Design, synthesis and biological evaluation of novel podophyllotoxin derivatives bearing 4β-disulfide/trisulfide bond as cytotoxic agents

Article abstract of DOI:10.1039/c5ra12837d

A novel series of C-4β-disulfide/trisulfide-containing podophyllotoxin derivatives were designed, synthesized, and biologically evaluated for their cytotoxic activities against human cancer cell lines, including KB (Mouth Epidermal Carcinoma Cells) and KB/VCR (Vincristine-resistant Mouth Epidermal Carcinoma Cells). Most of these compounds exhibited promising moderate to good cytotoxic activities. In particular, some of them displayed even superior activities to that of etoposide, especially for KB/VCR cell lines, indicating that introduction of the disulfide/trisulfide moiety would be beneficial for overcoming the multi-drug resistant limitation of etoposide. Moreover, the metabolic evaluation of the most promising compound was further performed to reveal that disulfide bond can be stable in human plasma over 8 hours, indicating good potential of these compounds for in vivo anti-cancer activities.

Full text of DOI:10.1039/c5ra12837d

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DOI: 10.1039/C5RA12837D
Journal Name
ARTICLE
Design, Synthesis and Biological Evaluation of Novel
Podophyllotoxin Derivatives Bearing 4β-Disulfide/trisulfide Bond
meiReceived 00th January 20xx,
Accepted 00th January 20xx
as Cytotoxic Agents†
Shi-Jun Zhu+, Hua-Zhou Ying+, Yan Wu, Ni Qiu, Tao Liu, Bo Yang, Xiao-Wu Dong*, Yong-Zhou Hu*
DOI: 10.1039/x0xx00000x
A
novel series of C-4β-disulfide/trisulfide-containing podophyllotoxin derivatives were designed, synthesized, and
www.rsc.org/
biologically evaluated for their cytotoxic activities against human cancer cell lines, including KB (Mouth Epidermal
Carcinoma Cells)and KB/VCR (Vincristine-resistantMouth Epidermal Carcinoma Cells). Most of these compounds exhibited
promising moderate to good cytotoxic activities. In particular, some of them displayed even superior activities to that of
etoposide, especially for KB/VCRcell lines, indicating that introduction of disulfide/trisulfide moiety would be benefical for
overcoming the multi-drug resistant limitation of etoposide. Moreover, the metabolic evaluation of the most promising
compound was further performed toreveal that difulfide bond can be stable in human plasma over 8 hours, indicating a
good prospect of these compounds for in-vivo anti-cancer activities.
A
O
1 Introduction
OMe
HO
O
S
S
MeO
S
S
S
OH
Disulfide/trisulfide moiety (−S−S−/−S−S−S−) has been
demonstrated to possess diverse functionalities, such as,
switch for protein function,1 conversion of reactive oxygen
S
S
S
S
N
O
N
H
S
O
NH2
Thiarubrine A
.-
species (O2 , H2O2 and HO.),2 and crucial role in targeting
Leinamycin
Varacin
tumors.3 They frequently occurred in
a
variety of
O
O
OC(O)NH2
OMe
O
R
S S
H
7
natural/synthetic compounds4 (i.e. leinamycin,5 thiarubrines,6
varacin,7 and esperamicins8) (Fig. 1A) and the emerging role of
disulfide and multisulfide therapeutic agents have been well
recognized. For instance, Vyas et. al.9a and kono et. al.9b
reported the mitomycin disulfides M-1a and M-1b, which were
modified from mitomycin C by replacing the C(7) amine with
aminoethylene disulfide group. The resulting M-1a and M-1b
exhibited superior biological profiles compared with
mitomycin C, which represented 10~100-folds greater
cytotoxicity in tumor cell lines and more efficient cellular
uptake.
SugarO
NHCO2Me
OSugar
N
NH
HO
N
S
NO2
M-1a: R=
MeSSS
(CH2)2N(H)C(O)(CH2)2C(H)(NH2)CO2H
NO2
M-1b: R=
Esperamicins
O
HO
O
B
O
N
(Me)2
N
N
HN
O
O
O
O
O
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
GL-331
OH
NK611
Etoposide (VP-16, Fig. 2), an analogue of podophyllotoxin, has
been widely used for treatment of numerous solid tumors (e.g.
lung, ovarian and testicular cancer) and hematological cancer
(e.g. lymphoma).10 Nevertheless, there still are some
undesirable side effects (e.g. myelosuppression, neurotoxicity)
TOP53
Fig.1. The structures of Disulfide/trisulfide-containing natural/synthetic compounds (A)
and etoposide analogues (B).
and drug-resistance problems that have occurred in the clinical
application.11 With aim to address these existing problems, a
number of etoposide derivatives have been designed and
synthesized,
subsequently,
their
structure-activities
relationships were extensively explored, revealing that C-4
moiety on ring C of etoposide would be extremely tolerant for
anti-cancer activities.12 As
a consequence, a variety of
podophyllotoxinderivatives, such as TOP53,13 GL-33114 and
NK61115 were disclosed (Fig. 1B), some of which showed
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Journal Name
C4-side chains (Fig. 2)
Herein, we designed and synthesized
a
variety of novel
disulfide/trisulfide-containing 4β-podophyllotoxin derivatives (17a-
y, 18a-e and 22a-f). To our knowledge, this is the first time
disulphide/trisulfide moiety was introduced into podophyllotoxin.
All of the prepared compounds were tested for their cytotoxic
activities against KB and KB/VCR cell lines. Subsequently, their
preliminary tructure-activity relationships (SARs) were explored in-
depth to investigate the effect of the linker (i.e. disulfide and
trisulfide) and various side chains. Moreover, the metabolism
stability in the human plasma was carried out to evaluate the
practical application value in further development of compound
17I, which showed the most promise in in-vitro study.
Fig.2.The design of disulfide/trisulfide-containing 4β-podophyllotoxin derivatives.
superior biological activities compared with etoposide, especially
for multi-resistant cancer cells. In our previous study, we also
reported a series of 4β-anilino substituted podophyllotoxin
derivatives (e.g. WL-001, Fig. 2) with potent cytotoxic activities
2 Results and discussion
2.1 Chemistry
Considering that disulfide bonds can be efficiently formed by
(IC50 = 1.91, 8.48 μM) against KB and KB/VCR cancer cells, oxidation of two different free thiol compounds, the synthesis
respectively, which is superior to that of VP-16 (IC50 = 4.61,
83.4 μM).16 Nevertheless, toxicity was observed during in-vivo
study, thereby hindering the further development of these
compounds and promoting us to discover other novel
podophyllotoxin derivatives as anti-cancer agents. In this context,
due to the multi-functionality and good biological compatibility of
disulfide/trisulfide bonds, we envisioned that it would act as a
promising alternative linker between podophyllotoxin and various
of disulfide-containing podophyllotoxin derivatives 17a
18a were divided into two part, including thiol-containing
side chain moieties 3a 5a 7a and 13a (Scheme 1) and
4β-mercapto-4'-O-demethyl-4-desoxypodophyllotoxin 15
(Scheme 2). Thiol compounds 3a and 5a were obtained via
nucleophilic reaction of thiourea with chloride-substituted
alkyl compounds 2a or 4a , which were acquired by the
-y and
-
e
-j
,
-
c,
-
e
-c
-j
-c
-
j
-c
Scheme 1 The synthetic route of thiol-containing sidechain moieties 3a-j, 5a-c and 7a-e and 13a-c. Reagents and conditions: (a)1-bromo-3-chloropropane, K2CO3, CH3CN, r.t.; (b)
.
thiourea, KI, EtOH, reflux; (c) NaOH aq. reflux; (d) 1-bromo-2-chloroethane, K2CO3, CH3CN, r. t.; (e) HOBt, EDC, DCM; (f) thiolacetic acid, BF3 Et2O; (g) CH3ONa, CH3OH; (h) cation
exchange resin; (i) 33% HBr in AcOH, DCM; (j) NaSH.9H2O, CS2, DMF.
2 | J. Name., 2012, 00, 1-3
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ARTICLE
O
O
OH
O
O
HO
I
OH
SH
S
S
R
O
O
O
O
d
c
O
O
O
O
b
O
a
O
O
O
O
O
O
O
O
O
O
O
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
H3CO
OCH3
OH
OH
15
OH
16
OH
OH
14
17a-y
N
O
17a R=
N
N
N
O
N
N
17p R=
17v' R=
17i R=
N
NBoc
N
17b R=
17c R=
e
O
O
N
17j R=
17q R=
17r' R=
17v R=
17w R=
S
N
NH
17k R=
O
N
N
N
O
17d' R=
OCH3
OTBDMS
N
N
N
f
NHBoc
O
17l R=
O
O
17r R=
e
N
17x' R=
17x R=
17d' R=
17e R=
OCH3
N
OH
NBoc
17m R=
NH2
OTBDMS
OH
f
O
17s' R=
17s R=
17t R=
N
N
OH
OTBDMS
17f R=
17n' R=
17n R=
e
NHBoc
NH2
O
f
N
NH
17g' R=
N
OH
e
17y' R=
17y R=
N
OBDMS
OH
17g R=
17h' R=
N
O
N
O
f
17o' R=
17o R=
N
N
NBoc
NH
O
TBDMSO
N
f
e
N
O
17u R=
N
17h R=
HO
N
OH
OH
HO
S
S
S
S
R
O
O
S
S
R
O
18a R=
18b R=
18c R=
O
O
O
O
S
O
HO
O
O
g
O
O
OH
18d R=
18e R=
HO
O
h or i
HO
O
O
OH
OH
O
O
O
HO
O
O
H3CO
OCH3
OH
H3CO
OCH3
OAc
OAc
H3CO
OCH3
AcO
OH
17a
HO
OH
O
OH
AcO
Scheme 2 The synthetic route of disulfide-containing 4β-podophyllotoxin derivatives 17a-y and 18a-e. Reagents and conditions: (a) NaI, TMSCl, CH3CN, 0 oC; (b) BaCO3,
.
