Synthesis and tumor cell growth inhibitory activity of biotinylated annonaceous acetogenins

Article abstract of DOI:10.1016/j.ejmech.2013.11.012

Nineteen biotinylated squamocin/bullatacin derivatives have been synthesized for targeted delivery to biotin receptor overexpressed tumor cells. Most biotinylated squamocin and bullatacin derivatives show similar in vitro cytotoxicity against the biotin receptor non-overexpressed L1210 cells as squamocin and bullatacin, respectively, while against biotin receptor overexpressed 4T1 and P815 tumor cells, several derivatives show significantly higher potency and better selectivity. Among all the synthesized compounds, 15,28-di-O-(6-biotinylamidohexanoyl)squamocin (16) is the most potent, which is 10 and 26 times more active than squamocin against 4T1 and P815 cells, respectively. Compound 16 also appears to be six and fifteen times more selective than squamocin towards 4T1 and P815 cells, respectively, against L1210 cells. The structure activity relationship analysis has revealed that the preferred site for biotinylation is different for squamocin and bullatacin, and it also depends on whether a linking spacer is present.

Full text of DOI:10.1016/j.ejmech.2013.11.012

European Journal of Medicinal Chemistry 71 (2014) 219e228
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and tumor cell growth inhibitory activity of biotinylated
annonaceous acetogenins
Jing-Fang Shi , Ping Wu , Zi-Hua Jiang b **, Xiao-Yi Wei
a
,
c
a
,
a,
*
a
Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Xingke Road
23, Tianhe District, Guangzhou 510650, China
Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada
7
b
c
University of Chinese Academy of Sciences, Yuquanlu 19A, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 July 2013
Received in revised form
Nineteen biotinylated squamocin/bullatacin derivatives have been synthesized for targeted delivery to
biotin receptor overexpressed tumor cells. Most biotinylated squamocin and bullatacin derivatives show
similar in vitro cytotoxicity against the biotin receptor non-overexpressed L1210 cells as squamocin and
bullatacin, respectively, while against biotin receptor overexpressed 4T1 and P815 tumor cells, several
derivatives show significantly higher potency and better selectivity. Among all the synthesized com-
pounds, 15,28-di-O-(6-biotinylamidohexanoyl)squamocin (16) is the most potent, which is 10 and 26
times more active than squamocin against 4T1 and P815 cells, respectively. Compound 16 also appears to
be six and fifteen times more selective than squamocin towards 4T1 and P815 cells, respectively, against
L1210 cells. The structure activity relationship analysis has revealed that the preferred site for bio-
tinylation is different for squamocin and bullatacin, and it also depends on whether a linking spacer is
present.
30 October 2013
Accepted 7 November 2013
Available online 15 November 2013
Keywords:
Tumor-targeting drug delivery
Biotinylated annonaceous acetogenins
Biotinesquamocin conjugates
Biotinebullatacin conjugates
Cytotoxicity
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
potent complex I inhibitor known [10]. Bullatacin (2), one of the
most potent acetogenins, is effective in treating P388 [11] and
Annonaceous acetogenins (ACGs) are secondary metabolites
occurring only in some plants of Annonaceae family. They are de-
L1210 [12] murine leukemias in normal mice. It also displays high
cytotoxicity against A2780 [12] human ovarian carcinoma xeno-
rivatives of long-chain fatty acid (C32 or C34) bearing a terminal
g-
grafts in athymic mice at a dose of only 50 mg/kg per day which is
lactone ring and have been reported to potently inhibit the activity
of NADH-ubiquinone oxidoreductase (respiratory complex I) [1e4].
These long-chain fatty acid derivatives display impressive cyto-
toxicity against various tumor cell lines with an IC50 value ranging
from 10 M to 10 M [5,6], as well as in vivo antitumor effect [7].
Squamocin (1) and bullatacin (2) (Fig. 1) are two best known ex-
300 times more potent than the in vivo activity of taxol [12,13].
Both bullatacin and squamocin have significant cytotoxicity on
cancer cells and their antitumor effect has been demonstrated in
several tumor models; however, their lack of tumor specificity and
high toxicity toward normal cells prevents their use in clinic for
cancer treatment [14,15]. In order to enhance the therapeutic po-
tential and reduce systemic toxicity, selective targeting to cancer
cells is important. Targeted cancer therapies are currently being
developed for more effective treatments of cancer with fewer side
effects [16e18]. These therapies are thought to be less harmful to
normal cells by focusing on molecular and cellular changes that are
specific to cancer cells.
ꢀ
6
ꢀ14
amples of annonaceous acetogenins belonging to adjacent bis-THF
(
tetrahydrofuran) type of acetogenins [8]. They both possess a
terminal -unsaturated -lactone and a central polar part con-
a
,b
g
sisting of two THF rings and three secondary alcohol functional
groups. Squamocin (1) has good activity with clear inhibitory effect
on the growth of HepS tumor in mice (with an inhibition rate of 78%
at a dose of 30
m
g/kg) [9]. It has been shown that squamocin is more
Most tumor-specific delivery systems are based on carrier-
mediated delivery to selective tissues and organs through ligands
recognized by receptors or other address molecules which are
overexpressed on target cancer cells [19]. Several ligands such as
antibodies [20e22], peptides [23e25], vitamin [26e29], and
integrin [30,31] could be used as suitable targeting moieties in
targeted drug delivery systems [32,33].
potent than piericidin-A, previously considered to be the most
*
Corresponding author. Tel.: þ86 20 37252538.
*
* Corresponding author. Tel.: þ1 807 766 7171.
E-mail addresses: zjiang@lakeheadu.ca (Z.-H. Jiang), wxy@scbg.ac.cn (X.-Y. Wei).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.012

220
J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
Scheme 1. Reagents and conditions: (a) DCC, DMF, rt, N
NaHCO3, O-DMF, rt, 16 h, 50%.
2
, 16 h. (b) 3 -aminocaproic acid,
H
2
Fig. 1. Structures of squamocin and bullatacin.
hydroxyl groups. The biotinylating agent containing the spacer (3,
Scheme 1) can be readily prepared from biotin and
acid as reported [44].
The coupling between biotin/3 and squamocin/bullatacin is
mediated by dicyclohexylcarbodiimide (DCC) and N,N-dimethyl-4-
aminopyridien (DMAP). This coupling condition is mild and does
not affect the labile terminal a,b-unsaturated g-lactone function-
ality. Thus, a series of biotinylated squamocin and bullatacin de-
rivatives 4e22 were synthesized when biotin and biotin derivative
3
7
Biotin (vitamin B , vitamin H) belongs to a category of essential
micronutrients required for normal cellular functions [32,34]. It is a
growth promoter at cellular level and its content in tumors is
substantially higher than in normal tissues [35] due to the rapidly
growing nature of cancer cells. Consequently, the receptors
involved in the uptake of biotin are overexpressed in many cancer
cells, such as leukemia (L1210FR), ovarian (OV 2008, ID8), Colon
3
were treated with squamocin and bullatacin, respectively
(
Colo-26), mastocytoma (P815), lung (M109), renal (RENCA,
(Schemes 2 and 3). Although some selectivity was observed among
RD0995), and breast (4T1, JC, MMT06056) cancer cell lines [32,36].
Recently, biotin receptors have been explored for targeted delivery
of cytotoxic drugs to cancer cells by covalently linking the drug
molecule to biotin [34,37e40]. For example, Ojima and coworkers
demonstrated that biotinetaxoid conjugates exhibited much
higher potency against L1210FR cells that overexpress biotin re-
ceptors on cell surfaces than other two cell lines (L1210 and W138)
which do not overexpress biotin receptors on cell surfaces [32,41].
In order to improve the therapeutic potential of squamocin and
bullatacin for cancer therapy, we decide to covalently link biotin to
these molecules to generate biotineacetogenin conjugates such as
conjugate I (Fig. 2). Selective delivery of such conjugates to biotin
overexpressed cancer cells and subsequent uptake into cytosome
are anticipated to be mediated by biotin receptors. The targeting
component biotin and/or the linking spacer in conjugate I will be
readily cleaved to release the cytotoxic drug squamocin. Reported
here are the chemical synthesis of a series of biotinylated squa-
mocin and biotinylated bullatacin conjugates and their growth
inhibitory activity against cancer cells.
the secondary hydroxyl groups in both squamocin and bullatacin
toward acylation under this reaction condition, in most cases the
selectivity was low (Table 1). The reaction typically resulted in a
mixture of mono- and di-biotinylated derivatives when 1.1e1.5
equivalents of the acylating acid was used. After extensive purifi-
cation of the crude products by silica gel chromatography and
preparative HPLC, all products were purified and structurally
confirmed.
Structure characterization was established by the analysis of 1D
(1
H, C and DEPT) and 2D (COSY, HSQC and HMBC) NMR spectra,
13
1
13
and HR-ESIMS data. The H and C chemical shifts of the bio-
tinylated derivatives were compared with those of squamocin or
bullatacin. When the secondary hydroxyl group was esterified, the
chemical shifts of the carbon (C-4, C-15, C-24, and C-28) bearing
that hydroxyl group moved downfield (Table 2). Up to 3.6 ppm
downfield shift was observed for C-24 in compound 11 while the
shift for C-15 was relatively small (<1 ppm) in all acylated de-
rivatives. On the other hand, the chemical shifts of the neighboring
carbons (C-5, C-14, C-16, C-23, C-25, C-27, and C-29) moved upfield
(
in a magnitude of 1.5e3.7 ppm) upon esterification of the specific
2
. Results and discussion
1
hydroxyl group. In the case of H NMR data, a significant downfield
shift (>1.1 ppm) was observed for the proton directly attached to
the carbon bearing the hydroxyl group that is acylated (Table 3).
2.1. Synthesis and structure characterization
1
The H NMR chemical shifts of the protons observed here are
Generally, a tumor targeting drug delivery system consists of a
tumor recognition moiety and a cytotoxic warhead connected
directly or through a suitable linker to form a conjugate [42]. There
are three secondary alcohol functional groups in squamocin and
bullatacin, which play less important role in their activity [43]. In
this study we have designed two groups of squamocin/bullatacin
derivatives by attaching the biotin residue either (a) directly or (b)
through an -aminocaproic acid linking spacer to one of the free
3
Fig. 2. General structure of biotinesquamocin conjugate I and the presumed cleavage
Scheme 2. Synthesis of biotinylsquamocins 4e8 and biotinylbullatacins 9e11. Re-
mechanism to release the active drug.
2 2
agents and conditions: (a) Biotin, DCC, DMAP, DMF, CH Cl2, rt, N , 16 h.

J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
221
value of selectivity index represents better selectivity. The data
indicate that several biotineacetogenin conjugates (6, 8, 10, 12, 16,
18, 20, and 22) are more selective than their parent compounds
(
(
squamocin and bullatacin) towards biotin receptor overexpressed
4T1 and P815) cells when compared to biotin receptor non-
overexpressed (L1210) cells. As observed for the cytotoxic activity,
compound 16 shows the highest improvement in selectivity among
all synthesized derivatives, which is six and fifteen times more
selective than the unmodified squamocin towards 4T1 and P815
cells, respectively, against L1210 cells. With respect to the structure
activity relationship, we have examined the effect of substitution
pattern and the presence of the 6-amino-caproic acid residue as the
linking spacer between the biotin residue and the acetogenin
scaffold. Among the mono-biotinylated squamocin derivatives (4e
6), compound 6 with the biotin residue directly attached at the C-
28 hydroxyl group is significantly more active against 4T1 and P815
cells than the other two analogs 4 (esterified at C-15) and 5
esterified at C-24), suggesting that the C-28 hydroxyl group in
(
squamocin is the preferred site of attachment for biotin. Interest-
ingly, when the linking spacer is present (as in 12e14), the
preferred site of attachment in squamocin is the C-15 hydroxyl
group as indicated by the much higher potency of 12 (esterified at
C-15) than 13 or 14. In the case of bullatacin, attachment of biotin at
C-15 hydroxyl group (as in 10) results in the most active compound
among bullatacin derivatives 9e11. Similarly, when the linking
spacer is present (as in 18e20), the preferred site of attachment for
the biotinyl residue changes. Compound 18 (esterified at C-4) and
compound 20 (esterified at C-24) show significantly higher activity
than 19 (esterified at C-15). These data clearly indicate that the
preferred site for attaching the biotinyl residue depends on
whether or not the linking spacer is present for both squamocin
and bullatacin. These seemly unpredictable data may be correlated
to the difference in binding affinity of biotin receptor with specific
compound as well as the difference in hydrolytic rate of ester
bond(s) to release the active drug. Binding affinity between the
biotin receptor on cancer cells and the biotin residue on specific
compound may be affected by various structure features such as
the nature of the parent compound (squamocin or bullatacin), the
site at which the biotinylated residue is attached (C-4, C-15, C-24, or
C-28), and whether or not the spacer is present. Our data indicate
that suitable structural change in these biotinylated acetogenin
derivatives can have drastic effect on the potency as well as
selectivity of their growth inhibitory activity against cancer cells.
