Glucal-conjugated sterols as novel vascular leakage blocker: Structure-activity relationship focusing on the C17-side chain

Article abstract of DOI:10.1016/j.ejmech.2014.01.027

A series of glucal-conjugated sterols as novel vascular leakage blocker were identified through design, synthesis and biologically evaluation. In addition, the structure-activity relationship (SAR) of the glucal-conjugated sterols focusing on the C₁₇-side chain was also established. The sterol analogs linked with the rigid C₁₇-side chain side chains exhibited potent cell survival activities. In particular, analog 21l, which possesses a cyclopentyl oxime moiety, was shown to have excellent pharmacological effects on retinal vascular leakage in a diabetic mouse model.

Full text of DOI:10.1016/j.ejmech.2014.01.027

European Journal of Medicinal Chemistry 75 (2014) 184e194
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Glucal-conjugated sterols as novel vascular leakage blocker:
Structureeactivity relationship focusing on the C17-side chain
,
,
Kyeojin Kim a 1, Sony Maharjan b 1, Changjin Lim a, Nam-Jung Kim c, Vijayendra Agrawal b,
Young Taek Han d, Sujin Lee a, Hongchan An a, Hwayoung Yun a, Hyun-Jung Choi b,
**
Young-Guen Kwon b , Young-Ger Suh
,
a,
*
a College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
b Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea
c Department of Pharmacy, College of Pharmacy, Kyung Hee University, Seoul 130-701, Republic of Korea
d College of Pharmacy, Woosuk University, Wanju 565-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of glucal-conjugated sterols as novel vascular leakage blocker were identified through design,
synthesis and biologically evaluation. In addition, the structure-activity relationship (SAR) of the glucal-
conjugated sterols focusing on the C17-side chain was also established. The sterol analogs linked with the
rigid C17-side chain side chains exhibited potent cell survival activities. In particular, analog 21l, which
possesses a cyclopentyl oxime moiety, was shown to have excellent pharmacological effects on retinal
vascular leakage in a diabetic mouse model.
Received 27 August 2013
Received in revised form
8 January 2014
Accepted 14 January 2014
Available online 25 January 2014
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
Anti-apoptotic agents
Tight junction
Pregnenolone
Oxime
Ginsenoside Rk1
Diabetic retinopathy
1. Introduction
endothelial cells and the collapse of endothelial cell homeostasis
[1e4]. Homeostasis at the vascular cell level is maintained by an
The endothelium is a thin layer of endothelial cells, which
constitute the inner lining of blood vessels and form an interface
between circulating blood in the lumen and rest of the vessel wall.
The disintegration of the endothelium causes pathological symp-
toms such as diabetic retinopathy, which is a complication that
occurs primarily in patients with diabetes mellitus [1]. Vascular
leakage can be caused by the disruption of tight junctions between
appropriate balance between cell proliferation and apoptosis [5,6].
In particular, the excessive apoptosis of vascular endothelial cells
(VECs) can disrupt the integrity of vessels and the normal function
of the endothelium [7,8]. The loss of integrity and vascular
permeability lead to a macular edema, which is normally accom-
panied by diabetic retinopathy [9,10]. Nevertheless, there has been
little progress in the treatment of vascular leakage despite clinical
trials of small molecules such as calcium dobesilate, aspirin, and
antihistamines [1]. Laser surgery still remains the primary treat-
ment for vascular leakage [11]. Consequently, medications for the
treatment of retinal vascular leakage are still urgently needed.
We reported the potent anti-apoptotic activities of ginsenosides
Rg3 (1) and Rk1 (2), which were isolated from the root of genus, in
human umbilical vein endothelial cell (HUVEC) lines [12]. We also
reported the identification of novel anti-apoptotic agents, including
SAC-0504 (3) and SAC-0601 (4), that consist of a cholesterol scaffold
and a carbohydrate equivalent, in place of the protopanaxadiol
backbone and the carbohydrate, respectively, of Rk1 [13] (Fig. 1).
Abbreviations: HUVEC, human umbilical vein endothelial cell; TJs, tight
junction; PTSA, para-toluenesulfonic acid; DHP, 3,4-dihydro-2H-pyran; DBU, 1,8-
Diazabicyclo[5.4.0]undec-7-ene; HREC, human retina endothelial cell; MTT, (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; VEGF, vascular
endothelial growth factor; BRB, blood-retinal barrier.
* Corresponding author. Tel.: þ82 2 880 7875; fax: þ82 2 888 9122.
** Corresponding author. Tel.: þ82 2 2123 5697; fax: þ82 2 362 9897.
E-mail addresses: ygkwon@yonsei.ac.kr (Y.-G. Kwon), ygsuh@snu.ac.kr
(Y.-G. Suh).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.027

K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
185
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of the glucal conjugated sterols with modofied C17
-
side chain based on the structures of ginsenosides Rg3 (1) and Rk1 (2)
The analogs of 3 and 4 with the modified C17-side chains were
synthesized from 9 as outlined in Scheme 1. The tetrahydropyran
analog 5, which possesses a tert-alcohol in the side chain, was
prepared by the reaction of 9 with dihydropyran in the presence of
PTSA, followed by a diastereoselective Grignard reaction [20]. The
dihydropyran analog 6 was synthesized via Grignard reaction and
subsequent glycosidation with tri-O-acetyl-D-glucal [13,21]. We
also synthesized pyran-linked analogs with side chains that are
structurally similar to that of ginsenoside Rk1 (2). The elimination
of the tert-alcohol of analog 5 using Et3N and MsCl produced
regio/stereoisomers of 12, 14 which could not be separated by
column chromatography. Other analogs with side chains pos-
sessing double bonds were conveniently prepared from the com-
mon intermediate 15, which was prepared by the Wittig
olefination [22] of 9. Analog 7 was synthesized by the reaction of
15 with DHP in the presence of PTSA. The reaction of tri-O-acetyl-
Fig. 1. Natural and synthetic sterol analogs.
SAC-0601 exhibited not only potent anti-apoptotic activity but also
a protective effect against the disruption of tight junctions (TJs)
which leads to retinal vascular leakage [13,14]. Therefore, the
further development of chemotherapeutically useful sterol analogs
for the treatment of diabetic retinopathy was quite attractive to us
because of the high demand for drugs that maintain endothelial
integrity and because of the excellent protective effects of these
sterol analogs, which were previously identified by us, for the
endothelial system [13e15].
D-glucal with intermediate 15 in the presence of BF3$OEt2 in THF
provided analog 8.
2.1.2. Synthesis of the glucal-conjugated oxime analogs
Next, we prepared a variety of oxime ether analogs as variants
of the olefin-possessing analog 8. Initially, various hydroxyl
amines were prepared as described in Scheme 2. Most hydrox-
ylamine$HCl salts could be prepared from commercially available
N-hydroxyphthalimide (17), which was transformed into alkox-
yphthalimides 18aep using DBU and the corresponding alkyl
halide [23] or by the Mitsunobu reaction [24] in the presence of
the corresponding alkyl alcohols. Sequential imide cleavage of the
resulting imides using methyl hydrazine followed by hydrochlo-
ride salt formation afforded 19aep [18,23,24]. With the requisite
alkyloxyamine salts and the commercially available hydrox-
ylamine$HCl and benzylhydroxylamine$HCl salts in hand, we were
able to synthesize oxime analogs via a unified synthetic strategy
Furthermore, we reported the anti-apoptotic activities of the
carbohydrate equivalent-linked analogs of phytosterols including
sitosterol, sitostanol and stigmasterol [16]. Interestingly, the ac-
tivities of these compounds depended on the C17-side chain
attached to the D-ring of the phytosterols. These results also
revealed that the branched alkyl chain of the phytosterol plays an
important role in determining the biological activities. Thus, we
designed second-generation sterol analogs linked to carbohydrate
equivalent,
4,6-di-O-acetyl-2,3-dideoxyhex-2-enopyran.
