Synthesis and biological evaluation of novel cabazitaxel analogues

Article abstract of DOI:10.1016/j.bmc.2021.116224

Cabazitaxel is one of the most recently FDA-approved taxane anticancer agent. In view of the advantages in preclinical and clinical data of cabazitaxel over former toxoids, the synthesis and biological evaluation of novel cabazitaxel analogues were conducted. First, a novel semi-synthesis of cabazitaxel was described. This strategy is concise and efficient, which needs five steps from the 10-deacetylbaccatin III (10-DAB) moiety and a commercially available C13 side chain precursor with a 32% overall yield. Besides, this strategy avoids using many hazardous reagents that involved in the previously reported processes. Then, a panel of cabazitaxel analogues were prepared basing on this strategy. The cytotoxicity evaluations showed that the majority of these cabazitaxel analogues are potent against both A549 and KB cells and their corresponding drug-resistant cell lines KB/VCR, and A549/T, respectively. Further in vivo antitumor efficacies assessment of 7,10-di-O-methylthiomethyl (MTM) modified cabazitaxel (compounds 16 and 19) on SCID mice A549 xenograft model showed they both had similar antitumor activity to the cabazitaxel. Since compound 19 was observed causing more body wight loss on the mice than 16, these preliminary studies suggest 16 might be a potent drug candidate for further preclinical evaluation.

Full text of DOI:10.1016/j.bmc.2021.116224

Bioorg. Med. Chem. 41 (2021) 116224
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel cabazitaxel analogues
,
*
b
,
,
Sumei Rena 1, Minmin Zhangc 1, Yujie Wanga, Jia Guoa, Junfei Wanga, Yingxia Lia,
,
Ning Dinga
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b School of Pharmaceutical Sciences, Shenzhen University Health Science Center, Shenzhen University, Shenzhen 518060, Guangdong, China
c Division of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi
Road, Shanghai 201203, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cabazitaxel is one of the most recently FDA-approved taxane anticancer agent. In view of the advantages in
preclinical and clinical data of cabazitaxel over former toxoids, the synthesis and biological evaluation of novel
cabazitaxel analogues were conducted. First, a novel semi-synthesis of cabazitaxel was described. This strategy is
concise and efficient, which needs five steps from the 10-deacetylbaccatin III (10-DAB) moiety and a commer-
cially available C13 side chain precursor with a 32% overall yield. Besides, this strategy avoids using many
hazardous reagents that involved in the previously reported processes. Then, a panel of cabazitaxel analogues
were prepared basing on this strategy. The cytotoxicity evaluations showed that the majority of these cabazitaxel
analogues are potent against both A549 and KB cells and their corresponding drug-resistant cell lines KB/VCR,
and A549/T, respectively. Further in vivo antitumor efficacies assessment of 7,10-di-O-methylthiomethyl (MTM)
modified cabazitaxel (compounds 16 and 19) on SCID mice A549 xenograft model showed they both had similar
antitumor activity to the cabazitaxel. Since compound 19 was observed causing more body wight loss on the
mice than 16, these preliminary studies suggest 16 might be a potent drug candidate for further preclinical
evaluation.
Cabazitaxel
Docetaxel
Taxel
Anticancer
Semi-synthesis
10-Deacetylbaccatin III
Cytotoxicity
1. Introduction
because of its poor affinity to P-glycoprotein due to the presence of
methoxy groups at C7 and C10.1 The extra methoxy groups also provide
Paclitaxel and docetaxel are two successful anticancer drugs and
have been used in chemotherapy for a variety of cancer types for a long
time. However, despite the hope and promises that these taxoids have
engendered, their utility is hampered by clinic limitations such as ac-
quired or intrinsic drug resistance of tumors, poor CNS activity, allergic
reactions, and unfavorable toxicity profiles.1–4 Therefore, the develop-
ment of new taxoid anticancer agents with fewer side effects, superior
pharmacological properties, improved activities, and against drug-
resistant human cancers is still attactive.2
cabazitaxel with a unique ability to cross the blood–brain barrier (BBB),
but the clinical advantages of this property have not been explored yet.6
The indication of cabazitaxel was approved by the FDA in combination
with prednisone for the second-line treatment of hormone-refractory
prostate cancer.5,7
In view of the advantages in preclinical and clinical data of cab-
azitaxel over former toxoids,6–8 and basing on our previous work on the
larotaxel analogues,2 we described here the semi-synthesis of cab-
azitaxel and a series of its analogues, as well as the investigations of their
effects on cytotoxicities toward A594 and KB tumor cell lines in vitro and
in vivo.
Cabazitaxel, developed by Rhone-Poulenc Rorer (now Sanofi), is one
of the most recently FDA-approved taxane anticancer agents.1,5 Cab-
azitaxel is similar in structure to docetaxel (Fig. 1), with the only
structural difference being the methylation of the C7 and C10 hydroxyl
groups.4 Cabazitaxel was selected for development as a result of its ef-
ficacy against both docetaxel-sensitive and docetaxel-resistant tumors
* Corresponding author.
E-mail address: dingning@fudan.edu.cn (N. Ding).
1
These authors contributed equally.
https://doi.org/10.1016/j.bmc.2021.116224
Received 24 March 2021; Received in revised form 13 May 2021; Accepted 18 May 2021
Available online 23 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
2. Results and discussion
2.1. Chemical synthesis
2.1.1. Summary of the previously reported semi-synthesis of cabazitaxel
Currently, taxoids are usually obtained by semi-synthesis while the
coupling of a C13 side chain precursor with a pre-modified 10-deacetyl-
baccatin III (10-DAB) moiety in general, since the naturally available 10-
DAB possesses the exact tetracyclic diterpene skeleton of paclitaxel.9
So far most of the reported strategies for semi-synthesis of cab-
azitaxel adopted the procedure that preparation of key intermediate
7,10-dimethyl-10-DAB (1) first followed by esterification in position 13
by a C13 side chain precursor as outlined in Scheme 1.
Fig. 1. The structures of Docetaxel and Cabazitaxel.
US Pat. No. 596270510 (Route A) disclosed the direct methylation of
the two positions 7 and 10 by using methyl iodide or methyl sulfate in
Scheme 1. Representative synthesis of cabazitaxel.
2

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
the presence of an anionization agent such as one or more strong bases in
anhydrous medium. However, this process utilized much more excessive
of methyl iodide (as solvent) or methyl sulfate (16 equiv. over 10-DAB)
for efficient methylation. Another major disadvantage of this process
was that during the methylation of 7,10-di-OH, 13-O-methylated prod-
ucts was also being formed and the required di-O-methylated product 1
was isolated by extensive chromatography.
2.1.2. Optimized semi-synthesis of cabazitaxel
Our initial effort towards cabazitaxel adopted a very similar strategy
to route B that is described in Scheme 1. However, in our hand, we found
that there were several practical issues associated with this strategy. For
example, the key building block 7,10-dimethyl-10-DAB (1) had poor
solubility in many organic solvents, such as THF and DMF, which were
the most frequently used solvent in the subsequent coupling reaction
with the C13 side chain precursor, therefore reduced the coupling yield.
Then we turned to another strategy, which was similar to that as
described in Scheme 1 route D. But in this case, the simultaneous
methylations of the 7,10-di-OH were still painful, which needed exces-
sive of hazardous methyl iodide to ensure efficiency. To solve the above
two major practical issues, we described here a new concise and efficient
route to the synthesis of cabazitaxel.18
Alternatively, indirect methods for the synthesis of 1 that assisted by
silyl ether as temporary protecting groups were proved to be more
practical as represented by the process disclosed in US Pat. No. 5847170
(Route B).11 By using trimethylsilyl chloride (TESCl) in pyridine,
although the required silylated product 2 was obtained in only 40–50%
of yield, it was much easier for purification as compared with the above
direct methylation method. An improved process was disclosed in US
Pat. No. 8901327 B2,12 which avoided the use of hazardous reagents,
like pyridine in the silylation step and hydrogen fluoride triethylamine
complex (3HF.Et3N) in the deprotection step. However, the synthesis
time of this process required from a starting material to a final product
was too long.13 Specifically, deprotection of silyl from 13 position of 3
needed an excessive long reaction time of 48 h. To solve this technical
problem, US Pat. 9000193 B214 disclosed an optimized process, which
comprised the steps of selective protection of 7-OH of 10-DAB by a
silylating agent at a lower temperature, selective methylation of 10-OH,
deprotection of 7-O-silyl group, and methylation of 7-OH.
As shown in Scheme 2, 10-DAB was first treated with DMSO and
Ac2O in acetic acid at 70 ◦C. In this reaction mixture, two methyl-
thiomethyl (MTM) groups were introduced on the 7,10-di-OH of 10-DAB
by a Pummerer reaction, and at the same time, the 13-OH was oxidized
to a ketone (12, 85%). Subsequently, the 13-ketone on compound 12
was reduced to a hydroxyl by NaBH4 with desired C-configuration (13,
75%). It should be noted that, unlike 7,10-dimethyl-10-DAB (1), com-
pound 13 has very good solubility in many organic solvents. With bac-
catin derivative 13 in hand, the “Ojima–Holton coupling” was employed
to construct the taxoids scaffold. By coupling of the enantiomerically
pure β-lactam (3R,4S)-1419,20 with 13 in the presence of LiHMDS fol-
lowed by removal of TBS (t-butyldimethylsilyl) provide 16 smoothly
(77% over 2 steps). Finally, the two MTM groups were reduced to two
methyls efficiently by Raney-Ni/H2, affording cabazitaxel (66%). This
strategy is very concise and efficient, which only needs five steps from
10-DAB with a 32% overall yield. Besides, this strategy avoids using
many hazardous reagents that were involved in the previously reported
processes.
Xia et al.15 reported another indirect strategy (Route C, through
compound 5 to reach 7), which employed benzyloxycarbonyls (Cbz) and
trimethylsilyl (TMS) as temporarily protection groups for 7,10-di-OH
and 13-OH, respectively.
Alternative strategies differing from the above ones were that
introduction of the side chain into the 10-DAB moiety before methyl-
ation of the 7,10-di-OH.15 The major advantage of this kind of strategies
was to avoid the temporary protection for 13-OH. For example, US Pat.
9567312 B2 16provided a representative method (Route D) for the
preparation of cabazitaxel, which used trichloroethyl carbonate (Troc)
as temporary protection groups to simultaneously block the 7,10-di-OH
of 10-DAB (compound 8) followed by an introduction of the side chain
on the 13-OH by “Ojima–Holton coupling”4,17 to provide compound 10.