In comparing the mono-biotinylated derivatives with the di-
biotinylated compounds (4e6 vs. 7 and 8; 12e14 vs. 15e17; 18e
20 vs. 21 and 22), we have only analyzed the di-biotinylated com-
pounds in which one of the two biotinyl residues is attached at the
most preferred site of attachment. In most cases the addition of the
second biotinyl residue does not result in further improvement in
potency. In the case of compound 16 (di-biotinylated at C-15 and C-
28), however, the second biotinyl residue does provide noticeable
additional benefit. Compound 16 is up to three times more potent
and two times more selective than 12 (mono-biotinylated at C-15),
and as a result 16 is the most potent and most selective compound
among all squamocin derivatives described in this study.
Scheme 3. Synthesis of (6-biotinylamidohexanoyl)squamocins 12e17 and (6-
biotinylamidohexanoyl)bullatacins 18e22. Reagents and conditions: (a) 3, DCC,
DMAP, DMF, CH Cl2, rt, N , 16 h.
2 2
consistent with those reported for aminoacyl triester derivatives of
squamocin [45].
2.2. Biological evaluation
To evaluate the tumor-targeting potential of the synthesized
biotinylated squamocin and bullatacin derivatives toward biotin
receptor (þ) cancer cells, we studied the cytotoxicity of these
compounds against three cancer cell lines: L1210 (leukemia), 4T1
(
breast cancer), and P815 (mastocytoma). Both 4T1 and P815 cells
overexpress biotin receptors while L1210 cells do not [27,31]. The
IC50 values of these biotinylated compounds (4e22) are presented
in Table 4 in comparison with the data obtained for the non-
biotinylated squamocin (1) and bullatacin (2). Overall, the major-
ity of these derivatives show similar or improved activity against all
three cancer cells when compared to their parent squamocin (1)
and bullatacin (2), indicating that the attachment of one or two
biotinyl moieties does not adversely affect their growth inhibitory
activity in general. Against L1210 cells, most biotinylated squamo-
cin or bullatacin derivatives show similar activity as squamocin or
bullatacin, respectively. In contrast, against biotin receptor (þ) 4T1
and P815 cells, a good number of biotinylated squamocin or bul-
latacin derivatives show significantly higher potency than squa-
mocin or bullatacin. For example, compound 16 is over 10 and 26
times more active than squamocin (1) against 4T1 and P815 cells,
respectively. In the case of bullatacin (2), the effect of biotinylation
on the potency of activity appears to be of less extent. The most
active biotinylated bullatacin derivative (20) is three and eight
times more potent than bullatacin against 4T1 and P815 cells,
respectively. These data clearly show that biotinylation of squa-
mocin and bullatacin can result in significant improvement of their
activity in biotin receptor (þ) cancer cells, which is probably the
result of an enhanced uptake of such molecules medicated by biotin
receptors.
Regarding the effect of linker, compound 12 (biotinylated at C-
15 with the linker) is almost four and ten times more potent than 4
(biotinylated at C-15 without the linker) against 4T1 cells and P815
cells, respectively, while the improvement in selectivity towards
4T1 and P815 cells is sixty and eight times, respectively, against
L1210 cells. As for bullatacin derivatives, compound 18 (bio-
tinylated at C-4 with the linker) and compound 20 (biotinylated at
C-24 with the linker) are twelve to forty nine times more active and
eight to thirty four times more selective than 9 and 11 towards 4T1
cells and P815 cells. These data indicate that the linking spacer may
In order to show whether or not biotinylated derivatives display
higher selectivity towards biotin receptor overexpressed cancer
cells, we have defined the selectivity index for each compound for
both 4T1 cells and P815 cells against L1210 cells (Table 4). For
example, the selectivity index of squamocin for 4T1 cells is the ratio
of IC50 against L1210 cells over the IC50 against 4T1 cells. A higher

222
J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
Table 1
Yield of biotinylation reaction of squamocin and bullatacin.
biotinylation of acetogenins is a viable strategy to increase the
potency and selectivity of their growth inhibitory activity against
biotin receptor (þ) cancer cells. Our data support the earlier
observation that biotin molecule can be linked to anticancer drugs
for tumor-targeting purpose. Further studies are under way to
evaluate the toxicity and in vivo antitumor effect of some of the
compounds described in this paper.
Compound
Squamocin
Biotinylating agent (equiv.)
Biotin (1.1)
Product
Yield (%)
4
5
6
7
8
9
6.6
11.3
20.1
0.9
2.0
8.4
3.7
2.6
7.3
8.8
6.3
2.5
2.2
1.6
7.3
6.6
11.1
1.1
1.6
Bullatacin
Biotin (1.2)
1
1
0
1
3. Experimental
Squamocin
3 (1.1)
12
3.1. Reagents and instrumentation
1
1
1
1
1
3
4
5
6
7
Squamocin and bullatacin were obtained in our previous
chemical investigation on the seeds of Annona squamosa [48].
Biotin and -aminocaproic acid were purchased from YuanYe Bio-
3
Bullatacin
3 (1.5)
18
technology Co., Ltd (Shanghai, China). N,N-Dicyclohexyl carbodii-
mide (DCC), N-hydroxysuccinimide (NHS), 4-dimethylamino-pyri-
dine (DMAP), and succinic anhydride were purchased from Aladdin
Chemical Co., Ltd (Shanghai, China). Potassium hydroxide was ob-
tained from DaMao Chemical Reagent Factory (Tianjin, China).
Anhydrous sodium sulfate and citric acid were purchased from
Guangzhou Reagent Factory (Guangzhou, China). Optical rotations
were obtained on a PerkineElmer 341 polarimeter with MeOH as
1
2
2
2
9
0
1
2
improve the cytotoxicity and selectivity of squamocin and bulla-
tacin derivatives against cancer cells.
In conclusion, we have prepared nineteen biotinylated de-
rivatives of squamocin and bullatacin and evaluated their growth
inhibitory activity with three cancer cell lines, including L1210 cells
and two other biotin receptor (þ) 4T1 and P815 cancer cells. Most
biotinylated squamocin and bullatacin derivatives show similar
potency of cytotoxicity against L1210 cells as squamocin and bul-
latacin, respectively. Against biotin receptor (þ) 4T1 and P815
cancer cells, several derivatives show significantly higher potency
and better selectivity than squamocin and bullatacin. For squa-
mocin, the most preferred site to attach biotin residue is C-28 hy-
droxyl group while that to attach the 6-amino-caproic acid spacer-
bearing biotin residue is C-15 hydroxyl group. In the case of bul-
latacin, the preferred site is changed to C-15 for attaching biotin
1
13
solvent. The H and C NMR spectra were collected on a Bruker
1
13
DRX-400 instrument at 400 ( H) and 100 ( C) MHz or a Bruker
Avance-600 instrument at 600 ( H) and 150 ( C) MHz. The 2D
NMR ( He H COSY, HSQC, and HMBC) spectra were recorded on a
1
13
1
1
Bruker Avance-600 instrument. Chemical shifts are reported as
ppm (d units) relative to tetramethylsilane (TMS) as an internal
standard and the coupling constants as J in hertz. The splitting
pattern abbreviations are as follows: s ¼ singlet, d ¼ doublet,
dd ¼ double doublet, t ¼ triplet, m ¼ multiplet. ESIMS data were
obtained on an MDS SCIEX API 2000 LC/MS instrument. HRESIMS
data were obtained on a Bruker maXis Q-TOF mass spectrometer.
Preparative HPLC was run on a Shimadzu LC-6A pump and a Shi-
madzu RID-10A refractive index detector and all separations were
carried out with an XTerra Prep MS C18 column (19 ꢁ 300 mm,
residue and C-4/C-24 for
-3 aminocaproic acid spacer-containing
biotin residue, respectively. Overall, we have demonstrated that
10 mm) at a flow rate of 5 mL/min except where otherwise stated.
Table 2
Selected 13C chemical shifts (150 MHz, CDCl
) of squamocin (1), bullatacin (2) and their biotinylated derivatives 4e22.
3
Compound
C
d (ppm)
3
4
5
14
15
16
23
24
25
27
28
29
Squamocin (1)a
33.12
30.88
33.50
33.44
31.08
33.41
30.65
33.21
33.26
30.71
30.77
33.33
33.23
33.41
30.90
33.42
33.27
30.66
33.25
30.72
33.36
74.09
74.56
74.23
74.30
74.82
74.06
75.04
74.16
74.03
75.13
75.17
74.18
74.08
74.11
74.46
74.02
74.15
75.01
74.11
75.07
74.10
83.36
80.41
83.57
83.45
80.57
83.31
80.05
83.40
83.34
80.33
80.20
83.04
83.20
83.24
80.40
83.17
83.28
80.03
83.24
80.07
83.34
82.15
83.10
80.88
82.36
82.34
80.53
82.57
80.72
82.24
80.42
82.79
80.53
82.75
82.31
83.26
80.54
82.32
82.68
80.54
82.26
80.57
71.51
71.43
75.16
71.55
71.63
74.77
71.51
74.93
71.42
74.82
71.64
74.71
71.34
71.39
71.43
74.97
71.30
71.42
74.74
71.43
74.63
32.49
32.79
31.47
32.50
32.86
30.81
32.60
31.24
32.47
31.06d
32.62
31.73
32.38
32.53
32.89
30.95
32.46
32.53
30.81
32.57
30.64
37.48
37.45
37.76
34.45
34.53
34.27
37.42
37.64
34.49
37.67
34.51
34.42
c
c
c
c
c
c
71.60
71.74
71.63
74.30
74.24
74.1
71.55
71.40
74.16
71.51
74.07
73.88
37.23c
37.38c
4
5
6
7
8
c
37.20
34.45c
c
34.53
34.27c
37.20
37.04
34.27
37.29c
1
1
1
1
1
1
2
3
4
5
6
7
d
c
c
c
c
34.30
34.36c
b
Bullatacin (2)
33.23
29.90
33.38
33.31
29.90
33.22
33.25
29.99
30.01
69.91
72.02
69.94
69.91
71.88
69.79
69.84
71.83
71.81
37.34
34.14
37.45
37.38
34.12
37.35
37.36
34.12
34.12
9
1
1
1
1
2
2
2
0
1
8
9
0
1
2
a
Data from Fujimoto et al. [46].
Data from Hui et al. [47].
Assignments interchangeble in the same row.
b
c,d

J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
223
Table 3
(Scheme 2). Upon completion of the reaction, the solvent was
removed under reduced pressure. The residue was dissolved in
ethyl acetate (EtOAc) and washed with 0.5% HCl, saturated aqueous
Selected 1H chemical shifts (600 MHz, CDCl
their biotinylated derivatives 4e22.
