Synthesis and antitumor activity of novel steroidal imidazolium salt derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.02.025

Sixty-one novel steroidal imidazolium salt derivatives were synthesized and evaluated in vitro against a panel of human tumor cell lines. The results showed that diosgenin?imidazolium salt derivatives displayed much higher cytotoxic activities than choles

Full text of DOI:10.1016/j.ejmech.2019.02.025

Accepted Manuscript
Synthesis and antitumor activity of novel steroidal imidazolium salt derivatives
Guogang Deng, Bei Zhou, Jing Wang, Zhuo Chen, Liang Gong, Yaxiao Gong,
Dongmei Wu, Yan Li, Hongbin Zhang, Xiaodong Yang
PII:
S0223-5234(19)30139-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.02.025
EJMECH 11117
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2018
Revised Date: 25 January 2019
Accepted Date: 8 February 2019
Please cite this article as: G. Deng, B. Zhou, J. Wang, Z. Chen, L. Gong, Y. Gong, D. Wu, Y. Li,
H. Zhang, X. Yang, Synthesis and antitumor activity of novel steroidal imidazolium salt derivatives,
European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.02.025.
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ACCEPTED MANUSCRIPT
Graphical abstract
Steroids
Diosgenin, Cholesterol, Dehydroepiandrosterone
Imidazolium salts
Molecular hybridization
61 Hybrid Compounds
Best potent
hybrid compound
O
H
O
H
H
Br
H
H
tumor cell lines IC50, µM
Br
N
N
HL-60
A549
0.49
0.44
0.67
0.73
0.79
SMMC-7721
MCF-7
SW480
a30

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Synthesis and antitumor activity of novel steroidal imidazolium salt
derivatives
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Guogang Denga, Bei Zhoua,c, Jing Wanga, Zhuo Chena, Liang Gongb, Yaxiao Gongb, Dongmei Wub, Yan
Lib,*, Hongbin Zhanga,*, Xiaodong Yanga,*
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aKey Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education and Yunnan Province,
School of Chemical Science and Technology, Yunnan University, Kunming, 650091, P. R. China.
bState Key Laboratory for Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Science, Kunming, 650204, P. R. China.
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cKey Laboratory for Forest Resources Conservation and Utilisation in the Southwest Mountains of China,
Ministry of Education, Southwest Forestry University, Kunming, China
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* Corresponding author. Tel.: +86-871-65031119; fax.: +86-871-65035538. E-mail: xdyang@ynu.edu.cn (X. Yang);
zhanghb@ynu.edu.cn (H. Zhang); liyanb@mail.kib.ac.cn (Y. Li)
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Abstract—Sixty-one novel steroidal imidazolium salt derivatives were synthesized and evaluated in vitro
against a panel of human tumor cell lines. The results showed that diosgenin‒imidazolium salt derivatives
displayed
much
higher
cytotoxic
activities
than
cholesterol‒imidazolium
salts
and
dehydroepiandrosterone‒imidazolium salts. The SARs results suggested that the existence of substituted 5,6-
dimethyl-benzimidazoles or benzimidazole ring and substitution of the imidazolyl-3α-position with a 2-
bromobenzyl or 2-naphthylmethyl group could be critical for promoting cytotoxic activity.
Diosgenin‒imidazolium salt a30 was found to be the most potent compound with IC50 values of 0.44–0.79 µM
against five human tumor cell lines. Compound a24 showed inhibitory activity selectively against SMMC-7721
cell lines with IC50 value of 0.21 µM and 54-fold more sensitive to DDP. Moreover, compound a30 inhibited
cell proliferation through inducing the G0/G1 cell cycle arrest and apoptosis in SMMC-7721 cells.
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Keywords: Diosgenin; Cholesterol; Dehydroepiandrosterone; Imidazolium salt; Antitumor activities; Structure–
activity relationships.
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1. Introduction
Steroids are an important family of polycyclic molecules with diverse structures, which are widely existed in
plants, animals and humans.[1, 2] Naturally occurring steroids exhibit a wide range of interesting biological
activities. They have been traditionally used as physiological regulators and hormones, as well as antitumour,
cardiotonic, diuretic and local anaesthetic agents.[3-6] Steroid-based drugs possess a broad range of clinical
treatments and remain one of the highest marketed classes of pharmaceuticals.[7] Recently, the structurally
modified steroids have attracted a great deal of attention, particularly the incorporation of heterocycles into the
steroid core.[8, 9] As shown in Fig. 1, steroidal pyridine Abiraterone has been used for the clinical therapeutics
of advanced prostate cancers[10], while steroidal spiro aminooxazoline (SSA) was prepared as potential
antiproliferative drug against human breast and colon carcinoma cells (T-47D and WiDr).[8]
On the other hand, N-heterocycles are of great interest to medicinal chemists because of their widespread
occurrence in active natural products and drug molecules.[10-14] Of them, imidazolium salts have obtained
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significant concerns owing to their important and extensive biological and pharmacological activities,[15-19]
particularly antitumor activity.[20-23] For instance, two novel imidazolium salts, Lepidiline A and B (Fig. 1),
presented effective cytotoxic activity against human tumor cell lines (UMUC3, PACA2, MDA231, and
FDIGROV).[24] In this context, our group has long been devoted to the synthesis of novel imidazolium salt
derivatives and their potential antitumor activity, aiming to discover new steroid-based antitumor agents.[25-30]
One representative example is the imidazolium salt NMIB (Fig. 1) which exerts potent in vitro and in vivo
antitumor activity.[31, 32] Further mechanism research verified that these imidazolium salt derivatives induced
the cell cycle arrest and apoptosis in tumor cells.[33-35] In previous structure-activity relationships (SARs)
studies, we found that the existence of substituted benzimidazole (such as 5,6-dimethyl-benzimidazole) ring and
substitution of the imidazolyl-3-position with a substituted benzyl or phenacyl group (such as 2-bromobenzyl or
2-naphthylacyl) could be vital for antitumor activity, which provides the valuable information for further
rational design and synthesis of imidazolium salts.
Imidazolium salts
Steroidal heterocycles
N
N
N
Cl
H
R
H
H
H
H
Lepidiline A R = H
Lepidiline B R = Me
Abiraterone
NHBu
HO
N
O
O
N
N
H
Br
HO
SSA
NMIB
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Fig. 1 Representative structures of antitumour activity steroidal derivatives and imidazolium salts.
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Molecular hybridization has played an important role in drug discovery during the past two decades. Thus, it
is clear that new pharmacologically interesting hybrid compounds will directly benefit the pharmaceutical
industry.[36-39] In view of the potential anticancer activity of steroidal derivatives and imidazolium salts, we
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launched the synthesis of hybridizing compounds bearing steroidal moieties and imidazolium salts. Although
17-(1-benzimidazole) substituted steroidal derivative was prepared and found to display anticancer activity,[40]
and some steroidal derivatives bearing substituted pyrazoles and triazoles pyrazoles and triazoles were
synthesized as neuroactive steroids,[41] to the best of our knowledge, no reports regarding synthesis and
biological activity for steroidal imidazolium salt hybrid derivatives have been reported. With this in mind, we
turned our attention to synthesis and antitumor activity of a series of novel steroidal imidazolium salt derivatives
and report our results herein.
2. Results and discussion
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2.1 Chemistry
To synthesize the steroidal imidazolium salts, we used various substituted imidazole derivatives that were
alkylated with activated 3-hydroxyl steroids, which were synthesized from abundant steroids (diosgenin,
cholesterol and dehydroepiandrosterone) in nature. As shown in Scheme 1, commercially available steroids
diosgenin a1 or cholesterol b1 was transformed via the mesylate derivatives (a2/b2) to give the respective 6α-
imidazolyl steroidal derivative (a3/b3), 3α-imidazolyl steroidal derivative (a4-a6/b4-b6) and 3β-imidazolyl
steroidal derivative (a7/b7) by refluxing with various substituted imidazole (imidazole, benzimidazole or 5,6-
dimethyl-benzimidazole) under toluene with 7-48% yields (two steps). The nucleophilic substitution reactions
generally led to three stereoisomers products. Major product was 6α-imidazolyl steroidal product (such as a3/b3,
42%/48%), and minor products were 3α-imidazolyl steroidal product (such as a4/b4, 15%/18%) and 3β-
imidazolyl steroidal product (such as a7/b7, 8%/7%). Thus, some stereoisomers (3α- or 3β-imidazolyl steroidal
derivative) were synthesized in relatively larger quantities. Finally, forty-nine diosgenin‒imidazolium salt
derivatives a8-a35 and cholesterol‒imidazolium salt derivatives b8-b28 were synthesized from coupling of
imidazolyl substituted steroidal derivatives (a3-a7/b3-b7) with various alkyl and phenacyl bromides at excellent
yields (67–98%).
As shown in Scheme 2, after 3β-OH group was protected with tert-butyl(dimethyl)silyl of commercial steroid
dehydroepiandrosterone c1, treatment of c2 with (R)-(+)-2-methyl-2-propanesulfinamide in the presence of
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tetraethyl titanate led to corresponding imine, then reduction of imine gave the 17β-tert-butylsulfinamido
derivative c3 in 82% yield (two steps). Subsequently, acid mediated deprotection of N-tert-butanesulfinyl and
OH group yielded amine c4 in 92% yield. Based on our previous synthetic method,[42] an imidazole ring were
installed at C-17 to give key intermediate 17β-imidazolyl steroidal derivative c5. 3β-OH group was further
protected with acetyl to give 3β-AcO-17β-imidazolyl steroid c6. Finally, twelve dehydroepiandrosterone‒
imidazolium salt derivatives c7-c12 and c13-c18 were synthesized from coupling of imidazolyl steroids c5 and
c6 with various alkyl and phenacyl bromides at 75–97% and 72–96% yields, respectively. The structures and
yields of all new steroidal imidazolyl derivative and steroidal imidazolium salt derivatives are shown in Table 1.
