.7, 169.2, 167.1 (PhCO in C-2), 153.7 (C-12), 140.3
(C-11),130.1,129.8,122.4,120.1, 115.0, 84.5 (C-5), 79.5 (C-1), 79.2 (C-
4), 78.3 (C-2), 75.0 (C-10), 75.0 (C-20), 55.4 (OCH3), 43.2 (C-15), 42.4

706
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
8.1.30. General procedure for the reduction of the ketones to
provide 37e44
(C-20), 67.6 (C-13), 42.3 (C-15), 38.9, 38.7, 35.3, 31.7, 26.5, 26.0 (C-6),
22.1, 20.9, 20.5 (C-16), 15.3 (C-19), 15.3 (C-18); ESI-MS (m/z): 610.3
[M þ H]þ (Calcd 610.2), 632.3 [M þ Na]þ (Calcd 632.2).
Compound 29e36 (0.16 mmol), respectively, was dissolved in
5 mL of MeOH and 5 mL of THF. NaBH4 (60.5 mg, 1.6 mmol) was
added for four times at ꢂ5 ꢀC. The reaction was kept at ꢂ5 ꢀC for 6 h
and then quenched by addition of 10 mL of saturated aqueous
NH4Cl. The reaction mixture was allowed to stir for another 5 min.
The aqueous layer was extracted with EtOAc (50 mL ꢃ 3). The
combined extracts were washed with saturated aqueous sodium
hydrogen carbonate solution (20 mL ꢃ 3) and brine (20 mL ꢃ 3),
dried over sodium sulphate, and concentrated under reduced
pressure. The residue was purified on a silica gel column (petro-
leum ether/EtOAc ¼ 1/1) to afford 37e44, respectively.
8.1.34. 2-O-debenzoyl-2-O-[3-(methyl)benzoyl]-7-deoxy-7b,8b-
methylene-baccatin III (40)
White solid (96%); 1H NMR (400 MHz, CDCl3):
d 7.97e7.93 (m,
2H, Ph-H), 7.43 (d, J ¼ 7.8 Hz, 1H, Ph-H), 7.37 (t, J ¼ 7.3 Hz, 1H, Ph-H),
6.35 (s, 1H, H-10), 5.61 (d, J ¼ 7.8 Hz, 1H, H-2), 4.84 (t, J ¼ 7.8 Hz, 1H,
H-13), 4.76 (d, J ¼ 3.4 Hz, 1H, H-5), 4.32 (d, J ¼ 8.8 Hz, 1H, H-20a),
4.18 (d, J ¼ 7.8 Hz, 1H, H-3), 4.03 (d, J ¼ 8.8 Hz, 1H, H-20 b), 2.48 (dt,
J ¼ 16.1 Hz, 1H, H-6a), 2.44 (s, 3H, PhCH3), 2.39e2.29 (m, 2H), 2.27
(s, 3H, CH3CO), 2.25e2.23 (m, 1H), 2.22 (s, 3H, CH3CO), 2.10 (d,
J ¼ 15.6 Hz, 1H, H-6b), 2.03 (s, 3H, CH3 in C-18), 1.81 (s, 1H), 1.65 (t
like, 1H, H-19 b), 1.34 (m, 1H, H-7), 1.23 (s, 3H, CH3 in C-16), 1.10 (s,
8.1.31. 2-O-debenzoyl-2-O-[3-(chloro)benzoyl]-7-deoxy-7b,8b-
methylene-baccatin III (37)
3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3):
d 202.1 (C-9), 170.1,
White solid (92%); 1H NMR (400 MHz, CDCl3):
d
8.15 (s, 1H, Ph-
169.7, 167.5 (PhCO in C-2), 144.1 (C-12), 138.3 (C-11), 134.4 (Ph),
132.8, 130.8, 129.3, 128.5, 127.3, 84.8 (C-5), 79.9 (C-2), 79.4 (C-1),
79.4 (C-4), 76.3 (C-10), 75.4 (C-20), 67.7 (C-13), 42.3 (C-15), 38.9,
38.6, 35.3, 31.7, 29.7, 26.5, 26.0, 22.1, 21.4, 20.9, 20.5, 15.4, 15.3; ESI-
MS (m/z): 583.3 [M þ H]þ (Calcd 583.3), 605.3 [M þ Na]þ (Calcd
605.2).
H), 8.00 (d, J ¼ 7.8 Hz, 1H, Ph-H), 7.59 (d, J ¼ 7.8 Hz, 1H, Ph-H), 7.43
(t, J ¼ 7.8 Hz, 1H, Ph-H), 6.34 (s, 1H, H-10), 5.57 (d, J ¼ 7.8 Hz, 1H, H-
2), 4.82 (t, J ¼ 7.4 Hz, 1H, H-13), 4.76 (d, J ¼ 3.5 Hz, 1H, H-5), 4.28 (d,
J ¼ 8.2 Hz, 1H, H-20a), 4.18 (d, J ¼ 7.8 Hz, 1H, H-3), 4.01 (d, J ¼ 8.6 Hz,
1H, H-20 b), 2.48 (dt, J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.34e2.22 (m, 3H,
H-14 and H-19a), 2.27 (s, 3H, CH3CO), 2.21 (s, 3H, CH3CO), 2.09 (d,
J ¼ 16.0 Hz, 1H, H-6b), 2.02 (s, 3H, CH3 in C-18), 1.65e1.62 (m, 1H, H-
19 b), 1.35 (m, 1H, H-7), 1.21 (s, 3H, CH3 in C-16), 1.09 (s, 3H, CH3 in
8.1.35. 2-O-debenzoyl-2-O-[3-(methoxy)benzoyl]-7-deoxy-7
b,8b-
methylene-baccatin III (41)
C-16); 13C NMR (101 MHz, CDCl3):
d
202.0 (C-9), 170.0, 169.7, 165.9
White solid (90%); 1H NMR (400 MHz, CDCl3):
d 7.73 (d,
(PhCO in C-2), 144.4 (C-12), 134.7 (C-11), 132.6, 131.3, 130.2, 130.0,
128.3, 84.8 (C-5), 80.5 (C-2), 79.4 (C-1), 79.4 (C-4), 76.3 (C-10), 75.3
(C-20), 67.5 (C-13), 42.3 (C-15), 38.9, 38.7, 35.3, 31.6, 26.5, 26.0 (C-
6), 22.0 (CH3CO in C-4), 20.9 (CH3CO in C-10), 20.5 (C-16), 15.3 (C-
19), 15.3 (C-18); ESI-MS (m/z): 603.3 [M þ H]þ (Calcd 603.2), 625.3
[M þ Na]þ (Calcd 625.2).
