Efficient synthesis of new antiproliferative steroidal hybrids using the molecular hybridization approach

Article abstract of DOI:10.1016/j.ejmech.2016.04.024

A series of steroidal hybrids with different terminal bioactive scaffolds were synthesized using the molecular hybridization approach and further evaluated for their antiproliferative activity against several cancer cell lines of different origins using t

Full text of DOI:10.1016/j.ejmech.2016.04.024

Accepted Manuscript
Efficient synthesis of new antiproliferative steroidal hybrids using the molecular
hybridization approach
Bin Yu, Ping-Ping Qi, Xiao-Jing Shi, Ruilei Huang, Hao Guo, Yi-Chao Zheng, De-
Quan Yu, Hong-Min Liu
PII:
S0223-5234(16)30309-9
10.1016/j.ejmech.2016.04.024
EJMECH 8539
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 November 2015
Revised Date: 14 March 2016
Accepted Date: 8 April 2016
Please cite this article as: B. Yu, P.-P. Qi, X.-J. Shi, R. Huang, H. Guo, Y.-C. Zheng, D.-Q. Yu, H.-M. Liu,
Efficient synthesis of new antiproliferative steroidal hybrids using the molecular hybridization approach,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.04.024.
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ACCEPTED MANUSCRIPT
Steroidal hybrids were synthesized and evaluated for their antiproliferative activity. Compound
12g potently inhibited growth of SH-SY5Y cells possibly through the inactivation of LSD1,
arrested cell cycle at G2/M phase, induced apoptosis and decreased MMP. Docking simulations
were performed to rationalize the potency toward LSD1.

ACCEPTED MANUSCRIPT
Efficient synthesis of new antiproliferative steroidal hybrids using the molecular
hybridization approach
Bin Yua, †, Ping-Ping Qia, †, Xiao-Jing Shia, †, Ruilei Huangb, Hao Guoc, Yi-Chao Zhenga, De-Quan Yua, *
Hong-Min Liua,
,
a
School of Pharmaceutical Sciences & Collaborative Innovation Center of New Drug Research
and Safety Evaluation, Zhengzhou University, Zhengzhou 450001, China
b School of Pharmaceutical Sciences, Central South University, Changsha 410000, China
c Chongqing Institute of Forensic Science, Chongqing 400021, China
Abstract: A series of steroidal hybrids with different terminal bioactive scaffolds were synthesized
using the molecular hybridization approach and further evaluated for their antiproliferative
activity against several cancer cell lines of different origins using the MTT assay. The preliminary
results indicated that compounds 12a-h with the terminal isatin motif were remarkably sensitive
to SH-SY5Y cells, thereby exerting potent growth inhibition in vitro. This selectivity is possibly
attributed to LSD1 inactivation (IC50 = 3.18 µM). Besides, we also found that the chloro atom at
the 7-position on the isatin core was beneficial for the activity through the SARs studies. Among
this series, compound 12g showed the best inhibitory activity (IC50 = 4.06 µM) against SH-SY5Y
cells, which was comparable to that of 5-Fu. Compound 12g arrested cell cycle at G2/M phase,
induced apoptosis accompanied with decrease of mitochondrial membrane potential, and
inhibited LSD1 potently (IC50 = 3.18 µM). Docking studies showed that compound 12g formed
interactions with surrounding amino acid residues and the steroid nucleus occupied the tubular
hydrophobic cavity of the active site. Compounds 13-18 represented weak to moderate activity
against the tested cancer cell lines. The steroidal dimer 20 and the structurally simplified
non-steroidal dimer 21 were found to be devoid of the inhibitory activity.
Keywords: Steroids; Molecular hybridization; Antiproliferative activity; Apoptosis; Cell cycle arrest;
LSD1 inactivation; Docking simulations
1. Introduction
The molecular hybridization generally refers to the incorporation of two or more bioactive
fragments into one molecule through suitable linkers. These new hybrids are always endowed
with improved activity or new biological properties compared to their individual components.
Molecular hybridization, as an emerging concept in drug discovery, has recently gained increasing
attention among medicinal community with several successful examples reported by different
groups (Fig. 1) [1]. For example, the hybridization of indolinone with tyrosine kinase inhibitor
Axitinib developed by Pfizer yielded the first potent PLK4 inhibitor CFI-400945, which has
advanced into phase I clinical trials for cancer therapy [2, 3]. Besides, Solaja et al. recently
reported that steroidal 4-amino quinolines are potent, dual-target inhibitors of the botulinum
neurotoxin serotype A metalloprotease and P. falciparum Malaria (Ki = 0.103 µM) [4]. Based on
the principle of molecular hybridization, our group recently synthesized two types of selective
Corresponding authors: Prof. Hong-Min Liu (liuhm@zzu.edu.cn); De-Quan Yu (dqyu@imm.ac.cn); †These
authors contributed equally to this work.
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LSD1 inhibitors, namely the 1, 2, 3-triazole-dithiocarbamates [5, 6] and pyrimidine-thiourea
hybrids [7] as orally active anticancer candidates (IC50 = 0.89 and 4.01 µM against MGC-803,
respectively).
Fig. 1 Representative bioactive molecules designed based on the molecular hybridization
principle
Dehydroepiandrosterone (abbreviated as DHEA), an endogenous steroid secreted by the adrenal
cortex, is able to inhibit the proliferation of human cancer cells in vitro and in vivo by targeting
PI3K/Akt signaling pathway [8]. Given its anticancer potential and readily commercial availability,
DHEA was chosen as a starting point for designing new steroidal hybrids with anticancer
properties. In order to increase the structural diversity and further investigate the structural
requirements for better potency, several different well-known bioactive scaffolds, such as isatins
[9-11], chalcones [12] and coumarins [6], were incorporated into the designed hybrids through
the molecular hybridization approach. In addition, steroidal hybrids with the terminal phenoxy
groups and anilines were also synthesized to compare the activity. As for the spacers, several
linkers like polyethylene glycol (PEG) [13], dithiocarbamate [12, 14] and 1, 2, 3-triazole [15, 16]
have been extensively used in drug design and, more importantly, have been proved to be crucial
for the activity [13, 15]. We herein employed the triazole bridge to link DHEA and biologically
active scaffolds mentioned above to generate new steroidal hybrids considering the diverse
biological properties and synthetic accessibility of triazoles. We previously reported a novel
[1,2,4] triazolo [1,5-a] pyrimidine-based phenyl-linked steroid dimer, which represented excellent
antiproliferative activity against human cancer cell lines and low toxicity toward human normal
cells [17]. As a follow-up work, we designed another new steroid dimer as well using the
5-fluorouracil (5-FU) as the bioactive center based on the molecular hybridization approach. As
part of our efforts toward finding new steroidal derivatives with anticancer activity [18-23] , this
design, as shown in Fig. 2, would help us gain a deeper insight into the structural requirements
for identifying potent steroid-based anticancer agents.
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Fig. 2 Designed steroidal hybrids in this work
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of dehydroandrosterone azidoacetate 2
The target molecules can be synthesized from the corresponding alkynes and steroidal azide 2
through the click chemistry. The synthesis of the steroidal azide 2 is shown in Scheme 1.
Bromoacetylation of DHEA under Schotten-Baumann conditions [24] afforded the corresponding
bromide 1 in 87 % yield, which then reacted with NaN3 in DMSO to give the steroidal azide 2 in
85 % yield. The steroidal azide 2 was used directly for next step after simple aqueous work-up
procedure. In the first step, a two-phase solvent system (DCM/H2O) was used; the base NaHCO3
in aqueous phase neutralized the generated HBr, while the starting materials and the product
were in the organic phase. Besides, this condition avoided unwanted side reactions of bromide 1
with H2O.
Scheme 1 Synthesis of the steroidal azide 2. (a) Bromoacetyl bromide, NaHCO3, DCM/H2O (1/1), rt;
(b) NaN3, DMSO, rt-50 oC.
2.1.2. Synthesis of alkyne building blocks 5-11
To increase the structural diversity and further explore the structural requirements, the
divergent synthesis focusing on the variation of bioactive scaffolds was employed. Considering
the broad and promising bioactivities of chalcones [25-28], coumarins [29-31] and isatin
derivatives [9, 10, 32], these scaffolds as illustrated in Fig. 2 were introduced to the steroid
nucleus using the molecular hybridization approach. Besides, the phenoxy group and anilines
were also introduced as the terminal motifs with an aim of comparing the activity with those
hybrids having different bioactive scaffolds. Another consideration of this design is that phenoxy
groups and anilines could act differently a H-bond acceptors and donors, respectively to interact
with potential targets. Such design could provide us more details about the structural
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requirements for better potency, ultimately helping us design more potent steroid-based
anticancer agents.
The synthesis of alkyne building blocks is shown in Fig. 3. Treatment of 4-hydroxybenzaldehyde
and 4-hydroxyacetophenone with propargyl bromide in the presence of K2CO3 gave compounds
3 and 4, respectively, which then underwent the KF/Al2O3-catalyzed Claisen-Schmidt
condensations with aromatic aldehydes and acetophenones, affording substituted chalcones
5a-e and 6a-b. Similarly, propargyl substituted isatins 7a-h, coumarins 8a-c, phenols 9a-c and
anilines 10a-c were conveniently synthesized in the presence of K2CO3 in DMF or acetone. In
order to construct 5-Fu linked steroid dimer, dipropargyl substituted 5-Fu 11 was also
synthesized.
o
Fig. 3 Synthesis of alkyne building blocks. (a) K2CO3, DMF, rt; (b) K2CO3, acetone, rt-50 C; (c)
Al2O3/KF, EtOH, 50 oC.
2.1.3. Synthesis of steroidal hybrids 12-18
With the azide and alkyne building blocks in hand, the designed steroidal hybrids were efficiently
synthesized through the standard CuAAC reactions in moderate to good yields (Scheme 2).
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O
O
H
H
a
N
N
N
O
+
O
H
H
H
H
Alkyne
building block
N3
O
O
Steroidal hybrids
O
O
12a R = H
12b R = 5-Cl
12d R = 5-F
12f R = 4-Br
12h R = 7-Cl
O
12c R = 5-Br
12e R = 4-Cl
N
N
N
N
O
12g R = 4,7-diCl
R
O
O
O
X
13a X = H
13b X = F
N
O
N
N
O
O
R
O
14a R = 4-Br
14c R = 4-Me
14e R = 4-NO2
14b R = 3,4-diCl
14d R = 3,4-diF
N
O
N
O
N
O
O
O
O
X
N
N
O
N
N
O
O
N
N
O
O
O
O
O
O
O
15a X = H
15b X = Me
16
O
O
R
R
N
N
O
NH
N
O
N
N
O
N
O
O
17a = 2-NO2 17b= 4-Br 17c = 4-Me
18a = 4-MeO 18b = 4-Br 18c = 3-NO2
Scheme 2 Synthesis of steroidal hybrids. (a) CuSO4·5H2O, sodium ascorbate, THF/H2O (1/1), rt.