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O
O
H
H
H
H
H
R1
MsCl, Et3N
DCM, 0 oC to rt
2 h
HO
diosgenin a1
H
HN
N
H
Toluene
110 oC, 24-36 h
two steps, 7-48%
MsO
H
a2/b2
H
H
H
HO
cholesterol b1
H
H
H
H
H
H
N
N
N
N
N
N
R1
a7/b7
a3/b3
a4-a6/b4-b6
imidazole ring
imidazole
benzimidazole
Toluene
R2 Br 110 oC, 24-48 h
67-98%
5,6-dimethylbenzimidazole
R2 = phenacyl, alkyl
H
H
H
H
H
H
R2
R2
N
N
R1
N
N
N
Br
Br
N
Br
R2
a8-a13/b8-b13
a14-a31/b14-b23
a32-a35/b24-b28
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Scheme 1 Synthesis of diosgenin‒ and cholesterol‒imidazolium salt derivatives a8-a35/b8-b28.
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Scheme 2 Synthesis of dehydroepiandrosterone‒imidazolium salt derivatives c7-c18.
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Table 1
Structures and yields of steroidal imidazolyl derivatives and steroidal imidazolium salt derivatives
Entry Compound Steroid
No.
Imidazole ring
R2
Molecular
formula
Yields (%)
C30H44N2O2
C30H44N2O2
C34H46N2O2
C36H50N2O2
C30H44N2O2
C30H48N2
42%
15%
9%
1
2
3
4
5
6
7
8
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
cholesterol
cholesterol
cholesterol
cholesterol
imidazole
–
–
–
–
–
–
–
–
–
a3
a4
a5
a6
a7
b3
b4
b5
imidazole
benzimidazole
5,6-dimethyl-benzimidazole
imidazole
10%
8%
48%
18%
10%
9%
imidazole
C30H48N2
imidazole
C34H50N2
benzimidazole
C36H54N2
9
5,6-dimethyl-benzimidazole
imidazole
b6
C30H48N2
7%
10
11
12
13
14
15
16
17
cholesterol
dehydroepiandrosterone
dehydroepiandrosterone
–
–
–
b7
C22H32N2O
C24H34N2O2
68%
86%
93%
92%
97%
98%
82%
imidazole
c5
imidazole
c6
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
imidazole
phenacyl
a8
C38H51BrN2O3
C39H53BrN2O4
C38H50Br2N2O3
imidazole
4-methoxyphenacyl
3-bromophenacyl
2-naphthylacyl
2-bromobenzyl
a9
imidazole
a10
a11
a12
imidazole
C42H53BrN2O3
C37H50Br2N2O2
C41H53BrN2O2
imidazole
18
19
20
21
diosgenin
diosgenin
diosgenin
diosgenin
imidazole
imidazole
imidazole
imidazole
2-naphthylmethyl
phenacyl
75%
72%
83%
73%
a13
a14
a15
a16
C38H51BrN2O3
C39H53BrN2O4
C38H50Br2N2O3
4-methoxyphenacyl
3-bromophenacyl
22
23
24
diosgenin
diosgenin
diosgenin
imidazole
imidazole
imidazole
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
94%
88%
90%
a17
a18
a19
C42H53BrN2O3
C37H50Br2N2O2
C41H53BrN2O2
25
26
27
28
29
30
31
32
33
34
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
benzimidazole
benzimidazole
benzimidazole
benzimidazole
benzimidazole
benzimidazole
phenacyl
74%
72%
79%
90%
96%
a20
a21
a22
a23
a24
a25
a26
a27
a28
a29
C42H53BrN2O3
C43H55BrN2O4
C42H52Br2N2O3
C46H55BrN2O3
C41H52Br2N2O2
4-methoxyphenacyl
3-bromophenacyl
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
C45H55BrN2O2 87%
5,6-dimethyl-benzimidazole phenacyl
79%
72%
86%
87%
C44H57BrN2O3
C45H59BrN2O4
C44H56Br2N2O3
C48H59BrN2O3
5,6-dimethyl-benzimidazole 4-methoxyphenacyl
5,6-dimethyl-benzimidazole 3-bromophenacyl
5,6-dimethyl-benzimidazole 2-naphthylacyl
5,6-dimethyl-benzimidazole 2-bromobenzyl
5,6-dimethyl-benzimidazole 2-naphthylmethyl
35
36
37
38
39
40
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
diosgenin
68%
94%
69%
92%
87%
89%
a30
a31
a32
a33
a34
a35
C42H53Br2N2O2
C47H59BrN2O2
C38H51BrN2O3
C39H53BrN2O4
C42H53BrN2O3
C37H50Br2N2O2
imidazole
imidazole
imidazole
imidazole
phenacyl
4-methoxyphenacyl
2-naphthylacyl
2-bromobenzyl
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cholesterol
cholesterol
cholesterol
imidazole
imidazole
imidazole
phenacyl
78%
81%
78%
b8
C38H55BrN2O
C39H57BrN2O2
C38H54Br2N2O
4-methoxyphenacyl
3-bromophenacyl
b9
b10
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45
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48
49
50
51
52
53
54
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56
57
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61
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cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
cholesterol
imidazole
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
phenacyl
84%
67%
75%
85%
76%
79%
93%
90%
97%
86%
78%
82%
74%
89%
95%
84%
78%
72%
75%
89%
89%
97%
78%
87%
92%
94%
96%
93%
92%
72%
b11
b12
b13
b14
b15
b16
b17
b18
b19
b20
b21
b22
b23
b24
b25
b26
b27
b28
c7
C42H57BrN2O
C37H54Br2N2
C41H57BrN2
imidazole
imidazole
imidazole
C38H55BrN2O
C39H57BrN2O2
C38H54Br2N2O
imidazole
4-methoxyphenacyl
3-bromophenacyl
2-naphthylacyl
2-bromobenzyl
phenacyl
imidazole
imidazole
C42H57BrN2O
C37H54Br2N2
imidazole
benzimidazole
benzimidazole
benzimidazole
C42H57BrN2O
4-methoxyphenacyl
2-bromobenzyl
C43H59BrN2O2
C41H56Br2N2
5,6-dimethyl-benzimidazole phenacyl
C44H61BrN2O
C43H60Br2N2
C38H55BrN2O
C39H57BrN2O2
C42H57BrN2O
C37H54Br2N2
5,6-dimethyl-benzimidazole 2-bromobenzyl
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
imidazole
phenacyl
4-methoxyphenacyl
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
phenacyl
cholesterol
C41H57BrN2
dehydroepiandrosterone
C30H39BrN2O2
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
dehydroepiandrosterone
C31H41BrN2O3
C30H38Br2N2O2
C34H41BrN2O2
C29H38Br2N2O
C33H41BrN2O
C32H41BrN2O3
C33H43BrN2O4
C32H40Br2N2O3
C36H43BrN2O3
C31H40Br2N2O2
C35H43BrN2O2
4-methoxyphenacyl
3-bromophenacyl
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
phenacyl
c8
c9
c10
c11
c12
c13
c14
c15
c16
c17
c18
4-methoxyphenacyl
3-bromophenacyl
2-naphthylacyl
2-bromobenzyl
2-naphthylmethyl
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113
114
115
To confirm the structures of the steroidal imidazolium salt derivatives, compound c11 was selected as a
representative compound and characterized using X-ray crystallography (CCDC 1880928),[43] as shown in Fig.
2.
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Fig. 2 X-ray crystal structure of compound c11.
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2.2 Biological evaluation and structure-activity relationship analysis
The cytotoxic potential for the seventy-three synthesized steroidal imidazolyl derivatives and steroidal
imidazolium salt derivatives against five human cancer cell lines including myeloid leukaemia (HL-60), liver
carcinoma (SMMC-7721), lung carcinoma (A-549), breast carcinoma (MCF-7) and colon carcinoma (SW480)
were determined using the MTS assay. DDP (Cisplatin), as well as diosgenin (a1), cholesterol (b1),
dehydroepiandrosterone (c1), imidazole, benzimidazole and 5,6-dimethyl-benzimidazole, were chosen as
positive controls. The results were listed in Table 2.
Table 2
Cytotoxic activities of steroidal imidazolyl derivatives and steroidal imidazolium salt derivatives in vitrob (IC50, µMa)
Entry
1
Compound No.