Then after the removal of the Trocs, methylations were conducted effi-
ciently. However, this process still utilized excessive of hazardous
methyl iodide.
2.1.3. Design and Semi-synthesis of cabazitaxel analogues
A large number of paclitaxel and docetaxel analogues have been
synthesized and evaluated on different kinds of tumor models. So far, a
useful body of SARs of them has become available.1,21–24 Briefly, these
SAR studies indicated that the C3′-phenyl group was not an essential
component for their potent activity, which can be replaced by an alkenyl
or alkyl group.19,25–29 SAR studies also suggested that meta-substituted
S
S
S
S
O
O
OH
O
O
O
O
O
O
O
OH
O
HO
HO
O
DMSO, Ac2O, HOAc
NaBH4, CH3OH/THF
75%
HO
O
HO
O
70 o
C
HO
O
85%
O
O
O
O
O
O
O
O
O
12
13
10-DAB
+
S
O
O
O
O
S
OCH3
O
O
O
O
O
O
NH
O
NH
O
OCH3
O
O
O
LiHMDS, THF, - 50 o
C
N
O
Raney-Ni, H2, EtOH
66%
O
OH
OR
HO
O
HO
O
OTBS
O
O
O
O
O
O
Cabazitaxel
15 R =TBS
16 R = H
14
TBAF/HOAc, THF
77% over 2 steps
Scheme 2. Our synthetic strategy for cabazitaxel.
3

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
2-benzoates of paclitaxel often showed enhanced cytotoxicities against
drug-resistant human cancer cell lines, especially those substituted by 2-
meta-MeO and 2-meta-N3.30,31 In addition, fluorine(s) has been strate-
gically incorporated into paclitaxel and docetaxel molecules, and the
effects of these molecules on the cytotoxicity exhibited substantially
better in vitro potency than paclitaxel and docetaxel against many
human cancer cell lines.32,33
with 13 (23, 60%). After desilylation (24, 73%), the two MTM groups,
two fluorine and the carbon–carbon double bonds in 24 were simulta-
neously reduced by Raney-Ni to provide 25 in one pot (90%). The
synthesis of 3′-difluoromethyl-cabazitaxel (29) was achieved by
coupling of (3R,4S)–2232,34 with 13 to afford 27 (83%), followed by
desilylation and reduction of the MTMs (68% over 2 steps).
The syntheses of meta-substituted 2-benzoates of cabazitaxel were
depicted in Scheme 4. Intermediate 15 was first reduced by Raney-Ni to
form 2′-O-TBS cabazitaxel 30 (76%). The 2-O-benzoyl group of 30 was
then selectively removed by Triton B at a low temperature35 to provide
alcohol 31 (56%), which upon esterification of the exposed C2-OH with
3-substituted benzoic acids assisted by coupling reagents (DCC/
DMAP)36 delivered compounds 32 and 34. After removal of the TBS
protecting groups on 32 and 34, the desired cabazitaxel analogues 33
and 35 were prepared. The sulfone derivative of cabazitaxel 36 was
prepared through oxidation of intermediate 16 by mCPBA (m-chlor-
operoxybenzoic acid) in dichloromethane smoothly (86%).
Based on this knowledge, we intended to prepare some cabazitaxel
analogues that modified on C3′ by an alkenyl/alkyl group (20, 21, 24,
25, 29), including those fluorine(s)-incorporating molecules (24, 29)
and those modified on 2-OH by meta-N3/meta-OMe substituted benzo-
ates (33, 35).
Modifications on C7/C10 position of paclitaxel or docetaxel25 often
made compounds more potent than the parent drugs against drug-
resistant human cancer cell lines, probably because proper structural
changes at these positions can reduce the affinity to P-glycoprotein. That
is one of the reasons that cabazitaxel is superior to former toxoids.
Therefore, we decided to evaluate some of the C7/C10-MTM groups
containing intermediate (19, 24, 28) and the sulfone derivative of
cabazitaxel (36).
2.2. Cytotoxicity evaluations.
Based on our new strategy for the semi-synthesis of cabazitaxel, the
designed analogues of cabazitaxel were synthesized. The syntheses of
C3′-modified analogues of cabazitaxel were outlines in Scheme 3.
Enantiomerically pure β-lactam (3R,4S)-1723,31 was employed to couple
with 13, affording the 3′-alkenyl cabazitaxel 18 (85%). After removal of
the TBS group on C2′-OH (87%), the two MTM groups were reduced to
methyls by Raney-Ni (21, 70%). The 3′-alkenyl group in some of the
compound 21 was further reduced to alkyl by Raney-Ni to afford 20
(15%). The 3′-ethyl cabazitaxel 25 was synthesized by a similar method,
which used the (3R,4S)–2233 as the corresponding β-lactam to couple
Although docetaxel and cabazitaxel are successful against certain
cancer types, they are potentially broad-spectrum anticancer drugs in
chemotherapy for a variety of cancer types. In this respect, we would
like to know the cytotoxicities of the cabazitaxel analogues against some
common cancer cell lines in the labs at the early stage of the studies.
Thus, based on our previous work on the larotaxel2, another new-gen-
eration toxoid, the cabazitaxel analogues were evaluated for their in
vitro cytotoxicity against A549 and KB cell lines and their corresponding
drug-resistant cell lines KB/VCR, and A549/T, respectively. Results are
summarized in Table 1. The IC50 values were determined through 72 h
Scheme 3. Synthesis of C3′-modified analogues of cabazitaxel.
4

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
Scheme 4. Synthesis of meta-substituted 2-benzoates of cabazitaxel and the sulfone derivative of cabazitaxel.
exposure of the testing compounds to the cancer cells employing the
methods previously developed.31
microtubules, which results in the arrest of the cell division cycle mainly
at the G2/M stage, leading to apoptosis through the cell-signaling
cascade.37 Thus, flow cytometry analysis was carried out and the re-
sults showed that compounds 16 and 19 arrested the cell division cycle
mainly at the G2/M stage on A549 cell lines (Fig 2). These results
indicated that compounds 16 and 19 might act as inhibitors for micro-
tubules depolymerization like other taxoids. The exact mechanism is
under investigation.
Docetaxel and cabazitaxel are selected as the positive controls.
Docetaxel does possess excellent activities on the drug-sensitive cell
lines, both A549 and KB, whereas, the activities reduced on the corre-
sponding drug-resistant cell lines as expected. Cabazitaxel performed
much better activities on both drug-sensitive and drug-resistant cell
lines and a strikingly decreased R/S ratio, which is an excellent indicator
of the level of drug resistance associated with drugs.
Previous SAR studies based on paclitaxel revealed that the phenyl
group at the C-3′ position is not strictly required for the activity. As
shown in Table 1, all the cabazitaxel analogues modified on this position
(20, 21, 25, 29) exhibited very similar cytotoxicity on the drug-sensitive
cell lines, both A549 and KB, and their corresponding drug-resistant cell
lines KB/VCR, and A549/T, respectively, to that of cabazitaxel. The
above results showed that the C3′ position appeared to tolerate a di-
versity of alkenyl-containing groups. Modified on C2 benzoyl by 2-meta-
N3, 2-meta-Cl and 2-meta-MeO often exhibited similar or enhanced
cytotoxicity based on paclitaxel SARs. Here the results showed that the
C2-modified cabazitaxel analogues (33, 35) were as active as cab-
azitaxel on both A549 and KB cells, as well as KB/VCR, and A549/T. It
should be noted that the 7,10-di-O-methylthiomethyl (MTM) com-
pounds (16, 19, and 24) are a quite novel class of toxoids. Initially, they
are synthetic intermediates in our synthetic strategy for cabazitaxel.
Since the modifications on 7, 10 positions have been shown successful
based on other taxoids, we decided to evaluate these compounds. To our
delight, all the three compounds, especially 16 and 19, performed
excellent cytotoxicity toward all the tested cells including the drug-
resistant ones. The R/S ratios are comparable with those of cabazitaxel.
2.4. In vivo antitumor efficacies of 16 and 19
Based on the interesting structures and the excellent results of 16 and
19 in the cytotoxicity evaluations, they were subjected to the subsequent
in vivo antitumor efficacies evaluations. The purpose of this in vivo
experiment is to gain information about the preliminary pharmacolog-
ical effects and general toxicity on the mice, which will facilitate the
following further investigations if necessary. To assess the in vivo anti-
tumor efficacies of 16 and 19, SCID mice A549 xenograft model were
established by subcutaneously injecting A549 cells in the logarithmic
phase into the left armpit of the mice. Compounds 16 and 19 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. Results are summarized in Fig. 3. In vivo antitumor efficacies in-
vestigations showed that compound 19 could significantly inhibit tumor
growth from the fifth day of administration, with the tumor growth
inhibition rate (TGI) of 61.7% and 82.2% at the doses of 5 and 10 mg/kg,
respectively. However, 19 at doses of 5 and 10 mg/kg caused more body
weight loss. The cause of body weight loss requires further toxicological
investigation. Compound 16 at a dose of 10 mg/kg inhibited tumor
growth as efficient as compound 19 at a dose of 5 mg/kg but it caused
less body weight loss, which indicated 16 might have a better thera-
peutic window. 5 mg/kg administration of 16 had a very similar influ-
ence on the tumor growth, TGI, and body wight to that of 5 mg/kg
2.3. Cell cycle distribution analysis of compounds 16 and 19
It is well known that taxoids inhibit depolymerization of
5

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
Table 1
Cytotoxicity of cabazitaxel analogues (IC50 nM).a
Compound
R1
R2
H
R3
R4
A549
A549/Tb
R/Sd
KB
KB/VCRc
R/Sd
21
OCH3
OCH3
5 ± 0.7
25 ± 5
5.0
20 ± 9
320 ± 4
16.0
20
H
OCH3
OCH3
8 ± 0.7
68 ± 4
8.5
17 ± 9
515 ± 8
30.0
25
29
H
H
OCH3
OCH3
OCH3
OCH3
15 ± 2
8 ± 1
324 ± 3
20 ± 0.7
21.6
2.5
19 ± 2
911 ± 16
47.9
4.3
281 ± 156
1221 ± 16
33
35
16
19
Ph
Ph
Ph
OCH3
N3
OCH3
OCH3
8 ± 0
47 ± 3
32 ± 2
31 ± 0.8
23 ± 5
5.9
2.7
2.8
2.9
20 ± 4
13 ± 4
9 ± 1
252 ± 11
315 ± 14
78 ± 5
12.6
24.2
8.7
OCH3
OCH3
12 ± 0
11 ± 2
8 ± 1
H
OMTM
OMTM
OMTM
OMTM
H
4 ± 1
31 ± 0.8
7.8
24
36
H
H
OMTM
OMTM
17 ± 4
154 ± 6
9.1
23 ± 2
>2500
227 ± 9
>2500
9.9
Ph
53 ± 11
1953 ± 13
36.9
–
Cabazitaxel
Docetaxel
Ph
Ph
H
H
OCH3
OCH3
15 ± 6
62.5 ± 7
4.2
20 ± 2
155.5 ± 7
265.7 ± 7
7.8
–OH
–OH
19.5 ± 6
836.2 ± 8
42.9
17.5 ± 3
15.2
aConcentration of compound that inhibits 50% (IC50, nM) of the growth of human tumor cell line after a 72 h drug exposure.
bA549/T: Paclitaxol resistant adenocarcinomic human alveolar basal epithelial cells (Pgpþ).
cKB/VCR: Vincristine resistant KB human oral epidermoid carcinoma cell line (Pgpþ).
dResistance factor (IC50 for drug resistant cell line, R)/(IC50 for drug-sensitive cell line, S).