) of squamocin (1), bullatacin (2) and
3
Compound
d
H
(ppm)
NaHCO
layer was then dried over anhydrous Na
rated under reduced pressure. The crude product was separated by
silica gel column chromatography (CC) using CHCl eMeOH (98:2,
97:3, 96:4, 95:5, 93:7, and 9:1; v/v) to afford five fractions with F-I
solution and saturated aqueous NaCl solution. The organic
3
4
15
24
28
2
SO , filtered and evapo-
4
Squamocin (1)a
3.33
4.83
3.38
3.39
4.83
3.38
4.87
3.38
3.39
4.84
3.76
3.85
4.93
3.82
3.79
4.93
3.84
4.94
3.82
4.95
3.84
4.94
3.83
3.85
3.85
4.99
3.85
3.82e3.93
4.99
3.83
4.99
3.52
3.59
3.56
4.88
4.88
4.84
3.58
3.55
4.87
3.53
4.83e4.89
4.83
4
5
6
7
8
3
(
1.027 g) and F-II (254 mg) as the fractions of target products. F-I
200 mg) was further separated by HPLC using MeOHeH O (83:17,
¼ 61 min)
and a sub-fraction which was purified by HPLC using MeCNeH
(70:30, v/v) to afford 5 (43 mg, 11.3%, t
¼ 95 min) and 6 (76 mg,
0.1%, t
(
2
v/v) at a flow rate of 5 mL/min to afford 4 (25 mg, 6.6%, t
R
12
13
14
15
16
17
2
O
R
2
R
¼ 104 min). F-II (254 mg) was dissolved in MeOH and the
4.83e4.89
3.38
3.38
solution yielded a precipitate which was collected by filtration to
b
give 8 (21 mg, 0.9%). The mother liquor was purified by HPLC with a
Bullatacin (2)
9
3.80
Ò
5.11
3.40
Nova-pak HR C18 column using MeOHeH
2
O (83:17, v/v) at a flow
¼ 51 min).
10
11
18
19
20
21
22
3.84
4.85
rate of 5 mL/min to afford 7 (50 mg, 2%, t
R
3.84
3.38
5.10
3.40
3
.3.1. 15-O-Biotinylsquamocin (4)
3.82e3.93
3.84
4.88
3.38
2
0
ꢀ1
A yellowish waxy solid; [
a]
D
þ 51.4 (c 0.1, MeOH); IR cm
5.11
4.89
(KBr): 3200e3500, 3083, 2923, 2853, 1755, 1705, 1466, 1373, 1319,
5.11
3.39
1
1254, 1186, 1148, 1117; H NMR (600 MHz, CDCl ): 6.99 (1H, br s,
3
d
a
0
0
Data from Fujimoto et al. [46].
Data from Hui et al. [47].
H-35), 6.36 (1H, br s, C7 eNH), 5.53 (1H, br s, C8 eNH), 5.00 (1H, m,
b
0
0
H-36), 4.83 (1H, m, H-15), 4.50 (1H, m, H-8 ), 4.32 (1H, m, H-7 ),
.89e3.95 (3H, m, H-16, H-20, H-23), 3.85 (1H, m, H-24), 3.79 (1H,
3
0
0
m, H-19), 3.59 (1H, m, H-28), 3.19 (1H, m, H-6 ), 2.92 (1H, m, H-9 a),
2
H-3), 1.90e2.02 (4H, m), 1.58e1.81 (8H, m), 1.45e1.58 (10H, m),
3.2. Preparation of 6-biotinylaminocaproic acid (3)
0
0
.74 (1H, d, J ¼ 12.6 Hz, H-9 b), 2.36 (2H, m, H-2 ), 2.25 (2H, t, J ¼ 7.4,
Biotin (2 g, 8.2 mmol) and NHS (924 mg, 8 mmol) were dissolved
13
1.21e1.38 (32H, m), 1.41 (3H, m, H-37), 0.88 (3H, m, H-34); C NMR
in 60 mL DMF. Then DCC (1.85 g, 9 mmol) was added and the
mixture was stirred at room temperature for 16 h (Scheme 1). The
resulting precipitate (N,N-dicyclohexylurea, DCU) was removed by
filtration and the filtrate (75 mL) was concentrated to small volume
0
0
(150 MHz, CDCl ): d 173.93 (C-1), 173.50 (C-1 ), 164.34 (C-10 ),
3
148.94 (C-35), 134.31 (C-2), 83.10 (C-23), 82.20 (C-20), 81.53 (C-19),
8
6
0.41 (C-16), 77.44 (C-36), 74.56 (C-15), 71.74 (C-28), 71.43 (C-24),
0 0 0 0
1.49 (C-7 ), 60.26 (C-8 ), 54.93 (C-6 ), 40.63 (C-9 ), 37.45 and 37.38
(
10 mL) under reduced pressure. Diethyl ether (200 mL) was then
added and the resulting precipitate was collected by filtration and
dried in the presence of CaH under vacuum to provide N-
hydroxysuccinimidobiotin (3.09 g) which was used in the next step.
-Aminocaproic acid (966 mg, 8.4 mmol) was dissolved in 30 mL
M aqueous NaHCO solution. Then N-hydroxysuccinimidobiotin
3.09 g) in 35 mL DMF was added dropwise to the solution and the
0
(C-27, C-29), 34.87 (C-2 ), 32.79 (C-25), 31.88 (C-32), 30.88 (C-14),
2
2
2
9.59 (2C), 29.56, 29.52, 29.50, 29.49, 29.42, 29.30, 29.19, 29.02,
8.22, 28.13, 28.12, 27.48, 27.42, 25.72, 25.46, 25.18 (2C), 24.43,
2
2.64 (C-33), 22.18 (C-26), 19.22 (C-37), 14.11 (C-34); HR-ESIMS m/
3
þ
z: 849.5633 [M þ H] (calcd for C47
81 2 9
H N O S, 849.5657).
1
(
3
3
.3.2. 24-O-Biotinylsquamocin (5)
mixture was stirred at room temperature for 16 h. The solution was
concentrated under reduced pressure to remove partial solvent and
20
ꢀ1
A yellowish waxy solid; [
a]
D
þ 32.1 (c 0.4, MeOH); IR cm
(KBr): 3200e3500, 3083, 2924, 2853, 1751, 1700, 1646, 1464, 1374,
1
6
50 mL aqueous citric acid (100 g/L) was then added and stirred at
1
1
6
4
319, 1272, 1116; H NMR (600 MHz, CDCl ) : 7.00 (1H, m, H-35),
3
d
ꢂ
0
C for 30 min. The precipitate was collected by filtration and
0
0
.12 (1H, br s, C7 eNH), 5.56 (1H, br s, C8 eNH), 5.00 (1H, m, H-36),
washed with distilled water. The precipitate was dissolved in a
mixture of isopropanol and water (8:2, v/v) and kept at 4 C to give
0
0
.93 (1H, m, H-24), 4.51 (1H, s, H-8 ), 4.31 (1H, m, H-9 ), 4.02 (1H, m,
ꢂ
H-23), 3.80e3.88 (3H, m, H-16, H-19, H-20), 3.56 (1H, m, H-28),
ꢀ1
pure 3 (1.455 g, 50%) as a yellowish crystal; IR cm (KBr): 3200e
500, 3070, 2932, 2859, 1696, 1644, 1543, 1475, 1391, 1324, 1265,
0
3
.38 (1H, m, H-15), 3.16 (1H, m, H-6 ), 2.91 (1H, dd, J ¼ 12.8, 5.0 Hz,
3
0
0
0
H-9 a), 2.75 (1H, d, J ¼ 12.8 Hz, H-9 b), 2.32 (2H, m, H-2 ), 2.24 (2H,
1
1118; H NMR (600 MHz, DMSO-d
6
):
d
7.75 (1H, t, J ¼ 5.4 Hz, C6e
m, H-3), 1.90e2.02 (4H, m), 1.58e1.81 (8H, m), 1.45e1.58 (10H, m),
NH), 6.45 (1H, s, C13eNH), 6.38 (1H, s, C14eNH), 4.35e4.27 (1H, m,
H-14), 4.12 (1H, m, H-13), 3.09 (1H, m, H-12), 3.00 (1H, m, H-6) 2.82
1
.21e1.38 (32H, m), 1.41 (3H, d, J ¼ 6.0 Hz, H-37), 0.88 (3H, t,
13
J ¼ 7.0 Hz, H-34); C NMR (150 MHz, CDCl
3
): d 174.05 (C-1), 173.61
(
(
1H, dd, J ¼ 12.4, 5.1 Hz, H-15a), 2.57 (1H, d, J ¼ 12.4 Hz, H-15b), 2.19
0
0
(C-1 ), 163.92 (C-10 ), 149.05 (C-35), 134.49 (C-2), 83.57 (C-16),
2H, t, J ¼ 7.4 Hz, H-2), 2.04 (2H, t, J ¼ 7.3 Hz, H-8), 1.20e1.67 (14H,
8
2.48 (C-20), 81.91 (C-19), 80.88 (C-23), 77.56 (C-36), 75.16 (C-24),
13
m). C NMR (150 MHz, DMSO-d
6
):
d
171.84 (C-1, C-7),162.74 (C-16),
0 0 0
4.23 (C-15), 71.63 (C-28), 62.21 (C-7 ), 60.32 (C-8 ), 55.48 (C-6 ),
7
4
3
2
61.06 (C-13), 59.20 (C-14), 55.44 (C-12), 39.68 (C-15), 38.25 (C-6),
0
0
0.70 (C-9 ), 37.76 and 37.20 (C-27, C-29), 34.36 (C-2 ), 33.50 (C-14),
2.03 (C-32), 31.47 (C-25), 29.90, 29.78 (2C), 29.75, 29.67, 29.56,
9.46, 29.33, 29.06, 28.64, 28.52, 28.46, 28.22, 28.07, 27.57 (2C),
3
5.21 (C-8), 29.05 (C-2), 28.92, 28.21, 28.04, 26.04, 25.73, 25.36.
3
.3. Preparation of biotinylsquamocins (4e8)
25.83, 25.33 (2C), 24.85, 22.79 (C-33), 21.67 (C-26), 19.38 (C-37),
þ
14.25 (C-34); HR-ESIMS m/z: 849.5639 [M þ H] (calcd for
Squamocin (1.522 g, 2.45 mmol) was treated with biotin
C H
47 81
N
2
O
9
S, 849.5657).
(
4
673 mg, 2.76 mmol) using dicyclohexylcarbodiimide (DCC, 947 mg,
.6 mmol), 4-dimethylaminopryidine (DMAP, 36 mg, 0.3 mmol) as
catalytic agent in dry N,N-dimethylformamide (DMF, 4 mL) and
CH Cl (16 mL) under N atmosphere at room temperature for 16 h
3.3.3. 28-O-Biotinylsquamocin (6)
2
0
ꢀ1
A yellowish waxy solid; [
a
]
D
þ 32.9 (c 1.4, MeOH); IR cm
2
2
2
(KBr): 3200e3500, 3075, 2925, 2855, 1755, 1697, 1462, 1375, 1319,

224
J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
Table 4
29.69, 29.64 (2C), 29.43, 29.31, 29.13, 28.50, 28.34, 28.32, 28.22,
7.72, 27.55 (2C), 25.56, 25.51, 25.31 (2C), 25.26, 24.98, 24.84, 22.71
Cytotoxic activity (IC50) and selectivity index of biotinylated derivatives of squa-
mocin and bullatacin.