2.1.4. Synthesis of dimers 20 and 21
We previously found that steroid dimers possessed excellent antiproliferative activity and low
toxicity to normal cells [17]. Inspired by this finding, we also synthesized a new steroid dimer 20
using the 5-Fu as the bioactive center. Besides, structurally simplified compound 21 was
synthesized as well from compound 11 and 2-chlorobenzyl azide 19 to compare the
antiproliferative activity (Scheme 3).
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Scheme 3 Synthesis of dimers 20 and 21. (a) CuSO4·5H2O, sodium ascorbate, THF/H2O (1/1), rt.
2.2. Antiproliferative activity
In continuation with our efforts toward the identification of novel steroidal compounds with
anticancer potential, we evaluated the antiproliferative activity of steroidal hybrids 12a-h, 13a-b,
14a-e, 15a-b, 16, 17a-c, 18a-c, steroid dimer 20 and compound 21 against several cancer cell
lines of different origins (MCF-7, U87, SH-SY5Y, MGC-803 and EC109) using the MTT assay. The
well-known anticancer drug 5-fluorouracil (5-Fu) works principally through irreversible inhibition
of thymidylate synthase, while some steroidal derivatives such as estradiol, progesterone, DHEA,
testosterone have also been reported to be able to markedly inhibit thymidylate synthase [33].
Due to the similar mode of action, 5-Fu was used as the reference drug in the MTT assay. The
preliminary results are listed in Table 1.
Table 1 In vitro antiproliferative activity of steroidal hybrids
IC50 (µM)
Compound
MCF-7
> 128
> 128
> 128
> 128
> 128
U87
SH-SY5Y
MGC-803
> 128
EC109
12a
12b
12c
12d
12e
45.61±1.66
47.89±1.68
39.21±1.59
91.13±1.96
33.07±1.52
8.62±0.94
7.32±0.87
10.3±1.01
9.25±0.97
18.51±1.27
> 128
57.77±1.32
32.49±1.51
58.85±1.77
> 128
61.72±1.79
44.11±1.65
> 128
123.99±2.09
12f
12g
12h
13a
13b
> 128
21.31±0.58
9.57±0.98
20.82±1.32
> 128
> 128
26.38±1.42
5.95±0.77
8.74±0.94
> 128
78.35±1.31
20.77±1.73
84.61±1.93
117.59±2.07
> 128
32.25±1.51
13.29±1.12
> 128
4.06±0.61
14.6±1.16
75.0±1.88
46.45±1.67
91.47±1.97
> 128
> 128
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14a
111.88±2.05
> 128
> 128
72.56±1.86
36.64±1.56
50.06±1.70
42.07±1.62
> 128
> 128
79.81±1.33
> 128
14b
14c
14d
14e
15a
15b
16
> 128
> 128
73.34±1.87
92.58±1.97
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
99.46±2.00
> 128
> 128
> 128
43.71±1.64
47.43±1.68
40.02±1.6
> 128
54.85±1.74
49.47±1.70
> 128
> 128
> 128
> 128
> 128
> 128
> 128
> 128
17a
17b
17c
18a
18b
18c
20
45.12±1.65
93.18±1.97
38.6±1.59
21.27±1.05
27.6±1.39
45.2±1.12
> 128
65±1.81
51.34±1.71
48.23±1.68
21.61±1.34
11.56±1.06
19.42±1.35
28.62±1.46
> 128
49.84±1.70
20.99±1.32
23.89±1.38
24.73±0.85
37.12±1.43
55.43±1.84
> 128
30.13±1.48
18.46±1.14
96.53±1.42
86.72±0.89
> 128
22.72±1.36
26.95±1.43
69.76±1.84
59.41±1.77
> 128
> 128
> 128
21
> 128
> 128
> 128
> 128
> 128
5-Fu
7.61±1.31
5.61±0.37
3.26±0.46
1.25±0.22
3.17±0.43
As shown in Table 1, these hybrids displayed different antiproliferative activities against different
cancer cell lines with the IC50 values ranging from 4.06 to > 128 µM. For compounds 12a-h with
the terminal isatin scaffold, their antiproliferative activities varied significantly toward different
cancer cell lines. For SH-SY5Y cells, compounds 12a-e and 12g-h showed excellent inhibition of
cell growth (IC50 < 20 µM), a slight difference in inhibiting growth of SH-SY5Y cells was observed
for compounds with different substitution patterns. Among them, compound 12g with the 4, 7
-dichloro group showed the best antiproliferative activity with an IC50 value of 4.06 µM, which
was comparable to that of 5-Fu (IC50 = 3.26 µM), while compounds 12e (4-Cl) and 12h (7-Cl)
exhibited slightly decreased inhibitory activity with the IC50 values of 18.51 and 14.6 µM,
respectively. However, compound 12f with a bromine atom at the 4-positin was found to be
devoid of the activity (IC50 > 128 µM). Besides, compounds 12g-h showed moderate to good
antiproliferative activity against the tested cancer cell lines. By contrast, compounds 12a-f acted
differently toward different cancer cell lines. Most of these compounds had moderate inhibitory
activity against U87, MGC-803 and EC109 cells, while no activity was observed toward MCF-7
cells for compounds 12a-f (IC50 > 128 µM). This observation suggests that these compounds may
act through targeting specific biological targets that are overexpressed in certain cells. By
comparing the activity of compounds 12e and 12g-h, we may conclude that 7-chloro or 4,
7-dichloro substituent is beneficial for improving the activity, at least against SH-SY5Y cells.
Another interesting finding is that SH-SY5Y cells were more sensitive to compounds 12a-h, which
is probably attributed to the isatin motif. This observation is consistent with the recent findings
by Hou et al. [34, 35]. They reported that isatin itself can significantly inhibit the growth of
SH-SY5Y cells in vitro and in vivo and induce apoptosis via the mitochondrial pathway. In addition
to aforementioned optimizations, further modifications could be carried out to design more
potent compounds based on the high reactivity of C3 carbonyl group of isatin. This finding
together with the preliminary mechanism reported by Hou et al. [34, 35] would provide a basis
for designing more potent molecules selectively targeting SH-SY5Y cells.
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By contrast, compounds 13a-b and 14a-e with the terminal chalcone scaffold in place of the isatin
motif showed moderate but remarkably decreased inhibitory activity against SH-SY5Y cells with
the IC50 values more than 36.64 µM compared to compounds 12a-h. Compounds 13a-b and
14a-e exerted weak or no activity toward other cancer cell lines regardless of their substituent
patterns and the nature of substituents. Similarly, compounds 15a-b and 16 with the terminal
coumarin scaffold exhibited moderate inhibitory activity against U87 and SH-SY5Y cells with the
IC50 values ranging from 40.02 to > 128 µM, while no activity was observed for MCF-7, MGC-803
and EC109 cells (IC50 > 128 µM). The difference may be attributed to the poor membrane
permeability of compounds with the chalcone and coumarin scaffolds. Interestingly, when the
phenoxy and aniline groups were introduced instead of the well-known bioactive chalcone and
coumarin scaffolds, compounds 17 and 18 showed significantly improved growth inhibition
toward MCF-7, MGC-803 and EC109 cells. Among them, compound 18a was the most potent one
with an IC50 value of 11.56 µM against MGC-803. However, compounds 18a-c with the terminal
aniline group exhibited weak or no activity against U87 and SH-SY5Y cells.
5-Fu is a frequently used chemotherapeutic agent for the treatment of solid tumors acting
through inhibiting thymidylate synthase. However, its severe side effects such as the high toxicity,
poor tumor selectivity, etc, to some extent, have restricted its clinical applications [36].
Therefore, the design and synthesis of new 5-Fu derivatives is necessary to reduce the side
effects, while retaining its anticancer potency. There is evidence that N1 and/or N3 substituted
5-Fu derivatives represent improved antitumor activity and lower toxicity [37]. Additionally,
DHEA has also been proved to be able to markedly inhibit thymidylate synthase [33]. So the
incorporation of DHEA and 5-Fu into one molecule through suitable linkage would be an effective
strategy to design new hybrids with synergistic antitumor effects. On the basis of our previous
observations that steroid dimers have excellent antiproliferative activity against cancer cell lines
and low toxicity toward non-cancerous cells [17], herein we designed another new steroidal
dimer 20, incorporating 5-Fu and DHEA into one molecule through the triazole linkage. In order
to compare the activity, the non-steroidal dimer 21 was also designed and synthesized using the
2-nitrophenyl group in place of DHEA nucleus. Unfortunately, these two dimers 20 and 21 did
not show any inhibitory activity toward the tested cancer cells regardless of their substituents
attached (IC50 > 128 µM). This finding may suggest that it is not a feasible strategy to design
potent anticancer agents through installing the triazole group to both nitrogen atoms of 5-Fu.
From above discussions, we can conclude that most of these hybrids exhibited weak to moderate
or even no activity against the tested cell lines. However, we observed that compounds 12a-h
with the terminal isatin core were more sensitive to SH-SY5Y cells with excellent inhibitory
activity. Among them, compound 12g was the most potent one with an IC50 value of 4.06 µM.
These findings provided us some useful information regarding the structural requirements for
better potency and would help us design more potent small molecules selectively targeting
SH-SY5Y cells.
2.3. Analysis of cell cycle distribution
Tumor cells, relative to normal cells, always grow uncontrollably because of the overexpression of
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cyclins or inactivation of critical CDKIs. Targeting the cell cycle of tumor cells has been recognized
as a promising strategy for cancer therapy [38]. To determine the effect of our designed steroidal
hybrids on the cell cycle, compound 12g was chosen for this purpose considering its excellent
growth inhibition toward SH-SY5Y cells. After treating SH-SY5Y cells with compound 12g at
different concentrations (0, 1.25, 2.5, 5.0 µM) for 24 h, cells were then fixed and stained with PI
for flow cytometry analysis. As shown in Fig. 4B, compound 12g arrested cell cycle at G2/M phase
in a concentration-dependent manner, along with decrease of cells in other phases. A
representative example is shown in Fig. 4A. The percentage of cells at G2/M phase was increased
to 27.74 % at high concentration (5.0 µM) from 14.53 % for the control group.