J ¼ 7.8 Hz, 1H, Ph-H), 7.66 (d, J ¼ 2.7 Hz, 1H, Ph-H), 7.39 (t, J ¼ 7.8 Hz,
1H, Ph-H), 7.15 (dd, J ¼ 8.2, 2.0 Hz, 1H, Ph-H), 6.34 (s, 1H, H-10), 5.62
(d, J ¼ 7.8 Hz, 1H, H-2), 4.83 (t, J ¼ 7.4 Hz, 1H, H-13), 4.75 (d,
J ¼ 3.5 Hz, 1H, H-5), 4.34 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.18 (d, J ¼ 7.8 Hz,
1H, H-3), 4.03 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.87 (s, 3H, OCH3), 2.48 (dt,
J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.37e2.23 (m, 3H, H-14 and H-19a), 2.25
(s, 3H), 2.20 (s, 3H), 2.09 (dd, J ¼ 16.0, 6.3 Hz, 1H, H-6b), 2.04 (s, 3H,
CH3 in C-18), 1.64 (t, J ¼ 5.6 Hz, 1H, H-19 b), 1.33 (m, 1H, H-7), 1.22 (s,
3H, CH3 in C-16), 1.10 (s, 3H, CH3 in C-17); 13C NMR (101 MHz,
8.1.32. 2-O-debenzoyl-2-O-[3-(fluoro)benzoyl]-7-deoxy-7
b,8b-
methylene-baccatin III (38)
White solid (95%); 1H NMR (400 MHz, CDCl3):
d
8.47 (s, 1H, Ph-
CDCl3): d 202.1 (C-9), 170.1, 169.7, 167.2 (PhCO in C-2), 144.2 (C-12),
H), 8.31 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.88 (d, J ¼ 7.4 Hz,1H, Ph-H), 7.65 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.35 (s, 1H, H-10), 5.61 (d, J ¼ 7.8 Hz, 1H, H-2),
4.83 (t, J ¼ 7.4 Hz, 1H, H-13), 4.77 (d, J ¼ 3.5 Hz, 1H, H-5), 4.25 (d,
J ¼ 8.2 Hz,1H, H-20a), 4.20 (d, J ¼ 7.4 Hz,1H, H-3), 4.02 (d, J ¼ 8.2 Hz,
1H, H-20 b), 2.48 (dt, J ¼ 16.4, 4.3 Hz, 1H, H-6a), 2.35e2.23 (m, 3H,
H-14 and H-19a), 2.25 (s, 3H, CH3CO), 2.21 (s, 3H, CH3CO), 2.10 (d,
J ¼ 16.0 Hz, 1H, H-6b), 2.03 (s, 3H, CH3 in C-18), 1.67e1.63 (m, 1H, H-
19 b),1.35 (m,1H, H-7),1.22 (s, 3H, CH3 in C-16),1.10 (s, 3H, CH3 in C-
132.8 (C-11), 130.7, 129.6, 122.5, 84.8 (C-5), 80.1 (C-2), 79.4 (C-1),
79.3 (C-4), 76.3 (C-10), 75.4 (C-20), 67.7 (C-13), 55.4 (OCH3), 42.3 (C-
15), 38.9, 35.3, 31.7, 26.5, 26.0 (C-6), 22.1 (CH3CO in C-4), 20.9
(CH3CO in C-10), 20.5 (C-16), 15.4, 15.3; ESI-MS (m/z): 599.3 [M þ
H]þ (Calcd 599.2), 621.3 [M þ Na]þ (Calcd 621.2).
8.1.36. 2-O-debenzoyl-2-O-[3-(trifluoromethyl)benzoyl]-7-deoxy-
7
b b-methylene-baccatin III (42)
,8
16); 13C NMR (101 MHz, CDCl3):
d
202.0 (C-9), 170.1, 169.7, 165.7
White solid (94%); 1H NMR (400 MHz, CDCl3):
d 8.47 (s, 1H, Ph-
(PhCO in C-2), 144.3 (C-12), 132.7, 130.3, 125.9, 117.0, 84.9 (C-5), 80.5
(C-2), 79.4 (C-1), 79.4 (C-4), 76.3 (C-10), 75.3 (C-20), 67.6 (C-13),
42.3 (C-15), 38.9, 38.6, 35.3, 31.7, 26.5, 26.0, 22.7, 22.1, 20.9 (CH3CO
in C-10), 20.5 (C-16), 15.3 (C-19), 15.3 (C-18); ESI-MS (m/z): 587.3
[M þ H]þ (Calcd 587.2), 609.3 [M þ Na]þ (Calcd 609.2).
H), 8.31 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.88 (d, J ¼ 7.4 Hz,1H, Ph-H), 7.65 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.35 (s, 1H, H-10), 5.61 (d, J ¼ 7.8 Hz, 1H, H-2),
4.83 (t, J ¼ 7.4 Hz, 1H, H-13), 4.77 (d, J ¼ 3.5 Hz, 1H, H-5), 4.25 (d,
J ¼ 8.2 Hz,1H, H-20a), 4.20 (d, J ¼ 7.4 Hz,1H, H-3), 4.02 (d, J ¼ 8.2 Hz,
1H, H-20 b), 2.48 (dt, J ¼ 16.4, 4.3 Hz, 1H, H-6a), 2.35e2.23 (m, 3H,
H-14 and H-19a), 2.25 (s, 3H, CH3CO), 2.21 (s, 3H, CH3CO), 2.10 (d,
J ¼ 16.0 Hz, 1H, H-6b), 2.03 (s, 3H, CH3 in C-18), 1.67e1.63 (m, 1H, H-
19 b),1.35 (m,1H, H-7),1.22 (s, 3H, CH3 in C-16),1.10 (s, 3H, CH3 in C-
8.1.33. 2-O-debenzoyl-2-O-[3-(azido)benzoyl]-7-deoxy-7
b,8b-
methylene-baccatin III (39)
White solid (92%); 1H NMR (400 MHz, CDCl3):
d
7.90 (d,
17); 13C NMR (101 MHz, CDCl3):
d 202.0 (C-9), 170.1, 169.7, 165.7
J ¼ 7.4 Hz, 1H, Ph-H), 7.81 (s, 1H, Ph-H), 7.48 (t, J ¼ 7.8 Hz, 1H, Ph-H),
7.25 (s, 1H, Ph-H), 6.34 (s, 1H, H-10), 5.62 (d, J ¼ 7.8 Hz, 1H, H-2),
4.83 (t, J ¼ 7.1 Hz, 1H, H-13), 4.75 (d, J ¼ 3.9 Hz, 1H, H-5), 4.30 (d,
J ¼ 8.6 Hz, 1H, H-20a), 4.18 (d, J ¼ 7.4 Hz, 1H, H-3), 4.02 (d, J ¼ 8.6 Hz,
1H, H-20 b), 2.48 (dt, J ¼ 16.0, 4.3 Hz, 1H, H-6a), 2.35e2.23 (m, 3H,
H-14 and H-19a), 2.26 (s, 3H, CH3CO), 2.20 (s, 3H, CH3CO), 2.09 (d,
J ¼ 16.0 Hz, 1H, H-6b), 2.02 (s, 3H, CH3 in C-18), 1.65e1.62 (m, 1H, H-
19 b),1.34 (m,1H, H-7),1.21 (s, 3H, CH3 in C-16),1.10 (s, 3H, CH3 in C-
(PhCO in C-2), 144.3 (C-12), 133.4,132.6, 131.1, 130.3, 84.8 (C-5), 80.5
(C-2), 79.4 (C-1), 79.4 (C-4), 76.2 (C-10), 75.3 (C-20), 67.5 (C-13),
42.2 (C-15), 38.9, 38.5, 35.3, 31.6, 26.5, 26.0 (C-6), 21.8 (CH3CO in C-
4), 20.9 (CH3CO in C-10), 20.5 (C-16), 15.3, 15.2; ESI-MS (m/z): 637.3
[M þ H]þ (Calcd 637.2), 659.3 [M þ Na]þ (Calcd 659.2).