HL-60
>20
SMMC-7721
>20
A-549
>20
MCF-7
>20
SW480
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16.44
>20
>20
>20
a1
2
>20
>20
>20
>20
b1
3
>20
>20
>20
>20
c1
4
imidazole
benzimidazole
>20
>20
>20
>20
5
>20
>20
>20
>20
6
5,6-dimethyl-benzimidazole >20
>20
>20
>20
7
8.33
>20
>20
>20
9.51
2.00
>20
>20
0.18
10.04
>20
10.29
>20
19.67
>20
a3
a4
a5
a6
a7
b3
b4
b5
b6
8
9
>20
>20
>20
10
11
12
13
14
15
>20
>20
>20
9.74
2.12
>20
9.92
11.65
>20
14.49
>20
>20
>20
>20
>20
＞
20
＞
20
>20
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>20
>20
>20
>20
>20
b7
>20
>20
>20
>20
>20
c5
>20
>20
>20
>20
>20
c6
2.69
1.25
1.28
1.03
1.24
1.22
1.09
1.25
1.25
0.96
0.43
1.01
1.19
1.22
1.36
1.10
0.34
1.02
0.44
0.74
0.98
0.45
0.49
0.42
1.18
0.94
1.37
0.92
1.53
1.52
1.42
2.41
5.53
1.64
6.85
6.00
9.91
10.63
1.59
0.88
7.38
7.32
7.28
3.46
4.58
2.45
7.15
1.51
1.71
1.89
0.95
1.15
1.81
1.45
1.75
1.33
0.21
0.88
0.90
0.44
1.39
0.57
0.44
0.59
2.14
1.61
5.49
2.27
1.29
1.10
1.22
1.23
1.16
1.04
1.49
2.90
1.70
1.88
0.94
1.65
7.76
7.18
7.03
7.27
6.60
4.92
7.30
1.94
1.55
3.34
1.22
1.12
2.55
2.45
1.77
1.32
4.47
1.25
9.57
5.02
7.15
5.53
0.67
5.12
1.86
1.21
3.49
2.99
7.28
6.68
7.84
8.53
7.65
7.67
8.42
13.22
7.22
8.82
7.36
7.51
5.50
4.49
5.21
2.26
3.86
1.95
5.18
1.47
1.43
2.27
1.41
1.09
2.49
2.11
1.53
1.67
2.14
2.31
2.91
2.31
2.78
3.05
0.73
1.90
5.66
1.96
5.83
1.88
6.98
6.54
8.70
8.00
10.83
8.04
8.41
5.25
8.51
>20
7.39
7.03
6.46
4.01
6.33
4.03
8.18
2.59
1.65
3.62
1.66
1.59
2.59
2.55
1.74
1.75
2.10
1.80
3.22
2.01
2.22
3.10
0.79
1.60
7.96
4.15
6.55
5.37
6.06
7.18
8.23
6.25
10.90
7.48
9.03
4.87
6.33
10.54
8.32
10.18
a8
a9
a10
a11
a12
a13
a14
a15
a16
a17
a18
a19
a20
a21
a22
a23
a24
a25
a26
a27
a28
a29
a30
a31
a32
a33
a34
a35
b8
b9
b10
b11
b12
b13
b14
b15
b16
b17
b18
b19
6.02
7.18
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79
4.41
4.47
0.28
6.96
7.73
7.97
7.26
2.35
5.39
>20
1.45
1.56
1.77
1.81
1.42
1.52
2.10
1.44
1.41
>20
7.66
7.31
8.11
8.44
8.10
8.75
9.65
8.05
6.95
>20
9.85
8.97
9.87
14.25
8.84
8.71
>20
10.10
10.60
10.44
13.16
6.56
10.66
11.28
7.75
9.29
>20
b20
b21
b22
b23
b24
b25
b26
b27
b28
c7
7.62
8.35
>20
>20
>20
>20
>20
>20
c8
6.42
2.00
6.30
2.68
6.42
2.20
4.35
1.26
1.48
1.22
2.11
14.90
3.50
7.63
4.41
12.03
4.31
5.47
1.75
3.83
2.45
11.27
>20
7.25
4.38
9.61
5.08
9.07
3.46
4.88
2.10
4.84
2.53
17.43
9.78
5.59
>20
c9
9.69
>20
c10
c11
c12
c13
c14
c15
c16
c17
c18
DDP
7.87
>20
6.47
11.12
6.06
7.26
4.45
6.87
5.64
17.05
10.70
7.20
4.39
6.52
4.74
6.94
80
a
Cytotoxicity as IC50 for each cell line, is the concentration of compound which reduced by 50% the optical density of
treated cells with respect to untreated cells using the MTS assay.
b Data represent the mean values of three independent determinations.
129
130
131
132
133
134
135
136
137
138
139
As displayed in Table 2, single steroidal compounds (diosgenin (a1), cholesterol (b1) and
dehydroepiandrosterone (c1)) and single imidazole compounds (imidazole, benzimidazole and 5,6-dimethyl-
benzimidazole), as controls, lacked activity against all tumor cell lines investigated at the concentration of 20
µM (Entries 1-6). The structures of imidazole and imidazolium salt in steroidal derivatives have an important
effect on the cytotoxic potential. Most of steroidal imidazolyl derivatives a3-a7/b3-b7/c5-c6 (Entries 7-18)
lacked activities against all tumor cell lines tested at the concentration of 20 µM, except a3, a7, b3 and b6 with
weak cytotoxic activities. Interestingly, compound b6, bearing a benzimidazole ring at position-3β of
cholesterol, showed obvious cytotoxic activity selectively against HL-60 cell lines with IC50 value of 0.18 µM.
However, their steroidal imidazolium salt derivatives (Entries 19-79) exhibited higher cytotoxic activities. For
three kinds of steroids, diosgenin‒imidazolium salt derivatives a8-a35 displayed the highest cytotoxic activities
12

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with IC50 values of 0.21–9.57 µM in vitro. Cholesterol‒imidazolium salt derivatives b8-b28 exhibited the higher
activities with IC50 values of 0.28–14.25 µM, while dehydroepiandrosterone‒imidazolium salt derivatives c7-
c18 showed the lowest activities with IC50 values of 1.22–14.90 µM or higher than 20 µM.
For the substituent’s position of imidazolium salt moiety in steroids, imidazolium salts at position-3α of
steroidal derivatives (a14-a31/b14-b23) possessed the highest cytotoxic activities and most of them exhibited
potent inhibitory activities. 3β-Imidazolium salt steroidal derivatives (a32-a35/b24-b28) and 6α-imidazolium
salt steroidal derivatives (a8-a13/b8-b13) showed medium or high inhibitory activities. In
dehydroepiandrosterone derivatives (c7-c18), imidazolium salts at position-17β of dehydroepiandrosterone
derivatives had the lowest activities, and imidazolium salts with 3β-AcO group (c7-c12) were better than with
3β-OH group (c13-c18).
Many literatures have reported that bioactivities of natural products and drug molecules have been closely
related to the stereo structures.[44-46] As shown in Table 2, the stereochemistry at position-3 in steroidal
imidazolium salt derivatives has a great influence on the cytotoxic potential. Although both isomers (3α- and
3β-) in steroidal derivatives possessed cytotoxic activities, imidazolium salts at position-3α displayed higher
inhibitory activities than 3β-isomer in most cell lines. For example, steroidal imidazolium salt a17 (3α-isomer)
and a18 (3α-isomer) showed higher inhibitory activities against SMMC-7721 cell lines with 2.9- and 2.4-fold
than a34 (3β-isomer) and a35 (3β-isomer), respectively. Similarly, steroidal imidazolium salt a18 (3α-isomer)
and b15 (3α-isomer) showed higher inhibitory activities against SW-480 cell lines with 3.2- and 2.2-fold than
a35 (3β-isomer) and b25 (3β-isomer), respectively.
In the case of imidazole ring, steroidal imidazolium salt derivatives (a8-a19/b8-b18/c7-c18) with imidazole
ring displayed medium inhibitory activities. Steroidal derivatives (a20-a25/b19-b21) with benzimidazole ring
possessed higher inhibitory activity. Interestingly, Steroidal derivatives (a26-a31/b22-b23) with 5,6-dimethyl-
benzimidazole rings exhibited significant inhibitory activities. Among them, diosgenin derivatives a26-a31,
with 5,6-dimethyl-benzimidazole rings, showed powerful inhibitory activity with IC50 values of 0.42-3.22 µM
against HL-60, A-549, MCF-7 and SW480 cell lines.
For the substituent at position-3 of imidazole ring, imidazolium salts with phenacyl substituent such as a8,
a14, a20, a26 and a32, as well as steroidal derivatives with 4-methoxyphenacyl or 3-bromophenacyl substituent
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such as a9, a10, a15, a16, a21, a22, a27, a28 and a33 had relative weak inhibitory activities against five tumor
cell lines. Meanwhile, imidazolium salts with 2-naphthylacyl substituent such as a11, a17, a23, a29 and a34
showed medium cytotoxic activities (IC50 = 0.44–9.57 µM). However, imidazolium salts with 2-bromobenzyl
substituent such as a12, a18, a24, a30 and a35, as well as 2-naphthylmethyl substituent such as a13, a19, a25
and a31 exhibited much higher inhibitory activity (IC50 = 0.21–6.60 µM). Notably, diosgenin‒imidazolium salt
a30, bearing a 2-bromobenzyl substituent at position-3 of 5,6-dimethyl-benzimidazole, was found to be the most
potent compound with IC50 values of 0.44–0.79 µM against five human tumor cell lines investigated.
Particularly, diosgenin‒imidazolium salt a24, with a 2-bromobenzyl substituent at position-3 of benzimidazole,
showed inhibitory activity selectively against SMMC-7721 cell lines with IC50 value of 0.21 µM and 54-fold
more sensitive to DDP.
The results suggest that the existence of substituted 5,6-dimethyl-benzimidazoles ring and substitution of the
imidazolyl-3α-position with a 2-bromobenzyl or 2-naphthylmethyl group could be critical for promoting
cytotoxic activity. Then, the structure-activity relationships (SARs) results were exemplified in Scheme 3.
14

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R2
Br
N
H
H
H
N
H
H
H
N
H
R2
R2
N
N
N
N
Br
H
H
Br
R1
R
N
Br
(4)
(2)
(3)
(1)
R2
Steroidal imidazolium salt derivatives:
diosgenin-imidazolium salts > cholesterol-imidazolium salts > dehydroepiandrosterone-imidazolium salts
Best
3α-imidazolium salts (2) > 6α-imidazolium salts (1) > 3β-imidazolium salts (3) > 17β-imidazolium salts (4)
Best
imidazole ring:
5,6-dimethyl-benzimidazole > benzimidazole > imidazole
Best
O
O
O
O
Br
R2 =
≈
>
>
>
>
Br
OMe
Best
Best Potent Compound
O
H
O
H
H
Br
H
H
tumor cell lines IC50, µM
Br
N
N
HL-60
A549
0.49
0.44
0.67
0.73
0.79
SMMC-7721
MCF-7
SW480
a30
181
182
Scheme 3 Structure-activity relationships of steroidal imidazolium salt derivatives.