Our
strategy focused on the C17-side chain modification of SAC-0504
(3) and SAC-0601 (4) based on the structures of ginsenosides
Rg3 (1) and Rk1 (2), which have tert-alcohol and exo-olefin in the
C
17-side chain, respectively. We initially investigated the effect of
[17e24]. The reaction of tri-O-acetyl- -glucal with 9 provided the
D
tert-alcohol on the assumption that tert-alcohol can be trans-
formed to the endo/exo-olefin via dehydration. In addition, we
further modified the C17-side chain by incorporating oxime ether
because we anticipated that the constrained conformation would
provide improved binding affinity to the target protein [17e19].
Insertion of heteroatom was also anticipated to reduce the high
lipophilic character of the analogs. Thus, commercially available
pregnenolone (9) was selected as an appropriate substrate for
synthesis of the newly designed analogs in terms of accessibility
and synthetic efficiency (Fig. 2).
key intermediate 20, which was transformed into a variety of
glucal-conjugated oxime analogs (21aer) by condensation with
the corresponding hydroxylamine$HCl salts in refluxing pyridine.
2.2. Biology
2.2.1. In vitro assay
Given that apoptosis is generally associated with the onset of
diabetic retinopathy [25,26], we investigated the anti-apoptotic
Fig. 2. Strategy for the development of novel sterol analogs based on SAC-0601.

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K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
Scheme 1. Synthesis of the glucal conjugated sterols with modofied C17-side chain based on the structures of ginsenosides Rg3 (1) and Rk1 (2). Reagents and conditions: (a) PTSA,
DHP, CH2Cl2, 57%; (b) Mg turning, 1-bromo-4-methyl pentane, Et2O, 15e18%; (c) tri-O-acetyl-D-glucal, BF3$OEt2, THF, 12e23%; (d) Et3N, MsCl, CH2Cl2, 40%; (e) PTSA, MeOH, 99%; (f) t-
BuOK, 16, benzene, reflux, 22%.
effect of the synthesized analogs using serum-deprived human
retina endothelial cells (HRECs). The anti-apoptotic activity assay
moiety for the endo-olefin group, which seems to induce an
appropriate conformation of the sterol analogs for higher binding
affinity and induce high solubility. As predicted, the oxime analog
21h exhibited an activity equipotent to that of the corresponding
alkyl analog 8 with improved solubility. We thoroughly investi-
gated the effects of the alkyl substituents of the oxime moiety on
the biological activities of oxime ether analogs. Generally, the
oxime analogs with short or medium-sized alkyl chains, such as
methyl, ethyl, isopropyl, allyl and propargyl groups, exhibited
slightly low anti-apoptotic activities. In contrast, the oxime ether
analogs with the butyl and pentyl substituents exhibited potent
activities. The analogs with branched chains (21h and 21j) were
more potent than the analogs with the corresponding linear chains.
The non-substituted oxime (21q) exhibited lower activity than 4,
and the analog with the 3,30-dimethylallyl oxime ether 21k,
exhibited a lower potency than the corresponding saturated analog
was conducted using the MTT colorimetric method at 10
mg/mL for
48 h as a preliminary study [27]. Noticeably, the 10 g/mL DMSO
m
stock solutions of SAC-0601 (4) and analogs 7 and 8 were prepared
as a suspension due to poor solubility whereas analogs 5, 6 and all
oxime ethers dissolved well in DMSO, which supports the increase
of solubility by an incorporation of hydroxyl group or oxime ether
moiety [28]. As shown in Fig. 3, some analogs with constrained side
chains exhibited more potent cell survival activities than the parent
analog 4. In particular, analogs such as 7 and 8 which possess an
endo-olefin were more potent than the analogs that possess a tert-
alcohol or an exo-olefin regardless of the carbohydrate equivalent
used. This result also implied that incorporation of the constrained
side chain enhances the anti-apoptotic activity. Based on the re-
sults, we focused on incorporation of oxime moiety, a bioisosteric
Scheme 2. Synthesis of the glucal-conjugated oxime analogs. Reaction conditions and reagents: (a) DBU, DMF, alkyl halide, 60 ꢀC; (b) diisopropyl azodicarboxylate, pyridine-3-
methanol, PPh3, CH2Cl2; (c) MeNHNH2, CH2Cl2; (d) BF3$OEt2, 3,4,6-tri-O-acetyl-D-glucal, CH2Cl2, 60%; (e) 19aer, pyridine, reflux.

K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
187
Fig. 3. Inhibitory effects of the oxime analogs on the apoptosis of HRECs. The cells were treated with 10 mg/mL for 48 h, 0 h: the viability of the cells cultured in medium without any
analog. Con: the viability of the cells cultured in medium containing DMSO used as a vehicle.
21h. Interestingly, the analogs possessing cycloalkyl oxime ethers
exhibited more potent activities than oxime ether 21h which had
an acyclic alkyl substituent. The oxime ethers (21l, n and r) with
cyclohexyl and benzyl substituents exhibited the most potent ac-
tivities. However, analog 21p, which had a heterocyclic substituent,
did not have inhibitory activity [29].
endothelial cells. These TJs are made up of many junctional proteins
with intracellular partners and actin filaments, which are engaged
in multiple interactions to regulate endothelial permeability [30e
38]. Therefore, we used an assay that probes the actin cytoskel-
eton, one of the major TJ proteins in endothelial cells used to
evaluate the ability of compounds to maintain the integrity of TJs
[31e38]. Vascular endothelial growth factor (VEGF), which has
been reported to be a key mediator of BRB collapse in diabetic
retinopathy and other retinal ischemic diseases [39e42], was uti-
lized to disrupt the stability of TJ proteins and to alter the actin
filament distribution [30]. A preliminary assay using analogs 6 and
8 showed clear reductions in the formation of actin stress fibers at
Analogs 21l, n and r exhibited dose-dependent anti-apoptotic
activities in the MTT assay, as shown in Fig. 4.
The oxime analogs were further tested for their protective effect
against TJ disruption [14], which leads to retinal vascular leakage.
These compounds were evaluated using an assay based on VEGF-
induced endothelial cell permeability, which can be assessed by
the formation of actin stress fibers. The blood-retinal barrier (BRB)
is composed of inner (iBRB) and outer (oBRB) barriers [30]. Many
clinical studies have shown that the major site that becomes
permeable in diabetic retinopathy is the iBRB [14,31,32]. The iBRB is
maintained by the TJs between adjacent retinal capillary
concentrations of 10e20
mg/mL. Accordingly, other oxime ether
analogs were evaluated at a concentration of 10
mg/mL. Interest-
ingly, analogs 21l, n and r, which exhibited potent anti-apoptotic
activities, also significantly reduced the formation of the stress fi-
bers in HRECs, consequently leading to the stabilization of the
Fig. 4. Dose-dependent inhibitory effects of 21l, n, and r on the apoptosis of HRECs. Cells were treated with concentrations from 0.1 to 10 mg/mL for 48 h.