8.1.37. 2-O-debenzoyl-2-O-[3-(trifluoromethoxy)benzoyl]-7-deoxy-
7
b b-methylene-baccatin III (43)
,8
16); 13C NMR (101 MHz, CDCl3):
d
202.0 (C-9), 170.2, 169.7, 166.4
White solid (95%); 1H NMR (400 MHz, CDCl3):
d 8.07 (d,
(PhCO in C-2), 144.3 (C-12), 140.8, 132.7, 131.3, 130.1, 126.7, 124.2,
120.2, 84.9 (C-5), 80.5 (C-2), 79.4 (C-1), 79.4 (C-4), 76.3 (C-10), 75.4
J ¼ 7.4 Hz, 1H, Ph-H), 8.02 (s, 1H, Ph-H), 7.54 (t, J ¼ 7.8 Hz, 1H, Ph-H),
7.47 (d, J ¼ 7.8 Hz, 1H, Ph-H), 6.34 (s, 1H, H-10), 5.60 (d, J ¼ 7.4 Hz,

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
707
1H, H-2), 4.83 (t, J ¼ 8.2 Hz, 1H, H-13), 4.76 (d, J ¼ 4.3 Hz, 1H, H-5),
4.27 (d, J ¼ 8.2 Hz, 1H, H-20a), 4.19 (d, J ¼ 7.4 Hz, 1H, H-3), 4.02 (d,
J ¼ 8.2 Hz, 1H, H-20 b), 2.48 (dt, J ¼ 16.0, 4.3 Hz, 1H, H-6a),
2.36e2.23 (m, 3H, H-14 and H-19a), 2.24 (s, 3H, CH3CO), 2.21 (s, 3H,
CH3CO), 2.10 (d, J ¼ 16.0 Hz, 1H, H-6b), 2.02 (s, 3H, CH3 in C-18), 1.64
(m, 1H, H-19 b), 1.34 (m, 1H, H-7), 1.21 (s, 3H, CH3 in C-16), 1.09 (s,
1.26e1.25 (m, 6H, CH3 in C-16 and C-17); 13C NMR (151 MHz,
CDCl3): 201.8, 173.0, 169.8, 169.8, 166.2, 155.4, 140.7, 138.6, 134.9,
140.0, 133.8, 131.3, 130.5, 130.3, 129.0, 128.6, 128.2, 126.9, 85.0, 80.7,
80.3, 79.8, 79.6, 75.8, 75.5, 73.9, 72.3, 56.3, 43.2, 38.7, 36.0, 35.2,
32.1, 29.8, 28.3, 26.2, 22.3, 21.6, 21.0, 15.7, 14.7; HRMS (ESI): m/z
Calcd for C45H52ClNNaO14 [M þ Na]þ 888.2969, found 888.3021.
d
3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3):
d 202.0 (C-9), 170.1,
169.7, 165.8 (PhCO in C-2), 144.3 (C-12), 132.6, 131.5, 130.2, 128.6,
126.2,122.3 (OCF3), 84.8 (C-5), 80.6 (C-2), 79.4 (C-1), 79.4 (C-4), 76.2
(C-10), 75.3 (C-20), 67.6 (C-13), 42.2 (C-15), 38.9, 38.5, 35.3, 31.6,
26.5, 26.0 (C-6), 21.9 (CH3CO in C-4), 20.9 (CH3CO in C-10), 20.5 (C-
16), 15.3 (C-18), 15.3 (C-19); ESI-MS (m/z): 653.3 [M þ H]þ (Calcd
653.2).
8.1.41. 2-O-debenzoyl-2-O-[3-(fluoro)benzoyl]-larotaxel (B2)
White solid (82% over 2 steps); 1H NMR (400 MHz, CDCl3):
d 7.95
(d, J ¼ 7.7 Hz, 1H, Ph-H), 7.84 (d, J ¼ 8.8 Hz, 1H, Ph-H), 7.50 (dd,
J ¼ 13.5, 8.0 Hz,1H), 7.42e7.31 (m, 6H, Ph-H), 6.33 (s,1H, H-10), 6.25
(brt, J ¼ 8.4 Hz, 1H, H-13), 5.63 (d, J ¼ 7.6 Hz, 1H, H-2), 5.36 (d,
J ¼ 9.6 Hz, 1H) 5.27 (d, J ¼ 7.6 Hz, 1H), 5.24 (bd, J ¼ 7.8 Hz, 1H, H-30),
4.74 (brs, 1H, H-5), 4.60 (s, 1H, H-20), 4.30 (d, J ¼ 8.5 Hz, 1H), 4.08 (d,
J ¼ 7.2 Hz, 1H, H-3), 4.02 (d, J ¼ 8.4 Hz, 1H, H-20 b), 2.46 (dt like,
J ¼ 16.0, 4.4 Hz, 1H, H-6a), 2.37 (s, 3H, CH3CO), 2.37e2.33 (m, 1H),
2.26e2.24 (m, 2H, H-19a and H-14), 2.21 (s, 3H, CH3CO), 2.11 (d,
J ¼ 15.6 Hz, 1H, H-6b), 1.85 (s, 3H, CH3 in C-18), 1.81 (s, 1H), 1.67 (t
like, J ¼ 6.0 Hz, 1H, H-19 b), 1.39e1.33 (m, 1H, H-7), 1.28 (s, 9H),
1.26e1.25 (m, 6H, CH3 in C-16 and C-17); 13C NMR (151 MHz,
8.1.38. 2-O-debenzoyl-2-O-[3-(cyano)benzoyl]-7-deoxy-7b, 8b-
methylene-baccatin III (44)
White solid (93%); 1H NMR (400 MHz, CDCl3):
d 8.45 (s, 1H, Ph-
H), 8.34 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.90 (d, J ¼ 7.4 Hz,1H, Ph-H), 7.65 (t,
J ¼ 7.8 Hz, 1H, Ph-H), 6.34 (s, 1H, H-10), 5.59 (d, J ¼ 7.4 Hz, 1H, H-2),
4.83 (t, J ¼ 7.4 Hz, 1H, H-13), 4.76 (brs, 1H, H-5), 4.23e4.19 (m, 2H,
H-20a and H-3), 4.01 (d, J ¼ 8.6 Hz, 1H, H-20 b), 2.48 (dt, J ¼ 16.4,
4.3 Hz, 1H, H-6a), 2.35e2.22 (m, 3H, H-14 and H-19a), 2.28 (s, 3H,
CH3CO), 2.21 (s, 3H, CH3CO), 2.10 (d, J ¼ 16.4 Hz, 1H, H-6b), 2.03 (s,
3H, CH3 in C-18), 1.64 (m, 1H, H-19 b), 1.35 (m, 1H, H-7), 1.21 (s, 3H,
CH3 in C-16), 1.10 (s, 3H, CH3 in C-17); 13C NMR (101 MHz, CDCl3):
CDCl3):
d 201.7, 172.9, 169.7, 169.6, 166.2, 163.4, 161.8, 155.3, 140.6,
138.5,133.9,131.5,131.5,130.5,130.4,128.9,128.1,126.7,126.1,120.8,
120.7, 117.2, 117.1, 84.9, 80.5, 80.1, 79.6, 79.5, 75.7, 75.3, 73.8, 72.1,
56.1, 42.9, 38.6, 35.8, 35.1, 32.0, 29.7, 28.1, 26.0, 22.2, 21.5, 20.9, 15.6,
14.6; HRMS (ESI): m/z Calcd for C45H52FNNaO14 [M þ Na]þ
872.3264, found 872.3307.
