Synthesis and antiproliferative activity of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-one with a C-21 hydrophilic substituent

Article abstract of DOI:10.1002/hc.21372

A series of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-ones 7-15 possessing C-21 hydrophilic substituents were synthesized from the corresponding C21-bromo steroid 6 in 24%-89% yields. The hydrophilic groups include amino group from Delépine reaction, hydroxyl group from the hydrolysis by cesium formate in anhydrous MeOH, and amino acids, hydrazinecarboxamide, piperazine, and imidazolyl from nucleophilic substitution. Acylation and O-glycosylation of the resulting C-21 hydroxylsteroid also afforded the corresponding acylated and glycosylated products in excellent yield (77%-89%). Among the compounds, aminosteroid 7 and piperazinyl steroid 14 were most potent against the proliferation of human prostate cancer PC-3 cells with IC₅₀ values of 42 and 82?μmol?L^?1, respectively.

Full text of DOI:10.1002/hc.21372

Received: 14 March 2017
DOI: 10.1002/hc.21372
Revised: 20 April 2017
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R E S E A R C H A R T I C L E
Synthesis and antiproliferative activity of 3α-hydroxyl-3β-
methoxymethyl-5α-pregnan-20-one with a C-21 hydrophilic
substituent
Shuo-En Tsai1,2,*
Jang-Chang Lee1,*
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Naoto Uramaru3
Hiroyuki Takayama4
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Guan-Jhong Huang5
Fung Fuh Wong1
1School of Pharmacy, China Medical
University, Taichung, Taiwan
2Ph.D. Program for Biotech Pharmaceutical
Industry, China Medical University,
Taichung, Taiwan
3Department of Environmental
Science, Nihon Pharmaceutical
University, Komuro Inamachi Kit-adachi-
gun, Saitama-ken, Japan
4Department of Environmental
Science, Nihon Pharmaceutical
University, Komuro Inamachi Kit-adachi-
gun, Saitama-ken, Japan
Abstract
A series of 3α-hydroxyl-3β-methoxymethyl-5α-pregnan-20-ones 7-15 possessing
C-21 hydrophilic substituents were synthesized from the corresponding C21-bromo
steroid 6 in 24%-89% yields. The hydrophilic groups include amino group from
Delépine reaction, hydroxyl group from the hydrolysis by cesium formate in anhy-
drous MeOH, and amino acids, hydrazinecarboxamide, piperazine, and imidazolyl
from nucleophilic substitution. Acylation and O-glycosylation of the resulting C-21
hydroxylsteroid also afforded the corresponding acylated and glycosylated products
in excellent yield (77%-89%). Among the compounds, aminosteroid 7 and piperazi-
nyl steroid 14 were most potent against the proliferation of human prostate cancer
PC-3 cells with IC50 values of 42 and 82 μmol L−1, respectively.
5School of Chinese Pharmaceutical
Sciences and Chinese Medicine
Resources, College of Biopharmaceutical
and Food Sciences, China Medical
University, Taichung, Taiwan
Correspondence
Fung Fuh Wong, School of Pharmacy, China
Medical University, Taichung, Taiwan.
Emails: wongfungfuh@yahoo.com.tw,
ffwong@mail.cmu.edu.tw
Funding information
the Tsuzuki Institute for Traditional
Medicine; Technology of the Republic of
China, Grant/Award Number: MOST 105-
2113-M-039-001
the treatment of autoimmune disorders, breast and prostate
cancers, cardiovascular diseases, and osteoarthritis, etc.[1–3]
1
INTRODUCTION
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Steroidal compounds attract much attention because of
their special biological activity.[1] They can regulate a vari-
ety of biological process and thus be developed as drugs for
Aside from being obtained from the natural sources, most
of the steroidal pharmaceuticals were semi-synthetic com-
pounds by connecting a special functionality to the core
structure.[1,4,5] Among the steroids, pregnan-20-ones with a
C-21 modification are reported to show improved or different
activities when appropriate moieties are introduced.[6]
*These authors contributed equally to this work.
Contract grant sponsor: the Tsuzuki Institute for Traditional Medicine.
Contract grant sponsor: Technology of the Republic of China.
Contract grant number: MOST 105-2113-M-039-001.
Endogenous steroids (1-3, Figure 1) are reported as
the positive allosteric GABAA receptor modulators.[7,8]
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Heteroatom Chemistry. 2017;28:e21372.
https://doi.org/10.1002/hc.21372
wileyonlinelibrary.com/journal/hc
© 2017 Wiley Periodicals, Inc.
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O
21
O
O
21
21
R
R
MeO
FIGURE 1 Structures of endogenous
steroids as the endogenous GABAA receptor
positive allosteric modulators
HO
HO
HO
4. R = H, 5. R = imidazolyl
1. R = H 2. R = OH
3
3α-Hydroxyl-3β-methoxymethyl-5α-pregnan-20-one is a neu-
roactive steroid that shows potent allosteric modulatory activ-
ity of the GABAA receptor.[9,10] Further derivation of 4 on its
C-21 position produces steroid 5 with a hydrophilic imidazolyl
group showing in vivo activity.[9] The conversion of 4 to 5 ex-
emplifies the use of hydrophilic group to enhance the potency
and pharmacokinetic properties in steroid compounds. Except
for the basic imidazolyl group,[11] amino acids,[12] heterocyclic
moieties,[13] sugar furanoses,[14] and high polarity salts[15] are
frequently connected to the scaffold of a lead in the medicinal
chemistry research paradigm. These groups can enhance the
potency by forming hydrogen bonds or dipole-dipole interac-
tions and improve the pharmacokinetic properties by increas-
ing the water solubility of the lead compounds.[11–15]
Research has been directed towards estrogen receptors and
their modulators due to their involvement in the progression of
cancer in humans.[16,17] Estrogen receptors have been of par-
ticular interest due to their prominence in breast and prostate
cancer.[18,19] In breast cancer, high EERγ mRNA has been as-
sociated with unfavorable cancer prognoses while high EERα
has been associated with more favorable cancer prognoses.[20]
Investigation into EERγ in breast cancer is still in progress.