H2O/Acetone, r.t.; (c) H2S saturated DCM, BF3 Et2O, pyridine, -15 oC; (d) 1-chlorobenzotriazole, benzotriazole, -78 oC,appropriate thiol-containing side chain moiety, DCM; (e) HCl
.
saturated EtOAc, DCM; (g) BF3 Et2O, CH3CN; (f) CHCl3, r.t.; (g) 2-thenaldehyde, ZnCl2, N2; (h) TsOH,1,1-Dimethoxyethane, acetonitrile.
substitution reaction of 1a
bromo-2-chloroethane, and successively alkali hydrolysis in mercaptoacetic acid, respectively. Then, deprotection of
presence of sodium hydroxide aqueous solution. On the other 12 in presence of CH3ONa/CH3OH furnished thiol-containing
hand, compounds 7a were prepared by directly sugars 13a and 13c, respectively. Similarly, the hydroxyl-
condensation of mercaptoacetic acid with appropriate acetylated thiol galactose 13b was obtained by bromination of
-j
with 1-bromo-3-chloropropane/1- precursors
8
or 11 with boron trifluoride diethyl etherate and
9
and
-e
substituted amines 6a
-e
. Preparation of glycosylthioacetates
9
per-O-acetylated galactose
9
and then sulfurization by sodium
and 12 were archived by reaction of per-O-acetylated sugar hydrosulfide.
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SH
O
O
O
S
S
S
R
O
O
O
O
O
O
O
O
O
O
H3 CO
OCH3
O
OH
a
b
20
NH
N
S
N
N
S
S
R
O
c
O
H3CO
OCH3
20
21a-f
19
OH
22a-f
N
O
21d, 22d R=
21e, 22e R=
21a, 22a R=
21b, 22b R=
OH
OH
O
OH
OH
HO
OH
N
N
NBoc
NH
21c, 22c R=
21f, 22f' R=
22f R=
OH
O
O
d
Scheme 3 The synthetic route of trisulfide-containing 4β-podophyllotoxinderivatives 22a-22f. Reagents and conditions: (a) S2Cl2, DMF; (b) DCM, appropriate thiol-containing side
chain moiety, Et3N, r. t.; (c) DCM, r. t.;(d) HCl saturated EtOAc, DCM.
Meanwhile, the synthesis of 4β-hydroxy-4'-O-demethyl-4- compound 22f was obtained by deprotection of 22f' in acidic
desoxypodophyllotoxin 15 was furnished by the C4-iodination condition.
of etoposide, and subsequently hydrolysis in presence of
barium carbonate. Then, 4β-mercapto-4'-O-demethyl-4- 2.2 Cytotoxic activities against KB and KB/VCR cells
desoxypodophyllotoxin 16 was prepared with high
All of target compounds 17a-y, 18a-e and 22a-f were tested
stereospecificity by the reaction of 15 and H2S in the presence
for in vitro cytotoxic activities against KB and vincristine-
resistant KB/VCR cell lines. The results of them together with
etoposide are summarized in Table 1 and Table S1 (ESI†). In
general, most of the synthesized disulfide/trisulfide derivatives
exhibited moderate to potent cytotoxic activities, especially for
KB cell lines, which were much better than that of etoposide.
Comparing the cytotoxic activities against KB cell lines of
disulfide and trisulfide series, in general, the disulfide
derivatives 17a-y and 18a-e were more potent than trisulfide
derivatives 22a-f. As exemplified in compounds 17e and 22a
with the same 4β-side chain, compound 17e showed 2.7-folds
of BF3•Et2O.17 On one hand, target compounds 17a-c
,
17e
17w and intermediates 17d' 17g'
17v' 17x'
,
17f
,
17i-m
,
17p
,
17q
,
17t
,
17u
,
,
,
h',
17n'
,
17o'
,
17r'
,
17s'
,
,
,
17y' were obtained by
oxidation of 16 with appropriate thiol compounds in
dichloromethane.18 And the protection groups (-Boc and -
TBDMS) of the obtained intermediates were easily removed by
HCl saturated EtOAc and BF3•Et2O, respectively, to afford
target compounds 17d
17y On the other hand, the sugar-containing target
compounds 18a were synthesized by reacting of thiol-
containing sugars 13a with 2-mercaptopyridine-activated 4β-
mercapto-4'-O-demethyl-4-desoxypodophyllotoxin 17a
Furthermore, compounds 18d were obtained by acetalation
, 17g-h, 17n, 17o, 17r, 17s, 17v, 17x and
.
-c
-c
more potent cytotoxic activity against KB cell than that of 22a
.
.
Similar SAR rules were observed in comparison with 17i and
22d. Insight into the effect of different 4β-substitution (e.g.
heteroaryl, alkyl, heteroalkyl and glycosyl) of podophyllotoxin
on cytotoxic activities reveals that the compounds 17a-c with
,
e
of 18b by thiophenecarboxaldehyde and acetaldehyde
dimethyl acetal, respectively.
Besides,
the
synthesis
of
trisulfide-containing
heteroaryl group
showed preferable activities, with IC50 value less than 1 μM
(IC50 of 17a 17b 17c against KB were 0.30, 0.17 and 0.82 μM,
(o-pyridine, p-pyridine and o-thiophen)
podophyllotoxin derivatives 22a
-f was illustrated in Scheme 3.
Initially, the preparation of 2,2'-thiobis(isoindoline-1,3-dione)
20 was achieved according to the literature.19 Then, N-
,
,
respectively), which were significantly more potent than
compounds 17d-y and 18a-e with alkyl/heteroalkyl/glycosyl
group at 4β-position. Although cytotoxic activities against KB
cell were not affected by the length and rigidity of linker
between the disulfide bond and amine moieties, for example,
disulfanylisoindolinediones 21a
exchange reaction of 20 with 4β-mercapto-4'-O-demethyl-4-
desoxypodophyllotoxin 16 and subsequently used to
-f were synthesized by the thiol
,
synthesize compounds 22a-e and 22f' via second thiol
exchange reaction and deprotection if necessary.20 And
the compounds with
n
-propyl-linker (e.g.17h and 17i) showed
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COMMUNICATION
22d
22e
22f
-(CH2)3-
7.65±0.31 2.91±0.28
9.37±1.07 18.0±0.65
7.72±2.75 6.39±0.37
0.38
1.92
0.83
Table 1 Cytotoxic activities of target compounds against
selected human cancer cell lines.
R
-
S
S
X
S
S
S
X
R
O
O
O
O
-
O
O
aIncubation time for cytotoxic assay is 72 hours; b Resistance factor was calculated
as a ratio of the IC50 value of KB/VCR cells to that of KB cells.
O
O
H3CO
OCH3
H3CO
OCH3
OH
17a-y,18a-e
OH
22a-f
almost equivalent cytotoxic activities to compounds with
ethyl-linker (e.g.17s and 17t) and compounds with acetyl-
linker (e.g. 17v).
IC50 (μM)
KB/VCR
Compd.
X
R
-
KB
RFa
Although etoposide is widely used as therapy for cancer
patients, the fact remains that tumors often acquire resistance
to etoposide. Thus, with aim to determine whether there are
Etoposide
-
-
2.27±0.58 16.8±0.11 7.40
0.3±0.05 9.1±0.67 30.3
0.17±0.06 7.54±0.48 44.3
0.82±0.11 5.2±2.72 6.34
17a
drug resistance of compounds 17a-y, 18a-e, and 22a-f as well
17b
17c
-
-
as etoposide in multi-drug resistant cell lines (KB/VCR),
resistance factor (RF), a ratio of cytotoxic activity against KB
and KB/VCR of tested compounds were calculated as an
evaluation criterion for their anti-resistance profiles. To our
delight, most of the synthesized compounds, except for
pyridine derivatives 17a,b and glycosyl series 18a-e, displayed
superior cytotoxic activities against KB/VCR to that of
etoposide, demonstrating that replacement of 4β-D-
17d
-
7.19±1.59 16.9±0.86 2.35
10.2±2.42 13.8±0.8 1.35
10.2±2.11 18.4±2.15 1.80
4.97±1.2 11.9±0.47 2.39
H
17e
17f
17g
17h
17i
-(CH2)2-
-(CH2)2-
-(CH2)2-
-(CH2)3-
-(CH2)3-
-OH
-NH2
1.05±0.24 4.12±0.43 3.92
1.52±0.48 6.44±0.43 4.24
3.72±0.48 6.44±0.43 1.73
glucopyranose by
a variety of disulfide/trisulfide-linked
17j
17k
17l
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
-(CH2)3-
alkyl/heteroalkyl were beneficial for overcoming drug-
resistance. In particular, RF values of several disulfide-
4.22±0.99 2.98±0.44 0.71
2.90±0.72 0.97±0.03 0.33
3.72±1.22 10.0±0.69 2.69
1.25±0.98 4.88±1.10 3.90
containing derivatives 17j, 17k, 17l, 17p and 17q (RF value:
17m
17n
1.73, 0.71, 0.33, 1.20 and 0.72 respectively) were significantly
improved when comparing with etoposide (RF value: 7.40),
while the cytotoxic activities of these compounds are more
potent than that of etoposide. It should be noted that
compound 17l with piperidine moiety exhibited comparable
cytotoxic activities against KB and significantly more potent
cytotoxic activities against KB/VCR, with IC50 values of 2.90 μM
(KB) and 0.97μΜ (KB/VCR), respectively. In addition, 22a-f
bearing trisulfide showed significantly more potent cytotoxic
activities against KB/VCR with RF value ranging from 0.38 to
2.24, although they possessed comparable or slightly weaker
cytotoxic activities against KB cells in comparison with
etoposide. For example, compound 22d exhibited remarkably
sensitive cytotoxic activity against KB/VCR, which is almost 5.8-
folds more potent than etoposide. It is revealed that tri-sulfide
bond would be an excellent linker for chemical modification on
4β-podophyllotoxin to overcome the multi-drug resistant
limitation of etoposide.