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K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
Fig. 5. Assay of oxime analogs based on the stabilization of the cortical actin ring in HRECs. HRECs were cultured on gelatin-coated dishes for 3 days. Confluent cells were serum-
starved in the serum-free media for 3 h, and then pretreated with analogs (10 mg/mL) for 60 min, followed by VEGF treatment (20 ng/mL) for 60 min. The cells were then fixed, and
actin was stained with rhodamineephalloidin.
cortical actin ring (Fig. 5). These compounds exhibited more potent
activities than the parent analog 4.
VEGF-induced permeability by analog 21l was examined using a
mouse model (Fig. 8) because it has been reported that the retinal
permeability caused by an increase in VEGF leads to retinopathy in
the diabetic mouse model [37]. After 24 h of intravitreal adminis-
tration of VEGF and analog 21l in mice, the retinas were isolated,
and the extent of extravasation of FITC-dextran was determined by
fluorescein angiography. As anticipated, VEGF-induced retinal
vascular leakage was hardly observed after co-injection with analog
21l. Inspired by the results of the VEGF-induced permeability assay,
we evaluated the inhibitory activity of analog 21l against retinal
vascular leakage caused by diabetic retinopathy in the diabetic
mouse model. Diabetes was induced in mice by daily intraperito-
neal injections of streptozotocin (Sigma) for four consecutive days
[42]. Three days after the last streptozotocin injection, mice whose
blood glucose concentrations exceeded 30 mg/L were used in this
experiment. Analog 21l reduced vascular leakage to the normal
We further evaluated analogs 21l, n and r using an occludin-
based assay. In this assay, normal HRECs retain the polygonal
shape and the linear pattern of occludin at the cell border and
consequently have intact TJs. However, this pattern is disrupted by
VEGF treatment [37]. As shown in Figs. 6 and 7, the oxime analogs
enhanced the occludin integrity in the VEGF-treated HRECs. These
cell-based assay results indicated that the oxime analogs prevent
the apoptosis of endothelial cells and consequently maintain the TJs
between endothelial cells.
2.2.2. In vivo assay
Based on the results of the cell-based assays, analog 21l was
selected for evaluation of its preventive effect against retinal
vascular leakage in an in vivo model. Initially, the prevention of
Fig. 6. Changes in the occludin pattern caused by the selected oxime analogs. HRECs were cultured on gelatin-coated dishes for 3 days. The cells were pretreated for 60 min with
21l, n, and r (10 g/mL) then stimulated with VEGF (20 ng/mL) for 60 min. The cells were then fixed, and occludin was labeled with a rabbit-anti-occludin antibody.
m

K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
189
Fig. 7. Changes in the occludin pattern caused by analog 21l. HRECs were grown to confluence and starved with serum free media for 3 h. (A) Cells were treated with various
concentration of 4 and 21l for 60 min followed by the stimulation of VEGF (20 ng/mL) for 60 min. (B) Cells were treated with 4 (10 g/mL) and 21l (10 g/mL) for 60 min followed by
m
m
the stimulation of VEGF (50 ng/mL) for 60 min. White arrow shows the disruption of the tight junction protein ZO-1 by treatment of VEGF. Blue arrow shows the disruption or
disappearance of the ZO-1 staining. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
level, as shown in Fig. 9A. In addition, the decreased occludin
expression in the retinas of diabetic mice was restored by 21l
treatment, as shown in Fig. 9B. Overall, the results of the cell-based
and in vivo assays confirmed that analog 21l prevents retinal vessel
leakage in a diabetic model as the results of its anti-apoptotic ac-
tivities and protective effects on TJs.
compounds, analog 21l was the most potent and exhibited prom-
ising in vivo activities for the treatment of vascular disorders. We
also established the structureeactivity relationship, focusing on the
constrained C17-side chains. Currently, further studies on the mo-
lecular mechanism and the functional genomics of the sterol ana-
logs are in progress.
3. Conclusion
4. Experimental protocols
In conclusion, we identified a series of glucal-conjugated sterols
with the constrained C17-side chains as novel vascular leakage
blocker through design, synthesis and biologically evaluation. An-
alogs 21l, n and r exhibited excellent anti-apoptotic activities and TJ
protective activities in the in vitro and in vivo models. Among these
4.1. Chemistry
Unless noted otherwise, all starting materials and reagents were
obtained from commercial suppliers and were used without further
purification. Tetrahydrofuran and Et2O were distilled from sodium
benzophenone ketyl. Dichloromethane and triethylamine were
freshly distilled from calcium hydride. All solvents used for routine
isolation of products and chromatography were reagent grade and
glass distilled. Reaction flasks were dried at 100 ꢀC before use, and
air- and moisture-sensitive reactions were performed under argon
atmosphere. Flash column chromatography was performed using
silica gel 60 (230e400 mesh, Merck) with the indicated solvents.
Thin-layer chromatography was performed using 0.25 mm silica
gel plates (Merck). Mass spectra were obtained using a VG trio-2
GCeMS instrument, and high resolution mass spectra were ob-
tained using a JEOL JMS-AX 505WA unit. 1H NMR spectra were
recorded on a JEOL JMS-LA 300, Bruker Analytik ADVANCE digital
400, ADVANCE digital 500 spectrometer in deuteriochloroform
(CDCl3). Chemical shifts are expressed in parts per million (ppm, d)
Fig. 8. Inhibitory effect of 21l against VEGF-induced retinal endothelial permeability in
downfield from tetramethylsilane and are referenced to the
deuterated solvent (CHCl3). 1H NMR data were reported in the order
of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet and/or multiple resonances), number of
protons, and coupling constant in hertz (Hz).
a mouse model. VEGF (50 ng) and/or 21l (10
mg/mL) was intravitreally administered to
one eye, and the vehicle (DMSO) with the same total volume of 2
mL was administered
to the contralateral eye. FITC-dextran was injected after 24 h into the ventricle. The
level of leakage was quantified using Image J. Permeabilities are presented as the
mean ꢁ S.D; ***P < 0.001.

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K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
Fig. 9. (A) Effect of 21l on diabetes-induced vascular retinal leakage. Diabetic mice were treated with 21l (10
mg/mL) by intravitreal injection in one eye. The contralateral eye was
treated with the same volume (2 L) of the vehicle (DMSO). FITC-dextran was injected after 24 h into the ventricle, and the retina was photographed. (B) Analog 21l restored the
m
normal occludin level in the eyes of a diabetic mouse, as assessed by Western blot analysis.