2
(
C-33), 22.24 (C-26), 19.36 (C-37), 14.21 (C-34); HR-ESIMS m/z:
þ
Compound
IC50
(
mM)
Selectivity
index
1075.6410 [M þ H] (calcd for C57
95 4 11 2
H N O S , 1075.6433).
a
L1210
4T1
P815
4T1
P815
3
.3.5. 24,28-Di-O-biotinylsquamocin (8)
2
0
ꢀ1
Squamocin (1) 1.35 ꢃ 0.04
0.36 ꢃ 0.03
19.80 ꢃ 1.18
>20
>20
3.75 0.068
A yellowish waxy solid; [
a
]
þ 35.3 (c 0.2, MeOH); IR cm
D
4
5
6
7
8
1
1
1
1
1
1
0.72 ꢃ 0.02
0.96 ꢃ 0.05
2.91 ꢃ 0.20
0.63 ꢃ 0.04
0.25 0.036
1.52 0.048
16.94 0.62
0.26 0.063
19.62 0.28
14.94 0.48
0.90 0.069
0.10 0.034
2.47 0.90
22.57 1.05
0.63 0.059
21.09 0.67
2.18 0.071
32.62 0.88
3.47 0.29
39.62 2.39
6.10 0.092
29.46 2.66
1.92 0.10
26.73 1.96
(KBr): 3200e3500, 3100, 2927, 2853, 1717, 1660, 1460, 1382, 1323,
1
1269, 1228, 1161, 1117; H NMR (600 MHz, CDCl ): 6.99 (1H, m, H-
3
3
d
0.83 ꢃ 0.02 0.049 ꢃ 0.004 1.34 ꢃ 0.07
0
00
5), 6.53 and 6.18 (each 1H, br s, C7 eNH, C7 eNH), 5.94 and 5.61
1.25 ꢃ 0.25
5.69 ꢃ 0.36
4.87 ꢃ 0.63
0.29 ꢃ 0.03
>20
>20
0
00
(each 1H, br s, C8 eNH, C8 eNH), 5.00 (1H, m, H-36), 4.93 (1H, m,
H-24), 4.84 (1H, m, H-28), 4.50 (2H, s, H-8 , H-8 ), 4.32 (2H, m, H-7 ,
H-7 ), 4.01 (1H, m, H-23), 3.86 (2H, m, H-19, H-20), 3.82 (1H, m, H-
2
3
4
5
6
7
1.15 ꢃ 0.16 0.077 ꢃ 0.002 2.41 ꢃ 0.03
0
00
0
1.38 ꢃ 0.09
0.68 ꢃ 0.03
0.84 ꢃ 0.07
1.54 ꢃ 0.04
6.57 ꢃ 0.45
0.34 ꢃ 0.03
>20
>20
0.93 ꢃ 0.004
0.75 ꢃ 0.02
>20
00
0 00
16), 3.38 (1H, m, H-15), 3.16 (2H, m, H-6 , H-6 ), 2.90 (2H, dd,
0
00
0.79 ꢃ 0.06 0.035 ꢃ 0.01
J ¼ 12.8, 5.0 Hz, H-9 a, H-9 a), 2.74 and 2.73 (each 1H, d, J ¼ 12.8 Hz,
0
00
0
00
1.18 ꢃ 0.18
2.32 ꢃ 0.10
1.42 ꢃ 0.01
1.87 ꢃ 0.18
0.11 ꢃ 0.01
0.65 ꢃ 0.03
H-9 b, H-9 b), 2.32 (4H, m, H-2 , H-2 ), 2.25 (2H, m, H-3), 1.90e2.02
4H, m),1.58e1.78 (13H, m), 1.45e1.57 (12H, m),1.21e1.38 (30H, m),
Bullatacin (2)
9
3.48 ꢃ 0.51
(
>20
13
1
.41 (3H, d, J ¼ 6.0 Hz, H-37), 0.87 (3H, t, J ¼ 6.9 Hz, H-34); C NMR
10
11
18
19
20
21
22
1.37 ꢃ 0.07 0.042 ꢃ 0.01
1.55 ꢃ 0.03
>20
0 00
173.94 (C-1), 173.61 and 173.53 (C-1 , C-1 ),
(150 MHz, CDCl
3
):
d
00
5.79 ꢃ 0.19
1.67 ꢃ 0.31
0
2.10 ꢃ 0.10 0.053 ꢃ 0.001 0.88 ꢃ 0.06
1.83 ꢃ 0.26 0.30 ꢃ 0.06 >20
164.05 (C-10 , C-10 ), 148.93 (C-35), 134.30 (C-2), 83.31 (C-16),
8
2.31 (C-20), 81.74 (C-19), 80.53 (C-23), 77.44 (C-36), 74.77 (C-24),
1.09 ꢃ 0.07 0.037 ꢃ 0.003 0.41 ꢃ 0.02
0 00
4.06 (C-15, C-28), 62.09 and 61.87 (C-7 , C-7 ), 60.25 and 60.13 (C-
7
8
3
3
2
2
2.04 ꢃ 0.03
1.06 ꢃ 0.08
>20
0.75 ꢃ 0.02
0
00
0
00
0
00
, C-8 ), 55.39 and 55.34 (C-6 , C-6 ), 40.62 and 40.56 (C-9 , C-9 ),
1.47 ꢃ 0.12 0.055 ꢃ 0.01
0
00
4.27 (C-27, C-29), 34.09 (C-2 , C-2 ), 33.41 (C-14), 31.74 (C-32),
0.81 (C-25), 29.74, 29.62 (2C), 29.61, 29.51, 29.31, 29.18 (2C), 28.89,
8.41, 28.38, 28.35, 28.09, 28.06, 27.54, 27.40 (2C), 25.71, 25.32,
a
Selectivity index for a specific compound towards 4T1 or P815 cells is defined as
the ratio of its IC50 against L1210 cells to its IC50 against respective cells.
5.17 (2C), 24.98, 24.83, 22.59 (C-33), 21.43 (C-26), 19.23 (C-37),
1
þ
1
5
4
262, 1199, 1118; H NMR (600 MHz, CDCl
3
) :
d
7.00 (1H, m, H-35),
14.09 (C-34); HR-ESIMS m/z: 1075.6401 [M þ H] (calcd for
0
0
.99 (1H, br s, C7 eNH), 5.60 (1H, br s, C8 eNH), 5.00 (1H, m, H-36),
57 95 4 11 2
C H N O S , 1075.6433).
0
0
.88 (1H, m, H-28), 4.51 (1H, m, H-8 ), 4.32 (1H, m, H-7 ), 3.91 (2H,
m, H-19, H-20), 3.82 (3H, m, H-16, H-23, H-24), 3.39 (1H, m, H-15),
3
.4. Preparation of biotinylbullatacins (9e11)
0
0
3
.16 (1H, m, H-6 ), 2.91 (1H, dd, J ¼ 12.8, 5.0 Hz, H-9 a), 2.75 (1H, d,
0
0
J ¼ 12.8 Hz, H-9 b), 2.30 (2H, m, H-2 ), 2.24 (2H, m, H-3), 1.90e2.02
4H, m), 1.78e1.83 (1H, m), 1.63e1.61 (3H, m), 1.45e1.58 (14H, m),
.21e1.35 (32H, m), 1.41 (3H, d, J ¼ 6.0 Hz, H-37), 0.87 (3H, t,
Biotinylated bulltacin derivatives were synthesized using the
(
1
same method as described for biotinylated squamocin derivatives
Scheme 2). Briefly, bullatacin (280 mg, 0.45 mmol), biotin (132 mg,
.54 mmol) and DMAP (6 mg, 0.05 mmol) were dissolved in 4 mL
CH Cl and 0.5 mL DMF. DCC (185 mg, 0.9 mmol) in 1 mL DMF was
(
0
13
J ¼ 6.9 Hz, H-34); C NMR (150 MHz, CDCl
3
):
C-1 ), 163.96 (C-10 ), 149.08 (C-35), 134.47 (C-2), 83.45 (C-16),
3.05 (C-19), 82.66 (C-20), 82.36 (C-23), 77.59 (C-36), 74.30 (C-15,
d 174.09 (C-1), 173.77
0
0
(
8
2
2
slowly added and the mixture was stirred for 16 h. After removal of
the solvent, the resulting residue was separated by CC over silica gel
usingCHCl
fractions (F
using MeOHeH
and a sub-fraction (57 mg) which was further separated by CC over
silica gel (200e300 mesh) using petroleum ethereisopropanol (4:1,
v/v) to afford 9 (32 mg, 8.4%) and 10 (14 mg, 3.7%).
0
0
0
C-28), 71.55 (C-24), 62.08 (C-7 ), 60.34 (C-8 ), 55.56 (C-6 ), 40.69 (C-
9
0
0
), 34.45 (C-27, C-29), 34.34 (C-2 ), 33.44 (C-14), 32.50 (C-25), 31.89
3
eMeOH(98:2, 97:3, 96:4, 95:5, 93:7, 9:1;v/v)toafford ten
eF10). F and F was combined and separated by HPLC
O (83:17, v/v) to afford 11 (10 mg, 2.6%, t
(
C-32), 29.91, 29.77 (2C), 29.75, 29.66, 29.46, 29.33 (2C), 29.08,
1
6
7
2
2
8.58, 28.49, 28.33, 27.56 (2C), 25.82, 25.48, 25.33 (2C), 25.04,
2
R
¼ 151 min)
4.99, 22.58 (C-33), 22.06 (C-26), 19.21 (C-37), 14.08 (C-34); HR-
þ
ESIMS m/z: 849.5640 [M þ H] (calcd for C47
H
81
N
2
O
9
S, 849.5657).
3
.3.4. 15,28-Di-O-biotinylsquamocin (7)
2
0
ꢀ1
A white powder; [
a
]
D
þ 44.6 (c 0.2, MeOH); IR cm (KBr):
3.4.1. 4-O-Biotinylbullatacin (9)
2
0
ꢀ1
1
3
200e3500, 3100, 2927, 2854, 1698, 1463, 1377, 1321, 1266, 1186,
A yellowish powder; [
3100e3500, 2925, 2853, 1730, 1464, 1376, 1319, 1190, 1119; H NMR
a
]
D
þ 17.3 (c 0.2, MeOH); IR cm (KBr):
1
1117; H NMR (600 MHz, CDCl
3
):
d
7.00 (1H, m, H-35), 6.62 and 6.47
0
00
0
0
(
each 1H, br s, C7 eNH, C7 eNH), 6.14 and 5.77 (each 1H, br s, C8 e
(600 MHz, CDCl
3
):
d
7.10 (1H, m, H-35), 5.69 (1H, br s, C7 eNH), 5.39
0
0
0
NH, C8 eNH), 5.00 (1H, m, H-36), 4.88 (1H, m, H-28), 4.83 (1H, m,
H-15), 4.50 (2H, m, H-8 , H-8 ), 4.32 (2H, m, H-7 , H-7 ), 3.95 (1H, m,
H-16), 3.90 (2H, m, H-20, H-23), 3.79 (2H, m, H-19, H-24), 3.16 (2H,
(1H, br s, C8 eNH), 5.11 (1H, m, H-4), 5.01 (1H, m, H-36), 4.51 (1H,
0
00
0
00
0
0
m, H-8 ), 4.32 (1H, m, H-7 ), 3.91e3.96 (2H, m, H-19, H-20), 3.84e
3.88 (3H, m, H-16, H-23, H-24), 3.40 (1H, m, H-15), 3.15 (1H, m, H-
0
00
0
00
0
0
0
m, H-6 , H-6 ), 2.90 (2H, dd, J ¼ 12.7, 4.9 Hz, H-9 a, H-9 a), 2.73 and
6 ), 2.92 (1H, dd, J ¼ 12.8, 5.0 Hz, H-9 a), 2.75 (1H, d, J ¼ 12.8 Hz, H-
0
00
0
00
0
2
2
.72 (each 1H, d, J ¼ 12.7, H-9 b, H-9 b), 2.31 (4H, m, H-2 , H-2 ),
.24 (2H, m, H-3), 1.90e2.02 (4H, m), 1.63e1.81 (9H, m), 1.45e1.62
9 b), 2.57e2.50 (2H, m, H-3), 2.30 (2H, m, H-2 ), 1.87e2.02 (3H, m),
1.77e1.83 (1H, m), 1.69e1.76 (1H, m), 1.54e1.68 (8H, m), 1.42e1.53
(
14H, m), 1.21e1.38 (32H, m), 1.41 (3H, d, J ¼ 6.8 Hz, H-37), 0.87 (3H,
(5H, m), 1.21e1.38 (36H, m), 1.40 (3H, d, J ¼ 6.8 Hz, H-37), 0.87 (3H,
13
13
m, H-34); C NMR (150 MHz, CDCl
1
1
7
6
6
2
3
):
73.64 (C-1 , C-1 ), 164.66 and 164.33 (C-10 , C-10 ), 149.07 (C-35),
34.43 (C-2), 83.39 (C-20), 82.34 (C-23), 81.79 (C-19), 80.57 (C-16),
7.56 (C-36), 74.82 (C-15), 74.24 (C-28), 71.63 (C-24), 62.14 and
d
174.04 (C-1), 173.80 and
t, J ¼ 7.0 Hz, H-34); C NMR (150 MHz, CDCl
3
): d 173.58 (C-1),
0
00
0
00
0
0
173.24 (C-1 ), 163.56 (C-10 ), 151.10 (C-35), 130.21 (C-2), 83.24 (C-
16), 82.88 (C-19), 82.55 (C-20), 82.31 (C-23), 77.64 (C-36), 74.11 (C-
0 0 0
15), 72.02 (C-4), 71.39 (C-24), 62.01 (C-7 ), 60.19 (C-8 ), 55.42 (C-6 ),
0
00
0
00
0
0
1.63 (C-7 , C-7 ), 60.47 and 60.29 (C-8 , C-8 ), 55.62 and 55.29 (C-
40.54 (C-9 ), 34.14 (C-5, C-2 ), 33.41 (C-14), 32.53 (C-25), 31.92 (C-
32), 29.90 (C-3), 29.72, 29.70, 29.63 (2C), 29.58, 29.54, 29.54, 29.48,
29.44, 29.34, 29.32, 28.96, 28.92, 28.45, 28.41, 28.34, 26.09, 25.66,
0
0
00
0
00
, C-6 ), 40.74 (C-9 , C-9 ), 34.97 and 34.53 (C-27, C-29), 34.37 (C-
, C-2 ), 32.86 (C-25), 31.87 (C-32), 31.08 (C-14), 29.75, 29.72 (2C),
00

J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
225
2
5.27, 24.84, 24.55, 22.69 (C-33), 19.02 (C-37), 14.12 (C-34); HR-
3.5.1. 15-O-(6-Biotinylamidohexanoyl)squamocin (12)
þ
20
ꢀ1
ESIM m/z: 849.5650 [M þ H] (calcd for C47
H
81
N
2
O
9
S, 849.5657).