17o
17p
-(CH2)3-
-(CH2)3-
1.12±0.65 13.8±0.89 12.3
1.81±0.46 2.18±0.61 1.20
17q
17r
-(CH2)3-
-(CH2)2-
2.02±0.55 1.46±0.02 0.72
6.77±1.66 14.3±1.66 2.11
17s
17t
-(CH2)2--
-(CH2)2-
1.36±0.42 12.5±0.54 9.19
2.66±0.13 17.7±1.21 6.65
17u
17v
17w
17x
17y
-
-
-
-
-
8.15±2.86 19.8±0.63 2.43
3.31±0.29 7.26±0.28 2.19
28.9±3.63
13.6±2.92
>50
>50
/
/
12.1±1.29 10.8±0.50 0.89
1.12±0.17 9.96±1.33 8.89
18a
18b
18c
-
-
-
2.3 Metabolic stability study
6.68±2.32 21.3±0.42 3.19
10.3±2.79 23.2±0.43 2.25
To investigate the metabolic stability 4β-disulfide-containing
podophyllotoxin derivatives, compound 17l (the most
promising compound) was chosen and undergone
preliminarily evaluation in vitro for the stability in human
plasma As shown in Fig.3, the concentration of compound 17l
a
18d
18e
-
-
7.58±0.45 24.7±2.69 3.26
8.40±1.87 26.7±0.57 3.18
.
was significantly decreased (c.a. 60%) in the first two hours,
then reaching a stable state up to 8 hours. It is indicated that
the disulfide would be stable in further in-vivo development,
revealing a good prospect of these compounds for in-vivo anti-
cancer activities.
H
22a
22b
-(CH2)2-
-(CH2)2-
27.6±4.07
20.8±1.6
0.75
-OH
6.15±0.12 7.97±2.21 1.30
22c
-CH2-
5.14±1.76 11.5±1.61 2.24
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4.1.8General procedure for the synthesis of 17
Method A for compounds 17a-c, 17d', 17e-f and 17g': the
commercial thiols (0.4 mmol) dissolved in CH2Cl2(2 mL) was
added
dropwise to
a
stirred solution
of 1-
chlorobenzotriazole(92 mg, 0.6 mmol) and benzotriazole (48
mg, 0.4 mmol) in CH2Cl2 (10 mL) under N2 at -78 °C. The
solution was allowed to warm to -20 °C and stirred for 2 hours.
Compound 16 17(250 mg, 0.6 mmol) was added slowly at -20
°C, and then allowed to warm to 0 °C for 3 hours. The reaction
was quenched at 0 °C with a solution of Na2S2O3 (0.05 g in 2
mL H2O) and aq. NaHCO3 (2 mL) with rapid stirring over 20
min, and then extracted with CH2Cl2 (3×50 mL).The combined
organic layer was dried over anhydrous Na2SO4, and
evaporated in vacuo. The residue was purified by silica column
chromatography using DCM-EtOAc mixtures as eluent to give
Fig.3. Metabolic stability in in vitro human plasma of Compound 17l
3 Conclusion
Owing to some problems that still exists for practical use of
etoposide in clinical settings, the development of a series of
the desired product 17a-g
.
novel
4β-disulfide/trisulfide-4'-O-demethyl-4-desoxypodo-
Method B for compounds 17h', 17i-m, 17n', 17o', 17p, 17q, 17r',
17s', 17t-u, 17v', 17w, 17x', 17y':Compound or (0.4 mmol)
phyllotoxin derivatives was disclosed in present study. To be of
interest, some of the compounds exhibited better cytotoxic
activities than etoposide, not only for sensitive cancer cell lines
(KB cells), but also for MDR cancer cell lines (KB/VCR cells). To
our knowledge, this is the first time that biologically
compatible disulfide/trisulfide bonds were introduced to the
podophyllotoxin backbone. In addition to the excellent
cytotoxic activities against KB and KB/VCR cells, 17I has further
shown remarkable results in the metabolic stability evaluation.
Further in-vitro evaluation of drug likeness properties and in-
vivo anticancer activity of 17I are currently in progress in our
laboratory.
3
5
dissolved in CH2Cl2 (2mL) was added dropwise to a stirred
solution of 1-chlorobenzotriazole (123 mg, 0.8 mmol) and
benzotriazole (48 mg, 0.4 mmol) in CH2Cl2 (20 mL) under N2 at
-78 °C. The solution was stirred for 30 minutes and then added
thiourea (91 mg, 1.2 mmol) with stirring for another 30
minutes. Compound 1617 (250 mg, 0.6 mmol) dissolved in
CH2Cl2 (2mL) was added at -78 °C and the mixture was allowed
to slowly warm to room temperature and stirred for 18 hours.
The reaction was added water (100ml) and then extracted
with CH2Cl2 (3×50 mL). The organic layer was washed with
statured NaCl and dried over anhydrous Na2SO4, then
evaporated in vacuo. The residue was purified by column
chromatography on silica gel using EtOAc–DCM mixtures as
4 Experimental section
eluent to give the desired product 17h-y
.
4.1 Chemistry
Deprotection method for 17d', 17g'-h', 17s' and 17v': the
obtained corresponding compound was immediately dissolved
in DCM (5 mL), and HCl saturated EtOAc (1 mL) was added at 0
°C. The resulting mixture was allowed to warm to room
temperature, and stirred until the disappearance of starting
material. The mixture was evaporated, and the residue was
added saturated NaHCO3 aqueous solution, and extracted with
DCM. The organic layer was washed with brine, and
condensed in vacuo to give desired product.
Commercially available starting materials, reagents, and dry
solvents were used as supplied. Melting points were obtained
on a B-540 Büchi melting-point apparatus and are uncorrected.
1H NMR spectra were recorded on a 500 MHz or 400 MHz, 13C
NMR were recorded on a 125 MHz and 100 MHz spectromter,
respectively (chemical shifts are given in ppm (d) relative to
TMS as internal standard, coupling constants (J) are in hertz
(Hz), and signals are designated as follows: s, singlet; d,
doublet; t, triplet; m, multiplet; br s, broad singlet, etc.). Mass
spectral data were obtained on an Esquire-LC-00075
spectrometer. High resolution mass spectra were measured on
an Agilent 1290 HPLC-6224 Time of Fight Mass Spectrometer.
Deprotection method for 17n', 17o', 17r' 17x'and 17y':the
obtained corresponding compound was dissolved in CH3CN (5
.
mL) and then was added BF3 Et2O (5 drops) in ice bath. After
The synthesis of side chain intermediates 2-5, 7, 9, 10, 13, 20,
the disappearance of starting material, the mixture was
and 21 were presented in Electronic Supplementary Material
evaporated in vacuo. The residue was added water and
(ESI).
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COMMUNICATION
4.1.8.5 4β-Ethyldisulfanyl-4'-O-demethyl-4-desoxypodophyllotoxin
17e
extracted with DCM. After washed with brine, dried over
anhydrous Na2SO4, the organic layer was evaporated to give
desired product.
Reagent: ethanethiol (25 mg, 0.4 mmol). The product was
obtained as a white solid, yield: 61%, mp: 237.5-239.8oC; 1H
NMR (500 MHz, CDCl3) δ 6.93 (s, 1H, 5-H), 6.46 (s, 1H, 8-H),
6.27 (s, 2H, 2′, 6′-H), 5.97 (dd, J = 12.0, 1.0 Hz, 2H, -OCH2O-),
4.55 (d, J = 5.0 Hz, 1H, 1-H), 4.45-4.43 (m, 2H, 11-H), 4.39 (d, J
= 4.5 Hz, 1H, 4-H), 3.78 (s, 6H,3′,5′-OCH3), 3.36-3.32 (m,1H, 2-
H), 3.19-3.14 (m, 1H, 3-H), 2.82 (q, J = 7.5 Hz, 2H, 1′′-H), 1.42 (t,
J = 7.5 Hz, 3H, 2′′-H); 13C NMR (125 MHz, CDCl3) δ 174.63,
148.25, 147.28, 146.50, 134.20, 132.75, 131.13, 127.94,
110.40, 110.23, 108.09, 101.69, 70.17, 56.60, 54.71, 43.70,
41.82, 37.58, 33.41, 14.54; ESI-MS: m/z [M+H]+477.
4.1.8.1
4β-(Pyridin-2-yldisulfanyl)-4'-O-demethyl-4-desoxypodo
phyllotoxin 17a
Reagent: pyridine-2-thiol (44.4 mg, 0.4 mmol). The product
was obtained as a white solid, yield:43%, mp: 214.8-216.2oC;
1H NMR (500 MHz, CDCl3) δ 8.65 (d, J = 5.0 Hz, 1H, 6′′-H), 7.81
(t, J = 6.0 Hz, 1H, 4′′-H), 7.67 (d, J = 8.0 Hz, 1H, 3′′-H), 7.30 (t, J =
5.0 Hz, 1H, 5′′-H), 7.22 (s, 1H,5-H), 6.47 (s, 1H,8-H), 6.26 (s, 2H,
2′, 6′-H), 5.96 (dd, J = 18.5, 1.5 Hz, 2H, -OCH2O-), 4.78 (d, J = 4.0
Hz, 1H, 1-H), 4.59 (d, J = 5.0 Hz, 1H, 4-H), 4.55-4.48 (m, 2H, 11-
H), 3.76 (s, 6H, 3′,5′-OCH3), 3.36 (dd, J =13.5, 5.0Hz, 1H, 2-H),
3.23-3.14 (m, 1H, 3-H); ESI-MS: m/z [M+H]+ 526.
