Design, Synthesis, and Biological Evaluation of Novel Selenium-Containing Isocombretastatins and Phenstatins as Antitumor Agents

Article abstract of DOI:10.1021/acs.jmedchem.7b00480

Two series of structurally related organoselenium compounds designed by fusing the anticancer agent methyl(phenyl)selane into the tubulin polymerization inhibitors isocombretastatins or phenstatins were synthesized and evaluated for antiproliferative acti

Full text of DOI:10.1021/acs.jmedchem.7b00480

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Article
Design, Synthesis, and Biological Evaluation of Novel Selenium-
containing Isocombretastatins and Phenstatins as Antitumor Agents
Yanqing Pang, Baijiao An, Lanlan Lou, Junsheng Zhang, Jun Yan, Ling Huang, Xingshu Li, and Sheng Yin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00480 • Publication Date (Web): 09 Aug 2017
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Design, Synthesis, and Biological Evaluation of Novel
Selenium-containing Isocombretastatins and Phenstatins as
Antitumor Agents
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Yanqing Pang,† Baijiao An,† Lanlan Lou, Junsheng Zhang, Jun Yan, Ling Huang, Xingshu Li,*
and Sheng Yin*
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006,
China
ABSTRACT: Two series of structurallyꢀrelated organoselenium compounds
designed by fusing the anticancer agent methyl(phenyl)selane into the tubulin
polymerization inhibitors isocombretastatins or phenstatins were synthesized and
evaluated for antiproliferative activity. Most of these selenium containing hybrids
exhibited potent cytotoxicity against a panel of cancel cell lines, with IC50 values in
the submicromolar concentration range. Among them, 11a, the 3ꢀmethylseleno
derivative of isocombretastatin Aꢀ4 (isoCAꢀ4), represented the most active compound
with IC50 values of 2−34 nM against twelve cancer cell lines, including two
drugꢀresistant cell lines. Importantly, its phosphate salt, 11ab, inhibited tumor growth
in xenograft mice models with inhibitory rate of 72.9% without apparent toxicity,
which was better than the reference compounds isoCAꢀ4P (inhibitory rate: 52.2%)
and CAꢀ4P (inhibitory rate: 47.6%). Mechanistic studies revealed that 11a is a potent
tubulin polymerization inhibitor, which could arrest cell cycle at G /M phase and
2
induce apoptosis along with the decrease of mitochondrial membrane potential. In
summary, 11a could serve as a promising lead for the development of highly efficient
anticancer agents.
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INTRODUCTION
The interfering with tubulin dynamics becomes a wellꢀverified strategy for the
development of highly efficient anticancer drugs because microtubules play a pivotal
role in essential cellular processes including the movement of organelles, intracellular
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transportation, and the formation and maintenance of cell shape. Natural products
capable of interfering with the assembly or disassembly of microtubules have drawn
much attention in the last decades, and a large number of natural tubulin modulators
have been reported as anticancer agents. For instance, paclitaxel, currently used in the
treatment of ovarian and breast cancer, could promote the polymerization of
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microtubules, resulting in highly stable, nonfunctional assembled microtubules,
while the vinca alkaloids (e.g. vincristine, vinblastine, etc.), dolastatins, and
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colchicine are wellꢀknown antitumor agents inhibiting microtubule assembly.
However, these complex molecules have limitations resulting from high toxicity,
difficulty of synthesis, and some of them become rapidly prone to resistance
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phenomena. Among the large class of natural tubulin polymerization inhibitors,
combretastatin Aꢀ4 (CAꢀ4), isolated from the African willow tree Combretum caffrum,
represents currently the simplest structure and its soluble prodrug, CAꢀ4P, has been
approved as orphan drug for the treatment of anaplastic thyroid cancer by both the
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, 6
FDA and European Medicines Agency. However, the Zꢀnatural stilbene compound
is prone to doubleꢀbond isomerization during storage and administration, leading to
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the Eꢀisomer, which displayed a dramatically reduced activity. Thus, the drug
discovery campaign based on the rational design of CAꢀ4 derivatives has been
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extensively carried out in the last few decades, which led to the synthesis of more than
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8000 analogues.
Among them, isocombretastatin Aꢀ4 (isoCAꢀ4),
a
1
,1ꢀdiarylethylene isomer of CAꢀ4, and phenstatin, a ketone bioisostere of isoCAꢀ4,
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turned out to be the most promising antitubulin agents with comparable in vitro
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activity to that of CAꢀ4, although the in vivo antitumor effects of these compounds
have not been investigated yet.
Selenium (Se) compounds have attracted a vast interest recently as promising
chemoꢀpreventive agents, and several organoselenium compounds have been shown
to inhibit cancer cell growth in various xenograft rodent models for different cancers
types, as well as to have synergistic effects in combination with chemotherapeutic
1
0,
11
drugs.
Among them, the arylselenium compounds, such as
10
12
11
methyl(phenyl)selane, benzeneselenol, and methylselenocysteine represent a
group of simple but wellꢀstudied anticancer molecules, which exert their antitumor
effects by induction of apoptosis, inhibition of angiogenesis, and modulation of AKT
11, 13
and COX pathways.
Despite the impressive number of CAꢀ4 analogues have been reported, only a few
CAꢀ4 analogues containing selenium atoms as spacer groups between the aromatic
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rings (diaryl selenide, 3,4ꢀdiarylꢀ1,2,5ꢀselenadiazol, and selenoxides ) have been
studied. However, these compounds only exhibited similar antiproliferative activity to
CAꢀ4 in vitro, without any further evaluation in vivo, and the introduction of selenium
atoms on the aromatic rings has never been studied. Considering all of the
aboveꢀmentioned information, we hypothesized that assembly of isocombretastatins
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or phenstatins with methyl(phenyl)selane in the same molecule could be a valid
shortcut in the development of new anticancer agents.
To fuse these two scaffolds, there are three potential sites that can be modified (Cꢀ3,
Cꢀ4 and Cꢀ4'). Firstly, we have tried to introduce a selenium atom on Cꢀ4 position of
Aꢀring, but the reaction hardly took place because of the stereoꢀhindrance of two
orthoꢀpositioned methoxy groups. Then, the introduction of a selenium atom on Cꢀ4'
position of Bꢀring was conducted. However, the the cytotoxicity of the resulting
derivatives did not show too much improvements (see Table 1). Thus, the
modification was conducted on the Cꢀ3 of ring A, and two series of
structurallyꢀrelated seleniumꢀcontaining molecules were designed, synthesized, and
evaluated for their antitumor potential in the current study.