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184
185
186
187
2.3 Steroidal imidazolium salt a30 induces G0/G1 cell cycle arrest and apoptosis in SMMC-7721 cells
To determine whether the proliferation inhibitory effect of imidazolium salt a30 was caused by cell cycle
arrest, propidium iodide (PI) staining and flow cytometry analysis of cells was performed in SMMC-7721 cells
treated with indicated concentrations of imidazolium salt a30 (0, 0.5, 1 µM). As shown in Fig. 3, imidazolium
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salt a30 induced G0/G1 cell cycle arrest in a dose dependent manner, while the G0/1 phase cell population
increased to 73.61 % and 78.10 % in cells treated with 2 µM and 4 µM imidazolium salt a30 as compared to
control showing 62.58 %. Inversely, S phase and G2/M phase cell population were respectively decreased to
14.22% and 4.67 % in 4 µM imidazolium salt a30 treated group as compared to control having 24.50 % and
8.72% , while the proportion of sub-G1 phase cells showed no significant change.
194
Treatment
Cells (%)
sub-G1
G0/G1
S
G2/M
DMSO
2.33±0.17
2.40±0.23
2.43±0.12
62.58±0.35
73.61±0.50
78.10±1.15
24.50±0.05
19.23±0.54
14.22±0.36
8.72±0.42
3.49±0.35
4.67±0.05
Compound a30 (0.5 µM)
Compound a30 (1 µM)
195
196
197
198
199
Fig. 3 Compound a30 induces G0/G1 phase arrest in SMMC-7721 cells. (A) Cells were treated with 0.5 and 1 µM of
compound a30 for 24 h. Cell cycle was determined by PI staining and cell cytometry. (B) The percentages of cells in
different phases were quantified. At least three independent experiments were performed and data of one representative
experiment is shown.
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201
202
203
204
205
The steroidal imidazolium salt a30 induced cell apoptosis was determined with Annexin V-FITC/PI double-
labeled cell cytometry. As shown in Fig. 4, after treatment of cells with imidazolium salt a30 at 1 µM and 2 µM
for 48 h, cell apoptosis in SMMC-7721 cells remarkably elevated to 19.07 % and 65.59 %, respectively. The
data suggested that illustrated that steroidal imidazolium salt a30 inhibited cell proliferation through induction
of G0/G1 cell cycle arrest and apoptosis of the SMMC-7721 cells.
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209
Fig. 4 Compound a30 caused significant apoptosis of SMMC-7721 cells. (A) Cells were treated with 1 and 2 µM
compound imidazolium salt a30 for 48 h. Cell apoptosis was determined by Annexin V-FITC/PI double-staining assay. (B)
The quantification of cell apoptosis.
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211
3. Conclusion
212
213
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216
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218
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221
222
223
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225
In summary, a number of novel steroidal imidazolium salt derivatives synthesized in this work proved to be
potent antitumor agents. The results showed that diosgenin‒imidazolium salt derivatives displayed much higher
cytotoxic activities than holesterol‒imidazolium salts and dehydroepiandrosterone‒imidazolium salts. The
SARs results suggested that the existence of substituted 5,6-dimethyl-benzimidazoles or benzimidazole ring and
substitution of the imidazolyl-3α-position with a 2-bromobenzyl or 2-naphthylmethyl group could be critical for
promoting cytotoxic activity. The diosgenin‒imidazolium salt derivatives a18, a19, a24, a25, a30 and a31, with
5,6-dimethyl-benzimidazoles ring and a 2-bromobenzyl or 2-naphthylmethyl group at imidazolyl-3-position,
exhibited powerful inhibitory activity. Particularly, diosgenin‒imidazolium salt a30 was found to be the most
potent compound with IC50 values of 0.44–0.79 µM against five human tumor cell lines investigated.
Diosgenin‒imidazolium salt a24 showed inhibitory activity selectively against SMMC-7721 cell lines with IC50
value of 0.21 µM and 54-fold more sensitive to DDP. Compound a30 can induce the G0/G1 phase cell cycle
arrest and apoptosis in SMMC-7721 cells. The steroidal imidazolium salts derivatives a18, a19, a24, a25, a30
and a31 could serve as a new starting point to explore promising lead compounds for the development of new
anticancer agents.
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227
4. Experimental Section
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4.1. Chemistry
4.1.1. General
229
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235
236
237
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239
240
241
242
243
244
245
246
247
248
249
250
251
Melting points were obtained on a XT-4 melting-point apparatus and were uncorrected. Proton nuclear
magnetic resonance (1H-NMR) spectra were recorded on a Bruker Avance 400 spectrometer at 400 MHz.
Carbon-13 nuclear magnetic resonance (13C-NMR) was recorded on Bruker Avance 400 spectrometer at 100
MHz. Chemical shifts are reported as δ values in parts per million (ppm) relative to tetramethylsilane (TMS) for
all recorded NMR spectra. Low-resolution Mass spectra were recorded on a VG Auto Spec-3000 magnetic
sector MS spectrometer. High Resolution Mass spectra were taken on AB QSTAR Pulsar mass spectrometer.
Silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qingdao
Marine Chemical Company (China). All air- or moisture-sensitive reactions were conducted under an argon
atmosphere. Starting materials and reagents used in reactions were obtained commercially from Acros, Aldrich,
Fluka and were used without purification, unless otherwise indicated.
4.1.2. Synthesis of compounds a3-a7/b3-b7
To a solution of diosgenin a1 (4.2 g, 10.0 mmol)/cholesterol b1 (3.9 g, 10.0 mmol) in dichloromethane (30.0
o
mL) was added methanesulfonyl chloride (0.9 mL, 12.0 mmol) and triethylamine (2.8 mL, 20.0 mmol) at 0 C.
The resulting mixture was stirred at room temperature for 12 h. After quenching the reaction with water (50.0
mL), the layers were separated. The organic phase was dried over anhydrous Na2SO4 and concentrated, and used
for the next synthetic step. A mixture of the previous methanesulfonate and imidazole or substituted imidazole
(30.0 mmol) was stirred in toluene (20.0 ml) at reflux for 24–36 h (monitored by TLC). After cooling to room
temperature, the solvent was concentrated, and the residue was diluted with EtOAc (20.0 mL). The organic layer
was washed with water (20.0 mL) and brine (20.0 mL), dried over anhydrous Na2SO4 and concentrated. The
o
residue was purified by column chromatography (silica gel, petroleum ether 60–90 C : ethyl acetate =
3:1→1:1) to afford a3-a7/b3-b7 in 7–48% yield (two steps) as yellow or white powder.
4.1.2.1 1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosahydro-2H-spiro[cyclopropa[1',
252
253
7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazole
（
a3
）
.
Yield 42%. White
o
1
powder, m.p. 199 – 201 C. IR νmax (cm-1): 3424, 2949, 1454, 1378, 1299, 1073, 982, 850, 819, 738, 662. H
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255
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257
258
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260
NMR (400 MHz, Chloroform-d) δ 7.77 (s, 1H), 7.11 (s, 1H), 7.02 (s, 1H), 4.43 (q, J = 8.0 Hz, 1H), 3.55 (t, J =
3.2 Hz, 1H), 3.49 – 3.44 (m, 1H), 3.37 (t, J = 10.8 Hz, 1H), 2.36 (dt, J = 14.4, 2.8 Hz, 1H), 2.14 – 2.08 (m, 1H),
1.88 – 1.68 (m, 5H), 1.66 – 1.54 (m, 7H), 1.47 – 1.11 (m, 8H), 0.99 – 0.90 (m, 6H), 0.78 (d, J = 6.0 Hz, 3H),
0.73 (s, 3H), 0.70 (s, 3H) ppm. 13C NMR (100 MHz, Chloroform-d) δ 136.5, 128.5, 117.9, 109.3, 80.6, 66.9,
62.2, 58.7, 55.8, 48.0, 43.2, 41.6, 40.7, 39.9, 35.2, 34.7, 33.4, 31.6, 31.4, 30.4, 30.3, 28.8, 24.5, 24.3, 22.3, 19.7,
17.1, 16.6, 14.5, 14.4 ppm.HRMS (ESI-TOF) m/z Calcd for C30H44N2O2 [M+H]+ 465.3476, found 465.3477.
4.1.2.2 1-((4R,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,8a,8b,9,11a,
261
262
12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-1H-imidazole
o
（
a4
）
.
Yield 15%. White powder, m.p. 199 – 201 C. IR νmax (cm-1): 3424, 2949, 1453, 1376, 1242, 1109,
263
264
265
266
267
268
269
270
1069, 980, 899, 662. 1H NMR (400 MHz, Chloroform-d) δ 7.55 (s, 1H), 7.05 (s, 1H), 6.98 (s, 1H), 5.42 (d, J =
5.6. Hz, 1H), 4.42 (q, J = 7.6 Hz, 1H), 3.92 – 3.84 (m, 1H), 3.49 – 3.45 (m, 1H), 3.37 (t, J = 10.8 Hz, 1H), 2.65
– 2.57 (m, 1H), 2.45 – 2.40 (m, 1H), 2.03 – 1.98 (m, 4H), 1.84 – 1.74 (m, 4H), 1.69 – 1.42 (m, 10H), 1.31 – 1.16
(m, 4H), 1.09 (s, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.80 – 0.78 (m, 6H) ppm. 13C NMR (100 MHz, Chloroform-d) δ
139.7, 135.3, 129.1, 122.6, 116.8, 109.3, 80.7, 66.9, 62.1, 57.5, 56.5, 50.1, 41.6, 40.5, 40.3, 39.7, 38.0, 36.8,
32.0, 31.8, 31.4, 31.3, 30.3, 29.9, 28.8, 20.8, 19.4, 17.1, 16.3, 14.5 ppm. HRMS (ESI-TOF) m/z Calcd for
C30H44N2O2 [M+H]+ 465.3476, found 465.3475.