In prostate cancer, high EERα and low EERγ correlated with
poorer outcomes.[21] Steroid compounds are an important
component of treatment in these hormone related cancers.
In this study, we synthesized a series of 3α-hydroxyl-3β-
methoxymethyl-5α-pregnan-20-ones 5-15 with C-21 hydro-
philic substituents and their antiproliferative activities against
the proliferation of prostate cancer cells PC-3 (Figure 2).
Among the derivatives, steroid 14 with C-21 piperazinyl pos-
sessed the most potent, showing an IC50 value of 42 μmol L−1.
successfully provided 7 in 87% yield. For the synthesis of C-21
hydroxyl steroid, cesium formate (HCO2Cs) in MeOH was used
for the hydrolysis of 6.[23] The reaction successfully provided
compound 8a with the retention of the C-17β configuration
with 85% yield and a C-17α diastereomer 8b with 2.0% yield.
As we reported in our previous study, various alkali formates
were used for the hydrolysis of α-bromocarbonyl compounds.
Cesium formate was the best reagent and provided the high
selectivity yield to provide 8a as the major diastereoisomer.[23]
The hydroxyl group at the C-21 position of 8a was suit-
able for further derivation due to its good reactivity toward
acylation and alkylation (see Scheme 2). For the acylation
of 8a, we chose isoxazole-5-carbonyl chloride for the C-21
hydroxyl group following the reported procedure.[24] The
reaction provided the acylated product 9 with a 89% yield.
For the preparation of alkylated steroid, we prepared glyco-
sylated product 10 for the sugar moiety which was expected
to greatly enhance the water solubility.[25] We first prepared
tetra-O-acetyl-α-galactopyranosyl-1-bromide 16 from ga-
lactopyranose following the reported procedure involving
acetylation and bromination.[26] Steroid 8a was reacted to-
wards compound 16 in the presence Ag2CO3 to obtain the
acetylated adductor without isolation. The acetylated inter-
mediate was hydrolyzed in methanolic K2CO3 at reflux for
4.0 hour to give the corresponding glycosylated steroid 10 in
77% total yield (see Scheme 2).[25,26]
As the amino acid possessed the good hydrophilic prop-
erties to promote the solubility in water, several of the drugs
were considered to introduce the functional amino acid
groups to the main structure for modification of the biolog-
ical activity.[27] As a result, we turned back to use steroid
6 to react with different nucleophiles possessing hydro-
philic properties at the C-21 position for further derivation
(see Table 1). At first, we gifted amino acids including D-
Ornithine hydrochloride[28] and L-cysteine[29] towards C-21
bromosteroid 6 in presence of hindered amine including N,N-
diisopropylethylamine or triethylamine in DMF or EtOH to
obtain the corresponding C-21 amino acid-steroid adducts 11
and 12 in 72% and 24% yields, respectively (see Table 1).
Based on the experimental results, we found the lower yield
was observed in L-cysteine as a nucleophile because of the
poor solubility in EtOH solution. On the other hands, we also
reacted aminoguanidine, which is a highly reactive nucleo-
phile that reacts with α- or β-dicarbonyl compounds in nu-
merous biological molecules (pyridoxal phosphate, pyruvate,
2
2.1
RESULTS AND DISCUSSION
Chemistry
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21-Bromo-3α-hydroxy-3β-methoxymethyl-5α-pregnan-2
0-one (6) was synthesized in five steps as the starting mate-
rial by following our previously published procedure.[9] C-21
Imidazoyl steroid 5 was also synthesized by our published lit-
erature by treating compound 6 with lithium imidazole at −5 to
0°C for 10 minute under N2 atmosphere (Scheme 1).[9,10] For
the synthesis of steroid 7 with a C-21 amino group, we used the
Delépine reaction by treating 6 with hexamethylenetetramine
in MeOH at room temperature for 48 hour.[22] The reaction

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HO
O
O
O
O
21
OH
OH
O
10.
9.
11.
O
N
H
OH
R
N
17
NH2
O
OH
MeO
HO
O
N
N X
H
N
NH2
13.
12.
6. R = Br, 7. R = NH2
8a. C-17 , R = OH
N
H
S
OH
NH
β
NH2
14. X = H, 15. dimer
8b. C-17 , R = OH
α
FIGURE 2 Structures of hydrophilic steroids 6-15
N
N
O
21
LiN
N
-5 oC, THF
(81%)
MeO
HO
5
O
N
O
O
21
Br
N
N
NH2
N
MeO
MeO
HO
MeOH
(87%)
HO
6
7
21
OH
17
HCO2Cs
MeOH
MeO
HO
SCHEME 1 Synthesis of C-21
imidazolyl steroid 5, C-21 amino steroid 7,
and steroids containing C-21 hydroxyl 8a
and 8b
8a. C-17 , 8b. C-17
β
α
O
O
O
O
Cl
N
O
O
N
Et3N, THF
MeO
HO
reflux, 89%
O
9
OH
HO
OAc
OAc
.