Fig. 4 Effect of compound 12g on the cell cycle distribution of SH-SY5Y cells. Cells were treated at
different concentrations (0, 1.25, 2.5, 5.0 µM) for 24 h. Then the cells were fixed and stained with
PI to analyze DNA content by flow cytometry. The experiments were performed three times. (A)
Representative example. (B) Quantitative analysis of cell cycle distribution induced by compound
12g. **P < 0.01 was considered statistically significant.
2.4. Apoptosis and possible mechanism involved
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The excellent inhibitory activity of compound 12g against SH-SY5Y cells promoted us to further
investigate its apoptotic effect by a biparametric cytofluorimetric analysis using propidium iodide
(PI) and Annexin V-FITC. After treating SH-SY5Y cells with compound 12g at different
concentrations (0, 1.25, 2.5, 5.0, 10.0 20.0 µM) for 24 h, cells were labeled with two dyes, and
then the flow cytometry was employed to detect the resulting red (PI) and green (FITC)
fluorescence. As illustrated in Fig. 5B, compound 12g induced apoptosis of SH-SY5Y cells in a
concentration-dependent manner, especially the late apoptosis. Specifically, the percentage of
apoptotic cells was about 9 % for the control group. When treated at 10 or 20 µM, a complete
apoptosis was observed with the percentage of apoptosis up to 98.7 % and 98.3 %, respectively.
The percentage of the late apoptosis was remarkably increased to 65.7 % when treated at 20 µM
from 5.7 % for the control group.
Fig. 5 Apoptotic effect of compound 12g on SH-SY5Y cells. Apoptotic cells were detected with
Annexin V-FITC/PI double staining after incubation at the indicated concentrations for 24 h. The
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lower left quadrants represent live cells, the lower right quadrants are for early apoptotic cells,
upper right quadrants are for late apoptotic cells, while the upper left quadrants represent cells
damaged during the procedure. (A) Representative experiment. (B) Quantitative analysis of
apoptotic cells. **P < 0.01 was considered statistically significant. The experiments were
performed three times.
The decrease of mitochondrial membrane potential (MMP) through multiple mechanisms
including deficiency of oxidizable substrates for the mitochondria, blockage of respiration, or
uncoupling of the inner membrane, followed by cytochrome c release from the mitochondria and
expression changes of apoptosis-related proteins (e.g. Bax, Bcl-2, p53, etc) have been observed
during apoptosis [39]. The remarkable apoptosis induced by compound 12g promoted us to
explore whether this compound had an effect on the MMP. SH-SY5Y cells were incubated at the
indicated concentrations for 24 h and then stained and analyzed by flow cytometry. As shown in
Fig. 6, compound 12g caused the MMP change in a concentration-dependent manner. The
percentage of cells at 10 µM was 40.1 %, significantly higher than that of the control group
(5.1 %). A similar result was obtained previously in our group, showing that steroids can decrease
the MMP, promote the cytochrome c release, and induce expression changes of related proteins
[17]. Therefore, we did not duplicate this work to analyze the expression of proteins by Western
blotting analysis but turned our attention to the selectivity observed toward SH-SY5Y cells.
Previously, Hou and co-workers proved that isatin can induce apoptosis and inhibit the growth of
SH-SY5Y neuroblastoma cells via the mitochondrial pathway [34, 35]. However, the detailed
molecular mechanism remains unclear. In SH-SY5Y cells, the lysine-specific demethylase 1 (LSD1)
is always overexpressed. Inhibition of LSD1 has been proved to be capable of inhibiting growth of
tumors expressing LSD1 in vitro and in vivo [40]. As part of our ongoing efforts toward identifying
novel LSD1 inactivators for cancer therapy [5-7, 41], we are interested in investigating whether
the observed selectivity of compounds 12a-h toward SH-SY5Y cells is due to the inhibition of
LSD1. To our delight, compound 12g inhibited LSD1 at low micromolar level (IC50 = 3.18 µM). This
finding suggests that compounds 12a-h may inhibit growth of SH-SY5Y cells by inactivating LSD1.
To the best of our knowledge, this is the first report about steroid-based LSD1 inhibitors. Further
mechanistic studies are ongoing in our laboratories and will be reported in due course.
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Fig. 6 Changes of mitochondrial membrane potential induced by compound 12g. SH-SY5Y cells
were incubated at the indicated concentrations for 24 h. The cells were stained with JC-1 and
then analyzed by flow cytometry.
2.5. Molecular docking studies
In view of the observed good inhibition of compound 12g toward LSD1, docking simulations were
performed to rationalize the potency using the MOE2014 software (PDB code: 2H94) [49]. As
shown in Fig. 7A, the C3 carbonyl oxygen of the isatin motif formed hydrogen bonds with Ala309
and Glu308 residues, while the chlorine atom attached to the C4 position of the isatin motif
formed C-Cl···H interaction with Val590, which may explain the observed importance of the C4
chlorine atom for the activity. Additionally, the phenyl ring of the isatin motif formed arene-H
interactions with Ala309 and Leu625, respectively. The triazole ring also contributed to the
activity by forming hydrogen bond with the backbone carbonyl oxygen of Thr624 and arene-H
interactions with Gly287 and Arg316 residues. As shown in Fig. 7B, the steroid nucleus of
compound 12g (shown in orange stick) well occupied the tubular hydrophobic cavity in the active
site of LSD1, while compound 22 (LSD1 IC50 > 10 µM) failed to occupy the hydrophobic cavity,
highlighting the importance of the steroid nucleus of compound 12g in the LSD1 inhibition and
also explaining the weak activity of compound 22. The docking results may help us gain a deeper
insight into the SARs studies and provide basis for further optimization.
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Fig. 7 (A) Predicted binding modes of compound 12g in the FAD-binding site of LSD1. FAD-binding
site residues are colored in gray while compound 12g is colored in blue and green, respectively.
Hydrogen bonds between the protein and compound 12g are shown in magenta dash line and
arene-H interactions are shown in cyan; (B) Putative binding modes of compound 12g (shown in
green stick) and compound 22 (shown in orange stick) in the active sites of LSD1 on the pocket
surface (green represents for hydrophobic area, blue represents for mild polar area and magenta
for H-bonding).
3. Conclusions
In summary, we designed and synthesized a series of steroidal hybrids with different terminal
bioactive scaffolds through the molecular hybridization strategy. The MTT assay showed that
most of these hybrids had weak to moderate activity against the tested cancer cell lines.
However, steroidal hybrids 12a-h with the terminal isatin moiety were more sensitive toward
SH-SY5Y cells possibly because of the inhibition of LSD1. Besides, compounds 12g-h with 4,
7-dichloro and 7-chloro groups, respectively also showed moderate to good inhibitory activity
against all the tested cancer cell lines. Compound 12g arrested cell cycle at G2/M phase, induced
apoptosis, accompanied the decrease of mitochondrial membrane potential, and inhibited LSD1
potently. Docking simulations indicated that the steroid nucleus played a determinant role in
LSD1 inactivation. Additionally, steroid dimer 20 and the structurally simplified compound 21 did
not show any inhibitory activity against the tested cancer cells. Further mechanistic studies on
the selectivity toward SH-SY5Y cells are ongoing in our laboratories and will be reported in due
course.
4. Experimental section
4.1. General
Reagents were purchased and used without further purification. Thin layer chromatography (TLC)
was carried out on glass plates coated with silica gel and visualized by UV light (254 nm). Melting
points were determined on an X-5 micromelting apparatus. All the NMR spectra were recorded
with a Bruker DPX 400 MHz spectrometer with TMS as the internal standard in CDCl3 or DMSO-d6.
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Chemical shifts are given as δ ppm values relative to TMS (Most of the peaks due to the steroidal
skeleton are merged and could not be differentiated. Thus, the δ values of only those peaks that
distinguish the product and could easily be differentiated are reported). High-resolution mass
spectra of all new compounds were recorded on a Waters Micromass Q-T of Micromass
spectrometer by electrospray ionization (ESI). CAUTION: Particular care should be taken to avoid
grinding sodium azide and acidifying mixtures containing sodium azide because the azide ion can
react with acids to form the extremely explosive, volatile and toxic hydrazoic acid.
Besides, sodium azide can also form highly explosive salts with many transition metals.
Halogenated solvents such as dichloromethane and chloroform should be avoided for sodium
azide-involved reactions and subsequent work-up procedures because such solvents can form
extremely explosive di- and triazidomethanes with sodium azide [42, 43].
4.2. Synthesis of bromoacetyl DHEA 1
To a solution of DHEA (1.0 g, 3.47 mmol, 1.0 eq) and NaHCO3 (582 mg, 6.94 mmol, 2.0 eq) in a
mixture of DCM (10 mL) and H2O (10 mL) was added bromoacetyl bromide (934 μL, 10.41 mmol,
3.0 eq) dropwise at room temperature. The mixture was then stirred for about 6 h. Upon
completion, the organic layer was separated and the aqueous later was extracted with DCM, the
combined organic layers were washed with brine and dried over MgSO4. The solvent was
removed under vacuum. The residue was then recrystallized from EtOH to afford the white solid,
1.24 g, yield: 87%, m. p.: 156.1-157.3 oC, Rf = 0.60 (petroleum ether/ethyl acetate = 2/1), 1H NMR
(400 MHz, CDCl3) δ 5.44 (d, J = 4.9 Hz, 1H), 4.70 (tdd, J = 10.7, 6.5, 4.2 Hz, 1H), 3.83 (s, 2H), 1.07 (s,
3H), 0.91 (s, 3H).13C NMR (100 MHz, CDCl3) δ 166.68, 139.47, 122.33, 75.85, 51.68, 50.10, 47.52,
37.73, 36.81, 36.71, 35.84, 31.45, 31.39, 30.78, 27.43, 26.34, 21.88, 20.33, 19.34, 13.55. HRMS
(ESI): m/z calcd for C21H29BrNaO3 (M + Na)+, 431.1198; found, 431.1180 (Br79) and 433.1161 (Br81)
4.3. Synthesis of azidoacetyl DHEA 2
To a solution of bromoacetyl DHEA (compound 1, 500 mg, 1.23 mmol, 1.0 eq) in DMSO (10 mL)
was added NaN3 (96 mg, 1.48 mmol, 1.2 eq) at room temperature, and then the mixture was
o
heated to 50 C and kept at this temperature for about 3 h. When cooled to room temperature,
EtOAc and H2O were added to the above mixture, the aqueous layer was extracted with EtOAc,
the combined layers were washed with H2O for several times to remove the DMSO, and then
washed with brine, dried over MgSO4 and evaporated to give the product, which was pure
enough and used directly for next step without further purification. White solid, 385 mg, yield:
o
1
85 %, m. p.: 144.9-146.5 C, Rf = 0.60 (petroleum ether/ethyl acetate = 2/1). H NMR (400 MHz,
CDCl3) δ 5.42 (d, J = 4.7 Hz, 1H), 4.72 (tt, J = 10.9, 5.3 Hz, 1H), 3.84 (s, 2H), 1.05 (s, 3H), 0.88 (s,
3H).13C NMR (100 MHz, CDCl3) δ 167.72, 139.41, 122.39, 75.61, 51.67, 50.50, 50.09, 47.51, 37.94,
36.82, 36.70, 35.83, 31.43, 31.38, 30.77, 27.63, 21.88, 20.32, 19.32, 13.55. HRMS (ESI): m/z calcd
for C21H29N3NaO3 (M + Na)+, 394.2107; found, 394.2095.