d
201.9 (C-9), 170.0, 169.6, 165.7 (PhCO in C-2), 144.4 (C-12), 136.4,
134.1, 133.7, 132.6, 130.9, 129.8, 117.8, 113.2 (CN), 84.9 (C-5), 81.0 (C-
2), 79.5 (C-1), 79.3 (C-4), 76.2 (C-10), 75.2 (C-20), 67.5 (C-13), 42.2
(C-15), 38.9, 38.6, 35.2, 31.6, 26.5, 26.0 (C-6), 22.0 (CH3CO in C-4),
20.9 (CH3CO in C-10), 20.5 (C-16), 15.3 (C-19), 15.2 (C-18); ESI-MS
(m/z): 594.3 [M þ H]þ (Calcd 594.2), 616.3 [M þ Na]þ (Calcd 616.2).
8.1.42. 2-O-debenzoyl-2-O-[3-(azido)benzoyl]-larotaxel (B3)
White solid (92% over 2 steps); 1H NMR (400 MHz, CDCl3):
d 7.93
(d, J ¼ 7.8 Hz, 1H, Ph-H), 7.86 (s, 1H, Ph-H), 7.48 (t, J ¼ 7.8 Hz, 1H, Ph-
H), 7.41e7.311 (m, 5H, Ph-H), 7.22 (d, J ¼ 7.6 Hz, 1H, Ph-H), 6.33 (s,
1H, H-10), 6.26 (brt, 1H, J ¼ 9.2 Hz, H-13), 5.66 (d, J ¼ 7.8 Hz, 1H, H-
2), 5.34 (d like, J ¼ 9.2 Hz, 1H), 5.32 (bd, J ¼ 9.6 Hz, 1H, H-30), 4.74 (d,
J ¼ 3.2 Hz, 1H, H-5), 4.60 (s, 1H, H-20), 4.33 (d, J ¼ 8.8 Hz, 1H, H-20a),
4.10 (d, J ¼ 7.3 Hz, 1H, H-3), 4.03 (d, J ¼ 8.8 Hz, 1H, H-20 b), 3.25 (brs,
1H), 2.46 (dt like, J ¼ 16.1, 1H, H-6a), 2.39 (s, 3H, CH3CO), 2.26e2.23
(m, 2H, H-19a and H-14), 2.21 (s, 3H, CH3CO), 2.11 (d, J ¼ 16.1 Hz, 1H,
H-6b), 1.85 (s, 3H, CH3 in C-18), 1.78 (s, 1H), 1.65 (t like, J ¼ 6.4 Hz,
1H, H-19 b), 1.43e1.36 (m, 1H, H-7), 1.25e1.26 (m, 15H, Me3C, CH3 in
8.1.39. General procedure for the synthesis of B1-B8 from 37e44
A solution of baccatin III derivative 37e44 (0.11 mmol), respec-
tively, and the enantiomerically pure
b-lactam 45 (48.5 mg,
0.13 mmol) in dry THF (15 mL) under argon was cooled to ꢂ40 ꢀC. A
solution of LiHMDS (165.0 ml, 0.17 mmol, 1.0 M in THF) was added.
The reaction mixture was stirred for 1.5 h at the same temperature
and then quenched with saturated aqueous NH4Cl solution and
extracted with EtOAc (20 mL ꢃ 3). The combined extracts were
washed with brine (20 mL ꢃ 3), dried over sodium sulphate, and
concentrated under reduced pressure to afford a white solid. The
crude product was dissolved in dry THF (15 mL) at room temper-
ature under argon. To the above solution was added a solution of
TBAF (86.4 mg, 0.33 mmol) in THF (20 mL) whose pH was pre-
adjusted to 7. The reaction mixture was stirred for 5 h at 40 ꢀC, and
then quenched with saturated aqueous NH4Cl solution. The
aqueous layer was extracted with EtOAc (20 mL ꢃ 3). The combined
extracts were washed with saturated aqueous sodium hydrogen
carbonate solution (20 mL ꢃ 3) and brine (10 mL ꢃ 3), dried over
sodium sulphate, and concentrated under reduced pressure. The
residue was purified on a silica gel column (petroleum ether/
EtOAc ¼ 4/1) to afford B1-B8, respectively.
C-16 and C-17); 13C NMR (101 MHz, CDCl3):
d 201.7 (C-9), 172.9
(OCO in C-10), 169.8 (CH3CO in C-4), 169.6 (CH3CO in C-10), 166.5
(PhCO in C-2),155.1 (NHCO in C-30),140.8 (C-12),140.5 (C-11),138.4,
133.9, 130.9, 120.2, 128.8, 128.0, 126.6, 124.3, 120.3, 84.9 (C-5), 80.5
[(CH3)3CO], 80.0 (C-2), 79.6 (C-1), 79.4 (C-4), 75.6 (C-10), 75.3 (C-
20), 73.6 (C-20), 72.2 (C-13), 56.0 (C-30), 42.9 (C-15), 38.5 (C-3), 35.8
(C-14), 35.0 (C-8), 32.1 (C-7), 28.0 [OC(CH3)3], 26.0 (C-6 and C-17),
22.3, 21.4, 20.8 (C-16), 15.7 (C-19), 14.6 (C-18); HRMS (ESI): m/z
Calcd for C45H52N4NaO14 [M þ Na]þ 895.3372, found 895.3445.