1
O
MeO
O
O
O
OH
OH
16
OAc
OAc
Br
HO
, Ag2CO3
HO
MeO
HO
8a
2. K2CO3, MeOH, reflux, 77%
10
SCHEME 2 Synthesis of acylated steroid 9 and glycosylated steroid 10

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TABLE 1 Reaction of bromosteroid 6 with different nucleophiles to generate the C-21 substituted steroids 11–13
O
O
Nu
Br
HNu
MeO
HO
MeO
HO
conditions
11 13
-
6
Entry
HNu used
Condition
Product
11
Yield (%)
1
i-Pr2NEt, DMF reflux
72
O
H
N
H
OH
NH2
2
3
NEt3, EtOH
12
13
24
51
O
HS
OH
NH2
NHEt2, DMF
NH
H
N
H
N
H
NH2
glucose, malondialdehyde, and others) to form the substituted
3-amino-1,2,4-triazine derivatives[30] with 6 in CH2Cl2 using
NHEt2 as the base to provide the desired 13 in 51% yield.
Piperazine possesses two NH groups capable of substi-
tuting the C-21 bromo atom of steroid 6. The reaction would
provide the mono-substituted 14 and the di-substituted 15 as
the products (see Table 2 and Scheme 3). We focused on the
preparation of 14 because it leaves a free NH group suitable
for further derivation and its structure resembles 4 that could
be used for structure-activity study. Application of the tradi-
tional method by heating of 6 with the different equivalent
amounts (1.0, 2.0, and 3.0 equiv) of piperazine in neat con-
dition provided 15 as the major product, similar to the result
of using an imidazole for the preparation of 4 (entries 1-3 in
Table 2).[9] However, these reactions provided the dimeric 15
as the major product and the yields did not vary significantly
with the amount of piperazine used (~70% yield). To dimin-
ish the formation of 15, we tried to react lithium piperazide as
the nucelophile with steroid 6 in anhydrous THF (entries 4 in
Table 2). The reaction provided the mono-substituted steroid
14 as the major product in 89% yield and the di-substituted 15
2.2
In vitro cytotoxic activity against
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human prostate PC-3 cell line
The antiproliferative activities of the steroids with C-21 hydro-
philic moiety was evaluated on human prostate PC-3 cell using
MTS assay and their IC50 values are presented in Table 3.31
All the steroids (4, 5 and 7-14) reported in this work were more
active (IC50: 42-485 μmol L−1) than the C-21 unsubstituted
4 and progesterone (IC50 > 1000 μmol L−1). Steroids with
simple C-21 substituents amino (7), hydroxyl (8a), and pip-
erazinyl (14) were more potent than steroids with C-21 ester
(10), pyranose (9), amino acid (12 and 13), and imidazole (4).
Among the compounds, basic compound C-21 piperazinyl 14
was most active to inhibit the proliferation of PC3 cells with
IC50 value of 42 μmol L−1 followed by C-21 amino steroid
7 (IC50: 82 μmol L−1). Except for exhibiting better activity,
these compounds were more soluble than we expected.
In conclusion, we have developed a series of routes for
the synthesis of the 3α-hydroxyl-3β-methoxymethyl-5α-
pregnan-20-one derivatives 7-15 with hydrophilic group at
the C-21 positon. The key steps were the amination by the
hindered Delépine agent, the improvement of hydrolysis re-
action by cesium formate (HCO2Cs) in anhydrous MeOH
solution, and the substituted introduction of hydrophilic nuc-
leophiles including amino acid and aminoguanidine into the
core structure. Moreover, a convenient and efficient method
1
in 11% yield (detected by H NMR). The purification of 14
was achieved by recrystallization in MeOH/diisopropyl ether.
The pure 14 was obtained in 72% yield. In this method, the
yield of dimeric 15 was low and could be easily removed by
recrystallization.

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TABLE 2 Conversion of steroid 6 to
the mono-piperazinoyl 14 and di-
piperazinoyl 15 by piperazine or lithium
piperazide
Yields (%)a
Entry
Reagent
T (°C)
Time (h)
14
15
1
(1.0 equiv)
(2.0 equiv)
(3.0 equiv)
(1.2 equiv)
Reflux
>120
24
76
HN
NH
NH
NH
NH
2
3
4
Reflux
>120
48
28
30
89
72
70
11
HN
HN
LiN
Reflux
−5 to 0°C
1.0
aThe yields were detected by 1H NMR.