4.1.2.3 1-((4R,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,8a,8b,9,11a,
271
272
12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-1H-benzo[d]
imidazole
（a5）.
Yield 9%. White powder, m.p. 225 – 227 oC. IR νmax (cm-1): 3416, 2948, 1484, 1456, 1386,
273
274
275
276
277
278
1280, 1052, 1008, 901, 747. 1H NMR (400 MHz, Chloroform-d) δ 8.02 (s, 1H), 7.83 – 7.80 (m, 1H), 7.44 – 7.42
(m, 1H), 7.29 – 7.27 (m, 2H), 5.47 (d, J = 3.2 Hz, 1H), 4.44 (q, J = 7.4 Hz, 1H), 4.21 – 4.14 (m, 1H), 3.41 –
3.30 (m, 1H), 2.77 (t, J = 12.8 Hz, 1H), 2.57 – 2.51 (m, 1H), 2.17 – 1.99 (m, 5H), 1.91 – 1.85 (m, 1H), 1.83 –
1.77 (m, 2H), 1.73 – 1.57 (m, 7H), 1.52 – 1.22 (m, 6H), 1.16 (s, 3H), 1.13 – 1.06 (m, 2H), 0.99 (d, J = 6.8 Hz,
13
3H), 0.82 – 0.79 (m, 6H) ppm. C NMR (100 MHz, CDCl3) δ 143.9, 140.2, 139.8, 133.4, 122.8, 122.6, 122.1,
120.5, 110., 109.3, 80.8, 66.9, 62.1, 56.5, 56.2, 50.1, 41.6, 40.3, 39.7, 39.3, 38.2, 37.0, 32.0, 31.8, 31.4, 30.3,
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281
28.8, 20.8, 19.6, 17.2, 16.3, 14.6 ppm. HRMS (ESI-TOF) m/z Calcd for C34H46N2O2 [M+H]+ 515.3632, found
515.3631.
4.1.2.4 5,6-dimethyl-1-((4R,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,
8a,8b,9,11a,12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-1H-
282
283
benzo[d]imidazole
（a6）.
Yield 10%. Pale yellow powder, m.p. 211 – 213 oC. IR νmax (cm-1): 3439, 2941,
284
285
286
287
288
289
290
291
292
1486, 1458, 1374, 1223, 1171, 1050, 980, 901. 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.56 (s, 1H),
7.17 (s, 1H), 5.46 (d, J = 5.6 Hz, 1H), 4.47 – 4.40 (m, 1H), 4.15 – 4.09 (m, 1H), 3.50 – 3.46 (m, 1H), 3.41 – 3.32
(m, 1H), 2.78 – 2.70 (m, 1H), 2.54 – 2.49 (m, 1H), 2.39 (s, 3H), 2.37 (s, 3H), 2.11 – 1.98 (m, 6H), 1.91 – 1.87
(m, 1H), 1.82 – 1.76 (m, 2H), 1.72 – 1.56 (m, 7H), 1.51 – 1.41 (m, 2H), 1.36 – 1.22 (m, 4H), 1.15 (s, 3H), 1.01
– 0.98 (m, 3H), 0.82 – 0.79 (m, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 142.6, 140.1, 139.5, 132.1, 131.8,
131.1, 129.2, 128.3, 125.4, 122.8, 120.5, 110.3, 109.9, 109.4, 81.0, 67.0, 62.2, 56.6, 56.2, 50.3, 41.8, 40.4, 39.9,
39.5, 38.4, 37.1, 32.2, 32.0, 31.5, 30.4, 29.0, 21.0, 20.8, 20.4, 19.7, 17.3, 16.5, 14.7 ppm. HRMS (ESI-TOF) m/z
Calcd for C36H50N2O2 [M+H]+ 543.3945, found 543.3943.
4.1.2.5 1-((4S,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,8a,8b,9,11a,
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12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-1H-imidazole
o
（
a7
）
.
Yield 8%. White powder, m.p. 195 – 197 C. IR νmax (cm-1): 3422, 2950, 1618, 1456, 1378, 1241,
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1215, 1079, 1052, 982, 899, 661. 1H NMR (400 MHz, Chloroform-d) δ 7.69 (s, 1H), 7.00 (d, J = 10.2 Hz, 2H),
5.48 (dd, J = 4.8, 2.0 Hz, 1H), 4.44 – 4.35 (m, 2H), 3.49 – 3.45 (m, 1H), 3.37 (t, J = 10.8 Hz, 1H), 2.91 – 2.83
(m, 1H), 2.51 (dt, J = 15.6, 2.4 Hz, 1H), 2.10 – 1.96 (m, 3H), 1.88 – 1.82 (m, 2H), 1.79 – 1.57 (m, 9H), 1.49 –
1.39 (m, 3H), 1.33 – 1.24 (m, 1H), 1.15 – 1.00 (m, 7H), 0.96 (d, J = 6.8 Hz, 3H), 0.79 – 0.77 (m, 6H) ppm. 13C
NMR (100 MHz, CDCl3) δ 138.7, 136.8, 128.4, 123.7, 118.7, 109.3, 80.8, 66.8, 62.0, 56.4, 53.0, 49.7, 41.6,
40.2, 39.6, 37.1, 35.9, 32.3, 32.0, 31.8, 31.4, 31.2, 30.3, 28.8, 28.5, 20.4, 19.4, 17.1, 16.3, 14.5 ppm. HRMS
(ESI-TOF) m/z Calcd for C30H44N2O2 [M+H]+ 465.3476, found 465.3476.
4.1.2.6
1-((1aR,3aR,5aR,10S,10aR)-3a,5a-dimethyl-6-((R)-6-methylheptan-2-yl)hexadecahydro
b3 Yield 48%.
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cyclopenta[a]cyclopropa[2,3]cyclopenta[1,2-f]naphthalen-10-yl)-1H-imidazole
（
）.
Brown powder, m.p. 99 – 101 oC. IR νmax (cm-1): 3424, 2938, 1469, 1383, 1223, 1112, 1079, 906, 828, 718, 664.
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1H NMR (400 MHz, Chloroform-d) δ 7.78 (s, 1H), 7.16 (s, 1H), 7.03 (s, 1H), 5.29 (s, 1H), 3.54 (s, 1H), 2.39 –
2.31 (m, 1H), 2.00 – 1.95 (m, 1H), 1.89 – 1.78 (m, 2H), 1.72 – 1.67 (m, 1H), 1.61 – 1.47 (m, 5H), 1.41 – 1.25
(m, 6H), 1.23 – 1.19 (m, 1H), 1.17 – 1.07 (m, 7H), 1.01 – 0.92 (m, 4H), 0.89 (d, J = 6.4 Hz, 3H), 0.85 (dd, J =
6.8, 2.0 Hz, 6H), 0.69 (s, 3H), 0.62 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 136.6, 128.5, 118.2, 58.9, 56.3,
56.2, 48.1, 43.2, 42.8, 40.1, 39.6, 36.2, 35.9, 35.4, 34.7, 33.6, 30.9, 28.3, 28.1, 24.7, 24.4, 24.3, 23.9, 22.9, 22.7,
22.6, 19.8, 18.8, 14.4, 12.3 ppm. HRMS (ESI-TOF) m/z Calcd for C30H48N2 [M+1]+ 437.389, found 437.389.
4.1.2.7 1-((3R,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,
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16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-1H-imidazole （b4）. Yield 18%. Brown
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powder, m.p. 136 – 138 oC. IR νmax (cm-1): 3435, 2937, 1724, 1434, 1417, 1239, 1117, 1027, 905, 823, 744, 664,
611. 1H NMR (400 MHz, Chloroform-d) δ 7.55 (s, 1H), 7.05 (s, 1H), 6.98 (s, 1H), 5.43 – 5.42 (m, 1H), 3.92 –
3.84 (m, 1H), 2.65 – 2.58 (m, 1H), 2.44 – 2.39 (m, 1H), 2.06 – 1.79 (m, 7H), 1.61 – 1.46 (m, 6H), 1.39 – 1.31
(m, 3H), 1.29 – 1.22 (m, 2H), 1.19 – 1.11 (m, 5H), 1.07 (s, 3H), 1.04 – 0.96 (m, 3H), 0.92 (d, J = 6.4 Hz, 3H),
0.86 (dd, J = 6.8, 2.0 Hz, 6H), 0.69 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 139.8, 135.4, 129.2, 123.0,
116.9, 57.7, 56.8, 56.3, 50.3, 42.4, 40.7, 39.8, 39.6, 38.1, 36.8, 36.3, 35.9, 32.0, 31.9, 30.1, 28.4, 28.2, 24.4,
24.0, 23.0, 22.7, 21.1, 19.5, 18.9, 12.0 ppm. HRMS (ESI-TOF) m/z Calcd for C30H48N2 [M+H]+ 437.389, found
437.389.
4.1.2.8 1-((3R,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,
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16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-1H-benzo[d]imidazole （b5）. Yield 10%.
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Pale yellow powder, m.p. 209 – 211 oC. IR νmax (cm-1): 3429, 2941, 1496, 1466, 1439, 1383, 1234, 1113, 1071,
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819, 729, 662. H NMR (400 MHz, Chloroform-d) δ 8.00 (s, 1H), 7.81 – 7.77 (m, 1H), 7.43 – 7.39 (m, 1H),
7.27 – 7.24 (m, 2H), 5.45 – 5.44 (m, 1H), 4.20 – 4.12 (m, 1H), 2.79 – 2.72 (m, 1H), 2.54 – 2.49 (m, 1H), 2.16 –
1.99 (m, 5H), 1.86 – 1.79 (m, 1H), 1.63 – 1.48 (m, 6H), 1.37 – 1.18 (m, 7H), 1.13 (s, 3H), 1.11 – 0.96 (m, 7H),
0.92 (d, J = 6.4 Hz, 3H), 0.85 (dd, J = 6.6, 1.8 Hz, 6H), 0.69 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 144.1,
140.3, 139.8, 133.5, 123.2, 122.7, 122.2, 120.6, 110.2, 56.9, 56.4, 56.3, 50.3, 42.5, 39.8, 39.6, 39.5, 38.4, 37.0,
36.3, 35.9, 32.0, 32.0, 29.0, 28.4, 28.2, 24.4, 24.0, 23.0, 22.7, 21.2, 19.7, 18.9, 12.0 ppm. HRMS (ESI-TOF) m/z
Calcd for C34H50N2 [M+H]+ 487.4047, found 487.4048.