4.1.8.6
4β-(2-Hydroxyethyl)disulfanyl)-4'-O-demethyl-4-desoxy
podophyllotoxin 17f
4.1.8.2
4β-(Pyridin-4-yldisulfanyl)-4'-O-demethyl-4-desoxypodo
Reagent: 2-mercaptoethanol (31.2 mg, 0.4 mmol). The product
was obtained as a white solid, yield: 40%, mp: 221.9-223.7oC;
1H NMR (500 MHz, CDCl3) δ 6.92 (s, 1H, 5-H), 6.47 (s, 1H, 8-H),
6.27 (s, 2H, 2′, 6′-H), 5.99 (dd, J = 11.5, 1.5 Hz, 2H, -OCH2O-),
4.56 (d, J = 5.0 Hz, 1H, 1-H), 4.48 (d, J = 4.5 Hz 1H, 4-H), 4.45-
4.40 (m, 2H, 11-H), 4.02-3.96 (m, 2H, 2′′-H), 3.78 (s, 6H, 3′,5′-
OCH3), 3.34 (dd, J = 14.0, 5.5 Hz, 1H, 2-H), 3.18-3.16 (m, 1H, 3-
H), 2.98 (t, J = 5.0, 2H, 1′′-H);13C NMR (125 MHz, CDCl3) δ
174.55, 148.36, 147.34, 146.53, 134.25, 132.87, 131.04,
127.59, 110.36, 110.28, 108.10, 101.74, 70.07, 60.51, 56.62,
54.66, 43.69, 42.16, 41.85, 37.56; ESI-MS: m/z [M+H]+ 493.
phyllotoxin 17b
Reagent: pyridine-4-thiol (44.4 mg, 0.4 mmol). The product
was obtained as a white solid, yield: 55%, mp: 173.1-175.8oC;
1H NMR (500 MHz, CDCl3) δ 8.58 (d, J = 4.0 Hz, 2H, pyridine),
7.50 (d, J = 5.5 Hz, 2H, pyridine), 6.81 (s, 1H, 5-H), 6.47 (s, 1H,
8-H), 6.24 (s, 2H, 2′, 6′-H), 5.96 (d, J = 20.5 Hz, 2H, -OCH2O-),
4.59 (d, J = 5.0 Hz, 1H, 1-H), 4.47 (d, J = 9.0 Hz, 2H, 11-H), 4.43
(d, J = 4.0 Hz, 1H, 4-H), 3.74 (s, 6H, 3′,5′-OCH3), 3.41 (dd, J =
14.0, 5.5 Hz, 1H, 2-H), 3.22-3.14 (m, 1H, 3-H);13C NMR (125
MHz, CDCl3) δ 174.14, 149.86, 148.72, 148.06, 147.38, 146.58,
134.33, 133.17, 130.75, 126.45, 120.89, 110.39, 110.10,
107.98, 101.85, 69.66, 56.56, 54.78,43.72, 41.86, 37.48; ESI-
MS: m/z [M+H]+ 526.
4.1.8.7
4β-((2-Aminoethyl)disulfanyl)-4'-O-demethyl-4-desoxy
podophyllotoxin 17g
Reagent:tert-butyl (2-mercaptoethyl)carbamate (70.8 mg, 0.4
mmol). The product was obtained as a white solid in two steps,
total yield: 36%, mp: 193.3-194.7oC; 1H NMR (500 MHz, CDCl3)
δ 6.95 (s, 1H,5-H), 6.46 (s, 1H,8-H), 6.27 (s, 2H, 2′, 6′-H), 5.98
(d, J = 4.5 Hz, 2H, -OCH2O-), 4.55 (d, J = 5.0 Hz, 1H,1-H), 4.46-
4.39 (m, 3H, 3-H, 11-H), 3.78 (s, 6H, 3′,5′-OCH3), 3.34 (dd, J =
14.0, 5.5 Hz, 1H,2-H), 3.18-3.16 (m, 3H,3-H, 2′′-H), 2.98-2.91
(m, 2H, 1′′-H); ESI-MS: m/z [M+H]+492.
4.1.8.3 4β-(Thiophen-2-yldisulfanyl)-4'-O-demethyl-4-desoxypodo
phyllotoxin 17c
Reagent: thiophene-2-thiol (46.4 mg, 0.4 mmol). The product
was obtained as a white solid, yield:64%, mp: 223.2-225.1oC;
1H NMR (500 MHz, CDCl3) δ 7.52 (dd, J = 5.5, 1.0 Hz,
1H,thiophene), 7.28 (dd, J = 3.5, 1.0 Hz, 1H, thiophene), 7.07
(dd, J = 5.5, 3.5 Hz, 1H, thiophene), 6.64 (s, 1H, 5-H), 6.43 (s,
1H, 8-H), 6.27 (s, 2H, 2′, 6′-H), 5.94 (dd, 2H, J =14.0, 1.5 Hz, -
OCH2O-), 5.41 (s, 1H, -OH), 4.56 (d, J = 3.5 Hz, 1H, 1-H), 4.53 (d,
J = 4.0 Hz, 1H, 4-H), 4.47-4.44 (m, 1H, 11-H), 4.36-4.33 (m, 1H,
11-H), 3.78 (s, 6H, 3′,5′-OCH3), 3.23-3.18 (m, 2H, 2-H, 3-H) ;13C
NMR (125 MHz, CDCl3) δ 174.52, 148.27, 147.27, 146.53,
134.23, 132.86, 131.01, 127.75, 110.31, 110.25, 108.06,
101.70, 70.08, 66.94, 57.82 , 56.57, 54.27, 53.63, 43.67, 41.82,
37.53, 36.67;ESI-MS: m/z [M+H]+ 531.
4.1.8.8 4β-((3-(Piperazin-1-yl)propyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17h
Reagent:3a (104.8 mg, 0.4 mmol). The product was obtained
as a white solid in two steps, total yield: 27%, mp: 179.8-181.6
1
oC; H NMR (500 MHz, DMSO-d6) δ 7.12 (s, 1H, 5-H), 6.48 (s,
1H, 8-H), 6.19 (s, 2H, 2′, 6′-H), 6.04 (d, J = 11.0 Hz, 2H, -OCH2O-
), 5.76 (s, 1H, -OH), 4.76 (d, J = 3.5 Hz, 1H, 1-H), 4.54 (t, J = 7.5
Hz, 1H, 11-H), 4.49 (d, J = 5.0 Hz, 1H, 4-H), 4.27 (t, J = 9.0 Hz,
1H, 11-H), 3.77-3.72 (m, 4H,piperazine), 3.63 (s, 6H, 3′,5′-
OCH3), 3.30-3.24 (m, 7H, 2-H, 3′′-H, piperazine), 3.10-3.02 (m,
2H, 1′′-H), 2.98-2.90 (m, 1H, 3-H), 2.23-2.21 (m, 2H, 2′′-H); ESI-
MS: m/z [M+H]+575.
4.1.8.4
4β-(L-AlanineN-[(1,1-
Dimethylethoxy)carbonyl]methylester) -4'-O-demethyl-4-desoxy-
podophyllotoxin 17d
Reagent: BocCysOMe (94 mg, 0.4 mmol).The product was
obtained as a white solid in two steps, total yield: 44%,mp:
195.4-197.2 oC;1H NMR (500 MHz, CDCl3) δ 6.95 (s, 1H, 5-H),
6.46 (d, J = 4.5 Hz, 1H, 8-H), 6.27 (s, 2H, 2′, 6′-H), 5.98 (dd, J =
10.5 Hz,1.5Hz, 2H, -OCH2O-), 4.55 (d, J = 5.0 Hz, 1H, 1-H), 4.51
(d, J = 4.5Hz, 1H, 4-H), 4.45-4.42 (m, 1H, 11-H), 4.40-4.36 (m,
1H, 11-H), 4.14 (s, 1H, 2′′-H), 3.77 (s, 3H, 4′′-H), 3.76 (s, 6H,
3′,5′-OCH3), 3.34 (dd, J = 14.0, 5.0 Hz, 1H, 2-H), , 3.20-3.15 (m,
1H, 3-H), 3.13 (t, J = 4.5 Hz, 2H,1′′-H); ESI-MS: m/z [M+H]+ 550.
4.1.8.9
4β-((3-Morpholinopropyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17i
Reagent:3b (64.4 mg, 0.4 mmol). The product was obtained as
a white solid, yield: 34%, mp: 169.8-171.5 oC; 1H NMR (500
MHz, CDCl3) δ 6.90 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, 2H, 2′,
6′-H), 5.99 (d, J = 11.0 Hz, 2H,-OCH2O-), 4.55 (d, J = 5.0 Hz, 1H,
1-H), 4.44-4.40 (m, 3H, 4, 11-H), 3.77 (s, 6H, 3′,5′-OCH3), 3.76-
3.75 (m,4H,morpholine), 3.36 (dd, J = 14.0, 5.0 Hz, 1H, 2-H),
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Journal Name
3.18-3.14 (m, 1H, 3-H), 2.86-2.79 (m, 2H, 3′′-H), 2.59-2.48 (m, 2′′-H);HR-MS (ESI+): calculated for C30H36N2O8S2 [M+H]+:
6H, 1′′-H, morpholine), 2.01-1.94 (m, 2H, 2′′-H); ESI-MS: m/z 617.1986, found: m/z = 617.1987 [M+H]+.
[M+H]+ 576.
4.1.8.14
4β-((4-Piperidinolpropyl)disulfanyl)-4'-O-demethyl-4-
4.1.8.10
4β-((N-Methylpiperazine-1-propyl)disulfanyl)-4'-O-
desoxypodophyllotoxin 17n
demethyl-4-desoxypodophyllotoxin 17j
Reagent: 3g (44.4 mg, 0.4 mmol). The product was obtained as
Reagent: 3c (69.6 mg, 0.4 mmol). The product was obtained as a red-white solid in two steps, total yield: 10%, mp: 235.5-
1
1
a yellow-white solid, yield: 33%, mp: 149.5-150.6oC; H NMR 236.3oC; H NMR (500 MHz, CDCl3) δ 6.92 (s, 1H, 5-H), 6.46 (s,
(500 MHz, CDCl3) δ 6.91 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, 1H, 8-H), 6.27 (s, 2H, 2′, 6′-H), 5.98 (dd, J = 9.5 Hz, 1.0Hz, 2H, -
2H, 2′, 6′-H), 5.98 (d, J = 11.0 Hz, 2H, -OCH2O-), 5.30 (s, 1H, - OCH2O-), 4.55 (d, J = 5.0 Hz, 1H, 1-H), 4.44-4.42 (m, 3H, 4-H,
OH), 4.55 (d, J = 5.0 Hz, 1H, 1-H), 4.44 (d, J = 9.0 Hz, 2H, 11-H), 11-H), 3.78 (s, 6H, 3′,5′-OCH3), 3.36 (dd, J = 14.0, 5.5 Hz, 1H, 2-
4.41 (d, J = 4.0 Hz, 1H, 4-H), 3.77 (s, 6H，3′,5′-OCH3), 3.33 (dd, H), 3.20-3.13 (m, 1H, 3-H), 3.12-3.06 (m, 2H,piperidine), 2.92-
J = 14.0, 5.0 Hz, 1H, 2-H), 3.19-3.12 (m, 1H, 3-H), 2.86-2.80 (m, 2.81 (m, 2H, 3′′-H), 2.75-2.56 (m, 5H,piperidine), 2.32-2.20 (m,
2H, 3′′-H), 2.64-2.47 (m, 10H, 1′′-H,piperazine), 2.33 (s, 3H, - 2H, 2′′-H), 2.10-2.00 (m, 2H, piperidine), 1.28-1.23 (m, 2H, 1′′-
CH3), 1.96-1.92 (m, 2H, 2′′-H) ;13C NMR (125 MHz, CDCl3) H); HR-MS (ESI+): calculated for C25H39N1O8S2 [M+H]+:
δ174.57, 148.23, 147.24, 146.58, 134.27, 132.78, 131.02, 590.1877, found: m/z = 590.1625[M+H]+.