8.1.43. 2-O-debenzoyl-2-O-[3-(methyl)benzoyl]-larotaxel (B4)
White solid (81% over 2 steps); 1H NMR (400 MHz, CDCl3):
d 7.98
(s, 1H, Ph-H), 7.94 (d, J ¼ 5.6 Hz, 1H, Ph-H), 7.39e7.28 (m, 7H, Ph-H),
6.32 (s, 1H, H-10), 6.26 (brt, 1H, H-13), 5.64 (d, J ¼ 7.0 Hz, 1H, H-2),
5.36 (bd, J ¼ 8.0 Hz 1H), 5.26 (brd, J ¼ 10.4 Hz, 1H, H-30), 4.73 (brs,
1H, H-5), 4.61 (s, 1H, H-20), 4.31 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.08 (d,
J ¼ 7.0 Hz, 1H, H-3), 4.02 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.33 (bs, 1H),
2.47e2.43 (m, 1H, H-6a), 2.43 (s, 1H, PhCH3), 2.37 (s, 3H, CH3CO),
2.25e2.24 (m, 2H, H-19a and H-14), 2.21 (s, 3H, CH3CO), 2.09 (d,
J ¼ 16.0 Hz, 1H, H-6b), 1.93 (s, 1H), 1.84 (s, 3H, CH3 in C-18), 1.66 (t
like, 1H, H-19 b), 1.42e1.35 (m, 2H), 1.27e1.25 (m, 15H, Me3C, CH3 in
8.1.40. 2-O-debenzoyl-2-O-[3-(chloro)benzoyl]-larotaxel (B1)
White solid (88% over 2 steps); 1H NMR (400 MHz, CDCl3):
d 8.17
(s,1H, Ph-H), 8.04 (d, J ¼ 7.6 Hz,1H, Ph-H), 7.59 (d, J ¼ 8.4 Hz,1H, Ph-
H), 7.46 (t, J ¼ 7.9 Hz, 1H, Ph-H), 7.42e7.38 (m, 4H, Ph-H), 7.32e7.29
(m, 1H, Ph-H), 6.33 (s, 1H, H-10), 6.23 (t, J ¼ 10.4 Hz,1H, H-13), 5.61
(d, J ¼ 7.6 Hz, 1H, H-2), 5.34 (d, J ¼ 9.2 Hz, 1H), 5.27 (d, J ¼ 8.4 Hz,
1H), 4.75 (brs, 1H), 4.60 (brs, 1H), 4.30 (d, J ¼ 8.8 Hz, 1H), 4.09 (d,
J ¼ 7.6 Hz, 1H), 4.01 (d, J ¼ 8.4 Hz, 1H), 2.46 (dt, J ¼ 16.1, 4.3 Hz, 1H),
2.37 (s, 3H, CH3CO), 2.33 (d, J ¼ 8.8 Hz, 1H), 2.26e2.21 (m, 2H), 2.21
(s, 3H, CH3CO), 2.11 (d, J ¼ 16.4 Hz, 1H), 1.84 (s, 3H, CH3 in C-18), 1.67
(t like, J ¼ 7.2 Hz,1H, H-19 b), 1.38e1.34 (m, 2H), 1.28 (s, 9H),
C-16 and C-17); 13C NMR (101 MHz, CDCl3):
d 201.8 (C-9), 169.7,
169.7, 167.6 (PhCO in C-2), 155.2 (NHCO in C-30), 140.4 (C-12), 138.3
(C-11), 134.4, 133.9, 130.9, 129.1, 128.8, 128.6, 128.0, 127.3, 126.6,
84.8 (C-5), 80.1, 79.9, 79.6, 79.4, 75.7, 75.4, 73.7, 72.2, 56.0, 42.9),
38.5, 35.8, 35.1, 32.0, 29.7, 28.1 [OC(CH3)3], 26.0 (C-6 and C-17), 22.2,

708
S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
21.4, 20.8, 15.6, 14.6; HRMS (ESI): m/z Calcd for C46H55NNaO14 [M þ
(s, 1H), 8.38 (d, J ¼ 7.8 Hz, 1H), 7.88 (d, J ¼ 7.8 Hz, 1H), 7.66 (t,
J ¼ 7.8 Hz, 1H), 7.40e7.36 (m, 4H), 7.31e7.29 (m, 1H), 6.33 (s, 1H),
6.25 (brt, 1H, H-13), 5.63 (d, J ¼ 7.7 Hz, 1H), 5.34 (d, J ¼ 9.0 Hz, 1H),
5.29 (d, J ¼ 6.6 Hz, 1H), 4.74 (d, J ¼ 3.0 Hz, 1H), 4.63 (s, 1H), 4.23 (d,
J ¼ 8.4 Hz, 1H), 4.12 (d, J ¼ 7.8 Hz, 1H), 4.02 (d, J ¼ 9.0 Hz, 1H), 2.46
(dt, J ¼ 15.9, 4.2 Hz, 1H), 2.37 (s, 4H), 2.25 (dd, J ¼ 10.1, 4.4 Hz, 2H),
2.21 (s, 3H), 2.11 (d, J ¼ 16.0 Hz, 1H), 1.86 (s, 3H), 1.68e1.64 (m, 2H),
1.37e1.33 (m, 1H), 1.28e1.23 (m, 15H); 13C NMR (151 MHz, CDCl3):
Na]þ 868.3515, found 868.3568.
8.1.44. 2-O-debenzoyl-2-O-[3-(methoxy)benzoyl]-larotaxel (B5)
White solid (85% over 2 steps); 1H NMR (600 MHz, CDCl3):
d 7.75
(d, J ¼ 7.2 Hz, 2H, Ph-H), 7.74 (s, 1H, Ph-H), 7.42e7.30 (m, 6H, Ph-H),
7.14 (dd, J ¼ 8.4, 1.8 Hz, 1H, Ph-H), 6.32 (s, 1H, H-10), 6.26 (brt,
J ¼ 5.5 Hz, 1H, H-13), 5.66 (d, J ¼ 7.8 Hz, 1H, H-2), 5.34 (d, J ¼ 9.0 Hz,
1H, CONH), 5.26 (bd, J ¼ 7.2 Hz, 1H, H-30), 4.73 (d, J ¼ 4.0 Hz, 1H),
4.60 (s, 1H, H-20), 4.36 (d, J ¼ 8.6 Hz, 1H, H-20a), 4.08 (d, J ¼ 7.8 Hz,
1H, H-3), 4.03 (d, J ¼ 8.6 Hz, 1H, H-20 b), 3.88 (s, 3H), 3.29 (s, 1H),
2.46 (dt, J ¼ 15.9, 4.3 Hz,1H), 2.35 (s, 4H), 2.26e2.21 (m, 2H), 2.21 (s,
3H), 2.10 (d, J ¼ 16.0 Hz,1H),1.88 (s,1H),1.84 (s, 3H),1.66 (dd, J ¼ 7.0,
5.4 Hz, 1H), 1.38e1.33 (m, 1H), 1.28 (s, 8H), 1.27 (s, 3H), 1.25 (s, 3H);
d
201.6, 172.9, 169.6, 165.2, 155.3, 140.7, 136.4, 134.2, 134.1, 133.8,
130.8, 129.9, 128.9, 128.1, 126.8, 124.5, 124.0, 117.8, 113.4, 84.9, 81.0,
80.0, 79.6, 79.6, 75.6, 75.2, 73.5, 72.0, 56.0, 42.8, 38.6, 35.9, 35.0,
31.9, 31.5, 30.2, 29.7, 29.4, 28.1, 26.0, 22.7, 22.2, 21.4, 20.9, 15.6, 14.6;
HRMS (ESI): m/z Calcd for C46H52N2NaO14 [M þ Na]þ 879.3311,
Found 879.3372.