4.1.1. Synthetic procedure for the glucal conjugated sterols with
modofied C17-side chain based on the structures of ginsenosides Rg3
(1) and Rk1 (2)
reaction mixture was stirred until the bubbling was over at ambient
temperature. The reaction mixture was quenched with 2 N HCl, and
diluted with EtOAc. The organic phase was washed with H2O and
brine, dried over MgSO4, and concentrated in vacuo. Purification of
the residue via flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10 to 1:5) afforded 96 mg (15%) of the desired alcohol
4.1.1.1. (2S)-2-((3S,10R,13S,17S)-10,13-Dimethyl-3-(tetrahydro-2H-
pyran-2-yloxy)-2,3,4,7,8, 9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)-6-methylheptan-2-ol (5). To a so-
lution of Magnesium turning (22 mg, 0.92 mmol) with catalytic
amount of iodine in diethylether (1 mL) was added 1-bromo-4-
methyl pentane (0.17 mL, 1.15 mmol). The reaction mixture was
stirred under argon gas until complete consumption of the Mag-
nesium turning at ambient temperature. To a reaction mixture was
added 10 (306 mg, 0.76 mmol) in diethylether (1 mL) at ambient
temperature. The reaction mixture was stirred until the bubbling
was over at ambient temperature. The reaction mixture was
quenched with 2 N HCl, and diluted with EtOAc. The organic phase
was washed with H2O and brine, dried over MgSO4, and concen-
trated in vacuo. Purification of the residue via flash column chro-
matography on silica gel (EtOAc:n-Hexane ¼ 1:10 to 1:5) afforded
100 mg (27%) of the desired alcohol 5: 1H NMR (CDCl3, 500 MHz)
11: 1H NMR (CDCl3, 300 MHz)
d
5.35 (d, J ¼ 5.3 Hz, 1H), 3.53 (m, 1H),
2.32e2.24 (m, 2H), 2.11e0.86 (m, 42H). LR-MS (FAB) m/z 425
(M þ Naþ)
4.1.1.4. 22-((3S,10R,13S,17R)-10,13-Dimethyl-17-(6-methylhept-1-en-
2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta
[a]phenanthren-3-yloxy)tetrahydro-2H-pyran (12). To a solution of
the above alcohol 5 (50 mg, 0.1 mmol) in CH2Cl2 (3 mL) was added
Et3N (0.07 mL, 0.5 mmol) and methanesulfonyl chloride (0.015 mL,
0.2 mmol) at 0 ꢀC. The reaction mixture was stirred at 40 ꢀC for
overnight. The reaction mixture was quenched with saturated
aqueous NaHCO3 and diluted with CH2Cl2. The organic phase was
washed with H2O and brine, dried over MgSO4, and concentrated in
vacuo. Purification of the residue via flash column chromatography
on silica gel (EtOAc:n-Hexane ¼ 1:20) afforded 18.5 mg (39%) of 12:
d
5.33 (t, J ¼ 6.3 Hz, 1H), 4.70 (s, 1H), 3.90 (m, 1H), 3.53e3.45 (m,
2H), 2.35e0.82 (m, 49H). HRMS (FAB) calcd for C32H54O3Na
(M þ Naþ): 509.3971. Found 509.3952.
1H NMR (CDCl3, 500 MHz)
d 5.34 (m, 1H), 4.84 (s, 1H), 4.74 (s, 1H),
4.70 (m, 1H), 3.90 (m, 1H), 3.53e3.45 (m, 2H), 2.34e0.52 (m, 45H).
4.1.1.2. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((S)-2-hydroxy-6-
methylheptan-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (6). To a solution of 11 (Fluka, H6378)
4.1.1.5. (3S,10R,13S,17R)-10,13-Dimethyl-17-(6-methylhept-1-en-2-
yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta
[a]phenanthren-3-ol (13). To a solution of the above 12 (18 mg,
0.037 mmol) in MeOH (1.5 mL) was added PTSA (1.9 mg,
0.01 mmol). The reaction mixture was stirred for overnight. The
reaction mixture was quenched with H2O and diluted with EtOAc.
The organic phase was washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo. Purification of the residue via
flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10) afforded 14 mg (99%) of 13: 1H NMR (CDCl3,
(43 mg, 0.11 mmol) with tri-O-acetyl-D-glucal (81 mg, 0.30 mmol)
in diethylether (5 mL) was added boron trifluoride diethyl etherate
(0.012 mL, 0.10 mmol) at 0 ꢀC. The reaction mixture was stirred for
1 h at ambient temperature. The reaction mixture was quenched
with saturated aqueous NaHCO3 and diluted with EtOAc. The
organic phase was washed with H2O and brine, dried over MgSO4,
and concentrated in vacuo. Purification of the residue via flash
column chromatography on silica gel (EtOAc:n-Hexane ¼ 1:10)
300 MHz)
d
5.34 (m, 1H), 4.79 (d, J ¼ 28.2, 1H), 3.51 (m, 1H), 2.25e
afforded 7.6 mg (12%) of 6: 1H NMR (CDCl3, 500 MHz)
d
5.87e5.79
0.52 (m, 41H). LR-MS (FAB) m/z 385 (M þ Hþ).
(dd, J ¼ 27.1, 10.2 Hz, 2H), 5.38 (m, 1H), 5.27 (m, 1H), 5.16 (s, 1H),
4.24e4.09 (m, 3H), 3.55 (m, 1H), 2.41e0.76 (m, 47H), 0.69 (d, 1H,
J ¼ 6.75 Hz), 0.54(m, 1H). The mass spectral data of analog 6 gives
the same value of analog 8, because of dehydration.
4.1.1.6. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17R)-10,13-dimethyl-17-
(6-methylhept-1-en-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (14). To a solution of the above 12
(18 mg, 0.037 mmol) in MeOH (1.5 mL) was added PTSA (1.9 mg,
0.01 mmol). The reaction mixture was stirred for overnight. The
reaction mixture was quenched with H2O and diluted with EtOAc.
The organic phase was washed with H2O and brine, dried over
MgSO4, and concentrated in vacuo. Purification of the residue via
flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10) afforded 14 mg (99%) of 13. To a solution of 13
4.1.1.3. (3S,10R,13S,17S)-17-((S)-2-Hydroxy-6-methylheptan-2-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 (11). To a solution of Magnesium
turning (46 mg, 1.9 mmol) with catalytic amount of iodine in
diethylether (1 mL) was added 1-bromo-4-methyl pentane
(0.345 mL, 2.37 mmol). The reaction mixture was stirred under
argon gas until complete consumption of the Magnesium turning at
ambient temperature. To a reaction mixture was added 9 (500 mg,
1.58 mmol) in diethylether (1 mL) at ambient temperature. The
(16.6 mg, 0.043 mmol) with tri-O-acetyl-
D-glucal (35 mg,
0.13 mmol) in diethylether (2 mL) was added boron trifluoride

K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
191
diethyl etherate (0.016 mL, 0.13 mmol) at 0 ꢀC. The reaction mixture
was stirred for overnight at ambient temperature. The reaction
mixture was quenched with saturated aqueous NaHCO3 and diluted
with EtOAc. The organic phase was washed with H2O and brine,
dried over MgSO4, and concentrated in vacuo. Purification of the
residue via flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10) afforded 6.9 mg (27%) of 14: 1H NMR (CDCl3,
residue via flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10) afforded the corresponding oxime analogs.
4.1.2.1. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(methox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21a). Compound 21a (48 mg, 85%)
was afforded from 54 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
500 MHz)
d 5.86e5.78 (m, 2H), 5.35 (m, 1H), 5.28e5.13 (m, 3H),
4.24e4.09 (m, 3H), 3.59e3.51 (m, 1H), 2.41e0.52 (m, 46H). HRMS
d
5.91e5.76 (m, 2H), 5.33 (m, 1H), 5.26 (m, 1H), 5.14 (s, 1H), 4.24e
4.07 (m, 3H), 3.80 (s, 3H), 3.58e3.48 (m, 1H), 2.42e0.93 (m, 32H),
0.60 (s, 3H); 13C NMR (CDCl3, 600 MHz)
170.8, 170.3, 157.4, 140.8,
(FAB) calcd for C32H52O2Na (M þ Naþ): 491.3965. Found 491.3875.
d
4.1.1.7. 2-((3S,10R,13S,17R)-10,13-Dimethyl-17-((E)-6-methylhept-2-
en-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo-
penta[a]phenanthren-3-yloxy)tetrahydro-2H-pyran (7). To a solu-
tion of isohexyl triphenylphosphonium bromide, (900 mg,
2.1 mmol) in benzene (8 mL) was added potassium tert-butoxide
1 M in THF solution (2.1 mL, 2.1 mmol) under Ar atmosphere. The
reaction mixture was refluxed for 25 min, then pregnenolone
(200 mg, 0.63 mmol) in benzene (2 mL) was added to the solution.