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Figure 1. Design strategy of seleniumꢀcontaining isocombretastatins and phenstatins
RESULTS AND DISCUSSION
Chemistry. The synthetic route of the seleniumꢀphenstatin hybrids 10a−i was
summarized in Scheme 1. The reaction of 2ꢀmethoxyphenol 1 with NBS in the
solution of acetonitrile provided 4ꢀbromoꢀ2ꢀmethoxyphenol 2, and then, the nitration
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of 2 gave intermediate 3, which reacted with methyl iodide to produce
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ꢀbromoꢀ1,2ꢀdimethoxyꢀ3ꢀnitrobenzene 4. The reduction of 4 with iron powder
1
8
afforded the amino intermediate 5, which was diazotized by the hydrochloric acid
and sodium nitrite, then followed by reaction with potassium selenocyanante to
produce 6. In the presence of potassium hydroxide in methanol, 6 was converted to
diselenide 7, which reacted with sodium borohydride and methyl iodide to afford the
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1
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key intermediate 8. The reaction of 8 with different benzaldehyde analogues
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afforded diarylmethanols 9a−i. Finally, target compounds 10a−i were obtained by
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the oxidation of 9a−i with 2ꢀiodoxybenzoic acid in good yields.
a
Scheme 1. Synthesis of compounds 10a−i
a
Reagents and reaction conditions: (a) NBS, CH
3
CN; (b) 65% HNO
3
, AcOH; (C) CH
3
I, KOH, TBAI, DMF; (d)
, CH I, EtOH; (h)
iron powder, AcOH, EtOH; (e) NaNO
2
, 10% HCl, KSeCN; (f) KOH, CH
3
OH; (g) NaBH
4
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nꢀBuLi, anhydrous THF; (i) IBX, THF.
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The seleniumꢀisoCA hybrids 11a−i were synthesized commodiously by employing
Wittig reactions between 10a−i and methyltriphenylphosphonium bromide (Scheme
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).
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Scheme 2. Synthesis of compounds 11a−i
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Reagents and reaction conditions: Methyltriphenylphosphonium bromide, nꢀBuLi, anhydrous THF.
To investigate the effect of different substituting position of selenium on the activity,
compounds 17 and 18 with the selenium atom on the Cꢀ4′ position of the Bꢀring were
synthesized using the same procedures as those of 10a−i and 11a−i apart from the use
of 4ꢀbromoaniline (12) as the starting materials (Scheme 3).
a
Scheme 3. Synthesis of compounds 17 and 18
a
2 3 4 3
Reagents and reaction conditions: (a) NaNO , 10% HCl, KSeCN; (b) KOH, CH OH; (c) NaBH , CH I, EtOH; (d)
nꢀBuLi, anhydrous THF; (e) IBX, THF.(f) methyltriphenylphosphonium bromide, nꢀBuLi, anhydrous THF.
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Considering the bioavailability of drugs largely depends on their water solubility,
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1ab, the disodium phosphate salt of 11a, was synthesized for the in vivo test. As
shown in Scheme 4, the esterification reaction between 11a and diethyl phosphite
afforded 11aa, which was deprotected in trimethylsilyl bromide then followed by
neutralization with NaOH to give the target compound 11ab.
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a
Scheme 4. Synthesis of compound 11ab
a
3 4 2 2 2 2
Reagents and reaction conditions: (a) diethyl phosphite, Et N, CCl , CH Cl ; (b) TMSBr, anhydrous CH Cl ; (c)
NaOH, MeOH.
BIOLOGY
Antiproliferative Activities and the SARs. To evaluate the synergistic effects of
these seleniumꢀcontaining hybrids, benzophenone derivative 10a, bearing a
methylselanyl to replace the 3ꢀmethoxyl of phenstatin, was first synthesized and
screened for antiproliferative activity against a panel of five different cancer cell lines
[
A549 (human nonꢀsmall cell lung carcinoma), MDAMB231 (human breast
carcinoma), HEPG2 (human hepatoma carcinoma), LOVO and RKO (human
colorectal carcinoma)]. As shown in Table 1, 10a exhibited pronounced activity with
IC50 values ranging from 24 to 79 nM, being more potent than the prototype
compound phenstatin. In order to investigate the structuresꢀactivity relationships of
these seleniumꢀphenstatin hybrids, compounds 10b−10i with different substituents at
Cꢀ2′, Cꢀ3′, and Cꢀ5′ of ring B (R
1
, R
2 3
, and R ) were prepared and evaluated for their
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antiproliferative activities. Compound 10b with the presence of a fluorine atom at
Cꢀ3′ (R = F), displayed similar activity to those of 10a (R = OH) and 10e (R = H),
2
2
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with IC50 values ranging from 33 to 59 nM. When fluorine atom was replaced by
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chlorine or bromine atom, the activities decreased dramatically as shown in 10c (R =
2
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Cl, IC50 = 253 ~ 1534 nM) and 10d (R = Br, IC50 = 169 ~ 1752 nM). These results
suggested that the large hetero atoms at Cꢀ3′ might be adverse to the activity.
Compounds 10f−10h provide less potent activity than those of 10a or 10e, indicating
that the excess of methoxy groups at B ring was unfavorable to the activity, especially
2
the presence of methoxy group at Cꢀ3′ (R = OMe). Similarly, the presence of methyl
at Cꢀ3′ (R = Me) also led to the dramatically decrease of the activity, as 10i only
2
provided a poor activity with IC50 values ranging from 997 to 5281 nM.
Inspired by above results, seleniumꢀisoCA hybrids 11a−i were synthesized and
evaluated. As shown in Table 1, It is interesting that the replacement of the carbonyl
group with a terminal double bond led to the different effect on the activity, as the
increased activity (11a vs 10a, 11c vs 10c, and 11i vs 10i) and decreased activity (10h
vs 11h) were both observed in these pairs of compounds. Within 11a−i, similar SARs
were observed as seleniumꢀphenstatin series and 11a turned out to be the most active
compound with the IC50 values of 2.2-11 nM toward five cancer cell lines.
Compounds 17 and 18 with the selenium atom on the Cꢀ4′ position also exhibited
pronounced activity (17, IC50: 66 ~ 296 nM; 18, IC50: 97 ~ 386 nM). However,
comparing with their Cꢀ3 selenium counterparts 10e and 11e, the generally weaker
activities of 17 and 18 indicated that the substitution of selenium on Cꢀ3 is beneficial.