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4.1.2.9
1-((3R,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,
15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-5,6-dimethyl-1H-benzo[d]imidazole
o
（
b6
）
.
Yield 9%. White powder, m.p. 241 – 243 C. IR νmax (cm-1): 3432, 2932, 1614, 1484, 1456, 1373,
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1285, 1160, 1006, 889, 861, 741. 1H NMR (400 MHz, Chloroform-d) δ 7.90 (s, 1H), 7.56 (s, 1H), 7.18 (s, 1H),
5.48 – 5.45 (m, 1H), 4.15 – 4.07 (m, 1H), 2.77 – 2.71 (m, 1H), 2.53 – 2.49 (m, 1H), 2.39 (s, 3H), 2.37 (s, 3H),
2.11 – 2.01 (m, 5H), 1.88 – 1.81 (m, 1H), 1.64 – 1.49 (m, 6H), 1.38 – 1.18 (m, 9H), 1.13 (s, 3H), 1.11 – 1.01 (m,
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5H), 0.93 (d, J = 6.4 Hz, 3H), 0.87 (dd, J = 6.6, 1.8 Hz, 6H), 0.71 (s, 3H) ppm. C NMR (100 MHz, CDCl3) δ
142.6, 140.0, 139.5, 132.1, 131.8, 131.0, 123.1, 120.5, 110.3, 56.9, 56.3, 56.2, 53.5, 50.4, 42.5, 39.9, 39.7, 39.5,
38.4, 37.0, 36.3, 35.9, 32.0, 32.0, 29.0, 28.4, 28.2, 24.4, 24.0, 23.0, 22.7, 21.2, 20.8, 20.4, 19.7, 18.9, 12.0 ppm.
HRMS (ESI-TOF) m/z Calcd for C36H54N2 [M+H]+ 515.436, found 515.4361.
4.1.2.10 1-((3S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,
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16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)-1H-imidazole (b7). Yield 7%. Pale brown
powder, m.p. 150 – 152 oC. IR νmax (cm-1): 3439, 2933, 1625, 1485, 1464, 1383, 1223, 872, 838, 618. 1H NMR
(400 MHz, Chloroform-d) δ 7.71 (s, 1H), 7.02 (s, 1H), 7.00 (s, 1H), 5.50 – 5.47 (m, 1H), 4.38 – 4.36 (m, 1H),
2.91 – 2.84 (m, 1H), 2.53 – 2.49 (m, 1H), 2.11 – 1.96 (m, 4H), 1.88 – 1.82 (m, 2H), 1.65 – 1.57 (m, 3H), 1.53 –
1.42 (m, 4H), 1.40 – 1.21 (m, 5H), 1.16 – 1.09 (m, 5H), 1.07 (s, 3H), 1.03 – 0.97 (m, 3H), 0.90 (d, J = 6.4 Hz,
3H), 0.86 (dd, J = 6.6, 2.0 Hz, 6H), 0.67 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 138.7, 136.9, 128.5, 124.2,
118.9, 56.8, 56.2, 53.3, 49.9, 42.4, 39.7, 39.6, 37.2, 36.3, 36.1, 35.9, 32.5, 32.1, 31.8, 28.6, 28.3, 28.1, 24.4,
24.0, 23.0, 22.7, 20.8, 19.5, 18.8, 12.0 ppm. HRMS (ESI-TOF) m/z Calcd for C30H48N2 [M+H]+ 437.389, found
437.389.
4.1.3. Synthesis of (3S,8R,9S,10R,13S,14S)-3-((tert-butyldimethylsilyl)oxy)-10,13-dimethyl-1,2,3,4,7,8,9,10,
11,12,13,14,15,16-tetradecahydro-17H-cyclopenta[a]phenanthren-17-one (c2)
To a solution of dehydroepiandrosterone c1 (2.9 g, 10.0 mmol) in dichloromethane (30.0 mL) was added tert-
Butyldimethylsilyl chloride (1.8 g, 12.0 mmol) and imidazole (1.4 g, 20.0 mmol) at 0 oC. The resulting mixture
was stirred at room temperature for 8 h. After quenching the reaction with water (50.0 mL), the layers were
separated. The organic layer was washed with water (20.0 mL) and brine (20.0 mL), dried over anhydrous
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Na2SO4 and concentrated. The residue was purified by column chromatography (silica gel, petroleum ether 60–
90 oC : ethyl acetate = 20:1) to afford c2 (3.96 g, 99%) as white powder.
o
Yield 99%. White powder, m.p. 153 – 155 C. IR νmax (cm-1): 3423, 2934, 1494, 1382, 1303, 1238, 1082, 811,
662. 1H NMR (400 MHz, Chloroform-d) δ 5.34 (d, J = 4.8 Hz, 1H), 3.52 – 3.44 (m, 1H), 2.46 (dd, J = 19.2, 8.8
Hz, 1H), 2.28 (t, J = 12.8 Hz, 1H), 2.21 – 2.16 (m, 1H), 2.13 – 2.04 (m, 2H), 1.98 – 1.91 (m, 1H), 1.86 – 1.79
(m, 2H), 1.75 – 1.60 (m, 4H), 1.58 – 1.42 (m, 3H), 1.31 – 1.25 (m, 2H), 1.09 – 0.95 (m, 5H), 0.89 (s, 12H), 0.06
(s, 6H). 13C NMR (100 MHz, CDCl3) δ 221.3, 141.9, 120.5, 72.6, 52.0, 50.5, 47.7, 42.9, 37.5, 36.9, 36.0, 32.16,
31.7, 31.6, 31.0, 26.1, 22.0, 20.5, 19.6, 18.4, 13.7, -4.4 ppm. HRMS (ESI-TOF) m/z Calcd for C25H42O2Si
[M+H]+ 425.2846, found 425.2846.
4.1.4. Synthesis of N-((3S,8R,9S,10R,13S,14S,17S)-3-((tert-butyldimethylsilyl)oxy)-10,13-dimethyl-2,3,4,7,8,9,
10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylpropane-2-sulfinamide
(c3)
To a mixture of ketone c2 (2.0 g, 5.0 mmol) and (R)-(+)-tert-Butylsulfinamide (909.0 mg, 7.5 mmol) in
tetrahydrofuran (30.0 mL) was added titanium (IV) ethoxide (2.1 mL, 10.0 mmol). Heat the resulting mixture at
reflux and monitor the reaction progress by TLC. After cooling to room temperature, methyl alcohol (20.0 mL)
o
was added to the mixture and cooled to 0 C. To the mixture was then added sodium borohydride (2.8 g, 75.0
o
mmol), and the resulting solution was stirres at 0 C for 5 h. The reaction mixture was filtered through a small
pad of Celite to remove Ti salts. The filtrate was treated with EtOAc (100.0 mL), saturated sodium potassium
tartrate (50.0 mL), and brine (50.0 mL) and the mixture stirred at room temperature for 1 h. The mixture was
filtered through a pad of Celite and the organic layer was dried over anhydrous Na2SO4 and concentrated. The
o
residue was purified by column chromatography (silica gel, petroleum ether 60–90 C : ethyl acetate = 3:1) to
afford c3 (2.1 g, 82%, two steps) as white powder.
Yield 82%. White powder, m.p. 221 – 223 oC. IR νmax (cm-1): 3431, 2930, 1598, 1474, 1375, 1068, 976, 799. 1H
NMR (400 MHz, Chloroform-d) δ 5.37 – 5.27 (m, 1H), 3.50 – 3.45 (m, 1H), 3.15 (q, J = 8.8 Hz, 1H), 2.91 (d, J
= 7.8 Hz, 1H), 2.29 – 2.14 (m, 3H), 2.02 – 1.94 (m, 1H), 1.83 – 1.64 (m, 5H), 1.59 – 1.37 (m, 6H), 1.21 (s, 9H),
1.14 – 1.03 (m, 2H), 1.02 – 0.94 (m, 5H), 0.88 (s, 9H), 0.69 (s, 3H), 0.05 (s, 6H). 13C NMR (100 MHz, CDCl3) δ
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141.7, 121.0, 72.7, 66.9, 55.9, 52.8, 50.5, 43.3, 42.9, 37.5, 37.1, 36.8, 32.2, 31.7, 31.2, 26.1, 24.0, 22.9, 20.7,
19.6, 18.4, 11.8, -4.4 ppm. HRMS (ESI-TOF) m/z Calcd for C29H53NO2SSi [M+H]+ 530.3458, found 530.3457.