O
Br
MeO
HO
6
piperazine
THF
OH
MeO
O
O
N
N
+
NH
MeO
N
MeO
O
HO
14
15
HO
SCHEME 3 Synthesis of mono-C-21 piperazinoyl steroid 14 and di-piperazinoyl steroid 15
for the introduction of piperazine functionality to was devel-
oped via lithium imidazole with the advantage of high yield
and low amount of dimeric by-product. Based on the growth
inhibitory results against human prostate cancer cells, this
indicated that compound C-21 piperazinyl 14 possessed the
significant inhibitory activity (42 μmol L−1) to inhibit the
growth of PC-3 cells.
xymethyl-5α-pregnan-20-one (6) was prepared according
to the reported procedure.[9] Flash column chromatography
was carried out on silica gel (230-400 mesh). Analytical
thin-layer chromatography (TLC) was performed on pre-
coated plates (silica gel 60 F-254), purchased from Merck
Inc. Mixtures of ethyl acetate and hexanes were used as elu-
ants. Purification by gravity column chromatography was
carried out by use of Merck Reagents Silica Gel 60 (particle
size 0.063-0.200 mm, 70-230 mesh ASTM). Infrared (IR)
spectra were measured on a Bomem Michelson Series FT-
IR spectrometer. The wavenumbers reported are referenced
to the polystyrene 1601 cm−1 absorption. Absorption inten-
sities are recorded by the following abbreviations: s, strong;
m, medium; w, weak. Proton NMR spectra were obtained
on a Bruker-300 (300 MHz) or a Bruker-200 (200 MHz)
3
3.1
EXPERIMENTAL
General
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All chemicals were reagent grade and used as purchased.
All reactions were carried out under nitrogen atmosphere
and monitored by TLC. 21-Bromo-3α-hydroxy-3β-metho

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TABLE 3 Antiproliferative activities of steroids with C-21
(10 mL) and saturated aqueous NaHCO3 (10 mL ×2), dried
over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography on
silica gel to give 7 (1.96 g, 5.19 mmol) in 87% yield: TLC Rf
0.13 (50% EtOAc in hexanes); mp 86-88°C; 1H NMR (CDCl3,
400 MHz) δ 0.62 (s, 3 H, CH3), 0.74 (s, 3 H, CH3), 1.18-2.65
(m, 24 H), 3.16 (s, 2 H, CH2), 3.37 (s, 3 H, CH3), 4.07 (d,
2 H, J=4.0 Hz, CH2); 13C NMR (CDCl3, 100 MHz) δ 11.13,
13.62, 20.87, 23.38, 24.39, 28.28, 30.11, 31.81, 33.23, 35.47,
35.95, 36.99, 38.88, 40.10, 44.97, 49.48, 53.83, 56.68, 59.33,
60.27, 71.97, 81.86, 202.26; IR (KBr) 3572 (br, NH2), 3434
(br, OH), 2966 (m), 2928 (m), 2851 (m), 1711 (s, C=O), 1474
(m), 1386 (m), 1193 (m), 1112 (s, C–O) cm−1; EI-MS m/z
(relative intensity) 378 (M—31, 6), 353 (32), 352 (21), 351
(100), 269 (34), 161 (11), 119 (10), 107 (13), 95 (13), 81 (23),
79 (12), 55 (12); Anal. Calcd for C23H39NO3: C, 73.17; H,
10.41; N, 3.71%; found: C, 73.15; H, 10.39; N, 3.68%.
hydrophilic substituents
21
O
R
MeO
HO
Compound
ID
IC50
Entry
R
(μmol L−1 a
)
1
2
3
4
5
6
7
8
4
5
7
8a
9
10
11
12
13
14
15
H
>1000
645
82
140
369
485
228
285
381
42
Imidazolyl
–NH2
–OH
Isooxazole-5-carbonyl
D-galactopyranoside
Ornithinyl
Cysteino
Synthesis of 3α-hydroxy-21-(1′-hydroxy)-3β-
methoxymethyl-5α,17β-pregnan-20-one (8a) and 3α-
hydroxy-21-(1′-hydroxy)-3β-methoxymethyl-5α,17α-
pregnan-20-one (8b)[23]
9
10
11
Aminoguanyl
Piperazinyl
A solution of 3 (1.0 equiv) and cesium formate (HCO2Cs,
3.0 equiv) in dry MeOH (10 mL) was heated at reflux for
2.0 hour. The solution was filtered to remove the excess
amount of HCO2Cs and the filtrate was concentrated under
reduced pressure. The residue was purified by column chro-
matography on silica gel to give the corresponding 8a and 8b
in 85% and 2% yields, respectively.
Di-substituted piperazinyl 383
aThe IC50 values were averaged from two independent
dose-response curves; the variation was generally <15%.
spectrometer by use of CDCl3 or DMSO-d6 as solvent.
Carbon-13 NMR spectra were obtained on a Bruker-300
(75 MHz) spectrometer by use of CDCl3 as solvent.
Carbon-13 chemical shifts are referenced to the center of
the CDCl3 triplet (δ 77.0 ppm). Multiplicities are recorded
by the following abbreviations: s, singlet; d, doublet; t, tri-
plet; q, quartet; m, multiplet; J, coupling constant (hertz).
Elemental analyses were carried out on a Heraeus CHN–O
RAPID element analyzer. Purity of all products was >99%
from the results of HPLC analysis.
1
For 8a: TLC Rf 0.29 (50% EtOAc in hexane); H NMR
(CDCl3, 200 MHz) δ 0.60 (s, 3 H, CH3), 0.72 (s, 3 H, CH3),
1.23-1.42 (m, 24 H), 3.15 (s, 2 H, CH2), 3.36 (s, 3 H, CH3),
3.97 (d, 2 H, J=4.0 Hz, CH2); 13C NMR (CDCl3, 50 MHz) δ
11.20, 13.64, 20.94, 22.86, 24.53, 28.37, 30.18, 31.92, 33.29,
35.51, 36.01, 37.06, 38.94, 40.16, 53.92, 56.89, 59.23, 59.41,
71.03, 78.73, 81.90, 208.26; IR (KBr) 3431 (br, OH), 2976
(m), 2923 (m), 2839 (m), 1711 (s, C=O), 1479 (m), 1368 (m),
1290 (m), 1235 (m), 1095 (m, C–O) cm−1; EI-MS m/z (rela-
tive intensity) 347 (M—31, 6), 334 (22), 333 (100), 301 (7),
269 (16), 120 (17), 107 (32), 95 (47), 55 (74); HRMS Calcd
for C23H38O4 378.2770, found 378.2765.