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a
Table 1. Antiproliferative Activities of 10a
−
i and 11a
−
i against Human Cancer Cell Lines
10
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12
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17
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b
IC50 (nM)
HEPG2
Compd
A549
55 ± 4
MDAMB231
LOVO
53 ± 14
RKO
1
0a
24 ± 7
34 ± 8
31 ± 4
79 ± 18
59 ± 6
10b
33 ± 8
47 ± 11
57 ± 9
1
1
1
0c
0d
0e
949 ±12
368 ± 36
31 ± 8
865 ± 14
435 ± 15
57 ± 5
1534 ± 290
655 ± 98
85 ± 9
189 ± 15
1752 ± 93
145 ± 14
523 ± 28
766 ± 10
921 ± 38
3150 ± 340
9 ± 3
253 ± 70
169 ± 15
47 ± 7
10f
2746 ± 200
285 ± 52
172 ± 12
1728 ± 15
3.9 ± 1
4257 ± 400
1016 ± 130
741 ± 74
997 ± 80
2.2 ± 1
4338 ± 220
1086 ± 130
288 ± 29
2296 ± 220
3 ± 1
874 ± 60
306 ± 30
235 ± 60
5281 ± 510
11 ± 5
1
1
1
0g
0h
0i
11a
1b
1c
1d
11e
1f
1g
1h
11i
1
29 ± 3
24 ± 3
87 ± 5
33 ± 3
82 ± 9
1
196 ± 20
236 ± 15
72 ± 22
1380 ± 150
265 ± 70
473 ± 9
141 ± 35
69 ± 2
83 ± 8
298 ± 60
691 ± 50
126 ± 70
6299 ± 750
473 ± 51
1350 ± 260
607 ± 61
77 ± 2
254 ± 30
171 ± 16
91 ± 9
451 ± 53
343 ± 75
107 ± 14
1036 ± 250
216 ± 49
3516 ± 310
245 ± 26
66 ± 3
1
667 ± 100
96 ± 23
1
3825 ± 780
344 ± 80
3324 ± 150
599 ± 47
65 ± 3
128 ± 18
299 ± 152
3145 ± 510
496 ± 55
296 ± 18
279 ± 6
1
1
1
1
7
8
97 ± 2
178 ± 6
386 ± 33
108 ± 4
216 ± 23
72 ± 18
phenstatin
IsoCAꢀ4
CAꢀ4
179 ± 30
3.5 ± 1
62 ± 6
122 ± 50
3.7 ± 2
8.5 ± 2
18.3 ± 3
3.8 ± 1
c
c
c
4 ± 1
4.1 ± 2
3.9 ± 1
2.4 ± 1
9.8 ± 2
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental Section. IC50 values are indicated as the mean ± SD (standard error) of at least three independent
c
experiments. Values were also reported as 9 ± 2, 3, and 2.1 ± 0.5 nM in literatures 22, 9a, and 23, respectively.
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1a Inhibits another Five Human Cancer Cell Lines. As the most potent compound
found in the initial cytotoxicity screening, 11a was selected for further evaluation
against another five different cancer cell lines [MCF7 (human breast carcinoma),
HELA (human cervical carcinoma), HCT116 (human colon carcinoma), MGC803
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(human gastric adenocarcinoma), and A2780 (human ovarian carcinoma)]. As shown
in Table 2, 11a also exhibited excellent antiproliferative activity with IC50 values in
the low nanomolar range (IC50 = 3 ~ 27 nM). All these data suggested that 11a was a
potential anticancer agent worthy of further study.
a
Table 2. Antiproliferative Activities of 11a on another Five Human Cancer Cell Lines
b
IC50 (nM)
Compd
MCF7
27 ± 6
HELA
3 ± 0.2
6 ± 0.1
3.8 ± 0.2
HCT116
12 ± 0.2
24 ± 0.5
7 ± 0.1
MGC803
9± 0.4
A2780
6 ± 0.1
11a
IsoCAꢀ4
48 ± 0.9
14 ± 0.5
19.8 ± 0.2
8.9 ± 0.1
26 ± 0.6
12 ± 0.3
CAꢀ4
a
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the
b
Experimental Section. IC50 values are indicated as the mean ± SD (standard error) of at least three independent
experiments.
Antiproliferative Activity of 11a against Drug-Resistant Cancer Cell Lines.
Multidrug resistance (MDR) is a major obstacle for successful treatment of cancer, as
rapidly acquired drug resistance often results in clinical chemotherapy failure. To
evaluate the antiꢀMDR potential of 11a, the cisplatinꢀresistant cell line A549/CDDP
and the doxorubicinꢀresistant cell line HEPG2/DOX were selected for
antiproliferative study. As shown in Table 3, 11a inhibited A549/CDDP and
HEGP2/DOX with IC50 values being at 34.4 and 20.3 nM, respectively. The small
resistant factor values (RF: 8.8 and 6.8, respectively) suggested that 11a possessed
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potent antiproliferative activities towards drugꢀresistant cancer cell lines, which
deserves further study.
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a
Table 3. Antiproliferative Activities of 11a towards Human DrugꢀResistant Cancer Cell Lines
b
IC50 (nM)
compd
Resistant
factor
Resistant
A549/CDDP
A549
HEPG2/DOX
HEPG2
b
b
factor
11a
34.4 ± 0.9
69.1 ± 0.4
3.9 ± 1
8.8
20.3 ± 0.6
38.6 ± 0.2
3 ± 1
6.8
2.1
IsoCAꢀ4
3.5 ± 0.1
19.7
18.3 ± 0.3
a
b
Data are presented as the mean ± SE from the doseꢀresponse cures of at least three independent experiments.
Resistant factor = (human drugꢀresistance cells IC50) / (human cancer sensitive cells IC50).
1
1a Inhibits Microtubule Polymerization. To elucidate whether 11a targets the
tubulinꢀmicrotubule system, the in vitro tubulin polymerization inhibition activity of
1
1a was evaluated by using the method originally described by D. Bonne et al. with
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some modifications.
As shown in Figure 2, the increased fluorescence intensity
with time of the purified and unpolymerized tubulin control samples indicated that
tubulin polymerization had occurred. After tubulin was incubated with 11a at various
concentrations ranging from 0.3125 to 7.5
ꢀM, the increased tendency of the
fluorescence intensity was obviously slowed down as compared with the control,
indicating that 11a inhibited the tubulin polymerization. Additionally, the results also
indicated
an IC50 value of 1.38 ± 0.09
compound isoCAꢀ4 (IC = 2.76 ± 0.11
t
hat 11a inhibited tubulin polymerization in a doseꢀdependent manner, with
ꢀ
M (Figure 2A), being stronger than the reference
ꢀΜ, Figure 2B).
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Figure 2. Compound 11a inhibits microtubule polymerization. Purified tubulin protein at 10 mM in a reaction
buffer was incubated at 37℃ in the absence (control) or presence of 11a (A) and isoCAꢀ4 (B) at the indicated
concentrations (ranging from 0.3125 to 7.5 ꢀM and 0.75 to 10 ꢁM, respectively). The experiments were performed
three times, and the results of representative experiments are shown.The IC50 value of isoCAꢀ4 was also reported
as 2.2 ꢀΜ in literature 9a.