4.1.8.15
4β-((3-(2-(Hydroxymethyl)piperidin-1-
127.83, 110.42, 110.21, 108.09, 101.67, 70.10, 56.75, 56.57,
55.08, 54.43, 53.05, 45.98, 43.67, 41.81, 37.55, 26.38; ESI-MS:
m/z [M+H]+ 589.
yl)propyl)disulfanyl) -4'-O-demethyl-4-desoxypodophyllotoxin 17o
Reagent: 3h (121.3 mg, 0.4 mmol). The product was obtained
as a yellow-white solid in two steps, total yield: 12%, mp:
211.9-213.6oC; 1H NMR (500 MHz, CDCl3) δ 6.97 (d, J = 13.0 Hz,
4.1.8.11 4β-((3-(Pyrrolidin-1-yl)propyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17k
Reagent: 3d (58.1 mg, 0.4 mmol). The product was obtained as 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, 2H, 2′, 6′-H), 5.97 (s, 2H, -
o
1
a yellow-white solid, yield: 41%, mp: 159.4-160.7 C; H NMR OCH2O-), 5.30(s, 1H, -OH), 4.54 (d, J = 5.0 Hz, 2H, 1-H), 4.51 (d,
(500 MHz, CDCl3) δ 6.99 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, J = 4.0 Hz, 1H, 4-H), 4.48 (dt, J = 19.0, 9.0 Hz, 2H, 11-H), 3.78 (s,
2H, 2′, 6′-H), 5.98 (s, 2H, -OCH2O-), 4.55-4.53 (m, 2H, 1-H, 4-H), 6H, 3′,5′-OCH3), 3.37-3.29 (m, 1H, 2-H, CH2OH,), 3.24-3.12 (m,
4.47-4.38 (m, 2H, 11-H), 3.78 (s, 6H,3′,5′-OCH3), 3.35 (dd, J = 2H,CH2OH, 3-H), 2.96-2.82 (m, 4H, 1′′, 3′′-H), 2.28-2.08 (m, 2H,
14.0, 5.5Hz, 1H, 2-H), 3.22-3.08 (m, 5H, 3-H, pyrrolidine), 3.03- 2′′-H, piperdine), 1.88-1.72 (m, 6H, piperdine); ESI-MS: m/z
2.98 (m, 2H, 3′′-H), 2.93-2.86 (m, 2H, 1′′-H), 2.39-2.22 (m, 2H, [M+H]+604.
4.1.8.16 4β-((3-(Diethylamino)propyl)disulfanyl)-4'-O-demethyl-4-
2′′-H), 2.14-2.08 (m, 4H, pyrrolidine); HR-MS (ESI+): calculated
for C28H33N1O7S2 [M+H]+: 560.1771, found: m/z =560.1770.
4.1.8.12. 4β-((3-(Piperidin-1-yl)propyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17p
Reagent: 3i (58.8 mg, 0.4 mmol). The product was obtained as
a white solid, yield: 26%, mp: 159.1-161.2oC; 1H NMR (500
desoxypodophyllotoxin 17l
Reagent: 3e (63.6 mg, 0.4 mmol). The product was obtained as MHz, CDCl3) δ 6.94 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, 2H, 2′,
a white solid, yield: 38%, mp: 153.2-155.5oC; 1H NMR (500 6′-H), 5.98 (dd, J = 4.5, 1.0 Hz, 2H, -OCH2O-), 4.55 (d, J = 5.5 Hz,
MHz, CDCl3) δ 6.94 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s, 2H, 2′, 1H, 1-H), 4.49 (d, J = 4.5 Hz, 1H, 4-H), 4.45 (dd, J = 19.0, 8.0 Hz,
6′-H), 5.98 (dd, J = 10.0 Hz, 1.0Hz, 2H, -OCH2O-), 5.30 (s, 1H, - 2H, 11-H), 3.78 (s, 6H, 3′,5′-OCH3), 3.36 (dd, J = 14.0, 5.5 Hz,
OH), 4.55 (d, J = 5.5 Hz, 1H, 1-H), 4.46-4.42 (m, 3H, 4, 11-H), 1H, 2-H), 3.22-3.13 (m, 1H, 3-H), 2.93-2.84 (m, 8H, 1′′, 3′′-H, 4′′-
3.78 (s, 6H, 3′,5′-OCH3), 3.36 (dd, J = 14.0, 5.0 Hz, 1H, 2-H), H, 4′′′-H), 2.23-2.08 (m, 2H, 2′′-H), 1.27 (t, J = 7.0 Hz, 6H, 5′′,5′′′-
3.20-3.12 (m, 1H, 3-H), 2.89-2.80 (m, 2H, 3′′-H), 2.64-2.40 (m, H)
；
13C NMR (125 MHz, CDCl3) δ 174.56, 148.34, 147.33,
4H, piperidine), 1.76-1.44 (m, 10H, 1′′-H, 2′′, piperidine);13C 146.53, 134.19, 132.86, 131.03, 127.63, 110.47, 110.23,
NMR (125 MHz, CDCl3) δ 174.64, 148.25, 147.28, 146.53, 108.01, 101.74, 70.06, 56.59, 54.30, 50.70, 46.84, 43.70, 41.81,
134.21, 132.77, 131.11, 127.88, 110.49, 110.21, 108.06, 37.54, 36.82, 10.27; HR-MS (ESI+): calculated for C28H33N1O8S2
101.69, 70.16, 57.61, 56.59, 54.71, 54.41, 43.71, 41.84, 37.80, [M+H]+:562.1928, found: m/z = 562.1919.
4.1.8.17
4β-((3-(Diisopropylamino)propyl)disulfanyl)-4'-O-
37.58, 29.83, 26.33, 25.89, 24.41;HR-MS (ESI+): calculated for
C29H35N1O7S2 [M+H]+: 574.1928, found: m/z 574.1923.
demethyl-4-desoxypodophyllotoxin 17q
4.1.8.13
demethyl-4-desoxypodophyllotoxin 17m
Reagent: 3
a yellow-white solid, yield: 21%, mp: 159.8-161.5oC; H NMR 2H, 2′, 6′-H), 5.98 (s, 2H, -OCH2O-), 4.56 (d, J = 5.5 Hz, 1H, 1-H),
(500 MHz, CDCl3) δ 6.91 (s, 1H, 5-H), 6.47 (s, 1H, 8-H), 6.27 (s, 4.53 (d, J = 4.5 Hz, 1H, 4-H), 4.47 (dt, J = 19.0, 9.0 Hz, 2H, 11-H),
2H, 2′, 6′-H), 5.98 (dd, J = 9.0, 1.0 Hz, 2H,-OCH2O-), 4.55 (d, J = 3.78 (s, 6H, 3′,5′-OCH3), 3.70-3.61 (m, 2H, 4′′, 5′′-H), 3.36 (dd, J
5.5 Hz, 1H, 1-H), 4.44 (br s, 1H, 4-H), 4.43 (dd, J = 10.0, 3.5 Hz, = 14.0, 5.0 Hz, 1H, 2-H), 3.24-3.15 (m, 1H, 3-H), 3.11-3.02 (m,
2H, 11-H), 3.78 (s, 6H,3′,5′-OCH3), 3.72-3.59 (m, 2H,piperazine), 2H, 3′′-H), 2.97-2.82 (m, 2H, 1′′-H), 2.59-2.46 (m, 2H, 2′′-H),
4β-((3-(4-Acetylpiperazin-1-yl)propyl)disulfanyl)-4'-O-
Reagent: 3j (70.1 mg, 0.4 mmol). The product was obtained as
a yellow-white solid, yield: 6%, mp: 167.7-169.3oC; 1H NMR
f
(81.6 mg, 0.4 mmol). The product was obtained as (500 MHz, CDCl3) δ 6.95 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.27 (s,
1
3.58-3.46 (m, 2H，piperazine), 3.36 (dd, J = 14.0, 5.5 Hz, 1H, 2- 1.57-1.40 (m, 12H, 6′′,7′′,8′′, 9′′-H); HR-MS (ESI+): calculated for
H), 3.20-3.12 (m, 1H, 3-H), 2.91-2.79 (m, 2H, 3′′-H), 2.58-2.38 C30H39N1O7S2 [M+H]+: 590.2241, found: m/z = 590.0812.