4.4. General procedure for the preparation of propargyl substituted isatin derivatives 7a-h
To a solution of isatin derivative (0.68 mmol, 1.0 eq) in DMF (4 mL) was added K2CO3 (0.68 mmol,
1.0 eq) at room temperature, the mixture was stirred for about 30 min, and then the propargyl
bromide (0.75 mmol, 1.1 eq) was added dropwise. The mixture was stirred for 6-10 h at room
temperature depending on the isatin derivative used. Upon completion, EtOAc and H2O were
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added. The aqueous layer was extracted with EtOAc; the combined organic layers were washed
with H2O for several times to remove the DMF, and then washed with brine, dried over MgSO4
and evaporated to give the products.
o
4.4.1. N-Propargyl isatin 7a, jacinth solid, yield: 82 %, m. p.: 159.3-160.5 C, Rf =0.59 (petroleum
ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.66 (dd, J = 12.3, 4.5 Hz, 2H), 7.25 – 7.06
(m, 2H), 4.54 (d, J = 2.5 Hz, 2H), 2.32 (t, J = 2.5 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 182.55,
157.17, 149.62, 138.46, 125.47, 124.22, 117.68, 111.12, 75.69, 73.36, 29.46.
4.4.2. N-Propargyl-5-chloroisatin 7b, saffron solid, yield: 75 %, m. p.: 168.4-169.7 oC, Rf =
1
0.61(Petroleum ether/ethyl acetate = 2/1). H NMR (400 MHz, CDCl3) δ 7.70 – 7.57 (m, 2H), 7.13
(dd, J = 7.9, 1.0 Hz, 1H), 4.56 (d, J = 2.5 Hz, 2H), 2.35 (t, J = 2.5 Hz, 1H).13C NMR (100 MHz, CDCl3)
δ 181.57, 156.60, 147.85, 137.83, 130.12, 125.36, 118.49, 112.48, 75.29, 73.77, 29.61.
o
4.4.3. N-Propargyl-5-bromoisatin 7c, red solid, yield: 86 %, m. p.: 160.3-162.0 C, Rf = 0.42
(petroleum ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.77 (m, 2H), 7.08 (dd, J = 7.9,
0.9 Hz, 1H), 4.55 (d, J = 2.5 Hz, 2H), 2.35 (t, J = 2.5 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 181.40,
156.42, 148.29, 140.67, 128.24, 118.84, 117.17, 112.87, 75.25, 73.80, 29.60.
o
4.4.4. N-Propargyl-5-fluoroisatin 7d, red solid, yield: 73 %, m. p.: 128.5-129.9 C, Rf = 0.60
(petroleum ether/ethyl acetate = 2/1).1H NMR (400 MHz, CDCl3) δ 7.47 – 7.31 (m, 2H), 7.14 (dd, J
= 8.5, 3.6 Hz, 1H), 4.55 (d, J = 2.5 Hz, 2H), 2.35 (t, J = 2.5 Hz, 1H).13C NMR (100 MHz, CDCl3) δ
182.02 (JC-F = 2.2 Hz), 159.54 (JC-F = 246.4 Hz), 156.94 (JC-F = 1.5 Hz), 145.68 (JC-F = 2.1 Hz), 124.85
(JC-F = 24.2 Hz), 118.28 (JC-F = 7.1 Hz), 112.54 (JC-F = 11.5 Hz), 112.38 (JC-F = 5.6 Hz), 75.41, 73.68,
29.58.
o
4.4.5. N-Propargyl-4-chloroisatin 7e, saffron solid, yield: 89 %, m. p.: 148.3-149.8 C, Rf = 0.31
(petroleum ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.58 (t, J = 8.1 Hz, 1H), 7.14 (d,
J = 8.2 Hz, 1H), 7.06 (d, J = 7.9 Hz, 1H), 4.56 (d, J = 2.5 Hz, 2H), 2.35 (t, J = 2.5 Hz, 1H).13C NMR
(100 MHz, CDCl3) δ 179.41, 156.27, 150.64, 138.62, 133.96, 125.79, 114.80, 109.38, 75.38, 73.69,
29.65.
o
4.4.6. N-Propargyl-4-bromoisatin 7f, yellow solid, yield: 89 %, m. p.: 153.1-154.9 C, Rf = 0.26
(petroleum ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.49 (t, J = 8.0 Hz, 1H), 7.33 (d,
J = 8.1 Hz, 1H), 7.11 (d, J = 7.9 Hz, 1H), 4.57 (d, J = 2.5 Hz, 2H), 2.34 (t, J = 2.5 Hz, 1H).13C NMR
(100 MHz, CDCl3) δ 179.90, 156.21, 151.10, 138.42, 128.93, 121.74, 116.52, 109.86, 75.36, 73.67,
30.94, 29.53.
4.4.7. N-Propargyl-4, 7-dichloroisatin 7g, saffron solid, yield: 76 %, m. p.: 151.8-153.3 oC, Rf = 0.52
(petroleum ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 8.7 Hz, 1H), 7.10 (d,
J = 8.7 Hz, 1H), 4.94 (d, J = 2.4 Hz, 2H), 2.34 (t, J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 178.68,
157.00, 146.19, 140.48, 133.05, 126.79, 117.16, 116.11, 77.22, 73.27, 31.94.
4.4.8. N-Propargyl-7-chloroisatin 7f, brown solid, yield: 88 %, m. p.: 151.7-152.4 oC, Rf =
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0.47(petroleum ether/ethyl acetate = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.67 – 7.54 (m, 2H), 7.22 –
7.09 (m, 1H), 4.93 (d, J = 2.5 Hz, 2H), 2.33 (t, J = 2.5 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 181.76,
157.88, 145.28, 140.55, 125.21, 124.18, 120.60, 117.95, 77.50, 72.94, 31.84.
4.5. Preparation of propargyloxy substituted chalcones 5a-e and 6a-b
4.5.1. Synthesis of 4-propargyloxy benzaldehyde 4
To a stirred solution of 4-hydroxybenzaldehyde (500 mg, 4.10 mmol, 1.0 eq) in DMF (10 mL) was
added K2CO3 (566 mg, 4.10 mmol, 1.0 eq). 30 min later, propargyl bromide (654 μL, 8.20 mmol,
2.0 eq) was added dropwise at room temperature. The mixture was stirred for about 6-10 h.
Upon completion, EtOAc and H2O were added, the aqueous layer was extracted with EtOAc, the
combined organic layers were washed with H2O for several times to remove the DMF, and the
organic layers were then washed with brine, dried over MgSO4 and evaporated to give the
o
product. White solid, yield: 77 %, m. p.: 82.3-83.4 C, Rf = 0.55 (petroleum ether/ethyl acetate =
2/1). 1H NMR (400 MHz, CDCl3) δ 9.93 (s, 1H), 7.88 (d, J = 8.7 Hz, 2H), 7.12 (d, J = 8.7 Hz, 2H), 4.81
(d, J = 2.3 Hz, 2H), 2.60 (t, J = 2.3 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 190.79, 162.38, 131.91,
130.61, 115.19, 77.55, 76.38, 55.96.
4.5.2. General procedure for the synthesis of 4-propargyloxy chalcones 6a-b
To a solution of aldehyde (0.66 mmol, 1.0 eq) and acetophenone (0.79 mmol, 1.2 eq) in EtOH (4
mL) was added KF/Al2O3 (0.66 mmol, 1.0 eq), which was prepared following our previous reported
method [17]. The mixture was kept at 50 oC for 5-6 h. Upon completion, the catalyst was filtered
and the residue was thoroughly washed with DCM, the solvent was removed under reduced
pressure to afford the products, which were used without further purification. Compounds 6a-b
have been reported previously [44], so the NMR data are not given here.
4.5.3. Synthesis of 4-propargyloxy acetophenone 3
To a solution of 4-hydroxyacetophenone (700 mg, 5.14 mmol, 1.0 eq) in DMF (12 mL) was added
K2CO3 (696 mg, 5.14 mmol, 1.0 eq) at room temperature, 30 min later, propargyl bromide (805 µL,
o
10.28 mmol, 2.0 eq) was added dropwise. The mixture was stirred for 10 h at 35 C. Upon
completion, EtOAc and H2O were added, the aqueous layer was extracted with EtOAc, the
combined organic layers were washed with H2O for several times to remove DMF, and the organic
layers were washed with brine, dried over MgSO4 and evaporated to give the residue, which was
o
then purified by column chromatography. Light yellow solid, yield: 79 %, m. p.: 77.2-78.0 C, Rf =
1
0.52 (petroleum ether/ethyl acetate = 2/1). H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 9.0 Hz, 1H),
7.04 (d, J = 9.0 Hz, 1H), 4.78 (d, J = 2.4 Hz, 1H), 2.70 – 2.44 (m, 4H, -CH3 and ≡CH).13C NMR (100
MHz, CDCl3) δ 196.74, 161.28, 131.06, 130.53, 114.58, 77.76, 76.16, 55.85, 26.40.
4.5.4. General procedure for the synthesis of 4-propargyloxy chalcones 5a-e
To a solution of aldehyde (0.60 mmol, 1.05 eq) and acetophenone (0.57 mmol, 1.0 eq) in EtOH (4
o
mL) was added KF/Al2O3 (0.57 mmol, 1.0 eq), the mixture was kept at 50 C for 6-8 h. Upon
completion, the catalyst was filtered and the residue was washed with DCM, the solvent was
removed under reduced pressure to afford the residues, which were recrystallized from EtOH to
give the products.
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4.5.4.1. (E)-3-(4-bromophenyl)-1-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one 5a, light yellow
o
1
solid, yield: 86 %, m. p.: 134.5-136.0 C, Rf = 0.46(petroleum ether/ethyl acetate = 4/1). H NMR
(400 MHz, CDCl3) δ 8.14 – 7.98 (m, 2H), 7.76 (d, J = 15.6 Hz, 1H), 7.61 – 7.49 (m, 5H), 7.15 – 7.05
(m, 2H), 4.81 (d, J = 2.4 Hz, 2H), 2.59 (t, J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 188.42,
161.34, 142.75, 133.96, 132.20, 131.67, 130.76, 129.74, 124.63, 122.33, 114.79, 77.74, 76.22,
55.90.