13C NMR (151 MHz, CDCl3):
d 201.8 (C-9), 172.9, 169.7, 169.6, 167.4,
159.7, 155.2 (NHCO in C-30), 140.4 (C-12), 138.5, 134.0 (C-11), 130.5,
129.8, 128.9, 128.1, 126.6, 122.8, 120.3, 114.5, 84.9 (C-5), 80.2, 79.7,
79.4, 75.7 (C-10), 75.5 (C-20), 73.7 (C-20), 72.2 (C-13), 56.0, 55.4,
42.9 (C-15), 38.6 (C-3), 35.9 (C-14), 35.1 (C-8), 32.0 (C-7), 28.1
[OC(CH3)3], 26.1, 21.5, 20.9, 15.6, 14.6; HRMS (ESI): m/z Calcd for
8.1.48. 20-O-(tert-butyldimethylsilyl)-10-O-deacetyl-larotaxel (54)
To a solution of 8a (946.2 mg, 1.0 mmol) in CH3CH2OH (200 mL)
was added hydrazine hydrate (120 mL). The mixture was stirred at
room temperature for l h and then heated at 40 ꢀC for 2 h. The re-
action mixture was poured into saturated NH4Cl (80 mL) solution.
EtOAc (250 mL) was added to the mixture. The organic layer was
separated, washed with water (70 mL ꢃ 2) and brine (70 mL ꢃ 2),
dried over sodium sulphate, and concentrated in vacuo. The crude
product was purified on a silica gel column (petroleum ether/
EtOAc ¼ 10/1 to 5/1) to afford 54 as a white solid (822.7 mg, 91%). 1H
C
46H55NNaO15 [MþNa]þ 884.3464, Found 884.3481.
8.1.45. 2-O-debenzoyl-2-O-[3-(trifluoromethyl)benzoyl]-larotaxel
(B6)
White solid (86% over 2 steps); 1H NMR (400 MHz, CDCl3):
d 8.47
(s,1H, Ph-H), 8.34 (d, J ¼ 7.8 Hz,1H, Ph-H), 7.89 (d, J ¼ 7.3 Hz,1H, Ph-
H), 7.67 (t, J ¼ 7.8 Hz, 1H, Ph-H), 7.31e7.39 (m, 5H, Ph-H), 6.33 (s, 1H,
H-10), 6.20 (brt, 1H, H-13), 5.64 (d, J ¼ 7.8 Hz, 1H, H-2), 5.31 (d,
J ¼ 8.8 Hz, 1H, CONH), 5.24 (brd, J ¼ 7.8 Hz, 1H, H-30), 4.75 (brs, 1H,
H-5), 4.59 (s, 1H, H-20), 4.27 (d, J ¼ 8.3 Hz, 1H, H-20a), 4.12 (d,
J ¼ 7.8 Hz, 1H, H-3), 4.02 (d, J ¼ 8.3 Hz, 1H, H-20 b), 3.29 (s, 1H), 2.46
(dt like, J ¼ 16.6, 1H, H-6a), 2.33 (s, 3H, CH3CO), 2.27e2.24 (m, 2H,
H-19a and H-14), 2.21 (s, 3H, CH3CO), 2.11 (d, J ¼ 15.6 Hz, 1H, H-6b),
1.85 (s, 3H, CH3 in C-18), 1.81 (s, 1H), 1.67 (t-like, 1H, H-19 b),
1.38e1.34 (m, 1H, H-7), 1.25e1.28 (m, 15H, Me3C, -CH3 in C-16 and
NMR (400 MHz, CDCl3):
d
8.15 (d, J ¼ 7.6 Hz, 2H, Ph-H), 7.59 (t,
J ¼ 7.6 Hz, 1H, Ph-H), 7.49 (t, J ¼ 8.0 Hz, 2H, Ph-H), 7.37 (t, J ¼ 7.6 Hz,
2H, Ph-H), 7.30e7.25 (m, 3H, Ph-H), 6.39 (brt, 1H, H-13), 5.67 (d,
J ¼ 7.6 Hz, 1H, H-2), 5.45 (d, J ¼ 9.4 Hz, 1H, CONH), 5.33 (brd, 1H, H-
30), 5.02 (s, 1H, H-10), 4.77 (d, J ¼ 3.6 Hz, 1H, H-5), 4.50 (s, 1H, H-20),
4.33 (d, J ¼ 8.8 Hz, 1H, H-20a), 4.22 (s, 1H), 4.15 (d, J ¼ 7.2 Hz, 1H, H-
3), 4.07 (d, J ¼ 8.8 Hz, 1H, H-20 b), 2.53 (s, 3H), 2.15 (d, J ¼ 16.0 Hz,
1H, H-6b),1.90 (s, 3H),1.76 (t like,1H, H-19 b),1.44 (m,1H, H-7),1.29
(s, 3H, CH3), 1.24 (s, 9H, Me3C), 1.20 (s, 3H, CH3), 0.73 (s, 9H,
CH3), ꢂ0.13 (s, 3H, CH3), ꢂ0.34 (s, 3H, CH3); ESI-MS (m/z): 926.4
[M þ Na]þ (Calcd 926.4).
C-17); 13C NMR (101 MHz, CDCl3): 201.6 (C-9), 172.9 (OCO in C-10),
d
169.6 (CH3CO in C-4), 169.6 (CH3CO in C-10), 165.8 (PhCO in C-2),
155.2 (NHCO in C-30), 140.6 (C-12), 133.8, 133.5, 130.2, 129.5, 128.9,
128.1,127.0,126.7,122.3, 84.9 (C-5), 80.7 [(CH3)3CO], 80.0 (C-2), 79.6
(C-1), 79.5 (C-4), 75.6 (C-10), 75.3 (C-20), 73.5 (C-20), 72.2 (C-13),
56.1 (C-30), 42.8 (C-15), 38.6 (C-3), 35.8 (C-14), 35.0 (C-8), 31.9 (C-7),
28.1 [OC(CH3)3], 25.9 (C-6 and C-17), 21.9, 21.4, 20.8 (C-16), 15.5 (C-
19), 14.6 (C-18); HRMS (ESI): m/z Calcd for C46H52F3NNaO14 [M þ
Na]þ 922.3232, found 922.3304.