To study whether 11a could disrupt the microtubule dynamics in living cells,
immunofluorescence assay in A549 cells were performed, and isoCAꢀ4 was used as
the reference compound. As shown in Figure 3, in the control group, the microtubule
network in the A549 cells exhibited normal arrangement and organization,
characterized by regularly assembled, normal filiform microtubules wrapped around
the uncondensed cell nucleus. After exposure to 11a for 8 h, the spindle formation
demonstrated distinct abnormalities and was heavily disrupted. Especially, when the
concentration of 11a was increased to 10 nM, the microtubule spindles wrapped
around the cell nucleus have significantly shrunk and a number of dotted disorder
formations were easily observed (Figure 3A). In contrast, cells treated with 10 nM of
isoCAꢀ4 for 8 h still retained a spindle shaped microtubule network but showed slight
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changes on the shape of the nucleus (Figure 3B). When the incubation time was
extended to 24 h, the multipolarization of the spindle and multinucleation phenomena
were found in both groups treated with 10 nM samples. These morphological
microtubule changes indicated that 11a dramatically disrupted the microtubule
organization at nanomolar concentrations, which might eventually lead to cell cycle
disorder.
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Figure 3. Compound 11a (A) and isoCAꢀ4 (B) disrupted the organization of the cellular microtubule network at
indicated concentrations. A549 cells were plated in confocal dishes and incubated with DMSO or 11a at the 5, 10
nM for 8 h and 24 h, followed by direct microscopy. The detection of the fixed and stained cells was performed
with an LSM 570 laser confocal microscope (Carl Zeiss, Germany). The experiments were performed three times,
and the results of the representative experiments were shown. Scale bar: 20 ꢀm.
11a Induces Mitochondrial Dysfunction. A prelusion of cell apoptosis was the
mitochondrial membrane depolarization, which usually leads to the decrease of
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mitochondrial membrane potential (MMP, ꢂΨm). The change of MMP could be
detected by flow cytometry analysis or a fluorescence microscope. To explore
whether 11a could induce mitochondrial dysfunction, quantitative mitochondrial
membrane potentials (MMPs) assay of JCꢀ1 staining mitochondria in A549 cells was
performed. As shown in Figure 4A, the cells of control group have wellꢀdefined
integral mitochondrial membranes (monomers: aggregates = 0.62 : 99.18), and after
cells exposed to 1 nM of 11a, the red fluorescence intensity (JCꢀ1 aggregates, cells
with high mitochondrial membrane potentials) decreased to 86.88%, and the green
fluorescence intensity (JCꢀ1 monomers, low mitochondrial membrane potentials)
correspondingly increased from 0.62% to 12.78%. When increasing the concentration
of 11a to 5 nM and 10 nM, the red fluorescence intensity decreased to 57.63% and
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.14%, respectively, and the green fluorescence intensity correspondingly increased to
42.63% and 90.78%. These phenomena were also verified by the fluorescence
microscopy assay, in which the treatment of 11a induced the increase of the
monomers and the decrease of the aggregates (Figure 4B). Taken together, 11a could
induce MMP collapse and mitochondrial dysfunction, which eventually triggers
apoptotic cell death.
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Figure 4. Compound 11a decreased the mitochondrial membrane potential in a doseꢀdependent manner. The A549
cells were treated with 11a at the indicated concentration (1, 5, and 10 nM) or DMSO (0.01%) for 24 h, followed
by incubation with the fluorescence probe JCꢀ1 for 30 min. Then, the cells were analyzed by flow cytometry (A) or
fluorescence microscope (B). The experiments were performed at least three times, and the results of the
representative experiments. Scale bar: 20 ꢀm.
2 2
11a Induces G /M Phase Arrest and Regulates the Expression of G /M-Related
Proteins. As most tubulin destabilizing agents could disrupt the regulated cell cycle
distribution, a flow cytometry analysis was performed to determine the arrest effect of
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1a on the G
in Figure 5, 11a effectively arrested cells in the G
timeꢀdependent manner. When A549 cells were treated with 11a from 1 nM to 10 nM
for 12 h, the population of cells in G /M phase dramatically increased as compared to
2
/M transition, a rigorously regulated process in the cell cycle. As shown
2
/M phase in a doseꢀ and
2
that of vehicle (DMSO) group (Figure 5A), along with concomitant losses in the G1
phase. This phenomenon was more obvious after 24 h incubation, when nearly 50%
2
cells were arrested at G /M phase at the concentration of 1 nM, and about 70% cells
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were arrested at concentrations of 5 nM or 10 nM. It is known that the activation of
Cdc2 kinase controlled by Cyclinb1 binding and Cdc25C phosphorylation could
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regulate eukaryotic cell entry into mitosis, leading to the cell division.
Thus, the
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association between 11aꢀinduced G /M arrest and the alterations of these protein
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expression were investigated by western blot analysis. As shown in Figure 5C, after
A549 cells were treated with 11a at the concentrations of 1, 5, and 10 nM for 12h, the
expression of Cdc25C, Cdc2 and Cyclinb1 protein distinctly decreased in doseꢀ and
timeꢀdependent manners. The phenomenon was more obvious when the treatment
time was extended to 24 h. These results, which were consistent with the cell cycle
analysis and the intracellular microtubule immunofluorescence assay, further
illustrated the basic mechanism of 11a on the cell cycle arrest.
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Figure 5. Cell cycle arrest effect of 11a. The A549 cells were treated with compound 11a at 1, 5, or 10 nM for 12 h
(
A) or 24 h (B). The expression of proteins Cyclin B1, Cdc25c, and Cdc2 were analysed by the western blot (C).
The experiments were performed three times, and the results of representative experiments are shown.
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1
1a Induces Apoptosis via the Regulation of Apoptosis-Related Proteins
Expression. According to aboveꢀmentioned cell cycle analysis, we suppose that 11a
might induce the apoptosis of A549 cells. To confirm this, A549 cells were treated
with 1 nM to 10 nM of 11a for 24 h or 48h, and then, the cells was harvested and
stained with Annexin VꢀFITC and propidium iodide (PI), and analyzed by flow
cytometry. As shown in Figure 6A, when the cells were incubated with 11a at 1, 5,
and 10 nM for 24 h, the percentages of early and late apoptosis cells were 3.64%,
3
3.88%, and 50.93%, respectively, being elevated from the control group (0.82%).