4.5.4.2. (E)-3-(3,4-dichlorophenyl)-1-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one 5b, light
yellow solid, yield: 87 %, m. p.: 143.2-144.6 oC, Rf = 0.44 (petroleum ether/ethyl acetate = 4/1). 1H
NMR (400 MHz, CDCl3) δ 8.14 – 8.01 (m, 2H), 7.77 – 7.74 (m, 1H), 7.74 – 7.67 (d, J = 16.0 Hz, 1H),
7.58 – 7.52 (d, J = 16.0 Hz, 1H), 7.52 – 7.50 (m, 1H), 7.50 – 7.45 (m, 1H), 7.16 – 7.06 (m, 2H), 4.81
(d, J = 2.4 Hz, 2H), 2.59 (t, J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 188.03, 161.46, 141.26,
135.11, 134.22, 133.29, 131.45, 130.94, 130.81, 129.67, 127.50, 123.31, 114.84, 77.70, 76.25,
55.92.
4.5.4.3. (E)-1-(4-(prop-2-yn-1-yloxy)phenyl)-3-(p-tolyl)prop-2-en-1-one 5c, light yellow solid, yield:
o
1
70 %, m. p.: 102.5-103.3 C, Rf = 0.38 (petroleum ether/ethyl acetate = 4/1). H NMR (400 MHz,
CDCl3) δ 8.14 – 8.00 (m, 2H), 7.82 (d, J = 15.6 Hz, 1H), 7.62 – 7.52 (m, 2H), 7.51 (s, 1H), 7.25 (d, J =
8.0 Hz, 2H), 7.13 – 7.04 (m, 2H), 4.81 (d, J = 2.4 Hz, 2H), 2.59 (t, J = 2.4 Hz, 1H), 2.42 (s, 3H).13C
NMR (100 MHz, CDCl3) δ 188.86, 161.13, 144.30, 140.92, 132.30, 132.02, 130.69, 129.70, 128.42,
114.70, 77.82, 76.16, 55.88, 21.54.
4.5.4.4. (E)-3-(3,4-difluorophenyl)-1-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one 5d, light
yellow solid, yield: 70 %, m. p.: 150.5-152.2 oC, Rf = 0.41 (petroleum ether/ethyl acetate = 4/1). 1H
NMR (400 MHz, CDCl3) δ 8.13 – 8.01 (m, 2H), 7.72 (d, J = 15.6 Hz, 1H), 7.54 – 7.44 (m, 2H), 7.44 –
7.34 (m, 1H), 7.28 – 7.16 (m, 1H), 7.16 – 7.06 (m, 2H), 4.81 (d, J = 2.4 Hz, 2H), 2.59 (t, J = 2.4 Hz,
1H).13C NMR (100 MHz, CDCl3) δ 188.12, 161.41, 141.70, 131.52, 130.77, 125.28, 122.67, 117.97,
117.79, 116.50, 116.33, 114.83, 100.00, 77.71, 76.24, 55.91.
4.5.4.5. (E)-3-(4-nitrophenyl)-1-(4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one 5e. yellow solid,
yield: 86 %, m. p.: 172.0-1173.2 oC, Rf = 0.32 (petroleum ether/ethyl acetate = 4/1). 1H NMR (400
MHz, CDCl3) δ 8.31 (d, J = 8.7 Hz, 2H), 8.09 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.81 (d, J =
8.7 Hz, 2H), 7.67 (d, J = 15.7 Hz, 1H), 7.12 (d, J = 8.9 Hz, 2H), 4.82 (d, J = 2.4 Hz, 1H), 2.60 (t, J = 2.4
Hz, 1H).13C NMR (100 MHz, CDCl3) δ 187.82, 161.65, 141.23, 140.93, 131.23, 130.91, 128.87,
125.57, 124.23, 114.94, 77.64, 76.31, 55.94.
4.6. General procedure for the preparation of propargyloxy substituted coumarins 8a-c
To a stirred solution of coumarin derivative (0.60 mmol, 1.0 eq) and K2CO3 (0.90 mmol, 1.5 eq) in
acetone (3 mL) was added propargyl bromide (0.72 mmol, 1.2 eq) at room temperature, and then
o
the mixture was kept at 50 C for 4-10 h. Upon completion, K2CO3 was filtered and the solvent
was removed under vacuum to give the residue, which was recrystallized from EtOAc to give the
corresponding product. Compounds 8a-c are known compounds [45-47] and therefore the
spectra data of compound 8a is only given here. White solid, yield: 26 %, m. p.: 119.1-120.5 oC, Rf
= 0.12 (petroleum ether/ ethyl acetate = 6/1). 1H NMR (400 MHz, CDCl3) δ 7.67 (d, J = 9.5 Hz, 1H),
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7.43 (d, J = 8.5 Hz, 1H), 7.03 – 6.86 (m, 2H), 6.31 (d, J = 9.5 Hz, 1H), 4.79 (d, J = 2.4 Hz, 2H), 2.60 (t,
J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 161.02, 160.55, 155.67, 143.28, 128.84, 113.70,
113.20, 113.09, 102.15, 76.58, 56.22.
4.7. General procedure for the preparation of substituted propargyl phenyl ethers 9a-c
To a stirred solution of substituted phenol (1.44mmol, 1.0 eq) and K2CO3 (1.44 mmol, 1.0 eq) in
acetone (4 mL) was added propargyl bromide (1.73 mmol, 1.2 eq) dropwise at room temperature,
the mixture was kept for 5-9 h under reflux. Upon completion, K2CO3 was filtered and the solvent
was removed under reduced pressure to give the residue, which was used directly for next step
without further purification. Compounds 9a-c are known compounds [48], so only the NMR data
o
of compound 9a is given below. Light yellow solid, yield: 66 %, m. p.: 77.5-78.2 C, Rf =0.30
1
(petroleum ether/ethyl acetate = 6/1). H NMR (400 MHz, CDCl3) δ 7.88 (m, 1H), 7.68 – 7.53 (m,
1H), 7.34 – 7.23 (m, 1H), 7.20 – 7.04 (m, 1H), 4.88 (d, J = 2.4 Hz, 2H), 2.61 (t, J = 2.4 Hz, 1H).13C
NMR (100 MHz, CDCl3) δ 150.77, 140.41, 133.95, 125.76, 121.45, 115.51, 77.19, 77.07, 57.22.
4.8. General procedure for the preparation of substituted propargyl anilines 10a-c
To a solution of substituted amine (1.58 mmol, 1.0 eq) in DMF (4 mL) were added K2CO3 (1.58
mmol, 1.0 eq) and propargyl bromide (1.58 mmol, 1.0 eq) at room temperature. The mixture was
o
stirred at the indicated temperature (r. t. - 50 C) for 6 h-3 days depending on the amine used.
Upon completion, EtOAc and H2O were added, the aqueous layer was extracted with EtOAc, the
combined organic layers were washed with H2O for several times to remove the DMF, and the
organic layers were washed with brine, dried over MgSO4 and evaporated to give the residue,
which was purified by column chromatography.
4.8.1. 4-Methoxy-N-(prop-2-yn-1-yl) aniline 10a, light yellow solid, yield: 43 %, m. p.: 45.6-46.2oC,
Rf = 0.44 (petroleum ether/ethyl acetate = 4/1). 1H NMR (400 MHz, CDCl3) δ 6.91 – 6.78 (d, J = 8.0
Hz, 2H), 6.76 – 6.65 (d, J = 8.0 Hz, 2H), 3.93 (d, J = 2.4 Hz, 2H), 3.82 – 3.73 (s, 3H), 3.63 (br, 1H),
2.24 (t, J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 152.99, 140.91, 115.13, 114.82, 81.36, 71.24,
55.72, 34.60.
o
4.8.2. 4-Bromo-N-(prop-2-yn-1-yl) aniline 10b, light yellow solid, yield: 29 %, m. p.: 32.5-33.1 C,
1
Rf = 0.61 (petroleum ether/ethyl acetate = 3/1). H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.9 Hz,
2H), 6.60 (d, J = 8.9 Hz, 2H), 3.94 (s, 3H, -NH- and –CH2-), 2.25 (t, J = 1.6 Hz, 1H).13C NMR (100
MHz, CDCl3) δ 145.81, 131.99, 115.13, 110.44, 80.46, 71.57, 33.60.
o
4.8.3. 3-Nitro-N-(prop-2-yn-1-yl) aniline 10c, yellow solid, yield: 32 %, m. p.: 86.2-87.8 C, Rf =
1
0.46 (petroleum ether/ethyl acetate = 3/1). H NMR (400 MHz, CDCl3) δ 7.65 (dd, J = 8.1, 1.5 Hz,
1H), 7.53 (t, J = 2.2 Hz, 1H), 7.36 (t, J = 8.1 Hz, 1H), 6.99 (dd, J = 8.1, 2.0 Hz, 1H), 4.28 (br, 1H), 4.04
(dd, J = 6.1, 2.4 Hz, 2H), 2.29 (t, J = 2.4 Hz, 1H).13C NMR (100 MHz, CDCl3) δ 147.59, 129.82,
119.37, 113.23, 107.32, 79.62, 72.11, 33.43.
4.9. General procedure for the preparation of steroidal hybrids 12-18
To a solution of azide 2 (0.27 mmol, 1.0 eq) and alkyne (0.27 mmol, 1.0 eq) in a mixture of THF (2
mL) and H2O (2 mL) were added sodium ascorbate (0.14 mmol, 0.5 eq) and copper sulfate
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pentahydrate (0.11 mmol, 0.4 eq). The mixture was stirred overnight at room temperature. Upon
completion, the mixture was filtered and the filtrate was evaporated to give the residue, which
was then subjected to column chromatography to afford the corresponding product.
o
4.9.1. Compound 12a, yellow solid, yield: 50 %, m. p.: 191.3-192.8 C, Rf = 0.13 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.64 (t, J = 7.7 Hz, 1H), 7.58 (d,
J = 7.2 Hz, 1H), 7.15 (m, 2H), 5.38 (s, 1H), 5.36 (s, 2H), 5.01 (s, 2H), 4.53 (d, J = 5.2 Hz, 1H), 0.99 (s,
3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 183.51, 167.00, 158.28, 150.59, 139.73, 138.53,
125.57, 124.95, 123.86, 122.54, 118.07, 111.64, 75.44, 51.25, 51.00, 49.95, 47.28, 37.90, 36.73,
36.66, 35.76, 35.45, 31.59, 31.32, 30.65, 27.62, 21.89, 20.35, 19.42, 13.65. HRMS (ESI): m/z calcd
for C32H37N4O5 (M + H)+, 557.2764; found, 557.2650.
o
4.9.2. Compound 12b, yellow solid, yield: 58 %, m. p.: 140.1-141.9 C, Rf = 0.35 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.64 (s,
1H), 7.20 (d, J = 8.2 Hz, 1H), 5.36 (s, 3H), 5.01 (s, 2H), 4.53 (s, 1H), 0.99 (s, 3H), 0.81 (s, 3H).13C
NMR (100 MHz, DMSO-d6) δ 220.09, 182.38, 166.95, 158.06, 149.05, 141.82, 139.71, 137.36,
128.10, 125.58, 124.44, 122.54, 119.51, 113.33, 75.43, 67.49, 51.25, 51.03, 49.95, 47.28, 37.89,
36.71, 36.66, 35.76, 35.55, 31.59, 31.33, 30.66, 27.61, 25.60, 21.90, 20.35, 19.38, 19.03, 13.65.