3.1.1
Synthesis of 3α-hydroxyl-3β-
|
methoxymethyl-5α-pregnan-20-one with a C-21
For 8b: TLC Rf 0.31 (50% EtOAc in hexane); mp 126-
128°C; 1H NMR (CDCl3, 200 MHz) δ 0.58 (s, 3 H, CH3), 0.71
(s, 3 H, CH3), 1.16-2.42 (m, 24 H), 3.14 (s, 2 H, CH2), 3.34 (s,
3 H, CH3), 4.14 (d, 2 H, J=4.0 Hz, CH2); 13C NMR (CDCl3,
50 MHz) δ 11.19, 13.57, 20.86, 22.91, 24.48, 28.34, 30.19,
31.89, 33.29, 35.50, 36.01, 37.05, 38.78, 40.15, 45.00, 53.91,
56.81, 59.32, 59.40, 69.42, 71.00, 81.89, 210.44; IR (KBr)
3431 (br, OH), 2923 (s), 2839 (m), 1711 (s, C=O), 1095 (m,
C–O), 1479 (m), 1368 (m), 1290 (m), 1235 (m) cm−1; EIMS
m/z (relative intensity) 347 (M—31, 17), 334 (43), 333 (100),
301 (20), 269 (34), 109 (14), 95 (23), 81 (25), 55 (19); HRMS
Calcd for C23H38O4 378.2770, found 378.2766.
hydrophilic substituent 7-14
Synthesis of 21-amino-3α-hydroxyl-3β-
methoxymethyl-5α-pregnan-20-one (7)[22]
A solution of 21-bromo-3α-hydroxy-3β-methoxymethyl-5α-
pregnan-20-one (6, 2.32 g, 5.96 mmol, 1.0 equiv) and hexa-
methylenetetramine (836 mg, 5.96 mmol, 1.0 equiv) in an-
hydrous CH2Cl2 (10 mL) was stirred at room temperature for
24 hour. The solution was slowly added with MeOH (7.0 mL),
37% conc. HCl solution (1.0 mL), and MeOH (8.0 mL) until
the suspensions formed. The resulting solution was stand
for 3 days, diluted with CH2Cl2 (10 mL), washed with water

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3α-hydroxy-3β-methoxymethyl-20-oxo-5α-pregnan-21-yl-
D-galactopyranoside tetra-acetate in 85% yield. A solution of
3α-hydroxy-3β-methoxymethyl-20-oxo-5α-pregnan-21-yl-
D-galactopyranoside tetra-acetate (0.780 g, 1.10 mmol, 1.0
equiv) and K2CO3 (0.231 g, 1.67 mmol, 1.5 equiv) in MeOH
(20 mL) was stirred at reflux for 4.0 hour. The solution was
acidified with acetic acid to pH ~5. The resulting suspension
was collected by filtration and purified by column chromatog-
raphy on silica gel to give pure 10 in 92% yield: TLC Rf 0.3
(50% EtOAc in hexane); 1H NMR (CDCl3, 200 MHz) δ 0.60
(s, 3 H, CH3), 0.74 (s, 3 H, CH3), 1.19-1.95 (m, 24 H), 3.11
(s, 2 H, CH2), 3.31 (s, 3 H, CH3), 3.45-3.78 (m, 6 H), 4.18-
4.47 (m, 2 H); 13C NMR (CDCl3, 50 MHz) δ 11.17, 13.74,
20.59, 20.78, 20.86, 20.92, 22.64, 31.86, 35.47, 35.47, 35.97,
40.10, 44.82, 53.88, 56.83, 58.82, 59.36, 61.25, 66.92, 68.39,
70.67, 70.79, 71.05, 90.53, 91.88, 100.23, 128.78, 130.89,
169.42, 169.83, 170.09, 170.19, 170.35, 170.71, 207.05; IR
(KBr) 3668 (br, OH), 2360 (m), 2328 (m), 1714 (s, C=O),
1070 (m, C–O) cm−1; Anal. Calcd for C29H48O9: C, 63.86; H,
8.80%. Found: C, 63.91; H, 8.76%.
Synthesis of 21-(isoxazole-5-carboxyl)-3α-hydroxy-3β-
methoxymethyl-5α-pregnan-20-one (9)[24]
A solution of compound 8a (1.04 g, 2.75 mmol, 1.0 equiv),
isoxazole-5-carbonyl chloride (434 mg, 3.30 mmol, 1.2
equiv), and triethylamine (NEt3, 841 mg, 8.31 mmol, 3.0
equiv) in anhydrous THF (10 mL) was heated at reflux
for 4.0 hour. The solution was concentrated under reduced
pressure and the residue was added with CH2Cl2 (20 mL).