Fig. 2. The A549 cells were arrested at G2/M after treatment of compounds 16 and 19. Cell cycle distribution was analyzed by fow cytometry (FACS Calibur,
BD) after A549 cells were treated by indicated compounds at concentration of 40 nM for 24 h.
administration of cabazitaxel. In summary, both compounds 16 and 19
showed similar in vivo antitumor activity to cabazitaxel on SCID mice
A549 xenograft model. Compound 19 at tested doses showed stronger
toxicity than 16 and cabazitaxel. These preliminary studies suggest 16
might be a potent drug candidate for further preclinical evaluation. The
investigations of compound 16 on its abilities to cross the BBB and
binding to the P-glycoprotein will be reported in due course.
vitro. The above results showed that the C3′ position on cabazitaxel
appeared to tolerate a diversity of alkenyl-containing groups. Modified
on C2 benzoyl by 2-meta-N3, and 2-meta-MeO exhibited similar cyto-
toxicity to that of cabazitaxel. The 7,10-di-O-methylthiomethyl (MTM)
compounds (16 and 19) are a quite novel class of toxoids. The flow
cytometry analysis indicated that compounds 16 and 19 might act as
inhibitors for microtubules depolymerization like other toxoids. Further
in vivo antitumor efficacies assessment of 16 and 19 on SCID mice A549
xenograft model showed they both had better antitumor activity than
cabazitaxel. Since compound 19 caused more body weight loss on the
mice than 16, these preliminary studies suggest 16 might be a potent
drug candidate for further preclinical evaluation.
3. Conclusion
In summary, we described here the semi-synthesis of cabazitaxel and
a series of its analogues, as well as the investigations of their effects on
cytotoxicities toward A594 and KB tumor cell lines, and their corre-
sponding drug-resistant cell lines KB/VCR, and A549/T, respectively, in
6

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
Fig. 3. In vivo antitumor efficacies of 16 and 19 on tumor growth in A549 xenograft model. (A) The 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 = 8 for Positive drug group,
n = 7 for other groups). Points indicate mean tumor volumes (mm3), relative tumor proliferation rate (%) or mean body weights (g).
4. Experimental section
4.1.1. 13-Oxo-7, 10-methylthiomethyl-10-deacetylbaccatin III (12)
To a solution of 10-deacetylbaccatin III (60.0 g, 110.2 mmol) in
DMSO (420 mL) was added acetic anhydride (420 mL) and acetic acid
(96 mL). Then the reaction mixture was stirred for 12 h at 70 ◦C. The
reaction mixture was concentrated in vacuo. EtOAc (1500 mL) was
added to the residue and washed with saturated aqueous sodium
hydrogen carbonate solution (300 mL × 6), water (200 mL × 3) and
brine (200 mL × 3), dried over sodium sulphate, and and concentrated
under reduced pressure to afford 12 as a light yellow foam solid (62.1 g,
85%). 1H NMR (400 MHz, CDCl3) δ 8.06 (d, 2H, J = 7.0 Hz, Ph-H), 7.62
(t, 1H, J = 7.6 Hz, Ph-H), 7.49 (t, 2H, J = 7.6 Hz, Ph-H), 5.80 (s, 1H, H-
10), 5.67 (d, 1H, J = 6.7 Hz, H-2), 4.94 (d, 1H, J = 9.2 Hz, H-5), 4.84 (d,
1H, J = 11.6 Hz, CH3SCHHO), 4.73 (d, 1H, J = 11.6 Hz, CH3SCHHO),
4.69 (d, 1H, J = 11.9 Hz, CH3SCHHO), 4.59 (d, 1H, J = 11.9 Hz,
CH3SCHHO), 4.33 (d, 1H, J = 8.3 Hz, H-20a), 4.28 (dd, 1H, J = 10.4, 6.7
Hz, H-7), 4.12 (d, 1H, J = 8.8 Hz, H-20b), 3.95 (d, 1H, J = 6.7 Hz, H-3),
2.93 (d, 1H, J = 19.9 Hz, H-14a), 2.82–2.76 (m, 1H, H-6a), 2.66 (d, 1H,
J = 19.6 Hz, H-14b), 2.21 (s, 6H, CH3SCH2O in C-7 and C-10), 2.20 (s,
3H, CH3CO), 2.17 (s, 3H, H-3, CH3 in C-18), 1.85–1.79 (m, 1H, H-6b),
1.71 (s, 3H, CH3 in C-19), 1.26 (s, 3H, CH3), 1.21 (s, 3H, CH3). ESI-MS
(m/z) 663.3 [M+H]+(Calcd 663.2).
4.1. General procedure
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 Purification System (PS-
MD-5, Innovation Technology, USA). Reactions were magnetically stir-
red and monitored by thin layer chromatography (TLC) with silica gel
plates (60F-254) with UV light, visualized by spraying with a solution of
(NH4)6Mo7O24⋅H2O (25 gLꢀ 1) in 5% sulfuric acid in ethanol followed by
charring. Flash column chromatography was performed with silica gel
(200–300 meshes) with the indicated solvent system and preparative
thin layer chromatography was performed on silica gel F254 glass plates
(layer thick 400–500 mm). Nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker 400 MHz or Bruker Avance II 600 spec-
trometer 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 tetramethylsilane (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.
4.1.2. 7, 10-Methylthiomethyl-10-deacetylbaccatin III (13)
To a solution of 1 (61.6 g, 92.9 mmol) in dry MeOH (1000 mL) and
THF (200 mL) was added NaBH4 (35.1 g, 929.0 mmol) slowly at 0 ◦C
over a period of 1 h. The reaction mixture was warmed to room tem-
perature and stirring continued for 4 h and then quenched with satu-
rated aqueous NH4Cl solution and concentrated in vacuo. EtOAc (1000
mL) was added to the residue and washed with 1 mol/L HCl (300 mL ×
3), saturated aqueous sodium hydrogen carbonate solution (300 mL × 3)
7

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
and brine (200 mL × 3), dried over sodium sulphate, and and concen-
trated under reduced pressure. The residue was purified on a silica gel
column (petroleum ether/EtOAc = 5/1) to afford 13 as a white foam
solid (46.3 g, 75%). 1H NMR (400 MHz, CDCl3) δ 8.10 (d, 2H, J = 8.2 Hz,
Ph-H), 7.60 (t, 1H, J = 7.0 Hz, Ph-H), 7.48 (t, 2H, J = 7.4 Hz, Ph-H), 5.80
(m, 2H, H-10 and H-2), 4.99 (d, 1H, J = 9.2 Hz, H-5),4.88 (t, 1H, J = 7.8
Hz, H-13), 4.74 (m, 2H, CH3SCH2O), 4.68 (d, 1H, J = 11.7 Hz,
CH3SCHHO), 4.61 (d, 1H, J = 11.7 Hz, CH3SCHHO), 4.33–4.27 (m, 2H,
H-20a, H-7), 4.16 (d, 1H, J = 8.2 Hz, H-20b), 3.94 (d, 1H, J = 6.7 Hz, H-
3), 2.81–2.78 (m, 2H, H-6a, H-14a), 2.66 (d, 1H, J = 19.6 Hz, H-14b),
2.29 (s, 3H, CH3O), 2.21–2.17 (m, 9H), 1.87–1.81 (m, 2H, H-6b and H-
14b), 1.73 (s, 3H, CH3 in C-19), 1.18 (s, 3H, CH3), 1.06 (s, 3H, CH3). ESI-
MS (m/z) 665.2 [M+H]+(Calcd 665.2).
2H, Ph-H), 7.42–7.31 (m, 5H, Ph-H), 6.21 (t, 1H, J = 6.0, 12.0 Hz, H-13),
5.63 (d, 1H, J = 6.0 Hz, H-2), 5.47 (d, 1H, J = 6.0 Hz, CONH), 5.27 (m,
1H, H-3′), 4.97 (d, 1H, J = 12.0 Hz, H-5), 4.80 (s, 1H, H-10), 4.63 (m,
1H, H-2′), 4.29 (d, 1H, J = 12.0 Hz, H-20b), 4.17 (d, 1H, J = 12.0 Hz, H-
20a), 3.86 (dd, 1H, J = 12.0, 6.0 Hz, H-7), 3.81 (d, 1H, J = 6.0 Hz, H-3),
3.56–3.45 (m, 1H, 2′–OH), 3.45 (s, 3H, CH3O in C-10), 3.30 (s, 3H, CH3O
in C-7), 2.68–2.72 (m, 1H, CH2 H-6b), 2.36 (s, 3H, CH3CO in C-4),
2.25–2.32 (m, 2H, CH2 in C-14), 1.88 (s, 3H, 18-CH3), 1.79 (m, 1H, H-
6a), 1.71 (s, 3H, 19-CH3), 1.71 (s, 1H, 1-OH), 1.36 (s, 9H, C(CH3)3), 1.21
(s, 3H, 17-CH3), 1.20 (s, 3H, 16-CH3); 13C NMR (100 MHz, CDCl3) δ
204.9, 172.7, 170.4, 167.0, 155.3, 138.7, 138.3, 135.6, 133.6, 130.1,
129.2, 128.8, 128.6, 128.1, 126.8, 84.1, 82.6, 81.7, 80.7, 80.2, 78.7,
76.5, 74.5, 73.7, 72.6, 57.3, 57.0, 56.9, 56.2, 53.4, 47.4, 43.3, 35.2,
32.1, 28.2, 26.8, 22.7, 20.7, 14.6, 10.3. ESI-MS (m/z): 836.3 [M+H]+
(Calcd 835.4) 858.3 [M+Na]+, 875.2 [M+K]+.