HRMS (ESI): m/z calcd for C32H36ClN4O5 (M + H)+, 591.2374; found, 591.2375.
o
4.9.3. Compound 12c, yellow solid, yield: 58 %, m. p.: 141.9-143.4 C, Rf = 0.28 (petroleum
1
ether/acetone = 2/1). H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.82 (dd, J = 8.4, 2.1 Hz, 1H),
7.74 (d, J = 2.1 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 5.38 (d, J = 4.7 Hz, 1H), 5.36 (s, 2H), 5.00 (s, 2H),
4.53 (tt, J = 10.5, 5.2 Hz, 1H), 0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.09,
182.23, 166.94, 157.90, 149.42, 140.17, 139.71, 127.17, 125.58, 122.54, 119.88, 115.63, 113.76,
75.43, 67.49, 51.25, 49.95, 47.28, 37.89, 36.71, 36.66, 35.76, 35.54, 31.59, 31.33, 30.66, 27.61,
25.60, 21.89, 20.36, 19.39, 13.66. HRMS (ESI): m/z calcd for C32H36BrN4O5 (M + H)+, 635.1869;
found, 635.1859.
4.9.4. Compound 12d, saffron solid, yield: 68 %, m. p.: 117.5-118.8 oC, Rf = 0.30 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.59 – 7.40 (m, 2H), 7.22 (dd, J
= 21.6, 6.2 Hz, 1H), 5.37 (m, 3H), 5.01 (s, 2H), 4.54 (s, 1H), 0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100
MHz, DMSO-d6) δ 220.11, 182.88, 166.98, 158.32, 156.83, 146.79, 141.87, 139.71, 125.58,
124.32, 122.54, 113.00, 112.08, 111.83, 75.43, 51.25, 51.01, 49.95, 47.28, 37.89, 36.72, 36.66,
35.76, 35.51, 31.59, 31.32, 30.65, 27.62, 21.89, 20.35, 19.39, 13.65. HRMS (ESI): m/z calcd for
C32H36FN4O5 (M + H)+, 575.2670; found, 575.2673.
o
4.9.5. Compound 12e, yellow solid, yield: 66 %, m. p.: 218.5-220.1 C, Rf = 0.19 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.61 (t, J = 8.1 Hz, 1H), 7.14 (t,
J = 8.5 Hz, 2H), 5.39 (d, J = 4.2 Hz, 1H), 5.35 (s, 2H), 5.01 (s, 2H), 4.54 (dd, J = 11.2, 5.7 Hz, 1H),
0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.09, 180.30, 166.96, 157.64, 151.85,
141.84, 139.72, 139.05, 131.49, 125.56, 124.80, 122.54, 115.15, 110.35, 75.44, 51.25, 51.01,
49.95, 47.28, 37.89, 36.72, 36.66, 35.76, 35.67, 31.59, 31.33, 30.65, 27.62, 21.89, 20.36, 19.40,
13.66. HRMS (ESI): m/z calcd for C32H36ClN4O5 (M + H)+, 591.2374; found, 591.2376.
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o
4.9.6. Compound 12f, yellow solid, yield: 70 %, m. p.: 220.9-222.3 C, Rf = 0.24 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.30 (d,
J = 8.0 Hz, 1H), 7.16 (d, J = 7.7 Hz, 1H), 5.42 – 5.37 (m, 1H), 5.35 (s, 2H), 5.01 (s, 2H), 4.60 – 4.44
(m, 1H), 0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 180.84, 166.94, 157.59, 152.27,
139.71, 138.97, 127.92, 122.54, 119.85, 116.76, 110.76, 75.43, 56.49, 51.26, 51.03, 49.96, 47.28,
37.88, 36.72, 36.66, 35.75, 35.57, 31.59, 31.32, 31.16, 30.65, 27.61, 21.89, 20.36, 19.40, 19.03,
13.65. HRMS (ESI): m/z calcd for C32H36BrN4O5 (M + H)+, 635.1869; found, 635.1872.
o
4.9.7. Compound 12g, yellow solid, yield: 65 %, m. p.: 132.1-133.9 C, Rf = 0.22 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.19 (s, 1H), 7.61 (d, J = 8.7 Hz, 1H), 7.20 (d,
J = 8.7 Hz, 1H), 5.38 (d, J = 4.9 Hz, 1H), 5.36 (s, 2H), 5.32 (s, 2H), 4.63 – 4.44 (m, 1H), 1.00 (s, 3H),
0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.10, 179.12, 166.96, 158.63, 146.86, 140.28,
139.71, 130.79, 129.37, 128.67, 126.23, 125.78, 124.93, 122.54, 118.10, 115.09, 75.39, 55.38,
51.25, 51.06, 49.95, 47.28, 37.95, 37.87, 36.72, 36.66, 35.76, 31.59, 31.33, 31.16, 30.65, 27.60,
21.89, 20.36, 19.40, 13.65. HRMS (ESI): m/z calcd for C32H35Cl2N4O5 (M + H)+, 625.1985; found,
625.1986.
o
4.9.8. Compound 12h, yellow solid, yield: 51 %, m. p.: 164.2-165.7 C, Rf = 0.17 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.18 (s, 1H), 7.62 (t, J = 7.9 Hz, 2H), 7.16 (t, J
= 7.7 Hz, 1H), 5.39 (d, J = 4.8 Hz, 1H), 5.35 (s, 2H), 5.30 (s, 2H), 4.54 (dt, J = 10.6, 5.4 Hz, 1H), 1.00
(s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.11, 182.24, 167.00, 159.32, 145.72,
140.01, 139.73, 125.31, 124.86, 124.04, 122.54, 121.62, 116.47, 75.39, 51.25, 51.03, 49.95, 47.28,
37.85, 36.67, 35.76, 31.59, 31.33, 31.17, 30.65, 27.60, 21.90, 20.36, 19.42, 13.66. HRMS
(ESI): m/z calcd for C32H36ClN4O5 (M + H)+, 591.2374; found, 591.2377.
o
4.9.9. Compound 13a, light yellow solid, yield: 75 %, m. p.: 186.0-187.5 C, Rf = 0.29 (petroleum
ether/acetone = 2/1), 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 8.16 (d, J = 7.5 Hz, 2H), 7.88 (d,
J = 8.6 Hz, 2H), 7.82 (s, 1H), 7.75 (s, 1H), 7.68 (dd, J = 15.6, 8.5 Hz, 1H), 7.58 (t, J = 7.5 Hz, 2H),
7.14 (d, J = 8.5 Hz, 2H), 5.41 (s, 2H), 5.39 (s, 1H), 5.30 (s, 2H), 4.66 – 4.45 (m, 1H), 0.97 (s, 3H),
0.76 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.10, 189.38, 167.02, 160.46, 144.46, 142.91,
139.68, 138.24, 133.44, 131.29, 129.22, 128.90, 128.08, 126.61, 122.55, 120.05, 115.66, 75.45,
67.49, 61.51, 51.22, 51.04, 49.93, 47.25, 37.89, 36.65, 35.74, 31.56, 31.29, 30.64, 27.62, 21.85,
20.34, 19.37, 13.61. HRMS (ESI): m/z calcd for C39H42Cl2N3O5 (M + H)+, 702.2502; found, 702.2484.
o
4.9.10. Compound 13b, yellow solid, yield: 54 %, m. p.: 167.4-169.1 C, Rf = 0.27 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.27 (s, 3H), 7.80 (m, 4H), 7.40 (s, 2H), 7.14
(d, J = 7.0 Hz, 2H), 5.41 (s, 3H), 5.30 (s, 2H), 4.54 (s, 1H), 0.97 (s, 3H), 0.76 (s, 3H).13C NMR (100
MHz, DMSO-d6) δ 187.90, 167.02, 164.19, 160.50, 144.63, 142.93, 139.68, 134.94, 131.94,
131.35, 128.05, 126.61, 122.55, 119.81, 116.32, 116.11, 115.66, 99.99, 75.46, 61.52, 51.22, 51.04,
49.93, 47.24, 37.89, 36.65, 35.75, 31.56, 31.28, 30.64, 27.61, 21.84, 20.34, 19.37, 13.59. HRMS
(ESI): m/z calcd for C39H43FN3O5 (M + H)+, 652.3187; found, 652.3184.
o
4.9.11. Compound 14a, gray solid, yield: 33 %, m. p.: 204.6-205.8 C, Rf = 0.44 (petroleum
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ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.02 (d,
J = 15.5 Hz, 1H), 7.87 (d, J = 8.2 Hz, 2H), 7.76 – 7.57 (m, 3H), 7.21 (d, J = 8.6 Hz, 2H), 5.41 (s, 2H),
5.35 (s, 3H), 4.54 (s, 1H), 0.97 (s, 3H), 0.77 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.07, 187.61,
167.00, 162.41, 142.74, 142.26, 139.68, 134.61, 132.33, 131.44, 131.21, 131.02, 126.68, 124.26,
123.18, 122.55, 115.29, 75.46, 61.68, 51.22, 51.05, 49.93, 47.25, 37.88, 36.64, 35.74, 31.56, 31.29,
31.17, 30.63, 27.62, 21.85, 20.34, 19.40, 13.61. HRMS (ESI): m/z calcd for C39H43BrN3O5 (M + H)+,
712.2386; found, 712.2364 (Br79) and 714.2352 (Br81).