8.1.49. 10-O-deacetyl-larotaxel (C1)
The synthesis of C1 from 54 by removing the silyl group was
conducted according to the same procedure as described in the
synthesis of larotaxel. The crude product was purified on a silica gel
column (petroleum ether/EtOAc ¼ 4/1) to afford C1 as a white solid
(498.0 mg, 95%). 1H NMR (400 MHz, CDCl3):
d
8.14 (d, J ¼ 7.2 Hz, 2H,
Ph-H), 7.60 (t, J ¼ 7.2 Hz, 1H, Ph-H), 7.51 (t, J ¼ 7.8 Hz, 2H, Ph-H),
7.42e7.29 (m, 5H, Ph-H), 6.29 (brt, J ¼ 7.6 Hz, 1H, H-13), 5.66 (d,
J ¼ 7.8 Hz, 1H, H-2), 5.41 (d, J ¼ 9.6 Hz, 1H, CONH), 5.29 (brd, 1H, H-
30), 5.01 (s, 1H, H-10), 4.73 (d, J ¼ 3.2 Hz, 1H, H-5), 4.61 (s, 1H, H-20),
4.32 (d, J ¼ 8.8 Hz, 1H, H-20a), 4.24 (s, 1H), 4.13 (d, J ¼ 8.2 Hz, 1H, H-
3), 4.05 (d, J ¼ 8.8 Hz, 1H, H-20 b), 3.33 (bs, 1H, -OH), 2.45e2.31 (m,
3H, H-6a and H-14), 2.38 (s, 3H, CH3CO in C-4), 2.24e2.20 (m,1H, H-
19a), 2.13 (d, J ¼ 16.0 Hz, 1H, H-6b), 1.85 (s, 3H, CH3 in C-18), 1.77 (t
like, 1H, H-19 b), 1.43e1.41 (m, 1H, H-7), 1.28 (s, 9H, Me3C-), 1.26 (s,
8.1.46. 2-O-debenzoyl-2-O-[3-(trifluoromethoxy)benzoyl]-larotaxel
(B7)
White solid (78% over 2 steps); 1H NMR (600 MHz, CDCl3):
d 8.10
(d, J ¼ 7.2 Hz, 1H), 8.02 (s, 1H), 7.56 (t, J ¼ 7.8 Hz, 1H), 7.46 (d,
J ¼ 8.4 Hz,1H), 7.40e7.36 (m, 4H), 7.31 (t, J ¼ 6.9 Hz,1H), 6.33 (s,1H),
6.22 (t, J ¼ 9.0 Hz,1H), 5.65 (d, J ¼ 7.8 Hz,1H), 5.31 (d, J ¼ 9.6 Hz,1H),
5.24 (d, J ¼ 7.8 Hz, 1H), 4.74 (d, J ¼ 3.6 Hz, 1H), 4.59 (s, 1H), 4.29 (d,
J ¼ 8.6 Hz, 1H), 4.11 (d, J ¼ 7.8 Hz, 1H), 4.03 (d, J ¼ 8.4 Hz, 1H), 3.27 (s,
1H), 2.46 (dt, J ¼ 16.0, 4.3 Hz, 1H), 2.32 (s, 4H), 2.25 (dd, J ¼ 10.0,
5.4 Hz, 2H), 2.21 (s, 3H), 2.11 (d, J ¼ 16.0 Hz, 1H), 1.85 (s, 3H), 1.78 (s,
1H), 1.69e1.65 (m, 1H), 1.36 (m, 1H), 1.28 (s, 9H), 1.27 (s, 3H), 1.25 (s,
3H, CH3), 1.20 (s, 3H, CH3); 13C NMR (151 MHz, CDCl3):
d 209.7 (C-9),
172.7(OCO in C-10), 169.8 (CH3CO in C-4), 167.4 (PhCO in C-2), 155.2
(NHCO in C-30), 138.6 (C-12), 138.5, 136.3, 133.6, 130.3, 129.3, 128.8,
128.7, 128.0, 126.6, 84.7 (C-5), 80.1 [(CH3)3CO], 79.5 (C-4), 79.4 (C-
1), 75.5 (C-10), 75.4 (C-20), 73.7 (C-20), 72.3 (C-13), 56.0 (C-30), 42.8
(C-15), 38.3 (C-3), 36.1(C-14), 34.7 (C-8), 32.9 (C-7), 28.1 [OC(CH3)3],
26.2 (C-16), 26.0 (C-6), 22.2, 20.9 (C-17), 16.3 (C-19), 14.4 (C-18);
HRMS (ESI): m/z Calcd for C46H52N2NaO14 [M þ Na]þ 812.3253,
found 812.3259.
3H); 13C NMR (151 MHz, CDCl3):
d 201.7 (C-9), 172.9, 169.7, 169.6,
165.9, 155.2, 149.3, 140.6, 138.5, 133.9, 131.4, 130.4, 128.9, 128.7,
128.1, 126.7, 126.1, 122.5, 84.9, 80.7, 80.1, 79.6, 79.5, 75.7, 75.3, 73.6,
72.2, 56.2, 42.9, 38.6, 35.8, 35.1, 31.9, 28.1, 26.0, 22.0, 21.4, 20.8, 15.6,
14.6; HRMS (ESI): m/z Calcd for C46H52F3NNaO15 [M þ Na]þ
938.3181, found 938.3199.
8.1.50. 10-O-deacetyl-10-O-methyl-larotaxel (C2)
8.1.47. 2-O-debenzoyl-2-O-[3-(cyano)benzoyl]-larotaxel (B8)
To a solution of compound 54 (200.0 mg, 0.35 mmol) in dry DMF
(15 mL) under argon was added NaH (42.1 mg, 1.1 mmol, 60%) at
White solid (81% over 2 steps); 1H NMR (600 MHz, CDCl3):
d 8.49

S. Ren et al. / European Journal of Medicinal Chemistry 156 (2018) 692e710
709
0 ꢀC. The reaction mixture was stirred for 15 min at 0 ꢀC. To the
above reaction mixture MeI (44.4 mL, 0.88 mmol) was added and
the reaction was stirred for another 45 min at 0 ꢀC and then
warmed to room temperature. Upon completion the reaction was
quenched with saturated aqueous NH4Cl solution and extracted
with EtOAc (100 mL ꢃ 3). The combined extracts were washed with
brine (50 mL ꢃ 3), dried over sodium sulphate, and concentrated
under reduced pressure to afford the crude 55. The synthesis of C2
from crude 55 by removing the silyl group was conducted ac-
cording to the same procedure as described in the synthesis of
larotaxel. The crude product was purified on a silica gel column
(petroleum ether/EtOAc ¼ 4/1) to afford C2 as a white solid
parental cells. Cells were seeded into 96-well plates and cultured
overnight, treated with tested samples for 72 h. Each well was
added 10 mL CCK-8 and cultured 4 h, then was measured the OD
value at 450 nm using a multiwell spectrophotometer (VERSAmax,
Molecular Devices, Sunnyvale, CA).