4.1.3. 2′-O-(tert-butyldimethylsilyl)- 7,10-O-demethyl-7, 10-O-
(methylthiomethyl)cabazitaxel (15)
A solution of 13 (44.0 g, 66.2 mmo1) and racemic 3-(t-butyldime-
thylsilyloxy)-β-lactam 14 (37.5 g, 99.3 mmol) in dry THF (300 mL)
under argon was cooled to ꢀ 50 ◦C. To the above mixture a solution of
LiHMDS (99.3 mL, 99.3 mmol, 1.0 M in THF) was added. The reaction
mixture was stirred for 3 h at the same temperature and then quenched
with saturated aqueous NH4Cl solution and extracted with EtOAc (500
mL × 3). The combined extracts were washed with brine (300 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure to
afford crude 15.
4.2.1. 2′-O-(tert-butyldimethylsilyl)-3′-dephenyl-3′-(2-methylprop-1-enyl)-
7,10-O-demethyl-7, 10-O-(methylthiomethyl)cabazitaxel (18)
A solution of 13 (1.0 g, 1.5 mmo1) and racemic 3-(t-butyldime-
thylsilyloxy)-β-lactam 17 (817.9 mg, 2.3 mmol) in dry THF (30 mL)
under argon was cooled to ꢀ 50 ◦C. To the above mixture a solution of
LiHMDS (2.3 mL, 2.3 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 (100
mL × 3). The combined extracts were washed with brine (50 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure .
The residue was purified on a silica gel column (petroleum ether/EtOAc
= 6/1) to afford 18 as a white solid (1.3 g, 85%). 1H NMR (400 MHz,
CDCl3) δ 8.10 (d, 2H, J = 7.3 Hz, Ph-H), 7.61 (t, 1H, J = 7.5 Hz, Ph-H),
7.47 (t, 2H, J = 7.7 Hz, Ph-H), 6.16 (t, 1H, J = 9.0 Hz, H-13), 5.67 (d, 1H,
J = 7.1 Hz, H-2), 5.59 (s, 1H, H-10), 5.25 (d, J = 8.5 Hz, 1H, CH = C),
4.97 (d, 1H, J = 8.3 Hz, H-5), 4.78–4.59 (m, 6H, H-3′, CONH, OCH2S ×
2), 4.32 (d, J = 8.4 Hz, 1H, H-20), 4.26 (q, J = 6.8 Hz, 2H, H-7, H-2′),
4.20 (d, 1H, J = 8.5 Hz, H-20), 3.90 (d, 1H, J = 6.9 Hz, H-3), 2.88–2.78
(m, 1H, H-6a), 2.42 (s, 3H, CH3O), 2.29 (d, 2H, J = 7.3 Hz), 2.20 (s, 3H,
CH3S), 2.16 (s, 3H, CH3S), 2.01 (s, 3H, CH3), 1.92–1.82 (m, 1H, H-6b),
1.79 (s, 3H, CH3), 1.74 (s, 3H, CH3), 1.36 (s, 9H, (CH3)3CO), 1.21 (s, 3H,
CH3), 1.19 (s, 3H, CH3), 0.96 (s, 9H, (CH3)3CSi), 0.15 (s, 3H, CH3), 0.11
(s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 204.6, 171.9, 170.1, 167.0,
–78.9, 78.6, 77.6, 76.5, 75.1, 74.9, 73.8, 72.0, 71.7, 60.4, 57.1, 47.1,
43.3, 35.3, 33.0, 28.3, 27.8, 26.4, 25.8, 25.7, 22.7, 21.1, 21.0, 18.6,
18.4, 15.5, 14.3, 14.2, 14.1, 14.0, 13.9, 10.8, ꢀ 4.8, ꢀ 5.3. ESI-MS (m/z)
1020.5 [M+H]+(Calcd 1020.5), 1042.3 [M+Na]+(Calcd 1042.4)
4.1.4. 7, 10-O-demethyl-7, 10-O-(methylthiomethyl)cabazitaxel (16)
To a solution of the above crude product 15 (64.0 g, 61.4 mmol) in
dry THF (300 mL) at room temperature were added a solution of TBAF
(48.2 g, 184.2 mmol) in THF (100 mL) under argon whose pH was pre-
adjusted to 7 by HOAc. The reaction mixture was stirred for 1 h at room
temperature, and then quenched with saturated aqueous NH4Cl solu-
tion. The aqueous layer was extracted with EtOAc (500 mL × 3). The
combined extracts were washed with saturated aqueous sodium
hydrogen carbonate solution (200 mL × 3) and brine (200 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure.
The residue was purified on a silica gel column (petroleum ether/EtOAc
1
= 2/1) to afford 16 as a white solid (43.9 g, 77% over two steps). H
NMR (400 MHz, CDCl3) δ 8.10 (d, J = 7.5 Hz, 2H, Ph-H), 7.61 (t, J = 7.1
Hz, 1H, Ph-H), 7.49 (t, J = 7.3 Hz, 2H, Ph-H), 7.40–7.31 (m, 4H, Ph-H),
6.22 (t, J = 8.7 Hz, 1H, H-13), 5.66 (d, J = 6.5 Hz, 1H, H-2), 5.59 (s,
1H,), 5.42 (d, J = 10.0 Hz, 1H), 5.28 (d, J = 7.7 Hz, 1H), 4.95 (d, J = 9.3
Hz, 1H), 4.77–4.59 (m, 5H), 4.31 (d, J = 8.6 Hz, 1H), 4.24 (dd, J = 9.7,
6.5 Hz, 1H), 4.17 (d, J = 8.5 Hz, 1H), 3.86 (d, J = 6.7 Hz, 1H), 3.43 (d, J
= 4.4 Hz, 1H, 2′–OH), 2.87–2.72 (m, 1H), 2.37 (s, 3H, CH3CO), 2.29 (d,
J = 8.4 Hz, 2H), 2.21 (s, 3H, CH3S), 2.15 (s, 3H, CH3S), 2.00 (s, 3H, CH3-
18), 1.86 (t like, 1H), 1.74 (s, 3H, CH3-17), 1.70 (s, 1H, 1-OH), 1.36 (s,
9H, Me3C-), 1.22 (s, 3H, CH3), 1.21 (s, 3H, CH3); 13C NMR (150 MHz,
CDCl3) δ 204.6, 172.8, 170.4, 167.0, 155.3, 139.7, 138.5, 134.6, 133.7,
133.7, 130.2, 129.2, 128.8, 128.7, 128.1, 126.8, 83.9, 81.4, 80.2, 78.7,
77.8, 76.6, 74.7, 73.8, 72.6, 72.3, 60.4, 57.3, 56.2, 47.2, 43.3, 35.3,
33.0, 28.2, 26.7, 22.7, 21.1, 20.7, 15.7, 14.3, 13.9, 10.7. HRMS (ESI) m/
z calcd for C47H61NO14S2 [M+Na]+ 950.3426, Found 950.3434.
4.2.2. 3′-Dephenyl-3′-(2-methylprop-1-enyl)-7, 10-O-demethyl-7, 10-O-
(methylthiomethyl)cabazitaxel (19)
To a solution of 18 (1.2 g, 1.2 mmo1) in dry THF (30 mL) at room
temperature were added a solution of TBAF (941.3 mg, 3.6 mmol) in
THF (10 mL) under argon whose pH was pre-adjusted to 7 by HOAc. The
reaction mixture was stirred for 25 min at room temperature, and then
quenched with saturated aqueous NH4Cl solution. The aqueous layer
was extracted with EtOAc (90 mL × 3). The combined extracts were
washed with saturated aqueous sodium hydrogen carbonate solution
(40 mL × 3) and brine (40 mL × 3), dried over sodium sulphate, and
concentrated under reduced pressure. The residue was purified on a
silica gel column (petroleum ether/EtOAc = 4/1) to afford 19 as a white
solid (858.0 mg, 78%). 1H NMR (600 MHz, CDCl3) δ 8.10 (d, J = 7.2 Hz,
2H, Ph-H), 7.60 (t, J = 7.4 Hz, 1H, Ph-H), 7.47 (t, J = 7.8 Hz, 2H, Ph-H),
6.16 (t, J = 8.6 Hz, 1H, H-13), 5.67 (d, J = 7.0 Hz, 1H, H-2), 5.60 (s, 1H),
5.33 (d, J = 8.4 Hz, 1H), 4.97 (d, J = 8.4 Hz, 1H), 4.85 (d, J = 8.8 Hz,
1H), 4.78–4.74 (m, 2H), 4.72 (d, J = 11.7 Hz, 1H), 4.66 (d, J = 11.8 Hz,
1H), 4.60 (d, J = 11.8 Hz, 1H), 4.31 (d, J = 8.5 Hz, 1H), 4.26–4.24 (m,
1H), 4.23 (dd, J = 6.9, 2.7 Hz, 1H), 4.19 (d, J = 8.5 Hz, 1H), 3.88 (d, J =
6.9 Hz, 1H), 3.46 (d, J = 6.5 Hz, 1H, 2′–OH), 2.79 (ddd, J = 14.5, 9.7,
6.7 Hz, 1H), 2.39 (dd, J = 15.3, 9.5 Hz, 1H), 2.36 (s, 3H, CH3CO), 2.32
(dd, J = 14.9, 9.0 Hz, 1H), 2.20 (s, 3H, CH3S), 2.15 (s, 3H, CH3S), 2.06 (s,
4.2. Cabazitaxel
Compound 16 (42.0 g, 45.3 mmol) was dissolved in EtOH (1000 mL)
and then Raneynickel (70 g) was added. After stirred for 12 h at 50 ◦C
under 3 atmosphere pressure of hydrogen gas, the mixture was filtered
and washed with EtOAc (400 mL), then the filtrate was concentrated.