4.1.5. Synthesis of (3S,8R,9S,10R,13S,14S,17S)-17-amino-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol (c4)
To a solution of secondary amine c3 (1.0 g, 2.0 mmol) in methyl alcohol/dichloromethane = 2:1 (30.0 mL)
was added 2N HCl in dioxane (12.0 mL). The resulting mixture was stirred at room temperature for 4 h. The
crude reaction was diluted with methyl alcohol (20.0 mL) and filtered. The filtrate was concentrated and the
solid obtained was washed with petroleum ether (3 × 10.0 ml), then dried to afford c4 (534.8 mg, 92%) as white
powder.
o
Yield 92%. White powder, m.p. 144 – 146 C. IR νmax (cm-1): 3450, 2947, 1747, 1472, 1369, 1254, 1091, 887,
1
837, 774. H NMR (400 MHz, Methanol-d4) δ 5.37 – 5.35 (m, 1H), 3.44 – 3.35 (m, 1H), 3.05 (t, J = 9.2 Hz,
1H), 2.28 – 2.13 (m, 3H), 2.05 – 1.96 (m, 2H), 1.89 (dt, J = 13.2, 3.2 Hz, 1H), 1.83 – 1.74 (m, 2H), 1.71 – 1.46
13
(m, 6H), 1.43 – 1.32 (m, 1H), 1.27 – 1.09 (m, 3H), 1.05 (s, 3H), 1.03 – 0.98 (m, 1H), 0.85 (s, 3H). C NMR
(100 MHz, MeOD) δ 140.9, 120.6, 70.9, 60.2, 52.5, 50.0, 41.6, 41.6, 37.1, 36.3, 35.7, 31.6, 31.1, 30.8, 26.1,
23.3, 20.1, 18.5, 10.4 ppm. HRMS (ESI-TOF) m/z Calcd for C19H31NO [M+H]+ 290.2478, found 290.2478.
4.1.6. Synthesis of (3S,10R,13S,17S)-17-(1H-imidazol-1-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ol (c5)
To a solution of amine c4 (500.0 mg, 1.7 mmol) in methyl alcohol (20 mL) was added 40% glyoxal (1.1 mL,
8.5 mmol), 35% formaldehyde (0.9 mL, 8.5 mmol) and 25% ammonium hydroxide (0.6 mL, 8.5 mmol). Heat
the resulting mixture at reflux and monitor the reaction progress by TLC. After cooling to room temperature, the
solvent was concentrated, and the residue was diluted with EtOAc (20.0 mL). The organic layer was washed
with water (20.0 mL) and brine (20.0 mL), dried over anhydrous Na2SO4 and concentrated. The residue was
purified by column chromatography (silica gel, ethyl acetate) to afford c5 (400.0 mg, 68%) as pale yellow
powder.
o
Yield 68%. Pale yellow powder, m.p. 282 – 284 C. IR νmax (cm-1): 3423, 2925, 1497, 1353, 1225, 1067, 811,
737, 663. 1H NMR (400 MHz, Chloroform-d) δ 7.52 (s, 1H), 7.03 (s, 1H), 6.94 (s, 1H), 5.35 (d, J = 5.4 Hz, 1H),
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3.94 (t, J = 9.6 Hz, 1H), 3.56 – 3.48 (m, 1H), 2.32 – 2.15 (m, 5H), 2.07 – 2.00 (m, 1H), 1.86 – 1.72 (m, 4H),
1.63 – 1.34 (m, 6H), 1.28 – 1.08 (m, 3H), 1.00 (s, 3H), 0.57 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 141.2,
136.6, 128.6, 121.1, 118.8, 71.6, 67.9, 53.1, 50.3, 43.9, 42.4, 37.4, 36.9, 36.7, 32.2, 31.7, 31.6, 26.2, 23.4, 20.8,
19.5, 12.0 ppm. HRMS (ESI-TOF) m/z Calcd for C22H32N2O [M+H]+ 341.2587, found 341.2587.
4.1.7. Synthesis of (3S,10R,13S,17S)-17-(1H-imidazol-1-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,
17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl acetate (c6)
To a solution of alcohol c5 (200.0 mg, 0.6 mmol) in dichloromethane (20.0 mL) was added acetic anhydride
(85.0 µL, 0.9 mmol), triethylamine (250.0 µL, 1.8 mmol) and 4-dimethylaminopyridine (3.6 mg, 3 mol%) at 0
oC. The resulting mixture was stirred at room temperature for 2 h. After quenching the reaction with saturated
sodium bicarbonate (20.0 mL), the layers were separated. The organic layer was washed with water (20.0 mL)
and brine (20.0 mL), dried over anhydrous Na2SO4 and concentrated. The residue was purified by column
o
chromatography (silica gel, petroleum ether 60–90 C : ethyl acetate = 1:1) to afford c6 (193.2 mg, 86%) as
white powder.
o
Yield 86%. Pale yellow powder, m.p. 166 – 168 C. IR νmax (cm-1): 3439, 2930, 1472, 1383, 1361, 1254, 1073,
887, 839, 773. 1H NMR (400 MHz, Chloroform-d) δ 7.50 (s, 1H), 7.01 (s, 1H), 6.93 (s, 1H), 5.36 (d, J = 4.8 Hz,
1H), 4.62 – 4.54 (m, 1H), 3.92 (t, J = 9.6 Hz, 1H), 2.35 – 2.10 (m, 4H), 2.05 – 1.98 (m, 4H), 1.87 – 1.70 (m,
13
4H), 1.62 – 1.32 (m, 6H), 1.28 – 1.09 (m, 3H), 0.99 (s, 4H), 0.55 (s, 3H) ppm. C NMR (100 MHz, CDCl3) δ
170.6, 139.9, 136.6, 128.6, 122.1, 118.7, 73.8, 67.8, 52.9, 50.1, 43.8, 38.1, 37.0, 36.8, 36.7, 32.0, 31.5, 27.7,
26.2, 23.4, 21.5, 20.6, 19.4, 12.0 ppm. HRMS (ESI-TOF) m/z Calcd for C24H34N2O2 [M+H]+ 383.2695, found
383.2695.
4.1.8. General procedure for the preparation of steroidal imidazolium salts a8-a35/b8-b28/c7-c18
A mixture of imidazole compounds a3-a7/b3-b7/c5-c6 (1.0 mmol) and phenacyl bromide or alkyl halide (1.2
mmol) was stirred in toluene (10.0 ml) at reflux for 24-48 h. An insoluble substance was formed. After
completion of the reaction as indicated by TLC, the precipitate was filtered through a small pad of Celite, and
washed with toluene (3 × 10.0 ml), then dried to afford imidazolium salts a8-a35/b8-b28/c7-c18 in 67–98%
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[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium bromide (a8).
Yield 93%. White powder, m.p. 280 – 282 oC. IR νmax (cm-1): 3540, 3438, 2953, 2927, 1700, 1449, 1226, 1154,
1050, 1003, 809, 754, 618. 1H NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 8.09 – 8.07 (m, 2H), 7.93 (s, 1H),
7.80 – 7.76 (m, 2H), 7.67 – 7.63 (m, 2H), 6.12 (d, J = 2.84 Hz, 2H), 4.33 (q, J = 7.2 Hz, 1H), 4.09 (s, 1H), 3.43
– 3.40 (m, 1H), 3.21 (t, J = 10.8 Hz, 1H), 2.43 – 2.40 (m, 1H), 2.07 – 2.01 (m, 1H), 1.87 – 1.78 (m, 2H), 1.72 –
1.32 (m, 14H), 1.30 – 1.13 (m, 4H), 1.01 (d, J = 7.2 Hz, 1H), 0.98 – 0.95 (m, 2H), 0.91 (d, J = 6.8 Hz, 3H), 0.73
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– 0.70 (m, 9H) ppm. C NMR (100 MHz, DMSO-d6) δ 191.7, 137.4, 135.0, 134.1, 129.6, 128.6, 124.2, 121.6,
108.8, 80.7, 66.4, 62.4, 61.7, 56.1, 55.3, 47.3, 43.2, 41.6, 34.7, 33.3, 33.2, 31.7, 31.5, 30.3, 29.9, 29.0, 24.5,
24.2, 22.3, 19.7, 17.6, 16.7, 15.2, 14.1 ppm. HRMS (ESI-TOF) m/z Calcd for C38H51N2O3 [M-Br]+ 583.3894,
found 585.3893.
4.1.8.2
3-(2-(4-methoxyphenyl)-2-oxoethyl)-1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosa
hydro-2H-spiro[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium
bromide (a9). Yield 92%. White powder, m.p. 248 – 250 oC. IR νmax (cm-1): 3448, 2953, 2928, 1685,1604, 1269,
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1243, 1176, 1153, 984, 600. H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 8.06 – 8.04 (m, 2H), 7.92 (s, 1H),
7.77 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 6.06 – 6.03 (m, 2H), 4.33 (q, J = 7.2 Hz, 1H), 4.08 (s, 1H), 3.89 (s, 3H),
3.44 – 3.40 (m, 1H), 3.21 (t, J = 10.8 Hz, 1H), 2.41 (d, J = 12.8 Hz, 1H), 2.07 – 2.01 (m, 1H), 1.86 – 1.78 (m,
2H), 1.71 – 1.47 (m, 10H), 1.45 – 1.35 (m, 4H), 1.30 – 1.12 (m, 4H), 1.01 (d, J = 7.2 Hz, 1H), 097 – 0.95 (m,
2H), 0.91 (d, J = 6.8 Hz, 3H), 0.74 – 0.70 (m, 9H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 190.0, 164.5, 137.4,
131.0, 126.9, 124.6, 121.5, 114.8, 108.8, 80.7, 66.4, 62.4, 61.6, 56.2, 55.7, 55.3, 47.3, 43.2, 41.6, 34.7, 33.3,
31.7, 31.5, 30.3, 29.9, 29.0, 24.5, 24.2, 22.3, 19.7, 17.6, 16.7, 15.1, 14.1 ppm. HRMS (ESI-TOF) m/z Calcd for
C39H53N2O4 [M-Br]+ 613.4, found 613.4001.