After 48 h incubation at 1, 5, and 10 nM, the percentages of early and late apoptotic
cells were strikingly increased to 18.65%, 40.42%, and 63.01%, respectively ( Figure
29
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B). The Bclꢀ2 family proteins are critical regulators of cell apoptosis, which can
either promote the release of cytochrome c and further induce cell apoptosis (e.g. Bax,
Bid, Bim, and Bad), or antagonize the proꢀapoptotic proteins to prevent apoptosis (e.g.
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Bclꢀ2, Bclꢀxl, and Bclꢀw).
To determine the apoptosisꢀassociated protein
alterations, the expression of Bad, Bclꢀ2, and Bclꢀxl were analyzed. As shown in
Figure 6D, when A549 cells were treated with 11a at different concentrations (1, 5,
and 10 nM) for 24 h or 48 h, the level of Bad was efficiently upꢀregulated, whereas
the level of antiꢀapoptotic proteins (Bclꢀ2, Bclꢀxl) was downꢀregulated significantly.
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These results demonstrated that 11a induced the cell cycle arrest and eventually led to
cell apoptosis.
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Figure 6. Compound 11a induced A549 cell apoptosis. The A549 cells were treated with compound 11a at 1, 5, or
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0 nM for 24 h (A) or 48 h (B). The percentages of cells in each stage of cell apoptosis were quantified by flow
cytometry: (upper left quadrant) necrosis cells; (upper right quadrant) lateꢀapoptotic cells;(bottom left quadrant)
live cells; and (bottom right quadrant) early apoptotic cells, total apoptosis cells percentage were obtained by
EXPO32 ADC analysis software (C) And anlysed the expression of proteins Bad, Bclꢀ2, and Bclꢀxl by the western
blot (D). The experiments were performed three times, and the results of representative experiments are shown.
Metabolic Stability of 11a. To provide some basic information of drugꢀlike
properties, the metabolic stability of 11a with rat liver microsomes was performed,
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and CAꢀ4 and isoCAꢀ4 were used as the reference compounds. These compounds
were incubated with rat liver microsomes in vitro at 37 °C for an indicated time in
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buffer solution containing NADPH regenerating system, and the residues were
determined by HPLC. As shown in Table 4, the remaining percentage of 11a at each
time point was higher than that of CAꢀ4 but lower than that of isoCAꢀ4. After 150 min
incubation while 11a and isoCAꢀ4 still remained 25.8% and 34.3%, respectively,
CAꢀ4 couldn’t be detected. A short halfꢀlife of pharmaceutical agents might result in
low bioavailability in vivo and the frequent administration with a high dose, which
might cause the drug accumulation and produce related toxicity. The halfꢀlives of 11a
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(t1/2 = 74.5 min), isoCAꢀ4 (t1/2 = 101.9 min), and CAꢀ4 (t1/2 < 60 min) indicated that
1
1a possessed a comparable metabolic stability to that of isoCAꢀ4, being greater than
CAꢀ4, which encouraged us to carry out further studies in vivo.
Table 4. Metabolic Stability of 11a, CAꢀ4, and isoCAꢀ4.
Remains %
Compd
1a
Time (min)
30
60
90
120
150
t
1/2
1
81.1%
67.2%
85.7%
62.3%
26.4%
77.1%
44.1%
22.5%
67.3%
33.4%
19.9%
49.8%
25.8%
74.5
a
b
CAꢀ4
NT
ND
IsoCAꢀ4
34.3%
101.9
a
b
NT: not tested because of the fast metabolic rate of CAꢀ4. ND: not determined.
Antitumor Effect of 11ab in vivo. To determine the in vivo antitumor effect of 11a,
nude mouse A549 xenograft models were established by subcutaneously injecting
A549 cells in the logarithmic phase into the right armpit of the mice. Considering the
bioavailability of drugs largely depends on their water solubility, 11ab, the disodium
phosphate salt of 11a was synthesized as the prodrug for the in vivo test. The mice
were randomly allocated to four groups with nine of each (vehicleꢀtreated,
CAꢀ4Pꢀtreated, isoCAꢀ4Pꢀtreated, and 11abꢀtreated groups). After the tumor volume
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was 100ꢀ200 mm in each group, 11ab, CAꢀ4P, and isoCAꢀ4P were intraperitoneally
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injected at a dose of 30 mg/kg to the mice every other day for the entire observation
period. As showed in Figure 7, at the end of the observation period, 11ab, CAꢀ4P, and
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isoCAꢀ4P groups gave 467.21, 926.50 and 829.79 mm of the mean final tumor
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volume, respectively, comparing the 1775.45 mm of the vehicle group. The average
tumor weight of the 11abꢀtreated group was 0.432 g (inhibitory rate: 72.9%)
comparing with 1.595 g of the vehicle group, which was less than that of
CAꢀ4Pꢀtreated group (0.835 g, inhibitory rate: 47.6%) and isoCAꢀ4Pꢀtreated group
(0.765 g, inhibitory rate: 52.2%). It also can be seen from Figure 7E that all the
groups gave nearly the same body weights, indicating the low toxicity of 11ab toward
mice.
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Figure 7. Efficacy of 11ab against A549 xenografts. (A, B) The images of sacrificed mice and excised tumours in
each group. (C) The tumour volume of mice in each group during observation period. (D) The weight of the
excised tumors in each group. (E) The body weight of the mice in each group at the end of the observation period.
The data were presented as the mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001, significantly different compared
with the control by t test, n = 9. Data were not in conformity with Mauchly’s test of sphericity, and
Greenhouse−Geisser was performed to correction.
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CONCLUSIONS
The multifactorial mechanistic characteristics of cancer results the possibility of
incorporating a sequential, multifactorial approach to the multifaceted disease.
Inspired by this, in the current study, two series of structurallyꢀrelated organoselenium
compounds were obtained by fusing the anticancer agent methyl(phenyl)selane into
the tubulin polymerization inhibitors isocombretastatins and phenstatins, respectively.
Most of these compounds exhibited potent antiproliferative activity against five
human cancer cell lines, with IC50 values in the submicromolar concentration range.
Among them, the optimal compound 11a displayed excellent activities in twelve
human cancer cell lines, including two drugꢀresistant cancer cell lines. Importantly, its
phosphate salt, 11ab exhibited obviously synergistic antitumor effects in A549
xenograft model in vivo without apparent toxicity, being better than the reference
compound isoCAꢀ4P. In addition, the in vivo antitumor effect of isoCAꢀ4P was
investigated for the first time. Finally, the mechanistic studies including the action of
11a on tubulin polymerization, cellꢀcycle arrest, cell apoptosis effect, mitochondrial
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dysfunction and the morphological alterations of A549 cell were also performed. All
of these results demonstrated that the introduction of selenium atom at the Cꢀ3 of
isoCAꢀ4 elevated its anticancer effect, probably by mediating on several mechanisms,
which make 11a a promising lead for further anticancerꢀdrug development.