The residue was diluted with EtOAc (700 mL), and washed with brine
(100 mL × 3). The organic layer was dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified on a silica gel
column (petroleum ether/EtOAc = 3/1) to afford Cabazitaxel as a white
amorphous solid (24.9 g, 66%). 1H NMR (400 MHz, CDCl3) δ 8.09 (d, J
= 7.6 Hz, 2H, Ph-H), 7.61 (t, J = 7.4 Hz, 1H, Ph-H), 7.49 (t, J = 7.7 Hz,
8

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
3H, CH3-18), 1.86 (ddd, J = 14.2, 10.6, 1.9 Hz, 1H), 1.78 (s, 3H, CH3-
18), 1.77 (s, 3H), 1.71 (s, 1H, 1-OH), 1.75 (s, 3H), 1.37 (s, 9H, Me3C-),
1.22 (s, 3H), 1.20 (s, 3H); 13C NMR (150 MHz, CDCl3) δ 204.6, 172.9,
170.3, 166.9, 155.4, 140.0, 137.9, 134.4, 133.6, 130.1, 129.3, 128.6,
120.7, 83.9), 81.3, 79.9, 78.8, 77.8, 76.6, 76.6, 74.7, 73.8, 73.8, 72.4,
72.3, 57.3, 51.6, 47.2, 43.3, 35.4, 32.9, 28.3, 26.5, 25.7, 22.4, 20.7,
18.6, 15.7, 14.4, 13.9, 10.7. HRMS (ESI) m/z calcd for C45H63NO14S2
[M+Na]+ 928.3582, Found 928.3587.
4.32 (bs, 2H, H-2′ and H-20), 4.29–4.21 (m, 1H, H-7), 4.19 (d, J = 8.0
Hz, 1H, H-20), 3.89 (d, J = 7.0 Hz, 1H, H-3), 2.82–2.72 (m, 1H, H-6),
2.41 (s, 3H, CH3CO), 2.37–2.32 (m, 1H, H-14), 2.24–2.19 (m, 1H, H-14),
2.20 (s, 3H, CH3S), 2.17 (s, 3H, CH3S), 2.02 (s, 3H, CH3-18), 1.90–1.83
(m, 1H, H-6), 1.74 (s, 3H, CH3-19), 1.33 (s, 9H, Me3C-), 1.21 (s, 3H, CH3-
16), 1.20 (s, 3H, CH3-17), 0.97 (s, 9H, (CH3)3CSi), 0.17 (s, 3H, CH3),
0.12 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) δ 204.7, 171.3, 170.4,
167.2, 155.0, 140.3, 134.3, 133.8, 130.4, 129.4, 128.9, 84.1, 81.4, 80.4,
79.0, 77.8, 77.4, 76.8, 76.7, 75.0, 74.0, 72.3, 72.2, 72.0, 60.6, 57.3,
47.2, 43.5, 35.4, 33.1, 29.9, 28.3, 28.2, 26.7, 25.8, 22.7, 21.2, 18.6,
15.8, 14.2, 14.2, 11.0, ꢀ 4.6, ꢀ 5.3. ESI-MS (m/z) 1028.4 [M+H]+(Calcd
1028.4), 1050.3 [M+Na]+(Calcd 1050.4).
4.2.3. 3′-Dephenyl-3′-(2-methylprop-1-enyl)-7, 10-O-dimethyl cabazitaxel
(21) and 3′-Dephenyl-3′-(2-methylpropyl)-7, 10-O-dimethyl cabazitaxel
(20)
To a solution of 19 (500.0 mg, 0.5 mmol) in 30 mL EtOAc was added
Raney Nickel (5.0 g) under hydrogen atmosphere. The reaction mixture
was stirred for 20 h at room temperature. The reaction mixture was then
filtered through celite and concentrated in vacuo. The residue was pu-
rified on a silica gel column (petroleum ether/EtOAc = 1/1) to afford 21
as a white solid (314.4 mg, 70%) and 20 as a white solid (67.5 mg, 15%).
21:1)H NMR (400 MHz, CDCl3) δ 8.09 (d, J = 8.0 Hz, 2H, Ph-H), 7.60 (t,
J = 7.4 Hz, 1H, Ph-H), 7.47 (t, J = 7.5 Hz, 2H, Ph-H), 6.17 (t, J = 8.8 Hz,
1H, H-13), 5.64 (d, J = 7.1 Hz, 1H, H-2), 5.31 (d, J = 8.2 Hz, 1H), 4.99
(d, J = 9.2 Hz, 1H), 4.84–4.74 (m, 3H), 4.31 (d, J = 8.4 Hz, 1H), 4.23 (s,
1H), 4.18 (d, J = 8.3 Hz, 1H), 3.90–3.85 (m, 1H), 3.83 (d, J = 7.2 Hz,
1H), 3.46 (s, 4H, CH3O, 2′–OH), 3.31 (s, 3H, CH3O), 2.74–2.68 (m, 1H,
H-6), 2.42–2.25(m, 2H), 2.36 (s, 3H, CH3CO), 1.97 (s, 3H), 1.83–1.80
(m, 1H), 1.77 (s, 6H), 1.72 (s, 3H), 1.66 (s, 1H, 1-OH), 1.37 (s, 9H, Me3C-
, 1.25 (s, 3H), 1.21 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 205.2, 170.5,
167.1, 155.6, 146.3, 139.2, 135.6, 133.8, 130.3, 129.4, 128.8, 84.3,
82.8, 81.8, 80.9, 80.1, 78.9, 76.7, 74.8, 73.9, 72.6, 63.3, 57.4, 57.3,
57.1, 47.6, 43.4, 35.5, 32.3, 32.0, 29.9, 29.6, 28.4, 28.2, 26.9, 26.0,
25.9, 22.8, 22.7, 20.8, 18.8, 15.0, 14.3, 10.5. HRMS (ESI) m/z calcd for
4.2.5. 3′-Dephenyl-3′-(2, 2-difluorovinyl)-7, 10-O-demethyl-7, 10-O-
(methylthiomethyl)cabazitaxel (24)
To a solution of 23 (130.0 mg, 0.1 mmo1) in dry THF (5 mL) at room
temperature were added a solution of TBAF (78.4 mg, 0.3 mmol) in THF
(5 mL) under argon whose pH was pre-adjusted to 7 by HOAc. The re-
action mixture was stirred for 1 h at room temperature, and then
quenched with saturated aqueous NH4Cl solution. The aqueous layer
was extracted with EtOAc (50 mL × 3). The combined extracts were
washed with saturated aqueous sodium hydrogen carbonate solution
(20 mL × 3) and brine (20 mL × 3), dried over sodium sulphate, and
concentrated under reduced pressure. The residue was purified on a
silica gel column (petroleum ether/EtOAc = 3/1) to afford 24 as a white
solid (84.4 mg, 73%). 1H NMR (600 MHz, CDCl3) δ 8.11 (d, J = 7.5 Hz,
2H, Ph-H), 7.61 (t, J = 7.4 Hz, 1H, Ph-H), 7.50 (t, J = 7.6 Hz, 2H, Ph-H),
6.23 (t, J = 8.4 Hz, 1H, H-13), 5.67 (d, J = 7.0 Hz, 1H, H-2), 5.59 (s, 1H,
H-10), 4.97 (t, J = 8.6 Hz, 2H), 4.88 (brt, 1H), 4.76 (d, J = 11.7 Hz, 1H),
4.72 (d, J = 11.7 Hz, 1H), 4.67 (d, J = 11.8 Hz, 1H), 4.60 (d, J = 11.7 Hz,
1H), 4.56 (dd, J = 9.5, 1.3 Hz, 1H), 4.32 (d, J = 8.5 Hz, 1H), 4.30 (brs,
1H), 4.25 (dd, J = 10.5, 6.8 Hz, 1H), 3.87 (d, J = 6.9 Hz, 1H), 3.60 (s,
1H, 2′–OH), 2.79 (ddd, J = 14.5, 9.6, 6.7 Hz, 1H), 2.40 (s, 3H, CH3CO),
2.34–2.31 (m, 2H), 2.21 (s, 3H, CH3S), 2.16 (s, 3H, CH3S), 2.04 (s, 3H,
CH3-18), 1.86 (ddd, J = 14.1, 10.7, 1.9 Hz, 1H), 1.75 (s, 3H, CH3-19),
1.72 (s, 1H, 1-OH), 1.34 (s, 9H, Me3C-), 1.22 (s, 3H, CH3-16), 1.20 (s,
3H, CH3-17); 13C NMR (150 MHz, CDCl3) δ 204.5, 172.3, 170.5, 167.1,
154.9, 139.5, 134.7, 133.7, 133.7, 130.2, 129.2, 128.7, 83.9, 81.4, 80.5,
78.8, 77.8, 76.6, 76.5, 74.7, 73.8, 73.2, 72.4, 72.3, 57.3, 47.9, 47.2,
43.4, 35.2, 32.9, 28.2, 26.6, 22.4, 20.7, 15.7, 14.3, 13.9, 10.7. HRMS
(ESI) m/z calcd for C43H57F2NO14S2 [M+Na]+ 936.3081, Found
936.3086.
C
43H59NO14 [M+Na]+ 836.3828, Found 836.3836. 20:1H NMR (400
MHz, CDCl3) δ 8.10 (d, J = 7.5 Hz, 2H, Ph-H), 7.60 (t, J = 7.3 Hz, 1H, Ph-
H), 7.47 (t, J = 7.6 Hz, 2H, Ph-H), 6.18 (t, J = 9.0 Hz, 1H, H-13), 5.63 (d,
J = 6.9 Hz, 1H, H-2), 4.99 (d, J = 9.2 Hz, 1H, H-5), 4.82 (s, 1H, H-10),
4.62 (d, J = 9.5 Hz, 1H, NH), 4.31 (d, J = 8.4 Hz, 1H), 4.20–4.10 (m,
3H), 3.88 (dd, J = 11.0, 6.5 Hz, 1H), 3.83 (d, J = 7.0 Hz, 1H), 3.49 (s,
1H, 2′–OH), 3.45 (s, 3H, CH3O), 3.31 (s, 3H, CH3O), 2.71 (ddd, J = 14.8,
9.6, 6.6 Hz, 1H), 2.43–2.29 (m, 2H), 2.38 (s, 3H, CH3CO), 1.96 (s, 3H,
CH3-18), 1.83–1.77 (m, 1H), 1.72 (s, 3H, CH3-19), 1.68 (s, 1H, 1-OH),
1.65–1.53 (m, 2H), 1.38–1.33 (m, 1H), 1.34 (s, 9H, Me3C-), 1.25 (s, 3H,
CH3-16), 1.20 (s, 3H, CH3-17), 0.99 (d, J = 2.5 Hz, 3H, CH2CH(CH3)2),
0.97 (d, J = 2.3 Hz, 3H, CH2CH(CH3)2,13C NMR (100 MHz, CDCl3) δ
205.2, 174.0, 170.4, 167.1, 155.7, 139.2, 135.6, 133.8, 130.4, 129.5,
128.8, 84.3, 82.8, 81.8, 80.9, 80.0, 78.9, 76.7, 74.9, 73.3, 73.0, 57.5,
57.3, 57.0, 51.5, 47.6, 43.5, 41.3, 35.4, 32.3, 29.9, 28.4, 26.8, 24.9,
23.5, 22.8, 22.0, 21.0, 14.9, 10.6. HRMS (ESI) m/z calcd for C43H61NO14
[M+Na]+ 838.3984, Found 838.3992.