4.9.12. Compound 14b, light yellow solid, yield: 77 %, m. p.: 153.6-154.9 oC, Rf = 0.28 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.26 (m, 4H), 8.09 (d, J = 15.6 Hz, 1H), 7.88
(s, 1H), 7.82 – 7.57 (m, 2H), 7.22 (d, J = 7.1 Hz, 2H), 5.41 (s, 2H), 5.36 (s, 3H), 4.53 (s, 1H), 0.96 (s,
3H), 0.76 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 220.06, 187.42, 166.99, 162.50, 142.74, 140.85,
139.65, 136.21, 133.02, 132.30, 131.56, 131.43, 130.88, 130.54, 129.64, 126.69, 124.39, 122.54,
115.29, 75.45, 61.68, 55.39, 51.20, 51.05, 49.91, 47.23, 39.36, 37.86, 36.68, 36.62, 35.73, 31.54,
31.26, 31.17, 30.62, 27.60, 21.84, 20.33, 19.39, 13.57. HRMS (ESI): m/z calcd for C39H42Cl2N3O5
(M + H)+, 702.2502; found, 702.2484.
4.9.13. Compound 14c, light yellow solid, yield: 75 %, m. p.: 191.8-193.1 oC, Rf = 0.24 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.18 (d, J = 8.4 Hz, 2H), 7.92 (d,
J = 15.5 Hz, 1H), 7.79 (d, J = 7.7 Hz, 2H), 7.69 (d, J = 15.5 Hz, 1H), 7.28 (d, J = 7.6 Hz, 2H), 7.21 (d, J
= 8.6 Hz, 2H), 5.41 (s, 2H), 5.37 (s, 1H), 5.35 (s, 2H), 4.54 (s, 1H), 2.36 (s, 3H), 0.97 (s, 3H), 0.76 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 220.07, 187.69, 167.00, 162.26, 143.71, 142.77, 140.95,
139.68, 132.57, 131.31, 131.23, 129.99, 129.32, 126.67, 122.55, 121.30, 115.24, 75.46, 61.67,
55.39, 51.22, 51.05, 49.93, 47.25, 37.88, 36.64, 35.74, 31.56, 31.28, 31.16, 30.63, 27.61, 21.85,
21.55, 20.34, 19.39, 13.60. HRMS (ESI): m/z calcd for C40H46N3O5 (M + H)+, 648.3437; found,
648.3412.
o
4.9.14. Compound 14d, yellow solid, yield: 62 %, m. p.: 139.5-140.8 C, Rf = 0.23 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.28 (s, 1H), 8.20 (d, J = 8.7 Hz, 2H), 8.15 (d,
J = 10.6 Hz, 1H), 8.00 (d, J = 15.6 Hz, 1H), 7.74 (s, 1H), 7.69 (d, J = 15.6 Hz, 1H), 7.54 (dd, J = 18.7,
8.7 Hz, 1H), 7.22 (d, J = 8.6 Hz, 2H), 5.41 (s, 2H), 5.36 (s, 3H), 4.54 (s, 1H), 0.97 (s, 3H), 0.77 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 220.04, 187.46, 166.99, 162.47, 142.74, 141.33, 139.68,
133.31, 131.48, 130.95, 127.25, 126.68, 123.63, 122.53, 118.49, 117.28, 115.28, 75.46, 67.48,
61.70, 51.21, 51.05, 49.93, 47.23, 37.88, 36.63, 35.73, 31.55, 31.28, 30.62, 27.61, 25.59, 21.84,
20.33, 19.38, 13.58. HRMS (ESI): m/z calcd for C39H42F2N3O5 (M + H)+, 670.3093; found, 670.3097.
o
4.9.15. Compound 14e, yellow solid, yield: 57 %, m. p.: 130.5-132.3 C, Rf = 0.20 (petroleum
1
ether/acetone = 2/1). H NMR (400 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.29 (m, 2H), 8.26 – 8.13 (m,
5H), 7.80 (d, J = 15.6 Hz, 1H), 7.24 (d, J = 8.8 Hz, 2H), 5.42 (s, 2H), 5.38 (s, 1H), 5.36 (s, 2H), 4.63 –
4.45 (m, 1H), 0.98 (s, 3H), 0.77 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.06, 187.50, 167.02,
162.65, 148.46, 142.69, 141.83, 140.82, 139.69, 131.63, 130.78, 130.29, 126.71, 126.48, 124.39,
122.54, 115.36, 75.46, 61.72, 51.21, 51.04, 49.93, 47.24, 37.88, 36.70, 36.64, 35.73, 31.55, 31.29,
30.63, 27.62, 21.85, 20.33, 19.40, 13.59. HRMS (ESI): m/z calcd for C39H43N4O7 (M + H)+, 679.3132;
found, 679.3131.
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o
4.9.16. Compound 15a, white solid, yield: 18 %, m. p.: 129.5-130.8 C, Rf = 0.18 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 8.01 (d, J = 9.5 Hz, 1H), 7.65 (d,
J = 8.6 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 8.6, 2.4 Hz, 1H), 6.31 (d, J = 9.5 Hz, 1H), 5.42 (s,
2H), 5.40 (d, J = 5.1 Hz, 1H), 5.32 (s, 2H), 4.57 (dt, J = 10.9, 5.0 Hz, 1H), 0.99 (s, 3H), 0.81 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 220.09, 167.06, 161.52, 160.72, 155.78, 144.77, 142.46,
139.72, 129.99, 126.83, 122.56, 113.42, 113.16, 113.07, 102.06, 75.48, 61.97, 51.25, 51.02, 49.96,
47.28, 37.91, 36.73, 36.67, 35.76, 31.60, 31.33, 30.65, 27.64, 21.90, 20.35, 19.40, 13.66. HRMS
(ESI): m/z calcd for C33H38N3O6 (M + H)+, 572.2761; found, 572.2762.
4.9.17. Compound 15b, white solid, yield: 54 %, m. p.: 177.2-178.6 oC, Rf = 0.14 (petroleum ether/
acetone=2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.29 (s, 1H), 7.70 (d, J = 8.8 Hz, 1H), 7.16 (d, J = 2.5
Hz, 1H), 7.05 (dd, J = 8.8, 2.5 Hz, 1H), 6.23 (d, J = 1.1 Hz, 1H), 5.42 (s, 2H), 5.39 (d, J = 5.0 Hz, 1H),
5.32 (s, 2H), 4.64 – 4.47 (m, 1H), 2.40 (s, 4H), 0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz,
DMSO-d6) δ 220.10, 167.06, 161.43, 160.59, 155.14, 153.87, 142.49, 139.72, 126.98, 126.81,
122.54, 113.84, 113.12, 111.79, 102.08, 75.47, 61.94, 51.24, 51.01, 49.95, 47.28, 39.37, 37.90,
36.72, 36.66, 35.76, 31.59, 31.33, 31.16, 30.65, 27.64, 21.89, 20.34, 19.39, 18.60, 13.65. HRMS
(ESI): m/z calcd for C34H40N3O6 (M + H)+, 586.2917; found, 586.2919.
o
4.9.18. Compound 16, white solid, yield: 29 %, m. p.: 164.0-165.8 C, Rf = 0.20 (petroleum
1
ether/acetone = 2/1). H NMR (400 MHz, DMSO-d6) δ 8.40 (s, 1H), 7.74 (dd, J = 7.9, 1.5 Hz, 1H),
7.70 – 7.64 (m, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.38 – 7.31 (m, 1H), 6.19 (s, 1H), 5.48 (s, 2H), 5.45 (s,
2H), 5.41 (d, J = 4.8 Hz, 1H), 4.58 (tt, J = 10.4, 5.1 Hz, 1H), 1.00 (s, 3H), 0.81 (s, 3H).13C NMR (100
MHz, DMSO-d6) δ 167.06, 164.75, 161.99, 153.25, 141.47, 139.73, 133.32, 127.25, 124.71,
123.25, 122.57, 116.98, 115.56, 91.86, 75.52, 63.13, 51.25, 49.96, 47.28, 37.94, 36.74, 36.67,
35.76, 31.60, 31.33, 30.65, 27.67, 21.90, 20.36, 19.41, 13.66. HRMS (ESI): m/z calcd for
C33H38N3O6 (M + H)+, 572.2761; found, 572.2759.
4.9.19. Compound 17a, light brown solid, yield: 78 %, m. p.: 153.2-154.7 oC, Rf = 0.31 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.68 (t,
J = 7.5 Hz, 1H), 7.60 (d, J = 8.4 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 5.41 (d, 5H, overlapped), 4.57 (s,
1H), 1.01 (s, 3H), 0.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 167.06, 150.97, 142.30, 140.26,
139.76, 135.46, 134.74, 127.64, 126.80, 125.39, 122.55, 121.41, 116.02, 75.48, 62.89, 51.25,
51.02, 49.96, 47.29, 37.93, 36.75, 36.68, 35.76, 31.59, 31.33, 30.66, 27.66, 21.90, 20.36, 19.43,
13.66. HRMS (ESI): m/z calcd for C30H37N4O6 (M + H)+, 549.2713; found, 549.2713.
o
4.9.20. Compound 17b, yellow solid, yield: 80 %, m. p.: 176.9-178.2 C, Rf = 0.42 (petroleum
ether/acetone=2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.23 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.03 (d, J
= 8.8 Hz, 2H), 5.40 (s, 3H), 5.19 (s, 2H), 4.58 (dt, J = 10.6, 4.8 Hz, 1H), 1.02 (s, 3H), 0.81 (s, 3H).13C
NMR (100 MHz, DMSO-d6) δ 220.09, 167.09, 157.73, 142.89, 139.75, 132.61, 126.58, 122.56,
117.53, 112.76, 75.49, 61.63, 51.26, 50.98, 49.97, 47.28, 39.38, 37.94, 36.75, 36.68, 35.76, 31.60,
31.34, 30.66, 27.67, 21.90, 20.36, 19.43, 13.66. HRMS (ESI): m/z calcd for C30H37BrN3O4 (M + H)+,
582.1967; found, 582.1953.
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4.9.21. Compound 17c, yellow solid, yield: 69 %, m. p.: 177.8-179.5 oC, Rf = 0.97 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.21 (s, 1H), 7.10 (d, J = 8.3 Hz, 2H), 6.93 (d,
J = 8.3 Hz, 2H), 5.40 (s, 3H, overlapped), 5.13 (s, 2H), 4.58 (dd, J = 10.7, 5.7 Hz, 1H), 2.24 (s, 3H),
1.02 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.10, 167.12, 156.36, 139.77, 130.29,
129.95, 126.36, 122.56, 115.00, 75.48, 61.39, 51.26, 50.95, 49.97, 47.28, 37.95, 36.76, 36.68,
35.76, 31.60, 31.34, 30.66, 27.67, 21.90, 20.55, 20.36, 19.43, 13.66. HRMS (ESI): m/z calcd for
C31H40N3O4 (M + H)+, 518.3019; found, 518.3021.