The reaction mixture was refluxed for 3 h and cooled to room
temperature. Then the reaction mixture was quenched with H2O
and diluted with diethylether. The organic phase was washed with
H2O and brine, dried over MgSO4, and concentrated in vacuo. Pu-
rification of the residue via flash column chromatography on silica
gel (EtOAc:n-Hexane ¼ 1:10) afforded 54 mg (22%) of the 15. To a
solution of 15 (23 mg, 0.06 mmol) and p-toluenesulfonic acid (3 mg.
0.015 mmol) in CH2Cl2 (4 mL) was added 3,4-dihydro-2H-
pyran(0.04 mL, 0.45 mmol) at ambient temperature. The reaction
mixture was stirred for 3 h then quenched with H2O and diluted
with EtOAc. The organic phase was washed with H2O and brine,
dried over MgSO4, and concentrated in vacuo. Purification of the
residue via flash column chromatography on silica gel (EtOAc:n-
Hexane ¼ 1:10) afforded 16 mg (57%) of 7 :1H NMR (CDCl3,
128.9, 128.4, 121.6, 92.8, 78.1, 66.8, 65.4, 63.2, 61.1, 56.6, 56.2, 50.2,
43.6, 40.4, 38.6, 37.1, 36.7, 32.0, 31.8, 28.2, 24.2, 23.1, 21.0, 20.9, 20.8,
19.3, 15.6, 13.1. HR-MS (FAB) calcd for C32H48NO7 (M þ Hþ)
558.3431; found 558.3419.
4.1.2.2. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(ethox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21b). Compound 21b (46 mg, 96%)
was afforded from 44 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
d
5.91e5.76 (m, 2H), 5.33 (m, 1H), 5.26 (m, 1H), 5.14 (s, 1H), 4.24e
4.10 (m, 3H), 4.04 (q, J ¼ 20.9 Hz, 2H), 3.56 (m, 1H), 2.42e0.76 (m,
35H), 0.60 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8, 170.3, 157.0,
d
140.8, 128.9, 128.3, 121.7, 92.8, 78.1, 68.6, 66.8, 65.4, 63.2, 56.7, 56.2,
50.2, 43.6, 40.4, 38.6, 37.1, 36.7, 32.0, 31.8, 28.2, 24.2, 23.1, 21.0, 20.9,
20.8, 19.3, 15.7, 14.8, 13.2. HR-MS (FAB) calcd for C33H50NO7
(M þ Hþ) 572.3587; found 572.3580.
4.1.2.3. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-10,13-dimethyl-17-
((E)-1-(propoxyimino)ethyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21c). Compound 21c (54 mg, 100%)
was afforded from 48 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
500 MHz)
d
5.33 (t, J ¼ 2.4 Hz, 1H), 5.15 (t, J ¼ 6.9 Hz, 1H), 4.71 (m,
1H), 3.90 (m, 1H), 3.53e3.45 (m, 2H), 2.34e0.52 (m, 46H). HRMS
d
5.91e5.76 (m, 2H), 5.33 (d, J ¼ 5.1 Hz, 1H), 5.26 (dd, J ¼ 9.2, 1.1 Hz,
(FAB) calcd for C32H52O2Na (M þ Naþ): 491.3965. Found 491.3875.
1H), 5.15 (s,1H), 4.25e4.10 (m, 3H), 3.99e3.88 (m, 2H), 3.53 (m,1H),
2.42e0.82 (m, 37H), 0.60 (m, 3H); 13C NMR (CD3OD, 600 MHz)
4.1.1.8. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17R)-10,13-dimethyl-17-
((E)-6-methylhept-2-en-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (8). To a solution of 7 (317 mg,
d 173.2, 172.8, 159.2, 143.1, 130.6, 130.5, 123.5, 95.0, 80.4, 76.6, 69.2,
67.7, 65.3, 58.7, 58.3, 52.6, 45.7, 42.4, 40.7, 39.3, 38.7, 34.2, 33.7, 30.1,
26.1, 25.0, 24.4, 23.0, 21.6, 21.5, 20.6, 16.8, 14.5, 11.6. HR-MS (FAB)
calcd for C34H52NO7 (M þ Hþ) 586.3744; found 586.3734.
0.82 mmol) with tri-O-acetyl-D-glucal (672 mg, 2.47 mmol) in
diethylether (22 mL) was added boron trifluoride diethyl etherate
(0.3 mL, 2.39 mmol) at 0 ꢀC. The reaction mixture was stirred for 3 h
at ambient temperature. The reaction mixture was quenched with
saturated aqueous NaHCO3 and diluted with EtOAc. The organic
phase was washed with H2O and brine, dried over MgSO4, and
concentrated in vacuo. Purification of the residue via flash column
chromatography on silica gel (EtOAc:n-Hexane ¼ 1:10) afforded
4.1.2.4. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(iso-
propoxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21d). Compound 21d (32 mg, 84%)
was afforded from 34 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
d
5.91e5.77 (m, 2H), 5.34 (d, J ¼ 5.1 Hz, 1H), 5.27 (dd, J ¼ 9.4, 1.3 Hz,
1H), 5.15 (s, 1H), 4.31e4.10 (m, 4H), 3.54 (m, 1H), 2.42e0.85 (m,
38H), 0.61 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8, 170.3, 156.4,
270 mg (55%) of 6b: 1H NMR (CDCl3, 500 MHz)
d
5.83 (dd, J ¼ 27.3,
d
10.3 Hz, 2H), 5.34 (t, J ¼ 2.3 Hz, 1H), 5.26 (d, J ¼ 9.0 Hz, 1H), 5.15e
140.8, 128.9, 128.4, 121.7, 92.8, 78.1, 74.2, 66.8, 65.4, 63.2, 56.8, 56.2,
50.2, 43.6, 40.4, 38.6, 37.1, 36.7, 32.0, 31.8, 28.2, 24.3, 23.2, 21.8, 21.7,
21.0, 20.9, 20.8, 19.3, 15.8, 13.2. HR-MS (FAB) calcd for C34H52NO7
(M þ Hþ) 586.3744; found 586.3737.
5.14 (m, 2H), 4.25e4.14 (m, 3H), 3.54 (m, 1H), 2.51e0.80 (m, 44H),
0.52 (m, 2H); 13C NMR (CDCl3, 400 MHz)
d 170.8, 170.3, 140.8, 133.9,
128.9, 128.4, 125.8, 121.8, 92.8, 78.1, 66.8, 65.4, 63.2, 58.9, 56.2, 50.4,
43.4, 40.4, 39.2, 38.6, 37.2, 36.8, 32.2, 31.9, 28.2, 27.7, 25.9, 24.7, 24.3,
22.6, 22.5, 21.0, 20.9, 20.8, 19.3, 17.8, 12.9. HRMS (FAB) calcd for
4.1.2.5. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(allylox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21e). Compound 21e (63 mg, 86%)
was afforded from 66 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
C
37H56O6Na (M þ Naþ): 691.3975. Found 691.3961.