4.2.6. 3′-Dephenyl-3′-(ethyl)-7, 10-O-dimethyl cabazitaxel (25)
To a solution of 24 (60.0 mg, 65.6 μmol) in 30 mL EtOAc was added
Raney Nickel (1.1 g) under hydrogen atmosphere. The reaction mixture
was stirred for 15 h at room temperature. The reaction mixture was then
filtered through celite and concentrated in vacuo. The residue was pu-
rified on a silica gel column (petroleum ether/EtOAc = 1/1.5) to afford
25 as a white solid (46.5 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 8.11 (d,
J = 7.4 Hz, 2H, Ph-H), 7.61 (t, J = 7.4 Hz, 1H, Ph-H), 7.49 (t, J = 7.5 Hz,
2H, Ph-H), 6.20 (t, J = 8.6 Hz, 1H, H-13), 5.65 (d, J = 6.6 Hz, 1H, H-2),
5.00 (d, J = 9.3 Hz, 1H, H-5), 4.82 (s, 1H, H-10), 4.70 (d, J = 9.7 Hz, 1H,
NH), 4.33–4.29 (m, 2H, H-20, H-2′), 4.18 (d, J = 8.3 Hz, 1H, H-20), 3.96
(dd, J = 16.6, 6.5 Hz, 1H), 3.89–3.82 (m, 2H), 3.45 (s, 3H, CH3O), 3.31
(s, 3H, CH3O), 2.75–2.67 (m, 1H, H-6), 2.40 (s, 3H, CH3CO), 2.35–2.28
(m, 2H), 1.96 (s, 3H, CH3-18), 1.81 (t, J = 12.8 Hz, 2H), 1.72 (s, 3H,
CH3-19), 1.69 (s, 1H, 1-OH), 1.33 (s, 9H, Me3C-), 1.25 (s, 3H, CH3-16),
1.21 (s, 3H, CH3-17), 1.02 (t, J = 7.3 Hz, 3H, CH2CH3); 13C NMR (100
MHz, CDCl3) δ 205.2, 174.2, 170.6, 170.4, 167.2, 155.8, 139.2, 135.5,
133.8, 130.4, 129.4, 128.8, 84.3, 82.8, 81.9, 80.9, 80.0, 78.9, 74.8, 72.9,
71.9, 57.4, 57.3, 57.1, 54.6, 47.5, 43.5, 35.3, 32.3, 29.9, 28.4, 26.8,
25.3, 22.8, 21.0, 14.8, 14.3, 11.0, 10.5. HRMS (ESI) m/z calcd for
4.2.4. 2′-O-(tert-butyldimethylsilyl)-3′-dephenyl-3′-(2, 2-difluorovinyl)-7,
10-O-demethyl-7, 10-O- (methylthiomethyl) cabazitaxel (23)
A solution of 13 (160.0 mg, 0.2 mmo1) and racemic 3-(t-butyldi-
methylsilyloxy)-β-lactam 22 (109.1 mg, 0.3 mmol) in dry THF (10 mL)
under argon was cooled to ꢀ 50 ◦C. To the above mixture a solution of
LiHMDS (0.3 mL, 0.3 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 (50
mL × 3). The combined extracts were washed with brine (20 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure.
The residue was purified on a silica gel column (petroleum ether/EtOAc
1
= 6/1) to afford 23 as a white solid (148.5 mg, 60%). H NMR (400
MHz, CDCl3) δ 8.11 (d, J = 7.3 Hz, 2H, Ph-H), 7.61 (t, J = 7.4 Hz, 1H, Ph-
H), 7.51 (t, J = 7.5 Hz, 2H, Ph-H), 6.22 (t, J = 8.9 Hz, 1H, H-13), 5.69 (d,
J = 6.9 Hz, 1H, H-2), 5.58 (s, 1H, H-10), 4.97 (d, J = 9.4 Hz, 2H, CONH,
H-5), 4.88 (bs, 1H, H-3′), 4.74 (dd, J = 24.6, 11.7 Hz, OCH2S), 4.64 (dd,
J = 23.5, 11.8 Hz, OCH2S), 4.43 (dd, J = 24.6, 9.8 Hz, 1H, CH = CF2),
C
41H57NO14 [M+Na]+ 810.3671, Found 810.3679.
9

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
4.2.7. 2′-O-(tert-butyldimethylsilyl)-3′-dephenyl-3′-(difluromethyl)-7, 10-
O-demethyl-7, 10-O- (methylthiomethyl)cabazitaxel (27)
mixture was stirred for 15 h at room temperature. The reaction mixture
was then filtered through celite and concentrated in vacuo. The residue
was purified on a silica gel column (petroleum ether/EtOAc = 8/1) to
afford 30 as a white solid (1.4 g, 76%). 1H NMR (400 MHz, CDCl3) δ 8.11
(d, J = 7.4 Hz, 2H, Ph-H), 7.59 (t, J = 7.4 Hz, 1H, Ph-H), 7.48 (t, J = 7.6
Hz, 2H, Ph-H), 7.37 (t, J = 7.4 Hz, 2H, Ph-H), 7.30–7.26 (m, 3H, Ph-H),
6.31 (t, J = 9.2 Hz, 1H, H-13), 5.65 (d, J = 7.1 Hz, 1H, H-2), 5.45 (bd, J
= 9.8 Hz, 1H, CONH), 5.31 (bd, J = 8.9 Hz, 1H, H-3′), 5.00 (d, J = 10.0
Hz, 1H, H-5), 4.80 (s, 1H, H-10), 4.52 (s, 1H, H-2′), 4.32 (d, J = 8.3 Hz,
1H, H-20), 4.19 (d, J = 8.3 Hz, 1H, H-20), 3.94–3.83 (m, 2H, H-3 and H-
7), 3.45 (s, 3H, CH3O), 3.31 (s, 3H, CH3O), 2.76–2.62 (m, 1H, H-6), 2.56
(s, 3H, CH3CO), 2.40–2.30 (m, 1H, H-14), 2.22–2.14 (m, 1H, H-14), 1.95
(s, 3H, CH3-18), 1.84–1.76 (m, 1H, H-6), 1.72 (s, 3H, CH3-19), 1.32 (s,
9H, Me3C-), 1.24 (s, 3H, CH3-16), 1.20 (s, 3H, CH3-17), 0.75 (s, 9H,),
ꢀ 0.12 (s, 3H), ꢀ 0.30 (s, 3H) ; 13C NMR (100 MHz, CDCl3) δ 205.1,
171.8, 170.4, 167.2, 139.6, 135.2, 133.7, 130.3, 129.4, 128.8, 128.8,
127.9, 126.6, 84.4, 82.6, 81.8, 80.9, 80.2, 79.1, 76.7, 75.9, 74.9, 71.8,
57.4, 57.3, 56.9, 47.4, 43.6, 35.4, 32.2, 29.9, 28.3, 26.9, 25.7, 23.3,
21.4, 18.4, 14.7, 10.6, ꢀ 5.3, ꢀ 5.7. ESI-MS (m/z) 950.4 [M+H]+(Calcd
950.5), 972.5 [M+Na]+(Calcd 972.5).
A solution of 13 (200.0 mg, 0.3 mmo1) and racemic 3-(t-butyldi-
methylsilyloxy)-β-lactam 26 (175.8 mg, 0.5 mmol) in dry THF (10 mL)
under argon was cooled to ꢀ 50 ◦C. To the above mixture a solution of
LiHMDS (0.5 mL, 0.5 mmol, 1.0 M in THF) was added. The reaction
mixture was stirred for 3 h at the same temperature and then quenched
with saturated aqueous NH4Cl solution and extracted with EtOAc (30
mL × 3). The combined extracts were washed with brine (10 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure.
The residue was purified on a silica gel column (petroleum ether/EtOAc
1
= 6/1) to afford 27 as a white solid (253.7 mg, 83%). H NMR (400
MHz, CDCl3) δ 8.12 (d, J = 7.5 Hz, 2H, Ph-H), 7.61 (t, J = 7.5 Hz, 1H, Ph-
H), 7.51 (t, J = 7.5 Hz, 2H, Ph-H), 6.19 (t, J = 8.9 Hz, 1H, H-13),
5.92–5.60 (m, 2H, H-2 and CF2H), 5.58 (s, 1H, H-10), 5.02 (bd, J = 10.4
Hz, 1H, CONH), 4.95 (bd, J = 9.3 Hz, 1H, H-5), 4.78–4.62 (m, 5H, H-2′
and OCH2S × 2), 4.51–4.40 (m, 1H, H-3′), 4.32 (d, J = 8.1 Hz, 1H, H-20),
4.25 (dd, J = 10.2, 6.7 Hz, 1H, H-7), 4.18 (d, J = 8.1 Hz, 1H, H-20), 3.88
(d, J = 6.7 Hz, 1H, H-3), 2.84–2.73 (m, 1H, H-6), 2.41 (s, 3H, CH3CO),
2.35–2.28 (m, 1H, H-14), 2.26–2.18 (m, 1H, H-14), 2.20 (s, 3H, CH3S),
2.16 (s, 3H, CH3S), 2.02 (s, 3H, CH3-18), 1.90–1.82 (m, 1H, H-6), 1.74
(s, 3H, CH3-19), 1.32 (s, 9H, Me3C-), 1.20 (s, 3H, CH3-16), 1.17 (s, 3H,
CH3-17), 0.95 (s, 9H, (CH3)3CSi), 0.18 (s, 3H, CH3), 0.12 (s, 3H, CH3);
13C NMR (100 MHz, CDCl3) δ 204.7, 171.0, 170.5, 167.3, 155.4, 140.1,
134.4, 133.8, 130.4, 129.3, 128.9, 84.1, 81.4, 81.0, 78.9, 77.9, 76.7,
74.9, 74.0, 72.3, 72.2, 72.1, 62.9, 57.4, 47.3, 43.5, 35.3, 33.1, 29.9,
28.2, 26.6, 25.8, 22.9, 21.1, 18.4, 15.8, 14.2, 14.1, 11.0, ꢀ 4.5, ꢀ 5.5.