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EXPERIMENT SECTION
Chemistry. General Methods. Reagents used in the synthesis were obtained
commercially and used without further purification, unless otherwise specified. Flash
column chromatography was performed using silica gel (200−300 mesh) purchased
from Qingdao Haiyang Chemical Co. Ltd. The purity of the samples was determined
by highꢀperformance liquid chromatography (HPLC), conducted on a Shimadzu
LCꢀ20AT series system with a TCꢀC18 column (4.6 mm × 250 mm, 5 ꢁm). The
samples were eluted with a 60:40 acetonitrile/H
2
O mixture at a flow rate of 0.5
mL/min, and was detected at a wavelength of 254 nm. The purity of all biologically
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evaluated compounds is greater than 95%. The H NMR and C NMR spectra were
recorded using TMS as the internal standard on a Bruker BioSpin GmbH spectrometer
at 400 and 100 MHz, respectively, and the coupling constants are reported in hertz.
The highꢀresolution mass spectra were obtained using a Shimadzu LCMSꢀITꢀTOF
mass spectrometer.
4
ꢀbromoꢀ2ꢀmethoxyphenol (2). To a solution of guaiacol (124 mg, 1 mmol) in
CH CN (20 mL) at 0 °C, bromosuccinimide (180 mg, 1 mmol) was added. After
3
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stirring for 30 min, the mixture was quenched with saturated sodium thiosulfate and
then extracted with EtOAc for three times. The combined organic phase was washed
with brine, dried over anhydrous sodium sulfate and concentrated. The crude product
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was purified by column chromatography (PE/EtOAc, 10:1) to provide 2. White oil, 95%
1
yield. H NMR (400 MHz, CDCl
3
) δ 7.06 – 6.91 (m, 2H), 6.80 (d, J = 8.3 Hz, 1H),
5
.55 (s, 1H), 3.88 (s, 3H).
ꢀbromoꢀ2ꢀmethoxyꢀ6ꢀnitrophenol (
in acetic acid (5 mL), 65% HNO (0.097 mL, 1.2 mmol) was added in dropwise. After
1
7
4
3). To a stirred solution of 2 (203 mg, 1 mmol)
3
stirring at room temperature for 10 min, the mixture was poured into ice water. The
resulting precipitate was filtered off, washed with water, and dried under vacuum to
1
give the product, yellow solid, 96% yield. H NMR (400 MHz, CDCl
3
) δ 10.70 (s,
1
H), 7.86 (d, J = 2.2 Hz, 1H), 7.21 (d, J = 2.2 Hz, 1H), 3.95 (s, 3H).
5ꢀbromoꢀ1,2ꢀdimethoxyꢀ3ꢀnitrobenzene ( ). A mixture of 3 (248 mg, 1 mmol),
4
tetrabutylammonium iodide (369 mg, 1 mmol), and KOH (112 mg, 2 mmol) in 10 mL
of DMF was added iodomethane (141 mg, 1 mmol). The reaction mixture was
stirred at 40 °C and monitored by TLC. After the completion of the reaction (48 h),
ice water was added. The resulting precipitate was filtered off, washed with water,
1
and dried under vacuum to give the product, yellow solid, 78% yield. H NMR (400
MHz, CDCl ) δ 7.76 (d, J = 2.2 Hz, 1H), 7.49 (d, J = 2.2 Hz, 1H), 3.89 (d, J = 8.8 Hz,
3
6
H).
5ꢀbromoꢀ2,3ꢀdimethoxyaniline (
1
8
5
). Iron powder (280 mg, 5 mmol) was added to a
mixture of 4 (260 mg, 1 mmol) in EtOH (15 mL) and AcOH (8 mL). The reaction was
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stirred at rt for 3h, then neutralized with a saturated solution of NaHCO . After
extraction with EtOAc, the organic layer was washed with brine, dried over
anhydrous sodium sulfate, concentrated, and purified by column chromatography
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7
8
9
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9
0
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6
7
8
9
0
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(
PE/EtOAc, 5:1 ) to obtain 5. Yellow oil, 68% yield. H NMR (400 MHz, CDCl ) δ
3
6
.53 (d, J = 2.1 Hz, 1H), 6.45 (d, J = 2.1 Hz, 1H), 3.88 (s, 2H), 3.82 (d, J = 1.7 Hz,
H), 3.79 (d, J = 1.8 Hz, 3H).
3
5
ꢀbromoꢀ1,2ꢀdimethoxyꢀ3ꢀselenocyanatobenzene (6). To a stirred solution of 5 (232
mg, 1mmol) in 10% HCl (2.5 mL) at 0 °C, sodium nitrite (83 mg, 1.2 mmol) was
added and the reaction mixture was kept below 5 °C. After completion of the reaction,
saturated sodium acetate was added to adjust pH at 6.0. Then, the solution of
potassium selenocyanate (220 mg, 1.5 mmol) in water (1 mL) was added in dropwise.
The reaction mixture was stirred at rt for 1 h, and the resulting precipitate was filtered
off, washed with water, and dried under vacuum to give the product, orange solid, 78%
1
yield. H NMR (400 MHz, CDCl ) δ 7.33 (d, J = 2.0 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H),
3
3.90 (s, 3H), 3.87 (s, 3H).
1
ꢀ(5ꢀbromoꢀ2,3ꢀdimethoxyphenyl)ꢀ2ꢀ(4ꢀbromoꢀ2,6ꢀdimethoxyphenyl)diselane
KOH (112 mg, 2 mmol) was added to a solution of 6 (321 mg, 1 mmol) in MeOH
5mL). The mixture was stirred for 1h, and the resulting orangeꢀyellow precipitate
(7).
(
was filtered off, washed with MeOH, and dried under vacuum to give the product,
1
orangeꢀyellow solid, 72% yield. H NMR (400 MHz, CDCl
3
) δ 7.23 (d, J = 2.2 Hz,
1H), 6.92 (d, J = 2.1 Hz, 1H), 3.92 (s, 3H), 3.85 (s, 3H).