4.1.8.3
3-(2-(3-bromophenyl)-2-oxoethyl)-1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosa
hydro-2H-spiro[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium
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bromide (a10). Yield 97%. White powder, m.p. 268 – 270 C. IR νmax (cm-1): 3440, 2953, 2927, 1705, 1564,
1455, 1382, 1240, 1153, 1129, 1053, 984, 899, 675. 1H NMR (400 MHz, DMSO-d6) δ 9.22 (s, 1H), 8.20 (d, J =
11.2 Hz, 1H), 8.09 – 8.03 (m, 1H), 7.99 – 7.90 (m, 2H), 7.76 (d, J = 12.4 Hz, 1H), 7.64 – 87.57 (m, 1H), 6.11
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(d, J = 11.6 Hz, 2H), 4.32 (q, J = 6.8 Hz, 1H), 4.07 (d, J = 11.6 Hz, 1H), 3.42 (d, J = 11.6 Hz, 1H), 3.24 – 3.19
(m, 1H), 2.40 (t, J = 13.6 Hz, 1H), 2.04 – 2.01 (m, 1H), 1.84 – 1.77 (m, 2H), 1.69 – 1.34 (m, 13H), 1.26 – 1.12
(m, 4H), 1.02 – 0.85 (m, 7H), 0.73 – 0.66 (m, 9H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 190.9, 137.5, 137.4,
136.2, 131.9, 131.1, 127.6, 124.2, 122.8, 121.6, 108.8, 80.7, 66.4, 62.4, 61.7, 56.1, 55.3, 47.3, 43.2, 41.6, 34.6,
33.2, 31.7, 31.5, 30.2, 29.9, 28.9, 24.5, 24.2, 22.3, 19.7, 17.6, 16.7, 15.1, 14.0 ppm. HRMS (ESI-TOF) m/z
Calcd for C38H50BrN2O3 [M-Br]+ 661.2999, found 661.2997.
4.1.8.4
3-(2-(naphthalen-2-yl)-2-oxoethyl)-1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosa
hydro-2H-spiro[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium
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bromide (a11). Yield 98%. White powder, m.p. 265 – 267 C. IR νmax (cm-1): 3447, 2953, 1693, 1629, 1455,
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1153, 1126, 1053, 982, 898, 746. H NMR (400 MHz, DMSO-d6) δ 9.28 (d, J = 1.6 Hz, 1H), 8.88 (d, J = 1.6
Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.8 Hz, 1H), 8.08 – 8.04 (m, 2H), 7.96 (t, J = 1.6 Hz, 1H), 7.84 (t,
J = 1.6 Hz, 1H), 7.77 – 7.68 (m, 2H), 6.25 (s, 2H), 4.33 (q, J = 7.2 Hz, 1H), 4.10 (d, J = 3.2 Hz, 1H), 3.44 – 3.40
(m, 1H), 3.21 (t, J = 10.8 Hz, 1H), 2.45 – 2.42 (m, 1H), 2.08 – 2.02 (m, 1H), 1.86 – 1.78 (m, 2H), 1.70 – 1.53
(m, 8H), 1.48 – 1.36 (m, 4H), 1.32 – 1.12 (m, 5H), 1.03 – 0.95 (m, 4H), 0.91 (d, J = 6.8 Hz, 3H), 0.75 – 0.71 (m,
9H) ppm. 13C NMR (100 MHz, DMSO-d6) δ 191.6, 137.5, 136.0, 132.5, 131.4, 130.9, 130.1, 129.8, 129.3,
128.4, 127.9, 124.3, 123.6, 121.6, 108.8, 80.7, 66.4, 62.4, 61.7, 56.1, 55.3, 47.3, 43.2, 41.6, 34.7, 33.2, 31.7,
31.5, 30.2, 30.0, 29.0, 24.6, 24.2, 22.3, 19.7, 17.6, 16.7, 15.2, 14.1 ppm. HRMS (ESI-TOF) m/z Calcd for
C42H53N2O3 [M-Br]+ 633.4051, found 633.4051.
4.1.8.5
3-(2-bromobenzyl)-1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosahydro-2H-spiro
[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium bromide (a12).
Yield 82%. White powder, m.p. 220 – 222 oC. IR νmax (cm-1): 3426, 2952, 1629, 1452, 1149, 1051, 983, 749. 1H
NMR (400 MHz, DMSO-d6) δ 9.35 (s, 1H), 7.94 (s, 1H), 7.74 (d, J = 7.2 Hz, 2H), 7.49 – 7.45 (m, 1H), 7.39 –
7.36 (m, 1H), 7.30 (d, J = 7.2 Hz, 1H), 5.62 (s, 2H), 4.32 (q, J = 7.2 Hz, 1H), 4.04 (s, 1H), 3.44 – 3.40 (m, 1H),
3.22 (t, J = 10.8 Hz, 1H), 2.41 (d, J = 14.4 Hz, 1H), 2.06 – 1.98 (m, 1H), 1.85 – 1.77 (m, 2H), 1.70 – 1.46 (m,
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10H), 1.42 – 1.13 (m, 9H), 1.01 (d, J = 7.2 Hz, 1H), 0.97 – 0.88 (m, 7H), 0.69 (s, 3H), 0.63 (s, 3H) ppm. C
NMR (100 MHz, DMSO-d6) δ 136.7, 134.4, 133.6, 131.4, 130.8, 128.9, 123.2, 122.8, 122.7, 108.8, 80.7, 66.4,
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62.3, 61.8, 55.3, 52.7, 47.2, 43.1, 41.6, 34.5, 33.2, 31.7, 31.5, 30.3, 29.9, 29.0, 24.7, 24.7, 22.2, 19.7, 17.6, 16.8,
15.2, 14.0 ppm. HRMS (ESI-TOF) m/z Calcd for C37H50BrN2O2 [M-Br]+ 633.305, found 633.305.
4.1.8.6
3-(naphthalen-2-ylmethyl)-1-((2S,2aR,3aR,5aR,5'R,7aS,8S,9R)-5a,5',7a,8-tetramethylicosahydro-2H-
bromide
spiro[cyclopropa[1',7a']indeno[5',4':4,5]indeno[2,1-b]furan-9,2'-pyran]-2-yl)-1H-imidazol-3-ium
(a13). Yield 75%. White powder, m.p. 256 – 258 oC. IR νmax (cm-1): 3408, 2952, 1454, 1382,1148, 1126, 1099,
983, 899, 818, 759. 1H NMR (400 MHz, Chloroform-d) δ 10.25 (s, 1H), 8.06 (s, 1H), 7.87 – 7.78 (m, 3H), 7.62
(d, J = 8.4 Hz, 1H), 7.56 – 7.47 (m, 3H), 7.29 (d, J = 3.6 Hz, 1H), 6.05 – 5.86 (m, 2H), 4.40 (s, 1H), 3.94 (s,
1H), 3.48 – 3.27 (m, 2H), 2.41 – 2.11 (m, 3H), 1.84 – 1.33 (m, 15H), 1.28 – 1.02 (m, 7H), 0.98 – 0.94 (m, 4H),
0.80 – 0.78 (m, 2H), 0.66 – 0.62 (m, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 136.7, 133.3, 133.1, 131.2, 129.3,
128.7, 128.2, 127.7, 126.9, 126.7, 125.9, 121.6, 120.3, 109.6, 109.1, 80.4, 66.8, 65.1, 62.6, 62.1, 61.9, 55.3,
53.4, 47.4, 43.2, 42.0, 41.5, 40.6, 39.5, 34.3, 34.0, 33.1, 31.6, 31.4, 30.2, 30.2, 28.7, 27.0, 26.0, 25.7, 25.3, 24.0,
22.1, 19.9, 17.2, 16.4, 16.0, 14.7, 14.5, 14.3 ppm. HRMS (ESI-TOF) m/z Calcd for C41H53N2O2 [M-Br]+
605.4102, found 605.4103.
4.1.8.7 3-(2-oxo-2-phenylethyl)-1-((4R,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,6a,6b,6',7,8,
8a,8b,9,11a,12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-1H-imidazol-
3-ium bromide (a14). Yield 72%. Pale yellow powder, m.p. 265 – 267 C. IR νmax (cm-1): 3423, 2948, 1696,
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1599, 1512, 1375, 1355, 1238, 1171, 1056, 988, 833. 1H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 8.11 – 8.05
(m, 3H), 7.79 – 7.75 (m, 2H), 7.64 (t, J = 7.6 Hz, 2H), 6.08 (s, 2H), 5.47 (d, J = 4.8 Hz, 1H), 4.40 – 4.27 (m,
2H), 3.22 (t, J = 12.0 Hz 1H), 2.76 (t, J = 12.4 Hz, 1H), 2.06 – 1.90 (m, 5H), 1.86 – 1.77 (m, 2H), 1.74 – 1.67
(m, 2H), 1.65 – 1.46 (m, 7H), 1.36 – 1.27 (m, 2H), 1.21 – 1.14 (m, 3H), 1.11 (s, 3H), 1.02 – 0.91 (m, 6H), 0.76
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– 0.73 (m, 6H) ppm. C NMR (100 MHz, DMSO-d6) δ 191.8, 139.6, 136.8, 135.0, 134.1, 129.6, 128.6, 124.5,
123.0, 120.9, 80.6, 66.4, 62.3, 59.8, 56.2, 55.9, 49.9, 41.6, 38.8, 37.6, 36.8, 31.9, 31.4, 30.3, 28.9, 20.8, 19.5,
17.5, 16.6, 15.1 ppm. HRMS (ESI-TOF) m/z Calcd for C38H51N2O3 [M-Br]+ 583.3894, found 583.3894.
4.1.8.8 3-(2-(4-methoxyphenyl)-2-oxoethyl)-1-((4R,5'R,6aR,8aS,9S,10R)-5',6a,8a,9-tetramethyl-1,3,3',4,4',5,5',6,
6a,6b,6',7,8,8a,8b,9,11a,12,12a,12b-icosahydrospiro[naphtho[2',1':4,5]indeno[2,1-b]furan-10,2'-pyran]-4-yl)-
1H-imidazol-3-ium bromide (a15). Yield 83%. White powder, m.p. 280 – 282 C. IR νmax (cm-1): 3428, 2949,
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1703, 1563, 1452, 1224, 1166, 1051, 1007, 674. 1H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 8.12 – 8.04 (m,
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