ESI-MS (m/z) 1016.4 [M+H]+(Calcd 1016.4), 1038.3 [M+Na]+(Calcd
1038.4).
4.2.10. 2′-O-(tert-butyldimethylsilyl)-2-O-debenzoyl cabazitaxel (31)
To a solution of 30 (1.0 g, 1.1 mmol) in dry DCM (50 mL) at ꢀ 30 ◦C
were added Triton B (40% W/W in MeOH, 800 μL). The reaction mixture
was stirred for 5 min at ꢀ 50 ◦C, and then quenched with saturated
aqueous NH4Cl solution. The aqueous layer was extracted with DCM (50
mL × 3). The combined extracts were washed with brine (20 mL × 3),
dried over sodium sulphate, and concentrated under reduced pressure.
The residue was purified on a silica gel column (petroleum ether/EtOAc
1
= 2/1) to afford 31 as a white solid (498.6 mg, 56%). H NMR (400
4.2.8. 3′-Dephenyl-3′-(difluromethyl)-7, 10-O-dimethyl cabazitaxel (29)
To a solution of 27 (240.0 mg, 0.2 mmo1) in dry THF (15 mL) at
room temperature were added a solution of TBAF (156.9 mg, 0.6 mmol)
in THF (10 mL) under argon whose pH was pre-adjusted to 7 by HOAc.
The reaction mixture was stirred for 1 h at room temperature, and then
quenched with saturated aqueous NH4Cl solution. The aqueous layer
was extracted with EtOAc (60 mL × 3). The combined extracts were
washed with saturated aqueous sodium hydrogen carbonate solution
(20 mL × 3) and brine (20 mL × 3), dried over sodium sulphate, and
concentrated under reduced pressure to afford crude 28.
MHz, CDCl3) δ 7.35 (t, J = 7.0 Hz, 2H, Ph-H), 7.29 (d, J = 7.0 Hz, 1H, Ph-
H), 7.20 (d, J = 7.5 Hz, 2H, Ph-H), 6.23 (t, J = 9.0 Hz, 1H, H-13), 5.50
(bd, J = 9.9 Hz, 1H, CONH), 5.20 (d, J = 8.5 Hz, 1H, H-3′), 5.02 (bd, J =
9.4 Hz, 1H, H-5), 4.74 (s, 1H, H-10), 4.68–4.59 (m, 2H, H-20), 4.44 (s,
1H, H-2′), 3.90 (d, J = 3.4 Hz, 1H, H-3), 3.84 (dd, J = 9.8, 6.0 Hz, 1H, H-
7), 3.50 (d, J = 6.4 Hz, 1H, H-2), 3.41 (s, 3H, CH3O), 3.29 (s, 3H, CH3O),
2.75–2.62 (m, 1H, H-6), 2.41 (s, 3H, CH3CO), 2.24–2.08 (m, 2H, H-14),
1.90 (s, 3H, CH3-18), 1.80–1.66 (m, 1H, H-6), 1.68 (s, 3H, CH3-19), 1.42
(s, 9H, Me3C-), 1.23 (s, 3H, CH3-16), 1.07 (s, 3H, CH3-17), 0.74 (s, 9H,),
ꢀ 0.15 (s, 3H), ꢀ 0.32 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 205.7, 171.9,
170.0, 155.5, 139.3, 138.8, 135.4, 128.7, 127.9, 126.6, 84.0, 83.0, 82.7,
81.1, 80.3, 78.4, 78.1, 75.6, 74.6, 72.4, 57.3, 56.9, 47.4, 43.1, 35.7,
32.3, 29.9, 28.4, 26.4, 25.7, 23.3, 21.3, 18.4, 14.7, 10.8, ꢀ 5.3, ꢀ 5.7.
ESI-MS (m/z) 868.5 [M+Na]+(Calcd 868.4).
To a solution of the above crude product 28 (185.3 mg, 0.2 mmol) in
30 mL EtOAc was added Raney Nickel (4.0 g) under hydrogen atmo-
sphere. The reaction mixture was stirred for 12 h at room temperature.
The reaction mixture was then filtered through celite and concentrated
in vacuo. The residue was purified on a silica gel column (petroleum
ether/EtOAc = 1/1.5) to afford 29 as a white solid (129.8 mg, 68% over
two steps). 1H NMR (400 MHz, CDCl3) δ 8.11 (d, J = 7.0 Hz, 2H, Ph-H),
7.61 (t, J = 7.2 Hz, 1H, Ph-H), 7.50 (t, J = 7.7 Hz, 2H, Ph-H), 6.24 (t, J =
9.4 Hz, 1H, H-13), 5.89 (td, J = 54.6, 5.2 Hz, 1H, CF2H), 5.64 (d, J = 6.4
Hz, 1H, H-2), 5.08 (d, J = 9.8 Hz, 1H, CONH), 4.98 (d, J = 8.3 Hz, 1H, H-
5), 4.80 (s, 1H, H-10), 4.64 (s, 1H, H-2′), 4.49–4.44 (m, 1H, H-3′), 4.32
(d, J = 8.8 Hz, 1H, H-20), 4.17 (d, J = 8.1 Hz, 1H, H-20), 3.89–3.82 (m,
2H), 3.54 (s, 1H, 2′–OH), 3.46 (s, 3H, CH3O), 3.31 (s, 3H, CH3O),
2.76–2.66 (m, 1H), 2.41 (s, 3H, CH3CO), 2.32–2.25 (m, 2H), 1.95 (s, 3H,
CH3-18), 1.83–1.77 (m, 1H), 1.75 (s, 1H, 1-OH), 1.72 (s, 3H, CH3-17),
1.34 (s, 9H, Me3C-), 1.25 (s, 3H, CH3), 1.20 (s, 3H, CH3); 13C NMR (100
MHz, CDCl3) δ 204.9, 172.3, 170.6, 167.1, 155.1, 138.3, 135.8, 133.6,
130.2, 129.1, 128.7, 84.1, 82.6, 81.7, 81.0, 80.7, 78.7, 76.5, 74.5, 73.2,
68.4, 57.4, 57.1, 56.9, 47.4, 43.3, 35.1, 32.0, 29.7, 28.0, 26.7, 22.6,
20.8, 14.6, 10.4; 13F NMR (376 MHz, CDCl3) δ ꢀ 126.3 (ddd, J = 286.5,
56.4, 12.0 Hz, 1F), ꢀ 128.1 (ddd, J = 288.4, 56.4, 9.4 Hz, 1F). HRMS
(ESI) m/z calcd for C40H53F2NO14 [M+Na]+ 832.3326, Found 832.3320.
4.2.11. 2′-O-(tert-butyldimethylsilyl)-2-O-debenzoyl-2-O-[3-(methoxy)
benzoyl]cabazitaxel(32)
To a solution of 31 (200.0 mg, 0.2 mmol), 3-methoxybenzoic acid
(151.1 mg, 1.0 mmol) and DMAP (24.4 mg, 0.2 mmol) in toluene (20
mL) was added 1, 3-dicyclohexylcarbodiimide (DCC) (330.1 mg, 1.6
mmol). The reaction mixture was stirred at 65 ◦C for 48 h. The resulting
precipitate was filtered off and concentrated under reduced pressure to
afford crude 32.
4.2.12. 2-O-debenzoyl-2-O-[3-(methoxy)benzoyl]cabazitaxel(33)
The crude product 32 was dissolved in dry THF (15 mL) at room
temperature under argon. To the above solution was added a solution of
TBAF (156.9 mg, 0.6 mmol) in THF (10 mL) whose pH was pre-adjusted
to 7. The reaction mixture was stirred for 1 h at room temperature, and
then quenched with saturated aqueous NH4Cl solution. The aqueous
layer was extracted with EtOAc (20 mL × 3). The combined extracts
were washed with saturated aqueous sodium hydrogen carbonate so-
lution (10 mL × 3) and brine (10 mL × 3), dried over sodium sulphate,
and concentrated under reduced pressure. The residue was purified on a
silica gel column (petroleum ether/EtOAc = 1/1) to afford 33 as a white
solid (137.1 mg, 67% for two steps). 1H NMR (400 MHz, CDCl3) δ 7.69
4.2.9. 2′-O-(tert-butyldimethylsilyl) cabazitaxel (30)
To a solution of 15 (2.0 g, 1.9 mmol) in 150 mL EtOAc was added
Raney Nickel (20.0 g) under hydrogen atmosphere. The reaction
10

S. Ren et al.
Bioorganic & Medicinal Chemistry 41 (2021) 116224
(d, J = 7.0 Hz, 1H, Ph-H), 7.62 (s, 1H, Ph-H), 7.45 – 7.34 (m, 5H, Ph-H),
7.33 (d, J = 5.9 Hz, 1H, Ph-H), 7.14 (dd, J = 8.5, 1.7 Hz, 1H, Ph-H), 6.20
(t, J = 8.7 Hz, 1H, H-13), 5.62 (d, J = 7.0 Hz, 1H, H-2), 5.45 (d, J = 9.6
Hz, 1H, NH), 5.26 (d, J = 7.9 Hz, 1H, H-3′), 4.98 (d, J = 9.3 Hz, 1H), 4.80
(s, 1H), 4.62 (s, 1H), 4.34 (d, J = 8.3 Hz, 1H), 4.17 (d, J = 8.3 Hz, 1H),
3.87 (s, 3H, CH3O), 3.86–3.83 (m, 1H), 3.80 (d, J = 6.9 Hz, 1H), 3.45 (s,
3H, CH3O), 3.30 (s, 3H, CH3O), 2.74–2.68 (m, 1H), 2.35 (s, 3H, CH3CO),
2.31–2.19 (m, 2H), 1.87 (s, 3H, CH3-18), 1.84–1.75 (m, 2H), 1.71 (s,3H,
CH3-19), 1.36 (s, 9H, Me3C-), 1.25 (s, 3H, CH3-17), 1.21 (s, 3H, CH3-16);
13C NMR (100 MHz, CDCl3) δ 205.1, 172.8, 170.6, 167.0, 159.8, 155.5,
138.9, 135.7, 130.6, 129.8, 129.0, 128.2, 127.0, 122.7, 120.2, 114.8,
84.2, 82.8, 81.9, 80.9, 78.8, 76.7, 74.7, 73.9, 72.7, 57.5, 57.2, 57.1,
56.4, 55.6, 47.5, 43.4, 35.4, 32.3, 29.9, 28.4, 27.0, 22.9, 20.9, 14.8,
10.5. HRMS (ESI) m/z calcd for C46H59NO15 [M+Na]+ 888.3777, Found
888.3785.