A yellowish waxy solid; [
a
]
D
þ 25.1 (c 0.3, MeOH); IR cm
(
KBr): 3200e3500, 3100, 2928, 2856, 1730, 1650, 1555, 1474, 1462,
1
3.4.2. 15-O-Biotinylbullatacin (10)
1320, 1192; H NMR (600 MHz, CDCl
3
):
d
7.01 (1H, m, H-35), 6.58
2
0
ꢀ1
1
0
0
A yellowish powder; [
a
]
D
þ 13.9 (c 0.2, MeOH); IR cm (KBr):
(1H, br s, C6 eNH), 6.54 (1H, t, J ¼ 5.5 Hz, C13 eNH), 5.84 (1H, br s,
0
3100e3500, 2927, 2855, 1732, 1462, 1379, 1320, 1191; H NMR
C14 eNH), 5.01 (1H, m, H-36), 4.87 (1H, m, H-15), 4.52 (1H, m, H-
0
0
0
(
600 MHz, CDCl
3
):
d
7.20 (1H, br s, H-35), 6.14 (1H, br s, C7 eNH),
14 ), 4.32 (1H, m, H-13 ), 4.00 (1H, m, H-16), 3.90 (2H, m, H-20, H-
23), 3.84 (1H, m, H-24), 3.80 (1H, m, H-19), 3.58 (1H, m, H-28), 3.22
0
5
.06 (1H, m, H-36), 5.00 (1H, br s, C8 eNH), 4.85 (1H, m, H-15), 4.50
0
0
0
0
(
1H, m, H-8 ), 4.32 (1H, m, H-7 ), 3.95 (3H, m, H-16, H-20, H-23),
(2H, m, H-6 ), 3.15 (1H, m, H-12 ), 2.90 (1H, dd, J ¼ 12.8, 4.7 Hz, H-
0
0
0
0
0
3
.80e3.86 (3H, m, H-4, H-19, H-24), 3.20 (1H, m, H-6 ), 2.93 (1H, m,
15 a), 2.75 (1H, d, J ¼ 12.8 Hz, H-15 b), 2.32 (2H, m, H-8 ), 2.24 (2H,
0
0
H-9 a), 2.72 (1H, m, H-9 b), 2.52 (1H, m, H-3a), 2.40 (1H, m, H-3b),
2
m, H-3), 2.19 (2H, t, J ¼ 7.4 Hz, H-2 ), 1.92e2.02 (3H, m), 1.80e1.88
0
.30 (2H, m, H-2 ), 1.92e2.02 (4H, m), 1.67e1.80 (5H, m), 1.60e1.66
(2H, m), 1.71e1.78 (1H, m), 1.58e1.70 (9H, m), 1.47e1.57 (8H, m),
(
1
2H, m), 1.54e1.60 (3H, m), 1.42e1.53 (4H, m), 1.21e1.38 (36H, m),
1.35e1.46 (9H, m), 1.21e1.39 (27H, m), 1.41 (3H, d, J ¼ 6.0 Hz, H-37),
13
13
.43 (3H, d, J ¼ 6.7 Hz, H-37), 0.88 (3H, t, J ¼ 6.7 Hz, H-34); C NMR
0.88 (3H, t, J ¼ 6.9 Hz, H-34); C NMR (150 MHz, CDCl
3
): d 173.90
0
0
0
0
0
(
150 MHz, CDCl
3
):
d
174.62 (C-1), 173.42 (C-1 ), 163.71 (C-10 ),
(C-1), 173.51 (C-7 ), 173.26 (C-1 ), 164.02 (C-16 ), 148.91 (C-35),
134.18 (C-2), 82.57 (C-23), 82.02 (C-20), 81.72 (C-19), 80.05 (C-16),
1
8
6
3
2
2
2
8
51.81 (C-35), 131.22 (C-2), 83.26 (C-23), 82.22 (C-20), 81.59 (C-19),
0
0.40 (C-16), 77.98 (C-36), 74.46 (C-15), 71.43 (C-24), 69.94 (C-4),
77.38 (C-36), 75.04 (C-15), 71.55 (C-28), 71.51 (C-24), 61.77 (C-13 ),
0
0
0
0
0
0 0 0 0
1.39 (C-7 ), 60.16 (C-8 ), 54.76 (C-6 ), 40.67 (C-9 ), 37.45 (C-5),
60.18 (C-14 ), 55.62 (C-12 ), 40.46 (C-15 ), 39.12 (C-6 ), 37.42 and
0
0
4.91 (C-2 ), 33.38 (C-3), 32.89 (C-25), 31.93 (C-32), 30.90 (C-14),
37.26 (C-27, C-29), 35.88 (C-2 ), 34.29 (C-8 ), 32.60 (C-25), 31.79 (C-
32), 30.65 (C-14), 29.49 (2C), 29.47, 29.42 (2C), 29.33, 29.21, 29.12,
29.09, 28.65, 28.28, 28.17, 28.01 (2C), 27.32 (2C), 26.28, 25.63 (2C),
25.37, 25.08, 24.92, 24.60, 22.55 (C-33), 22.00 (C-26), 19.14 (C-37),
9.78, 29.67, 29.65, 29.61, 29.53 (2C), 29.46, 29.45, 29.42, 29.40,
9.36, 29.07, 28.17, 28.08 (2C), 27.34, 26.21, 25.61, 25.44, 25.16,
4.26, 22.70 (C-33), 19.12 (C-37), 14.12 (C-34); HR-ESIMS m/z:
þ
þ
49.5625 [M þ H] (calcd for C47
81
H N
2
O
9
S, 849.5657).
14.03 (C-34); HR-ESIMS m/z: 962.6484 [M þ H] (calcd for
53 91 3
C H N O10S, 962.6498).
3
.4.3. 24-O-Biotinylbullatacin (11)
2
0
ꢀ1
A yellowish waxy solid; [
a]
D
þ 24.0 (c 0.4, MeOH); IR cm
3.5.2. 24-O-(6-Biotinylamidohexanoyl)squamocin (13)
2
0
ꢀ1
(
KBr): 3200e3500, 3100, 2923, 2853, 1703, 1599, 1463, 1356, 1260,
A yellowish waxy solid; [
(KBr): 3200e3500, 3084, 2926, 2855, 1753, 1727, 1702, 1640, 1546,
1461, 1375, 1320, 1266, 1246, 1183; H NMR (600 MHz, CDCl
a
]
D
þ 30.1 (c 0.1, MeOH); IR cm
1
1186, 1118; H NMR (600 MHz, CDCl ): 7.19 (1H, m, H-35), 5.66
3
d
0
0
1
(
(
1H, br s, C7 eNH), 5.19 (1H, br s, C8 eNH), 5.06 (1H, m, H-36), 4.99
1H, m, H-24), 4.51 (1H, m, H-8 ), 4.31 (1H, m, H-7 ), 3.93 (1H, m, H-
3
):
0
0
0
d
6.99 (1H, m, H-35), 6.47 (1H, br s, C6 eNH), 6.42 (1H, t, J ¼ 5.5 Hz,
0
0
2
3
3), 3.81e3.87 (4H, m, H-4, H-16, H-19, H-20), 3.38 (1H, m, H-15),
C13 eNH), 5.60 (1H, br s, C14 eNH), 4.99 (1H, m, H-36), 4.94 (1H, m,
0
0
0
0
.19 (1H, m, H-6 ), 2.92 (1H, dd, J ¼ 12.8, 5.0 Hz, H-9 a), 2.75 (1H, d,
H-24), 4.51 (1H, m, H-14 ), 4.32 (1H, m, H-13 ), 4.01 (1H, m, H-23),
3.85 (2H, m, H-19, H-20), 3.80 (1H, m, H-16), 3.55 (1H, m, H-28),
0
J ¼ 12.8 Hz, H-9 b), 2.52 (1H, m, H-3a), 2.40 (1H, m, H-3b), 2.31 (2H,
0
0 0
3.38 (1H, m, H-15), 3.21 (2H, m, H-6 ), 3.15 (1H, m, H-12 ), 2.91 (1H,
m, H-2 ), 1.92e2.02 (4H, m), 1.42e1.80 (14H, m), 1.21e1.38 (36H, m),
13
0
0
0
1
.44 (3H, d, J ¼ 6.8 Hz, H-37), 0.88 (3H, t, J ¼ 7.0 Hz, H-34); C NMR
dd, J ¼ 12.8, 4.8 Hz, H-15 a), 2.74 (1H, d, J ¼ 12.8 Hz, H-15 b), 2.31
0
0
0
(
150 MHz, CDCl
51.75 (C-35), 131.19 (C-2), 83.17 (C-16), 82.82 (C-20), 81.82 (C-19),
0.54 (C-23), 77.93 (C-36), 74.97 (C-24), 74.02 (C-15), 69.91 (C-4),
3
):
d
174.59 (C-1), 173.46 (C-1 ), 163.38 (C-10 ),
(2H, m, H-8 ), 2.24 (2H, m, H-3), 2.19 (2H, t, J ¼ 7.4 Hz, H-2 ), 1.92e
1
8
6
3
2
2
2
8
2.02 (4H, m), 1.70e1.78 (1H, m), 1.59e1.70 (8H, m), 1.47e1.57 (9H,
m), 1.42e1.45 (5H, m), 1.21e1.39 (32H, m), 1.41 (3H, d, J ¼ 6.0 Hz, H-
0
0
0
0
13
2.03 (C-7 ), 60.14 (C-8 ), 55.01 (C-6 ), 40.55 (C-9 ), 37.38 (C-5),
37), 0.88 (3H, t, J ¼ 6.8 Hz, H-34); C NMR (150 MHz, CDCl
3
):
0
0
0
0
4.17 (C-2 ), 33.42 and 33.31 (C-14, C-3), 31.88 (C-32), 30.95 (C-25),
d 173.95 (C-1), 173.44 (C-7 ), 173.38 (C-1 ), 163.84 (C-16 ), 148.95 (C-
9.68, 29.62, 29.57, 29.53, 29.49, 29.47, 29.43, 29.30 (2C), 28.91,
8.84, 28.41, 28.40, 28.21, 27.85, 27.20, 26.04, 25.63, 25.54, 25.49,
35), 134.25 (C-2), 83.40 (C-16), 82.31 (C-20), 81.73 (C-19), 80.72 (C-
23), 77.44 (C-36), 74.93 (C-24), 74.16 (C-15), 71.40 (C-28), 61.79 (C-
0 0 0 0 0
13 ), 60.21 (C-14 ), 55.65 (C-12 ), 40.55 (C-15 ), 39.18 (C-6 ), 37.64
4.45, 22.65 (C-33), 19.08 (C-37), 14.10 (C-34); HR-ESIMS m/z:
þ
0
0
49.5646 [M þ H] (calcd for C47
H
81
N
2
O
9
S; 849.5657).