The mixture was washed with water (10 mL) and saturated
aqueous NaHCO3 (10 mL ×2). The organic layer was dried
over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography
on silica gel to give the corresponding 9 (1.16 g, 2.45 mmol)
1
in 89% yield: TLC Rf 0.3 (50% EtOAc in hexane) H NMR
(CDCl3, 200 MHz) δ 0.60 (s, 3 H, CH3), 0.74 (s, 3 H, CH3),
1.19-1.95 (m, 24 H), 3.11 (s, 2 H, CH2), 3.31 (s, 3 H, CH3),
3.45-3.78 (m, 6 H), 4.18-4.47 (m, 2H); 13C NMR (CDCl3,
50 MHz) δ 11.17, 13.74, 20.59, 20.78, 20.86, 20.92, 22.64,
31.86, 35.47, 35.47, 35.97, 40.10, 44.82, 53.88, 56.83, 58.82,
59.36, 61.25, 66.92, 68.39, 70.67, 70.79, 71.05, 90.53, 91.88,
100.23, 128.78, 130.89, 169.42, 169.83, 170.09, 170.19,
170.35, 170.71, 207.05; IR (KBr) 3668 (br, OH), 2360 (m),
2328 (m), 1714 (s, C=O), 1070 (m, C-O) cm−1; Anal. Calcd
for C27H39NO6: C, 68.47; H, 8.30; N, 2.96%. Found: C,
68.43; H, 8.29; N, 2.93%.
Synthesis of 21-ornithinyl-3α-hydroxy-3β-
methoxymethyl-5α-pregnan-20-one (11)[28]
A solution of 6 (0.25 g, 0.58 mmole, 1 equiv), D-ornithine
hydrochloride (108 mg, 0.64 mmol, 1.0 equiv), and N,N-
diisopropylethylamine (DIEA, 0.23 mL, 1.7 mmol, 2.2
equiv) in anhydrous DMF (15 mL) was heated at reflux for
6.0 hour. When the reaction was completed, the solution
was filtered and washed with brine (10 mL ×2). The organic
layer was concentrated under reduced pressure and the resi-
due was purified by column chromatography on silica gel to
give the desired 11 in 72% yield: TLC Rf 0.48 (10% MeOH
Synthesis of 3α-hydroxy-3β-methoxymethyl-20-oxo-
5α-pregnan-21-yl-D-galacto-pyranoside (10)[25,26]
A solution of β-D-galactose pentaacetate (2.00 g, 5.12 mmol,
1.0 equiv) in CH2Cl2 (20 mL) was slowly added with PBr3
(5.55 g, 20.5 mmol, 4.0 equiv) in ice bath. The reaction mix-
ture was stirred at room temperature for 5.0 days. The solu-
tion was slowly added with water (3 mL) and extracted with
CH2Cl2 (20 mL). The organic layer was washed with water
(10 mL) and saturated aqueous NaHCO3 (10 mL ×2), dried
over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography
on silica gel to give 16 (2.00 g, 4.86 mmol) in 95% yield:
1
in CH2Cl2); H NMR (CDCl3, 200 MHz) δ 0.57 (s, 3 H,
CH3), 0.67 (s, 3 H, CH3), 1.16-1.62 (m, 24 H), 2.78 (s, 4 H),
2.88 (s, 3 H, CH3), 3.11-3.27 (m, 5 H), 3.31 (s, 3 H); 13C
NMR (CDCl3, 50 MHz) δ 10.30, 11.15, 13.76, 20.87, 24.33,
28.27, 30.09, 31.77, 33.24, 35.44, 35.94, 36.50, 36.98,
38.95, 40.07, 42.35, 45.22, 48.59, 53.74, 56.69, 59.35,
60.94, 70.99, 81.89, 162.64; IR (KBr) 2966 (s), 2924 (s),
2848 (m), 1716 (s, C=O), 1120 (m) cm−1; Anal. Calcd for
C28H48N2O5: C, 68.26; H, 9.82; N, 5.69%. Found: C, 68.30;
H, 9.79; N, 5.65%.
1
TLC Rf 0.64 (50% EtOAc in hexanes); H NMR (CDCl3,
200 MHz) δ 1.97-2.11 (m, 12 H, OAc), 4.07-4.14 (m, 2 H),
4.45 (m, 1 H), 4.97-5.04 (m, 1H), 5.33-5.49 (m, 2 H), 6.66
(d, 1 H, C-1); 13C NMR (CDCl3, 50 MHz) δ 20.54, 20.70,
60.80, 66.95, 67.74, 67.96, 71.03, 88.09, 169.74, 169.88,
170.04, 170.30.
Synthesis of 21-cysteino-3α-hydroxy-3β-
methoxymethyl-5α-pregnan-20-one (12)[29]
A solution of 8a (0.50 g, 1.32 mmol, 1.0 equiv), Ag2CO3
(1.64 g, 5.95 mmol, 4.5 equiv), and 4 Ǻ Molecular sieve
(1.5 g) in anhydrous CH2Cl2 (20 mL) was stirred for 30 min-
ute. A solution of 16 (1.36 g, 3.31 mmol, 2.5 equiv) in an-
hydrous CH2Cl2 (10 mL) was slowly added to the reaction
mixture. The solution was heated at reflux for 2.0 hour. The
solution was filtered through Celite and the cake was washed
with CH2Cl2 (20 mL). The filtrate was dried over MgSO4,
filtered, and concentrated under reduced pressure to give the
A solution of 6 (0.1 g, 0.23 mmole, 1.0 equiv), L-cysteine
(281 mg,0.23 mmol,1.0equiv)andN,N-diisopropylethylamine
(DIEA, 0.23 mL, 1.72 mmol, 2.2 equiv) was stirred to dis-
solve in anhydrous EtOH (15 mL). The reaction mixture was
basified with aqueous NaOH to pH 11-12. The mixture was
stirred at room temperature for 2.0 hour. When the reaction
was completed, the solution was neutralized with 3% aqueous
HCl solution and filtered. The particle and washed with icy

8 of 9
TSAI eT Al.
|
EtOH and re-dissolved in a small amount of water (<5 mL).