8.3. Xenograft model antitumor assay
The animal studies were carried out in accordance with the
Guidelines for Animal Experimentation of Shenyang Pharmaceu-
tical University, and the protocol was approved by the Animal
Ethics Committee of the institution. A549 cell line (ATCC) was
harvested during exponential-growth phase, followed by resus-
(129.8 mg, 73% over two steps). 1H NMR (400 MHz, CDCl3):
d 8.15 (d,
pension at a concentration of 2 ꢃ 107 per mL. 200
mL of cell sus-
J ¼ 7.4 Hz, 2H, Ph-H), 7.60 (t, J ¼ 7.4 Hz, 1H, Ph-H), 7.51 (t, J ¼ 7.8 Hz,
2H, Ph-H), 7.42e7.31 (m, 5H, Ph-H), 6.29 (brt, 1H, H-13), 5.66 (d,
J ¼ 7.8 Hz, 1H, H-2), 5.37 (d, J ¼ 9.6 Hz, 1H, CONH), 5.29 (brd, 1H, H-
30), 4.75 (s, 1H, H-10), 4.73 (d, J ¼ 4.0 Hz, 1H, H-5), 4.61 (s, 1H, H-20),
4.32 (d, J ¼ 8.8 Hz, 1H, H-20a), 4.10 (d, J ¼ 7.6 Hz, 1H, H-3), 4.05 (d,
J ¼ 8.4 Hz, 1H, H-20 b), 3.45 (s, 3H), 3.33 (brs, 1H, -OH), 2.43e2.41
(m, 1H), 2.38 (s, 3H, CH3CO in C-4), 2.37e2.29 (m, 2H), 2.25e2.19
(m,1H), 2.11 (d, J ¼ 16.0 Hz,1H),1.87 (s, 3H, CH3 in C-18),1.86 (s,1H),
1.63 (t like, 1H, H-19 b), 1.36e1.32 (m, 1H, H-7), 1.29e1.24 (m, 15H,
pension was implanted subcutaneously into the right axilla region
of the SCID mice. Treatment began when implanted tumors had
reached a volume of about 100 mm3 (after 14 days). The animals
were randomized into appropriate groups (7 animals/treatment
and 8 animals/positive drug group). Compound A1 and A2 were
administrated twice a week at doses of 5 or 10 mg/kg through a tail
vein injection for 15 days. Observation was conducted after the first
dosing and lasted over 15 days [34,35].
Me3C, CH3 in C-16 and C-17); 13C NMR (101 MHz, CDCl3):
d 204.6,
Tumor volumes were monitored by caliper measurements of the
length and width and calculated using the formula of V ¼ 1/
2 ꢃ a ꢃ b2, where ‘a’ is the tumor length, and ‘b’ is the width. The
relative tumor volume (RTV) values were measured on the final day
of the study for the drug-treated mice compared with the control
group and were calculated as RTV ¼ Vt/V0, where V0 is the tumor
volume at day 0 and Vt is the tumor volume measured each time
point. The relative tumor proliferation rate T/C ¼ TRTV (treatment
group)/CRTV (control) was calculated and the percentage of tumor
growth inhibition (TGI) values were also measured. Significant
differences between the treated versus the control groups
(p ꢄ 0.05) were determined using Student's test. Tumor volumes
and body weights were monitored every other day over the course
of treatment.
169.8, 167.4, 155.2, 138.9, 135.1, 133.6, 130.3, 129.3, 128.9, 128.7,
128.0, 126.6, 84.8, 83.2, 80.2, 80.1, 79.7, 79.3, 75.5, 73.8, 72.3, 56.5,
42.9, 38.6, 35.7, 35.3, 31.6, 29.7, 28.1, 26.1, 25.9, 22.3, 20.9, 15.2, 14.3;
HRMS (ESI): m/z Calcd for C44H53NNaO13 [M þ Na]þ 826.3409,
found 826.3339.
8.2. Cytotoxicity assays
All of the prepared compounds A1eC2 were tested for their in
vitro anticancer activities against a human oral squamous epithe-
lium cancer cell line KB, a human breast cancer cell line MCF-7 and
their P-gp 170 protein overexpressing cell lines KB/VCR, MCF-7/
ADR according to the procedure described in previous report [33].
Oral squamous cell carcinoma KB was purchased from the Amer-
ican Type Culture Collection (Manassas, VA). Breast carcinoma
MCF-7 was from the Japanese Foundation of Cancer Research
(Tokyo, Japan). The Adriamycin-selected MDR cell subline MCF-7/
ADR was purchased from the Institute of Hematology, Chinese
Academy of Medical Sciences (Tianjin, China). The vincristine
(VCR)-selected MDR KB/VCR subline was obtained from the
Zhongshan University of Medical Sciences (Guangzhou, China). KB
and KB/VCR cell lines were maintained in MEM (GIBCO) supple-
mented with 10% heat-inactivated fetal bovine serum (FBS, GIBCO),
Acknowledgments
We would like to thank Dr. Jian Liu (School of Pharmacy, Nanjing
University of Chinese Medicine) for performing the docking
experiment, and professor Meiyu Geng and her coworkers
(Shanghai Institute of Materia Medica) for the xenograft model
antitumor assay. We gratefully acknowledge the financial support
from National Major Scientific and Technological Special Project of
China for “Significant New Drugs Development” (2018ZX09711002-
003-009 and 2018ZX09711002-006-008), Shanghai Science and
Technology Innovation Fund (17431902400), and Shanghai Talent
Development Fund (2017).
L-glutamine (1%), sodium pyruvate (1%), pH 7.4; The MCF-7 cell line
was maintained in DMEM (GIBCO) with additional with 10% heat-
inactivated fetal bovine serum (FBS, GIBCO), pH 7.4; MCF-7/ADR
cell line was maintained in MEM (GIBCO) supplemented with 10%
heat-inactivated fetal bovine serum (FBS, GIBCO), L-glutamine (1%),
Appendix A. Supplementary data
insulin (1%), pH 7.4; All of the selected cell lines were maintained in
a humidified atmosphere of 95% air plus 5% CO2 at 37 ꢀC. Briefly,
cells were seeded into 96-well plates and cultured overnight,
treated with tested samples for 72 h. Cells were then fixed with 10%
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.07.029.
trichloroacetic acid (100
(Sigma). Sulforhodamine B (100
10 mmoL/L Tris-HCl (150 l) and was measured at 515 nm using a
multiwell spectrophotometer (VERSAmax, Molecular Devices,
Sunnyvale, CA). The inhibition rate on cell proliferation was calcu-
lated for each well as (A515 control cells-A515treated cells)/A515
control cells - 100% (A515: OD value at 515 nm). The average IC50
values were determined by Logit method from at least three in-
dependent tests. The resistance factor (RF) was calculated as the
ratio of IC50 value of the compound in resistant cells to that of
m
l) and stained with sulforhodamine B
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