4.1.8.18
4β-((2-(3-Hydroxypiperidin-1-yl)ethyl)disulfanyl)-4'-O-
(m, 6H,piperazine, 1′′-H), 2.10 (s, 3H, -CH3), 2.02-1.91 (m, 2H,
demethyl-4-desoxypodophyllotoxin 17r
8 | J. Name., 2012, 00, 1-3
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Reagent: 5k (110.1 mg, 0.4 mmol). The product was obtained (500 MHz, CDCl3) δ 7.17 (s, 1H, 5-H), 6.43 (s, 1H, 8-H), 6.26 (s,
as a white solid, and the corresponding product was dissolved 2H, 2′, 6′-H), 5.96 (dd, J = 7.5, 1.0 Hz, 2H, -OCH2O-), 4.74 (d, J =
.
in CH3CN (5 mL) and BF3 Et2O (5 drops) was added with ice 4.0 Hz, 1H, 1-H), 4.52 (d, J = 5.0 Hz, 1H, 4-H), 4.46-4.36 (m, 2H,
bath. After the disappearance of starting material, the mixture 11-H), 3.76 (s, 6H, 3′,5′-OCH3), 3.73-3.68 (m, 4H,piperazine),
was evaporated in vacuo and the residue was added with DCM 3.52-3.49 (m, 2H, 1′′-H), 3.29 (dd, J = 14.0, 5.0 Hz, 1H, 2-H),
and H2O. The organic layer was washed with saturated NaCl 3.20-3.12 (m, 1H, 3-H), 2.96-2.88 (m, 4H, piperazine); HR-MS
solution, dried over anhyd Na2SO4, and evaporated to give 17r (ESI+): calculated for C27H30N2O8S2 [M+H]+
：575.1516, found:
1
as a white solid, total yield: 28%, mp: 244.8-246.3oC; H NMR m/z =575.0119[M+H]+.
4.1.8.23
4β-((2-Oxo-2-(4-piperidone-1-yl)ethyl)disulfanyl)-4'-O-
(500 MHz, CDCl3) δ 6.95 (d, J = 3.6 Hz, 1H, 5-H), 6.47 (s, 1H, 8-
H), 6.27 (s, 2H, 2'-H, 6'-H), 5.98 (d, J = 6.0 Hz, 2H,-OCH2O-), 4.55
(dd, J = 5.0, 2.5 Hz, 1H, 1-H), 4.46-4.40 (m, 3H, 4-H, 11-H), 3.90-
3.84 (m,piperidine), 3.78 (s, 6H, 3′,5′-OCH3), 3.38-3.34 (m, 1H,
2-H), 3.20-3.12 (m, 1H, 3-H), 2.96-2.88 (m, 2H, 1′′-H), 2.82-2.67
(m, 2H, 1′′-H), 2.63-2.51 (m, 3H, piperidine), 2.43-2.35 (m, 1H,
piperidine), 1.90-1.81 (m, 1H, piperidine), 1.66-1.52 (m, 3H,
piperidine); ESI-MS: m/z [M+H]+576.
demethyl-4-desoxypodophyllotoxin 17w
Reagent: 7c (69.2 mg, 0.4 mmol). The product was obtained as
a white solid, yield: 24%, mp: 155.3-156.8oC; 1H NMR (500
MHz, CDCl3) δ 7.18 (s, 1H, 5-H), 6.45 (s, 1H, 8-H), 6.26 (s, 2H, 2'-
H, 6'-H), 5.97 (d, J = 6.5 Hz, 2H, -OCH2O-), 5.47 (s, 1H, -OH),
4.76 (d, J = 3.5 Hz, 1H, 1-H), 4.55 (d, J = 5.0 Hz, 1H, 4-H), 4.50-
4.35 (m, 2H, 11-H), 3.90-3.84 (m, 2H, 1′′-H), 3.82-3.78 (m,
4H,piperidine), 3.76 (s, 6H, 3′,5′-OCH3), 3.30 (dd, J = 14.0, 5.0
Hz, 1H, 2-H), 3.24-3.12 (m, 1H, 3-H), 2.62-2.50 (m, 4H,
4.1.8.19 4β-((2-(Piperazin-1-yl)ethyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17s
Reagent:5a
(98.5 mg, 0.4 mmol). The product was obtained as piperidine); ESI-MS: m/z [M+H]+601.
a white solid, total yield: 33%, mp: 194.5-195.4 oC; 1H NMR
(500 MHz, CDCl3) δ 7.25 (s, 1H, 5-H), 6.48 (s, 1H, 8-H), 6.19 (s,
2H, 2′, 6′-H), 6.04 (d, J = 12.0, 2H, -OCH2O-), 4.76 (d, J = 3.5Hz,
1H, 1-H), 4.54-4.49 (m, 2H, 11-H), 4.27 (t, J = 9.0Hz, 1H, 2-H),
4.05 (q, J = 7.0 Hz, 1H, 3-H), 3.65-3.58 (m, 7H,4-H, 3′,5′-OCH3),
3.46-3.18 (m, 12H,piperazine, 1'', 2''-H), 2.78-2.62 (m, 2H, 2''-
H), 2.52-2.42 (m, 4H,piperazine); ESI-MS: m/z [M+H]+ 561.
4.1.8.24 4β-((2-(3-Hydroxypiperidin-1-yl)-2-oxoethyl)disulfanyl)-4'-
O-demethyl-4-desoxypodophyllotoxin 17x
Reagent: 7d (115.6 mg, 0.4 mmol). The product was obtained
1
as a white solid, total yield: 61%,mp: 189.3-190.2oC; H NMR
(500 MHz, CDCl3) δ 7.16 (m, 1H, 5-H), 6.43 (s, 1H, 8-H), 6.26 (d,
J = 2.8 Hz, 2H, 2'-H, 6'-H), 5.96 (d, J = 5.5 Hz, 2H, -OCH2O-), 5.43
(s, 1H, -OH), 4.81-4.74 (m, 1H, 1-H), 4.56-4.51(m, 1H, 4-H),
4.50-4.36 (m, 2H, 11-H), 3.91-3.83 (m, 7H,piperidine , 1′′-H),
3.76 (s, 6H, 3′,5′-OCH3), 3.29-2.23 (m, 1H, 2-H), 3.18-3.12 (m,
4.1.8.20
4β-((2-Morpholinoethyl)disulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 17t
Reagent: 5b (58.8 mg, 0.4 mmol). The product was obtained as 1H, 3-H), 1.97-1.88 (m, 3H, piperidine), 1.73-1.65 (m, 1H,
a white solid, yield: 42%, mp: 194.8-196.2oC; 1H NMR (500 piperidine), 1.56 (d, J = 4.0 Hz, 1H, piperidine); ESI-MS: m/z
MHz, CDCl3) δ 6.92 (s, 1H, 5-H), 6.47 (s, 1H, 8-H), 6.27 (s, 2H, 2′, [M+H]+590.
4.1.8.25 4β-((2-(4-Hydroxypiperidin-1-yl)-2-oxoethyl)disulfanyl)-4'-
6′-H), 5.98 (d, J = 11.5 Hz, 2H, -OCH2O-), 5.55 (s, 1H, -OH), 4.56
(d, J = 3.5 Hz, 1H, 1-H), 4.50-4.38 (m, 3H, 4,11-H), 3.77 (s, 6H,
3′,5′-OCH3), 3.74 (brs, 4H,morpholine), 3.37 (dd, J = 13.5, 4.5
Hz, 1H, 2-H), 3.21-3.10 (m, 1H, 3-H), 3.00-2.88 (m, 2H, 2′′-H),
2.82-2.65 (m, 2H, 1′′-H), 2.61-2.41 (m, 4H,morpholine);13C
NMR (125 MHz, CDCl3) δ 174.43, 148.31, 147.32, 146.52,
136.20, 134.57, 134.25, 132.81, 131.67, 130.98, 128.08,
127.47, 110.47, 110.07, 108.10, 101.65, 70.01, 56.64, 55.42,
43.68, 42.28, 37.36; ESI-MS: m/z [M+H]+ 562.
O-demethyl-4-desoxypodophyllotoxin 17y
Reagent:7e (115.6 mg, 0.4 mmol). The product was obtained
1
as a white solid, total yield: 21%, mp: 194.5-196.1oC; H NMR
(500 MHz, CDCl3) δ 7.19 (d, J = 5.9 Hz, 1H, 5-H), 6.44 (s, 1H, 8-
H), 6.27 (s, 2H, 2′, 6′-H), 5.96 (d, J = 6.5 Hz, 2H, -OCH2O-), 4.76
(d, J = 4.0 Hz, 1H,1-H), 4.54 (d, J = 5.0 Hz, 1H,4-H), 4.46-4.39
(m, 2H, 11-H), 4.14-3.97 (m,2H, 1′′-H), 3.78 (s, 6H, 3′,5′-OCH3),
3.77-3.74 (m, 2H,piperidine), 3.30 (dd, J = 14.0, 5.0 Hz, 1H, 2-
H), 3.21-3.13 (m, 1H, 3-H), 1.98-1.88 (m, 2H, piperidine), 1.86-
1.68 (m, 2H, piperidine), 1.68-1.49 (m, 3H,piperidine); ESI-MS:
4.1.8.21 4β-((2-Morpholino-2-oxoethyl)disulfanyl)-4'-O-demethyl-
4-desoxypodophyllotoxin 17u
Reagent: 7a (64.4 mg, 0.4 mmol). The product was obtained as m/z [M+H]+590.
a white solid, yield: 69%, mp: 141.2-142.7oC; 1H NMR (500
MHz, CDCl3) δ 7.16 (s, 1H,5-H), 6.44 (s, 1H,8-H), 6.27 (s, 2H, 2′,
6′-H), 5.97 (dd, J = 7.0, 1.0 Hz, 2H, -OCH2O-), 5.30 (s, 1H,-OH),
4.75 (d, J = 4.5 Hz, 1H,1-H), 4.54 (d, J = 5.0 Hz, 1H, 4-H), 4.47
(dt, J = 19.0,9.0 Hz, 2H,11-H), 3.78 (s, 6H, 3′,5′-OCH3), 3.75-3.67
(m, 8H, morpholine), 3.56-3.50 (m, 2H, 1′′-H), 3.30 (dd, J =
14.0, 5.0 Hz, 1H, 2-H), 3.21-3.15 (m, 1H, 3-H); HR-MS (ESI+):
calculated for C27H29N1O9S2 [M+H]+: 576.1356, found: m/z =
575.9867[M+H]+.
4.1.9 General procedure for synthesis of 18a-c
To a solution of thiol-containing glycosyl derivates 13
(0.1mmol) in DCM/CH3OH, and compound 17a (52.5 mg, 0.1
mmol) in chloroform (2 mL) was added. The resulting mixture
was stirred at room temperature overnight. Then, the solvent
was evaporated in vacuo, and the residue was purified
bycolumn chromatography on silica gel using DCM-Methanol
as eluent to give the corresponding product.