The solution was concentrated under reduced pressure and
purified by column chromatography on silica gel to give the
desired 12 in 24% yield: TLC Rf 0.33 (50% EtOAc in hex-
ane); mp 126-128°C; 1H NMR (CDCl3, 200 MHz) δ 0.58 (s,
3 H, CH3), 0.70 (s, 3 H, CH3), 1.15-2.00 (m, 28 H), 3.14 (s,
2 H, CH2), 3.34 (s, 3 H, CH3), 4.06-4.14 (m, 2 H); 13C NMR
(CDCl3, 50 MHz) δ 11.20, 13.57, 20.86, 22.91, 24.48, 28.35,
29.68, 30.18, 31.90, 33.29, 35.51, 35.91, 36.02, 37.05, 38.79,
40.16, 45.01, 56.81, 59.40, 69.41, 71.07, 81.91, 210.48; IR
(KBr) 2972 (m), 2922 (m), 2848 (m), 2320 (m), 2310 (m),
1749 (s, C=O), 1734 (m), 1716 (m), 1697 (m), 1683 (m), 1558
(m), 1521 (m) cm−1; EI-MS m/z (relative intensity) 481.1 (8),
334 (21), 333 (100), 319 (11), 269 (13); HRMS Calcd for
C26H43NO5S 481.2651, found 481.2655.
reduced pressure to give a mixture of mono-substituted prod-
uct 14 (89%) and dimeric product 15 (11%) as observed from
1H NMR). The crude product was crystallized from MeOH/
diisopropyl ether to provide the pure 14 (706 mg, 1.58 mmol)
as white solids in 72% yield: TLC Rf 0.10 (10% MeOH in
CH2Cl2); mp 168-170°C; 1H NMR (CDCl3, 400 MHz) δ 0.57
(s, 3 H, CH3), 0.69 (s, 3 H, CH3), 0.61-0.82 (m, 1 H), 0.85-
0.96 (m, 1 H), 1.06-1.40 (m, 12 H), 1.45-1.64 (m, 8 H), 1.84
(d, 2 H, J=8.8 Hz, CH2), 2.05-2.15 (m, 1 H), 2.40 (d, 4 H,
J=4.4 Hz, CH2), 2.55 (dd, 1 H, J=10.4 Hz), 2.88 (dd, 4 H,
J=4.8 Hz, CH2), 3.13 (s, 2 H, CH2), 2.40 (s, 3 H, CH3); 13
C
NMR (CDCl3, 100 MHz) δ 11.15, 13.60, 20.93, 23.01, 24.45,
28.35, 30.16, 31.89, 33.26, 35.47, 35.97, 37.04, 39.06, 40.14,
44.71, 45.78, 53.93, 54.63, 56.84, 59.36, 60.30, 69.66, 70.95,
81.90, 208.45; IR (KBr) 3244, 2932 (s), 2843 (m), 1721 (m),
1450 (m), 1385, 1346, 1273, 1192, 1126, 1011, 918, 860,
733 cm−1; EI-MS m/z (relative intensity) 446 (M+, 2), 443
(7), 154 (22), 136 (17), 111 (13), 97 (26), 77 (12); HRMS
Calcd for C27H46N2O3 446.3508, found 446.3505.
Synthesis of 21-aminoguanyl-3α-hydroxy-3β-
methoxymethyl-5α-pregnan-20-one (13)[30]
A solution of 21-bromo-3α-hydroxy-3β-methoxymethyl-5α-p
regnan-20-one (6, 1.0 g, 2.23 mmole, 1.0 equiv), aminoguan-
idine (0.34 g, 2.49 mmol, 1.1 equiv), and N,N-diethylamine
(0.26 mL, 2.49 mmol, 1.1 equiv) in anhydrous DMF (10 mL)
was stirred at room temperature for 8.0 hour. When the reac-
tion was completed, the solution was filtered and extracted
with CH2Cl2 (20 mL). The organic layer was washed with
water (10 mL), saturated aqueous NaCl (10 mL ×2), dried
over MgSO4, filtered, and concentrated under reduced pres-
sure. The residue was purified by column chromatography
on silica gel to give the desired 13 in 51% yield: TLC Rf 0.48
(10% MeOH in CH2Cl2); 1H NMR (CDCl3, 200 MHz) δ 0.58
(s, 3 H, CH3), 0.70 (s, 3 H, CH3), 1.00-1.98 (m, 24 H), 2.78
(s, 4 H), 2.88 (s, 3 H, CH3), 3.11-3.27 (m, 5 H), 3.31 (s, 3
H); 13C NMR (CDCl3, 50 MHz) δ 10.30, 11.15, 13.76, 20.87,
24.33, 28.27, 30.09, 31.77, 33.24, 35.44, 35.94, 36.50, 36.98,
38.95, 40.07, 42.35, 45.22, 48.59, 53.74, 56.69, 59.35, 60.94,
70.99, 81.89, 162.64; IR (KBr) 2968 (m), 2922 (m), 2374
(m), 2322 (m), 2310 (m), 1716 (s, C=O), 1699 (m), 1683
(m), 1521 (m), 1506 (m) cm−1; EI-MS m/z (relative intensity)
433.5 (M+, 1), 388 (2), 86 (100), 58 (2); HRMS Calcd for
C24H42N4O3 434.3257, found 434.3262.