Hz, 2H, Ph-H), 7.42–7.38 (m, 4H, Ph-H), 7.33 (t, J = 6.7 Hz, 1H, Ph-H),
6.23 (t, J = 8.2 Hz, 1H, H-13), 5.86 (s, 1H, H-10), 5.68 (d, J = 6.9 Hz, 1H,
H-2), 5.46 (d, J = 8.5 Hz, 1H, NH), 5.26 (d, J = 8.1 Hz, 1H), 4.94 (d, J =
9.2 Hz, 1H), 4.65–4.63 (m, 2H), 4.56 (d, J = 12.1 Hz, 1H), 4.44 (d, J =
12.0 Hz, 1H), 4.38 (d, J = 12.0 Hz, 1H), 4.35–4.33 (m, 1H), 4.31 (d, J =
8.5 Hz, 1H), 4.17 (d, J = 8.6 Hz, 1H), 3.76 (d, J = 6.8 Hz, 1H), 3.57 (s,
1H, 2′–OH), 2.98 (s, 3H), 2.86 (s, 3H), 2.75–2.70 (m, 1H), 2.40 (s, 3H,
CH3CO), 2.31 (d, J = 8.1 Hz, 2H), 1.97 (s, 3H, CH3-18), 1.91–1.86 (m,
1H), 1.86 (s, 1H, 1-OH), 1.78 (s, 3H, CH3-19), 1.35 (s, 9H, Me3C-), 1.24
(s, 3H, CH3-16), 1.20 (s, 3H, CH3-17); 13C NMR (150 MHz, CDCl3) δ
203.7, 173.0, 170.5, 166.9, 155.5, 142.5, 138.4, 133.8, 133.0, 130.2,
129.0, 128.9, 128.8, 128.1, 126.8, 83.3, 81.9, 81.4, 81.3, 80.9, 80.6,
80.3, 78.7, 77.3, 77.0, 76.8, 76.4, 74.4, 73.6, 72.4, 57.2, 56.3, 46.8,
43.2, 38.2, 38.0, 35.3, 32.6, 28.2, 26.5, 22.6, 20.9, 14.4, 10.4. HRMS
(ESI) m/z calcd for C47H61NO18S2 [M+Na]+ 1014.3222, Found
1014.3227.
4.2.13. 2′-O-(tert-butyldimethylsilyl)-2-O-debenzoyl-2-O-[3-(azido)
benzoyl]cabazitaxel(34)
4.3. Cytotoxicity assays
To a solution of 31 (200.0 mg, 0.2 mmol), 3-azidobenzoic acid
(163.1 mg, 1.0 mmol) and DMAP (24.4 mg, 0.2 mmol) in toluene (20
mL) was added 1, 3-dicyclohexylcarbodiimide (DCC) (330.1 mg, 1.6
mmol). The reaction mixture was stirred at 65 ◦C for 48 h. The resulting
precipitate was filtered off and concentrated under reduced pressure to
afford crude 34.
All of the prepared compounds were tested for their in vitro anti-
cancer activities against A549 lung adenocarcinoma cell line, a human
oral squamous epithelium cancer cell line KB and the taxol-resistant lung
adenocarcinoma cell line A549/T, the vincristine-resistant cell line KB/
VCR were conducted in Shanghai Institute of Materia Medica. Cells were
seeded into 96-well plates and cultured overnight, treated with tested
4.2.14. 2-O-debenzoyl-2-O-[3-(azido)benzoyl]cabazitaxel(35)
samples for 72 h. Each well was added 10 μL CCK-8 and cultured 4 h,
The crude product 34 was dissolved in dry THF (15 mL) at room
temperature under argon. To the above solution was added a solution of
TBAF (156.9 mg, 0.6 mmol) in THF (10 mL) whose pH was pre-adjusted
to 7. The reaction mixture was stirred for 1 h at room temperature, and
then quenched with saturated aqueous NH4Cl solution. The aqueous
layer was extracted with EtOAc (20 mL × 3). The combined extracts
were washed with saturated aqueous sodium hydrogen carbonate so-
lution (10 mL × 3) and brine (10 mL × 3), dried over sodium sulphate,
and concentrated under reduced pressure. The residue was purified on a
silica gel column (petroleum ether/EtOAc = 1/1) to afford 35 as a white
solid (132.1 mg, 68% for two steps). 1H NMR (400 MHz, CDCl3) δ 7.87
(d, J = 7.5 Hz, 1H, Ph-H), 7.79 (s, 1H, Ph-H), 7.48 (t, J = 7.9 Hz, 1H, Ph-
H), 7.42–7.38 (m, 4H, Ph-H), 7.34–7.31 (m, 1H, Ph-H), 7.26–7.23 (m,
1H, Ph-H), 6.18 (t, J = 8.7 Hz, 1H, H-13), 5.62 (d, J = 6.9 Hz, 1H, H-2),
5.42 (d, J = 8.5 Hz, 1H, NH), 5.24 (d, J = 9.2 Hz, 1H, H-3′), 4.99 (d, J =
8.9 Hz, 1H, H-5), 4.80 (s, 1H, H-10), 4.60 (s, 1H, H-2′), 4.32 (d, J = 8.4
Hz, 1H, H-20), 4.16 (d, J = 8.3 Hz, 1H, H-20), 3.86 (dd, J = 10.6, 6.8 Hz,
1H), 3.82 (d, J = 7.2 Hz, 1H), 3.45 (s, 3H, CH3O), 3.30 (s, 3H, CH3O),
2.71 (ddd, J = 14.5, 9.9, 6.7 Hz, 1H), 2.36 (s, 3H, CH3CO), 2.33–2.19 (m,
2H), 1.88 (s, 3H, CH3-18), 1.83–1.76 (m, 1H), 1.71 (s, 3H, CH3-19), 1.36
(s, 9H, Me3C-), 1.25 (s, 3H, CH3-16), 1.21 (s, 3H, CH3-17); 13C NMR
(101 MHz, CDCl3) δ 205.1, 173.0, 170.6, 166.1, 155.4, 140.8, 139.0,
138.5, 135.6, 131.0), 130.3, 129.0, 128.20, 127.0, 126.9, 124.4, 120.3,
84.3, 82.7, 81.8, 80.8, 80.4, 78.9, 76.59 (s), 75.08 (s), 73.77 (s), 72.79
(s), 57.47 (s), 57.21 (s), 57.01 (s), 56.40 (s), 47.5, 43.4, 35.3, 32.2, 29.9,
28.3, 26.9, 22.9, 20.8, 14.8, 10.5. HRMS (ESI) m/z calcd for
then was measured the OD value at 450 nm using a multiwell spectro-
photometer (VERSAmax, Molecular Devices, Sunnyvale, CA).
4.4. Cell cycle assays for flow cytometry
Cell cycle was analyzed using propidium iodide (PI) staining. Briefly,
A549 cells were plated at a density of 1.5 × 105 cells/well in the 6-well
plates. After incubation overnight, cells were then treated with vehicle
or compounds at a concentration of 40 nM and cultured at 37 ◦C for 24 h.
At the end of treatment, cells were collected and washed with PBS, then
fixed in 70% ethanol overnight at 4 ◦C. After that, cells were incubated
with a mixture of 10 µg/ml PI (Beyotime, ST511) and 20 µg/ml RNase A
(Beyotime, ST578) at 37 ◦C for 30 min. Cell cycle distribution was
analyzed by fow cytometry (FACS Calibur, BD) and the proportion of
cells in G1, S, and G2/M phases were analyzed by FlowJo software.
4.5. Xenograft model antitumor assay
The animal studies were carried out in accordance with the Guide-
lines for Animal Experimentation of Shenyang Pharmaceutical Univer-
sity, 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 resuspension at a concentration of 2 × 107
per mL. 200 μL of cell suspension was implanted subcutaneously into the
left 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.
C
45H56N4O14 [M+Na]+ 899.3685, Found 899.3689.
4.2.15. 7, 10-O-demethyl-7, 10-O-(methylsulfonylmethyl)cabazitaxel (36)
To a solution of 16 (300.0 mg, 0.8 mmol) in dry DCM (50 mL) at
room temperature were added 85% MCPBA (812.1 mg, 4.0 mmol) under
argon slowly. The reaction mixture was stirred for 7 h at room tem-
perature, and then quenched with saturated aqueous Na2SO3 solution.
The aqueous layer was extracted with DCM (50 mL × 3). The combined
extracts were washed with saturated aqueous sodium hydrogen car-
bonate solution (20 mL × 3) and brine (20 mL × 3), dried over sodium
sulphate, and concentrated under reduced pressure. The residue was
purified on a silica gel column (petroleum ether/EtOAc = 2/1) to afford
36 as a white solid (275.8 mg, 87%). 1H NMR (600 MHz, CDCl3) δ 8.09
(d, J = 7.6 Hz, 2H, Ph-H), 7.62 (t, J = 7.4 Hz, 1H, Ph-H), 7.50 (t, J = 7.7
Tumor volumes were monitored by caliper measurements of the
length and width and calculated using the formula of V = 1/2 × a × 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 = Vt/V0, where V0 is the tumor volume at day 0 and Vt is the
tumor volume measured each time point. The relative tumor prolifera-
tion rate T/C = TRTV (treatment group)/CRTV (control) was calculated
and the percentage of tumor growth inhibition (TGI) values were also
11

S. Ren et al.
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Declaration of Competing Interest
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chemistry, structure-activity relationships and conformational analysis. In: ACS
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The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
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We would like to thank professor Meiyu Geng and her coworkers
(Shanghai Institute of Materia Medica) for the xenograft model anti-
tumor assay. We gratefully acknowledge the financial support from
National Major Scientific and Technological Special Project of China for
“Significant New Drugs Development” (2018ZX09101004-003 and
2018ZX09101-004-006-008).
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