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(
5ꢀbromoꢀ2,3ꢀdimethoxyphenyl)(methyl)selane (
8
). To a solution of diselenide 6
(
590 mg, 1 mmol) in ethanol/water (1:1, 8 mL), NaBH
4
(76 mg, 2 mmol) was added
in portions, and the mixture was stirred for 20 min. Iodomethane (141 mg, 1 mmol)
was added and the reaction was stirred at rt for 1 h. After completion of the reaction,
water was added, and the mixture was extracted with ethyl acetate for three times. The
organic phase was washed with brine and dried over anhydrous sodium sulfate. The
evaporation of the solvents gave the crude products which were purified by silica gel
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1
column (PE/EtOAc, 10:1) to afford 8, colorless oil, 89% yield. H NMR (400 MHz,
CDCl ) δ 6.88 (q, J = 2.2 Hz, 2H), 3.84 (s, 3H), 3.83 (s, 3H), 2.26 (s, 3H).
3
20
General Procedures for the Preparation of 9a-i. Under an argon atmosphere, 8
(403 mg, 1.3 mmol) was dissolved in dry THF (20 mL) and then the solution was
cooled to ꢀ78 °C. NꢀBuLi (1.3 mmol) was slowly added to the mixture and stirred for
0.5 h. Then, substituted benzaldehyde (1 mmol) in THF solution was added in
dropwise. After stirred for 12 h, the reaction was quenched by the addition of
saturated ammonium chloride solution, and extracted with ethyl acetate. The organic
phase was dried over anhydrous sodium sulfate, and the solvent was removed under
vacuum. The residue was purified by flash column chromatography (PE/EtOAc, 3:1)
to give the according products.
5
ꢀ((3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(hydroxy)methyl)ꢀ2ꢀmethoxyphenol (9a).
1
Colorless oil, yield: 69%. H NMR (400 MHz, CDCl
3
) δ 6.92 (ddd, J = 20.1, 2.0, 1.1
Hz, 2H), 6.84 (ddd, J = 7.3, 2.0, 1.0 Hz, 1H), 6.79 (dd, J = 2.1, 1.0 Hz, 1H), 6.67 (d, J
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=
7.5 Hz, 1H), 5.98 – 5.94 (m, 1H), 5.92 (s, 1H), 3.90 (s, 3H), 3.85 (s, 6H), 2.22(s,
3
H).
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3ꢀfluoroꢀ4ꢀmethoxyphenyl)methanol (9b).
0
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0
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9
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9
0
1
Colorless oil, yield: 69%. H NMR (400 MHz, CDCl ) δ 7.05 (ddd, J = 7.5, 1.9, 0.9
3
Hz, 1H), 6.94 (dd, J = 2.0, 1.1 Hz, 1H), 6.92 – 6.86 (m, 2H), 6.86 – 6.80 (m, 1H),
5
2
.98 – 5.92 (m, 1H), 4.50 (d, J = 4.9 Hz, 1H), 3.90 (s, 3H), 3.84 (d, J = 4.9 Hz, 6H),
.23 (s, 3H).
(3ꢀchloroꢀ4ꢀmethoxyphenyl)(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)methanol (9c).
1
Colorless oil, yield: 68%. H NMR (400 MHz, CDCl ) δ 7.40 (d, J = 2.1 Hz, 1H),
3
7
.21 (dd, J = 8.6, 2.2 Hz, 1H), 6.89 (d, J = 8.3 Hz, 1H), 6.77 (dd, J = 18.3, 1.7 Hz,
2H), 5.71 (d, J = 3.4 Hz, 1H), 3.89 (s, 3H), 3.85 (s, 3H), 3.83 (s, 3H), 2.24 (s, 3H).
(3ꢀbromoꢀ4ꢀmethoxyphenyl)(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)methanol (9d)
1
Colorless oil, yield: 69%. H NMR (400 MHz, CDCl
3
) δ 7.37 (dd, J = 2.0, 1.0 Hz,
1
H), 7.22 (ddd, J = 7.5, 2.0, 1.0 Hz, 1H), 6.98 – 6.88 (m, 3H), 5.98 – 5.92 (m, 1H),
3.90 (d, J = 4.9 Hz, 6H), 3.85 (s, 3H), 2.22 (s, 3H).
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(4ꢀmethoxyphenyl)methanol
(9e).
1
Colorless oil, yield: 69%. H NMR (400 MHz, CDCl
3
) δ 7.31 – 7.26 (m, 2H), 6.92 –
6
.84 (m, 2H), 6.83 – 6.73 (m, 2H), 5.74 (d, J = 3.4 Hz, 1H), 2.22 (s, 3H).
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(2,3,4ꢀtrimethoxyphenyl)methanol
(9f).
1
Colorless oil, yield: 68%. H NMR (400 MHz, CDCl
3
) δ 6.91 (d, J = 8.6 Hz, 1H), 6.81
(d, J = 5.2 Hz, 2H), 6.64 (d, J = 8.5 Hz, 1H), 5.89 (d, J = 5.7 Hz, 1H), 3.85 (d, J = 5.2
Hz, 9H), 3.82 (s, 3H), 3.76 (s, 3H), 2.21 (s, 3H).
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(
3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3,4ꢀdimethoxyphenyl)methanol
(9g).
1
Colorless oil, yield: 68%. H NMR (400 MHz, CDCl
3
) δ 6.99 – 6.92 (m, 3H), 6.90
(
dd, J = 2.1, 1.1 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 5.98 – 5.92 (m, 1H), 3.92 – 3.83
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(
m, 12H), 2.22 (s, 3H).
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3,4,5ꢀtrimethoxyphenyl)methanol
(9h).
1
Colorless oil, yield: 71%. H NMR (400 MHz, CDCl
3
) δ 6.92 (ddd, J = 20.2, 2.0, 1.1
Hz, 2H), 6.62 (d, J = 0.9 Hz, 2H), 5.98 – 5.92 (m, 1H), 3.90 (s, 3H), 3.84 (d, J = 4.9
Hz, 12H), 2.24 (s, 3H).
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(4ꢀmethoxyꢀ3ꢀmethylphenyl)methanol (9i).
1
Colorless oil, yield: 70%. H NMR (400 MHz, CDCl
3
) δ 7.14 (d, J = 7.3 Hz, 2H),
6.82 (d, J = 1.7 Hz, 1H), 6.78 (d, J = 7.0 Hz, 2H), 5.71 (d, J = 3.1 Hz, 1H), 3.84 (s,
H), 3.82 (d, J = 1.8 Hz, 6H), 2.23 (s, 3H), 2.20 (s, 3H).
3
21
General procedures for the preparation of 10a-i. 2ꢀiodoxybenzoic acid (560
mg, 2 mmol) was added in portions to a solution of 9 (1 mmol) in THF (10 mL). The
reaction mixture was allowed to stirred for 3h, and the resulting precipitate was
filtered off. The filtrate was concentrated and purified by flash column
chromatography (PE/EtOAc, 3:1) to give the target compound.