4.1.2. General synthetic procedure for preparation of oxime analogs
(21aer)
O-Alkyl or aryl hydroxylamine$HCl (1.2 equiv) was added to a
solution of 20 (1 equiv) in pyridine (3 mL). The reaction mixture
was refluxed for 3 h, cooled to room temperature, and quenched
with 2 N HCl, and diluted with diethylether. The organic phase was
dried over MgSO4, and concentrated in vacuo. Purification of the
d
5.95 (m, 1H), 5.86e5.76 (m, 2H), 5.33 (m, 1H), 5.28e5.19 (m, 2H),
5.15e5.11 (m, 2H), 4.52e4.50 (m, 2H), 4.24e4.12 (m, 3H), 3.53 (m,
1H), 2.42e0.60 (m, 35H); 13C NMR (CDCl3, 400 MHz)
170.8, 170.3,
d
157.6, 140.8, 134.9, 128.9, 128.4, 121.7, 116.6, 92.8, 78.1, 74.2, 66.8,
65.4, 63.2, 56.7, 56.2, 50.2, 43.6, 40.4, 38.6, 37.1, 36.7, 32.0, 31.8, 28.2,

192
K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
24.2, 23.1, 21.0, 20.9, 20.8, 19.3, 15.8, 13.2. HR-MS (FAB) calcd for
C
28.2, 25.2, 24.2, 23.1, 22.7, 22.6, 21.0, 20.9, 20.8,19.3,15.7,13.2. HR-MS
34H50NO7 (M þ Hþ) 584.3587; found 584.3599.
(FAB) calcd for C36H56NO7 (M þ Hþ) 614.4057; found 614.4035.
4.1.2.6. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-10,13-dimethyl-17-
((E)-1-(prop-2-ynyloxyimino)ethyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21f). Compound 21f (53 mg, 88%)
was afforded from 55 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
4.1.2.11. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-10,13-dimethyl-17-
((E)-1-(3-methylbut-2-enyloxyimino)ethyl)-2,3,4,7,8,9,10,11,12,-
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-
3,6-dihydro-2H-pyran-2-yl)methyl acetate (21k). Compound 21k
(49 mg, 80%) was afforded from 53 mg of ketone 20: 1H NMR
d
5.92e5.76 (m, 2H), 5.33 (d, J ¼ 4.95 Hz, 1H), 5.26 (m, 1H), 5.15 (s,
1H), 4.61 (d, J ¼ 2.4 Hz, 2H), 4.25e4.05 (m, 3H), 3.68e3.41 (m, 1H),
2.42e0.80 (m, 33H), 0.62 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8,
(300 MHz, CDCl3) d 5.91e5.76 (m, 2H), 5.40e5.32 (m, 2H), 5.26 (dd,
J ¼ 9.2, 1.1 Hz, 1H), 5.14 (s, 1H), 4.50 (d, J ¼ 7.0 Hz, 2H), 4.24e4.10 (m,
d
3H), 3.53 (m, 1H), 2.42e0.76 (m, 38H), 0.60 (s, 3H); 13C NMR (CDCl3,
170.4, 159.1, 140.8, 128.9, 128.4, 121.7, 92.8, 80.5, 78.1, 73.6, 66.8,
65.4, 63.2, 60.8, 56.7, 56.2, 50.2, 43.7, 40.4, 38.6, 37.2, 36.7, 32.0, 31.8,
28.2, 24.2, 23.1, 21.0, 20.9, 20.8, 19.3, 15.9, 13.2. HR-MS (FAB) calcd
for C34H48NO7 (M þ Hþ) 582.3431; found 582.3423.
400 MHz) d 170.8, 170.3, 157.2, 140.8, 136.9, 128.9, 128.4, 121.7, 120.8,
92.8, 78.1, 70.1, 66.8, 65.4, 63.2, 56.8, 56.2, 50.2, 43.6, 40.4, 38.6, 37.2,
36.7, 32.0, 31.8, 28.2, 25.9, 24.2, 23.1, 21.0, 20.9, 20.8, 19.3, 18.2, 15.8,
13.1. HR-MS (FAB) calcd for C36H54NO7 (M þ Hþ) 612.3900; found
612.3908.
4.1.2.7. ((2R,3S)-3-Acetoxy-6-((3S,10R,13,17S)-17-((E)-1-(butox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21g). Compound 21g (44 mg, 88%)
was afforded from 44 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
4.1.2.12. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(cyclo-
pentyloxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,-
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-
3,6-dihydro-2H-pyran-2-yl)methyl acetate (21l). Compound 21l
(40 mg, 95%) was afforded from 37 mg of ketone 20: 1H NMR
d
5.33(d, J ¼ 5.0 Hz, 1H) 5.27 (m, 1H), 5.34 (m, 1H), 5.26 (m, 1H), 5.15
(s, 1H), 4.25e4.10 (m, 3H), 4.02e3.97 (m, 2H), 3.59e3.48 (m, 1H),
2.42e0.77 (m, 39H), 0.60 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8,
(300 MHz, CDCl3)
d
5.85 (d, J ¼ 10.2 Hz, 1H), 5.80 (d, J ¼ 10.6 Hz, 1H)
d
5.34 (d, J ¼ 2.6 Hz, 1H), 5.27 (d, J ¼ 9.0 Hz, 1H), 5.15 (s, 1H), 4.61 (m,
1H), 4.24e4.10 (m, 3H), 3.55 (m, 1H), 2.42e0.82 (m, 41H), 0.62 (s,
170.3, 156.9, 140.8, 128.9, 128.4, 121.7, 92.8, 78.1, 73.0, 66.8, 65.4,
63.2, 56.7, 56.2, 50.2, 43.6, 40.4, 38.6, 37.2, 36.7, 32.0, 31.8, 31.4, 28.2,
24.3, 23.1, 21.0, 20.9, 20.8, 19.3, 19.2, 15.7, 14.0, 13.2. HR-MS (FAB)
calcd for C35H54NO7 (M þ Hþ) 600.3900; found 600.3905.
3H); 13C NMR (CDCl3, 300 MHz)
d 170.8, 170.3, 156.9, 140.8, 128.9,
128.4, 121.7, 92.8, 84.0, 78.1, 66.9, 65.4, 63.2, 56.8, 56.2, 50.2, 43.6,
40.4, 38.7, 37.2, 36.7, 32.2, 32.1, 32.0, 31.8, 28.2, 24.3, 23.9, 23.8, 23.2,
21.0, 20.9, 20.8, 19.3, 15.8, 13.2. HR-MS (FAB) calcd for C36H54NO7
(M þ Hþ) 612.3900; found 612.3901.