4.9.22. Compound 18a, light brown solid, yield: 56 %, m. p.: 149.2-150.0 oC, Rf = 0.25 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 7.92 (s, 1H), 6.70 (d, J = 8.1 Hz, 2H), 6.59 (d,
J = 8.1 Hz, 2H), 5.66 (br, 1H), 5.40 (s, 1H), 5.34 (s, 2H), 4.55 (d, J = 5.5 Hz, 1H), 4.28 (s, 2H), 3.63 (s,
3H), 1.01 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.13, 167.16, 153.62, 139.76,
133.63, 122.54, 120.19, 114.94, 113.94, 75.36, 55.70, 51.24, 50.87, 49.95, 47.28, 37.94, 36.75,
36.67, 35.76, 31.59, 31.32, 30.65, 27.66, 21.90, 20.35, 19.42, 13.65. HRMS (ESI): m/z calcd for
C31H41N4O4 (M + H)+, 533.3128; found, 533.3129.
4.9.23. Compound 18b, yellowish solid, yield: 80 %, m. p.: 95.8-96.6 oC, Rf = 0.29 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 7.95 (s, 1H), 7.19 (d, J = 7.7 Hz, 2H), 6.60 (d,
J = 7.7 Hz, 2H), 6.39 (s, 1H), 5.41 (s, 1H), 5.34 (s, 2H), 4.55 (s, 1H), 4.31 (s, 2H), 1.01 (s, 3H), 0.81 (s,
3H).13C NMR (100 MHz, DMSO-d6) δ 220.13, 167.12, 148.02, 139.75, 131.79, 129.37, 128.68,
122.55, 114.73, 107.12, 75.37, 51.25, 50.89, 49.96, 47.28, 38.71, 37.94, 36.74, 36.67, 35.76, 31.59,
31.33, 30.66, 27.65, 21.90, 20.36, 19.42, 13.65. HRMS (ESI): m/z calcd for C30H38BrN4O3 (M + H)+,
581.2127; found, 581.2128.
4.9.24. Compound 18c, yellow solid, yield: 54 %, m. p.: 183.9-185.8 oC, Rf = 0.20 (petroleum
ether/acetone = 2/1). 1H NMR (400 MHz, DMSO-d6) δ 8.00 (s, 1H), 7.44 (t, J = 2.2 Hz, 1H), 7.35 (m,
2H), 7.12 – 7.02 (m, 1H), 6.94 (t, J = 5.9 Hz, 1H), 5.39 (d, J = 4.9 Hz, 1H), 5.35 (s, 2H), 4.54 (m, 1H),
4.43 (d, J = 5.6 Hz, 2H), 0.99 (s, 3H), 0.81 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 220.13, 167.11,
149.84, 149.23, 145.39, 139.73, 130.42, 124.72, 122.54, 119.01, 110.66, 106.19, 75.36, 51.24,
50.92, 49.95, 47.28, 38.52, 37.91, 36.73, 36.66, 35.76, 31.59, 31.32, 30.65, 27.63, 21.90, 20.35,
19.40, 13.65. HRMS (ESI): m/z calcd for C30H38N5O5 (M + H)+, 548.2873; found, 548.2873.
4.10. Synthesis of 5-Fu linked dimers 20 and 21
4.10.1. Synthesis of dipropargyl substituted 5-Fu 11
To a stirred solution of 5-Fu (100 mg, 0.77 mmol, 1.0 eq) in DMF (10 mL) was added K2CO3 (106
mg, 0.77 mmol, 1.0 eq) at room temperature. 30 min later, propargyl bromide (184 μL, 2.31
mmol, 3.0 eq) was added to the above solution. The mixture was stirred at room temperature for
1.5 h. Upon completion, EtOAc and H2O were added, the aqueous layer was extracted with EtOAc,
the combined organic layers were washed with H2O for several times to remove the DMF, and the
organic layers were washed with brine, dried over MgSO4 and evaporated to give the residue,
which was recrystallized from EtOH (2 mL) to give the product, white solid, yield: 51 %, m. p.:
96.3-97.8 oC, Rf = 0.33 (petroleum ether/acetone = 2/1). 1H NMR (400 MHz, CDCl3) δ 7.63 (d, JC-F
=
5.3 Hz, 1H), 4.76 (d, J = 2.3 Hz, 2H), 4.64 (d, J = 2.5 Hz, 2H), 2.59 (t, J = 2.6 Hz, 1H), 2.23 (t, J = 2.4
Hz, 1H).13C NMR (100 MHz, CDCl3) δ 156.08 (d, JC-F = 26.1 Hz), 148.83, 140.18 (d, JC-F = 237.4 Hz),
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125.16 (d, JC-F = 33.6 Hz), 77.13, 76.69, 75.33, 71.41, 38.07, 31.17.
4.10.2. Synthesis of 2-nitrobenzyl azide 19
2-Chlorobenzyl chloride (158 μL, 1.24 mmol, 1.0 eq) and NaN3 (97 mg, 1.49 mmol, 1.2 eq) were
o
dissolved in DMSO (4 mL) at room temperature. The mixture was kept for about 3 h at 30 C.
Upon completion, Et2O and H2O were added, the aqueous layer was extracted with Et2O, the
combined organic layers were washed with H2O for several times to remove the DMSO, and the
organic layers were washed with brine, dried over MgSO4 and evaporated to give the yellow oil,
1
Rf = 0.67 (petroleum ether/ethyl acetate = 4/1). H NMR (400 MHz, CDCl3) δ 7.56 – 7.38 (m, 2H),
7.38 (m, 2H), 4.52 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 133.79, 133.33, 130.05, 129.80, 129.67,
127.19, 52.29.
4.10.3. Synthesis of 5-Fu linked steroidal dimer 20
To a solution of azide 2 (180 mg, 0.48 mmol, 1.0 eq) and alkyne 11 (50 mg, 0.24 mmol, 0.5 eq) in
a mixture of THF (3 mL) and H2O (3 mL) were added sodium ascorbate (48mg, 0.24 mmol, 0.5 eq)
and copper sulfate pentahydrate (48 mg, 0.19 mmol, 0.4 eq). The mixture was stirred overnight at
room temperature. Upon completion, the mixture was filtered and the filtrate was evaporated to
the residue, which was subjected to column chromatography to afford the product. White solid,
o
1
yield: 38 %, m. p.: 183.8-185.1 C, Rf = 0.30 (petroleum ether/acetone = 2/1). H NMR (400 MHz,
DMSO-d6) δ 8.39 (s, 1H), 8.14 (s, 1H), 8.00 (s, 1H), 5.52 – 5.22 (m, 6H), 5.08 (s, 2H), 5.02 (s, 2H),
4.57 (s, 2H), 1.02 (s, 6H), 0.81 (s, 6H).13C NMR (101 MHz, DMSO-d6) δ 220.03, 167.06, 167.03,
157.00, 156.74, 149.69, 142.50, 142.35, 140.75, 139.77, 138.48, 129.66, 129.33, 125.81, 125.62,
122.54, 75.47, 75.43, 51.29, 50.97, 50.87, 49.99, 47.28, 44.34, 37.95, 36.77, 36.68, 35.75, 31.61,
31.34, 30.67, 27.67, 21.89, 20.37, 19.41, 13.64. HRMS (ESI): m/z calcd for C52H66FN8O8 (M + H)+,
949.4988; found, 949.4990.
4.10.4. Synthesis of 5-Fu linked non-steroidal dimer 21
To a solution of azide 19 (128 mg, 0.76 mmol, 2.0 eq) and alkyne 11 (79 mg, 0.38 mmol, 1.0 eq) in
a mixture of THF (2 mL) and H2O (2 mL) were added sodium ascorbate (76mg, 0.38 mmol, 1.0 eq)
and copper sulfate pentahydrate (76 mg, 0.30 mmol, 0.79 eq). The mixture was stirred overnight
at room temperature. Upon completion, the mixture was filtered and the filtrate was evaporated
to the residue, which was subjected to column chromatography to afford the product. White
o
1
solid, yield: 14 %, m. p.: 59.1-60.9 C, Rf = 0.10 (petroleum ether/acetone = 2/1). H NMR (400
MHz, DMSO-d6) δ 8.34 (d, J = 6.3 Hz, 1H), 8.17 (s, 1H), 8.03 (s, 1H), 7.52 (d, J = 7.6 Hz, 2H), 7.46 –
7.28 (m, 4H), 7.20 (dd, J = 16.1, 7.1 Hz, 2H), 5.69 (s, 2H), 5.66 (s, 2H), 5.06 (s, 2H), 5.00 (s, 2H).13C
NMR (100 MHz, DMSO-d6) δ 157.08, 156.82, 149.71, 142.64, 142.48, 140.75, 138.48, 133.71,
133.59, 133.09, 133.03, 130.99, 130.85, 130.74, 130.67, 130.10, 130.07, 129.69, 129.36, 128.21,
128.19, 124.80, 124.54, 51.10, 50.96, 44.32, 37.07, 31.16. HRMS (ESI): m/z calcd for C32H36ClN4O5
(M + H)+, 591.2374; found, 591.1072.
4.11. Biological testing
The MTT assay, analysis of cell cycle distribution, detection of apoptosis and mitochondrial
membrane potential were carried out following our previously reported method [17]. The assay
of LSD1 inhibition was performed according to the methods previously published [5]. Therefore,
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no details are given here.
4.12. Molecular docking studies
The protein complexes used for this study were obtained from protein data bank (PDB code:
2H94), and all water molecules were eliminated. Hydrogen and partial charges were added by the
protonate 3D program of MOE2014; Energy minimization of the ligands was carried out using
energy minimize program of MOE. Default parameter settings generated by the program of MOE
were used for docking.
Acknowledgement
We are grateful for the financial support from the Natural Science Foundation of China (No.
21372206) and the Development Foundation of Priority, Ministry of Education (No.
20134101130001). Bin Yu and Xiao-Jing Shi express their thanks to the outstanding PhD training
program of Zhengzhou University for financial support. Bin Yu designed this project, analyzed the
data and wrote the manuscript; Ping-Ping Qi performed the chemical synthesis of reported
compounds; Xiao-Jing Shi carried out most of the biology experiments; Ruilei Huang carried out
the docking studies; Dr. Yi-Chao Zheng performed the LSD1 inhibition assay; Hao Guo provided
MS analysis services of some compounds; Prof. De-Quan Yu and Prof. Hong-Min Liu, as
corresponding authors, proofread this manuscript and approved the final submission.
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