3.1.2
21-piperazinyl-3α-hydroxy-3β-
|
methoxymethyl-5α-pregnan-20-one dimeric (15)
A solution of 21-bromo-3α-hydroxy-3β-methoxymethyl-5α-p
regnan-20-one (6, 481 mg, 1.09 mmole, 1.0 equiv) and pip-
erazine (259 mg, 3.01 mmol, 3.0 equiv) in anhydrous DMF
(10 mL) was stirred at room temperature for 8.0 hour. When
the reaction was completed, the solution was filtered and ex-
tracted with CH2Cl2 (20 mL). The organic layer was washed
with water (10 mL ×2), saturated aqueous NaCl (10 mL ×2),
dried over MgSO4, filtered, and concentrated under reduced
pressure. The residue was purified by column chromatography
on silica gel to give the desired 15 in 70% yield as a light yel-
low solids: mp 126-128°C; 1H NMR (CDCl3, 200 MHz) δ 0.57
(s, 6 H, CH3), 0.71 (s, 6 H, CH3), 1.11-2.09 (m, 44 H), 2.09-
2.53 (m, 8 H), 3.13 (s, 4 H, CH2), 3.34 (s, 6 H, CH3); 13C NMR
(CDCl3, 50 MHz) δ 11.18, 13.63, 20.96, 23.05, 24.47, 28.38,
30.20, 31.92, 33.30, 35.50, 36.01, 37.08, 40.18, 44.75, 53.10,
53.96, 56.88, 59.40, 60.40, 69.05, 71.02, 81.91, 280.24; IR
(KBr) 2929 (s), 2845 (m), 2362 (m), 1716 (m), 1089 (m) cm−1;
HRMS Calcd for C50H82N2O6 806.6173, found 806.6170.
Synthesis of 21-piperazinyl-3α-hydroxy-3β-
methoxymethyl-5α-pregnan-20-one (14)
3.2
Antiproliferative
|
A solution of piperazine (0.23 g, 2.67 mmol, 1.2 equiv)
in THF (20 mL) was added with lithium hydride (27 mg,
3.4 mmol, 1.5 equiv), and the solution was heated at reflux
for 30 minute under N2. The solution was cooled and slowly
added with compound 6 (971 mg, 2.20 mmol, 1.0 equiv) in
THF (20 mL) at 0°C and stirred at that temperature for ad-
ditional 1.0 hour. The reaction mixture was quenched with
water (5.0 mL) and extracted with CH2Cl2 (20 mL). The
organic layer was washed with 15% aqueous NH4Cl, brine,
dried over MgSO4 (s), filtered, and concentrated under
3.2.1
Cell lines and inhibitory
|
concentration assay[31]
PC3 cell line was maintained in RPMI 1640 supplemented
with 10% fetal bovine serum and with 100 units/mL Penicillin
and 100 units/mL at 37°C in ahumidified atmosphere of 5%
CO2/95% air.
Logarithmic phase cells were seeded in a 96-well plate
and incubated overnight prior to addition of the designated

TSAI eT Al.
9 of 9
|
Whittemore, R. M. Woodward, N. C. Lan, K. W. Gee, M. B.
Bolger, J. Med. Chem. 1997, 40, 61.
compounds. After incubation with different concentrations of
the tested compounds for 72 hour, cells were incubated with
RPMI containing 0.5 mg/mL MTT for 2 hour. The conver-
sion of MTT to formazan by metabolically viable cells was
measured by the absorbance at 570 nm in a 96-well microtiter
plate reader. The percentage conversion by mock-treated con-
trol cells was used to evaluate the effect of the chemicals on
cell growth and to determine the concentration that inhibited
50% of growth (IC50).
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Höltje, N. A. Urbanetz, B. Brandt, H.-D. Höltje, B. C. Lippold,
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[15] A. T. M. Serajuddin, Adv. Drug Deliv. Rev. 2007, 59, 603.
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ACKNOWLEDGMENTS
[20] E. A. Ariazi, G. M. Clark, J. E. Mertz, Cancer Res. 2002, 62, 6510.
[21] T. Fujimura, S. Takahashi, T. Urano, N. Ijichi, K. Ikeda, J.
Kumagai, T. Murata, K. Takayama, K. Horie-Inoue, Y. Ouchi, M.
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We are grateful to the Tsuzuki Institute for Traditional
Medicine and Technology of the Republic of China (MOST
105-2113-M-039-001) for financial support.
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How to cite this article: Tsai S-E, Lee J-C, Uramaru
N, Takayama H, Huang G-J, Wong FF. Synthesis and
antiproliferative activity of 3α-hydroxyl-3β-
methoxymethyl-5α-pregnan-20-one with a C-21
hydrophilic substituent. Heteroatom Chem.
2017;28:e21372. https://doi.org/10.1002/hc.21372