and 37.04 (C-27, C-29), 35.94 (C-2 ), 34.37 (C-8 ), 33.21 (C-14), 31.86
C-32), 31.24 (C-25), 29.73, 29.60 (2C), 29.58, 29.49, 29.39, 29.29,
29.16 (2C), 28.90, 28.45, 28.35, 28.14, 27.99, 27.84, 27.37 (2C), 26.33,
(
3.5. Preparation of (6-biotinylamidohexanoyl)squamocins (12e17)
2
5.69, 25.63, 25.15 (2C), 24.64, 22.62 (C-33), 21.65 (C-26), 19.20 (C-
þ
Squamocin (528 mg, 0.85 mmol), 3 (321 mg, 0.9 mmol) and
37), 14.11 (C-34); HR-ESIMS m/z: 962.6487 [M þ H] (calcd for
DMAP (18 mg, 0.15 mmol) were dissolved in 7 mL dry DMF. DCC
346 mg, 1.3 mmol) in 2 mL dry DMF was slowly added and the
mixture was stirred under N atmosphere at room temperature for
6 h (Scheme 3). The resulting precipitate (DCU) was removed by
filtration and the filtrate was concentrated under reduced pressure.
The residue was dissolved in EtOAc and sequentially washed with
C
53
H
91
N
3
O
10S, 962.6498).
(
2
3.5.3. 28-O-(6-Biotinylamidohexanoyl)squamocin (14)
2
0
ꢀ1
1
A yellowish waxy solid; [
a
]
D
þ 24.9 (c 0.2, MeOH); IR cm
(KBr): 3200e3500, 3088, 2926, 2855, 1755, 1729, 1703, 1643, 1551,
1
1461, 1374, 1320, 1265, 1199; H NMR (600 MHz, CDCl
3
):
d
6.99 (1H,
0
0
0
.5% aqueous HCl solution, saturated aqueous NaHCO
saturated aqueous NaCl. The organic layer was dried over anhydrous
Na SO , filtered, and concentrated under reduced pressure. The
resulting mixture was separated by CC over silica gel using CHCl
MeOH (97:3, 95:5, 93:7, 9:1; v/v) to yield eleven fractions (F eF11).
eF was combined and further separated by HPLC using MeOHe
O (80:20, v/v) to afford 12 (59.8 mg, 7.3%, t
¼ 66 min),13 (71.6 mg,
.8%, t ¼ 95 min). F10eF11 was
¼ 82 min) and 14 (51.1 mg, 6.3%, t
combined and separated by HPLC using MeOHeH O (80:20, v/v) to
afford 15 (26 mg, 2.5%, t ¼ 138 min)
¼ 121 min), 16 (24.7 mg, 2.2%, t
and 17 (18 mg, 1.6%, t
¼ 151 min).
3
solution and
m, H-35), 6.43 (1H, br s, C6 eNH), 6.37 (1H, t, J ¼ 5.5 Hz, C13 eNH),
0
5.68 (1H, br s, C14 eNH), 5.00 (1H, m, H-36), 4.87 (1H, m, H-28),
0
0
2
4
4.51 (1H, m, H-14 ), 4.32 (1H, m, H-13 ), 3.91 (2H, m, H-19, H-20),
3.82 (3H, m, H-16, H-23, H-24), 3.39 (1H, m, H-15), 3.22 (2H, m, H-
3
e
0
0
0
1
6 ), 3.15 (1H, m, H-12 ), 2.91 (1H, dd, J ¼ 12.8, 4.9 Hz, H-15 a), 2.74
0
0
F
4
8
(1H, d, J ¼ 12.8 Hz, H-15 b), 2.30 (2H, m, H-8 ), 2.24 (2H, m, H-3),
0
H
8
2
R
2.19 (2H, t, J ¼ 7.4 Hz, H-2 ), 1.92e2.02 (3H, m), 1.84e1.91 (1H, m),
R
R
1.77e1.83 (1H, m), 1.69e1.76 (1H, m), 1.58e1.70 (8H, m), 1.46e1.57
(10H, m), 1.42e1.46 (3H, m), 1.21e1.38 (32H, m), 1.41 (3H, d,
2
13
R
R
J ¼ 6.0 Hz, H-37), 0.87 (3H, t, J ¼ 6.8 Hz, H-34); C NMR (150 MHz,
0 0 0
3
CDCl ): d 174,0 (C-1), 173.62 (C-7 ), 173.28 (C-1 ), 164.11 (C-16 ),
R

226
J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
1
48.95 (C-35), 134.33 (C-2), 83.34 (C-16), 82.90 (C-19), 82.53 (C-20),
2.24 (C-23), 77.45 (C-36), 74.16 (C-28), 74.03 (C-15), 71.42 (C-24),
3.5.6. 24,28-Di-O-(6-biotinylamidohexanoyl)squamocin (17)
2
0
ꢀ1
8
A yellowish waxy solid; [
a
]
D
þ 14.4 (c 0.4, MeOH); IR cm
0 0 0 0 0
1.83 (C-13 ), 60.26 (C-14 ), 55.68 (C-12 ), 40.57 (C-15 ), 39.25 (C-6 ),
6
3
3
2
(KBr): 3200e3500, 3100, 2929, 2857, 1725, 1649, 1551, 1460, 1308,
0
0
1
5.96 (C-2 ), 34.49, 34.27 and 34.21 (C-27, C-29, C-8 ), 33.26 (C-14),
2.47 (C-25), 31.73 (C-32), 29.76, 29.62 (2C), 29.60, 29.51, 29.31,
9.21, 29.18 (2C), 28.94, 28.41, 28.17, 28.08, 27.40 (2C), 26.43, 25.68
1315, 1194, 1117; H NMR (600 MHz, CDCl ): 7.01 (1H, s, H-35),
3
d
0
00
6.96 and 6.93 (each 1H, br s, C6 eNH, C6 eNH), 6.91 and 6.78 (each
0
00
0
00
1H, m, C13 eNH, C13 eNH), 5.90 (2H, m, C14 eNH, C14 eNH), 5.01
(
1
(
2C), 25.32, 25.17 (2C), 24.81, 24.73, 22.58 (C-33), 22.07 (C-26),
(1H, m, H-36), 4.94 (1H, m, H-24), 4.83 (1H, m, H-28), 4.52 (2H, m,
þ
0
00
0
00
9.23 (C-37), 14.08 (C-34); HR-ESIMS m/z: 962.6487 [M þ H]
H-14 , H-14 ), 4.32 (2H, m, H-13 , H-13 ), 4.00 (1H, m, H-23), 3.85
(2H, m, H-19, H-20), 3.80 (1H, m, H-16), 3.38 (1H, m, H-15), 3.22
calcd for C53 10S, 962.6498).
91 3
H N O
0 00 0 00 0
4H, m, H-6 , H-6 ), 3.15 (2H, m, H-12 , H-12 ), 2.90 (2H, m, H-15 a,
(
0
0
0
00
0
00
H-15 a), 2.75 (2H, m, H-15 b, H-15 b), 2.31 (4H, m, H-8 , H-8 ), 2.26
3
.5.4. 15,24-Di-O-(6-biotinylamidohexanoyl)squamocin (15)
0
00
(
2H, m, H-3), 2.21 (4H, m, H-2 , H-2 ), 1.90e2.01 (3H, m), 1.79e1.85
1H, m), 1.58e1.78 (15H, m), 1.46e1.57 (13H, m), 1.40e1.46 (5H, m),
2
0
A yellowish waxy solid; [
a
]
D
þ 33.6 (c 0.3, MeOH); IR (KBr,
(
ꢀ
1
cm ): 3200e3500, 3083, 2923, 2854, 1732, 1707, 1645, 1553, 1463,
1
m, H-35), 6.97 and 6.87 (each 1H, br s, C6 eNH, C6 eNH), 6.93 and
6
1
.21e1.38 (34H, m), 1.41 (3H, d, J ¼ 6.8 Hz, H-37), 0.87 (3H, m, H-
1
374, 1321, 1265, 1193, 1164; H NMR (600 MHz, CDCl
3
):
d 7.01 (1H,
13
3
34); C NMR (150 MHz, CDCl ): d 173.94 (C-1), 173.68 and 173.59
0
00
0
00
0
00
0
00
(C-7 , C-7 ), 173.45 (C-1 , C-1 ), 164.33 (C-16 , C-16 ), 148.91 (C-35),
0
00
.77 (each 1H, t, J ¼ 5.5 Hz, C13 eNH, C13 eNH), 6.09 and 5.92
1
34.34 (C-2), 83.04 (C-16), 82.37 (C-20), 81.73 (C-19), 80.53 (C-23),
0 00
(
each 1H, br s, C14 eNH, C14 eNH), 5.01 (1H, m, H-36), 4.95 (1H, m,
0
7.44 (C-36), 74.71 (C-24), 74.18 (C-15), 73.88 (C-28), 61.91 (C-13 ,
7
1
0
00
H-24), 4.84 (1H, m, H-15), 4.53 (2H, m, H-14 , H-14 ), 4.32 (2H, m,
H-13 , H-13 ), 3.94e4.01 (2H, m, H-16, H-23), 3.83 (2H, m, H-19, H-
2
H-12 ), 2.90 (2H, m, H-15 a, H-15 a), 2.74 (2H, m, H-15 b, H-15 b),
2
1
1
00 0 00 0 00 0
3 ), 60.24 (C-14 , 14 ), 55.88 and 55.82 (C-12 , C-12 ), 40.65 (C-15 ,
0
00
0
0
0
00
0
00
C-15 ), 39.29 (C-6 , C-6 ), 35.93 (C-2 , C-2 ), 34.42 and 34.36 (C-27,
C-29), 34.00 (C-8 , C-8 ), 33.33 (C-14), 31.73 (C-25, C-32), 29.75,
0
0
0
0
0), 3.53 (1H, m, H-28), 3.20 (4H, m, H-6 , H-6 ), 3.15 (2H, m, H-12 ,
0
00
0
0
0
00
0
00
2
2
2
2
9.63, 29.61 (2C), 29.52, 29.32, 29.32, 29.19 (2C), 29.16, 28.90, 28.47,
8.36, 28.26, 28.11, 28.08, 27.42 (2C), 26.51, 26.45, 25.80, 25.73,
5.67, 25.33, 25.19 (2C), 24.79, 24.76, 23.94, 22.58 (C-33), 21.63 (C-
6), 19.23 (C-37), 14.08 (C-34); HR-ESIMS m/z: 1301.8089 [M þ H]
0
00
0
00
.30 (4H, m, H-8 , H-8 ), 2.24 (2H, m, H-3), 2.20 (4H, m, H-2 , H-2 ),
.90e2.01 (4H, m), 1.58e1.78 (18H, m), 1.46e1.57 (13H, m), 1.40e
.46 (4H, m), 1.21e1.38 (32H, m), 1.41 (3H, d, J ¼ 6.8 Hz, H-37), 0.88
þ
1
3
(
7
3H, m, H-34); C NMR (150 MHz, CDCl
3
):
d
0
173.96 (C-1), 173.65 (C-
117 6 13 2
(calcd for C69H N O S , 1301.8115).