4.1.2.8. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(iso-
butoxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21h). Compound 21h (40 mg, 84%)
was afforded from 43 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
4.1.2.13. ((2R,3S)-3-Acetoxy-6-((10R,13S,17S)-17-((E)-1-(cyclo-
hexyloxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21m). Compound 21m (50 mg, 84%)
was afforded from 50 mg of ketone 20: 1H NMR (400 MHz, CDCl3)
d
5.91e5.76 (m, 2H), 5.33 (d, J ¼ 5.1 Hz, 1H), 5.26 (m, 1H), 5.15 (s,
1H), 4.25e4.10 (m, 3H), 3.85e3.71 (m, 2H), 3.53 (m, 1H), 2.42e0.77
(m, 39H), 0.60 (s, 3H); 13C NMR (CDCl3, 600 MHz)
170.8, 170.3,
d
d
5.85 (d, J ¼ 10.4 Hz,1H), 5.80 (m,1H), 5.34 (s,1H), 5.27 (d, J ¼ 9.2 Hz,
1H), 5.15 (s, 1H), 4.24e4.15 (m, 3H), 4.00 (m, 1H), 3.55 (m, 1H), 2.41e
0.84 (m, 42H), 0.61 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8, 170.3,
156.9,140.8,128.9, 128.4,121.7, 92.8. 79.9, 78.1, 66.8, 65.4, 63.2, 56.7,
56.2, 50.2, 43.7, 40.4, 38.6, 37.1, 36.7, 32.0, 31.8, 28.2, 28.0, 24.3, 23.1,
21.0, 20.9, 20.8, 19.3, 19.2, 19.2, 15.7, 13.2. HR-MS (FAB) calcd for
d
156.6, 140.8, 128.9, 128.4, 121.7, 92.8, 79.6, 78.1, 66.8, 65.4, 63.2, 56.9,
56.2, 50.2, 43.7, 40.4, 38.7, 37.2, 36.7, 32.1, 31.9, 31.8, 31.8, 28.2, 25.9,
24.3, 23.9, 23.9, 23.2, 21.0, 21.0, 20.9, 19.3, 15.9, 13.2. HR-MS (FAB)
calcd for C37H56NO7 (M þ Hþ) 626.4057; found 626.4050.
C
35H54NO7 (M þ Hþ) 600.3900; found 600.3917.
4.1.2.9. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-10,13-dimethyl-17-
((E)-1-(pentyloxyimino)ethyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21i). Compound 21i (40 mg, 84%)
was afforded from 43 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
4.1.2.14. ((2R,3S)-3-Acetoxy-6-((10R,13S,17S)-17-((E)-1-(cyclohex-2-
enyloxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21n). Compound 21n (47 mg, 80%)
was afforded from 50 mg of ketone 20: 1H NMR (400 MHz, CDCl3)
d
5.91e5.76 (m, 2H), 5.33 (d, J ¼ 4.8 Hz, 1H), 5.25 (m, 1H), 5.15 (s,
1H), 4.24e4.10 (m, 3H), 4.00e3.96 (m, 2H), 3.55 (m, 1H), 2.41e0.76
(m, 41H), 0.60 (s, 3H); 13C NMR (CDCl3, 400 MHz)
170.8, 170.3,
d
d
5.90e5.79 (m, 4H), 5.34 (d, J ¼ 4.7 Hz, 1H), 5.27 (d, J ¼ 9.1 Hz, 1H),
157.0, 140.7, 128.9, 128.3, 121.7, 92.8, 78.1, 73.3, 66.8, 65.3, 63.1, 56.7,
56.1, 50.1, 43.6, 40.3, 38.6, 37.1, 36.7, 32.0, 31.8, 28.9, 28.1, 28.1, 24.2,
23.1, 22.5, 21.0, 21.0, 20.8,19.3,15.8,14.0,13.2. HR-MS (FAB) calcd for
5.15 (s, 1H), 4.56 (br, 1H), 4.24e4.11 (m, 3H), 3.54 (m, 1H), 2.42e0.62
(m, 41H). HR-MS (FAB) calcd for C37H54NO7 (M þ Hþ) 624.3900;
found 624.3909.
C
36H56NO7 (M þ Hþ) 614.4057; found 614.4063.
4.1.2.15. ((2R,3S)-3-Acetoxy-6-((10R,13S,17S)-17-((E)-1-(cyclo-
hexylmethoxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,-
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-
3,6-dihydro-2H-pyran-2-yl)methyl acetate (21o). Compound 21o
(50 mg, 82%) was afforded from 50 mg of ketone 20: 1H NMR
4.1.2.10. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(iso-
pentyloxyimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,-
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-
3,6-dihydro-2H-pyran-2-yl)methyl acetate (21j). Compound 21j
(48 mg, 100%) was afforded from 41 mg of ketone 20: 1H NMR
(400 MHz, CDCl3)
d
5.86 (d, J ¼ 10.5 Hz, 1H), 5.80 (m, 1H), 5.35 (d,
(300 MHz, CDCl3)
d
5.91e5.76 (m, 2H), 5.33 (d, J ¼ 5.0 Hz, 1H), 5.26
J ¼ 4.9 Hz, 1H), 5.28 (m, 1H), 5.16 (s, 1H), 4.24e4.14 (m, 3H), 3.86e
(m,1H), 5.14 (m,1H), 4.28e4.12 (m, 3H), 4.02 (t, J ¼ 13.5 Hz, 2H), 3.53
3.76 (m, 2H), 3.55 (m, 1H), 2.41e0.82 (m, 43H), 0.61 (s, 3H); 13C NMR
(m, 1H), 2.42e0.82 (m, 41H), 0.60 (s, 3H); 13C NMR (CDCl3, 600 MHz)
(CDCl3, 600 MHz) d 170.8, 170.3, 156.8, 140.8, 128.9, 128.4, 121.7, 92.8,
d
170.8, 170.3, 156.9, 140.8, 128.9, 128.4, 121.7, 92.8, 78.1, 71.8, 66.8,
78.9, 78.1, 66.8, 65.4, 63.2, 56.7, 56.2, 50.2, 43.7, 40.4, 38.7, 37.6, 37.2,
36.7, 32.0, 31.8, 29.9, 29.8, 28.2, 26.7, 25.9, 25.9, 24.3, 23.1, 21.0, 20.9,
65.4, 63.2, 56.7, 56.2, 50.2, 43.6, 40.4, 38.6, 38.0, 37.1, 36.7, 32.0, 31.8,

K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
193
20.8, 19.3, 15.7, 13.2. HR-MS (FAB) calcd for C38H58NO7 (M þ Hþ)
4.2.3. Animals
640.4213; found 640.4208.
Eightweeks-old C57BL/6mice (Orient Co., Seoul, Korea) were
used for all the experiments. All mice were maintained in a laminar
airflow cabinet under specific pathogen-free conditions. They
received food and water ad libitum and were maintained on a 12 h
alternating light/dark cycle. All facilities are approved by AAALAC
(Association of Assessment and Accreditation of Laboratory Animal
Care), and all animal experiments were conducted under the
institutional guide-lines established for the Animal Core Facility at
Yonsei University, College of Medicine, Seoul, Korea.
4.1.2.16. ((2R,3S)-3-Acetoxy-6-((10R,13S,17S)-10,13-dimethyl-17-((E)-
1-(pyridin-3-ylmethoxyimino)ethyl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21p). Compound 21p (195 mg, 60%)
was afforded from 270 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
d
8.61 (s, 1H), 8.54 (d, J ¼ 4.4 Hz, 1H), 7.74 (d, 1H, J ¼ 7.7 Hz), 7.32 (m,
1H), 5.90e5.80 (m, 2H), 5.36 (m,1H), 5.29 (d, J ¼ 9.2 Hz,1H), 5.18 (m,
1H), 5.10 (s, 2H), 4.27e4.10 (m, 3H), 3.56 (m, 1H), 2.40e0.54 (m,
35H); 13C NMR (CDCl3, 500 MHz)
d 170.7, 170.2, 158.5, 149.6, 148.8,
4.2.4. Diabetic retinopathy mouse model
140.7, 135.7, 134.1, 128.8, 128.3, 123.1, 121.6, 92.7, 78.0, 72.6, 66.8,
65.3, 63.1, 56.7, 56.1, 50.1, 43.6, 40.3, 38.6, 37.1, 36.6, 32.0, 31.7, 28.1,
24.1, 23.0, 20.9, 20.9, 20.8, 19.3, 16.0, 13.1. HR-MS (FAB) calcd for
Diabetes was induced in mice by daily intraperitoneal injection
of streptozotocin (Sigma) (90 mg/kg in 0.1 M sodium citrate buffer
[pH 4.5]) for 4 consecutive days as modified from Satofuka et al.