4.1.9.1 4β-((1-Galactosyl)disulfanyl)-4'-O-demethyl-4-desoxypodo
4.1.8.22
4β-((2-Oxo-2-(piperazin-1-yl)ethyl)disulfanyl)-4'-O-
phyllotoxin 18a
demethyl-4-desoxypodophyllotoxin 17v
Reagent:13a (19.6 mg, 0.1 mmol). The product was obtained
1
as a white solid, yield: 73%; mp: 171.3-172.8oC; H NMR (500
Reagent: 7b (104.0 mg, 0.4 mmol). The product was obtained
1
as a white solid, total yield: 36%, mp: 149.9-151.2oC; H NMR
MHz, MeOD-d4) δ 7.25 (s, 1H, 5-H), 6.41 (s, 1H, 8-H), 6.30 (s,
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Journal Name
2H, 2'-H, 6'-H), 5.94 (d, J = 2.5 Hz, 2H, -OCH2O-), 5.02 (d, J = 4.0 3-H); 13C NMR (125 MHz, Acetone) δ 174.67, 149.01, 147.92,
Hz, 1H, 1′-H), 4.67 (d, J = 10.0 Hz, 1H, 1-H), 4.52-4.43 (m, 3H, 4- 147.90, 141.50, 136.10, 133.78, 131.66, 129.65, 127.03,
H, 11-H), 3.94 (d, J = 3.0 Hz, 1H, Gla), 3.89-3.84 (m, 1H, Gla), 126.76, 126.61, 110.77, 110.53, 109.65, 102.51, 99.08, 92.79,
3.81-3.73 (m, 2H, Gla), 3.72 (s, 6H, 3′,5′-OCH3), 3.68 (d, J = 9.5 81.60, 75.75, 74.28, 71.41, 70.35, 69.03, 56.72, 56.64, 44.33,
Hz, 1H, Gla), 3.55 (dd, J = 9.0, 3.5 Hz, 1H, 2-H) , 3.39-3.32 (m, 42.04, 38.21; ESI-MS: m/z [M+H]+705.
4.1.11 4β-((2-Methylhexahydropyrano[3,2-d][1,3]dioxine-7,8-diol)
1H, Gla), 3.27-3.18 (m, 1H, 3-H)
；
13C NMR (125 MHz, MeOD) δ
disulfanyl)-4'-O-demethyl-4-desoxypodophyllotoxin 18e
175.76, 148.15, 147.16, 147.05, 134.48, 132.44, 131.04,
128.55, 110.41, 109.27, 108.16, 101.43, 93.98, 79.93, 74.89,
70.31, 69.80, 69.16, 61.49, 55.43, 55.30, 43.41, 41.39, 37.74;
ESI-MS: m/z [M+H]+611.
To a solution of compound 18b (37 mg, 0.06 mmol) and TsOH
(10 mg) in acetonitrile(10 mL), 1,1-Dimethoxyethane (0.15
mL,1.43 mmol) was added under N2 protection. The mixture
was allowed to stir at room temperature for 3 hours. After the
reaction completed, the mixture was added water and
4.1.9.2 4β-((1-Glucosyl)disulfanyl)-4'-O-demethyl-4-desoxypodo
phyllotoxin 18b
Reagent: 13c (19.6 mg, 0.4 mmol). The product was obtained extracted with DCM. The combined organic layer was washed
1
as a white solid, yield: 59%; mp: 175.6-176.6
MHz, Acetone-d6) δ 7.30 (s, 1H, 5-H), 7.14 (s, 1H, -OH), 6.46 (s, by column chromatography on on silica gel using DCM-EtOAc
1H 8-H), 6.31 (s, 2H, 2'-H, 6'-H), 6.00 (s, 2H, -OCH2O-), 5.11 as eluent to give the corresponding product 18e as a white
(d, J = 3.0 Hz, 1H, 1′-H), 4.78 (d, J = 9.5 Hz, 1H, 1-H), 4.59 (d, J = solid, yield: 69%; mp: 163.2-164.7oC; 1H NMR (500 MHz,
5.0 Hz, 1H 4-H), 4.56-4.54 (m, 2H, glu), 4.46-4.44 (m, 1H, 11- Acetone-d6) δ 7.14 (s, 1H, 5-H), 7.07 (s, 1H, -OH), 6.48 (s, 1H, 8-
℃; H NMR (500 with brine and condensed in vacuo. The residue was purified
，
，
H), 4.36-4.31 (m, 2H, glu, 11-H), 3.97 (d, J = 2.5 Hz, 1H, glu), H), 6.33 (s, 2H, 2'-H, 6'-H), 6.01 (d, J = 1.5 Hz, 2H, -OCH2O-),
3.88-3.83 (m, 1H, glu), 3.77-3.71 (m, 1H, glu), 3.68 (s, 6H, 3′,5′- 4.87 (d, J = 2.0 Hz, 1H, Glu), 4.82 (d, J = 9.5 Hz, 1H, Glu), 4.79-
OCH3), 3.60-3.56 (m, 1H, glu), 3.54-3.49 (m, 1H, glu), 3.48-3.40 4.76 (m, 2H, Glu), 4.70 (d, J = 4.5 Hz, 1H, 1-H), 4.54 (d, J = 4.0
(m, 2H, glu), 3.29-3.26 (m, 2H, 2-H, 3-H), 3.18-3.12 (m, 1H, glu) Hz ,1H, 4-H), 4.48-4.44 (m, 1H, 11-H), 4.36-3.31 (m, 1H, 11-H),
；
13C NMR (125 MHz, Acetone-d6) δ 174.80, 148.91, 147.88, 4.22-4.18 (m, 1H, 7′-H), 3.69 (s, 7H, 3′,5′-OCH3, Glu), 3.60-3.53
147.83, 136.09, 133.64, 131.81, 129.64, 111.52, 110.36, (m, 3H, Glu), 3.35-3.28 (m, 3H, Glu, 2-H, 3-H), 1.27 (d, J = 5.0
109.69, 102.39, 93.55, 82.26, 79.60, 73.78, 71.40, 70.46, 62.85, Hz, 3H, -CH3) ; ESI-MS: m/z [M+H]+637.
56.66, 56.35, 44.33, 42.03, 38.37; ESI-MS: m/z [M+NH4]+628.
4.1.15 General procedure for synthesis of 22a-f
4.1.9.3
desoxypodophyllotoxin 18c
Reagent: 13b (36.4 mg, 0.4 mmol). The product was obtained
4β-((1-Acetyl
galactosyl)disulfanyl)-4'-O-demethyl-4-
Method for compound 22a-e and 22f': The mixture of
compound 1617 (83mg, 0.2 mmol) and 21 (0.24 mmol) in DCM
(5 mL) was stirred at room temperature for 3 hours under N2
protection. Then, the solvent was evaporated in vacuo, the
residue was purified by column chromatography on silica gel
using PE-DCM-EtOAc as eluent to give the desired product.
1
as a white solid, yield: 51%; mp: 149.1-150.6℃; H NMR (500
MHz, CDCl3) δ 7.04 (s, 1H), 6.46 (s, 1H), 6.27 (s, 2H), 5.97 (dd, J
= 7.5, 1.0 Hz, 2H), 5.51 (d, J = 3.0 Hz, 1H), 5.29 (t, J = 10.0 Hz,
1H), 5.10 (dd, J = 10.0, 3.5 Hz, 1H), 4.81 (d, J = 10.0 Hz, 1H),
4.77 (d, J = 4.0 Hz, 1H), 4.53 (d, J = 5.0 Hz, 1H), 4.47 (dd, J = 8.5,
7.0 Hz, 1H), 4.37 (t, J = 9.5 Hz, 1H), 4.28 (dd, J = 11.5, 7.0 Hz,
1H), 4.23-4.18 (m, 1H), 4.16-4.08 (m, 2H), 3.77 (s, 6H), 3.24-
3.12 (m, 2H), 2.20 (s, 3H), 2.07 (s, 3H), 2.03 (s, 3H), 2.00 (s, 3H);
ESI-MS: m/z [M+Na]+801.
Deprotection method for 22f: The obtained 22f' was
immediately dissolved in DCM (5 mL), and HCl saturated EtOAc
(1 mL) was added at 0 °C. The mixture was allowed to stir at
room temperature until the starting material disappeared.
Then, the solvent was evaporated, added NaHCO3 saturated
aqueous solution, and extracted with DCM. The combined
organic layer was washed with bine, dried over anhydrous
Na2SO4, evaporated in vacuo to give compound 22f without
purification.
4.1.10 4β-((2-(Thiophen-2-yl)hexahydropyrano[3,2-d][1,3]dioxine-
7,8-diol)disulfanyl)-4'-O-demethyl-4-desoxypodophyllotoxin 18d
To a solution of compound 18b (68 mg, 0.11 mmol) in 2-
thenaldehyde (1 mL), dry ZnCl2 (34 mg) was added under N2
protection. After the reaction completed, the mixture was
added water and extracted with DCM. The combined organic
layer was washed with brine and condensed in vacuo. The
residue was purified by column chromatography on silica gel
using DCM-EtOAc as eluent to give 18d as a white solid, yield:
39%; mp: 168.7-170.2oC; 1H NMR (500 MHz, Acetone-d6) δ
7.48 (dd, J = 5.0, 1.0 Hz, 1H,thiophene), 7.19 (d, J = 3.0 Hz, 1H,
thiophene), 7.17 (s, 1H, 5-H), 7.14 (s, 1H, -OH), 7.03 (dd, J =
5.0, 3.5 Hz, 1H, thiophene), 6.49 (s, 1H, 8-H), 6.33 (s, 2H, 2'-H,
6'-H), 6.02 (dd, J = 3.0, 1.0 Hz, 2H, -OCH2O-), 5.93 (s, 1H, 7′-H),
4.92-4.88 (m, 4H, Glu), 4.56 (d, J = 4.0 Hz, 1H, 1-H), 4.50-4.46
(m, 1H, 4-H), 4.39-4.31 (m, 2H, 11-H), 3.85 (t, J = 10.1 Hz, 1H,
Glu), 3.81-3.72 (m, 2H,Glu), 3.69 (s, 6H, 3′,5′-OCH3), 3.66-3.62
(m, 1H,Glu), 3.59 (t, J = 9.5 Hz, 1H, Glu), 3.34-3.29 (m, 2H, 2-H,
4.1.15.1
4β-Ethyltrisulfanyl-4'-O-demethyl-4-desoxypodophyllo
toxin 22a
Reagent: 21a (57.4 mg, 0.24 mmol). The product was obtained
1
as a white solid, yield: 34%, mp: 243.5-244.3oC; H NMR (500
MHz, CDCl3) δ 6.93 (s, 1H, 5-H), 6.45 (s, 1H, 8-H), 6.28 (s, 2H, 2'-
H, 6'-H), 5.60 (dd, J = 14.0, 1.5 Hz, 2H, -OCH2O-), 5.42 (s, 1H, -
OH), 4.64 (d, J = 3.5 Hz, 1H, 1-H), 4.55 (d, J = 4.0 Hz, 1H, 4-H),
4.50-4.40 (m, 2H, 11-H), 3.78 (s, 6H,3′,5′-OCH3), 3.24-3.15 (m,
2H, 2-H, 3-H), 3.05 -2.93 (m, 2H, 1′′-H), 1.44 (t, J = 7.5 Hz, 3H,
2′′-H); ESI-MS: m/z[M+Na]+531.