0
00
0
00
00
, C-7 ), 173.47 (C-1 , C-1 ), 164.32 (C-16 , C-16 ), 148.98 (C-35),
134.29 (C-2), 81.96 (C-20), 81.28 (C-19), 80.42 (C-23), 80.33 (C-16),
7
7.46 (C-36), 75.13 (C-15), 74.82 (C-24), 71.51 (C-28), 61.85 and
3.6. Preparation of (6-biotinylamidohexanoyl)bullatacins (18e22)
To a solution of bullatacin (292 mg, 0.47 mmol), 3 (251 mg,
0.7 mmol), DMAP (11 mg, 0.09 mmol) in 4 mL CH Cl and 1 mL N-
2 2
dimethylformamide was added DCC (206 mg, 1 mmol) and stirred
for 16 h (Scheme 3). The crude products were separated by CC over
0 00 0 00
1.84 (C-13 , C-13 ), 60.31 and 60.25 (C-14 , C-14 ), 55.89 and 55.79
6
(
0 00 0 00 0 00
C-12 , C-12 ), 40.60 (C-15 , C-15 ), 39.24 and 39.21 (C-6 , C-6 ),
0
00
3
3
2
2
2
7.67 and 37.29 (C-27, C-29), 36.00 and 35.92 (C-2 , C-2 ), 34.44 and
0
00
4.40 (C-8 , C-8 ), 31.89 (C-32), 31.06 and 30.71 (C-25, C-14), 29.62,
9.59, 29.56, 29.51 (2C), 29.42, 29.31, 29.19, 29.12, 29.09, 28.40,
8.33, 28.25, 28.19, 28.15, 28.04, 27.42 (2C), 27.28, 26.38, 26.29,
5.90, 25.80, 25.73, 25.38, 25.18 (2C), 24.74, 24.72, 22.65 (C-33),
silica gel using CHCl
fractions. F eF was combined and separated by HPLC, elution with
MeOHeH O (80:20, v/v) to afford 18 (33 mg, 7.3%, t
(30 mg, 6.6%, t
¼ 125 min) and 20 (50 mg, 11.1%, t
and F
C18 column using MeOHeH
min to afford 21 (7 mg, 1.1%, t
¼ 52 min).
3
eMeOH (97:3, 95:5, 92:8; v/v) to afford 9
4
7
2
[
1.78 (C-26), 19.23 (C-37), 14.13 (C-34); HR-ESIMS m/z: 1301.8085
2
R
¼ 134 min), 19
M þ H]þ (calcd for C69
O
13
S
2
, 1301.8115).
¼ 146 min). F
117
H N
6
R
R
8
Ò
9
was combined and separated by HPLC with Nova-pak HR
2
O (80:20, v/v) at a flow rate of 5 mL/
¼ 42min), 22 (10 mg, 1.6%,
3.5.5. 15,28-Di-O-(6-biotinylamidohexanoyl)squamocin (16)
2
0
ꢀ1
R
A yellowish waxy solid; [
a]
D
þ 48.0 (c 0.2, MeOH); IR cm
t
R
(
KBr): 3200e3500, 3090, 2929, 2856, 1730, 1703, 1643, 1554, 1462,
1
1376, 1322, 1266, 1166; H NMR (600 MHz, CDCl
3
): d 7.01 (1H, m, H-
0
00
3
5), 6.91 and 6.88 (each 1H, br s, C6 eNH, C6 eNH), 6.77 and 6.72
3.6.1. 4-O-(6-Biotinylamidohexanoyl)bullatacin (18)
0
00
20
ꢀ1
(
each 1H, t, J ¼ 5.5 Hz, C13 eNH, C13 eNH), 6.01 and 5.94 (each 1H,
A yellowish waxy solid; [
a
]
D
þ 57.1 (c 0.1, MeOH); IR cm
0
00
m, C14 eNH, C14 eNH), 5.01 (1H, m, H-36), 4.83e4.89 (2H, m, H-
(KBr): 3200e3500, 3100, 2923, 2854, 1730, 1643, 1547, 1462, 1376,
0
00
0
00
1
15, H-28), 4.53 (2H, s, H-14 , H-14 ), 4.32 (2H, m, H-13 , H-13 ), 3.99
1320, 1189; H NMR (600 MHz, CDCl
3
):
d
7.11 (1H, m, H-35), 6.49
0
0
(
1H, m, H-16), 3.88 (1H, m, H-23), 3.84 (2H, m, H-19, H-24), 3.74
(1H, br s, C6 eNH), 6.40 (1H, t, J ¼ 5.5 Hz, C13 eNH), 5.72 (1H, br s,
0
00
0
00
0
0
(
1H, m, H-20), 3.22 (4H, m, H-6 , H-6 ), 3.15 (2H, m, H-12 , H-12 ),
C14 eNH), 5.10 (1H, m, H-4), 5.02 (1H, m, H-36), 4.52 (1H, m, H-14 ),
0
00
0
00
0
2
.90 (2H, m, H-15 a, H-15 a), 2.74 (2H, m, H-15 b, H-15 b), 2.33 (4H,
4.32 (1H, m, H-13 ), 3.93 (2H, m, H-19, H-20), 3.85 (3H, m, H-16, H-
0
00
0
00
0
0
m, H-8 , H-8 ), 2.25 (2H, m, H-3), 2.20 (4H, m, H-2 , H-2 ),1.90e2.01
3H, m), 1.79e1.86 (1H, m), 1.58e1.78 (15H, m), 1.46e1.57 (13H, m),
23, H-24), 3.40 (1H, m, H-15), 3.21 (2H, m, H-6 ), 3.15 (1H, m, H-12 ),
0
(
2.91 (1H, dd, J ¼ 12.8, 4.8 Hz, H-15 a), 2.74 (1H, d, J ¼ 12.8 Hz, H-
0
0
0
1
0
.40e1.46 (5H, m), 1.21e1.38 (34H, m), 1.41 (3H, d, J ¼ 6.8 Hz, H-37),
15 b), 2.61e2.47 (2H, m, H-3), 2.26 (2H, m, H-8 ), 2.20 (2H, m, H-2 ),
1.87e2.01 (4H, m), 1.46e1.84 (13H, m), 1.42e1.83 (10H, m), 1.21e
1.38 (32H, m),1.40 (3H, d, J ¼ 6.8 Hz, H-37), 0.88 (3H, t, J ¼ 6.8 Hz, H-
.88 (3H, m, H-34); 13C NMR (150 MHz, CDCl
):
d
173.96 (C-1),
3
00
0
00
0
0
173.70 and 173.58 (C-7 , C-7 ), 173.42 (C-1 , C-1 ), 164.35 (C-16 , C-
0
0
13
0
16 ), 149.00 (C-35), 134.28 (C-2), 82.79 (C-23), 82.12 (C-20), 81.84
34); C NMR (150 MHz, CDCl ): d 173.69 (C-1), 173.51 (C-1 ), 173.34
3
0
0
(
(
C-19), 80.20 (C-16), 77.47 (C-36), 75.17 (C-15), 74.07 (C-28), 71.64
C-24), 61.86 (C-13 , C-13 ), 60.30 (C-14 , C-14 ), 55.81 (C-12 , C-
(C-7 ), 163.98 (C-16 ), 151.33 (C-35), 130.10 (C-2), 83.28 (C-16), 82.84
(C-19), 82.57 (C-20), 82.32 (C-23), 77.69 (C-36), 74.15 (C-15), 71.88
0
00
0
00
0
0
0
0
00
0
00
0
0 0 0
(C-4), 71.30 (C-24), 61.90 (C-13 ), 60.37 (C-14 ), 55.60 (C-12 ), 40.51
12 ), 40.64 (C-15 , C-15 ), 39.26 and 39.21 (C-6 , C-6 ), 35.95 (C-2 ,
0
0
0
00
0
0
0
0
C-2 ), 34.51 and 34.30 (C-27, C-29), 34.36 (C-8 , C-8 ), 32.62 (C-25),
1.74 (C-32), 30.77 (C-14), 29.59 (2C), 29.57, 29.51 (3C), 29.30, 29.18
3C), 28.75, 28.38, 28.26, 28.22, 28.17, 28.07, 27.42 (2C), 26.44,
6.39, 25.76 (2C), 25.41, 25.36, 25.18 (2C), 25.12, 24.76, 24.71, 22.58
(C-19 ), 39.29 (C-6 ), 35.76 (C-2 ), 34.24 (C-8 ), 34.12 (C-5), 33.27 (C-
14), 32.46 (C-25), 31.91 (C-32), 29.90 (C-3), 29.72, 29.62 (2C), 29.58,
29.55, 29.47, 29.44, 29.33, 29.31, 29.11, 29.07, 28.97, 28.94, 28.41,
28.17, 28.11, 28.04, 26.38, 26.09, 25.73, 25.67, 25.26, 24.49 (2C),
22.69 (C-33), 19.00 (C-37), 14.14 (C-34); HR-ESIMS m/z: 962.6490
3
(
2
and 22.12 (C-33, C-26), 19.23 (C-37), 14.09 (C-34); HR-ESIMS m/z:
þ
[M þ H]þ (calcd for C53
1301.8083 [M þ H] (calcd for C69
H
117
N
6
O
13
2
S , 1301.8115).
91 3
H N O10S; 962.6498).

J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
00
227
0
0
0
0
00
0
00
3
.6.2. 15-O-(6-Biotinylamidohexanoyl)bullatacin (19)
12 ), 40.58 (C-15 , C-15 ), 39.21 (C-6 , C-6 ), 35.97 (C-2 , C-2 ),
2
0
ꢀ1
0
00
A yellowish waxy solid; [
a
]
D
þ 17.3 (c 0.2, MeOH); IR cm
34.35 (C-8 , C-8 ), 34.12 (C-5), 32.57 (C-25), 31.91 (C-32), 30.72 (C-
14), 29.99 (C-3), 29.75, 29.63 (2C), 29.60 (2C), 29.56, 29.53, 29.47,
29.44, 29.36 (2C), 29.24, 29.14, 29.09, 28.80, 28.37, 28.25, 28.06,
28.00, 26.39 (2C), 26.13, 25.79, 25.64, 25.59, 25.44, 25.23, 25.18,
24.66, 24.54, 22.69 (C-33), 18.99 (C-37), 14.15 (C-34); HR-ESIMS m/
(
KBr): 3200e3500, 3100, 2926, 2855, 1731, 1651, 1554, 1462, 1373,
1
1320, 1192; H NMR (600 MHz, CDCl
3
):
d
7.22 (1H, m, H-35), 6.40
0
0
(
1H, br s, C6 eNH), 6.39 (1H, t, J ¼ 5.5 Hz, C13 eNH), 5.68 (1H, br s,
0
C14 eNH), 5.06 (1H, m, H-36), 4.88 (1H, m, H-15), 4.52 (1H, m, H-
0
0
z: 1301.8079 [M þ H]þ (calcd for C69
1
4 ), 4.32 (1H, m, H-13 ), 4.03 (1H, m, H-16), 3.82e3.93 (5H, m, H-4,
117 6 13 2
H N O S , 1301.8115).