(2009). The blood glucose level was checked from tail vein blood
samples using blood glucose test meter (Gluco DrÔ, All Medicus)
everyday. Three days after last streptozotocin injection, mice were
declared diabetic when the blood glucose concentrations exceeded
300 mg/dl. These mice were intravitreally injected with 1, 5, 10
of analog in total volume of 2 L to one eye and equal volume of
vehicle (DMSO) to contralateral eye (n ¼ 5/group). 24 h post in-
jection, retina leakage was quantified using fluorescein angiog-
raphy and Evans blue.
C
37H51N2O7 (M þ Hþ) 635.3696; found 635.3701.
4.1.2.17. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(hydrox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21q). Compound 21q (43 mg, 74%)
was afforded from 43 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
mg
m
d
5.88 (d, J ¼ 10.3 Hz, 1H), 5.79 (m, 1H), 5.33 (d, J ¼ 4.8 Hz, 1H), 5.27
(d, J ¼ 9.3 Hz, 1H), 5.15 (s, 1H), 4.24e4.10 (m, 3H), 3.54 (m, 1H),
2.42e0.91 (m, 33H), 0.61 (s, 3H); 13C NMR (CDCl3, 600 MHz)
170.8,
d
170.3, 158.7, 140.8, 128.9, 128.4, 121.6, 92.8, 78.1, 66.9, 65.4, 63.2,
56.7, 56.1, 50.2, 43.8, 40.4, 38.6, 37.1, 36.7, 32.0, 31.7, 28.2, 24.2, 23.1,
21.0, 20.9, 20.8, 19.3, 15.1, 13.1. HR-MS (FAB) calcd for C31H46NO7
(M þ Hþ) 544.3274; found 544.3265.
4.2.5. Measurement of retinal leakage using fluorescein
angiography and Evans blue
For fluorescein angiography, mice were injected with 3 mg of
40-kDa FITC-dextran (Sigma Aldrich, St Louis, USA) into the left
ventricle and the dye was allowed to circulate for 5 min. The
eyes were enucleated and immediately fixed in 4% para-
formaldehyde for 45 min. The retinas were then dissected out, cut
in the Maltese cross-configuration, and flat-mounted onto glass
slides. The retinas were viewed under fluorescence microscope
(Zeiss; magnification, ꢂ200). Quantification of retinal vascular
leakage was done using Evans blue dye as modified from Scheppke
et al. (2008). After 24 h of analogs injection, Evans blue (Sigma) was
injected into the tail vein of diabetic mice and the dye was allowed
to circulate for 1 h. Then, the mice were sacrificed and eyes were
immediately enucleated for retina isolation as described above. The
dissected retinas were dried in vacuum and weighed. Evans blue
dye was extracted from the retinas in formamide (Bioneer; 0.2 mL/
retina) at 70 ꢀC for 18 h followed by centrifugation at 14,000 rpm
for 60 min. The absorbance of Evans blue dye in the supernatant
was measured at 620 nm. Retinal vascular permeability
was measured as: [retinal Evans blue concentration (mg/mL)/
retinal weight (mg)]/[blood Evans blue concentration (mg/
mL) ꢂ circulation time (h)].
4.1.2.18. ((2R,3S)-3-Acetoxy-6-((3S,10R,13S,17S)-17-((E)-1-(benzylox-
yimino)ethyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6-dihydro-
2H-pyran-2-yl)methyl acetate (21r). Compound 21r (57 mg, 81%)
was afforded from 59 mg of ketone 20: 1H NMR (300 MHz, CDCl3)
d
7.34e7.29 (m, 4H), 7.26 (m, 1H), 5.82 (d, J ¼ 12.6 Hz, 1H), 5.80 (dt,
J ¼ 8.2, 1.7 Hz, 1H), 5.34 (m, 1H), 5.26 (dd, J ¼ 7.6, 0.8 Hz, 1H), 5.16 (s,
1H), 5.06 (s, 2H), 4.24e4.08 (m, 3H), 3.54 (m, 1H), 2.40e0.82 (m,
32H), 0.56 (s, 3H); 13C NMR (CDCl3, 400 MHz)
d 170.8, 170.3, 157.9,
140.8, 138.7, 128.9, 128.4, 128.2, 128.2, 127.9, 127.9, 127.4, 121.7, 92.8,
78.1, 75.3, 66.8, 65.4, 63.2, 56.7, 56.2, 50.2, 43.7, 40.4, 38.7, 37.2, 36.7,
32.0, 31.8, 28.2, 24.2, 23.1, 21.0, 21.0, 20.9, 19.4, 16.1, 13.1. HR-MS
(FAB) calcd for C38H52NO7 (M þ Hþ) 634.3744; found 634.3727.
4.2. Bioassay material and method
4.2.1. Cell culture
Human retinal ECs (HRECs) were purchased from Applied Cell
Biology Research Institute (Kirkland, Wash.) and passages 2e7
were used for experiments. Cells were grown in 2% gelatin-coated
dishes and maintained in endothelial cell basal medium (EBM-2,
CC-3156) containing EGM-2-kit (CC-4176) (Clonetics, Lonza-
Walkersville) and 20% FBS.
4.2.6. Western blot analysis
HRECs were lysed in 200 mL RIPA buffer. Lysates were centri-
fuged at 14,000 rpm for 15 min, and the supernatant was collected.
Proteins were separated by 8% sodium dodecyl sulfateepoly-
acrylamide gel electrophoresis and transferred to nitrocellulose
membranes. Membranes were incubated with rabbit anti-occludin
antibody (Zymed Laboratories Inc., CA); and goat anti-rabbit-HRP
(Thermoscientific, USA) was used as the secondary antibody.
Detection was performed with ECL Western blotting detection kit
(Amersham, UK) according to the manufacturer’s instructions.
4.2.2. Immunofluorescence microscopy
HRECs were fixed in 3.7% formaldehyde for 20 min at room
temperature. After fixation, the cells were permeabilized with 0.1%
Triton X-100 in phosphate buffered saline for 15 min at 4 ꢀC. Cells
were incubated with rabbit anti-occludin antibody (Zymed Labo-
ratories Inc., CA) overnight at 4 ꢀC. The cells were then incubated
with Anti-rabbit-Alexa Fluor 488 (Molecular Probes) for 1 h at room
temperature. Actin filaments were visualized by staining with
rhodamineephalloidin (Molecular Probes, OR) for 30 min. Cells
were mounted using DAKO mounting reagent and observed by
fluorescence microscopy (Zeiss; magnification, ꢂ400).
4.2.7. Statistical analysis
Data are presented as mean ꢁ S.D, and statistical comparisons
between groups were performed by one-way ANOVA followed by
Student’s t test.

194
K. Kim et al. / European Journal of Medicinal Chemistry 75 (2014) 184e194
[19] S.A. Khan, A.M. Asiri, K. Saleem, Synthesis and biological evaluation of new
Acknowledgments
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16 (2012) 7e11.
This work is supported by a grant from the Bio & Medical
Technology Development Program of the National Research Foun-
dation of Korea (NRF) funded by the Ministry of Education , Science
and Technology (MEST) (NRF-2011-0019267) and by the National
Research Foundation of Korea (NRF) grant for the Global Core
Research Center (GCRC) funded by the Korea Government (MSIP)
(No. 2011-0030001).
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