4.1.15.2 4β-((2-Hydroxyethyl)trisulfanyl)-4'-O-demethyl-4-desoxy
podophyllotoxin 22b
Reagent: 21b (61.2 mg, 0.24 mmol). The product was obtained
1
as a white solid, yield: 59%, mp: 229.3-231.1oC; H NMR (500
10 | J. Name., 2012, 00, 1-3
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MHz, CDCl3) δ 6.94 (s, 1H, 5-H), 6.46 (s, 1H, 8-H), 6.28 (s, 2H, 2'- 4.2 Cytotoxic activities assay
H, 6'-H), 5.99 (dd, J = 14.0, 1.5 Hz, 2H, -OCH2O-), 5.43 (s, 1H, -
The cytotoxic activities of the tested compounds in KB and
KB/VCR cells were measured using the SRB (sulforhodamine B)
method. Cells were seeded in 96-well microtiter plates (at a
density of 4000 cells per well) for overnight attachment and
exposed to each of the test compound (1.0 ~ 100.0 µM) for 72
h. The SRB solution (5.0 mg/mL in RPIM 1640 medium; Sigma-
Aldrich) was added (20.0 µl/well), and plates were incubated
for a further 4 h at 37°C. The purple formazan crystals were
dissolved in 100.0 µL of DMSO. After 5 min, the plates were
read on an automated microplate spectrophotometer (Bio-Tek
Instruments, Winooski, VT) at 490 nm. Assays were performed
in triplicate and independently for three times. The
concentration of drug inhibiting 50% of cells (IC50) was
calculated using the software of dose-effect analysis.
OH), 4.67 (d, J = 4.0 Hz, 1H, 1-H), 4.55 (d, J = 4.5 Hz, 1H, 4-H),
4.47 (t, J = 7.0 Hz, 1H, 11-H), 4.40 (t, J = 9.5 Hz, 1H, 11-H), 4.01
(t, J = 5.5 Hz, 2H, 1′′-H), 3.78 (s, 6H,3′,5′-OCH3), 3.26-3.08 (m,
4H, 2-H, 3-H, 2′′-H); ESI-MS: m/z [M+H]+ 525.
4.1.15.3
4β-((2,3-Dihydroxypropyl)trisulfanyl)-4'-O-demethyl-4-
desoxypodophyllotoxin 22c
Reagent: 21c (68.4 mg, 0.24 mmol). The product was obtained
1
as a white solid, yield: 36%, mp: 251.5-252.9oC; H NMR (500
MHz, CDCl3) δ 6.93 (d, J = 23.0 Hz, 1H, 5-H), 6.46 (s, 1H, 8-H),
6.28 (s, 2H, 2'-H, 6'-H), 5.99 (d, J = 10.5 Hz, 2H, -OCH2O-), 5.43
(s, 1H, -OH), 4.67 (d, J = 3.5 Hz, 1H, 1-H), 4.56 (d, J = 3.5 Hz, 1H,
4-H), 4.47 (d, J = 6.5 Hz, 1H, 11-H), 4.40-4.36 (m, 2H, 11, 3′′-H),
3.86-3.83 (m, 1H, 3′′-H), 3.78 (s, 6H,3′,5′-OCH3), 3.69-3.63 (m,
1H, 2′-H), 3.25-3.00 (m, 4H, 2, 3, 1′′-H); HR-MS (ESI+):
calculated for C23H24O7S3 [M+NH4]+: 572.1077, found: m/z =
572.1084.
4.3 Evaluation of metabolism stability in human plasma
Compound 17l (2 mg) was dissolved in DMSO (20μL) and
acetonitrile (180μL) respectively, and then they were diluted
with phosphate buffer saline (PH = 7.4) to afford stock solution
(2 mg/mL, 1 mL). Human plasma was incubated at 37 °C for
15min. An aliquot (100 μL) of the stock solution was added to
the human plasma (100 μL), and the mixture was incubated at
37 °C. At different time intervals (maximum incubation = 8h),
the reaction was stopped by addition of 200μL acetonitrile
4.1.15.4 4β-((3-Morpholinopropyl)trisulfanyl) -4'-O-demethyl-4-
desoxypodophyllotoxin 22d
Reagent: 21d (81.1 mg, 0.24 mmol). The product was obtained
1
as a white solid, yield: 38%, mp: 180.3-181.5oC; H NMR (400
MHz, CDCl3) δ 6.84 (s, 1H, 5-H), 6.39 (s, 1H, 8-H), 6.20 (s, 2H, 2'-
H, 6'-H), 5.92 (dd, J = 8.4, 1.2 Hz, 2H, -OCH2O-), 4.48 (d, J = 5.2
Hz, 1H, 1-H), 4.38-4.33 (m, 3H, 4-H, 11-H), 3.71 (s, 6H,3′,5′-
OCH3), 3.70-3.64 (m, 4H, morpholine), 3.29 (dd, J = 13.8, 5.2
Hz, 1H, 2-H), 3.13-3.03 (m, 1H, 3-H), 2.83-2.72 (m, 2H, 2′′-H),
2.48-2.34 (m, 6H,morpholine, 3′′-H), 1.94-1.84 (m, 2H, 1′′-
H);HR-MS (ESI+) calculatedfor C28H33N1O8S3 [M+H]+: 608.1441,
found: m/z =608.1445 [M+H]+.
containing
nitrobenzene
(internal
standard).
After
precipitation of proteins, the samples were centrifuged at
14000 rpm for 8 min to pellet the precipitated protein. Then,
the supernatant (20 μL) of these aliquots was analysed via
HPLC to determine the residual amount of 17l. The absorbance
detector was set at a wavelength of 254nM, and the mobile
phase consisted of acetonitrile: water (containing 0.1%
trifluoroacetic acid) = 40:60 (v/v %). The mobile phase flow
rate was 1.0 mL/min, and the HPLC column (250 mm × 4.6
mm) was packed with 5 μm C18.
4.1.15.5 4β-(((2R,3S,4R,5R,6S)-3,4,5-Trihydroxy-6-(hydroxymethyl)
tetrahydro-2H-pyran-2-yl)trisulfanyl)-4'-O-demethyl-4-desoxypod
ophyllotoxin 22e
Reagent: 21e (89.5 mg, 0.24 mmol). The product was obtained
1
as a white solid, yield: 9 %, mp: 182.9-184.8oC; H NMR (400
MHz, Acetone) δ 7.30 (s, 1H, 5-H), 7.09 (s, 1H, Phenol), 6.46 (s,
1H, 8-H), 6.32 (s, 2H, 2'-H, 6'-H), 5.99 (s, 2H, -OCH2O-), 5.11 (d,
J = 1.6 Hz, 1H, 1′-H), 4.77 (d, J = 9.6 Hz, 1H, 1-H), 4.61-4.51 (m,
3H, 4-H,glu), 4.50-4.43 (m, 1H, 11-H), 4.39-4.28 (m, 1H, 11-H),
4.32 (d, J = 4.5 Hz, 1H, glu), 4.01 (ddd, J = 11.5, 5.0, 2.5 Hz, 1H,
glu), 3.85-3.80 (m, 1H, glu), 3.78-3.74 (m, 1H, glu), 3.68 (s, 6H,
3′,5′-OCH3), 3.60-3.39 (m, 5H, glu), 3.30-3.27 (m, 2H, 2, 3-H);
HR-MS (ESI+) calculatedfor C27H30O12S3 [M+H]+: 643.0972,
found: m/z=643.0968[M+H]+.
Acknowledgements
We thank Mrs. Jianyang Pan (Pharmaceutical Informatics
Institute, Zhejiang University) for performing the NMR
spectrometry. Zhejiang Provincial Natural Science Foundation
of China (LY13H300002) and Program for Zhejiang Leading
Team of S&T Innovation (2011R50014).
4.1.15.64β-((2-Oxo-2-(piperazin-1-yl)ethyl)trisulfanyl)-4'-O-
demethyl-4-desoxypodophyllotoxin 22f
Notes and references
Reagent: 21f (69.2 mg, 0.4 mmol). The product was obtained
1
1
2
3
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as a white solid, two steps yield: 41%, mp: 151.8-153.6oC; H
NMR (500 MHz, CDCl3) δ 6.96 (s, 1H, 5-H), 6.45 (s, 1H, 8-H),
6.28 (s, 2H, 2'-H, 6'-H), 5.99 (dd, J = 5.0, 1.0 Hz, 2H, -OCH2O-),
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DOI: 10.1039/C5RA12837D
Graphic abstract:
A novel series of podophyllotoxin derivatives bearing 4βꢀdisulfide/trisulfide were
designed, synthesized and biologically evaluated for their cytotoxic activities against
KB cells and KB/VCR cells.