0
0
H-19, H-20, H-23, H-24), 3.22 (2H, m, H-6 ), 3.15 (1H, m, H-12 ), 2.91
0
0
(
2
1H, dd, J ¼ 12.8, 4.8 Hz, H-15 a), 2.72 (1H, d, J ¼ 12.8 Hz, H-15 b),
3
.6.5. 4,24-Di-O-(6-biotinylamidohexanoyl)bullatacin (22)
A yellowish waxy solid; [
KBr): 3200e3500, 3100, 2927, 2856, 1728, 1648, 1552, 1461, 1383,
320, 1259, 1198, 1166; H NMR (600 MHz, CDCl
5), 6.72 (1H, br s, C6 eNH), 6.70 (2H, m, C13 eNH, C6 eNH ), 6.56
1H, m, C13 eNH), 5.86 (2H, m, C14 eNH, C14 eNH), 5.11 (1H, m,
0
.51 (1H, m, H-3a), 2.41 (1H, m, H-3b), 2.33 (2H, m, H-8 ), 2.20 (2H,
20
ꢀ1
a
]
D
þ 28.3 (c 0.4, MeOH); IR cm
0
m, H-2 ), 1.87e2.01 (3H, m), 1.79e1.89 (2H, m), 1.60e1.74 (12H, m),
(
1
3
(
1
3
d
.42e1.83 (10H, m), 1.21e1.38 (32H, m), 1.43 (3H, d, J ¼ 6.7 Hz, H-
1
3
): 7.11 (1H, m, H-
d
13
7), 0.88 (3H, t, J ¼ 6.8 Hz, H-34); C NMR (150 MHz, CDCl
3
):
0
0
00
0
0
0
0
174.70 (C-1), 173.57 (C-7 ), 173.43 (C-1 ), 164.00 (C-16 ), 151.91 (C-
5), 131.07 (C-2), 82.68 (C-23), 82.18 (C-20), 81.94 (C-19), 80.03 (C-
00
0
00
3
1
0
H-4), 5.01 (1H, m, H-36), 4.99 (1H, m, H-24), 4.52 (2H, m, H-14 , H-
6), 78.00 (C-36), 75.01 (C-15), 71.42 (C-24), 69.79 (C-4), 61.86 (C-
00 0 00
1
4 ), 4.32 (2H, m, H-13 , H-13 ), 4.03 (1H, m, H-23), 3.83 (3H, m, H-
0
0
0
0
0
1
5
3
2
2
3 ), 60.3 (C-14 ), 55.60 (C-12 ), 40.49 (C-15 ), 39.23 (C-6 ), 37.35 (C-
0 00
6, H-19, H-20), 3.39 (1H, m, H-15), 3.20 (4H, m, H-6 , H-6 ), 3.15
1
(
0
0
), 35.78 (C-2 ), 34.30 (C-8 ), 33.22 (C-3), 32.53 (C-25), 31.86 (C-32),
0.66 (C-14), 29.70, 29.58 (2C), 29.55, 29.48 (2C), 29.40, 29.35,
9.31 (2C), 29.09, 28.73, 28.31, 28.12, 27.99, 27.95, 26.31, 26.06,
0
00
0
00
2H, m, H-12 , H-12 ), 2.90 (2H, dd, J ¼ 12.8, 4.6 Hz, H-15 a, H-15 a),
0
00
00
2
(
1
.75 (2H, d, J ¼ 12.8 Hz, H-15 b, H-15 b), 2.51 (1H, m, H-3a), 2.41
0
0
00
1H, m, H-3b), 2.33 (4H, m, H-8 , H-8 ), 2.20 (4H, m, H-2 , H-2 ),
5.66, 25.59 (2C), 25.36, 24.62 (2C), 22.64 (C-33),19.06 (C-37),14.09
.90e2.01 (4H, m), 1.58e1.81 (17H, m), 1.46e1.57 (13H, m), 1.21e
þ
(
9
C-34); HR-ESIMS m/z: 962.6472 [M þ H] (calcd for C53
91 3
H N O10S,
1
3
7
.38 (37H, m),1.40 (3H, d, J ¼ 6.8 Hz, H-37), 0.88 (3H, t, J ¼ 6.8 Hz, H-
62.6498).
13
0
00
4); C NMR (150 MHz, CDCl
3
):
d
173.69e173.39 (C-1, C-1 , C-1 , C-
0
00
0
00
, C-7 ), 164.23 (C-16 , C-16 ), 151.33 (C-35), 130.10 (C-2), 83.34 (C-
6), 82.53 (C-20), 81.90 (C-19), 80.57 (C-23), 77.70 (C-36), 74.63 (C-
4), 74.10 (C-15), 71.81 (C-4), 61.92 (C-13 , C-13 ), 60.38 (C-14 , C-
3
.6.3. 24-O-(6-Biotinylamidohexanoyl)bullatacin (20)
1
2
0
ꢀ1
A yellowish waxy solid; [
a
]
D
þ 14.8 (c 0.1, MeOH). IR cm (KBr):
0
00
0
2
1
3
200e3500, 3100, 2924, 2854, 1723, 1653, 1548, 1463, 1375, 1320,
00
0
00
0
00
0
00
4 ), 55.76 (C-12 , C-12 ), 40.54 (C-15 , C-15 ), 39.23 (C-6 , C-6 ),
1
1189; H NMR (600 MHz, CDCl
3
):
d
7.22 (1H, m, H-35), 6.46 (2H, m,
0
00
0
00
3
(
2
2
2
5.79 (C-2 , C-2 ), 34.28 (C-8 , C-8 ), 34.12 (C-5), 33.36 (C-14), 31.91
0
0
0
C6 eNH, C13 eNH), 5.66 (1H, br s, C14 eNH), 5.06 (1H, m, H-36),
C-32), 30.64 (C-25), 30.01 (C-3), 29.68, 29.60 (2C), 29.54 (2C),
9.46, 29.37 (2C), 29.35, 29.23, 29.10 (2C), 28.91, 28.50, 28.45, 28.39,
8.21 (2C), 28.05, 28.01, 26.94, 26.39, 26.35, 25.80, 25.63, 25.59,
0
0
4
(
1
5
2
.99 (1H, m, H-24), 4.51 (1H, m, H-14 ), 4.32 (1H, m, H-13 ), 4.03
1H, m, H-23), 3.84 (4H, m, H-4, H-16, H-19, H-20), 3.38 (1H, m, H-
0
0
5), 3.21 (2H, m, H-6 ), 3.15 (1H, m, H-12 ), 2.91 (1H, dd, J ¼ 12.8,
5.21, 24.70 (2C), 24.54, 22.69 (C-32),18.99 (C-37),14.15 (C-34); HR-
0
0
.0 Hz, H-15 a), 2.75 (1H, d, J ¼ 12.8 Hz, H-15 b), 2.51 (1H, m, H-3a),
þ
ESIMS m/z: 1301.8086 [M þ H] (calcd for C69
117 6 13 2
H N O S ,
0
0
.41 (1H, m, H-3b), 2.31 (2H, m, H-8 ), 2.19 (2H, m, H-2 ), 1.87e2.01
1
301.8115).
(
1
4H, m), 1.57e1.80 (10H, m), 1.39e1.56 (10H, m), 1.32e1.39 (5H, m),
.21e1.31 (30H, m), 1.43 (3H, d, J ¼ 6.8 Hz, H-37), 0.88 (3H, t,
13
3.7. Cell culture
J ¼ 6.8 Hz, H-34); C NMR (150 MHz, CDCl
3
): d 174.72 (C-1), 173.64
C-7 ), 173.46 (C-1 ), 164.02 (C-16 ), 151.92 (C-35), 131.10 (C-2), 83.24
0
0
0
(
(
(
(
(
(
2
(
C
L1210, P815 and 4T1 cell lines were obtained from Kunming cell
bank, Chinese Academy of Sciences. L1210 and P815 cell lines were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invi-
trogen), supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (FBS) as well as 1% (v/v) penicillin and streptomycin (P/S).
T1 cell line was cultured in RPMI1640 (Invitrogen) cell culture
medium supplemented with 10% FBS and 1% penicillin and strep-
tomycin. All cell lines were maintained in a humidified atmosphere
C-16), 82.35 (C-20), 81.77 (C-19), 80.54 (C-23), 78.02 (C-36), 74.74
0
0
0
C-24), 74.11 (C-15), 69.84 (C-4), 61.96 (C-13 ), 60.38 (C-14 ), 55.61
0
0
0
C-12 ), 40.47 (C-15 ), 39.32 (C-6 ), 37.36 (C-5), 35.72 (C-2 ), 34.35
0
C-8 ), 33.25 (C-3, C-14), 31.81 (C-32), 30.81 (C-25), 29.64, 29.57
2C), 29.50 (5C), 29.31 (2C), 29.05, 28.87, 28.43, 28.38, 28.09, 27.97,
4
7.13, 26.33 (2C), 25.68, 25.57 (2C), 25.51, 24.62, 22.66 (C-33), 19.08
þ
C-37), 14.12 (C-34); HR-ESIMS m/z: 962.6479 [M þ H] (calcd for
53 91
H N
3
O10S, 962.6498).
ꢂ
of 5% CO
2
at 37 C.
3
.6.4. 4, 15-Di-O-(6-biotinylamidohexanoyl)bullatacin (21)
2
0
ꢀ1
A yellowish waxy solid; [
a]
D
þ 30.3 (c 1, MeOH); IR cm (KBr):
3.8. Cell viability assay
3
200e3500, 3100, 2929, 2856, 1700, 1645, 1555, 1464, 1377, 1321,
3
1
6
5
3
246, 1170, 1102; H NMR (600 MHz, CDCl
3
):
d
7.11 (1H, m, H-35),
L1210, 4T1 and P815 cells (1 ꢁ 10 /well) were seeded in flat-
bottomed 96 well microplates. Then squamocin/bullatacin and
biotinylated squamocin/bullatacin derivatives dissolved in DMSO
0
00
00
0
00
.72 (2H, br s, C6 eNH, C6 eNH), 6.56 (2H, m, C13 eNH, C13 eNH),
0
.86 (2H, m, C14 eNH, C14 eNH), 5.11 (1H, m, H-4), 5.01 (1H, m, H-
0
00
6), 4.89 (1H, m, H-15), 4.52 (2H, m, H-14 , H-14 ), 4.32 (2H, m, H-
(10, 2, 0.4, 0.08, 0.0016, 0.00032 mg/mL as concentration gradient)
were added to wells, respectively. The cells were incubated for
0
00
13 , H-13 ), 4.03 (1H, m, H-16), 3.79e3.94 (4H, m, H-19, H-20, H-23,
0
00
0
00
ꢂ
H-24), 3.20 (4H, m, H-6 , H-6 ), 3.14 (2H, m, H-12 , H-12 ), 2.91 (2H,
72 h at 37 C in CO
2
gas incubator and then treated with 3-(4,5-
0
00
0
dd, J ¼ 12.8, 4.6 Hz, H-15 a, H-15 a), 2.75 (2H, d, J ¼ 12.8 Hz, H-15 b,
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(20 L/well, 5 mg/mL). After another 4 h incubation, the medium
was removed and 150 L of DMSO was added to each well.
Absorbance in each well, including the blanks, was measured at
570 nm in a microtiter plate reader after the samples were swirled
gently. Experiments were repeated at least three times. Growth
inhibition rate was calculated as follows: inhibition rate
0
0
0
H-15 b), 2.51 (1H, m, H-3a), 2.41 (1H, m, H-3b), 2.33 (4H, m, H-8 ,
m
0
0
0
00
H-8 ), 2.20 (4H, m, H-2 , H-2 ), 1.90e2.01 (3H, m), 1.58e1.83 (10H,
m), 1.46e1.57 (21H, m), 1.21e1.38 (37H, m), 1.40 (3H, d, J ¼ 6.8 Hz,
m
13
H-37), 0.88 (3H, t, J ¼ 6.8 Hz, H-34); C NMR (150 MHz, CDCl
3
):
0
00
0
00
0
d
173.69 (C-1), 173.41e173.53 (C-1 , C-1 , C-7 , C-7 ), 164.27 (C-16 ,
0
0
C-16 ), 151.36 (C-35), 130.08 (C-2), 82.76 (C-19), 82.26 (C-23), 82.06
(
(
C-20), 80.07 (C-16), 77.71 (C-36), 75.07 (C-15), 71.83 (C-4), 71.43
C-24), 61.84 (C-13 , C-13 ), 60.27 (C-14 , C-14 ), 55.77 (C-12 , C-
(%) ¼ {1 ꢀ [
D
OD (compounds) ꢀ
D
OD (blank)]/[
D
OD (controls) ꢀ
0
00
0
00
0
D
OD (blank)]} ꢁ 100%.

228
J.-F. Shi et al. / European Journal of Medicinal Chemistry 71 (2014) 219e228
Acknowledgments
methotrexate-human serum albumin conjugated nanoparticles by targeting
with luteinizing hormone-releasing hormone (LHRH) peptide, Int. J. Mol. Sci.
2 (2011) 4591e4608.
1
This work was supported by the Knowledge Innovation Program
of the Chinese Academy of Sciences (Grant no. KSCX-EW-J-28). Z.-
H. Jiang is grateful to the Wang Kuang Cheng Foundation, the
Chinese Academy of Science for a travel grant.
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