(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3ꢀhydroxyꢀ4ꢀmethoxyphenyl)methanone
1
(10a). White solid, yield: 78%. H NMR (400 MHz, CDCl ) δ 7.45 (d, J = 2.1 Hz, 1H),
3
7
.39 (dd, J = 8.3, 2.1 Hz, 1H), 7.21 (q, J = 1.9 Hz, 2H), 6.92 (d, J = 8.4 Hz, 1H), 5.76
13
(d, J = 1.0 Hz, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.89 (s, 3H), 2.24 (s, 3H). C NMR
(100 MHz, CDCl
3
) δ 194.5, 151.8, 150.3, 149.5, 145.3, 134.5, 131.0, 127.6, 123.8,
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+
1
22.1, 116.2, 111.5, 109.7, 60.2, 56.13, 56.05, 5.1. HRMS (ESI) (m/z) [M+H] calcd
18 5
for C17H O
Se, 383.0393; found, 383.0408. Purity: 96.1% (by HPLC).
(
3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3ꢀfluoroꢀ4ꢀmethoxyphenyl)methanone
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
2
3
4
5
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8
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0
1
(
10b). White solid, yield: 76%. H NMR (400 MHz, CDCl ) δ 7.65 – 7.57 (m, 2H),
3
7
2
1
.23 – 7.17 (m, 2H), 7.03 (t, J = 8.4 Hz, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.90 (s, 3H),
13
3
.24 (s, 3H). C NMR (100 MHz, CDCl ) δ 193.3, 152.5, 151.0, 150.6, 149.7, 134.0,
30.4, 127.8, 127.5, 122.1, 117.8, 112.2, 111.5, 60.2, 56.3, 56.1, 5.1. HRMS (ESI)
+
(m/z) [M+H] calcd for C17
H
17
O
4
FSe, 385.0350; found, 385.0368. Purity: 96.3% (by
HPLC).
(
3ꢀchloroꢀ4ꢀmethoxyphenyl)(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)methanone
1
(10c). White solid, yield: 79%. H NMR (400 MHz, CDCl
3
) δ 7.90 (d, J = 2.2 Hz, 1H),
7
.74 (dd, J = 8.6, 2.1 Hz, 1H), 7.23 – 7.16 (m, 2H), 7.00 (d, J = 8.6 Hz, 1H), 4.00 (s,
13
3H), 3.96 (s, 3H), 3.90 (s, 3H), 2.24 (s, 3H). C NMR (100 MHz, CDCl
3
) δ 193.1,
1
58.4, 152.0, 149.9, 133.9, 132.3, 130.9, 130.5, 127.8, 122.7, 122.2, 111.6, 111.1,
+
17 4
60.2, 56.4, 56.1, 5.1. HRMS (ESI) (m/z) [M+H] calcd for C17H O ClSe, 401.0052;
found, 401.0068. Purity: 97.3% (by HPLC).
(3ꢀbromoꢀ4ꢀmethoxyphenyl)(3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)methanone
1
(10d).White solid, yield: 81%. H NMR (400 MHz, CDCl
3
) δ 8.07 (d, J = 2.1 Hz, 1H),
7.78 (dd, J = 8.5, 2.1 Hz, 1H), 7.24 – 7.15 (m, 2H), 6.97 (d, J = 8.6 Hz, 1H), 3.99 (s,
13
3
H), 3.96 (s, 3H), 3.90 (s, 3H), 2.24 (s, 3H). C NMR (100 MHz, CDCl
3
) δ 193.0,
159.3, 152.0, 149.9, 135.4, 133.9, 131.34, 131.25, 127.9, 122.3, 111.7, 111.6, 111.0,
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7
8
9
+
6
0.2, 56.5, 56.1, 5.1. HRMS (ESI) (m/z) [M+H] calcd for C17
H
17
O
4
SeBr, 444.9546;
found, 444. 9564. Purity: 96.5% (by HPLC).
(
3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(4ꢀmethoxyphenyl)methanone (10e). White
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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45
46
47
48
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56
57
58
59
60
1
solid, yield: 78%. H NMR (400 MHz, CDCl3) δ 7.82 (d, J = 8.4 Hz, 2H), 7.24 – 7.16
(
m, 2H), 6.97 (d, J = 8.5 Hz, 2H), 3.95 (s, 3H), 3.89 (d, J = 1.4 Hz, 6H), 2.23 (s,
13
3
H). C NMR (100 MHz, CDCl
3
) δ 194.3, 163.2, 151.8, 149.4, 134.6, 132.4, 130.2,
+
1
27.6, 122.1, 113.6, 111.5, 60.1, 56.0, 55.5, 5.0. HRMS (ESI) (m/z) [M+H] calcd for
17 18 4
C H O Se, 367.0444; found, 367. 0455. Purity: 95.4% (by HPLC).
(
3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(2,3,4ꢀtrimethoxyphenyl)methanone (10f).
1
White solid, yield: 80%. H NMR (400 MHz, CDCl
3
) δ 7.30 (d, J = 1.7 Hz, 1H), 7.23
(d, J = 1.7 Hz, 1H), 7.11 (d, J = 8.6 Hz, 1H), 6.73 (d, J = 8.6 Hz, 1H), 3.94 (d, J = 3.0
13
Hz, 6H), 3.90 (s, 3H), 3.88 (s, 3H), 3.79 (s, 3H), 2.20 (s, 3H). C NMR (100 MHz,
CDCl
3
) δ 194.1, 156.1, 152.6, 151.8, 150.3, 142.1, 134.6, 127.4, 126.3, 124.9, 122.8,
+
1
11.2, 106.7, 61.9, 61.0, 60.2, 56.1, 56.0, 5.0. HRMS (ESI) (m/z) [M+H] calcd for
19 22 6
C H O Se, 427.0655; found, 427.0670. Purity: 98.3% (by HPLC).
(
3,4ꢀdimethoxyꢀ5ꢀ(methylselanyl)phenyl)(3,4ꢀdimethoxyphenyl)methanone (10g).
1
White solid, yield: 78%. H NMR (400 MHz, CDCl
3
) δ 7.47 (s, 1H), 7.44 – 7.36 (m,
1
H), 7.22 (s, 2H), 6.91 (d, J = 8.3 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 6H), 3.90 (s, 3H),
13
2.25 (s, 3H). C NMR (100 MHz, CDCl ) δ 194.3, 153.0, 151.8, 149.4, 148.9, 134.6,
3
1
30.2, 127.6, 125.2, 122.0, 112.2, 111.5, 109.8, 60.1, 56.5, 56.1, 56.0, 5.0. HRMS
+
(ESI) (m/z) [M+H] calcd for C18
H
20
O
5
Se, 397.0550; found, 397.0571. Purity: 95.6%
(by HPLC).
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